The present application is an international application claiming the priority benefit of U.S. application Ser. No. 10/904,588, which was filed on Nov. 17, 2004, as a continuation-in-part application of U.S. application Ser. No. 09/828,313, which was filed on Apr. 6, 2001 claiming the priority benefit of U.S. Provisional Patent Application Ser. No. 60/196,001 filed Apr. 7, 2000. The U.S. application Ser. No. 10/904,588 is also a continuation-in-part application of U.S. application Ser. No. 10/292,408, which was filed on Nov. 12, 2002, claiming the priority benefit of U.S. Provisional Patent Application Ser. No. 60/346,096 filed Nov. 9, 2001. The entire contents of the applications are hereby incorporated by reference.
1. Field of the Invention
This invention relates generally to nucleic acid sequences encoding polypeptides that are associated with abiotic stress responses and abiotic stress tolerance in plants. In particular, this invention relates to nucleic acid sequences encoding polypeptides that confer drought, cold, and/or salt tolerance to plants.
2. Background Art
Abiotic environmental stresses, such as drought stress, salinity stress, heat stress, and cold stress, are major limiting factors of plant growth and productivity. Crop losses and crop yield losses of major crops such as soybean, rice, maize (corn), cotton, and wheat caused by these stresses represent a significant economic and political factor and contribute to food shortages in many underdeveloped countries.
Plants are typically exposed during their life cycle to conditions of reduced environmental water content. Most plants have evolved strategies to protect themselves against these conditions of desiccation. However, if the severity and duration of the drought conditions are too great, the effects on development, growth, and yield of most crop plants are profound. Continuous exposure to drought conditions causes major alterations in the plant metabolism, which ultimately lead to cell death and consequently yield losses.
Developing stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of these problems. However, traditional plant breeding strategies to develop new lines of plants that exhibit resistance (tolerance) to these types of stresses are relatively slow and require specific resistant lines for crossing with the desired line. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Additionally, the cellular processes leading to drought, cold, and salt tolerance in model drought- and/or salt-tolerant plants are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways. This multi-component nature of stress tolerance has not only made breeding for tolerance largely unsuccessful, but has also limited the ability to genetically engineer stress tolerant plants using biotechnological methods.
Drought stresses, heat stresses, cold stresses, and salt stresses have a common theme important for plant growth and that is water availability. As discussed above, most plants have evolved strategies to protect themselves against conditions of desiccation; however, if the severity and duration of the drought conditions are too great, the effects on plant development, growth and yield of most crop plants are profound. Furthermore, most of the crop plants are very susceptible to higher salt concentrations in the soil. Because high salt content in some soils results in less water being available for cell intake, high salt concentration has an effect on plants similar to the effect of drought on plants. Additionally, under freezing temperatures, plant cells lose water as a result of ice formation that starts in the apoplast and withdraws water from the symplast. A plant's molecular response mechanisms to each of these stress conditions are common, and protein kinases, such as casein kinases, play an essential role in these molecular mechanisms.
Protein kinases represent a superfamily, and the members of this superfamily catalyze the reversible transfer of a phosphate group of ATP to serine, threonine, and tyrosine amino acid side chains on target polypeptides. Protein kinases are primary elements in signaling processes in plants and have been reported to play crucial roles in perception and transduction of signals that allow a cell (and the plant) to respond to environmental stimuli. In particular, casein kinase I proteins are monomeric serine/threonine type protein kinases that contain a highly conserved central kinase domain. Members of this family have divergent N-terminal and C-terminal extensions. The N-terminal region is responsible for substrate recognition and the C-terminal extension is important for the interaction of the kinase with substrates. The C-terminal extension also is thought to be important for mediating regulation through autophosphorylation (Gross and Anderson, 1998 Cell Signal 10:699-711; Graves and Roach, 1995, J Biol Chem 270:21689-21694).
Although some genes that are involved in stress responses and water use efficiency in plants have been characterized, the characterization and cloning of plant genes that confer stress tolerance and water use efficiency remains largely incomplete and fragmented. For example, certain studies have indicated that drought and salt stress in some plants may be due to additive gene effects, in contrast to other research that indicates specific genes are transcriptionally activated in vegetative tissue of plants under osmotic stress conditions. Although it is generally assumed that stress-induced proteins have a role in tolerance, direct evidence is still lacking, and the functions of many stress-responsive genes are unknown.
There is a fundamental physiochemically-constrained trade-off, in all terrestrial photosynthetic organisms, between CO 2 absorption and water loss (Taiz and Zeiger 1991 Plant Physiology, Benjamin/Cummings Publishing Co, p 94). CO 2 needs to be in aqueous solution for the action of CO 2 fixation enzymes such as Rubisco (Ribulose 1,5-bisphosphate Carboxylase/Oxygenase) and PEPC (Phosphoenolpyruvate carboxylase). As a wet cell surface is required for CO 2 diffusion, evaporation will inevitably occur when the humidity is below 100% (Taiz and Zeiger 1991 Plant Physiology, Benjamin/Cummings Publishing Co p 257). Plants have numerous physiological mechanisms to reduce water loss (e.g. waxy cuticles, stomatal closure, leaf hairs, sunken stomatal pits). As these barriers do not discriminate between water and CO 2 flux, these water conservation measures will also act to increase resistance to CO 2 uptake (Kramer 1983 Water Relations of Plants, Academic Press p 305). Photosynthetic CO 2 uptake is absolutely required for plant growth and biomass accumulation in photoautotrophic plants. Water Use Efficiency (WUE) is a parameter frequently used to estimate the trade off between water consumption and CO 2 uptake/growth (Kramer 1983 Water Relations of Plants, Academic Press p 405). WUE has been defined and measured in multiple ways. One approach is to calculate the ratio of whole plant dry weight, to the weight of water consumed by the plant throughout its life (Chu et al 1992 Oecologia 89:580). Another variation is to use a shorter time interval when biomass accumulation and water use are measured (Mian et al 1998 Crop Sci. 38:390). Often measurements from restricted parts of the plant are used, for example, measuring only aerial growth and water use (Nienhuis et al 1994 Amer J Bot 81:943). WUE has also been defined as the ratio of CO 2 uptake to water vapor loss from a leaf or portion of a leaf, often measured over a very short time period (seconds/minutes) (Kramer 1983 Water Relations of Plants, Academic Press p 406). The ratio of 13 C/ 12 C fixed in plant tissue, and measured with an isotope ratio mass-spectrometer, has also been used to estimate WUE in plants using C3 photosynthesis (Martin et al 1999 Crop Sci. 1775).
An increase in WUE is informative about the relatively improved efficiency of growth and water consumption, but on its own it does not describe which of these two processes (or both) have changed. In selecting traits for improving crops, an increase in WUE due to a decrease in water use, without a change in growth would have particular merit in an irrigated agricultural system where the water input costs were high. An increase in WUE driven mainly by an increase in growth without a corresponding jump in water use would have applicability to all agricultural systems. In many agricultural systems where water supply is not limiting, an increase in growth, even if it came at the expense of an increased water use (i.e. no change in WUE), could also increase yield. Therefore new methods to increase both WUE and biomass accumulation are required to improve agricultural productivity. As WUE integrates many physiological processes relating to primary metabolism and water use, it is typically a highly polygenic trait with a large genotype by environment interaction (Richards et al 2002 Crop Sci 42:111). For these and other reasons few attempts to select for WUE changes in traditional breeding programs have been successful.
There is a need, therefore, to identify genes expressed in stress tolerant plants and plants that are efficient in water use that have the capacity to confer stress tolerance and water use efficiency to its host plant and to other plant species. Newly generated stress tolerant plants will have many advantages, such as an increased range in which the crop plants can be cultivated, by for example, decreasing the water requirements of a plant species. Other desirable advantages include increased resistance to lodging, the bending of shoots or stems in response to wind, rain, pests, or disease.
This invention fulfills in part the need to identify new, unique casein kinases capable of conferring stress tolerance to plants upon over-expression. The present invention describes a novel genus of Casein Kinase Stress-Related Polypeptides (CKSRPs) and CKSRP coding nucleic acids that are important for modulating a plant's response to an environmental stress. More particularly, overexpression of these CKSRP coding nucleic acids in a plant results in the plant's increased tolerance to an environmental stress.
Therefore, the present invention includes an isolated plant cell comprising a CKSRP coding nucleic acid, wherein expression of the nucleic acid sequence in the plant cell results in increased tolerance to environmental stress as compared to a wild type variety of the plant cell. Preferably, the CKSRP is from Physcomitrella patens, Saccharomyces cerevisiae , or Brassica napus . Namely, described herein are the Physcomitrella patens Casein Kinase-4 (PpCK-4 or EST 289), Physcomitrella patens Casein Kinase-1 (PpCK-1 or EST 194), Physcomitrella patens Casein Kinase-2 (PpCK-2 or EST 263), Physcomitrella patens Protein Kinase-4 (PpPK-4 or EST 142), Saccharomyces cerevisiae Casein Kinase-1 (ScCK-1 or ORF 760), Brassica napus Casein Kinase-1 (BnCK-1), Brassica napus Casein Kinase-2 (BnCK-2). Brassica napus Casein Kinase-3 (BnCK-3), Brassica napus Casein Kinase-4 (BnCK-4), and Brassica napus Casein Kinase-5 (BnCK-5).
The invention provides in some embodiments that the CKSRP and coding nucleic acid are those that are found in members of the genus Physcomitrella, Saccharomyces or Brassica . In another preferred embodiment, the nucleic acid and polypeptide are from a Physcomitrella patens or Brassica napus plant or a Saccharomyces cerevisiae yeast. The invention provides that the environmental stress can be salinity, drought, temperature, metal, chemical, pathogenic and oxidative stresses, or combinations thereof. In preferred embodiments, the environmental stress can be selected from one or more of the group consisting of drought, high salt, and low temperature.
The invention further provides a seed produced by a transgenic plant transformed by a CKSRP coding nucleic acid, wherein the plant is true breeding for increased tolerance to environmental stress as compared to a wild type variety of the plant.
The invention further provides an agricultural product produced by any of the below-described transgenic plants, plant parts, or seeds. The invention further provides an isolated CKSRP as described below. The invention further provides an isolated CKSRP coding nucleic acid, wherein the CKSRP coding nucleic acid codes for a CKSRP as described below.
The invention further provides an isolated recombinant expression vector comprising a CKSRP coding nucleic acid as described below, wherein expression of the vector in a host cell results in increased tolerance to environmental stress as compared to a wild type variety of the host cell. The invention further provides a host cell containing the vector and a plant containing the host cell.
The invention further provides a method of producing a transgenic plant with a CKSRP coding nucleic acid, wherein expression of the nucleic acid in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant comprising: (a) transforming a plant cell with an expression vector comprising a CKSRP coding nucleic acid, and (b) generating from the plant cell a transgenic plant with an increased tolerance to environmental stress as compared to a wild type variety of the plant. In preferred embodiments, the CKSRP and CKSRP coding nucleic acid are as described below.
The present invention further provides a method of identifying a novel CKSRP, comprising (a) raising a specific antibody response to a CKSRP, or fragment thereof, as described below; (b) screening putative CKSRP material with the antibody, wherein specific binding of the antibody to the material indicates the presence of a potentially novel CKSRP; and (c) identifying from the bound material a novel CKSRP in comparison to known CKSRP. Alternatively, hybridization with nucleic acid probes as described below can be used to identify novel CKSRP nucleic acids.
The present invention also provides methods of modifying stress tolerance of a plant comprising, modifying the expression of a CKSRP nucleic acid in the plant, wherein the CKSRP is as described below. The invention provides that this method can be performed such that the stress tolerance is either increased or decreased. Preferably, stress tolerance is increased in a plant via increasing expression of a CKSRP nucleic acid.
FIG. 1 shows the results of a drought stress test with over-expressing PpCK-1 transgenic plants and wild-type Arabidopsis lines. The transgenic lines display a tolerant phenotype. Individual transformant lines are shown.
FIG. 2 shows the results of a freezing stress test with over-expressing PpCK-1 transgenic plants and wild-type Arabidopsis lines. The transgenic lines display a tolerant phenotype. Individual transformant lines are shown.
FIG. 3 shows the results of a drought stress test with over-expressing PpCK-2 transgenic plants and wild-type Arabidopsis lines. The transgenic lines display a tolerant phenotype. Individual transformant lines are shown.
FIG. 4 shows a diagram illustrating the relative homology of the disclosed Physcomitrella patens and Saccharomyces cerevisiae casein kinases and other known casein kinases.
FIG. 5 shows an alignment of the amino acid sequences of the five disclosed Physcomitrella patens and Saccharomyces cerevisiae casein kinases with the amino acid sequences of other known casein kinases (SEQ ID NOS 10, 47-48, 6, 4, 2, 49-52, 8, and 53, respectively in order of appearance). Amino acid residues that are conserved among each of the sequences, and those amino acid residues that are either identical or similar over some or all of the sequences, are indicated with shading.
FIG. 6 shows a diagram illustrating the relative homology of the five disclosed Physcomitrella patens and Saccharomyces cerevisiae casein kinases with the disclosed Brassica napus , linseed, wheat, barley, sunflower and soybean casein kinases.
FIG. 7 shows an alignment of the amino acid sequence of the five disclosed Physcomitrella patens and Saccharomyces cerevisiae casein kinases with the disclosed Brassica napus , linseed, wheat, barley, sunflower and soybean casein kinases. For correlation of gene ID and SEQ ID NO see Table A. The figure also indicates the consensus sequence of casein kinase I based on the aligned sequences. Amino acid residues that are conserved among each of the sequences, and those amino acid residues that are either identical or similar over some or all of the sequences, are indicated with shading.
FIG. 8: PpPK-4, PpCK-4, PpCK-2 or PpCK-1 were overexpressed in Arabidopsis thaliana under the control of a constitutive promoter. The transgenic lines were assayed for relative water use efficiency (WUE), dry weight (DW), and plant water use (E) (% difference from controls).
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein. However, before the present compounds, compositions, and methods are disclosed and described, it is to be understood that this invention is not limited to specific nucleic acids, specific polypeptides, specific cell types, specific host cells, specific conditions, or specific methods, etc., as such may, of course, vary, and the numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. In particular, the designation of the amino acid sequences as polypeptide “Casein Kinase Stress-Related Polypeptides” (CKSRPs), in no way limits the functionality of those sequences.
The present invention describes a novel genus of CKSRPs and CKSRP coding nucleic acids that are important for modulating a plant's response to an environmental stress. More particularly, over-expression of these CKSRP coding nucleic acids in a plant results in the plant's increased tolerance to an environmental stress. Representative members of the CKSRP genus include, but are not limited to, PpCK-1, PpCK-2, PpCK-4, PpPK-4, ScCK-1, BnCK-1, BnCK-2, BnCK-3, BnCK-4, and BnCK-5. In a preferred embodiment, all members of the genus are biologically active casein kinases.
Accordingly, the present invention encompasses CKSRP polynucleotide and polypeptide sequences and their use for increasing a plant's tolerance to an environmental stress. In one embodiment, the CKSRP sequences are from a plant, preferably a Physcomitrella plant or a Brassica plant, and more preferably a Physcomitrella patens plant or a Brassica napus plant. In another embodiment, the CKSRP sequences include PpCK-1 (SEQ ID NOS:3 and 4), PpCK-2 (SEQ ID NOS:5 and 6), PpCK-4 (SEQ ID NOS:1 and 2), PpPK-4 (SEQ ID NOS:7 and 8), ScCK-1 (SEQ ID NOS:9 and 10), BnCK-1 (SEQ ID NOS:11 and 12), BnCK-2 (SEQ ID NOS:13 and 14), BnCK-3 (SEQ ID NOS:15 and 16), BnCK-4 (SEQ ID NOS:17 and 18), and BnCK-5 (SEQ ID NOS:19 and 20). The disclosed Physcomitrella patens CKSRP sequences and the disclosed Saccharomyces cerevisiae CKSRP sequence have significant percent identity to known casein kinases as is indicated in Table 1.
| TABLE 1 | ||||||||||
| ORF | ORF | EST | EST | EST | EST | EST | ||||
| 760 | 760 | EST | 142 | EST | 194 | 263 | 263 | EST | 289 | |
| Sim | Iden | 142 | Iden | 194 | Iden | Sim | Iden | 289 | Iden | |
| (%) | (%) | Sim (%) | (%) | Sim (%) | (%) | (%) | (%) | Sim (%) | (%) | |
| ORF 760 | 32.9 | 25.4 | 33.4 | 24.7 | 43.3 | 34.5 | 42.0 | 32.4 | ||
| AAB68417 | 50.9 | 41.2 | 36.3 | 26.0 | 36.8 | 26.5 | 42.7 | 32.8 | 41.9 | 31.4 |
| AAA35230 | 48.8 | 39.4 | 36.2 | 25.8 | 36.9 | 26.1 | 42.3 | 31.3 | 43.2 | 31.1 |
| EST 263 | 43.3 | 34.5 | 40.4 | 32.5 | 40.5 | 33.2 | 80.8 | 74.0 | ||
| EST 194 | 33.4 | 24.7 | 90.2 | 86.9 | 40.5 | 33.2 | 42.0 | 32.4 | ||
| EST 289 | 42.0 | 32.4 | 41.3 | 33.3 | 41.7 | 33.7 | 80.8 | 74.0 | ||
| AAH06490 | 43.7 | 33.4 | 44.2 | 34.6 | 43.0 | 33.8 | 63.7 | 53.8 | 63.9 | 53.4 |
| AAH03558 | 42.4 | 33.0 | 43.7 | 34.5 | 44.1 | 34.9 | 63.2 | 54.5 | 65.0 | 54.2 |
| AAH08717 | 39.7 | 31.5 | 50.7 | 40.0 | 51.7 | 40.7 | 53.3 | 45.8 | 54.1 | 46.7 |
| AAD26525 | 41.6 | 32.8 | 43.6 | 32.3 | 42.5 | 31.9 | 48.9 | 38.4 | 49.0 | 38.0 |
| EST 142 | 32.9 | 25.4 | 90.2 | 86.9 | 40.4 | 32.5 | 41.3 | 33.3 | ||
| AJ487966 | 44.8 | 33.6 | 40.1 | 30.7 | 40.5 | 30.9 | 72.0 | 58.6 | 68.7 | 55.9 |
The present invention provides a transgenic plant cell transformed by a CKSRP coding nucleic acid, wherein expression of the nucleic acid sequence in the plant cell results in increased tolerance to an environmental stress as compared to a wild type variety of the plant cell. The invention further provides transgenic plant parts and transgenic plants containing the plant cells described herein. Plant parts include, but are not limited to, stems, roots, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores, and the like. In one embodiment, the transgenic plant is male sterile. Also provided is a plant seed produced by a transgenic plant transformed by a CKSRP coding nucleic acid, wherein the seed contains the CKSRP coding nucleic acid, and wherein the plant is true breeding for increased tolerance to environmental stress as compared to a wild type variety of the plant. The invention further provides a seed produced by a transgenic plant expressing a CKSRP, wherein the seed contains the CKSRP, and wherein the plant is true breeding for increased tolerance to environmental stress as compared to a wild type variety of the plant. The invention also provides an agricultural product produced by any of the below-described transgenic plants, plant parts, and plant seeds. Agricultural products include, but are not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.
As used herein, the term “variety” refers to a group of plants within a species that share constant characters that separate them from the typical form and from other possible varieties within that species. While possessing at least one distinctive trait, a variety is also characterized by some variation between individuals within the variety, based primarily on the Mendelian segregation of traits among the progeny of succeeding generations. A variety is considered “true breeding” for a particular trait if it is genetically homozygous for that trait to the extent that, when the true-breeding variety is self-pollinated, a significant amount of independent segregation of the trait among the progeny is not observed. In the present invention, the trait arises from the transgenic expression of one or more DNA sequences introduced into a plant variety.
The present invention describes for the first time that the Physcomitrella patens CKSRPs, PpCK-1, PpCK-2, PpPK-4, and PpPK-4; Saccharomyces cerevisiae CKSRP ScCK-1; and Brassica napus CKSRPs, BnCK-1, BnCK-2, BnCK-3, BnCK-4, and BnCK-5 are useful for increasing a plant's tolerance to environmental stress. As used herein, the term polypeptide refers to a chain of at least four amino acids joined by peptide bonds. The chain may be linear, branched, circular, or combinations thereof. Accordingly, the present invention provides isolated CKSRPs selected from PpCK-1, PpCK-2, PpCK-4, PpPK-4, ScCK-1, BnCK-1, BnCK-2, BnCK-3, BnCK-4, and BnCK-5, and homologs thereof. In preferred embodiments, the CKSRP is selected from: 1) Physcomitrella patens Casein Kinase-1 (PpCK-1) polypeptide as defined in SEQ ID NO:4) Physcomitrella patens Casein Kinase-2 (PpCK-2) polypeptide as defined in SEQ ID NO:6) Physcomitrella patens Casein Kinase-4 (PpCK-4) polypeptide as defined in SEQ ID NO:1) Physcomitrella patens Protein Kinase-4 (PpPK-4) polypeptide as defined in SEQ ID NO:8) Saccharomyces cerevisiae Casein Kinase-1 (ScCK-1) polypeptide as defined in SEQ ID NO:10) Brassica napus Casein Kinase-1 (BnCK-1) polypeptide as defined in SEQ ID NO:12; Brassica napus Casein Kinase-2 (BnCK-2) polypeptide as defined in SEQ ID NO:14; Brassica napus Casein kinase-3 (BnCK-4) polypeptide as defined in SEQ ID NO:16; Brassica napus Casein kinase-4 (BnCK-4) polypeptide as defined in SEQ ID NO:18; Brassica napus Casein Kinase-5 (BnCK-5) polypeptide as defined in SEQ ID NO:20; and homologs and orthologs thereof. Homologs and orthologs of the amino acid sequences are defined below.
The CKSRPs of the present invention are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression vector (as described below), the expression vector is introduced into a host cell (as described below), and the CKSRP is expressed in the host cell. The CKSRP can then be isolated from the cells by an appropriate purification scheme using standard polypeptide purification techniques. For the purposes of the invention, the term “recombinant polynucleotide” refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering. Examples include any cloned polynucleotide, and polynucleotides that are linked or joined to heterologous sequences. The term “recombinant” does not refer to alterations to polynucleotides that result from naturally occurring events, such as spontaneous mutations. Alternative to recombinant expression, a CKSRP, or peptide thereof, can be synthesized chemically using standard peptide synthesis techniques. Moreover, native CKSRP can be isolated from cells (e.g., Physcomitrella patens, Saccharomyces cerevisiae , or Brassica napus cells), for example using an anti-CKSRP antibody, which can be produced by standard techniques utilizing a CKSRP or fragment thereof.
As used herein, the term “environmental stress” refers to sub-optimal conditions associated with salinity, drought, temperature, metal, chemical, pathogenic, and oxidative stresses, or combinations thereof. In preferred embodiments, the environmental stress can be selected from one or more of the group consisting of salinity, drought, or temperature, or combinations thereof, and in particular, can be selected from one or more of the group consisting of high salinity, low water content, or low temperature. It is also to be understood that as used in the specification and in the claims, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “a cell” can mean that at least one cell can be utilized. As also used herein, the term “water use efficiency” refers to the amount of organic matter produced by a plant divided by the amount of water used by the plant in producing it, i.e. the dry weight of a plant in relation to the plant's water use.
As also used herein, the term “nucleic acid” and “polynucleotide” refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. These terms also encompass untranslated sequence located at both the 3′ and 5′ ends of the coding region of the gene: at least about 1000 nucleotides of sequence upstream from the 5′ end of the coding region and at least about 200 nucleotides of sequence downstream from the 3′ end of the coding region of the gene. Less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2′-hydroxy in the ribose sugar group of the RNA can also be made. The antisense polynucleotides and ribozymes can consist entirely of ribonucleotides, or can contain mixed ribonucleotides and deoxyribonucleotides. The polynucleotides of the invention may be produced by any means, including genomic preparations, cDNA preparations, in vitro synthesis, RT-PCR, and in vitro or in vivo transcription.
An “isolated” nucleic acid molecule is one that is substantially separated from other nucleic acid molecules, which are present in the natural source of the nucleic acid (i.e., sequences encoding other polypeptides). Preferably, an “isolated” nucleic acid is free of some of the sequences, which naturally flank the nucleic acid (i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) in its naturally occurring replicon. For example, a cloned nucleic acid is considered isolated. In various embodiments, the isolated CKSRP nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g., a Physcomitrella patens cell, a Saccharomyces cerevisiae cell, or a Brassica napus cell). A nucleic acid is also considered isolated if it has been altered by human intervention, or placed in a locus or location that is not its natural site, or if it is introduced into a cell by agroinfection. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be free from some of the other cellular material with which it is naturally associated, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
Specifically excluded from the definition of “isolated nucleic acids” are: naturally-occurring chromosomes (such as chromosome spreads), artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies. Also specifically excluded are the above libraries wherein a specified nucleic acid makes up less than 5% of the number of nucleic acid inserts in the vector molecules. Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including whole cell preparations that are mechanically sheared or enzymatically digested). Even further specifically excluded are the whole cell preparations found as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis wherein the nucleic acid of the invention has not further been separated from the heterologous nucleic acids in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a P. patens CKSRP cDNA can be isolated from a P. patens library using all or a portion of one of the sequences disclosed herein. Moreover, a nucleic acid molecule encompassing all or a portion of one of the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence. For example, mRNA can be isolated from plant cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al., 1979, Biochemistry 18:5294-5299), and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19. A nucleic acid molecule of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecule so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to a CKSRP nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19. These cDNAs may comprise sequences encoding the CKSRPs, (i.e., the “coding region”), as well as 5′ untranslated sequences and 3′ untranslated sequences. Alternatively, the nucleic acid molecules of the present invention can comprise only the coding region of any of the sequences in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ. ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or can contain whole genomic fragments isolated from genomic DNA. The present invention also includes CKSRP coding nucleic acids that encode CKSRPs as described herein. Preferred is a CKSRP coding nucleic acid that encodes a CKSRP selected from the group consisting of PpCK-1 (SEQ ID NO:4), PpCK-2 (SEQ ID NO:6), PpCK-4 (SEQ ID NO:2), PpPK-4 (SEQ ID NO:8), ScCK-1 (SEQ ID NO:10), BnCK-1 (SEQ ID NO:12), BnCK-2 (SEQ ID NO:14), BnCK-3 (SEQ ID NO:16), BnCK-4 (SEQ ID NO:18), and BnCK-5 (SEQ ID NO:20).
Moreover, the nucleic acid molecule of the invention can comprise a portion of the coding region of one of the sequences in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19, for example, a fragment that can be used as a probe or primer or a fragment encoding a biologically active portion of a CKSRP. The nucleotide sequences determined from the cloning of the CKSRP genes from Physcomitrella patens, Saccharomyces cerevisiae , and Brassica napus allow for the generation of probes and primers designed for use in identifying and/or cloning CKSRP homologs in other cell types and organisms, as well as CKSRP homologs from other mosses and related species. The portion of the coding region can also encode a biologically active fragment of a CKSRP.
As used herein, the term “biologically active portion of” a CKSRP is intended to include a portion, e.g., a domain/motif, of a CKSRP that participates in modulation of stress tolerance in a plant, and more preferably, drought tolerance or salt tolerance. For the purposes of the present invention, modulation of stress tolerance refers to at least a 10% increase or decrease in the stress tolerance of a transgenic plant comprising a CKSRP expression cassette (or expression vector) as compared to the stress tolerance of a non-transgenic control plant. Methods for quantitating stress tolerance are provided at least in Example 7 below. In a preferred embodiment, the biologically active portion of a CKSRP increases a plant's tolerance to an environmental stress.
Biologically active portions of a CKSRP include peptides comprising amino acid sequences derived from the amino acid sequence of a CKSRP, e.g., an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or the amino acid sequence of a polypeptide identical to a CKSRP, which include fewer amino acids than a full length CKSRP or the full length polypeptide which is identical to a CKSRP, and exhibit at least one activity of a CKSRP. Typically, biologically active portions (e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100, or more amino acids in length) comprise a domain or motif with at least one activity of a CKSRP. Moreover, other biologically active portions, in which other regions of the polypeptide are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portion of a CKSRP includes one or more selected domains/motifs, or portions thereof, having biological activity such as the conserved central kinase domain, as is shown in FIGS. 5 and 7. In a preferred embodiment, the conserved central kinase domain comprises four conserved regions, wherein the first region commences with a glycine residue at position 1 and has a glycine at position 3 and a phenylalanine residue at position 5; the second region is downstream from the first region, commences with a valine residue at position 1, and has a lysine at position 4, a glutamate residue at position 6, a glutamine residue at position 14, a leucine residue at position 15, a glutamate residue at position 18, a tyrosine residue at position 22, a proline residue at position 32, a glycine residue at position 38, a asparagine residue at position 44, a leucine residue at positions 50 and 51, a glycine residue at position 52, a proline residue at position 53, a leucine residue at position 55, a leucine residue at position 58, a phenylalanine residue at position 59, a cysteine residue at position 62, a phenylalanine residue at position 66, a lysine residue at position 69, a threonine residue at position 70, a glutamine residue at position 77, a isoleucine residue at position 79, a histidine residue at position 86, an arginine residue at position 93, an aspartic acid residue at position 94, a lysine residue at position 96, a proline residue at position 97, a asparagine residue at position 99, a phenylalanine residue at position 100, and a leucine residue at position 110; the third region is downstream from the second region, commences with an aspartic acid residue at position 1, and has an alanine residue at position 5, a lysine residue at position 6, a tyrosine residue at position 8, an aspartic acid residue at position 10, a threonine residue at position 13, a histidine residue at position 16, a isoleucine residue at position 17, a proline residue at position 18, a tyrosine residue at position 19, a arginine residue at position 20, a lysine residue at position 23, a glycine residue at position 27, a threonine residue at position 28, a alanine residue at position 29, a arginine residue at position 30, a tyrosine residue at position 31, a serine residue at position 33, an asparagines residue at position 35, a histidine residue at position 37, a glycine residue at position 39, a glutamate residue at position 41, a serine residue at position 43, an arginine residue at position 44, an arginine residue at position 45, an aspartic acid residue at position 46, an aspartic acid residue at position 47, a glutamate residue at position 49, a glycine residue at position 52, a tyrosine residue at position 57, a phenylalanine residue at position 58, a leucine residue at position 63, a proline residue at position 64, a tryptophan residue at position 65, a glutamine residue at position 66, and a glycine residue at position 67, and the fourth region is downstream from the third region, commences with a proline residue at position 1, and has an arginine residue at position 12, a leucine residue at position 14, a phenylalanine residue at position 16, a proline residue at position 20, an aspartic acid residue at position 21, a tyrosine residue at position 22, an aspartic acid residue at position 41, an aspartic acid residue at position 45, and a tryptophan residue at position 46.
The invention also provides CKSRP chimeric or fusion polypeptides. As used herein, a CKSRP “chimeric polypeptide” or “fusion polypeptide” comprises a CKSRP operatively linked to a non-CKSRP. A CKSRP refers to a polypeptide having an amino acid sequence corresponding to a CKSRP, whereas a non-CKSRP refers to a polypeptide having an amino acid sequence corresponding to a polypeptide which is not substantially identical to the CKSRP, e.g., a polypeptide that is different from the CKSRP and is derived from the same or a different organism. Within the fusion polypeptide, the term “operatively linked” is intended to indicate that the CKSRP and the non-CKSRP are fused to each other so that both sequences fulfill the proposed function attributed to the sequence used. The non-CKSRP can be fused to the N-terminus or C-terminus of the CKSRP. For example, in one embodiment, the fusion polypeptide is a GST-CKSRP fusion polypeptide in which the CKSRP sequences are fused to the C-terminus of the GST sequences. Such fusion polypeptides can facilitate the purification of recombinant CKSRPs. In another embodiment, the fusion polypeptide is a CKSRP containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a CKSRP can be increased through use of a heterologous signal sequence.
Preferably, a CKSRP chimeric or fusion polypeptide of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and re-amplified to generate a chimeric gene sequence (See, for example, Current Protocols in Molecular Biology, ds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A CKSRP encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the CKSRP.
In addition to fragments and fusion polypeptides of the CKSRPs described herein, the present invention includes homologs and analogs of naturally occurring CKSRPs and CKSRP encoding nucleic acids in a plant. “Homologs” are defined herein as two nucleic acids or polypeptides that have similar, or “identical,” nucleotide or amino acid sequences, respectively. Homologs include allelic variants, orthologs, paralogs, agonists, and antagonists of CKSRPs as defined hereafter. The term “homolog” further encompasses nucleic acid molecules that differ from one of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19 (and portions thereof) due to degeneracy of the genetic code and thus encode the same CKSRP as that encoded by the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19. For example homologs of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19 are described in SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74 and SEQ ID NO: 76.
As used herein, a “naturally occurring” CKSRP refers to a CKSRP amino acid sequence that occurs in nature. Preferably, a naturally occurring CKSRP comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20.
An agonist of the CKSRP can retain substantially the same, or a subset, of the biological activities of the CKSRP. An antagonist of the CKSRP can inhibit one or more of the activities of the naturally occurring form of the CKSRP. For example, the CKSRP antagonist can competitively bind to a downstream or upstream member of the cell membrane component metabolic cascade that includes the CKSRP, or bind to a CKSRP that mediates transport of compounds across such membranes, thereby preventing translocation from taking place.
Nucleic acid molecules corresponding to natural allelic variants and analogs, orthologs, and paralogs of a CKSRP cDNA can be isolated based on their identity to the Physcomitrella patens, Saccharomyces cerevisiae , or Brassica napus CKSRP nucleic acids described herein using CKSRP cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. In an alternative embodiment, homologs of the CKSRP can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the CKSRP for CKSRP agonist or antagonist activity. In one embodiment, a variegated library of CKSRP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of CKSRP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential CKSRP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion polypeptides (e.g., for phage display) containing the set of CKSRP sequences therein. There are a variety of methods that can be used to produce libraries of potential CKSRP homologs from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene is then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential CKSRP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (See, e.g., Narang, S. A., 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323; Itakura et al., 1984, Science 198:1056; Ike et al., 1983, Nucleic Acid Res. 11:477).
In addition, libraries of fragments of the CKSRP coding regions can be used to generate a variegated population of CKSRP fragments for screening and subsequent selection of homologs of a CKSRP. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a CKSRP coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA, which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal, and internal fragments of various sizes of the CKSRP.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of CKSRP homologs. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify CKSRP homologs (Arkin and Yourvan, 1992, PNAS 89:7811-7815; Delgrave et al., 1993, Polypeptide Engineering 6(3):327-331). In another embodiment, cell based assays can be exploited to analyze a variegated CKSRP library, using methods well known in the art. The present invention further provides a method of identifying a novel CKSRP, comprising (a) raising a specific antibody response to a CKSRP, or a fragment thereof, as described herein; (b) screening putative CKSRP material with the antibody, wherein specific binding of the antibody to the material indicates the presence of a potentially novel CKSRP; and (c) analyzing the bound material in comparison to known CKSRP, to determine its novelty.
As stated above, the present invention includes CKSRPs and homologs thereof. To determine the percent sequence identity of two amino acid sequences (e.g., one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20, and a mutant form thereof), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one polypeptide for optimal alignment with the other polypeptide or nucleic acid). The amino acid residues at corresponding amino acid positions are then compared. When a position in one sequence (e.g., one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20) is occupied by the same amino acid residue as the corresponding position in the other sequence (e.g., a mutant form of the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20), then the molecules are identical at that position. The same type of comparison can be made between two nucleic acid sequences.
The percent sequence identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent sequence identity=numbers of identical positions/total numbers of positions×100). Preferably, the isolated amino acid homologs included in the present invention are at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%, or more identical to the core casein kinase domain containing the amino acid sequence shown as residues 2-304 of SEQ ID NO:2, residues 77-368 of SEQ ID NO:4, residues 2-294 of SEQ ID NO:6, residues 77-368 of SEQ ID NO:8, residues 77-336 of SEQ ID NO:10, residues 1-296 of SEQ ID NO:12, residues 1-296 of SEQ ID NO:14, residues 5-300 of SEQ ID NO:16, residues 1-295 of SEQ ID NO:18, or residues 25-327 of SEQ ID NO:20. In another embodiment, the isolated amino acid homolog of the present invention is encoded by a nucleic acid as defined by nucleotides at positions 4 to 912 of SEQ ID NO:1, nucleotides at positions 229 to 1104 of SEQ ID NO:3, nucleotides at positions 4 to 882 of SEQ ID NO:5, nucleotides at positions 229 to 1104 of SEQ ID NO:7, nucleotides at positions 229 to 1008 of SEQ ID NO:9, nucleotides at positions 1 to 888 of SEQ ID NO:11, nucleotides at positions 1 to 888 of SEQ ID NO:13, nucleotides at positions 13 to 900 of SEQ ID NO:15, nucleotides at positions 1 to 885 of SEQ ID NO:17, or nucleotides at positions 73 to 981 of SEQ ID NO:19.
Members of casein kinase I protein family have divergent N-terminal and C-terminal extensions. The N-terminal region is responsible for substrate recognition and the C-terminal region is important in the interaction with substrates and is thought to be important for mediating regulation through autophosphorylation (Gross and Anderson Cell Signal 1998 10:699-711; Graves and Roach J Biol Chem 1995 270:21689-21694). The amino acid sequence of Orf 760 (SEQ ID NO:10) contains two insertions not found in the other casein kinase I proteins, a 17 amino acid stretch from position 37 to 53 and a 16 amino acid stretch from position 165 to 180. The presence of these novel insertions could be important for function and the subsequent phenotype in transgenic lines overexpressing Orf 760. Casein kinase I proteins have been shown to modulate their activity by autophosphorylation of C-terminal serine and threonine residues (Graves and Roach J Biol Chem 1995 270:21689-21694). The alignment in FIG. 5 demonstrates that C-terminal regions are found in Orf 760, EST 263, and EST 289, after approximately 525 amino acids in the consensus alignment, that are absent from EST 142 and EST 194. However, unlike Orf 760, EST 263, and EST 289, as demonstrated in the alignment in FIG. 5, EST 142 and EST 194 both contain N-terminal region of approximately 72 amino acids. The presence or absence of these N-terminal and C-terminal regions define at least six classes for these casein kinase I proteins that function in stress response. The N-terminal region, or the C-terminal region, or the core kinase domain, or combinations of core kinase domains with different or homologous N-terminal and/or C-terminal extension regions or site-directed mutagenesis can be used to alter consensus autophosphorylation sites to generate better agronomic phenotypes. For example, a chimeric or fusion polypeptide can comprise residues 1-72 of EST142 (the N-terminal region of SEQ ID NO:8) fused with any of the core kinase domains shown in FIG. 5, or with any of the C-terminal domains shown in FIG. 5, or with both the core kinase and C-terminal domains of any of the polypeptides shown in FIG. 5. In another embodiment, residues 295-473 of EST289 (the C-terminal region of SEQ ID NO:6) can be combined with the core kinase domain of any of the polypeptides shown in FIG. 5, or with any of the N-terminal domains shown in FIG. 5, or with both the core kinase and N-terminal domains of any of the polypeptides shown in FIG. 5 to generate a chimeric or fusion polypeptide which would confer better agronomic phenotypes.
In another embodiment, the isolated amino acid homologs included in the present invention are at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%, or more identical to an entire amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20. For example homologs of the amino acid sequences SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 are described in SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75 and SEQ ID NO: 77.
In yet another embodiment, the isolated amino acid homologs included in the present invention are at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%, or more identical to an entire amino acid sequence encoded by a nucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or to their homologs e.g. as specified in SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74 or SEQ ID NO: 76.
In other embodiments, the CKSRP amino acid homologs have sequence identity over at least 15 contiguous amino acid residues, more preferably at least 25 contiguous amino acid residues, and most preferably at least 35 contiguous amino acid residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or with homologs of the amino acid sequences SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 as described in SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75 and SEQ ID NO: 77.
In another preferred embodiment, an isolated nucleic acid homolog of the invention comprises a nucleotide sequence which is at least about 40-60%, preferably at least about 60-70%, more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, or more identical to a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19, or to a portion comprising at least 60 consecutive nucleotides thereof. The preferable length of sequence comparison for nucleic acids is at least 75 nucleotides, more preferably at least 100 nucleotides, and most preferably the entire length of the coding region. It is even more preferable that the nucleic acid homologs encode proteins having homology with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20 over the central kinase domain shown in FIGS. 5 and 7.
It is further preferred that the isolated nucleic acid homolog of the invention encodes a CKSRP, or portion thereof, that is at least 70% identical to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20 and that functions as a modulator of an environmental stress response in a plant. In a more preferred embodiment, overexpression of the nucleic acid homolog in a plant increases the tolerance of the plant to an environmental stress. In a further preferred embodiment, the nucleic acid homolog encodes a CKSRP that functions as a casein kinase.
For the purposes of the invention, the percent sequence identity between two nucleic acid or polypeptide sequences is determined using the Vector NTI 6.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda, Md. 20814). A gap-opening penalty of 15 and a gap extension penalty of 6.66 are used for determining the percent identity of two nucleic acids. A gap-opening penalty of 10 and a gap extension penalty of 0.1 are used for determining the percent identity of two polypeptides. All other parameters are set at the default settings. For purposes of a multiple alignment (Clustal W algorithm), the gap-opening penalty is 10, and the gap extension penalty is 0.05 with blosum62 matrix. It is to be understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymidine nucleotide is equivalent to a uracil nucleotide.
In another aspect, the invention provides an isolated nucleic acid comprising a polynucleotide that hybridizes to the polynucleotide of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19 under stringent conditions. More particularly, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19, In other embodiments, the nucleic acid is at least 30, 50, 100, 250, or more nucleotides in length. Preferably, an isolated nucleic acid homolog of the invention comprises a nucleotide sequence which hybridizes under highly stringent conditions to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19 and functions as a modulator of stress tolerance in a plant. In a further preferred embodiment, overexpression of the isolated nucleic acid homolog in a plant increases a plant's tolerance to an environmental stress. In an even further preferred embodiment, the isolated nucleic acid homolog encodes a CKSRP that functions as a casein kinase.
As used herein with regard to hybridization for DNA to a DNA blot, the term “stringent conditions” refers to hybridization overnight at 60° C. in 10× Denhart's solution, 6×SSC, 0.5% SDS, and 100 μg/ml denatured salmon sperm DNA. Blots are washed sequentially at 62° C. for 30 minutes each time in 3×SSC/0.1% SDS, followed by 1×SSC/0.1% SDS, and finally 0.1×SSC/0.1% SDS. As also used herein, in a preferred embodiment, the phrase “highly stringent conditions” refers to hybridization in a 6×SSC solution at 65° C. In another embodiment, “highly stringent conditions” refers to hybridization overnight at 65° C. in 10×Denharts solution, 6×SSC, 0.5% SDS, and 100 μg/ml denatured salmon sperm DNA. Blots are washed sequentially at 65° C. for 30 minutes each time in 3×SSC/0.1% SDS, followed by 1×SSC/0.1% SDS, and finally 0.1×SSC/0.1% SDS. Methods for nucleic acid hybridizations are described in Meinkoth and Wahl, 1984, Anal. Biochem. 138:267-284; Current Protocols in Molecular Biology, Chapter 2, Ausubel et al. Eds., Greene Publishing and Wiley-Interscience, New York, 1995; and Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes, Part I, Chapter 2, Elsevier, N.Y., 1993. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent or highly stringent conditions to a sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19 corresponds to a naturally occurring nucleic acid molecule. As used herein, a “naturally occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural polypeptide). In one embodiment, the nucleic acid encodes a naturally occurring Physcomitrella patens CKSRP, a Saccharomyces cerevisiae CKSRP or a Brassica napus CKSRP.
Using the above-described methods, and others known to those of skill in the art, one of ordinary skill in the art can isolate homologs of the CKSRPs comprising amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20. Such homologs are specified for example in SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75 and SEQ ID NO: 77.
Table A correlates the gene ID with the SEQ ID NO in the sequence listing.
| TABLE A | ||||
| SEQ | ||||
| Sequence | Sequence | ID | ||
| Gene ID | type | nature | NO | Organism |
| EST263 | DNA | Full-length | 1 | Physcomitrella patens |
| EST263 | protein | Full-length | 2 | Physcomitrella patens |
| EST194 | DNA | Full-length | 3 | Physcomitrella patens |
| EST194 | protein | Full-length | 4 | Physcomitrella patens |
| EST289 | DNA | Full-length | 5 | Physcomitrella patens |
| EST289 | protein | Full-length | 6 | Physcomitrella patens |
| EST142 | DNA | Full-length | 7 | Physcomitrella patens |
| EST142 | protein | Full-length | 8 | Physcomitrella patens |
| ORF760 | DNA | Full-length | 9 | yeast |
| ORF760 | protein | Full-length | 10 | yeast |
| BN42723666 | DNA | Full-length | 11 | canola |
| BN42723666 | protein | Full-length | 12 | canola |
| BN51274564 | DNA | Full-length | 13 | canola |
| BN51274564 | protein | Full-length | 14 | canola |
| BN51362554 | DNA | Full-length | 15 | canola |
| BN51362554 | protein | Full-length | 16 | canola |
| BN51390516 | DNA | Full-length | 17 | canola |
| BN51390516 | protein | Full-length | 18 | canola |
| BN51392938 | DNA | Full-length | 19 | canola |
| BN51392938 | protein | Full-length | 20 | canola |
| AAB68417 | protein | Full-length | 47 | yeast |
| AAA35230 | protein | Full-length | 48 | yeast |
| AAH06490 | protein | Full-length | 49 | human |
| AAH03558 | protein | Full-length | 50 | human |
| AAH08717 | protein | Full-length | 51 | human |
| AAD26525 | protein | Full-length | 52 | human |
| AJ487966 | protein | Full-length | 53 | rice |
| HV62560799 | DNA | Full-length | 54 | barley |
| HV62560799 | protein | Full-length | 55 | barley |
| GM59629961 | DNA | Full-length | 56 | soybean |
| GM59629961 | protein | Full-length | 57 | soybean |
| GM59587121 | DNA | Full-length | 58 | soybean |
| GM59587121 | protein | Full-length | 59 | soybean |
| TA60000862 | DNA | Full-length | 60 | wheat |
| TA60000862 | protein | Full-length | 61 | wheat |
| GM59702429 | DNA | Full-length | 62 | soybean |
| GM59702429 | protein | Full-length | 63 | soybean |
| LU61714150 | DNA | Full-length | 64 | linseed |
| LU61714150 | protein | Full-length | 65 | linseed |
| BN42182310 | DNA | Full-length | 66 | canola |
| BN42182310 | protein | Full-length | 67 | canola |
| HA66640192 | DNA | Full-length | 68 | sunflower |
| HA66640192 | protein | Full-length | 69 | sunflower |
| GM59587863 | DNA | Full-length | 70 | soybean |
| GM59587863 | protein | Full-length | 71 | soybean |
| BN51270917 | DNA | Full-length | 72 | canola |
| BN51270917 | protein | Full-length | 73 | canola |
| BN51343700 | DNA | Full-length | 74 | canola |
| BN51343700 | protein | Full-length | 75 | canola |
| TA59828214 | DNA | Full-length | 76 | Wheat |
| TA59828214 | protein | Full-length | 77 | wheat |
One subset of these homologs is allelic variants. As used herein, the term “allelic variant” refers to a nucleotide sequence containing polymorphisms that lead to changes in the amino acid sequences of a CKSRP and that exist within a natural population (e.g., a plant species or variety). Such natural allelic variations can typically result in 1-5% variance in a CKSRP nucleic acid. Allelic variants can be identified by sequencing the nucleic acid sequence of interest in a number of different plants, which can be readily carried out by using hybridization probes to identify the same CKSRP genetic locus in those plants. Any and all such nucleic acid variations and resulting amino acid polymorphisms or variations in a CKSRP that are the result of natural allelic variation and that do not alter the functional activity of a CKSRP, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding CKSRPs from the same or other species such as CKSRP analogs, orthologs, and paralogs, are intended to be within the scope of the present invention. As used herein, the term “analogs” refers to two nucleic acids that have the same or similar function, but that have evolved separately in unrelated organisms. As used herein, the term “orthologs” refers to two nucleic acids from different species, but that have evolved from a common ancestral gene by speciation. Normally, orthologs encode polypeptides having the same or similar functions. As also used herein, the term “paralogs” refers to two nucleic acids that are related by duplication within a genome. Paralogs usually have different functions, but these functions may be related (Tatusov, R. L. et al., 1997, Science 278(5338):631-637). Analogs, orthologs, and paralogs of a naturally occurring CKSRP can differ from the naturally occurring CKSRP by post-translational modifications, by amino acid sequence differences, or by both. Post-translational modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation, and such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. In particular, orthologs of the invention will generally exhibit at least 80-85%, more preferably, 85-90% or 90-95%, and most preferably 95%, 96%, 97%, 98%, or even 99% identity, or 100% sequence identity, with all or part of a naturally occurring CKSRP amino acid sequence, and will exhibit a function similar to a CKSRP. Preferably, a CKSRP ortholog of the present invention functions as a modulator of an environmental stress response in a plant and/or functions as a casein kinase. More preferably, a CKSRP ortholog increases the stress tolerance of a plant. In one embodiment, the CKSRP orthologs maintain the ability to participate in the metabolism of compounds necessary for the construction of cellular membranes in a plant, or in the transport of molecules across these membranes.
In addition to naturally-occurring variants of a CKSRP sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into a nucleotide sequence of SEQ ID NO:1; SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19, thereby leading to changes in the amino acid sequence of the encoded CKSRP, without altering the functional activity of the CKSRP. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in a sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of one of the CKSRPs without altering the activity of said CKSRP, whereas an “essential” amino acid residue is required for CKSRP activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having CKSRP activity) may not be essential for activity and thus are likely to be amenable to alteration without altering CKSRP activity.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding CKSRPs that contain changes in amino acid residues that are not essential for CKSRP activity. Such CKSRPs differ in amino acid sequence from a sequence contained in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, yet retain at least one of the CKSRP activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least about 50% identical to the central protein kinase region of an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20. Preferably, the polypeptide encoded by the nucleic acid molecule is at least about 50-60% identical to the central protein kinase region of one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, more preferably at least about 60-70% identical to the central protein kinase region of one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, even more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95% identical to the central protein kinase region of one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, and most preferably at least about 96%, 97%, 98%, or 99% identical to the central protein kinase region of one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20. In another embodiment, the polypeptide encoded by the nucleic acid molecule is at least about 50-60% identical to one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, more preferably at least about 60-70% identical to one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, even more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95% identical to one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, and most preferably at least about 96%, 97%, 98%, or 99% identical to one of the sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20. The preferred CKSRP homologs of the present invention preferably participate in a stress tolerance response in a plant, or more particularly, participate in the transcription of a polypeptide involved in a stress tolerance response in a plant, and/or function as a casein kinase.
An isolated nucleic acid molecule encoding a CKSRP having sequence identity with a polypeptide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20 can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19, respectively, such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded polypeptide. Mutations can be introduced into one of the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a CKSRP is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a CKSRP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a CKSRP activity described herein to identify mutants that retain CKSRP activity. Following mutagenesis of one of the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19, the encoded polypeptide can be expressed recombinantly and the activity of the polypeptide can be determined by analyzing the stress tolerance of a plant expressing the polypeptide as described in Example 7.
Additionally, optimized CKSRP nucleic acids can be created. Preferably, an optimized CKSRP nucleic acid encodes a CKSRP that binds to a phosphate group and/or modulates a plant's tolerance to an environmental stress, and more preferably increases a plant's tolerance to an environmental stress upon its overexpression in the plant. As used herein, “optimized” refers to a nucleic acid that is genetically engineered to increase its expression in a given plant or animal. To provide plant optimized CKSRP nucleic acids, the DNA sequence of the gene can be modified to 1) comprise codons preferred by highly expressed plant genes; 2) comprise an A+T content in nucleotide base composition to that substantially found in plants; 3) form a plant initiation sequence; or 4) to eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA, or that form secondary structure hairpins or RNA splice sites. Increased expression of CKSRP nucleic acids in plants can be achieved by utilizing the distribution frequency of codon usage in plants in general or in a particular plant. Methods for optimizing nucleic acid expression in plants can be found in EPA 0359472; EPA 0385962; PCT Application No. WO 91/16432; U.S. Pat. No. 5,380,831; U.S. Pat. No. 5,436,391; Perlack et al., 1991, Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al., 1989, Nucleic Acids Res. 17:477-498.
As used herein, “frequency of preferred codon usage” refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. To determine the frequency of usage of a particular codon in a gene, the number of occurrences of that codon in the gene is divided by the total number of occurrences of all codons specifying the same amino acid in the gene. Similarly, the frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage in a large number of genes expressed by the host cell. It is preferable that this analysis be limited to genes that are highly expressed by the host cell. The percent deviation of the frequency of preferred codon usage for a synthetic gene from that employed by a host cell is calculated first by determining the percent deviation of the frequency of usage of a single codon from that of the host cell followed by obtaining the average deviation over all codons. As defined herein, this calculation includes unique codons (i.e., ATG and TGG). In general terms, the overall average deviation of the codon usage of an optimized gene from that of a host cell is calculated using the equation 1A=n=1 Z X n —Y n X n times 100 Z where X n =frequency of usage for codon n in the host cell; Y n =frequency of usage for codon n in the synthetic gene; n represents an individual codon that specifies an amino acid; and the total number of codons is Z. The overall deviation of the frequency of codon usage, A, for all amino acids should preferably be less than about 25%, and more preferably less than about 10%.
Hence, a CKSRP nucleic acid can be optimized such that its distribution frequency of codon usage deviates, preferably, no more than 25% from that of highly expressed plant genes and, more preferably, no more than about 10%. In addition, consideration is given to the percentage G+C content of the degenerate third base (monocotyledons appear to favor G+C in this position, whereas dicotyledons do not). It is also recognized that the XCG (where X is A, T, C, or G) nucleotide is the least preferred codon in dicots whereas the XTA codon is avoided in both monocots and dicots. Optimized CKSRP nucleic acids of this invention also preferably have CG and TA doublet avoidance indices closely approximating those of the chosen host plant (e.g., Physcomitrella patens, Brassica napus, Glycine max , or Oryza sativa ). More preferably these indices deviate from that of the host by no more than about 10-15%.
In addition to the nucleic acid molecules encoding the CKSRPs described above, another aspect of the invention pertains to isolated nucleic acid molecules that are antisense thereto. Antisense polynucleotides are thought to inhibit gene expression of a target polynucleotide by specifically binding the target polynucleotide and interfering with transcription, splicing, transport, translation, and/or stability of the target polynucleotide. Methods are described in the prior art for targeting the antisense polynucleotide to the chromosomal DNA, to a primary RNA transcript, or to a processed mRNA. Preferably, the target regions include splice sites, translation initiation codons, translation termination codons, and other sequences within the open reading frame.
The term “antisense,” for the purposes of the invention, refers to a nucleic acid comprising a polynucleotide that is sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA, so as to interfere with expression of the endogenous gene. “Complementary” polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other. The term “antisense nucleic acid” includes single stranded RNA as well as double-stranded DNA expression cassettes that can be transcribed to produce an antisense RNA. “Active” antisense nucleic acids are antisense RNA molecules that are capable of selectively hybridizing with a primary transcript or mRNA encoding a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20.
The antisense nucleic acid can be complementary to an entire CKSRP coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding a CKSRP. The term “coding region” refers to the region of the nucleotide sequence comprising codons that are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding a CKSRP. The term “noncoding region” refers to 5′ and 3′ sequences that flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions). The antisense nucleic acid molecule can be complementary to the entire coding region of CKSRP mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of CKSRP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of CKSRP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. Typically, the antisense molecules of the present invention comprise an RNA having 60-100% sequence identity with at least 14 consecutive nucleotides of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19, or a polynucleotide encoding a polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20. Preferably, the sequence identity will be at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, or 98%, and most preferably 99%.
An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al., 1987, Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
The antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a CKSRP to thereby inhibit expression of the polypeptide, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic (including plant) promoter are preferred.
As an alternative to antisense polynucleotides, ribozymes, sense polynucleotides, or double stranded RNA (dsRNA) can be used to reduce expression of a CKSRP polypeptide. As used herein, the term “ribozyme” refers to a catalytic RNA-based enzyme with ribonuclease activity that is capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which it has a complementary region. Ribozymes (e.g., hammerhead ribozymes described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can be used to catalytically cleave CKSRP mRNA transcripts to thereby inhibit translation of CKSRP mRNA. A ribozyme having specificity for a CKSRP-encoding nucleic acid can be designed based upon the nucleotide sequence of a CKSRP cDNA, as disclosed herein (i.e., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19) or on the basis of a heterologous sequence to be isolated according to methods taught in this invention. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a CKSRP-encoding mRNA. See, e.g., U.S. Pat. Nos. 4,987,071 and 5,116,742 to Cech et al. Alternatively, CKSRP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W., 1993, Science 261:1411-1418. In preferred embodiments, the ribozyme will contain a portion having at least 7, 8, 9, 10, 12, 14, 16, 18, or nucleotides, and more preferably 7 or 8 nucleotides, that have 100% complementarity to a portion of the target RNA. Methods for making ribozymes are known to those skilled in the art. See, e.g., U.S. Pat. Nos. 6,025,167; 5,773,260; and 5,496,698.
The term “dsRNA,” as used herein, refers to RNA hybrids comprising two strands of RNA. The dsRNAs can be linear or circular in structure. In a preferred embodiment, dsRNA is specific for a polynucleotide encoding either the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, or a polypeptide having at least 80% sequence identity with a polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20 over the central protein kinase domain. The hybridizing RNAs may be substantially or completely complementary. By “substantially complementary,” is meant that when the two hybridizing RNAs are optimally aligned using the BLAST program as described above, the hybridizing portions are at least 95% complementary. Preferably, the dsRNA will be at least 100 base pairs in length. Typically, the hybridizing RNAs will be of identical length with no over hanging 5′ or 3′ ends and no gaps. However, dsRNAs having 5′ or 3′ overhangs of up to 100 nucleotides may be used in the methods of the invention.
The dsRNA may comprise ribonucleotides, ribonucleotide analogs such as 2′-O-methyl ribosyl residues, or combinations thereof. See, e.g., U.S. Pat. Nos. 4,130,641 and 4,024,222. A dsRNA polyriboinosinic acid:polyribocytidylic acid is described in U.S. Pat. No. 4,283,393. Methods for making and using dsRNA are known in the art. One method comprises the simultaneous transcription of two complementary DNA strands, either in vivo, or in a single in vitro reaction mixture. See, e.g., U.S. Pat. No. 5,795,715. In one embodiment, dsRNA can be introduced into a plant or plant cell directly by standard transformation procedures. Alternatively, dsRNA can be expressed in a plant cell by transcribing two complementary RNAs.
Other methods for the inhibition of endogenous gene expression, such as triple helix formation (Moser et al., 1987, Science 238:645-650 and Cooney et al., 1988, Science 241:456-459) and co-suppression (Napoli et al., 1990, The Plant Cell 2:279-289) are known in the art. Partial and full-length cDNAs have been used for the co-suppression of endogenous plant genes. See, e.g., U.S. Pat. Nos. 4,801,340, 5,034,323, 5,231,020, and 5,283,184; Van der Kroll et al., 1990, The Plant Cell 2:291-299; Smith et al., 1990, Mol. Gen. Genetics 224:477-481; and Napoli et al., 1990, The Plant Cell 2:279-289. For sense suppression, it is believed that introduction of a sense polynucleotide blocks transcription of the corresponding target gene. The sense polynucleotide will have at least 65% sequence identity with the target plant gene or RNA. Preferably, the percent identity is at least 80%, 90%, 95%, or more. The introduced sense polynucleotide need not be full length relative to the target gene or transcript. Preferably, the sense polynucleotide will have at least 65% sequence identity with at least 100 consecutive nucleotides of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19. The regions of identity can comprise introns and/or exons and untranslated regions. The introduced sense polynucleotide may be present in the plant cell transiently, or may be stably integrated into a plant chromosome or extrachromosomal replicon.
Alternatively, CKSRP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a CKSRP nucleotide sequence (e.g., a CKSRP promoter and/or enhancer) to form triple helical structures that prevent transcription of a CKSRP gene in target cells. See generally, Helene, C., 1991, Anticancer Drug Des. 6(6):569-84; Helene, C. et al., 1992, Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15.
In addition to the CKSRP nucleic acids and polypeptides described above, the present invention encompasses these nucleic acids and polypeptides attached to a moiety. These moieties include, but are not limited to, detection moieties, hybridization moieties, purification moieties, delivery moieties, reaction moieties, binding moieties, and the like. A typical group of nucleic acids having moieties attached are probes and primers. Probes and primers typically comprise a substantially isolated oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50, or 75 consecutive nucleotides of a sense strand of one of the sequences set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19; an anti-sense sequence of one of the sequences set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19; or naturally occurring mutants thereof. Primers based on a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19 can be used in PCR reactions to clone CKSRP homologs. Probes based on the CKSRP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or substantially identical polypeptides. In preferred embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a genomic marker test kit for identifying cells which express a CKSRP, such as by measuring a level of a CKSRP-encoding nucleic acid, in a sample of cells, e.g., detecting CKSRP mRNA levels or determining whether a genomic CKSRP gene has been mutated or deleted.
In particular, a useful method to ascertain the level of transcription of the gene (an indicator of the amount of mRNA available for translation to the gene product) is to perform a Northern blot (For reference, see, for example, Ausubel et al., 1988, Current Protocols in Molecular Biology, Wiley: New York). The information from a Northern blot at least partially demonstrates the degree of transcription of the transformed gene. Total cellular RNA can be pre-pared from cells, tissues, or organs by several methods, all well-known in the art, such as that described in Bormann, E. R. et al., 1992, Mol. Microbiol. 6:317-326. To assess the presence or relative quantity of polypeptide translated from this mRNA, standard techniques, such as a Western blot, may be employed. These techniques are well known to one of ordinary skill in the art. (See, for example, Ausubel et al., 1988, Current Protocols in Molecular Biology, Wiley: New York).
The invention further provides an isolated recombinant expression vector comprising a CKSRP nucleic acid as described above, wherein expression of the vector in a host cell results in increased tolerance to environmental stress as compared to a wild type variety of the host cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover,