WO2012126347A1 - 耐盐基因及其应用 - Google Patents

耐盐基因及其应用 Download PDF

Info

Publication number
WO2012126347A1
WO2012126347A1 PCT/CN2012/072580 CN2012072580W WO2012126347A1 WO 2012126347 A1 WO2012126347 A1 WO 2012126347A1 CN 2012072580 W CN2012072580 W CN 2012072580W WO 2012126347 A1 WO2012126347 A1 WO 2012126347A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
amino acid
nucleotide sequence
acid sequence
salt
Prior art date
Application number
PCT/CN2012/072580
Other languages
English (en)
French (fr)
Inventor
谢旗
张译月
李刚
张华伟
Original Assignee
中国科学院遗传与发育生物学研究所
先正达参股股份有限公司
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
Application filed by 中国科学院遗传与发育生物学研究所, 先正达参股股份有限公司 filed Critical 中国科学院遗传与发育生物学研究所
Publication of WO2012126347A1 publication Critical patent/WO2012126347A1/zh

Links

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

Definitions

  • the present invention relates to salt tolerance genes and uses thereof.
  • the invention also relates to vectors, host cells and plants comprising the genes and methods of producing plants. This gene can be used to improve the tolerance of plants to salt stress environments. Background technique
  • Salt stress is one of the major abiotic stresses that limit plant growth and development and can cause significant losses in agriculture. Many efforts have been made to study the response mechanism of plants to salt stress, and it is expected that strains resistant to salt stress can be developed accordingly.
  • the Brassicaceae family (also known as the Brassicaceae) is a semi-salt of the halophyte (73 ⁇ 4e// continued g e//a ⁇ / ⁇ ), which is genetically a valuable model plant.
  • Small salt mustard and the model plant Arabidopsis thaliana ( ⁇ rab ic, w thaliana ⁇ in the same family. But unlike Arabidopsis, small salt mustard can tolerate extreme cold, drought and high salt conditions.
  • the inventors of the present invention constructed a cDNA library for the aerial parts and roots of salt-treated small salt mustard using a pGAD-GH shuttle vector capable of direct expression in S. cerevisiae cerev ⁇ ae. (Zhang YY et al., 2008, Comparison Analysis of Transcripts from the Halophyte Thellungiella halophila. Journal of Integr Plant Biol, 50 (10): 1327-1335).
  • the inventors of the present invention further found a clone showing significant salt tolerance, on the basis of which the ThLEAl group was isolated.
  • the inventors of the present invention surprisingly found that this gene can significantly improve yeast viability when ectopically expressed in salt-sensitive yeast cells, although there is no protein showing sequence homology to plant LEA in yeast.
  • ThLEAl can exhibit conservative salt tolerance in both non-halophytes and yeast, and can protect yeast and plant cells under salt stress. This not only greatly contributes to the study of plant salt stress response mechanism, but also enables people to impart salt tolerance to salt-sensitive species through the transformation and ectopic expression of ThLEAl gene, thereby producing conditions capable of salt stress.
  • the line that grows underneath. The inventors have thus completed the present invention. Summary of the invention
  • the present invention relates to an isolated polynucleotide comprising or consisting of a nucleotide sequence selected from the group consisting of:
  • SEQ ID NO: 1 or the nucleotide sequence shown in SEQ ID NO: 3;
  • nucleotide sequence which hybridizes to the complement of the nucleotide sequence of (1) under conditions of low stringency, moderate stringency, and preferably high stringency hybridization;
  • nucleotide sequence of (1) a nucleotide sequence of % or 98% or 99% identity;
  • nucleotide sequence encoding the same amino acid sequence as the nucleotide sequence of (1), but having a nucleotide sequence different in sequence;
  • nucleotide sequence encoding one of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 2, or, due to one or more (for example, 1-25, 1-20, 1-15, 1-10, 1-5,
  • the invention also provides an isolated polynucleotide comprising a nucleotide sequence substantially identical to the nucleotide sequence set forth in SEQ ID NO: 1.
  • the invention also relates to a construct comprising a polynucleotide of the invention.
  • the invention also relates to an expression cassette comprising a polynucleotide of the invention, and a regulatory element that modulates expression of said polynucleotide.
  • the regulatory element can, for example, be a promoter operably linked to the polynucleotide, including but not limited to a constitutive promoter, a tissue-specific promoter or an inducible promoter.
  • the regulatory element may also be, for example, an enhancer that enhances expression of the polynucleotide, a silencer that attenuates expression of the polynucleotide, a terminator that terminates transcription of the polynucleotide, and the like.
  • the invention also relates to vectors comprising the polynucleotides of the invention.
  • the vector may be a cloning vector or an expression vector for expressing the polynucleotide.
  • the invention also relates to a cell comprising a polynucleotide of the invention or a construct of the invention or an expression cassette of the invention or a vector of the invention.
  • the cell may be an animal cell, a plant cell, such as a plant cell, or a biological cell, such as a bacterial or fungal cell, such as a yeast cell.
  • the cells may be isolated, ex vivo, cultured, or part of a plant.
  • the invention further relates to a plant or plant part, a plant material, a plant seed comprising the cells of the invention.
  • the plant may be a plant of the family Polygonaceae, such as cabbage, rapeseed, cabbage, Arabidopsis or small salt mustard, or other plants, such as monocots such as rice, wheat, barley, corn, sorghum, sugar cane, oats. , or rye, etc., or other dicots such as tobacco, soybeans, hollyhocks, beets, peppers, potatoes, tomatoes, etc. It also relates to transgenic seeds from the plants.
  • the invention also relates to an isolated polypeptide (also referred to as a protein) comprising or consisting of an amino acid sequence selected from the group consisting of:
  • the invention also provides an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: Substantially identical amino acid sequences.
  • polynucleotides and polypeptides (also referred to as proteins) of the invention are capable of conferring tolerance to transgenic biological salts comprising the same.
  • the polynucleotides and polypeptides of the invention are capable of increasing the survival rate of their transgenic host cells in a salt-containing environment, or improving the growth of their transgenic organisms in a salt-containing environment.
  • the invention also relates to a method of producing a plant, the method comprising: regenerating a transgenic plant from a plant cell of the invention, or crossing a plant of the invention with another plant.
  • the other plant is preferably a salt tolerant plant.
  • the invention also relates to plants produced by the method of the invention.
  • the invention further relates to the use of a polynucleotide of the invention or a construct of the invention or an expression cassette of the invention or a vector of the invention for improving the salt tolerance of a plant.
  • the invention further relates to a method for improving the salt tolerance of a plant, the method comprising preparing a plant comprising a polynucleotide of the invention or a construct of the invention or an expression cassette of the invention or a vector of the invention, for example, the method may comprise The transgenic plant is regenerated from the plant cell of the invention or the plant of the invention is crossed with another plant.
  • the invention also relates to a method for increasing the salt tolerance of a plant, the method comprising preparing a plant comprising a polynucleotide of the invention or a construct of the invention or an expression cassette of the invention or a vector of the invention, for example, the method may comprise The transgenic plant is regenerated from the plant cell of the invention or the plant of the invention is crossed with another plant.
  • the invention further relates to the use of a polynucleotide of the invention or a construct of the invention or an expression cassette of the invention or a vector of the invention for increasing the salt tolerance of a plant.
  • Figure 1 shows the sequence analysis of ThLEAl.
  • the grey box indicates the conserved amino acid sequence present in the LEA1 superfamily. The start and stop codons are shown in bold.
  • Figure 2 shows the expression of ThLEAl in salt-treated small salt mustard seedlings.
  • Total RNA extracted from rosette leaves and roots of plants treated at different treatment times is shown. The number at the top indicates the processing time (hours).
  • the upper panel shows hybridization to a TTLL gene-specific probe.
  • the bottom panel shows the rRNA stained with indigo as a loading control.
  • Figure 3 shows the tolerance of yeast transformants to NaCl.
  • the growth of cultures of yeast transformed with empty vector (CK) and selected ThLEAl after serial dilution on SD plates containing different concentrations of NaCl or without NaCl is shown, the picture is after 8 days of growth.
  • Figure 4 shows the salt treated 35S-ThLEAl transgenic Arabidopsis thaliana.
  • A Schematic representation of a 35S nuller construct for overexpression of ThLEAl in plants.
  • B Quantitative data for germination on MS medium or MS medium containing 100 mM NaCl (right panel). The germination of the second sky vector control (grey bars) and 35S-ThLEAl plants (white bars) was calculated. The results were normalized to reference untreated controls.
  • SEQ ID NO:2 ThLEAl protein sequence
  • SEQ ID NO:3 ThLEAl cDNA sequence
  • LEA protein The LEA protein is the late embryogenesis abundant protein, which is a group of proteins present in different tissues and cell types of plants. This protein was first discovered because of their high level of accumulation in late embryonic development.
  • ThLEAl gene The ThLEAl gene is a gene encoding LEA-like protein found in the Arabidopsis thaliana, and is therefore named ThLEAl gene.
  • the longest cDNA of the gene, ORF (483 bp) encodes a 160 amino acid protein, the ThLEA protein. Its CDS is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2. The method of obtaining the ThLEAl gene will be described in detail in the Examples section below.
  • “Related” / "operably linked” refers to two physically or functionally related nucleic acid sequences. For example, if a promoter or regulatory DNA sequence and a DNA sequence encoding an RNA or protein are operably linked or positioned such that regulation of the DNA sequence will affect the expression level of the coding or structural DNA sequence, then the promoter or regulatory DNA sequence is encoded with the coding RNA. Or the DNA sequence of a protein is "related".
  • a “chimeric gene” is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operably linked to a nucleic acid sequence encoding or expressed as a protein, or to a nucleic acid sequence encoding or expressed as a protein, such that the regulatory nucleic acid sequence is regulated Transcription or expression of a related nucleic acid sequence.
  • the regulatory nucleic acid sequence of the chimeric gene is not normally operable as found in nature Link the relevant nucleic acid sequence.
  • Codon A nucleotide sequence that directly specifies the amino acid sequence of its protein product, which may be a DNA, cDNA, synthetic or recombinant nucleotide sequence.
  • the boundaries of the coding sequence are usually determined by an open reading frame. The 5th end of the open reading frame is defined by the start codon, and the 3rd end is defined by the stop codon.
  • the start codon is usually ATG, or it can be other start codons such as GTG or TTG. Stop codons include, for example, TAA, TAG, TGA.
  • cDNA refers to DNA complementary to an RNA molecule which is a single- or double-stranded DNA molecule reverse transcribed from a mature, spliced mRNA molecule derived from a eukaryotic cell by a reverse transcriptase. Thus, the cDNA does not contain any introns that may otherwise be present in the corresponding genomic DNA sequence.
  • nucleic acid coding sequences or amino acid sequences of different genes or proteins are aligned with each other, nucleic acids or amino acids "corresponding to" some counting positions are aligned with these positions, However, it is not necessary to be a nucleic acid or an amino acid in these precise numerical positions relative to the respective nucleic acid coding sequence or amino acid sequence of a particular LEA.
  • nucleic acid or amino acid in the particular LEA sequence corresponding to some of the counting positions of the reference LEA sequence is aligned with these positions of the reference LEA.
  • expression cassette means a nucleic acid sequence that directs expression of a particular nucleotide sequence suitable for expression in a host cell, comprising a regulatory element operably linked to a nucleotide sequence of interest.
  • the regulatory element may be a promoter, enhancer, silencer, terminator and/or other element that controls expression of the nucleotide sequence, such as a polyadenylation sequence or the like.
  • the expression cassette also contains the sequences required for proper translation of the nucleotide sequence.
  • An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its constituents is heterologous to at least one of its other components.
  • the expression cassette may also be naturally occurring, but an expression cassette for heterologous expression is obtained in recombinant form.
  • the expression cassette is heterologous to the host, i.e., the particular nucleic acid sequence of the expression cassette does not naturally occur in the host cell and must be introduced into the host cell or precursor of the host cell by a transformation event.
  • Expression of the nucleotide sequence in the expression cassette can be controlled by a constitutive promoter or an inducible promoter, wherein the inducible promoter initiates transcription only when the host cell is exposed to some specific external stimulus.
  • Expression of the nucleotide sequence in the expression cassette can also be controlled by a tissue-specific promoter. In the case of multicellular organisms, such as plants, the promoter may also be specific to the tissue, or organ or developmental stage. Paragraph specific.
  • a “gene” is a defined region within the genome that, in addition to the aforementioned encoding nucleic acid sequences, comprises other predominantly regulatory nucleic acid sequences which are responsible for the expression of the coding portion, i.e., transcriptional and translational control.
  • the gene may also contain other 5, and 3, untranslated sequences and terminating sequences. Further elements that may be present are, for example, introns.
  • a "heterologous" nucleic acid sequence is a nucleic acid sequence that is not naturally associated with the host cell into which it is introduced, and comprises multiple copies of a non-naturally occurring naturally occurring nucleic acid sequence.
  • Homologous recombination is the mutual exchange of nucleic acid fragments between homologous nucleic acid molecules.
  • nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence
  • nucleic acid sequence is "likely encoded" with the reference nucleic acid sequence.
  • isolated nucleic acid molecule or isolated protein is a nucleic acid molecule or protein that is artificially isolated from its natural environment and therefore is not a natural product.
  • the isolated nucleic acid molecule or protein may be present in purified form or may be present in a non-native environment, such as a recombinant host cell or a transgenic plant.
  • Natural refers to a gene that is present in the genome of an untransformed cell.
  • Naturally occurring is used to describe an object that can be found in nature, unlike an artificially produced object.
  • proteins or nucleotide sequences that are isolated from natural sources and that are not intentionally artificially modified in the laboratory, in organisms (including viruses), are “naturally occurring.”
  • Nucleic acid molecule or “nucleic acid sequence” is a single or double stranded DNA that can be isolated from any source or
  • nucleic acid molecule is a DNA fragment.
  • a "nucleic acid molecule” is also referred to as a polynucleotide molecule.
  • Plant is any plant, especially a seed plant, at any stage of development.
  • Plant cells are the structural and physiological units of plants, including protoplasts and cell walls.
  • the plant cells may be in the form of isolated single cells or cultured cells, or as a higher organized unit such as, for example, a plant tissue, a plant organ or a part of an entire plant.
  • Plant cell culture means a plant unit of various developmental stages such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryos, zygotes and embryo cultures.
  • Plant material means a leaf, stem, root, part of a flower or flower, fruit, pollen, egg cell, zygote, seed, cuttings, cell or tissue culture, or any other part or product of a plant.
  • Plant organs are parts of plants that are clearly and clearly structured and differentiated, such as roots, stems, leaves, flower buds or embryos.
  • plant tissue means a group of plant cells organized into structural and functional units. Any tissue including plants in plants or cultures. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any plant cell group that is organized into structural and/or functional units. The combined use or separate application of the term with any of the specific types of plant tissues listed above or included in the definition is not intended to exclude any other type of plant tissue.
  • a “promoter” is a non-translated DNA sequence upstream of the coding region that contains the binding site of RNA polymerase and initiates transcription of the DNA.
  • the promoter region may also contain other elements that are modulators of gene expression.
  • Protoplasts are isolated plant cells that have no cell walls or only a portion of the cell wall.
  • regulatory element refers to a sequence involved in the control of expression of a nucleotide sequence.
  • the regulatory element comprises a promoter and a termination signal operably linked to the nucleotide sequence of interest. Usually they also contain the sequences required for proper translation of the nucleotide sequence.
  • a "shuffled" nucleic acid is a nucleic acid produced by a shuffling method, such as any of the shuffling methods described herein.
  • the shuffling nucleic acid is produced by recombining two or more nucleic acids (or strings) manually and optionally cyclically (physically or physically).
  • one or more screening steps are utilized in the shuffling method to identify the nucleic acid of interest; the screening step can be performed before or after any recombination step.
  • the entire process of recombination and array selection can be iteratively repeated.
  • a reorganization may refer to the entire process of reorganization and array selection, or alternatively, may refer only to the recombination portion of the entire process.
  • substantially identical in the context of two nucleic acid or protein sequences means that when comparing and aligning to obtain maximum correspondence, as determined by one of the following sequence comparison algorithms or by visual inspection, having at least 60%, preferably Two or more sequences or subsequences of 80%, more preferably 85%, more preferably 90%, even more preferably 95% and most preferably at least 99% nucleotide or amino acid residue identity.
  • the substantial identity is present in a region of at least about 50 residues in length, more preferably in a region of at least about 100 residues, and most preferably, the sequences in at least about 150 residues are substantially identical.
  • the sequences are substantially identical throughout the length of the coding region.
  • substantially identical nucleic acid or protein sequences have substantially the same function.
  • sequence comparison typically, one sequence is compared to the detection sequence as a reference sequence.
  • the detection and reference sequences are entered into a computer, if necessary, the coordinates of the subsequence are specified, and the parameters of the sequence algorithm program are specified. Then, based on the selected program parameters, the sequence comparison algorithm will calculate the percent sequence identity of the test sequence relative to the reference sequence.
  • HSPs high score sequence pairs
  • the cumulative score is calculated using the parameters M (reward score for paired matching residues; always greater than zero) and N (penalty value for mismatching residues; always less than zero).
  • M forward score for paired matching residues; always greater than zero
  • N penalty value for mismatching residues; always less than zero.
  • the cumulative score is calculated using a score matrix.
  • the BLASTP program uses the word length value (W) 3 , the expected value (E) 10 and the BLOSUM62 score matrix (see, Henikoff & Henikoff, Proc. Nail. Acad. Sci. USA 89: 10915 (1989)) as the default. value.
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)).
  • One similarity measure provided by the BLAST algorithm is the smallest sum probability ( ⁇ ( ⁇ )), which provides an indication of the probability of a chance match between two nucleotide or amino acid sequences.
  • ⁇ ( ⁇ ) the smallest sum probability
  • a detection nucleic acid sequence is considered to be similar to a reference sequence if the minimum sum probability of the detection nucleic acid sequence compared to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences are substantially identical.
  • specific hybridization means that when the sequence is present in a complex mixture (e.g., total cellular) of DNA or RNA, under stringent conditions, the molecule binds only to a particular nucleotide sequence, forming a double helix or hybridization.
  • Base binding refers to complementary hybridization between a probe nucleic acid and a target nucleic acid, and contains fewer mismatches that can be tolerated by reducing the stringency of the hybridizing medium to achieve the desired detection of the target nucleic acid sequence.
  • Stringent hybridization conditions and “stringent hybrid rinsing conditions” in the context of nucleic acid hybridization assays such as Southern and Northern hybridization are sequence dependent and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. Nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 'Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York A large number of guidelines. In general, for specific sequences at defined ionic strength and pH, high stringency hybridization and rinsing conditions are selected to be lower than the thermal melting point (about 5 ° C. Typically, under “stringent conditions", The needle will hybridize to its target subsequence without hybridizing to other sequences.
  • D ' is (under defined ionic strength and pH conditions) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • very stringent conditions are selected to be equal to T m.
  • T m In a Southern or Northern blot An example of a stringent hybridization condition in which a complementary nucleic acid having more than 100 complementary residues is hybridized on the filter is 50% guanidinamide with 1 mg of heparin at 42 ° C, and the hybridization is carried out overnight.
  • High stringency rinsing conditions An example is 72 ° C, 0.15 M NaCl for about 15 minutes.
  • An example of a strict rinsing condition is rinsing at 65 ° C, 0.2 X SSC for 15 minutes (see, Sambrook, below, description of SSC buffer).
  • a low stringency rinse is performed prior to the sexual rinse to remove the background probe signal.
  • an example of a medium stringency rinse is 45 ° C, lx SSC rinse for 15 minutes.
  • an example of a low stringency rinse is 40 ° C, 4-6 X SSC rinse for 15 minutes.
  • strict Conditions are usually included at pH 7.0 to 8.3
  • the salt concentration of less than about 1.0 M Na ion usually from about 0.01 to 1.0 aa ion concentration (or other salt), usually at a temperature of at least about 30 °C.
  • Stringent conditions can also be obtained by the addition of destabilizing agents such as guanamide.
  • the signal to noise ratio is 2 ⁇ (or higher) independent of the value observed by the probe indicating detection of specific hybridization.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created with the maximum codon degeneracy allowed by the genetic code.
  • the following is an example of a hybridization/rinsing condition setting which can be used to clone a homologous nucleotide sequence substantially identical to the reference nucleotide sequence of the present invention:
  • the reference nucleotide sequence and the reference nucleotide sequence are preferably at 50°.
  • nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross-reactive or specifically associated with the protein encoded by the second nucleic acid. Therefore, the protein is usually substantially identical to the second protein, for example, two of which differ only by conservative substitutions.
  • “Synthesized” refers to a nucleotide sequence comprising structural features that are not found in the native sequence. For example, artificial sequences that are more closely related to the G+C content of the dicotyledonous and/or monocotyledonous genes and the normal codon distribution are synthesized.
  • Transformation is the process of introducing a heterologous nucleic acid into a host cell or organism.
  • transformation means the stable integration of a DNA molecule into the genome of a target organism.
  • Transformed/transgenic/recombinant refers to a host organism, such as a bacterium or a plant, into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the host genome or the nucleic acid molecule can also exist as an extrachromosomal molecule. Such extrachromosomal molecules can be autonomously replicated.
  • a transformed cell, tissue, or plant is understood to contain not only the final product of the transformation process, but also its transformation. Gene progeny.
  • non-transformed refers to a wild-type organism, such as a bacterium or a plant, that does not contain a heterologous nucleic acid molecule.
  • polynucleotide include single or double stranded DNA and RNA molecules, It may comprise one or more prokaryotic sequences, a cDNA sequence, a genomic DNA sequence comprising exons and introns, a chemically synthesized DNA and RNA sequence, and a sense and corresponding antisense strand.
  • At least one salt-tolerant protein of the invention is expressed in a higher organism, such as a plant.
  • the nucleotide sequence of the present invention can be inserted into an expression cassette, and then preferably, the expression cassette is stably integrated into the plant genome.
  • the nucleotide sequence is contained in a non-pathogenic self-replicating virus.
  • the nucleotide sequence is comprised in Agrobacterium.
  • Plants transformed according to the invention may be monocotyledonous or dicotyledonous, including but not limited to corn, wheat, barley, rye, sweet potato, beans, peas, chicory, lettuce, kale, broccoli, broccoli, turnip, radish , spinach, asparagus, onion, garlic, pepper, celery, winter squash, pumpkin, marijuana, zucchini, apple, pear, warm, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, Blackberry, Pineapple, avocado, Papaya, Mango, Banana, Soy, Tomato, Sorghum, Sugarcane, Beet, Sunflower, Rapeseed, Clover, Tobacco, Carrot, Cotton, Rice, Rice, Potato, Eggplant, Cucumber Mustard and woody plants such as conifers and deciduous trees.
  • the plant used in the present invention is a Polygonaceae plant.
  • the plant is a genus of Brassica, including black mustard CSra c « « gra ), European rape (Sraw ca ""jms"), ⁇ . Brassica oleraceae) . (Brassica rapa), Ethiopian Qi ( ⁇ ) Car carinata), ⁇ - ⁇ (Brassica juncea). The plant can also be ⁇ Other plants of the family.
  • nucleotide sequence Once the desired nucleotide sequence has been transformed into a particular plant species, it can be propagated in that species or transferred to other varieties of the same species, including commercial varieties, using conventional breeding techniques.
  • the nucleotide sequence of the invention is expressed in a transgenic plant, thereby causing a corresponding biosynthesis of a protein that confers salt tolerance to the plant in the transgenic plant.
  • transgenic plants with improved traits can be produced.
  • the nucleotide sequences of the invention may require modification and optimization. All organisms have specific codon usage preferences, as is known in the art, and their codons can be altered to maintain plant preference while maintaining the amino acids encoded by the nucleotide sequences of the present invention.
  • high levels of expression in plants can be best achieved from coding sequences having at least about 35%, preferably more than about 45%, more preferably more than 50%, and most preferably more than about 60% GC content.
  • the preferred gene sequences can be adequately expressed in monocot and dicot species, the sequences can be modified to accommodate the specific codon preferences and GC content preferences of monocots or dicots, as these preferences have proven to be Different (Murray et al, Nucl. Acids Res. 17: 477-498 (1989)).
  • the nucleotide sequence can be screened for the presence of an unconventional splice site that causes information truncation.
  • a synthetic gene can be prepared according to the method disclosed in U.S. Patent No. 5,625,136, which is incorporated herein by reference.
  • corn preferred codons i.e., single codons that most commonly encode that amino acid in corn
  • the maize preferred codon for a particular amino acid can be derived, for example, from a known genetic sequence of maize.
  • the corn codon usage of 28 genes of maize plants is taught in Murray et al, Nucleic Acids Research 17: 477-498 (1989), the disclosure of which is incorporated herein by reference.
  • nucleotide sequence can be optimized for expression in any plant. All or any part of a recognized gene sequence can be optimized or synthesized. That is, synthetic or partially optimized sequences can also be utilized.
  • nucleotide sequences of the invention For efficient initiation of translation, it may be desirable to modify the sequence adjacent to the starting valine. For example, they can be modified by including sequences that are known to be effective in plants. Joshi proposed a plant suitable Consensus sequence (NAR 15: 6643-6653 (1987)), Clonetech proposed a further translation initiation consensus sequence (1993/1994 catalog, page 210). These consensus sequences are suitable for use with the nucleotide sequences of the invention. A construct comprising the nucleotide sequence up to and including ATG (while keeping the second amino acid unmodified) or alternatively up to and including ATG (with the possibility of modifying the second amino acid of the transgene) This sequence was introduced.
  • the novel ThLEAl gene of the present invention may be operably associated with various promoters expressed in plants as its native sequence or as a synthetic sequence as described above, including constitutive, inducible, temporal regulation, developmental regulation, chemical regulation
  • the tissue is fused to a tissue-specific promoter to produce a recombinant DNA molecule, i.e., a chimeric gene.
  • the choice of promoter will vary with expression time and space requirements, and also depends on the target species.
  • the nucleotide sequence of the present invention can be expressed in leaves, main stems or stems, ears, inflorescences (e.g., spikes, panicles, cobs, etc.), roots, and/or seedlings.
  • dicotyledon promoters are selected for expression in dicots, monocots
  • the promoter is used for expression in monocots.
  • the origin of the selected promoter is not limited as long as the promoter functions to drive expression of the nucleotide sequence in the desired cell.
  • Preferred constitutive promoters include the CaMV 35S and 19S promoters (Fraley et al., U.S. Patent No. 5,352,605, issued Oct. 4, 1994). Further preferred promoters are derived from any of several actin genes expressed in most cell types.
  • the promoter expression cassette described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for expression of a ThLEAl group, which is particularly suitable for use in a monocot host. .
  • Another preferred constitutive promoter is derived from ubiquitin, another gene product known to accumulate in many cell types. From several species, such as sunflower (Binet et al, 1991. Plant Science 79: 87-94), maize (Christensen et al., 1989. Plant Molec. Biol. 12: 619-632) and Arabidopsis (Norris et al 1993. Plant) Molec. Biol. 21: 895-906) cloned the ubiquitin promoter, which can be used in transgenic plants.
  • the maize ubiquitin promoter in the transgenic monocot system has been developed, and its sequence and vector for the transformation of monocots are disclosed in the patent publication EP 0 342 926.
  • the ubiquitin promoter is suitable for the expression of new erect panicle genes in transgenic plants, particularly monocots.
  • ThLEAl protein expression in plants can be linked A suitable transcription terminator downstream of the heterologous nucleotide sequence.
  • terminators are available, and they are known in the art (e.g., tml derived from CaMV, E9 derived from rbcS). Any available terminator known to function in plants can be used in the present invention.
  • Encs enhanced expression sequences such as intron sequences (eg, derived from Adhl and bronzel) and viral leader sequences (eg, from TMV, MCMV, and AMV).
  • nucleotide sequences of the invention it is possible to target the expression of the nucleotide sequences of the invention to different cellular localizations in plants. In some cases, it may be desirable to localize in the cytosol, while in other cases it may be preferred to localize in some subcellular organelles.
  • the subcellular localization of the enzyme encoded by the transgene employs techniques well known in the art. Typically, DNA encoding a target peptide derived from a gene product known to target organelles is fused to the upstream of the nucleotide sequence. Many such target sequences for chloroplasts are known and are known to demonstrate their function in heterologous construction. Expression of a nucleotide sequence of the invention can also be targeted into the endoplasmic reticulum or vacuole of a host cell. Techniques for achieving these objects are well known in the art.
  • transformation vectors useful for plant transformation are known to those skilled in the art of plant transformation, and the nucleic acid molecules of the invention can be used in conjunction with any such vector.
  • the choice of vector will depend on the preferred transformation technique and target plant species used for transformation. For some target species, different antibiotic or herbicide selective markers may be preferred.
  • Selectable markers commonly used in transformation include genes that confer resistance to kanamycin and related antibiotics (Messing & Vierra., 1982. Gene 19: 259-268; and Bevan et al., 1983. Nature 304: 184-187 ), the bar gene conferring resistance to the herbicide phosphinothricin, (White et al, 1990. Nucl. Acids Res 18: 1062, and Spencer et al, 1990.
  • the choice of the selectable marker is not critical to the invention.
  • the nucleotide sequence of the invention is directly transformed into the plastid genome.
  • the main advantage of plastid transformation is that the plastid usually does not require substantial modification to express the bacterial gene, and the plastid expresses multiple open reading frames under the control of a single promoter.
  • U.S. patent The plastid transformation technique is described in detail in PCT Application No. WO 95/16783 and McBride et al., (1994) Proc. Nati. Acad. Sci. USA 91, 7301-730, 5,451,513, 5,545,817 and 5,545,818.
  • the basic techniques for chloroplast transformation include, for example, the use of bio-bombardment or protoplast transformation (eg, calcium chloride or PEG-mediated transformation) to ligate regions of the clonal plastid DNA flanking the gene of interest and the selectable marker into a suitable target tissue.
  • bio-bombardment or protoplast transformation eg, calcium chloride or PEG-mediated transformation
  • protoplast transformation eg, calcium chloride or PEG-mediated transformation
  • point mutations in the chloroplast 16S rRNA and rpsl2 genes that confer resistance to spectinomycin and/or streptomycin can be utilized as selection markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Nati. Acad. Sci. USA 87,8526-8530; Staub, J. M” and Maliga, P. (1992) Plant Cell 4, 39-45). This bombards about every 100 target blades. One-time frequency produces stable homologous transformants. The cloning sites present between these markers can be used to generate plastid targeting vectors for the introduction of foreign genes (Staub, JM, and Maliga, P.
  • nucleotide sequence of the invention is inserted into a plastid targeting vector and transformed into the desired plant host plastid genome.
  • a plant which is homogenous to the plastid genome containing the nucleotide sequence of the present invention is obtained, and preferably, the plant has the ability to express a nucleotide sequence at a high level.
  • Example Example 1 Large-scale identification and isolation of the 73 ⁇ 4LE ⁇ gene
  • the G19 strain is a salt-sensitive yeast strain in which the gene ENA1 encoding the plasma membrane Na + export pump is deleted, rendering the G19 yeast strain more sensitive to external Na + concentrations than wild-type yeast.
  • G19 yeast cells were transformed with the Gal4 promoter-derived AD fusion salt mustard cDNA and plated on selection medium containing 400 mM NaCl. Transformants capable of surviving on a salt-containing selection plate were selected. Of the 10 6 yeast transformants, more than 100 transformants were selected for subsequent salt tolerance confirmation, and the inserted cDNA was isolated for sequencing. Based on sequence analysis comparing homologs in other species, genes for putative salt tolerance candidate strains with intact or nearly intact ORFs were selected to confirm their ability to confer salt tolerance in plants.
  • ThLEAl A cDNA clone showing a considerable tolerance to high salt was selected. Sequence analysis revealed that the plant cDNA contained therein encoded an LEA-like protein. The gene was named ThLEAl.
  • the ThLEAl cDNA is 701 bp long (as shown in SEQ ID NO: 3) and has an open reading frame of up to 483 b (Fig. 1A) (as shown in SEQ ID ⁇ : 1).
  • the putative amino acid sequence consists of 160 amino acids, as shown in SEQ ID NO: 2, with a molecular weight of approximately 16.3 kDa and an isoelectric point of 9.19.
  • ThLEAl CDS Inserting full-length or substantially full-length ThLEAl CDS (SEQ ID NO: 1) into the S. cerevisiae/E. coli shuttle vector pGAD GH (Clontech) and inserting this under the control of the constitutive yeast promoter ADH1
  • the shuttle vector of SEQ ID NO: 1 was used to transform yeast G19 cells.
  • the empty vector was transferred to yeast cells as a control.
  • the vector can be transferred to yeast cells by a variety of methods well known in the art, for example, see the Methods In Yeast Genetics A Laboratory Course Manual (published in 1990, by Mark D. Rose et al.) 39- The method provided on page 52.
  • Select medium plate in SD (0.67% Bacto-yeast nitrogen base without amino acids, 2% glucose, 2% Bacto-agar, 0.062%) -Lea dropout) was selected and inoculated into liquid SD selection medium (0.67% amino acid-free bacteria - yeast with nitrogen-containing substrate, 2% glucose, 0.062%-Lea dropout), cultured at 30 ° C Until growth to the late log phase / stationary phase.
  • Saturated culture liquid SD The medium is diluted io, 100 or 1000 times. Two microliters of the diluted culture were spotted onto SD medium containing NaCl (300 mM, 350 mM, 400 mM) and without NaCl, cultured at 30 ° C for 8 days, and growth was recorded.
  • the transformed yeast cells have improved survival under salt stress conditions (see Figure 3). As can be seen from Fig. 3, when the salt content in the medium reached a toxic concentration to the yeast cells, the control yeast containing the empty vector grew poorly, and the yeast cells transformed into ThLEAl showed better salt tolerance and more. Good growth indicates that ThLEAl confers salt tolerance to yeast cells.
  • Example 3 Transcriptional response of ThLEAl to salt stress
  • This example uses the Shandong ecotype (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences).
  • the surface-sterilized seeds were germinated and grown at 22 ° C on MS medium (see table below) containing 2% sucrose and 0.8% agar. Seedlings in which 2-4 true leaves were grown were transferred to soil for cultivation and cultured at 24 ° C for 16 hours.
  • Five-week-old seedlings were subjected to stress treatment by irriting the seedlings with 500 mM NaCl (1 liter) and sampling at 0, 1, 4, 8, 12 and 24 hours (sampling from the time of saline).
  • RNA was isolated by lyophilizing and grinding the leaves and root samples of salt-treated small salt mustard in liquid nitrogen, essentially as Soni R and Murray A, 1994, Isolation of intact DNA and RNA from plant tissue.
  • the milled 1 OOrng plant powder was mixed with 600 ⁇ l of extraction buffer (50 mM Tris-HCl H 6.0, 10 mM EDTA, 2% SDS, 100 mM LiCl) and phenol/chloroform 1:1 heated at 65 ° C. Vortex and centrifuge at 12000 rpm for 15 minutes at 4 °C.
  • the supernatant was extracted twice with phenol and precipitated with an equal volume of 4 M LiCl, followed by centrifugation (12,000 rpm, 15 min). After centrifugation, the RNA pellet was resuspended in 50 ⁇ l buffer (10 mM Tris-Cl, pH 7.5. 1 mM EDTA.). The supernatant was again extracted with phenol, and precipitated by adding one tenth of a volume of 3 M NaAc and three volumes of absolute ethanol. The RNA was precipitated by centrifugation (12,000 rpm, 15 min), and the RNA pellet was washed with 75% ethanol and resuspended in 50 ⁇ l of TE buffer.
  • RNA was loaded into each lane. Labeling was performed using the Ready-primed standard "Amersham International" ⁇ ThLEAl full-length CDS with [a- 32 P] dCTP, using labeled fragments for hybridization and washing under high stringency conditions according to standard procedures. .
  • ThLEAl was strongly induced after 4 hours of treatment.
  • the accumulation of ThLEAl transcripts in the roots exceeds the aerial parts. This result indicates that ThLEAl is indeed a gene that can be induced by salt.
  • Example 4 Overexpression of ThLEAl in Arabidopsis and Improvement of Arabidopsis Salt Tolerance
  • ThLEAl was cloned into the modified binary vector pGreen (see Figure 4A). ), overexpressed in model plant Arabidopsis and tested for salt tolerance.
  • the cDNA fragment comprising SEQ ID NO: 1 was excised from the GAD GH vector and cloned into a modified pGreen binary vector (Hellens RP et al., 2000).
  • pGreen a versatile and flexible binary Ti vector for Agrobacterium-mQdiatQd plant transformation. Plant Mol Biol 42: 819-832) is placed under the control of the 35S double promoter (see Figure 4A), thereby performing in Arabidopsis Overexpression.
  • This construct was introduced into the EHA 105 strain of Agrobacterium tumefaciens (Agrobacterium t mefaciens) containing the Sou plasmid (Hellens RP et al., supra). The resulting bacteria are used for bacterial infiltration by plants (Bent AF and Clough SJ (1998) Agrocell germ-line transformation: transformation of Arabidopsis without tissue culture. In Plant Molecular Biology Manual, 2nd ed, SB Gelvin and RA Schilperoot, eds (Dordrecht, The Netherlands: Kluwer Academic Publishers): 1-14) Transformation of wild-type Arabidopsis thaliana (Colombia ecotype). Wild type Arabidopsis thaliana was transformed in the same manner using the empty vector pGreen as a control.
  • the obtained seeds were surface-sterilized with 10% bleach and washed 3 times with sterile water.
  • the sterile seeds were suspended in 0.15% agarose and plated onto MS medium containing 1.5% sugar.
  • 50 mg/mL kanamycin was added to the medium.
  • the plates were stacked in the dark, placed at 4 ° C for 2-4 days, then transferred to a tissue culture chamber at 24 ° C, and cultured in a 16 hour light period.
  • One week old plants were transferred to pots and transferred to a greenhouse of similar temperature and light conditions until seeds were formed.
  • To select homozygous lines the germination status of T2 seeds in the presence of kanamycin was analyzed. Only T3 plants with a separation rate of approximately 3:1 were used.
  • Salt stress treatment was carried out in pots, and 4 weeks old Arabidopsis plants were watered with NaCl solution once every 4 days, and the concentration of NaCl solution gradually increased, specifically 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 250 mM, a total of six Times. Then take pictures of the plants.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Description

耐盐基因及其应用 技术领域
本发明涉及耐盐基因及其应用。 本发明还涉及包含该基因的载体、 宿主 细胞和植物及生产植物的方法。 该基因可用于改善植物对盐胁迫环境的耐受 性。 背景技术
盐胁迫是限制植物生长和发育的主要非生物性压力之一， 在农业上可能 引起重大损失。 人们付出了许多努力来研究植物对盐胁迫的响应机制， 期望 能够据此开发出对盐胁迫耐受的品系。
一些生长在高盐环境中的植物种类进化出了复杂的方式来适应所处的环 境， 这些植物可以作为盐耐受机制研究的基础。 芸薹科 (Brassicaceae family) (又名十字花科)盐芥属的盐生植物小盐芥 (7¾e//續 g e//a Ζζα/ορΜα)在遗传上是 一种很有价值的模式植物。 小盐芥与模式植物拟南芥 (^rab i c， w thaliana^ 于同一个科。 但与拟南芥不同的是， 小盐芥能够耐受极端的寒冷、 干旱和高 盐条件。 特别是当处于高达 500 mM NaCl的高盐条件时， 小盐芥仍然能够耐 受并且完成生命周期 (Inan G等， 2004, Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles. Plant Physiol 135: 1718-1737)。这些属性使得小盐芥成为研究植物对盐胁迫的响应和耐受机制的 优良对象。 但是， 目前尚不清楚小盐芥的具体耐盐机制。
在先前的研究中， 本发明的发明人使用能够在酿酒酵母 (H aromyces cerev^ae)中直接进行表达的 pGAD-GH穿梭载体为经过盐处理的小盐芥的地 上部分和根部构建了 cDNA文库 (Zhang YY等， 2008, Comparison Analysis of Transcripts from the Halophyte Thellungiella halophila. Journal of Integr Plant Biol, 50 (10): 1327-1335)。 其后， 利用这种高效的酵母体系， 通过大规模的胁 迫 -耐受筛选从小盐芥中分离了能够提高盐耐受性的多个 cDNA克隆 (Chen AP 等， 2007 , Ectopic expression of ThCYP 1 , a stress-responsive cyclophilin gene from Thellungiella halophila, confers salt tolerance in fission yeast and tobacco cells. Plant Cell Rep, 26:237-245和 Liu N等, 2007 , Functional screening of salt stress-related genes from Thellungiella halophila using fission yeast system Physiol Plantarum 129: 671—678)。
本发明的发明人进一步发现了一个显示出显著盐耐受性的克隆， 以此为 基础分离了 ThLEAl 基^。 本发明的发明人惊讶地发现这种基因在盐敏感性 的酵母细胞中异位表达时能够显著改善酵母的存活率， 尽管在酵母中不存在 与植物 LEA显示出序列同源性的蛋白质。经过分析和实验还发现 ThLEAl无 论在非盐生植物中还是在酵母中都能够展现出保守的盐耐受性， 能够保护处 于盐胁迫条件下酵母和植物细胞。 这不仅对植物的盐胁迫响应机制研究有很 大帮助， 还让人们能够通过 ThLEAl基因的转化和异位表达而将盐耐受性赋 予本来对盐份敏感的物种， 从而产生能够在盐胁迫条件下生长的品系。 发明 人由此完成了本发明。 发明内容
本发明涉及分离的多核苷酸， 其包含选自下组的核苷酸序列或由该序列 组成：
(1) SEQ ID ΝΟ: 1或 SEQ ID NO: 3所示的核苷酸序列；
(2) 与（1)的核苷酸序列的互补序列在低等严格条件、 中等严格条件、 优 选高严格杂交条件下杂交的核苷酸序列；
(3) 与（1)的核苷酸序列具有至少 50%、 至少 60%、 至少 70%、 至少 75%、 优选至少 80%、更优选至少 85%、特别优选至少 90%、尤其是至少 95%或 98% 或 99%同一性的核苷酸序列；
(4) 与（1)的核苷酸序列编码相同氨基酸序列的蛋白质、 但在序列上不同 的核苷酸序列；
(5) 编码如下氨基酸序列之一的核苷酸序列： SEQ ID NO:2所示的氨基酸 序列， 或者， 由于一或多个 (例如 1-25个、 1-20个， 1-15个， 1-10个， 1-5个，
1-3个)氨基酸残基的替代、缺失和 /或插入而与 SEQ ID NO:2所示的氨基酸序 列不同的氨基酸序列， 或者， 与 SEQ ID NO:2 所示的氨基酸序列具有至少 50%、 至少 60%、 至少 70%、 至少 75%、 优选至少 80%、 更优选至少 85%、 更优选至少 90%、 尤其是至少 95%或 98%或 99%同一性的氨基酸序列；
(6) (1)-(5)任何一个的核苷酸序列的活性片段； 或
(7) 与（1)-(5)任何一个的核苷酸序列互补的核苷酸序列。 本发明还提供分离的多核苷酸， 其包含与 SEQ ID NO: l中所示的核苷酸 序列基本相同的核苷酸序列。
本发明还涉及构建体， 其包含本发明的多核苷酸。
本发明还涉及表达盒， 其包含本发明的多核苷酸， 以及对所述多核苷酸 表达起调控作用的调控元件。 所述调控元件例如可以为与所述多核苷酸可操 作地连接的启动子， 包括但不限于组成型启动子、 组织特异性启动子或诱导 型启动子。 所述调控元件还可以例如为增强所述多核苷酸表达的增强子、 减 弱所述多核苷酸表达的静默子、 终止所述多核苷酸转录的终止子等等。
本发明还涉及载体， 其包含本发明的多核苷酸。 所述载体可以是克隆载 体或者用于表达所述多核苷酸的表达载体。
本发明还涉及细胞， 其包含本发明的多核苷酸或本发明的构建体或本发 明的表达盒或本发明的载体。 所述细胞可以是动物细胞， 植物细胞， 例如芸 薹科植物细胞， 或者 生物细胞， 例如细菌或真菌细胞， 例如酵母细胞。 所 述细胞可以是分离的、 离体的、 培养的、 或者是植物的一部分 。
本发明还涉及植物或者植物部分， 植物材料， 植物种子， 其包含本发明 的细胞。 所述植物可以是芸薹科植物， 例如白菜、 油菜、 甘蓝、 拟南芥或小 盐芥等， 也可以是其它植物， 例如单子叶植物如水稻、 小麦、 大麦、 玉米、 高粱、 甘蔗、 燕麦、 或黑麦等， 或者其他双子叶植物如烟草、 大豆、 向曰葵、 甜菜、 辣椒、 马铃薯、 番茄等。 还涉及来自所述植物的转基因种子。
本发明还涉及分离的多肽 (也称蛋白质），其包含从如下组氨基酸序列中选 择的氨基酸序列或由该序列组成：
(1) SEQ ID NO:2中所示的氨基酸序列，
(2)由于一或多个 (例如 1-25个、 1-20个， 1-15个， 1-10个， 1-5个， 1-3 个)氨基酸残基的替代、 缺失和 /或插入而与 SEQ ID NO:2中所示的氨基酸序 列不同的氛基酸序列，
(3)与 SEQ ID NO:2中所示的氨基酸序列具有至少 50%、 至少 60%、 至少 70%、 至少 75%、 优选至少 80%、 更优选至少 85%、 特别优选至少 90%、 尤 其是至少 95%或 98%或 99%同一性的氨基酸序列，
(4) (1)或 (2)或 (3)所述氨基酸序列的活性片段， 和
(5) 本发明的多核苷酸分子编码的氨基酸序列。
本发明还提供分离的多肽， 其包含与 SEQ ID NO:2中所示的氨基酸序列 基本相同的氨基酸序列。
本发明的多核苷酸和多肽 (也称蛋白质）能够赋予包含它的转基因生物盐 耐受性。 优选地， 本发明的多核苷酸和多肽能够提高其转基因宿主细胞在含 盐环境下的存活率， 或改善其转基因生物在含盐环境下的生长状况。
本发明还涉及生产植物的方法， 该方法包括： 从本发明的植物细胞再生 转基因植物， 或者将本发明的植物与另一植物杂交。 优选地， 所述另一植物 优选是耐盐植物。
本发明还涉及本发明的方法生产的植物。
本发明还涉及本发明的多核苷酸或本发明的构建体或本发明的表达盒或 本发明的载体在改良植物耐盐性中的用途。 本发明还涉及改良植物耐盐性的 方法， 该方法包括制备含有本发明的多核苷酸或本发明的构建体或本发明的 表达盒或本发明的载体的植物， 例如， 所述方法可以包括从本发明的植物细 胞再生转基因植物或者将本发明的植物与另一植物杂交。
本发明还涉及提高植物耐盐性的方法， 该方法包括制备含有本发明的多 核苷酸或本发明的构建体或本发明的表达盒或本发明的载体的植物， 例如， 所述方法可以包括从本发明的植物细胞再生转基因植物或者将本发明的植物 与另一植物杂交。
本发明还涉及本发明的多核苷酸或本发明的构建体或本发明的表达盒或 本发明的载体在提高植物耐盐性中的用途。 附图简述
图 1为 ThLEAl的序列分析。来自小盐芥的 7¾L&^基因的核苷酸序列和 推定的氨基酸序列。 氨基酸残基以单字母编码表示。 灰色框指明了 LEA1超 家族中存在的保守氨基酸序列。 起始和终止密码子以粗体显示。
图 2为 ThLEAl在经盐处理的小盐芥幼苗中的表达。 显示了从经过不同 处理时间的植物的莲座叶和根部提取的总 RNA。 顶部的数字指明了处理时间 (小时)。 上方的小图显示与 TTLL 基因特异性探针的杂交。 底部小图显示了 作为上样对照的用亚曱蓝染色的 rRNA。
图 3显示了酵母转化体对 NaCl的耐受性。显示了转化有空载体 (CK)和所 选 ThLEAl的酵母的培养物在系列稀释之后在含有不同浓度 NaCl或不含 NaCl 的 SD平板上的生长情况， 图为生长 8日之后的照片。 图 4显示了经盐处理的 35S-ThLEAl转基因拟南芥。 （A)用于在植物中过 表达 ThLEAl的 35S默 动子构建体的示意图。（B)在 MS培养基或含有 lOOmM NaCl的 MS培养基上的萌发定量数据 (右侧小图）。 计算了在第 2天空载体对 照 (灰色条形)和 35S-ThLEAl植物 (白色条形）的萌发情况。 将结果参考未经处 理的对照进行标准化。 所示百分比为三次重复 (每次 40-80)的平均值± 80。 * 表示在 PO.01时极为显著 (斯氏 t检验)。 （C)4周龄植株的盐耐受性测定。 显 示了盐处理开始之后 24天时代表性植株的照片。
SE ID NO: 1： ThLEAl编码
Figure imgf000006_0001
CACTGGATAAo
SEQ ID NO:2: ThLEAl蛋白质序列
MQSMKETASNIAASAKSGMDKTKATLEEKAEKMKTQDPVEKQMAT
Figure imgf000006_0002
VGGGGTTGYGTGGGYTG c
SEQ ID NO:3: ThLEAl cDNA序列
AGGACTTTCGCTTTCAAAAAAAAAAGAAAAAAA 具体实施方式
"LEA蛋白 "： LEA蛋白即胚胎发育晚期丰富蛋白（late embryogenesis abundant protein), 其是存在于植物的不同组织和细胞类型中的一组蛋白质。 这种蛋白质最早被发现是因为它们在胚发育晚期的高水平积累。
"ThLEAl基因"： ThLEAl基因是发明人在小盐芥中发现的编码 LEA样 蛋白的基因，故此命名为 ThLEAl基因。该基因最长的 cDNA的 ORF ( 483bp ) 编码一种 160个氨基酸的蛋白质， 即 ThLEA蛋白。 其 CDS如 SEQ ID NO: 1 所示， 编码的蛋白质的氨基酸序列示于 SEQ ID NO: 2。 在下文的实施例部分 将详细描述获得 ThLEAl基因的方法。
"相关 "/"可操作地连接 "指两个物理或功能相关的核酸序列。 例如， 如 果启动子或调节 DNA序列和编码 RNA或蛋白质的 DNA序列可操作地连 接或定位以至于调节 DNA序列将影响编码或结构 DNA序列的表达水平， 那么称启动子或调节 DNA序列与编码 RNA或蛋白质的 DNA序列 "相关"。
"嵌合基因 "是重组核酸序列， 其中启动子或调节核酸序列可操作地连 接编码 mRNA或作为蛋白质表达的核酸序列， 或与编码 mRNA或作为蛋 白质表达的核酸序列相关， 使得调节核酸序列能调节相关核酸序列的转录 或表达。 嵌合基因的调节核酸序列不是如自然界中所发现的正常可操作地 连接相关核酸序列。
"编码序列"： 指直接指定其蛋白质产物的氨基酸序列的核苷酸序列， 其可以为 DNA、 cDNA、 合成的或重组的核苷酸序列。 编码序列的边界通 常由开放阅读框决定。 开放阅读框的 5，端由起始密码子界定， 而 3，端由终 止密码子界定。 起始密码子通常为 ATG, 或者也可以是其它起始密码子如 GTG或 TTG。 终止密码子包括例如 TAA、 TAG, TGA。
"cDNA"是指与 RNA分子互补的 DNA, 其是通过反转录酶从得自真核 细胞的成熟的、 经过剪接的 mRNA分子反转录而来的单链或双链的 DNA分 子。 如此， cDNA中不含本来可能存在于与之对应的基因组 DNA序列中的任 何内含子。
对应于：在本发明上下文中"对应于 "意指当不同 ^基因或蛋白质的 核酸编码序列或氨基酸序列互相比对时， "对应于"一些计数位置的核酸或 氨基酸是与这些位置比对，但不必是在相对于特定 LEA各自核酸编码序列 或氨基酸序列的这些精确数字位置中的核酸或氨基酸。 同样， 当特定 ^ 的编码或氨基酸序列与参照 LEA 的编码或氨基酸序列比对时， "对应于" 参照 LEA序列一些计数位置的该特定 LEA序列中核酸或氨基酸是与参照 LEA 的这些位置比对，但不必是在该特定 LEA蛋白质各自核酸编码序 列或氨基酸序列的这些精确数字位置中的核酸或氨基酸。
这里所用的"表达盒 "意指能指导适合宿主细胞中特定核苷酸序列表 达的核酸序列， 包含与目的核苷酸序列可操作地连接的调控元件。 所述调 控元件可以是启动子、 增强子、 静默子、 终止子和 /或其它控制所述核苷酸 序列表达的元件， 如聚腺苷酸化序列等。 通常， 表达盒也包含核苷酸序列 正确翻译所需的序列。 包含目的核苷酸序列的表达盒可以是嵌合的， 意指 至少其成分之一相对于至少它的其它成分之一是异源的。 表达盒也可以是 天然存在的， 但以重组形式获得用于异源表达的表达盒。 然而， 通常， 表 达盒相对于宿主是异源的， 即， 表达盒的特定核酸序列不天然出现在宿主 细胞中， 必须通过转化事件将其引入宿主细胞或宿主细胞的前体。 表达盒 中核苷酸序列的表达可以受组成型启动子或诱导型启动子控制， 其中仅当 宿主细胞暴露于一些特定外部刺激时， 所述诱导型启动子才起始转录。 表 达盒中的核苷酸序列的表达也可以受组织特异性启动子控制。 如果是多细 胞生物体的情况， 如植物， 启动子也可以是对特定组织， 或器官或发育阶 段特异的。
"基因 "是位于基因组内的限定区域， 除了前述编码核酸序列外， 包含 其它主要是调节性的核酸序列， 所述调节性核酸序列负责编码部分的表 达， 即转录和翻译控制。 基因也可以包含其它 5，和 3，非翻译序列和终止序 列。 进一步可以存在的元件是， 例如内含子。
"异源 "核酸序列是与其被引入的宿主细胞不天然相关的核酸序列， 包 含非天然存在的天然存在核酸序列的多拷贝。
"同源重组"是同源核酸分子间核酸片段的相互交换。
当核酸序列编码与参照核酸序列编码的多肽有相同氨基酸序列的多 肽时， 该核酸序列与参照核酸序列是"同类编码"。
"分离的"核酸分子或"分离的 "蛋白质是人工地与其天然环境分离而存 在， 因此不是天然产物的核酸分子或蛋白质。 分离的核酸分子或蛋白质可 以以纯化形式存在， 或者可以存在于非天然环境， 例如重组宿主细胞或转 基因植物中。
天然： 指在未转化细胞的基因组中存在的基因。
天然存在： 术语"天然存在"用于描述可以在自然界中发现的客体， 其 与人工产生的客体不同。 例如， 可以从自然来源分离， 并没有在实验室有 意进行人工修饰的、生物体（包括病毒）中存在的蛋白质或核苷酸序列是"天 然存在 "的。
"核酸分子"或"核酸序列"是可以从任何来源分离的单或双链 DNA或
RNA的线性片段。 在本发明上下文中， 优选地， 核酸分子是 DNA片段。
"核酸分子"也称多核苷酸分子。
"植物"是在任何发育阶段的任何植物， 特别是种子植物。
"植物细胞"是植物的结构和生理学单位， 包含原生质体和细胞壁。 植 物细胞可以是分离的单个细胞或培养细胞形式， 或作为高等有组织的单位 如， 例如植物组织， 植物器官或整个植物的一部分。
"植物细胞培养物"意指各种发育阶段的植物单位如， 例如原生质体， 细胞培养细胞， 植物组织中的细胞， 花粉， 花粉管， 胚珠， 胚嚢， 合子和 胚的培养物。
"植物材料"指叶， 茎， 根， 花或花的部分， 果实， 花粉， 卵细胞， 合 子， 种子， 插条， 细胞或组织培养物， 或植物的任何其它部分或产物。 "植物器官"是植物清楚的和明显结构化和分化的部分， 如根， 茎， 叶， 花蕾或胚。
这里所用的"植物组织"意指组织成结构和功能单位的一组植物细胞。 包括植物中或培养物中植物的任何组织。 该术语包括但不限于整个植物， 植物器官，植物种子，组织培养物和组织成结构和 /或功能单位的任何植物 细胞组。 该术语与以上列举的或该定义包含的任何特定类型植物组织的联 合应用或单独应用不意味排除任何其它类型植物组织。
"启动子"是编码区域上游非翻译的 DNA序列，其包含 RNA聚合酶的 结合位点， 并起始 DNA的转录。 启动子区域也可以包含作为基因表达调 节物的其它元件。
"原生质体"是没有细胞壁或仅有部分细胞壁的分离的植物细胞。
"调节元件"指参与控制核苷酸序列表达的序列。 调节元件包含可操作 地连接目的核苷酸序列的启动子和终止信号。 通常它们也包含核苷酸序列 正确翻译所需的序列。
"改组的"核酸是通过改组方法， 如这里所述的任何改组方法产生的核 酸。 通过人工的和可选地循环的方式（物理地或实际上）重组两个或多个核 酸 (或字符串）产生改组核酸。 一般地， 在改组方法中利用一步或多步筛选 步骤以鉴定目的核酸； 可以在任何重组步骤前或后进行该筛选步骤。 在一 些 (但不是所有）改组实施方式中， 期望在筛选前进行多轮重组以增加待筛 选库的多样性。 可选地， 可以循环重复重组和陣选的全部过程。 根据上下 文， 改组可以指重组和陣选的全部过程， 或可替代地， 可以仅指全部过程 的重组部分。
基本相同： 在两个核酸或蛋白质序列上下文中的短语"基本相同 "指当 比较和比对以获得最大对应时， 如利用下面序列比较算法之一或目测所测 定的， 具有至少 60%, 优选 80%, 更优选 85%, 更优选 90%, 甚至更优选 95 %和最优选至少 99%核苷酸或氨基酸残基同一性的两个或多个序列或亚 序列。 优选地， 基本同一性存在于至少约 50个残基长度的序列区域， 更 优选至少约 100个残基的区域上， 最优选地， 在至少约 150残基中的序列 基本相同。在特别优选的实施方式中，在编码区整个长度中序列基本相同。 而且， 基本相同的核酸或蛋白质序列具有基本相同的功能。
为了进行序列比较，通常，一个序列作为参照序列而与检测序列比较。 当利用序列比较算法时， 将检测和参照序列输入到计算机中， 如果必要的 话指定亚序列的坐标， 并指定序列算法程序的参数。 然后， 根据选定的程 序参数， 序列比较算法将计算出检测序列相对于参照序列的百分序列同一 性。
例如， 通过 Smith & Waterman, Adv. Appl. Math. 2: 482 (1981)的局部同 源性算法，通过 Needleman & Wimsch, J. Mol. Biol. 48: 443 (1970)的同源性 比对算法， 通过 Pearson & Lipman, Proc. Nat 7. Acad. Sci. USA 85 : 2444 ( 1988)的相似性检索方法， 通过这些算法的计算机化实施 (Wisconsin Genetics软件包中 GAP, BESTFIT, FASTA和 TFASTA , Genetics Computer Group, 575 Science Dr., Madison, WI)或通过目测（通常参见， Ausubel等， 下文)可以进行用于比较的序列的最佳比对。
适于测定百分序列同一性和序列相似性的一个算法例子是 BLAST算 法， 在 Altschul等， J. Mol. Biol. 215: 403-410 (1990)中描述了该算法。 通 过国家生物技术信息中心（http://www. Ncbi.nlm. nih. gov/)公众可得到进行 BLAST分析的软件。 该算法包括： 通过鉴定出查寻序列中长度为 W的短 字而首先鉴定出高分值序列对 (HSPs) , 所述短字在与数据库序列中相同长 度的字比对时匹配或满足一些正值阀值记分 Τ。 Τ 称为相邻字记分阈值 (Altschul等， 1990)。这些最初的邻近字命中作为开始查寻的线索去发现包 含它们的更长的 HSPs。 然后， 这些字命中将沿每个序列的两个方向尽可 能远的延伸， 直到积累比对分值不再增加。对于核苷酸序列， 用参数 M (成 对匹配残基的奖励分值;总是大于零)和 N (错配残基的罚分值；总是小于零） 计算积累分值。 对于氨基酸序列， 用记分矩阵计算积累分值。 当积累比对 分值从获得的最大值回落数量 X, 由于一个或更多负分值残基比对积累， 积累分值达到或低于零， 或两个序列的任一个到达终点时， 每个方向的字 命中延伸停止。 BLAST算法的参数 W, T和 X决定了比对的敏感性和速 度。 BLASTN程序（对于核苷酸序列）使用字长值 (W)l l , 期望值 (E) 10 , 截 断值 100 , M=5 , N=-4和两个链的比较为缺省值。对于氨基酸序列， BLASTP 程序使用字长值 (W)3 ,期望值 (E) 10和 BLOSUM62记分矩阵 (参见， Henikoff & Henikoff, Proc. Nail. Acad. Sci. USA 89： 10915 (1989))为缺省值。
除了计算百分序列同一性外， BLAST 算法也进行两个序列间相似性 的统计学分析（参见， 例如 Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993))。 BLAST 算法提供的一个相似性测定是最小和概率 (Ρ(Ν)) ,其提供了两个核苷酸或氨基酸序列间偶然出现匹配的概率的指示。 例如， 如果检测核酸序列与参照核酸序列比较的最小和概率少于约 0.1 , 更优选少于约 0.01 , 最优选少于约 0.001 , 那么认为检测核酸序列与参照 序列相似。
两个核酸序列基本相同的另一个指标是两个分子在严格条件下互相 杂交。 短语"特异性杂交"指当该序列存在于复杂混合物（例如， 总细胞 的） DNA或 RNA中时， 在严格条件下， 分子仅与特定核苷酸序列结合， 形 成双螺旋或杂交。 "基本结合"指探针核酸和靶核酸间互补杂交， 并且包含 较少的错配， 通过降低杂交介质的严格性可耐受所述的错配， 以实现靶核 酸序列的期望检测。
在核酸杂交试验如 Southern和 Northern杂交上下文中 "严格杂交条件" 和 "严格杂交漂洗条件 "是序列依赖性的， 并且在不同环境参数下是不同 的。 较长的序列在较高温度特异性杂交。 在 Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes ,第 I部分第 2章' Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York中可以发现 核酸杂交的大量指南。一般地， 对于在限定离子强度和 pH下的特定序列， 高严格性杂交和漂洗条件选择为低于热熔点 (丁„ 约 5 °C。 典型地， 在"严格 条件"下， 探针将与其靶亚序列杂交， 而不与其它序列杂交。
丁„1是 (在限定离子强度和 pH条件下）50%靶序列与完全匹配的探针杂 交时的温度。对于特定的探针，非常严格的条件选择为等于 Tm。在 Southern 或 Northern印迹中在滤膜上有多于 100个互补残基的互补核酸杂交的一个 严格杂交条件的例子是在 42°C , 具有 lmg肝素的 50%曱酰胺， 过夜进行 该杂交。 高严格性漂洗条件的例子是 72°C , 0.15M NaCl约 15分钟。 严格 漂洗条件的例子是在 65 °C , 0.2 X SSC漂洗 15分钟 (参见， Sambrook, 下 文， SSC 緩冲液的描述)。 通常， 在高严格性漂洗前进行低严格性漂洗以 除去背景探针信号。 对于例如多于 100个核苷酸的双螺旋而言， 中严格性 漂洗的例子是 45 °C , l x SSC漂洗 15分钟。对于例如多于 100个核苷酸的 双螺旋而言， 低严格性漂洗的例子是 40 °C , 4-6 X SSC漂洗 15分钟。 对于 短探针 (例如， 约 10到 50个核苷酸）， 严格条件通常包括在 pH7.0到 8.3 的少于约 1.0M Na离子的盐浓度， 通常约 0.01到 l.ONa离子浓度 (或其它 盐）， 通常温度至少是约 30°C。 通过添加去稳定剂如曱酰胺也可以获得严 格条件。 一般地， 在特定杂交测定中， 信噪音比就无关探针所观察到的值 高 2χ(或更高)表明特异杂交的检测。 在严格条件下不互相杂交的核酸如果 它们编码的蛋白质是基本相同的， 那么它们仍是基本相同的。 例如， 当用 遗传密码所允许的最大密码子简并性创造核酸拷贝时， 就会出现这种情 况。
下面是杂交 /漂洗条件设置的例子，所述条件可以用于克隆与本发明参 照核苷酸序列基本相同的同源核苷酸序列： 参照核苷酸序列与参照核苷酸 序列优选在 50°C , 7%十二烷基硫酸钠（SDS), 0.5M NaP04, ImM EDTA 中杂交， 在 50°C , 2 X SSC, 0.1% SDS中漂洗， 更期望在 50°C , 7%十二 烷基硫酸钠（SDS), 0.5M NaPO4, ImM EDTA中杂交， 在 50°C , 1 X SSC, 0.1% SDS中漂洗，更期望在 50°C , 7%十二烷基硫酸钠（SDS), 0.5M NaPO4, ImM EDTA中杂交， 在 50°C , 0.5 X SSC, 0.1% SDS中漂洗， 优选地， 在 50 °C , 7%十二烷基硫酸钠（SDS), 0.5M NaP04, ImM EDTA 中杂交， 在 50 °C , 0.1 X SSC, 0.1% SDS中漂洗， 更优选地， 在 50°C , 7%十二烷基硫 酸钠（SDS), 0.5M NaPO4, ImM EDTA中杂交， 在 65°C , 0.1 X SSC, 0.1% SDS中漂洗。
两个核酸序列或蛋白质基本相同的另一个指标是第一核酸编码的蛋 白质与第二核酸编码的蛋白质免疫交叉反应或特异结合。 因此， 蛋白质通 常与第二蛋白质基本相同， 例如， 其中两个蛋白质仅仅由于保守性置换而 不同。
"合成的 "指包含天然序列中不存在的结构特征的核苷酸序列。 例如， 称更密切地类似双子叶和 /或单子叶植物基因 G+C含量和正常密码子分布 的人工序列是合成的。
"转化 "是向宿主细胞或生物体中引入异源核酸的过程，特别地， "转化" 意指 DNA分子稳定整合进入目的生物体基因组中。
"转化的 /转基因的 /重组的 "指已经引入异源核酸分子的宿主生物体， 如细菌或植物。 核酸分子可以稳定地整合进入宿主基因组或者核酸分子也 可以作为染色体外分子存在。 这种染色体外分子可以是自主复制的。 转化 的细胞， 组织， 或植物理解为不仅包含转化过程的最终产物， 也包含其转 基因子代。 "非转化的"， "非转基因的"， 或"非重组的"宿主指不含有异源 核酸分子的野生型生物体， 例如细菌或植物。
本文所用的术语"多核苷酸"、 "多核苷酸分子"、 "多核苷酸序列"、 "编码 序列"、 "开放阅读框 (ORF)"等包括单链或双链的 DNA和 RNA分子， 可包含 一个或多个原核序列， cDNA序列，包含外显子和内含子的基因组 DNA序列， 化学合成的 DNA和 RNA序列， 以及有义和相应的反义链。
生产和操作本文公开的多核苷酸分子及寡核苷酸分子的方法是本领域技 术人员已知的， 并可按照已描述的重组技术 (参见 Maniatis 等， 1989, 分子克 隆， 实验室手册, 冷泉港实验室出版社， 冷泉港， 纽约； Ausubel等， 1989, 分子生物学当前技术, Greene Publishing Associates & Wiley Interscience , NY; Sambrook等， 1989, 分子克隆， 实验室手册, 第 2版， 冷泉港实验室出版社， 冷泉港，纽约； Innis等 (编）， 1995 , PCR策略, Academic Press, Inc. , San Diego; 和 Erlich (编）， 1992, PCR技术， 牛津大学出版社， New York)完成。 植物转化
在特别优选实施方式中， 在高等生物体例如植物中表达至少一种本发 明的赋予盐耐受性的蛋白。 可将本发明核苷酸序列插入到表达盒中， 然后 优选地，表达盒稳定整合在所述植物基因组中。在另一个优选实施方式中， 核苷酸序列包含在非致病自我复制的病毒中。 在另一个优选实施方式中， 核苷酸序列包含在农杆菌中。 按照本发明转化的植物可以是单子叶植物或 双子叶植物， 包括但不限于玉米， 小麦， 大麦， 黑麦， 甘薯， 豆， 豌豆， 菊苣， 莴苣， 甘蓝， 花椰菜， 花茎甘蓝， 芜菁， 萝卜， 菠菜， 芦笋， 洋葱， 大蒜， 胡椒， 芹菜， 笋瓜， 南瓜， 大麻， 夏南瓜， 苹果， 梨， 温椁， 瓜， 李子， 樱桃， 桃子， 油桃， 杏， 草莓， 葡萄， 木莓， 黑莓， 菠萝， 鳄梨， 蕃木瓜， 芒果， 香蕉， 大豆， 番茄， 高粱， 甘蔗， 甜菜， 向日葵， 菜籽油 菜， 三叶草， 烟草， 胡萝卜， 棉花， 苜蓿， 稻， 马铃薯， 茄子， 黄瓜， 拟 南芥属和木本植物如针叶树和落叶树。 特别优选的是水稻、 小麦、 大麦、 玉米、 燕麦、 黑麦、 甘蔗、 甜菜、 大豆、 马铃薯。 优选地， 用于本发明的植 物是芸薹科植物。优选地，所述植物是芸薹属植物， 包括黑芥 CSra c« « gra)、 欧洲油菜 (Sraw ca ""jms")、 ^^. Brassica oleraceae) . 克 (Brassica rapa)、 埃 塞俄比亚齐 (βΓβ β carinata)、 ^-^(Brassica juncea)。 所述植物也可以 芸 薹科的其它植物。
一旦已将期望的核苷酸序列转化进入特定植物物种中， 可以在该物种 中繁殖它或用常规育种技术将它转移进入相同物种的其它品种， 特别包括 商业品种中。
优选地， 在转基因植物中表达本发明的核苷酸序列， 由此在转基因植 物中引起相应的赋予植物耐盐性的蛋白的生物合成。 以这种方式， 可产生 具有改良性状的转基因植物。 为了在转基因植物中表达本发明核苷酸序 歹1 J , 本发明核苷酸序列可能需要修饰和优化。 所有生物体都有特定的密码 子使用偏爱性， 这是本领域已知的， 可以在保持本发明所述核苷酸序列编 码的氨基酸的同时改变其密码子以符合植物偏爱性。 而且， 从有至少约 35%, 优选多于约 45%, 更优选多于 50%, 最优选多于约 60%GC含量的 编码序列可以最好地实现植物中高水平的表达。 尽管可以在单子叶植物和 双子叶植物物种中充分地表达优选的基因序列， 但可以修饰序列以适应单 子叶植物或双子叶植物的特异密码子偏好和 GC含量偏好， 因为这些偏好 已被证明是不同的（Murray等， Nucl. Acids Res. 17: 477-498 (1989))。 此外， 可筛选核苷酸序列以寻找引起信息截断的非常规剪接位点的存在。 利用公 开专利申请 EP 0 385 962(Monsanto), EP 0 359 472(Lubrizol)和 WO 93/07278(Ciba-Geigy)中所述的方法， 用本领域熟知的位点定向诱变技术， PCR和合成基因构建进行在这些核苷酸序列中需要进行的所有改变，如上 述那些改变。
在本发明一个实施方式中， 根据这里并入作为参考文献的美国专利 5,625,136中公开的方法可制备合成基因。在该方法中， 利用了玉米优选的 密码子， 即最常编码玉米中那个氨基酸的单密码子。 特定氨基酸的玉米优 选密码子可以来源于， 例如玉米的已知基因序列。 在 Murray 等， Nucleic Acids Research 17: 477-498 (1989)中教导了玉米植物 28个基因的玉米密码 子使用， 这里并入这篇文献的公开内容为参考文献。
以这种方式可以优化核苷酸序列以便在任何植物中的表达。 公认基因 序列的所有或任何部分都可以优化或合成。 即， 也可以利用合成或部分优 化的序列。
为了翻译的有效起始，可能需要修饰邻近起始曱硫氨酸的序列。例如， 通过包含已知在植物中有效的序列可以修饰它们。 Joshi提出了植物适合的 共有序列（NAR 15: 6643-6653 (1987)), Clonetech提出了进一步的翻译起始 子共有序列（1993/1994 目录， 210页）。 这些共有序列适合与本发明核苷酸 序列一起使用。 向包含所述核苷酸序列， 直到和包含 ATG (同时保持第二 个氨基酸没有被修饰）或可替代地直到和包含 ATG后的 GTC (具有修饰转 基因第二个氨基酸的可能性)的构建物中引入该序列。
可将作为其天然序列或作为如上所述优化合成序列的本发明新的 ThLEAl 基因可操作地与在植物中表达的各种启动子， 包括组成型， 诱导 型， 时序调节， 发育调节， 化学调节， 组织优选和组织特异性启动子相融 合以制备重组 DNA分子， 即嵌合基因。 启动子的选择将随着表达时间和 空间需要而变化， 而且也取决于靶物种。 因此， 可以使用在叶， 主茎或茎 杆， 穗， 花序（例如， 穗状花序， 圓锥花序， 穗轴等）， 根， 和 /或幼苗中表 达本发明核苷酸序列。 尽管证明了来源于双子叶植物的许多启动子在单子 叶植物中是可起作用的， 反之亦然， 但是理想地， 选择双子叶植物启动子 用于双子叶植物中的表达， 单子叶植物的启动子用于单子叶植物中的表 达。 然而， 没有限制所选启动子的起源， 只要启动子起作用驱动期望细胞 中核苷酸序列的表达就足够了。
优选的组成型启动子包括 CaMV 35S和 19S启动子 (Fraley等， 1994 年 10月 4日公布的美国专利号 5,352,605)。另外优选的启动子来源于在大 多数细胞类型中表达的几种肌动蛋白基因的任何一种。 可以容易地修饰 McElroy等 (Mol. Gen. Genet. 231 : 150-160 (1991))所述的启动子表达盒以 用于 ThLEAl基 的表达, 该基因表达盒特别适合在单子叶植物宿主中使 用。
另一个优选的组成型启动子来源于泛素， 它是已知在许多细胞类型中 积累的另一种基因产物。 从几种物种， 例如向日葵 (Binet 等， 1991. Plant Science 79: 87-94) , 玉米（Christensen 等， 1989. Plant Molec. Biol. 12 ： 619-632)和拟南芥 (Norris等 1993. Plant Molec. Biol. 21 : 895-906)克隆了泛 素启动子， 可用于转基因植物中。 已发展了转基因单子叶植物系统中的玉 米泛素启动子， 并且在专利公布 EP 0 342 926中公开了其序列和用于单子 叶植物转化的载体。 泛素启动子适合在转基因植物， 特别是单子叶植物中 新直立密穗基因的表达。
除了适合启动子的选择外， 植物中 ThLEAl蛋白表达的构建可以连接 在异源核苷酸序列下游的适合的转录终止子。 可得到几种这种终止子， 并 且它们是本领域已知的（例如来源于 CaMV的 tml, 来源于 rbcS的 E9)。 任 何已知在植物中起作用的可得到的终止子都可以用在本发明中。 明增强表达的序列， 如内含子序列（例如来源于 Adhl和 bronzel)和病毒前 导序列（例如来源于 TMV,MCMV和 AMV)。
有可能优选将本发明核苷酸序列的表达靶向到植物中不同细胞定位。 在一些情况下， 可能期望定位在细胞溶质中， 而在其它情况下， 可能优选 定位在一些亚细胞细胞器中。 转基因所编码的酶的亚细胞定位采用了本领 域熟知的技术。 通常， 操作编码来源于已知靶向细胞器的基因产物的靶肽 的 DNA, 使之融合到核苷酸序列的上游。 已知用于叶绿体的许多这种靶 序列， 并且已知证明了它们在异源构建中的功能。 本发明核苷酸序列的表 达也可靶向到宿主细胞的内质网或液泡中。 实现上述这些目的的技术是本 领域熟知的。
可用于植物转化的大量转化载体是植物转化领域技术人员已知的， 并 且本发明的核酸分子可以与任何这种载体联合使用。 载体的选择将依赖于 用于转化的优选转化技术和靶植物物种。 对于一些靶物种， 可以优选不同 的抗生素或除草剂选择性标记。 通常用在转化中的选择性标记包括赋予对 卡那霉素和相关抗生素抗性的 基因（Messing & Vierra. , 1982. Gene 19: 259-268; 和 Bevan等， 1983. Nature 304: 184-187) , 赋予对除草剂膦丝菌素 抗性的 bar基因，（White等， 1990. Nucl. Acids Res 18: 1062,和 Spencer等， 1990. Theor. Appl. Genet 79: 625-631) , 赋予对抗生素潮霉素抗性的 hph基 @ (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931) , 和赋予对 methatrexate 抗性的 dhfr 基因（Bourouis 等， 1983. EMBO J. 2 (7) ： 1099-1 104) , 赋予对草甘磷抗性的 EPSPS基因（U. S.专利号: 4,940, 935和 5,188, 642) ,和提供代谢甘露糖能力的甘露糖 -6-磷酸异构酶基因（美国专利 号 5, 767, 378和 5,994, 629)。 然而， 选择性标记的选择对本发明不是至关 重要的。
在另一个优选的实施方式中， 将本发明的核苷酸序列直接转化到质体 基因组中。 质体转化的主要优点是质体通常不需要实质修饰就能表达细菌 基因， 并且质体能表达单启动子控制下的多个开放读框。 在美国专利 5,451 ,513、 5,545,817 和 5,545,818 中， PCT 申请号 WO 95/16783 中和 McBride等， （1994) Proc. Nati. Acad. Sci. USA 91 ,7301 -7305中详尽地描述 了质体转化技术。 叶绿体转化的基本技术包括例如利用生物轰击或原生质 体转化 (例如氯化钙或 PEG介导的转化)将位于目的基因和选择标记侧翼的 克隆质体 DNA的区域一起弓 1入适合的靶组织中。 1到 1.5kb侧翼区域， 称 为导向序列， 可促进与质体基因组的同源重组， 因此允许原质体系特定区 域的置换或修饰。 最初， 可利用在提供了对壮观霉素和 /或链霉素抗性的叶 绿体 16S rRNA 和 rpsl2 基因的点突变作为转化的选择标记（Svab, Z., Hajdukiewicz, P.,和 Maliga, P. (1990) Proc. Nati. Acad. Sci. USA 87,8526- 8530; Staub, J. M"和 Maliga, P. (1992) Plant Cell 4,39-45)。 这以约每 100次 靶叶片轰击 1次的频率产生了稳定的同质转化体。 这些标记间存在的克隆 位点可以用来产生用于导入外源基因的质体靶向载体（Staub, J. M.，和 Maliga, P. (1993) EMBO J. 12,601-606)。通过用显性选择标记，编码壮观霉 素去毒酶 (spectinomycin-cletoxifying enzyme)氨基糖苷类 -3，-腺苷醜转移酶 的细菌 aadA基因置换隐性 rRNA或 r-蛋白质抗生素抗性基因可获得转化 频率的显著增加（Svab, Z.和 Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90,913-917)。 以前， 该标记成功地用于高频率转化绿藻 Chlamyclomonas re " wri¾ 质体基因组 (； Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089)。 其它用于质体转化的选择标记是本领域已知的， 并且包含 在本发明范围内。 通常， 转化后需要约 15到 20个细胞分裂周期以达到同 质状态。 通过同源重组将基因插入每个植物细胞中存在的所有几千个环形 质体基因组拷贝中的质体表达利用了拷贝数大大高于核表达基因的优势， 使得表达水平可以容易地超过总可溶植物蛋白质的 10%。在优选实施方式 中， 将本发明核苷酸序列插入到质体靶向载体中， 并且转化进入期望的植 物宿主质体基因组中。 获得了对于含有本发明核苷酸序列的质体基因组而 言属同质的植物， 优选地， 该植物具有高水平地表达核苷酸序列的能力。
下列实施例只是举例描述本发明， 而不用来限制本发明的范围。 本领 域技术人员明白， 可以使用其他具体实施方案实现本发明的目的， 这些具 体实施方案同样包含在本发明的范围内。 实施例 实施例 1. 大规模鉴定和 7¾LE ^基因的分离
使用出芽酿酒酵母菌株 G19 {MATa leu2-3, 2-112, trpl-1, 簡3-3， ade2-l, his3-ll canl-100, 15( ) e 人 · // .'.'e 进行盐耐受基因的大规模鉴定。 G19 菌株是一种盐敏感性酵母菌株， 该菌株中缺失了编码质膜 Na+输出泵的基因 ENA1 , 致使 G19酵母菌株与野生型酵母相比对于外部 Na+浓度更加敏感。
为了进行盐耐受基因的大规模鉴定， 用 Gal4启动子衍生的 AD融合盐芥 cDNA转化 G19酵母细胞， 并涂布在含有 400 mM NaCl的选择培养基上。 选 出能够在含盐的选择平板上存活的转化体。 在 106个酵母转化体中， 选择了 超过 100个转化体进行后续盐耐受性确认，并分离出插入的 cDNA用于测序。 基于与其他物种中同源物进行比较的序列分析， 选择存在完整的或几乎完整 的 ORF 的推定盐耐受性候选株的基因以确认它们在植物中赋予盐耐受的能 力。
选出一个对高盐分展现出可观耐受性的 cDNA克隆。 序列分析显示其含 有的植物 cDNA编码一个 LEA样蛋白。 将该基因命名为 ThLEAl。 ThLEAl cDNA长 701 bp (如 SEQ ID NO:3所示）， 并且具有最长为 483 b 的开放阅 读框（图 1A) (如 SEQ ID ΝΟ: 1所示）。 推定的氨基酸序列由 160个氨基酸组 成， 如 SEQ ID NO:2所示， 分子量大约为 16.3 kDa, 等电点为 9.19。 实施例 2. ThLEAl的异位表达赋予酵母盐耐受性
将全长或基本上全长的 ThLEAl CDS(SEQ ID NO: 1)插入到酿酒酵母 /大 肠杆菌穿梭载体 pGAD GH (Clontech)中并在组成型酵母启动子 ADH1的调控 下， 将这种插入了 SEQ ID NO: l的穿梭载体用于转化酵母 G19细胞。 并将空 载体转入酵母细胞作为对照。 可以用本领域所熟知的各种方法将所述载体转 入酵母细胞中， 例如参见冷泉港实验手册 （Methods In Yeast Genetics A Laboratory Course Manual , 1990年出版， 由 MarkD.Rose等人编写） 39-52页 提供的方法。
在 SD 选择培养基平板 (0.67%不含氨基酸的细菌-酵母用含氮基底 (Bacto-yeast nitrogen base without amino acids)、 2%葡萄糖、 2%细菌用琼月旨 (Bacto-agar)、 0.062%-Lea dropout)上选出转化体并接种到液体 SD选择培养基 (0.67%不含氨基酸的细菌 -酵母用含氮基底、 2%葡萄糖、 0.062%-Lea dropout) 中， 在 30°C下培养， 直到生长至对数期晚期 /静止期。 将饱和培养物液体 SD 选择培养基稀释 io、 100或 1000倍。 将 2微升稀译后的培养物点样至含有 NaCl (300mM、 350mM、 400mM)和不含 NaCl的 SD培养基上， 30°C培养 8 天， 并记录生长情况。
转化后的酵母细胞在盐胁迫条件下具有改进的存活率 (参见图 3)。 从图 3 中可见， 当培养基中的盐含量对于酵母细胞达到有毒浓度时， 含有空载体的 对照酵母生长艮差， 而转入了 ThLEAl 的酵母细胞显示了更好的盐耐受性和 更好的生长， 表明 ThLEAl为酵母细胞赋予了盐耐受功能。 实施例 3. ThLEAl对盐胁迫的转录应答
为了研究 ThLEAl在盐条件下的应答， 对小盐芥幼苗进行盐处理并分析 了 TTLL&^基因的转录。
本实施例使用小盐芥山东生态型（Shandong ecotype)(中国科学院遗传与 发育生物学研究所)。 使表面灭菌后的种子在含有 2%蔗糖和 0.8%琼脂的 MS 培养基 (见下表)上 22°C萌发并生长。 将长出 2-4片真叶的幼苗转移至土壤中 培养， 并以 16小时光照期在 24°C下培养。 对五周龄的幼苗施加胁迫处理， 即用 500 mM NaCl (1升）浇灌幼苗， 并在第 0、 1、 4、 8、 12和 24小时 (从浇 盐水的时间开始取样)采样。
MS培养基成
使用 浓 成分
度 (mg/L)
硝酸钾 KN03 1900
硝酸铵 NH4N03 1650
大量元素
磷酸二氢钾 KH2P04 170
硫酸镁 MgS04.7H20 370
氯化钙 CaCl2.2H20 440
碘化钾 KI 0.83
硼酸 H3B03 6.2
微量元素
硫酸锰 MnS04.4H20 22.3
硫酸锌 ZnS04.7 H20 8.6
钼酸钠 Na2Mo04.2 H20 0.25 硫酸铜 CuS04.5 H20 0.025
氯化钴 CoCl2.6 H20 0.025
乙 二 胺 四 乙 酸 二 钠
37.3
铁盐 Na2.EDTA
硫酸亚铁 FeS04.7H20 27.8
肌醇 100
甘氨酸 2
有机成分 盐酸 ¾胺素 VB1 0.1
盐酸吡哆醇 VB6 0.5
烟酸 VB5或 VPP 0.5
糖 (sucrose) 30g/L
琼脂 (agar) 7 g/L 通过将经过盐处理的小盐芥的叶片和根样品在液氮中冻干并进行研磨来 分离总 RNA,方法基本如 Soni R和 Murray A， 1994, Isolation of intact DNA and RNA from plant tissue. Anal Biochem 218: 474-476 中所述。 将研磨得到的 1 OOrng植物粉末与 600μ1提取緩冲液 (50mMTris-HCl H 6.0, 10 mM EDTA, 2% SDS, 100 mM LiCl))以及在 65°C加热过的酚 /氯仿 1: 1混合， 涡旋振荡并在 4 °C以 12000rpm离心 15分钟。 将上清液用酚抽提 2次并用等体积的 4 M LiCl 沉淀， 再进行离心（12000rpm, 15min)。 离心之后， 将 RNA沉淀用 50μ1ΤΕ緩 冲液 (10 mM Tris-Cl, pH 7.5. 1 mM EDTA.)重悬。 再次用酚抽提上清液， 并加 入十分之一体积的 3M NaAc和 3倍体积的无水乙醇进行沉淀。离心（12000rpm, 15min) 沉淀 RNA后用 75%乙醇洗涤 RNA沉淀并在 50μ1 TE緩冲液中重悬。
对于 Northern 印迹， 将 15 微克总 RNA 上样至每个泳道中。 使用 Ready-primed标" ^己试剂盒 (Amersham International)^ ThLEAl 全长 CDS 以 [a-32P] dCTP标记，使用标记后的片段来进行探测。根据标准流程在高严格条 件下进行杂交和洗涤。
如图 2中所示， 在 4小时处理后， ThLEAl受到了强烈的诱导。 此外， ThLEAl的转录物在根中的积累超过了地上部分。这个结果指出 ThLEAl确实 是能够由盐诱导的基因。 实施例 4. ThLEAl在拟南芥中的过表达与拟南芥盐耐受性的改善 为了确认赋予植物盐耐受性的能力， 将 ThLEAl克隆至经过修饰的二元 载体 pGreen中（参见图 4A),在模式植物拟南芥中进行过表达并进行盐耐受测 定。
为了过表达选出的盐耐受相关性盐芥基因，将包含 SEQ ID NO: 1的 cDNA 片段从 GAD GH载体中切出，并克隆至带有修饰的 pGreen二元载体 (Hellens RP 等 , 2000 , pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mQdiatQd plant transformation. Plant Mol Biol 42: 819-832)中并 置于 35S双启动子的调控下 (参见图 4A) , 从而在拟南芥中进行过表达。
将这种构建体引入含有 Sou 质粒 (Hellens RP等，见上)的根癌土壤杆菌 {Agrobacterium t mefaciens)EHA 105菌株中。所得细菌用于通过植株真空渗入 法 (Bent AF 和 Clough SJ (1998) Agrobacterium germ-line transformation: transformation of Arabidopsis without tissue culture. In Plant Molecular Biology Manual, 2nd ed, S.B. Gelvin and R. A. Schilperoot, eds (Dordrecht, The Netherlands: Kluwer Academic Publishers): 1-14)转化野生型拟南芥 (哥伦比亚 生态型）。 并以空载体 pGreen作为对照， 以同样的方法转化野生型拟南芥。
将获得的种子用 10%漂白剂进行表面灭菌， 并用无菌水洗涤 3次。 将无 菌种子悬浮在 0.15%琼脂糖中并铺板到含有 1.5% 糖的 MS培养基上。 为了 选择转基因植物，向培养基中加入 50 mg/mL卡那霉素。将平板叠放在黑暗中， 于 4°C条件下放置 2-4天， 然后移入 24 °C的组织培养室， 以 16小时光照期进 行培养。 将 1周龄的植株转移至盆中， 移入温度和光照条件类似的温室， 直 至形成种子。 为了选择纯合品系，分析 T2代种子在卡那霉素存在下的萌发状 态。 只有分离率约为 3 : 1的 T3植株才被使用。
盐胁迫处理在盆中进行， 用 NaCl溶液浇灌 4周龄的拟南芥植株， 每 4 天一次， 且 NaCl溶液的浓度逐渐增加， 具体为 50mM、 100mM、 150mM、 200mM、 250mM、 250mM, 共六次。 之后为植物拍照。
转化了 ThLEAl 的拟南芥植物与使用空载体作为对照的植物相比， 在含 盐的平板上生长时或在土壤中生长并用 NaCl处理时，均显示出增强的盐耐受 性 (参见图 4B)。 35S-ThLEAl转基因植物在含盐培养基上的萌发率约为 46%, 远高于对照品系的约 22%。 在连续的盐处理之后， 35S- 7¾LE^转基因植物 的存活率约为 38%, 而空载体对照植物完全无法存活 (参见图 4C)。 这些结果 表明异位表达 7¾L&^能够改善拟南芥的盐耐受性。

Claims

权利要求
1. 分离的多核苷酸， 其包含选自下组的核苷酸序列：
(1) SEQ ID ΝΟ:1或 SEQ ID NO:3所示的核苷酸序列；
(2) 与（1)的核苷酸序列的互补序列在中等严格条件、 优选高严格杂交条 件下杂交的核苷酸序列；
(3) 与（1)的核苷酸序列具有至少 50%、至少 60%、至少 70%、至少 75%、 优选至少 80%、 更优选至少 85%、 更优选至少 90%、 尤其是至少 95%或 98% 或 99%同一性的核苷酸序列；
(4) 与（1)的核苷酸序列编码相同氨基酸序列的蛋白质、 但在序列上不同 的核苷酸序列；
(5) 编码如下氨基酸序列之一的核苷酸序列： SEQ ID NO:2中所示的氨 基酸序列， 或者， 由于一或多个 (例如 1-25个、 1-20个， 1-15个， 1-10个， 1-5个， 1-3个)氨基酸残基的替代、 缺失和 /或插入而与 SEQ ID NO:2所示的 氨基酸序列不同的氨基酸序列， 或者， 与 SEQ ID NO:2所示的氨基酸序列具 有至少 50%、 至少 60%、 至少 70%、 至少 75%、 优选至少 80%、 更优选至少 85%、更优选至少 90%、尤其是至少 95%或 98%或 99%同一性的氨基酸序列；
(6) (1)-(5)任何一个的核苷酸序列的活性片段； 或
(7) 与（1)-(5)任何一个的核苷酸序列互补的核苷酸序列。
2. 权利要求 1的多核苷酸， 其具有 SEQ ID NO: 1或 SEQ ID NO: 3中所 示的核苷酸序列。
3. 构建体， 其包含权利要求 1或 2的多核苷酸。
4. 表达盒，其包含权利要求 1或 2的多核苷酸以及调控所述多核苷酸表 达的调控元件。
5. 权利要求 4的表达盒，其中所述调控元件为与所述核苷酸序列可操作 连接的启动子。
6. 权利要求 5的表达盒， 其中所述启动子为组成型启动子、 组织特异性 启动子或诱导型启动子。
7. 载体， 其包含权利要求 1或 2的多核苷酸， 所述载体可以是克隆载 体或者用于表达所述多核苷酸的表达载体。
8. 权利要求 7的载体， 其中进一步包含终止子序列。
9. 细胞， 其包含权利要求 1或 2的多核苷酸或权利要求 3的构建体或 权利要求 4-6中任一项的表达盒或权利要求 7或 8的载体， 所述细胞可以是 动物细胞、 植物细胞或者微生物细胞， 例如大肠杆菌细胞， 优选植物细胞。
10. 转基因植物或者植物部分， 其包含权利要求 9的细胞， 其中所述植 物优选是农作物例如水稻、 小麦、 大麦、 玉米、 高粱、 甘蔗、 燕麦、 或黑麦， 或烟草、 大豆、 向日葵、 甜菜、 辣椒、 马铃薯、 番茄， 或来源于该植物的转 基因种子或植物材料。
11. 分离的多肽， 其包含从如下组氨基酸序列中选择的氨基酸序列： (l)SEQ ID NO:2所示的氨基酸序列，
(2)由于一或多个 (例如 1-25个、 1-20个， 1-15个， 1-10个， 1-5个， 1-3 个)氨基酸残基的替代、 缺失和 /或插入而与 SEQ ID NO:2中任何一个所示的 氛基酸序列不同的氛基酸序列，
(3)与 SEQ ID NO:2中任何一个所示的氨基酸序列具有至少 50%、 至少 60%、 至少 70%、 至少 75%、 优选至少 80%、 更优选至少 85%、 更优选至少 90%、 尤其是至少 95%或 98%或至少 99%同一性的氨基酸序列，
(4) (1)或 (2)或 (3)所述氨基酸序列的活性片段， 和
(5)权利要求 1或 2的多核苷酸分子编码的氨基酸序列。
12. 权利要求 11的多肽， 其包含 SEQ ID NO:2所示的氨基酸序列。
13. 生产盐耐受性植物的方法， 该方法包括： 从权利要求 9的植物细胞 再生转基因植物， 或者将权利要求 10的植物与另一植物杂交； 其中所述植物 优选是直立密穗植物， 其中所述植物优选是农作物例如水稻、 小麦、 大麦、 玉米、 高粱、 甘蔗、 燕麦、 或黑麦。
14. 权利要求 13的方法生产的植物或来源于该植物的转基因种子。
15. 赋予植物盐耐受性的方法， 该方法包括制备含有权利要求 1或 2的 多核苷酸或权利要求 3的构建体或权利要求 4-6中任一项的表达盒或权利要 求 7或 8的载体的植物或从权利要求 9的植物细胞再生转基因植物。
16. 权利要求 15的方法， 进一步包括将权利要求 15的植物与另一植物 杂交， 并筛选具有提高的盐耐受性的后代。
17. 权利要求 15或 16的方法， 其中所述植物是单子叶植物例如水稻、 小麦、 大麦、 玉米、 高粱、 甘蔗、 燕麦、 或黑麦， 或者是双子叶植物如白菜、 油菜、 甘蓝、 拟南芥、 小盐芥、 烟草、 大豆、 向日葵、 甜菜、 辣椒、 马铃薯、 番茄。
18. 增加高盐土地中植物产量的方法， 包括在所述土地上种植耐盐植物， 所述耐盐植物为权利要求 10或 14的植物、 或由权利要求 13或 15-17中任一 项的方法获得的植物， 其与种植不具有盐耐受性的同种植物时相比使单位面 积内的植物产量增加。
19. 权利要求 18的方法， 其中所述植物是单子叶植物例如水稻、 小麦、 大麦、 玉米、 高粱、 甘蔗、 燕麦、 或黑麦， 或者是双子叶植物如白菜、 油菜、 甘蓝、 拟南芥、 小盐芥、 烟草、 大豆、 向日葵、 甜菜、 辣椒、 马铃薯、 番茄。
20. 权利要求 1或 2的多核苷酸或权利要求 3的构建体或权利要求 4-6 中任一项的表达盒或权利要求 7或 8的载体在赋予植物盐耐受性中的用途。
21. 权利要求 20的用途， 其中所述植物是单子叶植物例如水稻、 小麦、 大麦、 玉米、 高粱、 甘蔗、 燕麦、 或黑麦， 或者是双子叶植物如白菜、 油菜、 甘蓝、 拟南芥、 小盐芥、 烟草、 大豆、 向日葵、 甜菜、 辣椒、 马铃薯、 番茄。
PCT/CN2012/072580 2011-03-22 2012-03-20 耐盐基因及其应用 WO2012126347A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2011100690925A CN102690830A (zh) 2011-03-22 2011-03-22 耐盐基因及其应用
CN201110069092.5 2011-03-22

Publications (1)

Publication Number Publication Date
WO2012126347A1 true WO2012126347A1 (zh) 2012-09-27

Family

ID=46856567

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/072580 WO2012126347A1 (zh) 2011-03-22 2012-03-20 耐盐基因及其应用

Country Status (2)

Country Link
CN (1) CN102690830A (zh)
WO (1) WO2012126347A1 (zh)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103044535A (zh) * 2012-12-18 2013-04-17 华南师范大学 高粱抗盐基因SbSAP14及其应用
CN104178495A (zh) * 2013-05-21 2014-12-03 中国科学院遗传与发育生物学研究所 耐盐抗旱基因及其编码的蛋白和应用
CN107787180B (zh) * 2013-09-11 2020-09-22 中国科学院植物研究所 转基因植物
WO2015042743A1 (zh) * 2013-09-26 2015-04-02 创世纪转基因技术有限公司 一种小盐芥钙调磷酸酶b类似蛋白cbl-8及其编码基因与应用
CN111733276B (zh) * 2020-07-17 2021-04-16 中国农业科学院作物科学研究所 耐盐基因及其应用
CN117186198A (zh) * 2022-05-30 2023-12-08 中国科学院遗传与发育生物学研究所 高粱SbMYB12蛋白质及其编码基因在调控植物耐盐性中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1793364A (zh) * 2005-11-11 2006-06-28 中国科学技术大学 盐芥的一个耐盐基因及其编码蛋白与应用
CN101747419A (zh) * 2008-12-08 2010-06-23 中国科学院遗传与发育生物学研究所 耐盐相关蛋白及其编码基因与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1793364A (zh) * 2005-11-11 2006-06-28 中国科学技术大学 盐芥的一个耐盐基因及其编码蛋白与应用
CN101747419A (zh) * 2008-12-08 2010-06-23 中国科学院遗传与发育生物学研究所 耐盐相关蛋白及其编码基因与应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 5 February 2008 (2008-02-05), XIE, Q ET AL.: "LEA-like protein[Eutrema halophilum]", Database accession no. ABY86896.1 *
DATABASE GENBANK [online] 5 February 2008 (2008-02-05), XIE, Q ET AL.: "Thellungiella halophila LEA- -like protein(LEAl) mRNA, complete cds", Database accession no. EU365627.1 *
DATABASE GENBANK 1 April 2005 (2005-04-01), DEEPTI TAYAL ET AL.: "Brassica carinata group I late embryogenesis abundant protein mRNA, complete cds", Database accession no. AY572959 *
LAN YING: "Cloning and functional analysis of drought and salinity tolerance of soybean of LEA genes", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 15 March 2005 (2005-03-15) *
ZHANG HUA: "Cloning, expression and function analysis of the BADH and LEA gene from Chorispora bungeana", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 15 April 2008 (2008-04-15) *

Also Published As

Publication number Publication date
CN102690830A (zh) 2012-09-26

Similar Documents

Publication Publication Date Title
EP1835028B1 (en) The transcription factor gene osnacx from rice and use for improving tolerance of plants to drought and salt thereof
CA2957986C (en) Biotic and abiotic stress tolerance in plants
Gong et al. GhAGP31, a cotton non‐classical arabinogalactan protein, is involved in response to cold stress during early seedling development
WO2007020638A2 (en) Methods of increasing abiotic stress tolerance and/or biomass in plants and plants generated thereby
US20210102218A1 (en) Expression of transcription regulators that provide heat tolerance
WO2015007240A1 (en) Transgenic maize
WO2012126347A1 (zh) 耐盐基因及其应用
US7238865B2 (en) SOS1 gene from halophila that confers salt tolerance
US10837026B2 (en) Drought and salt tolerant plants
WO2013091563A1 (en) Methods for improving crop yield
CN111606986B (zh) 一种抗旱耐盐相关蛋白及其相关生物材料与应用
CA2866169C (en) Environmental stress-resistant plant with high seed productivity and method for producing the same
WO2008043268A1 (en) Gmrd22-like genes and use thereof to protect against abiotic stress
AU2013214833B2 (en) HaHB11 provides improved plant yield and tolerance to abiotic stress
AU2009201381A1 (en) Hordeum vulgare NA+/H+antiporter (HVNHX1) gene, method for improving salt tolerance in plants by expressing HVNHX1 gene and transgenic plants with improved salt tolerance developed by using the method
US10414807B2 (en) Transcription factor genes and proteins from Helianthus annuus, and transgenic plants including the same
WO2024030824A2 (en) Plant regulatory sequences and expression cassettes
EP1673462A2 (en) Plant transcriptional regulators of abiotic stress
CN116751809A (zh) 耐盐相关的蛋白GmXTH32及其相关生物材料和应用
WO2008100112A1 (en) Transgenic plants exhibiting increased resistance to biotic and abiotic stresses or accelerated flowering time and methods for producing the same
WO2007029270A2 (en) Abiotic stress tolerant gene from avicennia marina encoding a protein
EP2357239A1 (en) Methods and means for a selectable marker system in plants

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12761219

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12761219

Country of ref document: EP

Kind code of ref document: A1