US20050066396A1 - Casein kinase stress-related polypeptides and methods of use in plants - Google Patents

Casein kinase stress-related polypeptides and methods of use in plants Download PDF

Info

Publication number
US20050066396A1
US20050066396A1 US10/904,588 US90458804A US2005066396A1 US 20050066396 A1 US20050066396 A1 US 20050066396A1 US 90458804 A US90458804 A US 90458804A US 2005066396 A1 US2005066396 A1 US 2005066396A1
Authority
US
United States
Prior art keywords
residue
seq
polypeptide
nucleic acid
plant cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/904,588
Other languages
English (en)
Inventor
Amber Shirley
Damian Allen
Nocha Van Thielen
Oswaldo da Costa e Silva
Ruoying Chen
Lori Mills
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF Plant Science GmbH
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/828,313 external-priority patent/US6867351B2/en
Priority claimed from US10/292,408 external-priority patent/US7176026B2/en
Application filed by Individual filed Critical Individual
Priority to US10/904,588 priority Critical patent/US20050066396A1/en
Publication of US20050066396A1 publication Critical patent/US20050066396A1/en
Assigned to BASF PLANT SCIENCE GMBH reassignment BASF PLANT SCIENCE GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ALLEN, DAMIAN, CHEN, RUOYING, MILLS, LORI, SHIRLEY, AMBER, VAN THIELEN, NOCHA E., DA COSTA E SILVA, OSWALDO
Priority to PCT/US2005/041522 priority patent/WO2006055631A2/fr
Priority to BRPI0517851-7A priority patent/BRPI0517851A/pt
Priority to AU2005307824A priority patent/AU2005307824A1/en
Priority to CA002587401A priority patent/CA2587401A1/fr
Priority to CNA2005800466941A priority patent/CN101102665A/zh
Priority to US11/667,820 priority patent/US20080052794A1/en
Priority to EP05849385A priority patent/EP1814378A4/fr
Priority to MX2007005802A priority patent/MX2007005802A/es
Priority to ARP050104850A priority patent/AR051503A1/es
Priority to US11/737,826 priority patent/US7399904B2/en
Priority to ZA200704387A priority patent/ZA200704387B/xx
Priority to US12/472,651 priority patent/US7795415B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • This invention relates generally to nucleic acid sequences encoding polypeptides that are associated with abiotic stress responses and abiotic stress tolerance in plants.
  • this invention relates to nucleic acid sequences encoding polypeptides that confer drought, cold, and/or salt tolerance to plants.
  • 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.
  • 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.
  • 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).
  • 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).
  • Rubisco Rabulose 1,5-bisphosphate Carboxylase/Oxygenase
  • PEPC Phosphoenolpyruvate carboxylase
  • 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 p305). 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 p405). WUE has been defined and measured in multiple ways.
  • WUE Water Use Efficiency
  • 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 p406). 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 C 3 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • CKSRPs Casein Kinase Stress-Related Polypeptides
  • 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.
  • the CKSRP is from Physcomitrella patens, Saccharomyces cerevisiae , or Brassica napus .
  • 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 Casei n 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 CKSRP and coding nucleic acid are those that are found in members of the genus Physcomitrella, Saccharomyces , or Brassica .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 casein kinases (SEQ ID NOS 10, 12, 14, 16, 18, 20, 8, 4, 6, and 2, respectively in order of appearance).
  • 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 is the drawing pertaining to Table 15: ScCK-1 (ORF 760) was overexpressed in Arabidopsis thaliana under the control of a constitutive promoter. The transgenic lines were assayed for dessication tolerance, measuring the average number of days of survival after the wild type control was dead (Table 7). *Please see table 15.
  • 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.
  • all members of the genus are biologically active casein kinases.
  • the present invention encompasses CKSRP polynucleotide and polypeptide sequences and their use for increasing a plant's tolerance to an environmental stress.
  • 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.
  • 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 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.
  • the transgenic plant is male sterile.
  • 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.
  • 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, PpCk-3, 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.
  • 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.
  • 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.
  • 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 Kin
  • the CKSRPs of the present invention are preferably produced by recombinant DNA techniques.
  • 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.
  • 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.
  • a CKSRP, or peptide thereof can be synthesized chemically using standard peptide synthesis techniques.
  • 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.
  • the term “environmental stress” refers to sub-optimal conditions associated with salinity, drought, temperature, metal, chemical, pathogenic, and oxidative stresses, or combinations thereof.
  • 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.
  • “a” or “an” can mean one or more, depending upon the context in which it is used.
  • reference to “a cell” can mean that at least one cell can be utilized.
  • 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.
  • 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.
  • 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.
  • RNA molecules 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).
  • 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.
  • 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 cerevisiase 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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. Russia, Fla.).
  • reverse transcriptase e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, 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.
  • oligonucleotides corresponding to a CKSRP nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • biologically active portions comprise a domain or motif with at least one activity of a CKSRP.
  • 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.
  • 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 .
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • a CKSRP chimeric or fusion polypeptide of the invention is produced by standard recombinant DNA techniques.
  • 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.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • 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, Eds. Ausubel et al. John Wiley & Sons: 1992).
  • 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
  • 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.
  • 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.
  • 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.
  • a “naturally occurring” CKSRP refers to a CKSRP amino acid sequence that occurs in nature.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an expression library can be derived which encodes N-terminal, C-terminal, and internal fragments of various sizes of the CKSRP.
  • 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).
  • 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.
  • the present invention includes CKSRPs and homologs thereof.
  • CKSRPs CKSRPs and homologs 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.
  • 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 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 residue
  • 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.
  • 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.
  • 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, or SEQ ID NO:19.
  • 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, or SEQ ID NO:20.
  • 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 .
  • 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.
  • overexpression of the nucleic acid homolog in a plant increases the tolerance of the plant to an environmental stress.
  • the nucleic acid homolog encodes a CKSRP that functions as a casein kinase.
  • 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.
  • the gap-opening penalty is 10
  • the gap extension penalty is 0.05 with blosum 62 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.
  • 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.
  • 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.
  • the nucleic acid is at least 30, 50, 100, 250, or more nucleotides in length.
  • 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.
  • overexpression of the isolated nucleic acid homolog in a plant increases a plant's tolerance to an environmental stress.
  • the isolated nucleic acid homolog encodes a CKSRP that functions as a casein kinase.
  • the term “stringent conditions” refers to hybridization overnight at 60° C. in 10 ⁇ Denhart's solution, 6 ⁇ SSC, 0.5% SDS, and 100 ⁇ g/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.
  • 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.
  • 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.
  • 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).
  • the nucleic acid encodes a naturally occurring Physcomitrella patens CKSRP, a Saccharomyces cerevisiae CKSRP or a Brassica napus CKSRP.
  • 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, or SEQ ID NO:20.
  • One subset of these homologs is allelic variants.
  • 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.
  • 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.
  • analogs refers to two nucleic acids that have the same or similar function, but that have evolved separately in unrelated organisms.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, may not be essential for activity and thus are likely to be amenable to alteration without altering CKSRP activity.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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:10, S
  • 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.
  • 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).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g.
  • a predicted nonessential amino acid residue in a CKSRP is preferably replaced with another amino acid residue from the same side chain family.
  • 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.
  • 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.
  • optimized CKSRP nucleic acids can be created.
  • 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.
  • “optimized” refers to a nucleic acid that is genetically engineered to increase its expression in a given plant or animal.
  • 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.
  • 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).
  • 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%.
  • 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%.
  • 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%.
  • 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.
  • the target regions include splice sites, translation initiation codons, translation termination codons, and other sequences within the open reading frame.
  • antisense 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.
  • 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.
  • 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.
  • the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding a CKSRP.
  • 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.
  • 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.
  • 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.
  • the sequence identity will be at least 70%, more preferably at least 75%, 80%, 85%,
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid 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.
  • 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′-methoxycar
  • 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).
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -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.
  • ribozymes As an alternative to antisense polynucleotides, ribozymes, sense polynucleotides, or double stranded RNA (dsRNA) can be used to reduce expression of a CKSRP polypeptide.
  • dsRNA double stranded RNA
  • 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
  • Ribozymes 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.
  • 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.
  • 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.
  • the ribozyme will contain a portion having at least 7, 8, 9, 10, 12, 14, 16, 18, or 20 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.
  • dsRNA refers to RNA hybrids comprising two strands of RNA.
  • the dsRNAs can be linear or circular in structure.
  • 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.
  • 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.
  • the dsRNA will be at least 100 base pairs in length.
  • the hybridizing RNAs will be of identical length with no over hanging 5′ or 3′ ends and no gaps.
  • 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.
  • dsRNA can be introduced into a plant or plant cell directly by standard transformation procedures.
  • dsRNA can be expressed in a plant cell by transcribing two complementary RNAs.
  • 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.
  • 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.
  • 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.
  • a CKSRP nucleotide sequence e.g., a CKSRP promoter and/or enhancer
  • the present invention encompasses these nucleic acids and polypeptides attached to a moiety.
  • 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.
  • 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.
  • a useful method to ascertain the level of transcription of the gene 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 prepared 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.
  • 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.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • 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, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • “operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) and Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, eds. Glick and Thompson, Chapter 7, 89-108, CRC Press: Boca Raton, Fla., including the references therein. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides or peptides, encoded by nucleic acids as described herein (e.g., CKSRPs, mutant forms of CKSRPs, fusion polypeptides, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of CKSRPs in prokaryotic or eukaryotic cells.
  • CKSRP genes can be expressed in bacterial cells such as C. glutamicum , insect cells (using baculovirus expression vectors), yeast and other fungal cells (See Romanos, M. A. et al., 1992, Foreign gene expression in yeast: a review, Yeast 8:423-488; van den Hondel, C. A. M. J. J. et al., 1991, Heterologous gene expression in filamentous fungi, in: More Gene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p.
  • telomeres Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press: San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide but also to the C-terminus or fused within suitable regions in the polypeptides.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of a recombinant polypeptide; 2) to increase the solubility of a recombinant polypeptide; and 3) to aid in the purification of a recombinant polypeptide by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin, and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S., 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.
  • GST glutathione S-transferase
  • the coding sequence of the CKSRP is cloned into a pGEX expression vector to create a vector encoding a fusion polypeptide comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X polypeptide.
  • the fusion polypeptide can be purified by affinity chromatography using glutathione-agarose resin. Recombinant CKSRP unfused to GST can be recovered by cleavage of the fusion polypeptide with thrombin.
  • Suitable inducible non-fusion E coli expression vectors include pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3) or HMS174 (DE3) from a resident y prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant polypeptide expression is to express the polypeptide in a host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128).
  • Another strategy is to alter the sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as C. glutamicum (Wada et al., 1992, Nucleic Acids Res. 20:2111-2118).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the CKSRP expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et al., 1987, EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
  • Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi include those detailed in: van den Hondel, C. A. M. J. J. & Punt, P.
  • the CKSRPs of the invention can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of polypeptides in cultured insect cells include the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989, Virology 170:31-39).
  • a CKSRP nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B., 1987, Nature 329:840) and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988, Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989, EMBO J.
  • promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss, 1990, Science 249:374-379) and the fetopolypeptide promoter (Campes and Tilghman, 1989, Genes Dev. 3:537-546).
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics or herbicides) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin, and methotrexate, or in plants that confer resistance towards an herbicide such as glyphosate, glufosinate, or imidazolinone.
  • Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a CKSRP or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by, for example, herbicide selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • herbicide selection e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die.
  • the CKSRPs are expressed in plants and plants cells such as unicellular plant cells (e.g. algae) (See Falciatore et al., 1999, Marine Biotechnology 1 (3):239-251 and references therein) and plant cells from higher plants (e.g., the spermatophytes, such as crop plants).
  • a CKSRP may be “introduced” into a plant cell by any means, including transfection, transformation or transduction, electroporation, particle bombardment, agroinfection, and the like.
  • One transformation method known to those of skill in the art is the dipping of a flowering plant into an Agrobacteria solution, wherein the Agrobacteria contain the CKSRP nucleic acid, followed by breeding of the transformed gametes.
  • biotic and abiotic stress tolerance is a general trait wished to be inherited into a wide variety of plants like maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed and canola, manihot, pepper, sunflower and tagetes, solanaceous plants like potato, tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil palm, coconut), perennial grasses, and forage crops, these crop plants are also preferred target plants for a genetic engineering as one further embodiment of the present invention.
  • Forage crops include, but are not limited to, Wheatgrass, Canarygrass, Bromegrass, Wildrye Grass, Bluegrass, Orchardgrass, Alfalfa, Salfoin, Birdsfoot Trefoil, Alsike Clover, Red Clover, and Sweet Clover.
  • transfection of a CKSRP into a plant is achieved by Agrobacterium mediated gene transfer.
  • Agrobacterium mediated plant transformation can be performed using for example the GV3101 (pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain. Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al., 1994, Nucl. Acids. Res. 13:4777-4788; Gelvin, Stanton B.
  • rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al., 1989, Plant Cell Report 8:238-242; De Block et al., 1989 , Plant Physiol. 91:694-701).
  • Agrobacterium and plant selection depend on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker.
  • Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994, Plant Cell Report 13:282-285. Additionally, transformation of soybean can be performed using for example a technique described in European Patent No. 0424 047, U.S. Pat. No. 5,322,783, European Patent No. 0397 687, U.S. Pat. No. 5,376,543, or U.S. Pat. No. 5,169,770.
  • Transformation of maize can be achieved by particle bombardment, polyethylene glycol mediated DNA uptake, or via the silicon carbide fiber technique. (See, for example, Freeling and Walbot “The maize handbook” Springer Verlag: New York (1993) ISBN 3-540-97826-7).
  • a specific example of maize transformation is found in U.S. Pat. No. 5,990,387, and a specific example of wheat transformation can be found in PCT Application No. WO 93/07256.
  • the introduced CKSRP may be maintained in the plant cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosomes.
  • the introduced CKSRP may be present on an extra-chromosomal non-replicating vector and may be transiently expressed or transiently active.
  • a homologous recombinant microorganism can be created wherein the CKSRP is integrated into a chromosome, a vector is prepared which contains at least a portion of a CKSRP gene into which a deletion, addition, or substitution has been introduced to thereby alter, e.g., functionally disrupt, the CKSRP gene.
  • the CKSRP gene is a Physcomitrella patens, Brassica napus, Glycine max , or Oryza sativa CKSRP gene, but it can be a homolog from a related plant or even from a mammalian, yeast, or insect source.
  • the vector is designed such that, upon homologous recombination, the endogenous CKSRP gene is functionally disrupted (i.e., no longer encodes a functional polypeptide; also referred to as a knock-out vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous CKSRP gene is mutated or otherwise altered but still encodes a functional polypeptide (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous CKSRP).
  • DNA-RNA hybrids can be used in a technique known as chimeraplasty (Cole-Strauss et al., 1999, Nucleic Acids Research 27(5):1323-1330 and Kmiec, 1999, Gene Therapy American Scientist 87(3):240-247). Homologous recombination procedures in Physcomitrella patens are also well known in the art and are contemplated for use herein.
  • the altered portion of the CKSRP gene is flanked at its 5′ and 3′ ends by an additional nucleic acid molecule of the CKSRP gene to allow for homologous recombination to occur between the exogenous CKSRP gene carried by the vector and an endogenous CKSRP gene, in a microorganism or plant.
  • the additional flanking CKSRP nucleic acid molecule is of sufficient length for successful homologous recombination with the endogenous gene.
  • several hundreds of base pairs up to kilobases of flanking DNA are included in the vector (See e.g., Thomas, K. R., and Capecchi, M.
  • the vector is introduced into a microorganism or plant cell (e.g., via polyethylene glycol mediated DNA), and cells in which the introduced CKSRP gene has homologously recombined with the endogenous CKSRP gene are selected using art-known techniques.
  • recombinant microorganisms can be produced that contain selected systems that allow for regulated expression of the introduced gene.
  • inclusion of a CKSRP gene on a vector placing it under control of the lac operon permits expression of the CKSRP gene only in the presence of IPTG.
  • Such regulatory systems are well known in the art.
  • the CKSRP polynucleotide preferably resides in a plant expression cassette.
  • a plant expression cassette preferably contains regulatory sequences capable of driving gene expression in plant cells that are operatively linked so that each sequence can fulfill its function, for example, termination of transcription by polyadenylation signals.
  • Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al., 1984, EMBO J.
  • a plant expression cassette preferably contains other operatively linked sequences like translational enhancers such as the overdrive-sequence containing the 5′-untranslated leader sequence from tobacco mosaic virus enhancing the polypeptide per RNA ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711).
  • Examples of plant expression vectors include those detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R., 1992, New plant binary vectors with selectable markers located proximal to the left border, Plant Mol. Biol.
  • Plant gene expression should be operatively linked to an appropriate promoter conferring gene expression in a timely, cell specific, or tissue specific manner.
  • Promoters useful in the expression cassettes of the invention include any promoter that is capable of initiating transcription in a plant cell. Such promoters include, but are not limited to, those that can be obtained from plants, plant viruses, and bacteria that contain genes that are expressed in plants, such as Agrobacterium and Rhizobium.
  • the promoter may be constitutive, inducible, developmental stage-preferred, cell type-preferred, tissue-preferred, or organ-preferred. Constitutive promoters are active under most conditions. Examples of constitutive promoters include the CaMV 19S and 35 S promoters (Odell et al., 1985, Nature 313:810-812), the sX CaMV 35S promoter (Kay et al., 1987, Science 236:1299-1302) the Sep1 promoter, the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171), the Arabidopsis actin promoter, the ubiquitan promoter (Christensen et al., 1989, Plant Molec. Biol.
  • promoters from the T-DNA of Agrobacterium such as mannopine synthase, nopaline synthase, and octopine synthase, the small subunit of ribulose biphosphate carboxylase (ssuRUBISCO) promoter, and the like.
  • Inducible promoters are preferentially active under certain environmental conditions, such as the presence or absence of a nutrient or metabolite, heat or cold, light, pathogen attack, anaerobic conditions, and the like.
  • the hsp80 promoter from Brassica is induced by heat shock
  • the PPDK promoter is induced by light
  • the PR-1 promoter from tobacco, Arabidopsis , and maize are inducible by infection with a pathogen
  • the Adh1 promoter is induced by hypoxia and cold stress.
  • Plant gene expression can also be facilitated via an inducible promoter (For review, see Gatz, 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108).
  • Chemically inducible promoters are especially suitable if gene expression is wanted to occur in a time specific manner.
  • Examples of such promoters are a salicylic acid inducible promoter (PCT Application No. WO 95/19443), a tetracycline inducible promoter (Gatz et al., 1992, Plant J. 2:397-404), and an ethanol inducible promoter (PCT Application No. WO 93/21334).
  • the inducible promoter is a stress-inducible promoter.
  • stress inducible promoters are preferentially active under one or more of the following stresses: sub-optimal conditions associated with salinity, drought, temperature, metal, chemical, pathogenic, and oxidative stresses.
  • Stress inducible promoters include, but are not limited to, Cor78 (Chak et al., 2000, Planta 210:875-883; Hovath et al., 1993, Plant Physiol. 103:1047-1053), Cor15a (Artus et al., 1996, PNAS 93(23):13404-09), Rci2A (Medina et al., 2001, Plant Physiol.
  • tissue and organ preferred promoters include those that are preferentially expressed in certain tissues or organs, such as leaves, roots, seeds, or xylem.
  • tissue preferred and organ preferred promoters include, but are not limited to fruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred, integument-preferred, tuber-preferred, stalk-preferred, pericarp-preferred, and leaf-preferred, stigma-preferred, pollen-preferred, anther-preferred, a petal-preferred, sepal-preferred, pedicel-preferred, silique-preferred, stem-preferred, root-preferred promoters, and the like.
  • Seed preferred promoters are preferentially expressed during seed development and/or germination.
  • seed preferred promoters can be embryo-preferred, endosperm preferred, and seed coat-preferred. See Thompson et al., 1989, BioEssays 10:108.
  • seed preferred promoters include, but are not limited to, cellulose synthase (celA), Cim1, gamma-zein, globulin-1, maize 19 kD zein (cZ19B1), and the like.
  • tissue-preferred or organ-preferred promoters include the napin-gene promoter from rapeseed (U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al., 1991, Mol. Gen. Genet. 225(3):459-67), the oleosin-promoter from Arabidopsis (PCT Application No. WO 98/45461), the phaseolin-promoter from Phaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4-promoter from Brassica (PCT Application No.
  • WO 91/13980 or the legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2(2):233-9), as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice, etc.
  • Suitable promoters to note are the Ipt2 or Ipt1-gene promoter from barley (PCT Application No. WO 95/15389 and PCT Application No. WO 95/23230) or those described in PCT Application No.
  • WO 99/16890 promoters from the barley hordein-gene, rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, oat glutelin gene, Sorghum kasirin-gene, and rye secalin gene).
  • promoters useful in the expression cassettes of the invention include, but are not limited to, the major chlorophyll a/b binding protein promoter, histone promoters, the Ap3 promoter, the ⁇ -conglycin promoter, the napin promoter, the soybean lectin promoter, the maize 15 kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter, the g-zein promoter, the waxy, shrunken 1, shrunken 2, and bronze promoters, the Zm13 promoter (U.S. Pat. No. 5,086,169), the maize polygalacturonase promoters (PG) (U.S. Pat. Nos. 5,412,085 and 5,545,546), and the SGB6 promoter (U.S. Pat. No. 5,470,359), as well as synthetic or other natural promoters.
  • the major chlorophyll a/b binding protein promoter include, but are not limited to, the major chlor
  • Additional flexibility in controlling heterologous gene expression in plants may be obtained by using DNA binding domains and response elements from heterologous sources (i.e., DNA binding domains from non-plant sources).
  • heterologous DNA binding domain is the LexA DNA binding domain (Brent and Ptashne, 1985, Cell 43:729-736).
  • the invention further provides a recombinant expression vector comprising a CKSRP DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to a CKSRP mRNA.
  • Regulatory sequences operatively linked to a nucleic acid molecule cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types. For instance, viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific, or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus wherein antisense nucleic acids are produced under the control of a high efficiency regulatory region.
  • the activity of the regulatory region can be determined by the cell type, into which the vector is introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but they also apply to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a CKSRP can be expressed in bacterial cells such as C.
  • glutamicum insect cells, fungal cells, or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), algae, ciliates, plant cells, fungi, or other microorganisms like C. glutamicum .
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells
  • algae ciliates
  • plant cells fungi, or other microorganisms like C. glutamicum .
  • Other suitable host cells are known to those skilled in the art.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a CKSRP.
  • the invention further provides methods for producing CKSRPs using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a CKSRP has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered CKSRP) in a suitable medium until the CKSRP is produced.
  • the method further comprises isolating CKSRPs from the medium or the host cell.
  • Another aspect of the invention pertains to isolated CKSRPs, and biologically active portions thereof.
  • An “isolated” or “purified” polypeptide or biologically active portion thereof is free of some of the cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of CKSRP in which the polypeptide is separated from some of the cellular components of the cells in which it is naturally or recombinantly produced.
  • the language “substantially free of cellular material” includes preparations of a CKSRP having less than about 30% (by dry weight) of non-CKSRP material (also referred to herein as a “contaminating polypeptide”), more preferably less than about 20% of non-CKSRP material, still more preferably less than about 10% of non-CKSRP material, and most preferably less than about 5% non-CKSRP material.
  • non-CKSRP material also referred to herein as a “contaminating polypeptide”
  • the CKSRP or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation.
  • culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation.
  • substantially free of chemical precursors or other chemicals includes preparations of CKSRP in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of a CKSRP having less than about 30% (by dry weight) of chemical precursors or non-CKSRP chemicals, more preferably less than about 20% chemical precursors or non-CKSRP chemicals, still more preferably less than about 10% chemical precursors or non-CKSRP chemicals, and most preferably less than about 5% chemical precursors or non-CKSRP chemicals.
  • isolated polypeptides, or biologically active portions thereof lack contaminating polypeptides from the same organism from which the CKSRP is derived.
  • polypeptides are produced by recombinant expression of, for example, a Physcomitrella patens or Brassica napus CKSRP in plants other than Physcomitrella patens or Brassica napus , or microorganisms such as C. glutamicum , ciliates, algae, or fungi.
  • a Physcomitrella patens or Brassica napus CKSRP in plants other than Physcomitrella patens or Brassica napus
  • microorganisms such as C. glutamicum , ciliates, algae, or fungi.
  • the nucleic acid molecules, polypeptides, polypeptide homologs, fusion polypeptides, primers, vectors, and host cells described herein can be used in one or more of the following methods:identification of Physcomitrella patens, Saccharomyces cerevisiae or Brassica napus and related organisms; mapping of genomes of organisms related to Physcomitrella patens, Saccharomyces cerevisiae or Brassica napus ; identification and localization of Physcomitrella patens, Saccharomyces cerevisiae or Brassica napus sequences of interest; evolutionary studies; determination of CKSRP regions required for function; modulation of a CKSRP activity; modulation of the metabolism of one or more cell functions; modulation of the transmembrane transport of one or more compounds; modulation of stress resistance; and modulation of expression of CKSRP nucleic acids.
  • the CKSRP functions as an active potassium transport protein.
  • the CKSRP functions as an active potassium transport
  • the moss Physcomitrella patens is related to other mosses, such as Ceratodon purpureus , that are capable of growth in the absence of light.
  • Mosses like Ceratodon and Physcomitrella share a high degree of sequence identity on the DNA sequence and polypeptide level allowing the use of heterologous screening of DNA molecules with probes evolving from other mosses or organisms, thus enabling the derivation of a consensus sequence suitable for heterologous screening or functional annotation and prediction of gene functions in third species.
  • the ability to identify such functions can therefore have significant relevance, e.g., prediction of substrate specificity of enzymes.
  • these nucleic acid molecules may serve as reference points for the mapping of moss genomes, or of genomes of related organisms.
  • the CKSRP nucleic acid molecules of the invention have a variety of uses. Most importantly, the nucleic acid and amino acid sequences of the present invention can be used to transform plants, thereby inducing tolerance to stresses such as drought, high salinity, and cold.
  • the present invention therefore provides a transgenic plant transformed by a CKSRP nucleic acid, wherein expression of the nucleic acid sequence in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant.
  • the transgenic plant can be a monocot or a dicot.
  • transgenic plant can be selected from maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes, solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass, and forage crops, for example.
  • the present invention describes using the expression of PpCk-1, PpCK-2, PpCK-4, and PpPK-4 of Physcomitrella patens ; ScCK-1 of Saccharomyces cerevisiae ; and BnCK-1, BnCK-2, BnCK-3, BnCK-4, and BnCK-5 of Brassica napusto engineer drought-tolerant, salt-tolerant, and/or cold-tolerant plants.
  • This strategy has herein been demonstrated for Arabidopsis thaliana , Rapeseed/Canola, soybeans, corn, and wheat, but its application is not restricted to these plants.
  • the invention provides a transgenic plant containing a CKSRP such as the PpCK-1 as defined in SEQ ID NO:4, PpCK-2 as defined in SEQ ID NO:6, PpCK-4 as defined in SEQ ID NO:2, PpPK-4 as defined in SEQ ID NO:8, ScCK-1 as defined in SEQ ID NO:10, BnCK-1 as defined in SEQ ID NO:12, BnCK-2 as defined in SEQ ID NO:14, BnCK-3 as defined in SEQ ID NO:16, BnCK-4 as defined in SEQ ID NO:18, and BnCK-5 as defined in SEQ ID NO:20, wherein the plant has an increased tolerance to an environmental stress selected from one or more of the group consisting of drought, increased salt, or decreased or increased temperature.
  • the environmental stress is drought or decreased temperature.
  • the invention provides a method of producing a transgenic plant with a CKSRP coding nucleic acid, wherein expression of the nucleic acid(s) in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant comprising: (a) introducing into a plant cell an expression vector comprising a CKSRP nucleic acid, and (b) generating from the plant cell a transgenic plant with a increased tolerance to environmental stress as compared to a wild type variety of the plant.
  • the plant cell includes, but is not limited to, a protoplast, gamete producing cell, and a cell that regenerates into a whole plant.
  • transgenic refers to any plant, plant cell, callus, plant tissue, or plant part, that contains all or part of at least one recombinant polynucleotide. In many cases, all or part of the recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations.
  • the CKSRP nucleic acid encodes a protein comprising 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 present invention also provides a method of modulating a plant's tolerance to an environmental stress comprising, modifying the expression of a CKSRP coding nucleic acid in the plant.
  • the plant's tolerance to the environmental stress can be increased or decreased as achieved by increasing or decreasing the expression of a CKSRP, respectively.
  • the plant's tolerance to the environmental stress is increased by increasing expression of a CKSRP.
  • Expression of a CKSRP can be modified by any method known to those of skill in the art. The methods of increasing expression of CKSRPs can be used wherein the plant is either transgenic or not transgenic.
  • the plant can be transformed with a vector containing any of the above described CKSRP coding nucleic acids, or the plant can be transformed with a promoter that directs expression of native CKSRP in the plant, for example.
  • a promoter can be tissue preferred, developmentally regulated, stress inducible, or a combination thereof.
  • non-transgenic plants can have native CKSRP expression modified by inducing a native promoter.
  • PpCK-1 as defined in SEQ ID NO:4, PpCK-2 as defined in SEQ ID NO:6, PpCK-4 as defined in SEQ ID NO:2, PpPK-4 as defined in SEQ ID NO:8, ScCK-1 as defined in SEQ ID NO:10, BnCK-1 as defined in SEQ ID NO:12, BnCK-2 as defined in SEQ ID NO:14, BnCK-3 as defined in SEQ ID NO:16, BnCK-4 as defined in SEQ ID NO:18, or BnCK-5 as defined in SEQ ID NO:20 in target plants can be accomplished by, but is not limited to, one of the following examples: (a) constitutive promoter, (b) stress-inducible promoter, (c) chemical-induced promoter, and (d) engineered promoter overexpression with, for example, zinc-finger derived transcription factors (Greisman and Pabo, 1997, Science 275:657).
  • transcription of the CKSRP is modulated using zinc-finger derived transcription factors (ZFPs) as described in Greisman and Pabo, 1997, Science 275:657 and manufactured by Sangamo Biosciences, Inc.
  • ZFPs zinc-finger derived transcription factors
  • These ZFPs comprise both a DNA recognition domain and a functional domain that causes activation or repression of a target nucleic acid such as a CKSRP nucleic acid. Therefore, activating and repressing ZFPs can be created that specifically recognize the CKSRP promoters described above and used to increase or decrease CKSRP expression in a plant, thereby modulating the stress tolerance of the plant.
  • the present invention also includes identification of the homologs of PpCK-1 as defined in SEQ ID NO:4, PpCK-2 as defined in SEQ ID NO:6, PpCK-4 as defined in SEQ ID NO:2, PpPK-4 as defined in SEQ ID NO:8, ScCK-1 as defined in SEQ ID NO:10, BnCK-1 as defined in SEQ ID NO:12, BnCK-2 as defined in SEQ ID NO:14, BnCK-3 as defined in SEQ ID NO:16, BnCK-4 as defined in SEQ ID NO:18, and BnCK-5 as defined in SEQ ID NO:20 in a target plant, as well as the homolog's promoter.
  • the invention also provides a method of increasing expression of a gene of interest within a host cell as compared to a wild type variety of the host cell, wherein the gene of interest is transcribed in response to a CKSRP, comprising: (a) transforming the host cell with an expression vector comprising a CKSRP coding nucleic acid, and (b) expressing the CKSRP within the host cell, thereby increasing the expression of the gene transcribed in response to the CKSRP, as compared to a wild type variety of the host cell.
  • these sequences can also be used to identify an organism as being Physcomitrella patens, Brassica napus, Saccharomyces cerevisiae , or a close relative thereof. Also, they may be used to identify the presence of Physcomitrella patens, Brassica napus, Saccharomyces cerevisiae , or a relative thereof in a mixed population of microorganisms.
  • the invention provides the nucleic acid sequences of a number of Physcomitrella patens, Brassica napus , and Saccharomyces cerevisiae genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a Physcomitrella patens, Brassica napus , or Saccharomyces cerevisiae gene that is unique to this organism, one can ascertain whether this organism is present.
  • nucleic acid and polypeptide molecules of the invention may serve as markers for specific regions of the genome. This has utility not only in the mapping of the genome, but also in functional studies of Physcomitrella patens, Brassica napus , or Saccharomyces cerevisiae polypeptides.
  • the Physcomitrella patens genome could be digested, and the fragments incubated with the DNA-binding polypeptide. Those fragments that bind the polypeptide may be additionally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels.
  • nucleic acid molecules of the invention may be sufficiently identical to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related mosses.
  • the CKSRP nucleic acid molecules of the invention are also useful for evolutionary and polypeptide structural studies.
  • the metabolic and transport processes in which the molecules of the invention participate are utilized by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the polypeptide that are essential for the functioning of the enzyme. This type of determination is of value for polypeptide engineering studies and may give an indication of what the polypeptide can tolerate in terms of mutagenesis without losing function.
  • Manipulation of the CKSRP nucleic acid molecules of the invention may result in the production of CKSRPs having functional differences from the wild-type CKSRPs.
  • These polypeptides may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.
  • the effect of the genetic modification in plants, C. glutamicum , fungi, algae, or ciliates on stress tolerance can be assessed by growing the modified microorganism or plant under less than suitable conditions and then analyzing the growth characteristics and/or metabolism of the plant.
  • Such analysis techniques are well known to one skilled in the art, and include dry weight, wet weight, polypeptide synthesis, carbohydrate synthesis, lipid synthesis, evapotranspiration rates, general plant and/or crop yield, flowering, reproduction, seed setting, root growth, respiration rates, photosynthesis rates, etc.
  • yeast expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into Saccharomyces cerevisiae using standard protocols. The resulting transgenic cells can then be assayed for fail or alteration of their tolerance to drought, salt, and temperature stresses.
  • plant expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into an appropriate plant cell such as Arabidopsis , soy, rape, maize, wheat, Medicago truncatula , etc., using standard protocols. The resulting transgenic cells and/or plants derived there from can then be assayed for fail or alteration of their tolerance to drought, salt, and temperature stresses.
  • CKSRP genes of the invention may also result in CKSRPs having altered activities, which indirectly impact the stress response and/or stress tolerance of algae, plants, ciliates, or fungi, or other microorganisms like C. glutamicum .
  • the normal biochemical processes of metabolism result in the production of a variety of products (e.g., hydrogen peroxide and other reactive oxygen species), which may actively interfere with these same metabolic processes.
  • peroxynitrite is known to nitrate tyrosine side chains, thereby inactivating some enzymes having tyrosine in the active site (Groves, J. T., 1999, Curr. Opin. Chem. Biol. 3(2):226-235).
  • CKSRPs of the invention While these products are typically excreted, cells can be genetically altered to transport more products than is typical for a wild-type cell. By optimizing the activity of one or more CKSRPs of the invention that are involved in the export of specific molecules, such as salt molecules, it may be possible to improve the stress tolerance of the cell.
  • sequences disclosed herein, or fragments thereof can be used to generate knockout mutations in the genomes of various organisms, such as bacteria, mammalian cells, yeast cells, and plant cells (Girke, T., 1998, The Plant Journal 15:39-48).
  • the resultant knockout cells can then be evaluated for their ability or capacity to tolerate various stress conditions, their response to various stress conditions, and the effect on the phenotype and/or genotype of the mutation.
  • For other methods of gene inactivation see U.S. Pat. No. 6,004,804 “Non-Chimeric Mutational Vectors” and Puttaraju et al., 1999, Spliceosome-mediated RNA trans-splicing as a tool for gene therapy, Nature Biotechnology 17:246-252.
  • nucleic acid and polypeptide molecules of the invention may be utilized to generate algae, ciliates, plants, fungi, or other microorganisms like C. glutamicum expressing mutated CKSRP nucleic acid and polypeptide molecules such that the stress tolerance is improved.
  • the present invention also provides antibodies that specifically bind to a CKSRP, or a portion thereof, as encoded by a nucleic acid described herein.
  • Antibodies can be made by many well-known methods (See, e.g., Harlow and Lane, “Antibodies; A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells can then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced. (See, for example, Kelly et al., 1992, Bio/Technology 10:163-167; Bebbington et al., 1992, Bio/Technology 10:169-175).
  • the phrases “selectively binds” and “specifically binds” with the polypeptide refer to a binding reaction that is determinative of the presence of the polypeptide in a heterogeneous population of polypeptides and other biologics.
  • the specified antibodies bound to a particular polypeptide do not bind in a significant amount to other polypeptides present in the sample.
  • Selective binding of an antibody under such conditions may require an antibody that is selected for its specificity for a particular polypeptide.
  • a variety of immunoassay formats may be used to select antibodies that selectively bind with a particular polypeptide. For example, solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a polypeptide. See Harlow and Lane, “Antibodies, A Laboratory Manual” Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
  • monoclonal antibodies from various hosts.
  • a description of techniques for preparing such monoclonal antibodies may be found in Stites et al., eds., “Basic and Clinical Immunology,” (Lange Medical Publications, Los Altos, Calif., Fourth Edition) and references cited therein, and in Harlow and Lane “Antibodies, A Laboratory Manual” Cold Spring Harbor Publications, New York, 1988.
  • the protonema developed from the haploid spore as a chloroplast-rich chloronema and chloroplast-low caulonema, on which buds formed after approximately 12 days. These grew to give gametophores bearing antheridia and archegonia. After fertilization, the diploid sporophyte with a short seta and the spore capsule resulted, in which the meiospores matured.
  • RNA and DNA isolation were cultured in aerated liquid cultures. The protonemas were comminuted every 9 days and transferred to fresh culture medium.
  • CTAB buffer 2% (w/v) N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8.0; 1.4 M NaCl; 20 mM EDTA; N-Laurylsarcosine buffer: 10% (w/v) N-laurylsarcosine; 100 mM Tris HCl pH 8.0; and 20 mM EDTA.
  • CTAB buffer 2% (w/v) N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8.0; 1.4 M NaCl; 20 mM EDTA; N-Laurylsarcosine buffer: 10% (w/v) N-laurylsarcosine; 100 mM Tris HCl pH 8.0; and 20 mM EDTA.
  • the plant material was triturated under liquid nitrogen in a mortar to give a fine powder and transferred to 2 ml Eppendorf vessels.
  • the frozen plant material was then covered with a layer of 1 ml of decomposition buffer (1 ml CTAB buffer, 100 ⁇ l of N-laurylsarcosine buffer, 20 ⁇ l of ⁇ -mercaptoethanol, and 10 ⁇ l of proteinase K solution, 10 mg/ml) and incubated at 60° C. for one hour with continuous shaking.
  • the homogenate obtained was distributed into two Eppendorf vessels (2 ml) and extracted twice by shaking with the same volume of chloroform/isoamyl alcohol (24:1).
  • phase separation centrifugation was carried out at 8000 ⁇ g and room temperature for 15 minutes in each case.
  • the DNA was then precipitated at ⁇ 70° C. for 30 minutes using ice-cold isopropanol.
  • the precipitated DNA was sedimented at 4° C. and 10,000 g for 30 minutes and resuspended in 180 ⁇ l of TE buffer (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
  • the DNA was treated with NaCl (1.2 M final concentration) and precipitated again at ⁇ 70° C. for 30 minutes using twice the volume of absolute ethanol.
  • the DNA was dried and subsequently taken up in 50 ⁇ l of H 2 O+RNAse (50 mg/ml final concentration). The DNA was dissolved overnight at 4° C., and the RNAse digestion was subsequently carried out at 37° C. for 1 hour. Storage of the DNA took place at 4° C.
  • RNA and poly-(A) + RNA and cDNA library construction from Physcomitrella patens .
  • both total RNA and poly-(A) + RNA were isolated.
  • the total RNA was obtained from wild-type 9 day old protonemata following the GTC-method (Reski et al., 1994, Mol. Gen. Genet., 244:352-359).
  • the Poly(A) + RNA was isolated using Dyna Beads® (Dynal, Oslo, Norway) following the instructions of the manufacturer's protocol. After determination of the concentration of the RNA or of the poly(A)+ RNA, the RNA was precipitated by addition of ⁇ fraction (1/10) ⁇ volumes of 3 M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at ⁇ 70° C.
  • first strand synthesis was achieved using Murine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany) and oligo-d(T)-primers, second strand synthesis by incubation with DNA polymerase 1, Klenow enzyme and RNAseH digestion at 12° C. (2 hours), 16° C. (1 hour), and 22° C. (1 hour). The reaction was stopped by incubation at 65° C. (10 minutes) and subsequently transferred to ice. Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche, Mannheim) at 37° C. (30 minutes). Nucleotides were removed by phenol/chloroform extraction and Sephadex G50 spin columns.
  • EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends by T4-DNA-ligase (Roche, 12° C., overnight) and phosphorylated by incubation with polynucleotide kinase (Roche, 37° C., 30 minutes). This mixture was subjected to separation on a low melting agarose gel.
  • DNA molecules larger than 300 base pairs were eluted from the gel, phenol extracted, concentrated on Elutip-D-columns (Schleicher and Schuell, Dassel, Germany), and were ligated to vector arms and packed into lambda ZAPII phages or lambda ZAP-Express phages using the Gigapack Gold Kit (Stratagene, Amsterdam, Netherlands) using material and following the instructions of the manufacturer.
  • cDNA libraries as described in Example 3 were used for DNA sequencing according to standard methods, and in particular, by the chain termination method using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Rothstadt, Germany). Random sequencing was carried out subsequent to preparative plasmid recovery from cDNA libraries via in vivo mass excision, retransformation, and subsequent plating of DH10B on agar plates (material and protocol details from Stratagene, Amsterdam, Netherlands). Plasmid DNA was prepared from overnight grown E.
  • Physcomitrella patens ORFs corresponding to PpCK-1, PpCK-2, PpCK-4, and PpPK-4.
  • the Physcomitrella patens partial cDNAs (ESTs) for partial PpCK-1, PpCK-2, PpCK-4, and PpPK-4 were identified in the Physcomitrella patens EST sequencing program using the program EST-MAX through BLAST analysis. These particular clones, which were found to encode Protein Kinases, were chosen for further analyses.
  • the cultures were treated prior to RNA isolation as follows: Salt Stress: 2, 6, 12, 24, 48 hours with 1-M NaCl-supplemented medium; Cold Stress: 4° C. for the same time points as for salt; Drought Stress: cultures were incubated on dry filter paper for the same time points as for salt.
  • RACE Protocol 5′ RACE Protocol.
  • the EST sequences of PpCK-1, PpCK-2, PpCK-4, and PpPK-4 identified from the database search as described in Example 5 were used to design oligos for RACE (See Table 6).
  • the extended sequence for these genes were obtained by performing Rapid Amplification of cDNA Ends polymerase chain reaction (RACE PCR) using the Advantage 2 PCR kit (Clontech Laboratories) and the SMART RACE cDNA amplification kit (Clontech Laboratories) using a Biometra T3 Thermocycler following the manufacturer's instructions.
  • Full-length Amplification Full-length clones corresponding to PpCK-1, PpCK-2, and PpCK-4 were obtained by performing polymerase chain reaction (PCR) with gene-specific primers (See Table 6) and the original EST as the template.
  • the conditions for the reaction were standard conditions with PWO DNA polymerase (Roche). PCR was performed according to standard conditions and to manufacturer's protocols (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Biometra T3 Thermocycler).
  • the parameters for the reaction were: five minutes at 94° C. followed by five cycles of one minute at 94° C., one minute at 50° C., and 1.5 minutes at 72° C. This was followed by 25 cycles of one minute at 94° C., one minute at 65° C., and 1.5 minutes at 72° C.
  • the amplified fragments were extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector (Invitrogen) following manufacturer's instructions. Recombinant vectors were transformed into Top10 cells (Invitrogen) using standard conditions (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
  • Transformed cells were selected for on LB agar containing 100 mg/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside), and 0.8 mg IPTG (isopropylthio-b-D-galactoside) grown overnight at 37° C.
  • White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 mg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions.
  • the ORF 760 gene from Saccharamyces cerevisiae encoding a casein kinase 1, was first described in European Patent Application No. 03022225.1 by Metanomics, Inc. filed Sep. 30, 2003.
  • the Metanomics patent application is hereby incorporated by reference in its entirety.
  • the ORF 760 gene was isolated using the standard protocol of Pfu DNA polymerase or a PfulTaq DNA polymerase mix (Herculase) for the amplification procedure. Amplified ORF fragments were analyzed by gel electrophoresis.
  • Each primer consists of a universal 5′ end and ORF specific 3′ end whereby the universal sequences differ for the forward and reverse primers (Forward primer sequence contained an EcoRI for yeast or SmaI for E. coli and the reverse primer sequence a SmaI for yease or SacI for E. coli ) allowing a unidirectional cloning.
  • PCR reactions for the amplification included: 1 ⁇ PCR buffer, 0.2 mM dNTP, 100 ng Saccharomyces cerevisiae genomic DNA (S288C) or 60 ng genomic DNA Escherichia coli K-12 (MGI 655), 25 pmol reverse primer, 2.5 u Pfu or Herculase DNA polymerase.
  • the conditions consisted of: 1 cycle for 3′ at 94° C.; followed by 25 cycles of 30′′ at 94° C., 30′′ at 55° C., and 5-6′ at 72° C.; followed by 1 cycle for 610′ at 72° C., then at 4° C. indefinitely.
  • the forward sequence for ScCK-1 (ORF 760) is 5′-GGAATTCCAGCTGACCACCA TGTCCCAACGATCTTCACMCAC-3′ (SEQ ID NO:33).
  • the reverse sequence for ScCK-1 (ORF 760) is 5′-GATCCCCGGGMTTGCCATGTCAAAAAAAAAGGMAAA GAGAAAAG-3′ (SEQ ID NO:34).
  • Identification of Brassica napus ORFs Corresponding to BnCK-1, BnCK-2, BnCK-3, BnCK-4, and BnCK-5 Tissue harvest, RNA isolation, and cDNA library construction.
  • Canola plants were grown under a variety of conditions and treatments, and different tissues were harvested at various developmental stages. Plant growth and harvesting were done in a strategic manner such that the probability of harvesting all expressable genes in at least one or more of the resulting libraries is maximized.
  • the mRNA was isolated as described in Example 3 from each of the collected samples, and cDNA libraries were constructed. No amplification steps were used in the library production process in order to minimize redundancy of genes within the sample and to retain expression information. All libraries were 3′ generated from mRNA purified on oligo dT columns. Colonies from the transformation of the cDNA library into E. coli were randomly picked and placed into microtiter plates.
  • Probe Hybridization Plasmid DNA was isolated from the E coli colonies and then spotted on membranes. A battery of 288 33 P radiolabeled 7-mer oligonucleotides were sequentially hybridized to these membranes. To increase throughput, duplicate membranes were processed. After each hybridization, a blot image was captured during a phosphorimage scan to generate a hybridization profile for each oligonucleotide. This raw data image was automatically transferred via LIMS to a computer. Absolute identity was maintained by barcoding for the image cassette, filter, and orientation within the cassette. The filters were then treated using relatively mild conditions to strip the bound probes and returned to the hybridization chambers for another round of hybridization. The hybridization and imaging cycle was repeated until the set of 288 oligomers was completed.
  • a profile was generated for each spot (representing a cDNA insert), as to which of the 288 33 P radiolabeled 7-mer oligonucleotides bound to that particular spot (cDNA insert), and to what degree.
  • This profile is defined as the signature generated from that clone.
  • Each clone's signature was compared with all other signatures generated from the same organism to identify clusters of related signatures. This process “sorts” all of the clones from an organism into clusters before sequencing.
  • the clones were sorted into various clusters based on their having identical or similar hybridization signatures.
  • a cluster should be indicative of the expression of an individual gene or gene family.
  • a by-product of this analysis is an expression profile for the abundance of each gene in a particular library.
  • One-path sequencing from the 5′ end was used to predict the function of the particular clones by similarity and motif searches in sequence databases.
  • the full-length DNA sequence of the Saccharomyces cerevisiae ScCK-1 was blasted against proprietary contig databases of canola at E value of E-10.
  • All the contig hits were analyzed for the putative full-length sequences, and the longest clones representing the putative full-length contigs were fully sequenced.
  • Five such contigs isolated from the proprietary contig databases are BnCK-1, BnCK-2, BnCK-3, BnCK-4, and BnCK-5.
  • the purified fragment was then digested with EcoRI (Roche), purified by agarose gel, and extracted via the QIAquick Gel Extraction kit (Qiagen) according to manufacturer's instructions.
  • the vector pBlueScript was digested with EcoRI and SmaI (Roche) according to manufacturer's instructions, and the resulting fragment was extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacturer's instructions.
  • the digested pBlueScript vector and the gentamycin cassette fragments were ligated with T4 DNA Ligase (Roche) according to manufacturer's instructions, joining the two respective EcoRI sites and joining the blunt-ended HindIII site with the SmaI site.
  • the recombinant vector (pGMBS) was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 ⁇ g/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) and 0.8 mg IPTG (isopropylthio- ⁇ -D-galactoside), grown overnight at 37° C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 ⁇ g/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Analyses of subsequent clones and restriction mapping were performed according to standard molecular biology techniques (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
  • Both the pGMBS vector and p1 bxSuperGUS vector were digested with XbaI and KpnI (Roche) according to manufacturer's instructions, excising the gentamycin cassette from pGMBS and producing the backbone from the p1 bxSuperGUS vector.
  • the resulting fragments were extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacturer's instructions. These two fragments were ligated with T4 DNA ligase (Roche) according to manufacturer's instructions.
  • the resulting recombinant vector (pBPS-JH001) was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 ⁇ g/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) and 0.8 mg IPTG (isopropylthio- ⁇ -D-galactoside), grown overnight at 37° C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 ⁇ g/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Analyses of subsequent clones and restriction mapping were performed according to standard molecular biology techniques (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
  • pBPS Binary vector construction: pBPS—SC022.
  • the plasmid construct pACGH101 was digested with PstI (Roche) and FseI (NEB) according to manufacturers' instructions. The fragment was purified by agarose gel and extracted via the Qiaex II DNA Extraction kit (Qiagen). This resulted in a vector fragment with the Arabidopsis Actin2 promoter with internal intron and the OCS3 terminator.
  • Plasmid DNA was recovered using the Qiaprep Spin Miniprep kit (Qiagen) and sequenced in both the 5′ and 3′ directions using standard conditions. Subsequent analysis of the sequence data using Vector NTI software revealed that there were not any PCR errors introduced in the NPTII gene sequence.
  • the NPT-Topo construct was then digested with PstI (Roche) and FseI (NEB) according to manufacturers' instructions.
  • the 0.9 kilobase fragment was purified on agarose gel and extracted by Qiaex II DNA Extraction kit (Qiagen).
  • the Pst/Fse insert fragment from NPT-Topo and the Pst/Fse vector fragment from pACGH101 were then ligated together using T4 DNA Ligase (Roche) following manufacturer's instructions.
  • the ligation reaction was then transformed into Top10 cells (Invitrogen) under standard conditions, creating pBPS-sc019 construct.
  • Colonies were selected on LB plates with 50 ⁇ g/ml kanamycin sulfate and grown overnight at 37° C. These colonies were then used to inoculate 2 ml LB media with 50 ⁇ g/ml kanamycin sulfate and grown overnight at 37° C. Plasmid DNA was recovered using the Qiaprep Spin Miniprep kit (Qiagen) following the manufacturer's instructions.
  • the pBPS—SC019 construct was digested with KpnI and BsaI (Roche) according to manufacturer's instructions. The fragment was purified via agarose gel and then extracted via the Qiaex II DNA Extraction kit (Qiagen) as per its instructions, resulting in a 3 kilobase Act-NPT cassette, which included the Arabidopsis Actin2 promoter with internal intron, the NPTII gene, and the OCS3 terminator.
  • the pBPS-JH001 vector was digested with SpeI and ApaI (Roche) and blunt-end filled with Klenow enzyme and 0.1 mM dNTPs (Roche) according to manufacturer's instructions. This produced a 10.1 kilobase vector fragment minus the Gentamycin cassette, which was recircularized by self-ligating with T4 DNA Ligase (Roche), and transformed into Top10 cells (Invitrogen) via standard conditions. Transformed cells were selected for on LB agar containing 50 ⁇ g/ml kanmycin sulfate and grown overnight at 37° C.
  • Colonies were then used to inoculate 2 ml of liquid LB containing 50 ⁇ g/ml kanamycin sulfate and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. The recircularized plasmid was then digested with KpnI (Roche) and extracted from agarose gel via the Qiaex II DNA Extraction kit (Qiagen) according to manufacturers' instructions.
  • the Act-NPT Kpn-cut insert and the Kpn-cut pBPS-JH001 recircularized vector were then ligated together using T4 DNA Ligase (Roche) and transformed into Top10 cells (Invitrogen) according to manufacturers' instructions.
  • the resulting construct, pBPS-SC022 now contained the Super Promoter, the GUS gene, the NOS terminator, and the Act-NPT cassette.
  • Transformed cells were selected for on LB agar containing 50 ⁇ g/ml kanmycin sulfate and grown overnight at 37° C. Colonies were then used to inoculate 2 ml of liquid LB containing 50 ⁇ g/ml kanamycin sulfate and grown overnight at 37° C.
  • Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. After confirmation of ligation success via restriction digests, pBPS-sc022 plasmid DNA was further propagated and recovered using the Plasmid Midiprep Kit (Qiagen) following the manufacturer's instructions.
  • the resulting recombinant vectors contained the corresponding casein kinase in the sense orientation under the control of the constitutive superpromoter.
  • TABLE 13 Listed are the names of the constructs of the Physcomitrella patens casein kinases used for plant transformation.
  • Enzymes used to Recombinant Enzymes used restrict binary Binary to generate binary vector Gene Vector gene fragment vector construct PpCK-1 pBPS- Xmal/Hpal Xmal/Ecl136 pBPS- SC022 SY012 PpCK-2 pBPS- Xmal/Hpal Xmal/Ecl136 pBPS- SC022 JYW034 PpCK-4 pBPS- Xmal/Hpal Xmal/Ecl136 pBPS- SC022 SY018 PpPK-4 pBPS- Xma/EcoRV Xmal/Ecl136 pBPS- JH001 LVM015
  • the ScCK-1 gene was subcloned into a binary vector 1bxbigResgen that is based on a modified pPZP binary vector backbone.
  • the vector comprised the kanamycin gene for bacterial selection (Hajukeiwicz et al., 1994, Plant Mol. Biol. 25:989-994) and the bar gene driven by the mas1 promoter on its T-DNA(Velten et al., 1984, EMBO J. 3:2723-2730; Mengiste, et al., 1997, Plant J., 12:945-948).
  • the T-DNA contained the strong double 35S promoter (Kay et al., 1987, Science 236:1299-1302) in front of a cloning cassette which was followed by the nos terminator (Depicker et al., J. Mol. Appl. Gen. 1 (6):561-573).
  • the cloning cassette consisted of the sequence: 5′-GGMTTCCAGCTGACCACCATGGC MTTCCCGGGGATC-3′ (SEQ ID NO:37).
  • Other selection systems and promoters are known in the art and are similarly capable of use in the present invention (e.g. AHAS marker, ubiquitin promoter (Callis et al., J. Biol. Chem.
  • the binary vector and the ORF 760 gene (100 ng) were digested with EcoRI and SmaI using the standard protocol provided by the supplier (MBI Fermentas, Germany).
  • the ORF 760 gene was purified using a Qiagen column (Qiagen, Hilden, Germany), and was ligated with the restriction digested binary vector (30 ng) using standard procedures (Maniatis et al.).
  • Agrobacterium Transformation The recombinant vectors were transformed into Agrobacterium tumefaciens C58C1 and PMP90 according to standard conditions (Hoefgen and Willmitzer, 1990; Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396).
  • Arabidopsis thaliana ecotype C24 were grown and transformed according to standard conditions (Bechtold, 1993, Acad. Sci. Paris. 316:1194-1199; Bent et al., 1994 , Science 265:1856-1860).
  • T1 seeds were sterilized according to standard protocols (Xiong et al., 1999, Plant Molecular Biology Reporter 17:159-170). Seeds were selected on 2 Murashige and Skoog media (MS) (Sigma-Aldrich), 0.6% agar and supplemented with 1% sucrose, and 2 ⁇ g/ml benomyl (Sigma-Aldrich). Seeds on plates were vernalized for four days at 4° C. The seeds were germinated in a climatic chamber at an air temperature of 22° C.
  • Transformed seedlings were selected after 14 days and transferred to ⁇ fraction (1/2) ⁇ MS media supplemented with 0.6% agar, 1% sucrose, and allowed to recover for five to seven days.
  • T1 seedlings were transferred to dry, sterile filter paper in a petri dish and allowed to desiccate for two hours at 80% RH (relative humidity) in a Sanyo Growth Cabinet MLR-350H, micromols m ⁇ 2 s ⁇ 1 (white light; Philips TL 65W/25 fluorescent tube). The RH was then decreased to 60%, and the seedlings were desiccated further for eight hours. Seedlings were then removed and placed on ⁇ fraction (1/2) ⁇ MS 0.6% agar plates supplemented with 2 ⁇ g/ml benomyl (Sigma-Aldrich) and scored after five days. The transgenic plants were then screened for their improved drought tolerance.
  • PpCK-1-overexpressing Arabidopsis thaliana plants showed a 50% survival rate to the drought stress (5 survivors from 10 stressed plants)
  • PpCK-2-overexpressing Arabidopsis thaliana plants showed a 52% survival rate to the drought stress (16 survivors from 31 stressed plants)
  • PpCK-4-overexpression Arabidopsis thaliana plants showed a 14% survival rate to the drought stress (1 survivors from 7 stressed plants), as compared to the 11% survival rate that was demonstrated by the untransformed control plants (1 survivor from 9 stressed plants).
  • Transgenic Arabidopsis plants comprising the ScCK-1 gene were screened for their tolerance to drought in three separate experiments. In the first experiment, the plants were subjected to a period of twelve days of drought conditions. After the twelve days, the transgenic plants were screened for their improved drought tolerance. Transgenic plants containing the ScCK-1 transgene (11 plants) retained viability, as shown by their turgid appearance and maintenance of green color, for an average of 2.2 days beyond the untransformed wild type control plant.
  • transgenic plants are screened for their improved cold tolerance, demonstrating that transgene expression confers cold tolerance.
  • PpCK-1-overexpressing Arabidopsis thaliana plants showed a 100% survival rate to the freeze stress (14 survivors from 14 stressed plants), as compared to the 2% survival rate that was demonstrated by the untransformed control plants (1 survivor from 48 stressed plants).
  • Salt Tolerance Screening Seedlings are transferred to filter paper soaked in ⁇ fraction (1/2) ⁇ MS and placed on ⁇ fraction (1/2) ⁇ MS 0.6% agar supplemented with 2 ⁇ g/ml benomyl the night before the salt tolerance screening.
  • the filter paper with the seedlings was moved to stacks of sterile filter paper, soaked in 50 mM NaCl, in a petri dish. After two hours, the filter paper with the seedlings was moved to stacks of sterile filter paper, soaked with 200 mM NaCl, in a petri dish.
  • the filter paper with the seedlings was moved to stacks of sterile filter paper, soaked in 600 mM NaCl, in a petri dish. After 10 hours, the seedlings were moved to petri dishes containing ⁇ fraction (1/2) ⁇ MS 0.6% agar supplemented with 2 ⁇ g/ml benomyl. The seedlings were scored after 5 days.
  • Transgenic plants overexpressing the transgene are screened for their improved salt tolerance demonstrating that transgene expression confers salt tolerance.
  • PpCK-1-overexpressing Arabidopsis thaliana plants showed a 0% survival rate to the freeze stress (0 survivors from 20 stressed plants)
  • PpCK-2-overexpressing Arabidopsis thaliana plants showed a 10% survival rate to the freeze stress (1 survivors from 10 stressed plants)
  • PpCK-4-overexpressing Arabidopsis thaliana plants showed a 0% survival rate to the freeze stress (0 survivors from 6 stressed plants), as compared to the 13% survival rate that was demonstrated by the untransformed control plants (3 survivor from 23 stressed plants).
  • the PpCK-1, PpCK-2, PpCK-4, and PpPK-4 genes were overexpressed in Arabidopsis thaliana under the control of a constitutive promoter.
  • the transgenic lines were assayed for water use efficiency (WUE), and some of the lines were also assayed for biomass after drought cycling (See Table 15).
  • SCO 24 represents the empty vector control
  • BPS C24 represents the Arabidopsis ecotype used for transformation.
  • DW indicates dry weight
  • E denotes plant water use.
  • the letters under the Assay column represent independent experiments.
  • EST 289 (PpCK-4) transgenic lines had significant increases in dry weight.
  • EST 142 (PpPK-4) transgenic lines had significant increases in WUE and biomass under drought cycling conditions.
  • the mean versus both of the controls for each paramenter was increased, 5-8% for WUE, 11-19% for DW, and 6-9% for E.
  • the variation in phenotype from gene to gene may be explained by variation in the level of transgene expression and the site of transgene insertion.
  • ScCK-1 (ORF 760) was overexpressed in Arabidopsis thaliana under the control of a constitutive promoter. The transgenic lines were assayed for dessication tolerance, measuring the average number of days of survival after the wild type control was dead (Table 7).* Please see drawing at FIG. 8 .
  • RNA samples taken as exemplified below. Two and one half microliters of the RNA sample was used in a 50 ⁇ l PCR reaction using Taq DNA polymerase (Roche Molecular Biochemicals) according to the manufacturer's instructions.
  • Binary vector plasmid with each gene cloned in was used as positive control, and the wild-type C24 genomic DNA was used as negative control in the PCR reactions.
  • Ten ⁇ l of the PCR reaction was analyzed on 0.8% agarose—ethidium bromide gel.
  • PpCK-1 The primers used in the reactions are: GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO: 38) GCGTTAACATGCCCATCTTCTCATACTCAGACC (SEQ ID NO: 39)
  • the PCR program was as following: 30 cycles of 1 minute at 94° C., 1 minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72° C. A 1.7 kb fragment was produced from the positive control and the transgenic plants.
  • PpCK-2 The primers used in the reactions are: GCTGACACGCCAAGCCTCGCTAGTC (SEQ ID NO: 40) GCGTTAACCTTAGGAATCGTATGGCAGAGAGCT (SEQ ID NO: 41)
  • the PCR program was as following: 30 cycles of 1 minute at 94° C., 1 minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72° C. A 1.9 kb fragment was produced from the positive control and the transgenic plants.
  • PpPK-4 The primers used in the reactions were: 5′ATCCCGGGAGGCATTGAACTACCTGGAG (SEQ ID NO: 42) TGAG3′ 5′GCGATATCGTTGAACTAGTAATCTGTGT (SEQ ID NO: 43) TAACTTTATC3′
  • the PCR program was as following: 30 cycles of 1 minute at 94° C., 1 minute at 62° C., and 4 minutes at 72° C., followed by 10 minutes at 72° C. A 1.7 kilobase fragment was produced from the positive control and the transgenic plants.
  • the transgenes were successfully amplified from the T1 transgenic lines, but not from the wild type C24. This result indicates that the T1 transgenic plants contain at least one copy of the transgenes. There was no indication of existence of either identical or very similar genes in the untransformed Arabidopsis thaliana control, which could be amplified by this method from the wild-type plants.
  • the embryo axis is examined to make sure that the meristematic region is not damaged.
  • the excised embryo axes are collected in a half-open sterile Petri dish and air-dried to a moisture content less than 20% (fresh weight) in a sealed Petri dish until further use.
  • Agrobacterium tumefaciens culture is prepared from a single colony in LB solid medium plus appropriate selection agents followed by growth of the single colony in liquid LB medium to an optical density at 600 nm of 0.8. Then, the bacteria culture is pelleted at 7000 rpm for 7 minutes at room temperature, and resuspended in MS (Murashige and Skoog, 1962) medium supplemented with 100 ⁇ M acetosyringone. Bacteria cultures are incubated in this pre-induction medium for 2 hours at room temperature before use. The axis of soybean zygotic seed embryos at approximately 15% moisture content are imbibed for 2 hours at room temperature with the pre-induced Agrobacterium suspension culture.
  • the embryos are removed from the imbibition culture and are transferred to Petri dishes containing solid MS medium supplemented with 2% sucrose and incubated for 2 days in the dark at room temperature. Alternatively, the embryos are placed on top of moistened (liquid MS medium) sterile filter paper in a Petri dish and incubated under the same conditions described above. After this period, the embryos are transferred to either solid or liquid MS medium supplemented with 500 mg/L carbenicillin or 300 mg/L cefotaxime to kill the Agrobacteria . The liquid medium is used to moisten the sterile filter paper. The embryos are incubated during 4 weeks at 25° C., under 150 ⁇ mol m ⁇ 2 sec ⁇ 1 and 12 hours photoperiod.
  • the seedlings produce roots, they are transferred to sterile metromix soil.
  • the medium of the in vitro plants is washed off before transferring the plants to soil.
  • the plants are kept under a plastic cover for 1 week to favor the acclimatization process.
  • the plants are transferred to a growth room where they are incubated at 25° C., under 150 ⁇ mol m ⁇ 2 sec ⁇ 1 light intensity and 12 hours photoperiod for about 80 days.
  • transgenic plants are screened for their improved drought, salt, and/or cold tolerance according to the screening method described in Example 9, demonstrating that transgene expression confers stress tolerance and/or increased water use efficiency.
  • transgenic plants are screened for their improved stress tolerance according to the screening method described in Example 9, demonstrating that transgene expression confers stress tolerance and/or increased water use efficiency.
  • Homologous genes can be used to identify homologous or heterologous genes from cDNA or genomic libraries.
  • Homologous genes e.g. full-length cDNA clones
  • 100,000 up to 1,000,000 recombinant bacteriophages are plated and transferred to nylon membranes.
  • DNA is immobilized on the membrane by, e.g., UV cross linking.
  • Hybridization is carried out at high stringency conditions. In aqueous solution, hybridization and washing is performed at an ionic strength of 1 M NaCl and a temperature of 68° C.
  • Hybridization probes are generated by, e.g., radioactive ( 32 P) nick transcription labeling (High Prime, Roche, Mannheim, Germany). Signals are detected by autoradiography.
  • Partially homologous or heterologous genes that are related but not identical can be identified in a manner analogous to the above-described procedure using low stringency hybridization and washing conditions.
  • the ionic strength is normally kept at 1 M NaCl while the temperature is progressively lowered from 68 to 42° C.
  • Radiolabeled oligonucleotides are prepared by phosphorylation of the 5-prime end of two complementary oligonucleotides with T4 polynucleotide kinase. The complementary oligonucleotides are annealed and ligated to form concatemers. The double stranded concatemers are then radiolabeled by, for example, nick transcription. Hybridization is normally performed at low stringency conditions using high oligonucleotide concentrations.
  • the temperature is lowered stepwise to 5-10° C. below the estimated oligonucleotide T m , or down to room temperature, followed by washing steps and autoradiography. Washing is performed with low stringency, such as 3 washing steps using 4 ⁇ SSC. Further details are described by Sambrook, J. et al., 1989, “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press or Ausubel, F. M. et al., 1994, “Current Protocols in Molecular Biology”, John Wiley & Sons.
  • c-DNA clones can be used to produce recombinant protein for example in E. coli (e.g. Qiagen QIAexpress pQE system). Recombinant proteins are then normally affinity purified via Ni-NTA affinity chromatography (Qiagen). Recombinant proteins are then used to produce specific antibodies for example by using standard techniques for rabbit immunization. Antibodies are affinity purified using a Ni-NTA column saturated with the recombinant antigen as described by Gu et al., 1994, BioTechniques 17:257-262.
  • the antibody can be used to screen expression cDNA libraries to identify homologous or heterologous genes via an immunological screening (Sambrook, J. et al., 1989, “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press or Ausubel, F. M. et al., 1994, “Current Protocols in Molecular Biology”, John Wiley & Sons).
  • In vivo Mutagenesis of microorganisms can be performed by passage of plasmid (or other vector) DNA through E. coli or other microorganisms (e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae ), which are impaired in their capabilities to maintain the integrity of their genetic information.
  • E. coli or other microorganisms e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae
  • Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp, W. D., 1996, DNA repair mechanisms, in: Escherichia coli and Salmonella , p.
  • DNA band-shift assays also called gel retardation assays
  • reporter gene assays such as that described in Kolmar, H. et al., 1995, EMBO J. 14:3895-3904 and references cited therein. Reporter gene test systems are well known and established for applications in both pro- and eukaryotic cells, using enzymes such as ⁇ -galactosidase, green fluorescent protein, and several others.
  • membrane-transport proteins The determination of activity of membrane-transport proteins can be performed according to techniques such as those described in Gennis, R. B., 1989, Pores, Channels and Transporters, in Biomembranes, Molecular Structure and Function, pp. 85-137, 199-234 and 270-322, Springer: Heidelberg.
  • Purification of the Desired Product from Transformed Organisms Recovery of the desired product from plant material (i.e., Physcomitrella patens or Arabidopsis thaliana ), fungi, algae, ciliates, C. glutamicum cells, or other bacterial cells transformed with the nucleic acid sequences described herein, or the supernatant of the above-described cultures can be performed by various methods well known in the art. If the desired product is not secreted from the cells, the cells can be harvested from the culture by low-speed centrifugation, and the cells can be lysed by standard techniques, such as mechanical force or sonification. Organs of plants can be separated mechanically from other tissue or organs.
  • cellular debris is removed by centrifugation, and the supernatant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from desired cells, then the cells are removed from the culture by low-speed centrifugation, and the supernate fraction is retained for further purification.
  • the supernatant fraction from either purification method is subjected to chromatography with a suitable resin, in which the desired molecule is either retained on a chromatography resin while many of the impurities in the sample are not, or where the impurities are retained by the resin, while the sample is not.
  • chromatography steps may be repeated as necessary, using the same or different chromatography resins.
  • One skilled in the art would be well-versed in the selection of appropriate chromatography resins and in their most efficacious application for a particular molecule to be purified.
  • the purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the product is maximized.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Botany (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)
US10/904,588 2000-04-07 2004-11-17 Casein kinase stress-related polypeptides and methods of use in plants Abandoned US20050066396A1 (en)

Priority Applications (13)

Application Number Priority Date Filing Date Title
US10/904,588 US20050066396A1 (en) 2000-04-07 2004-11-17 Casein kinase stress-related polypeptides and methods of use in plants
MX2007005802A MX2007005802A (es) 2004-11-17 2005-11-17 Polipeptidos de caseina quinasa relacionados con el estres y metodos de uso en plantas.
EP05849385A EP1814378A4 (fr) 2004-11-17 2005-11-17 Polypeptides de caseine kinase lies au stress et procede d'utilisation de ceux-ci dans des plantes
US11/667,820 US20080052794A1 (en) 2004-11-17 2005-11-17 Casein Kinase Stress-Related Polypeptides And Methods Of Use In Plants
CNA2005800466941A CN101102665A (zh) 2004-11-17 2005-11-17 酪蛋白激酶胁迫相关多肽及其用于植物的方法
AU2005307824A AU2005307824A1 (en) 2004-11-17 2005-11-17 Casein Kinase Stress-Related Polypeptides and methods of use in plants
BRPI0517851-7A BRPI0517851A (pt) 2004-11-17 2005-11-17 célula de planta transgênica, planta transgênica, semente, ácido nucleico isolado, vetor de expressão recombinante isolado, e, método de produzir uma planta transgênica
PCT/US2005/041522 WO2006055631A2 (fr) 2004-11-17 2005-11-17 Polypeptides de caseine kinase lies au stress et procede d'utilisation de ceux-ci dans des plantes
CA002587401A CA2587401A1 (fr) 2004-11-17 2005-11-17 Polypeptides de caseine kinase lies au stress et procede d'utilisation de ceux-ci dans des plantes
ARP050104850A AR051503A1 (es) 2004-11-17 2005-11-18 Polipeptidos de caseina quinasa relacionados con el estres y metodos de uso en plantas
US11/737,826 US7399904B2 (en) 2000-04-07 2007-04-20 Casein kinase stress-related polypeptides and methods of use in plants
ZA200704387A ZA200704387B (en) 2004-11-17 2007-05-29 Casein kinase stress-related polypeptides and methods of use in plants
US12/472,651 US7795415B2 (en) 2004-11-17 2009-05-27 Casein kinase stress-related polypeptides and methods of use in plants

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US19600100P 2000-04-07 2000-04-07
US09/828,313 US6867351B2 (en) 2000-04-07 2001-04-06 Protein kinase stress-related proteins and methods of use in plants
US34609601P 2001-11-09 2001-11-09
US10/292,408 US7176026B2 (en) 2001-11-09 2002-11-12 Protein kinase stress-related polypeptides and methods of use in plants
US10/904,588 US20050066396A1 (en) 2000-04-07 2004-11-17 Casein kinase stress-related polypeptides and methods of use in plants

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
US09/828,313 Continuation-In-Part US6867351B2 (en) 2000-04-07 2001-04-06 Protein kinase stress-related proteins and methods of use in plants
US10/292,408 Continuation-In-Part US7176026B2 (en) 2000-04-07 2002-11-12 Protein kinase stress-related polypeptides and methods of use in plants

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09/828,313 Continuation-In-Part US6867351B2 (en) 2000-04-07 2001-04-06 Protein kinase stress-related proteins and methods of use in plants
US11/737,826 Division US7399904B2 (en) 2000-04-07 2007-04-20 Casein kinase stress-related polypeptides and methods of use in plants

Publications (1)

Publication Number Publication Date
US20050066396A1 true US20050066396A1 (en) 2005-03-24

Family

ID=36407725

Family Applications (4)

Application Number Title Priority Date Filing Date
US10/904,588 Abandoned US20050066396A1 (en) 2000-04-07 2004-11-17 Casein kinase stress-related polypeptides and methods of use in plants
US11/667,820 Abandoned US20080052794A1 (en) 2004-11-17 2005-11-17 Casein Kinase Stress-Related Polypeptides And Methods Of Use In Plants
US11/737,826 Expired - Fee Related US7399904B2 (en) 2000-04-07 2007-04-20 Casein kinase stress-related polypeptides and methods of use in plants
US12/472,651 Expired - Fee Related US7795415B2 (en) 2004-11-17 2009-05-27 Casein kinase stress-related polypeptides and methods of use in plants

Family Applications After (3)

Application Number Title Priority Date Filing Date
US11/667,820 Abandoned US20080052794A1 (en) 2004-11-17 2005-11-17 Casein Kinase Stress-Related Polypeptides And Methods Of Use In Plants
US11/737,826 Expired - Fee Related US7399904B2 (en) 2000-04-07 2007-04-20 Casein kinase stress-related polypeptides and methods of use in plants
US12/472,651 Expired - Fee Related US7795415B2 (en) 2004-11-17 2009-05-27 Casein kinase stress-related polypeptides and methods of use in plants

Country Status (10)

Country Link
US (4) US20050066396A1 (fr)
EP (1) EP1814378A4 (fr)
CN (1) CN101102665A (fr)
AR (1) AR051503A1 (fr)
AU (1) AU2005307824A1 (fr)
BR (1) BRPI0517851A (fr)
CA (1) CA2587401A1 (fr)
MX (1) MX2007005802A (fr)
WO (1) WO2006055631A2 (fr)
ZA (1) ZA200704387B (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030182692A1 (en) * 2001-11-09 2003-09-25 Thielen Nocha Van Protein kinase stress-related polypeptides and methods of use in plants
US20070079400A1 (en) * 2000-04-07 2007-04-05 Damian Allen Protein kinase stress-related proteins and methods of use in plants
CN101701038B (zh) * 2009-10-29 2012-05-02 中国农业科学院作物科学研究所 植物低温生长相关蛋白及其编码基因与应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102311490B (zh) 2010-07-08 2014-10-22 中国科学院上海生命科学研究院 一种植物抗热基因JAZ5a及其应用
CA2839840A1 (fr) * 2011-07-07 2013-01-10 Keygene N.V. Utilisation de jaz5a pour l'amelioration de la resistance a la secheresse chez une plante
US8944163B2 (en) 2012-10-12 2015-02-03 Harris Corporation Method for hydrocarbon recovery using a water changing or driving agent with RF heating
US9101100B1 (en) 2014-04-30 2015-08-11 Ceres, Inc. Methods and materials for high throughput testing of transgene combinations

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030135877A1 (en) * 2001-03-16 2003-07-17 Haertel Heiko A. Sugar and lipid metabolism regulators in plants

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040031072A1 (en) * 1999-05-06 2004-02-12 La Rosa Thomas J. Soy nucleic acid molecules and other molecules associated with transcription plants and uses thereof for plant improvement
US20100293669A2 (en) * 1999-05-06 2010-11-18 Jingdong Liu Nucleic Acid Molecules and Other Molecules Associated with Plants and Uses Thereof for Plant Improvement
DE60141547D1 (de) * 2000-04-07 2010-04-22 Basf Plant Science Gmbh Stress-gekoppelte Protein-Phosphatase und ihre Verwendung in Pflanzen
WO2002052012A2 (fr) * 2000-12-22 2002-07-04 Cropdesign N.V. Genes de betterave a sucre impliques dans la tolerance aux stress
AR050453A1 (es) * 2004-08-11 2006-10-25 Basf Plant Science Gmbh Acidos nucleicos que confieren alteraciones en los lipidos y azucares en plantas ii

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030135877A1 (en) * 2001-03-16 2003-07-17 Haertel Heiko A. Sugar and lipid metabolism regulators in plants

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070079400A1 (en) * 2000-04-07 2007-04-05 Damian Allen Protein kinase stress-related proteins and methods of use in plants
US7442853B2 (en) 2000-04-07 2008-10-28 Basf Plant Science Gmbh Protein kinase stress-related proteins and methods of use in plants
US20030182692A1 (en) * 2001-11-09 2003-09-25 Thielen Nocha Van Protein kinase stress-related polypeptides and methods of use in plants
US7176026B2 (en) * 2001-11-09 2007-02-13 Basf Plant Science Gmbh Protein kinase stress-related polypeptides and methods of use in plants
CN101701038B (zh) * 2009-10-29 2012-05-02 中国农业科学院作物科学研究所 植物低温生长相关蛋白及其编码基因与应用

Also Published As

Publication number Publication date
EP1814378A4 (fr) 2008-09-24
US20080052794A1 (en) 2008-02-28
US7399904B2 (en) 2008-07-15
EP1814378A2 (fr) 2007-08-08
US7795415B2 (en) 2010-09-14
WO2006055631A3 (fr) 2007-05-18
CA2587401A1 (fr) 2006-05-26
AR051503A1 (es) 2007-01-17
CN101102665A (zh) 2008-01-09
MX2007005802A (es) 2007-07-16
AU2005307824A1 (en) 2006-05-26
BRPI0517851A (pt) 2008-10-21
US20080050820A1 (en) 2008-02-28
ZA200704387B (en) 2008-08-27
WO2006055631A2 (fr) 2006-05-26
US20090282580A1 (en) 2009-11-12

Similar Documents

Publication Publication Date Title
US7795415B2 (en) Casein kinase stress-related polypeptides and methods of use in plants
US7598431B2 (en) Protein kinase stress-related polypeptides and methods of use in plants
US7399903B2 (en) Vesicle trafficking stress-related polypeptides and methods of use in plants
US7880055B2 (en) Lecitin-like protein kinase stress-related polypeptides and methods of use in plants
AU2008229869B2 (en) Transcription factor stress-related polypeptides and methods of use in plants
US20030140380A1 (en) Amine oxidase stress-related polypeptides and methods of use in plants
AU2008212062B2 (en) Protein kinase stress-related polypeptides and methods of use in plants
US20030163850A1 (en) Ion transporter stress-related polypeptides and methods of use in plants
AU2011203289A1 (en) Protein kinase stress-related polypeptides and methods of use in plants
AU2002363445A1 (en) Protein kinase stress-related polypeptides and methods of use in plants
AU2002348367A1 (en) Transcription factor stress-related polypeptides and methods of use in plants

Legal Events

Date Code Title Description
AS Assignment

Owner name: BASF PLANT SCIENCE GMBH, GERMANY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SHIRLEY, AMBER;ALLEN, DAMIAN;VAN THIELEN, NOCHA E.;AND OTHERS;REEL/FRAME:015897/0216;SIGNING DATES FROM 20050406 TO 20050412

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION