WO2013166996A1 - 一种提高植物抗逆境能力的基因及其用途 - Google Patents

一种提高植物抗逆境能力的基因及其用途 Download PDF

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WO2013166996A1
WO2013166996A1 PCT/CN2013/075532 CN2013075532W WO2013166996A1 WO 2013166996 A1 WO2013166996 A1 WO 2013166996A1 CN 2013075532 W CN2013075532 W CN 2013075532W WO 2013166996 A1 WO2013166996 A1 WO 2013166996A1
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seq
plants
polypeptide
stress
polynucleotide
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PCT/CN2013/075532
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French (fr)
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张鹏
安冬
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中国科学院上海生命科学研究院
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    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention is in the field of biotechnology and botany; more specifically, the present invention relates to a gene for improving plant resistance to stress and uses thereof. Background technique
  • Plant stress resistance is the ability of plants to fight against adverse environments, such as drought, cold, and salt. Under natural conditions, due to various geographical locations, climatic conditions, and human activities, various adverse environments have been created, which exceed the range that plants can grow normally and develop, causing damage or even death. These environments that cause damage to plants are called stress or stress. The adaptability and resistance of plants to adverse environments is resistance or resistance.
  • adversity There are many types of adversity, including physical, chemical, and biological factors, which can be divided into two categories: biological stress and non-native stress.
  • the abiotic stresses that have an important impact on plants are mainly water (drought and drowning), temperature (high and low temperature), and saline-alkali physical and chemical stresses.
  • Low temperature stress is one of the main obstacle factors affecting the normal growth of plants.
  • researchers in the field have conducted more research on the cold resistance of plants, and explored the identification methods of cold resistance of plants.
  • the relationship between physiological and biochemical mechanisms of cold resistance and physiological and biochemical indexes of plants and cold resistance of plants are discussed.
  • an isolated polypeptide is provided, the polypeptide being selected from the group consisting of -
  • amino acid sequences shown by SEQ ID NO: 3 or SEQ ID NO: 4 e.g., 1-50, preferably 1-20, more preferably 1-10, more preferably 1
  • polypeptide is a polypeptide of the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4;
  • the polypeptide is relative to the amino acid sequence of SEQ ID NO: 3, the amino acid sequences of positions 40-53, 56-1, 12, and 114-1 are unchanged, and the other positions are one or more (such as 1-50). , preferably 1-20, more preferably 1-10, More preferably 1-5, such as 2, 3) amino acid residue substitutions, deletions or additions, and having the function of improving the plant's ability to resist stress, (a) derived polypeptide; or
  • the polypeptide is relative to SEQ ID NO: 4, positions 52-65, 68-124, 126-129 amino acid sequence unchanged and other positions pass one or more (such as 1-50, preferably 1-20, more preferably 1-10, more preferably 1-5, such as 2, 3) amino acid residue substitutions, deletions or additions, and have the function of improving the ability of plants to resist stress (b) a derived polypeptide.
  • the plant is selected from the group consisting of, but not limited to, Euphorbiaceae, Convolvulaceae, Solanaceae, Cruciferae, Gramineae, Woody plants.
  • the plant is selected from the group consisting of cassava, rubber, ramie, and sassafras of the family Euphorbiaceae; or the sweet potato into the sweet potato; or the potato of the Solanaceae; or the rice, wheat, barley, and corn of the grass family , sorghum, etc.; or Arabidopsis thaliana.
  • an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:
  • the polynucleotide encodes a polypeptide of the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.
  • polynucleotide in a preferred embodiment, the polynucleotide:
  • nucleotide sequence is as shown in SEQ ID NO: 1;
  • a vector comprising the polynucleotide is provided.
  • a genetically engineered host cell comprising the vector is provided.
  • a method of making a plant comprising transferring the polynucleotide into a plant is provided.
  • the method comprises:
  • step (1) (2) contacting a plant cell or tissue or organ with Agrobacterium in step (1) to transfer the polynucleotide into a plant cell and integrate it into the chromosome of the plant cell;
  • step (3) Regenerating the plant cell or tissue or organ in step (3).
  • a plant comprising the polynucleotide of any of the preceding claims is provided.
  • a method of preparing the polypeptide comprising - (a) cultivating the host cell under conditions suitable for expression; (b) isolating the polypeptide from the culture.
  • polypeptide or polynucleotide encoding the polypeptide for enhancing a plant's ability to resist stress or to prepare a substance which enhances the ability of a plant to resist stress.
  • the stress is a cold (stress) environment, a high salt (stress) environment, a drought (stress) environment, an oxidative (stress) environment, and an infiltration (stress) environment.
  • polypeptide or polynucleotide encoding the polypeptide is further used to:
  • SOD superoxide dismutase
  • CAT catalase
  • CBF signaling pathway genes such as FAD7, COR47 and GolS3
  • biomass eg fresh weight or dry weight
  • a cold environment eg low temperature environment around 10 °C
  • a method of increasing the ability of a plant to resist stress comprising: increasing the expression or activity of the polypeptide in the plant.
  • an agonist of the polypeptide or a gene encoding the same is provided.
  • a promoter that is induced by stress is provided, the promoter being selected from the group consisting of:
  • the promoter is selected from the group consisting of:
  • nucleotide sequences of 574-579, 716-720, 130-133, 417-420, 284-289, and 649-654 are not related to the nucleotide sequence of SEQ ID NO: 5. And other positions pass one or more (such as 1-50, preferably 1-20, more preferably 1-10, more preferably 1-5, such as 2, 3) bases a polynucleotide formed by substitution, deletion or addition, and having a function of inducing expression of a gene of interest under adverse conditions; or
  • a three-base substitution, deletion or addition of a base, and a polynucleotide having a function of inducing expression of a gene of interest under adverse conditions are provided.
  • the use of the promoter is provided for inducing expression of a gene of interest under adverse conditions.
  • a construct is provided, the construct comprising the promoter that is induced by stress.
  • the promoter downstream of the stress-inducible expression contains at least one multiple cloning site (eg, a cleavage site), and the promoter that is induced to be induced by the stress can be An operative linkage for insertion of a gene of interest.
  • the construct is an expression vector.
  • the construct contains the following operably linked elements:
  • the promoter and the gene of interest.
  • the gene of interest is a foreign gene.
  • the gene of interest is a structural gene.
  • the gene of interest encodes a protein having a specific function.
  • the gene of interest is located downstream of the promoter and is less than 2000 bp from the promoter (preferably, less than l OOObp; more preferably, less than 500 bp; most preferably, less than 300bp).
  • Figure 1 shows the results of a Southern blot showing that there may be two copies of the cassava CBF gene.
  • Figure 2 shows the alignment of the MeCBF1 and MeCBF2 nucleotide sequences (A) and MeCBFl and B MeCBF2 protein sequences.
  • NLS, AP2 and DSW are CBF conserved sequences.
  • Figure 3 shows the changes in the expression level of MeCBF1 under cold stress (A) and other stress conditions (B), and the tissue-specific expression of MeCBF1 (C).
  • GA-cold refers to the gene expression after 3 hours of GA pretreatment for 3 h and then treatment at 4 °C for 3 h.
  • Figure 4 shows the changes in the expression level of MeCBF2 under cold stress (A) and other stress conditions (B), and the tissue-specific expression of MeCBF2 (C).
  • GA-cold refers to the gene expression after 3 hours of GA pretreatment for 3 h and then treatment at 4 °C for 3 h.
  • Figure 5A shows the MeCBF1 and MeCBF2 onion epidermal cell localization assays.
  • Figure 5B shows the protoplast localization of MeCBF1 and MeCBF2 tobacco.
  • Figure 6 shows that MeCBF1 overexpresses Arabidopsis thaliana with enhanced cold tolerance.
  • Wild type Col-0 and 3 single copies Seeds expressing homozygous lines (SMl-13-3, SM1-14-19, SMl-19-10) were surface-sterilized and plated on minimal medium. After 4 days of vernalization at 4 ° C, they were placed in horizontal and vertical cultures in a 22 ° C and 10 ° C light incubator. The growth state of the seedlings was observed after a period of cultivation.
  • Figure ⁇ shows that MeCBF1 overexpressing Arabidopsis thaliana has a good growth under low temperature conditions.
  • Figure 8 shows the cold tolerance of Arabidopsis thaliana and wild-type Arabidopsis thaliana overexpressing meCBF1 at low temperature.
  • SM1-19 and SM1-17 are two T2 plants.
  • Figure 9 shows the anti-cold ability of Arabidopsis thaliana and wild-type Arabidopsis thaliana overexpressing MeCBF1 in a plate experiment.
  • Figure 10 shows that the normally germinated SM1 transgenic Arabidopsis thaliana is significantly salt tolerant compared to the wild type.
  • Figure 1 1 shows the cold-induced expression of the MeCBF1 promoter.
  • Figure 12 shows the growth state of BL21 (pET32a-MeCBF1) and BL21 (pET32a) recombinant prokaryotic expression strains on a resistance screening medium.
  • Figure 13 shows that MeCBF1 has a specific DNA binding activity.
  • the upper part of A is a partial structural diagram of the recombinant vector pGAO424-MeCBFl.
  • the middle middle figure in A is a partial structural diagram of the recombinant vector pHISi-3wDRE-HS/3wDRE-HA.
  • the lower part of A is a schematic diagram of the partial structure of the recombinant vector pHISi-3mDRE-HS2/3mDRE-HA2.
  • B shows the growth status of yeast cells after transfer to different recombinant vectors.
  • Figure 14 shows that the C-terminus of MeCBF1 has transcriptional activation activity.
  • Panel A is a schematic representation of some of the components of each recombinant vector used in the yeast hybrid system.
  • Panel B shows the color development results of each yeast transformant.
  • Figure 15 shows that MeCBF1 overexpressing Arabidopsis plants has higher salt tolerance.
  • Col-0 is wild type, and the other three lines (14-19, 19-10, 13-3) are pCAMBIA 1301-355:: MeCBFl independent homozygous strain system.
  • Figure 16 shows that MeCBF1 overexpressing Arabidopsis plants (SM1- 14-19, SM1-19-10 SM1-13-3) has a lower Na + concentration and a higher K + concentration.
  • FIG 17 shows that MeCBF1 overexpressing Arabidopsis plants (SM1- 14-19, SM1-19-10 SM1-13-3) has significant resistance to osmotic stress.
  • FIG. 18 shows that MeCBF1 overexpressing Arabidopsis plants has strong drought tolerance.
  • Figure 19 shows that MeCBF1 overexpressing Arabidopsis plants (SM1- 14-19, SM1-19-10 SM1-13-3) has significant antioxidant stress.
  • Figure 20 shows that MeCBF1 overexpresses Arabidopsis plants (SM1- 14-19, SM1-19-10 SM1-13-3) with a low accumulation of 3 ⁇ 40 2 content.
  • Figure 21 shows that MeCBF1 overexpressing Arabidopsis plants (SM1- 14-19, SM1-19-10 SM1-13-3) have higher SOD (A) and CAT enzyme activities (B) under low temperature treatment conditions.
  • Figure 22 shows Southern Blotting detection of the copy number of MeCBF1 overexpressing transgenic cassava. Lanel and Lane 2 proved that the corresponding plants were single-copy strains (SM1-1, SM1-2), and Lane3 proved that the corresponding plants were multi-copy strains (SMl-3).
  • Figure 23 shows the expression of CBF-target genes induced by MeCBF1 overexpressing cassava plants.
  • Figure 24 shows an increase in the expression level of MeCBF1 protein in MeCBF1 overexpressing cassava plants.
  • FIG 25 shows that MeCBF1 overexpressing cassava plants have higher resistance to low temperature when cultured in medium.
  • Figure 26 shows that MeCBF1 overexpressing cassava plants showed high resistance to low temperature when grown in the field.
  • Figure 27 shows that MeCBF1 overexpressing cassava field seedlings has higher chlorophyll (A) and proline (B) contents.
  • Figure 28 shows that MeCBF1 overexpressing cassava plants has a high ability to resist oxidative stress.
  • Figure 29 shows that MeCBF1 overexpressing cassava plants have a higher chlorophyll content.
  • FIG. 30 shows that MeCBF1 overexpressing cassava plants has strong resistance to water loss.
  • Figure 31 shows that MeCBF1 overexpressing cassava plants has a strong rehydration capacity.
  • FIG. 32 shows that MeCBF1 overexpressing cassava plants has strong resistance to water loss.
  • Figure 33 shows that MeCBF1 overexpressing cassava plants have strong drought resistance.
  • Figure 34 shows MeCBF1 overexpressing the cassava field phenotype.
  • the inventors have extensively studied for the first time to isolate a new useful gene for regulating plant resistance to stress.
  • the gene of the present invention can be excellently applied to the improvement of plant varieties, and the plant resistance to stress is enhanced.
  • the invention has important significance for prolonging the planting time, expanding the planting area (such as planting northward), and increasing plant yield.
  • the present invention has been completed on this basis.
  • the plant (or crop) suitable for use in the present invention as long as it is suitable for performing a gene transformation operation, or is suitable for a gene knockout operation such as various crops, flower plants, or forestry plants. Wait.
  • the plant may be, for example, without limitation: a dicot, a monocot, or a gymnosperm.
  • the plants include, but are not limited to: wheat, barley, rye, rice, corn, sorghum, beets, apples, pears, plums, peaches, apricots, cherries, strawberries, raspberries, blackberries, beans , lentils, peas, soybeans, rapeseed, mustard, poppy, olean, sunflower, coconut, castor oil plant, cocoa beans, peanuts, gourd, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, grape Pomelo, spinach, chicory, asparagus, cabbage, Chinese cabbage, pakchoi, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugar cane, tea, pepper, vine , ramie, bananas, natural rubber trees and ornamental plants.
  • the "plant” or “crop” includes but is not limited to: Euphorbiaceae (E ⁇ /wr aceae plant, Convolvulaceae, Solanaceae, Cruciferae, Gramineae, Woodyaceae) More preferably, the Euphorbiaceae plant includes but is not limited to: Esculenta), rubber, ramie, black mullet, etc.; said convolvulaceae plants include but are not limited to: sweet potato; said Solanaceae plants include, but are not limited to: potatoes; said grasses The materials include, but are not limited to, rice, wheat, barley, corn, sorghum, etc.; or the cruciferous plants include, but are not limited to, Arabidopsis thaliana.
  • Euphorbiaceae E ⁇ /wr aceae plant, Convolvulaceae, Solanaceae, Cruciferae, Gramineae, Woodyaceae
  • the Euphorbiaceae plant includes but is not limited to: Esculenta), rubber
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • an isolated polypeptide that increases the ability of a plant to resist stress means that the MeCBF protein is substantially free of other proteins, lipids, carbohydrates or other naturally associated with it. Other substances.
  • One skilled in the art can purify the MeCBF protein using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel.
  • the MeCBF protein may be a MeCBF1 protein and a MeCBF2 protein.
  • polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • polypeptides of the invention may be naturally purified products, either chemically synthesized or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells).
  • the polypeptide of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol.
  • Polypeptides of the invention may also or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of the MeCBF protein.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the MeCBF protein of the present invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example a polypeptide formed by fusion of a polyethylene glycol) or O) an amino acid sequence fused to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein) ).
  • conservative or non-conservative amino acid residues preferably conservative amino acid residues
  • substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a
  • MeCBF protein refers to a polypeptide having the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 which has the ability to enhance the resistance of plants or to increase the sensitivity of plants to salts.
  • the term also encompasses variant forms of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4, which have the ability to increase the resistance of the plant to enhance the sensitivity of the plant to salt.
  • variants include (but are not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, still more preferably 1 -8 or 1-5) amino acid deletions, insertions and/or substitutions, And adding one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and/or the N-terminus.
  • amino acids usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, still more preferably 1 -8 or 1-5) amino acid deletions, insertions and/or substitutions
  • adding one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and/or the N-terminus are examples of the function of the protein.
  • the term also encompasses active fragments and active derivatives of the MeCBF protein.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA capable of hybridizing to MeCBF protein DNA under high or low stringency conditions, And a polypeptide or protein obtained using an antiserum against the MeCBF protein.
  • the invention also provides other polypeptides, such as fusion proteins comprising a MeCBF protein or a fragment thereof. In addition to the nearly full length polypeptide, the present invention also encompasses soluble fragments of the MeCBF protein.
  • the fragment has at least about 20 contiguous amino acids of the MeCBF protein sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100.
  • a contiguous amino acid typically at least about 20 contiguous amino acids of the MeCBF protein sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100.
  • the invention also provides analogs of MeCBF proteins or polypeptides.
  • the difference between these analogs and the native MeCBF protein may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as by radiation or exposure to a mutagen to produce random mutagenesis, or by site-directed mutagenesis or other techniques known in molecular biology.
  • Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, ⁇ -amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • MeCBF protein conservative variant polypeptide means up to 20, preferably up to 10, more preferably up to 5, compared to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. Preferably, at most 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the present invention also provides a polynucleotide sequence encoding the MeCBF protein of the present invention or a conservative variant polypeptide thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genome
  • DNA or synthetic DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or may be a degenerate variant.
  • degenerate variant in the present invention refers to a protein encoding a protein having SEQ ID NO: 3 or SEQ ID NO: 4, but with SEQ ID NO: 1 or SEQ ID NO: 2. A sequence of nucleic acid sequences that differ in sequence.
  • Polynucleotides encoding the mature polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally Additional coding sequences) as well as non-coding sequences.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may also include a polynucleotide encoding additional and/or non-coding sequences.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C ; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 °C, etc.; or (3) at least 80% identity between the two sequences Preferably, the hybridization occurs at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of SEQ ID NO: 3.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate encoding MeCBF A polynucleotide of a protein.
  • the full-length nucleotide sequence of the MeCBF protein of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered using the vectors or MeCBF protein coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.
  • polynucleotide sequences of the present invention can be used to express or produce recombinant MeCBF proteins by conventional recombinant DNA techniques. Generally there are the following steps:
  • the MeCBF protein polynucleotide sequence can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art.
  • any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the MeCBF protein-encoding DNA sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or kanamycin or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or kanamycin or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
  • Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.
  • an enhancer sequence is inserted into the vector.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, used as a promoter to enhance transcription of a gene.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 . Conversion can also be carried out by electroporation if desired.
  • the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, rice immature embryo transformation method and the like.
  • Agrobacterium transformation or gene gun transformation such as leaf disc method, rice immature embryo transformation method and the like.
  • plants can be regenerated by conventional methods to obtain plants having altered traits.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties, if desired. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the recombinant MeCBF protein has many uses. For example, for screening antibodies, polypeptides or other ligands that promote or counteract the function of the MeCBF protein. Screening a polypeptide library with the expressed recombinant MeCBF protein can be used to find valuable polypeptide molecules that inhibit or stimulate the function of the MeCBF protein.
  • a part or all of the polynucleotide of the present invention can be immobilized as a probe on a microarray or a DNA chip (also referred to as "gene chip") for analyzing differential expression analysis of genes in tissues. Transcription products of MeCBF protein can also be detected by in vitro amplification of RNA-polymerase chain reaction (RT-PCR) using MeCBF protein-specific primers.
  • RT-PCR RNA-polymerase chain reaction
  • the invention also relates to a method of improving a crop comprising increasing the expression or protein activity of a MeCBF gene in said plant. This allows the plant to have superior resistance to stress (including: cold, high salt or dry environments).
  • MeCBF gene Methods for increasing the expression of the MeCBF gene are well known in the art. For example, you can carry the MeCBF code by transferring it
  • the expression construct of the gene allows the plant to overexpress MeCBF; or can enhance the expression of the MeCBF gene by driving with a strong promoter; or by an enhancer (such as the first intron of the rice waxy gene, the first intron of the Actin gene, etc.)
  • strong promoters suitable for use in the methods of the invention include, but are not limited to, the 35S promoter, the Ubi promoter of rice, maize, and the like.
  • RNAi RNA interference
  • knockout RNA silencing
  • a method for obtaining a plant having high expression of MeCBF is as follows:
  • step (1) (2) contacting the plant cell or tissue or organ with the Agrobacterium in step (1), thereby transferring the MeCBF protein DNA coding sequence into the plant cell and integrating it into the chromosome of the plant cell;
  • the method can be carried out by any suitable conventional means including reagents, temperature, pressure conditions and the like.
  • the invention also encompasses agonists of the MeCBF protein or a gene encoding the same. Because of the agonist of MeCBF
  • MeCBF The activity or expression of MeCBF, therefore, the agonist of MeCBF can also improve the plant's resistance to stress by affecting MeCBF, thereby achieving the purpose of trait improvement.
  • the agonist of MeCBF refers to any substance which can increase the activity of MeCBF, maintain the stability of MeCBF, promote the expression of MeCBF, prolong the effective action time of MeCBF, or promote the transcription and translation of MeC ⁇ P.
  • the invention is useful as a substance for improving the ability of plants to resist stress.
  • a MeC ⁇ P1 gene having a genomic sequence as shown in SEQ ID NO: 1 encoding a protein of 219 amino acids (SEQ ID NO: 3) is provided.
  • the MeCBF1 gene can provide a new way for plant resistance to stress improvement, and thus has great application prospects.
  • M e CBF2 gene genomic sequence as SEQ ID NO: 2 as shown, encodes a protein containing 231 amino acids (SEQ ID NO: 4).
  • the MeCAF2 gene can provide a new way for plant resistance to stress improvement, and thus has great application prospects. Promoter and its application
  • promoter or “promoter region (domain)” refers to a nucleic acid sequence that is typically present upstream (5') of the coding sequence of the gene of interest and is capable of directing transcription of the nucleic acid sequence into mRNA.
  • the promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.
  • the promoter or promoter region includes a variant of a promoter which may be a naturally occurring allelic variant or a non-naturally occurring variant. The variants include substitution variants, deletion variants, and insertion variants.
  • operably linked refers to the function of two or more nucleic acid regions or nucleic acid sequences.
  • Sexual spatial arrangement For example: The promoter region is placed at a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region such that the promoter region is "operably linked" to the nucleic acid sequence.
  • the invention provides a promoter, wherein the promoter is selected from the group consisting of:
  • a polynucleotide which is capable of hybridizing with a (1) defined polynucleotide sequence under stringent conditions and which has a function of inducing expression of a gene of interest under adverse conditions.
  • stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 °C, etc.; or (3) at least 80% identity between the two sequences Preferably, the hybridization occurs at least 90% or more, more preferably 95% or more. Moreover, the hybridizable polynucleotide also has a function of inducing expression of the gene of interest under adverse conditions.
  • Hybridization of polynucleotides is a technique well known to those skilled in the art, and the hybridization characteristics of a particular pair of nucleic acids indicate their similarity or identity. Accordingly, the present invention also relates to hybridization to the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6 and having at least 50%, preferably at least 60%, more preferably at least 70% between the two sequences. More preferably, at least 80%, more preferably at least 85%, more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99%) of the identity of the polynucleotide.
  • the promoter of the present invention induces expression of a gene of interest under adverse conditions, for example, inducing a gene of interest to be expressed in a cold environment.
  • the inventors have found that under the guidance of the promoter of the present invention, Me ⁇ or a gene can be specifically expressed under stress induction.
  • the promoter of the present invention can be operably linked to a gene of interest which can be exogenous (heterologous) relative to the promoter.
  • the gene of interest may generally be any nucleic acid sequence (preferably a structural nucleic acid sequence), preferably encoding a protein having a specific function.
  • the promoter of the present invention may also be operably linked to a modified gene sequence of interest which is exogenous (heterologous) relative to the promoter.
  • the gene of interest can be modified to produce a variety of desirable properties.
  • the gene of interest can be modified to increase expression, to alter post-translational modifications (such as phosphorylation sites), to transfer translation products to extracellular, to improve protein stability, to insert or delete cellular signals, and the like.
  • promoters and genes of interest can be designed to downregulate specific genes. This is typically accomplished by ligating the promoter to the sequence of the gene of interest, which sequence is directed in antisense orientation. Those of ordinary skill in the art are familiar with such antisense techniques. Any nucleic acid sequence can be modulated in this manner.
  • any of the aforementioned promoter and gene sequences of interest may be included in the construct, more specifically as a recombinant vector.
  • the recombinant vector generally comprises an operably linked (usually from 5' to 3' direction): a promoter that directs transcription of the gene of interest, and a gene of interest.
  • the recombinant vector may also include a 3 'transcription terminator, 3 'polymerization, if desired Nucleotide signals, other non-translated nucleic acid sequences, transport and targeting nucleic acid sequences, resistance selection markers, enhancers or operators.
  • the gene of interest is located downstream of the stress-inducible promoter and is less than 2000 bp from the promoter (preferably, less than l OOObp; more preferably, less than 500 bp; most preferably, Less than 300bp).
  • the recombinant vector may contain one or more other promoters in addition to the promoter of the present invention.
  • Such other promoters are, for example, tissue-specific, constitutive or inducible.
  • a vector comprising the appropriate promoter and gene of interest described above can be used to transform a suitable host cell to enable expression of the protein.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, 2002, or according to the manufacturer's recommended conditions. . Percentages and parts are by weight unless otherwise stated.
  • Cassava is a tropical and subtropical crop that is very sensitive to low temperatures.
  • the inventors In order to study the abiotic stress regulation mechanism of cassava (Manihot esculenta Crantz), the inventors first tried to clone the cassava-like sequence based on the CBF conserved sequence-AP2 domain, and then obtained the full-length sequence by 5' and 3'-RACE, but this The method was not successful.
  • the present inventors screened a cDNA library of mixed tissue of leaf and root of cassava after a drought treatment (full-length cDNA RIKEN library sequence reference Sakurai T, Plata G, Rodriguez-Zapata F, Seki M, Salcedo A, Toyoda A, Ishiwata A, Tohme J, Sakaki Y, Shinozaki K et ah Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response.
  • CBF is mainly present in various species in the gene family
  • this sequence is used as a probe to digest the cassava TMS60444 (Swiss Federal Institute of Technology) genome through three restriction enzymes (EcoRI, HindllK Xball).
  • the number of copies of the gene was examined by blotting and the results showed that the gene might have two copies ( Figure 1).
  • the homologous sequence, the present invention is also to another MeCBF cloned gene, designated M e C3 ⁇ 4F (cloning: We MeCBFl cloning primers, the PCR sequences obtained after sequencing the carrier is connected to the T- 18, the sequencing results It was found that the sequence was mainly MeCBF1, but there was another sequence.
  • MeCBF protein is involved in the cassava stress response pathway.
  • the present inventors cold-treated a cassava (TMS60444) seedling for one month at 4 °C for 0 hr, 2 hr, 4 hr, 9 hr, 25 hr, and detected the expression of MeCBF1 by Realtime-PCR (primer sequence: sense primer: TTCTTGGTCGCCTGATACCTG, antisense primer) : GCTTCTTGGGATAGCTGGAAG), MeCB ⁇ was found to be up-regulated at 2 hr, then decreased slightly after 4 hr, and then increased to 25 hr after 9 hr (this experiment has been repeated 3 times). The results indicate that MeCBF is induced by cold induction, suggesting that it participates in the cold signaling pathway. 3A.
  • MeCBF1 is down-regulated at 4 hr, suggesting that this gene is feedback-regulated by a certain mechanism, that is, if the gene is induced to reach a certain amount, the plant will initiate a feedback mechanism to inhibit the gene from continuing to up-regulate.
  • cold stress still exists, so the gene will continue to be up-regulated in order to exert anti-cold effect, and at this time it will be up-regulated to a higher degree than the first.
  • GA pretreatment significantly inhibits the expression of Me ⁇ gene, which may be a certain balance between plant growth and resistance stress: If the plant needs to thrive, it will reduce the tolerance to harsh environment, and in order to improve the ability to resist stress, it must be At the expense of normal growth; in other words, under appropriate environmental conditions, plants will complete a normal physiological cycle, while in an adverse environment, plants will survive at the expense of growth to resist the surrounding discomfort.
  • tissue-specific expression of this gene the inventors extracted RNA from different tissues of 4-5 strains of cassava seedlings in greenhouses, including very young apical buds (AP: apical buds) and young leaves (YL).
  • MeC3 ⁇ 4F2 under cold stress, various stress conditions and tissue-specific expression (primer: sense primer: atggatgttttctctcactaccc, antisense bow
  • Example 3 Localization of MeCSF Gene
  • MeCBF2-GSPl-l CGAGGAGCACTGCAACCGTGATCAGAT
  • MeCBF2-GSPl-2 GGGTC AGGGT AGTGAGAAAAC ATCC
  • MeCBF2-GSP2-l ATATACTCTATCTCTT TGGGCAGCTGT
  • MeCBF2-GSP2-2 ATGGA
  • MeCBFl promoter from the MeCBF2 promoter
  • ABRE is similar
  • K represents T or G
  • M represents A or C
  • B represents T or C or G
  • Y represents C or T
  • N represents an arbitrary nucleotide
  • V represents A or [or 0.
  • the inventors constructed the pA7-M e C ⁇ F7::GFP and pA7-MeC3 ⁇ 4F ::GFP vectors, and the pA7-GFP empty vector (purchased from TAKARA) as a negative control.
  • the Arabidopsis thaliana gene was used as a positive control to construct a pA7-AC ⁇ P4::GFP positive clone.
  • the vector construction method was as follows: The forward and reverse primers containing the Xhol and 3' ends containing the Spel restriction site were designed, and the target bands were obtained by PCR using the cassava and Arabidopsis genomes respectively.
  • pMD 18 T vector purchased from TAKARA
  • the target fragment was inserted into the T vector by double digestion with Xhol and Spel.
  • the clones with the correct sequencing were selected. During the sequencing process, one clone was inserted instead of MeCBF1, but MeCBF2, so the MeCBF2 transient expression vector was constructed at the same time.
  • the target band was obtained by Xhol and Spel double digestion and pA7-GFP (purified from Invitrogen).
  • the company which has been digested with Xhol and Spel, was ligated with T4 ligase. After the adaptor was transformed into Top 10 E. coli (purchased from Invitrogen), the monoclonal antibody was picked and verified by Xhol and Spel. Can be used for onion positioning experiments.
  • the primer sequences are as follows:
  • MeCBF1 and MeCBF2 green GFP fluorescence signals were mainly located in the nucleus except for the cell membrane, which showed similar cellular localization to AtCBF4 of Arabidopsis thaliana, while the fluorescent signal of pA7-GFP empty vector was located throughout the cell. (Fig. 5A). Therefore, it is believed that MeCBF1 and MeCBF2 are nuclear localization proteins.
  • MeCBF l pCAMBIA1301 -35S: : MeCBFl , abbreviated as SM1
  • SM1 MeCBF l
  • the construction of pCAMBIA1301 -35S: :MeCBF1 and the preparation of transgenic Arabidopsis are as follows: Design primers (atggatgttttctctcactaccc (SEQ ID NO: 1 1) and ttatacagaaaaactccataatgg (SEQ ID NO: 12)) according to the known MeCBF1 nucleotide information.
  • the target band (Example 1 method) was amplified by PCR using cold-treated TMS60444 cassava cDNA as a template. After the gel was recovered, it was ligated to the pMD 18 T vector vector. After the adaptor was transformed into Top 10 E. coli competent state, the monoclonal antibody was picked and then subjected to EcoRI. And Hindlll digestion confirmed that the target band has been inserted into the T vector and sent to the sequencing, and the sequence is Sall-ATG-MeCBF l -TAA-Xbal clone.
  • the clone was digested with Sail and Xbal and inserted into the 35 S promoter at the end of the engineered pCAMBIA1301 -35S (modification method: commercialized pCAMBIA1301 vector (purchased from Invitrogen), the other end
  • the NOS terminator X has been ligated by Sail and Xbal double-cutting.
  • the ligation solution is transformed into the competent state of Top l O E. coli, the monoclonal is picked, and then confirmed by Xhol and Spel digestion, the correct vector is the final vector, and the vector is transformed into the agricultural Bacillus sp. GV3101 (purchased from Invitrogen) can be used for Arabidopsis transformation after verification.
  • Arabidopsis transformation uses the classical flower dip method, which has been widely used.
  • Seeds of wild-type Col-0 and overexpressing lines (SMl-13-3, SM1-14-19, SM1-19-10) were surface-sterilized and plated on minimal medium. After 4 days of vernalization at 4 °C, the cells were cultured horizontally or vertically at 22 °C and 10 °C, and it was found that the growth state of SM1 strain was significantly better than that of wild type, as shown in Fig. 6. The fresh weight, dry weight, leaf length and leaf width of 20 seedlings of 30 days (d) were measured.
  • the present inventors analyzed the domain of the MeCBF1 protein, and the main domains of MeCBF1 are NLS (relative to the amino acid sequence of SEQ ID NO: 3, positions 40-53), AP2/EREBP (relative to the amino acid sequence of SEQ ID NO: 3, Blocks 56-1 12), DSW (relative to the amino acid sequence of SEQ ID NO: 3, positions 1 14-1 17), see Figure 2B, these domains are critical sites for proteins to exert anti-stress effects. Based on the above analysis, the inventors established a plurality of variant sequences of MeCBF1 protein, which are as follows:
  • MeCBF1-Ml variant Based on the MeCBF1 protein (SEQ ID NO: 3) sequence, in which the amino acid at position 11 is changed from L to I, the MeCBF1-Ml variant is obtained.
  • MeCBF1 protein SEQ ID NO: 3
  • amino acid at position 210 is changed from A to V
  • a MeCBF1-M2 variant is obtained.
  • MeCBF1-M3 variant Based on the MeCBF1 protein (SEQ ID NO: 3) sequence, in which the amino acids 1-3 were deleted, the MeCBF1-M3 variant was obtained.
  • MeCBF1-M4 variant Based on the MeCBF1 protein (SEQ ID NO: 3) sequence, in which amino acids 210-214 were deleted, the MeCBF1-M4 variant was obtained.
  • MeCBF1 protein CSEQ ID NO: 3 Based on the MeCBF1 protein CSEQ ID NO: 3) sequence, wherein 4 amino acids ⁇ were added to the C-terminus, a MeCBF1- ⁇ 5 variant was obtained.
  • the CDS sequence of the MeCBF1 gene shown in SEQ ID NO: 1 was first cloned into the multiple cloning site of the pCAMBIA1301-35S vector to obtain a recombinant vector containing the CDS. Then, according to the sequence of the protein variant designed above, a base mutation or a base deletion or a base addition is performed on the base of the recombinant vector encoding the amino acid of the corresponding site by using a conventional site-directed mutagenesis technique to obtain a corresponding variant. Recombinant vector.
  • the recombinant vector constructed above was transferred into Agrobacterium and transformed into Arabidopsis thaliana to obtain Arabidopsis thaliana transgenic plants Ml-Linel Ml-Line2 ; M2-Linel, M2-Line2 ; M3-Linel M3-Line2 ; M4-Linel , M4-Line2 ; M5-Linel M5-Une2.
  • Verification by plate experiments Seedlings germinated on MS minimal medium for about 14 days were treated with l lhr at -5 °C for 5 days to observe plant traits. It was found that most of the transgenic seedlings were green and maintained good viability compared with the wild type (white leaves).
  • Example 5 salt tolerance of MeCBF1
  • SM1 transgenic plants on NaCl medium were transferred the transgenic and wild-type Arabidopsis seeds to the 100 mM NaCl medium after germination for 4 days on normal medium, and found that after 16 days of culture, The transgenic seedlings were more stretched and the growth status was better than the control (Fig. 10).
  • the above results demonstrate that the SM1 transgenic shoots that have been normally germinated have better salt tolerance than the wild type.
  • Example 6 MeCBF1 and MeCSF2 promoters induce expression under stress
  • the MeCBFJ promoter contains multiple stress-inducing elements, while MeC ⁇ FV mRNA is induced by stress.
  • the inventors constructed a MeCBF1 promoter: :GUS vector.
  • the vector construction method was as follows: Primer was designed and Pstl and Ncol restriction sites were added at both ends, and the TMS60444 cassava seedling genome was used as template PCR to obtain the target band after the tapping recovery connection pMD 18T-vector (purchased from TAKARA), and then transformed into E.
  • MeCBFl -P-P GCCTGCAGTCAAAGTATATTTCGACTTC (SEQ ID NO: 13);
  • MeC5 7-P-N TACCATGGAGAAGTAGAGAGTGTGAGTG (SEQ ID NO: 14).
  • the vector constructed above was transformed into Arabidopsis thaliana, and at least 3 T3 homozygous lines were obtained.
  • Transgenic Arabidopsis seedlings were used for GUS staining after 4 °C treatment for different time, and GUS staining of Arabidopsis thaliana seedlings without cold treatment was used as a pair of gossip 1 *. °
  • the promoter of MeCBF1 (SEQ ID NO: 5) and the promoter of MeCBF2 (SEQ ID NO: 6) have multiple stress-related cis-acting elements.
  • a MeCBF1-based promoter (SEQ ID NO: 5) sequence in which the 6th base is mutated from C to C, the 25th base is mutated from C to G, and the 318th base is mutated from T to A, obtaining M e CS ⁇ 7-P l variant.
  • the 857th base was changed from T to A, and the 495-497 ATA was deleted, and the MeCB ⁇ -P2 variant was obtained.
  • Me(3 ⁇ 4F7 promoter) SEQ ID NO: 5
  • the 5' end plus the base AGTCC and the 3' end lacking 4 bases to obtain the MeCBFl -?3 variant.
  • the promoter based on MeCS CSEQ ID NO: 6 sequence, wherein the 40th base is mutated from A to T, the 243-246 base is deleted, and the 1465th is changed from C to G, and the MeCBF2-?4 variant is obtained.
  • a meCBF2-based promoter (SEQ ID NO: 6) sequence, wherein the 5' end adds 4 bases ATGG,
  • the 1576-1583 base is deleted, and the MeCBF2-?5 variant is obtained.
  • the above promoter variant was constructed into the MeCSW promoter::GUS vector, and the construction method was as follows: Primer was designed and both ends were added with Pstl and Ncol restriction sites, and the genome was used as template PCR to obtain the target band after the tapping recovery connection pMD 18T-vector (purchased from TAKARA), then transformed into E. coli competent state, and obtained positive clones. After sequencing, they were ligated into pCAMBIA1303 vector (obtained from CAMBIA) by Pstl and Ncol, and the ligation solution was transformed into E. coli to pick positive clones.
  • GV3101 Agrobacterium was transformed, and a positive clone was obtained, and then Arabidopsis thaliana was transformed according to the Arabidopsis transformation method.
  • the recombinant vector constructed above was transferred into Agrobacterium and transformed into Arabidopsis thaliana to obtain Arabidopsis transgenic plants PI-Line 1, P1-Line2; P2-Line 1 P2-Line2; M3-Line 1, P3-Line2 P4-Linel P4-Line2 ; P5-Linel P5-Line2.
  • the inventors constructed the MeCBF1 gene into the pET32a vector.
  • the primers required to construct the pET32a-M e C3 ⁇ 4F7 vector are as follows:
  • MeCBFl-EcoRl cggaattcATGGATGTTTTCTCTCTC (SEQ ID NO: 15)
  • MeCBFl -Hindlll ccaagcTTcTACAGAAAAACTCCAT (SEQ ID NO: 16)
  • the expression of the recombinant protein was induced by IPTG, and the growth of the recombinant strain (BL21) on LB medium containing different concentrations of NaCl and mititol was analyzed.
  • the specific method was as follows: Newly activated BL21 (pET32a-MeCBi ⁇ ) and control BL21 (pET32a) monoclonal were picked, inoculated into 5 ml of LB (Kan), and cultured overnight at 37 °C with shaking.
  • the overnight cultures were separately inoculated into LB (Kan) medium (10 ml) at a ratio of 1% until the OD was 0.5-0.8 (about 2 hr for culture), and the inducing agent 0.5 mmol/L IPTG was further cultured for 4 h and used for stress test.
  • Resistant LB plates containing different concentrations of NaCl and mannitol were taken, and the two strains were streaked on the plates, and cultured inverted for about 12 hours to observe the growth of the two bacteria.
  • MeCBF1 has obvious salt and drought resistance functions in the prokaryotic expression system.
  • the inventors also verified the variants of MeCBFl MeCBFl-Ml, MeCBFl-M2, MeCBFl-M3
  • MeCBFl-M4, MeCBFl -M5 have salt and drought resistance.
  • the coding genes were constructed into pET32a vector, transformed into BL21, and after the same culture as above, the bacteria were streaked on different concentrations of NaCl and mannitol resistant LB plates, and cultured in an inverted state to observe the growth of the bacteria. As a result, these bacteria were also able to grow normally, and the state was significantly better than that of the control BL21 (pET32a).
  • Example 8. MeCBF1 has specific DNA binding activity and transcriptional activation activity
  • MeCBF1 has DNA binding activity
  • the inventors firstly synthesized normal and mutated DRE/CRT sequences, which were named 3wDRE-HS/3wDRE-HA and 3mDRE-HS2/3mDRE-HA2, respectively.
  • the primer sequences are -
  • 3mDRE-HS2 GTGAATTCTAaatcaATTAaatcaATTAaatcaATTCTAGACT (SEQ ID NO: 19);
  • 3mDRE-HA2 AGTctagAATtgattTAATtgattTAATtgattTAGAATTCAC (SEQ ID NO: 20).
  • the positive and negative primer sequences were annealed into double strands by PCR, and named as wild type DRE/CRT and mutant DRE/CRT, respectively, and then ligated into pHISi vector (Clontech) by EcoRI and Xbal, and the recombinant vector construction was verified by sequencing. Correctly, recombinant vectors pHISi-3wDRE-HS/3wDRE-HA and pHISi-3mDRE-HS2/3mDRE-HA2 were obtained, respectively, as shown in Fig. 13A.
  • MeCBFl-Sail gagtcgaccTACAGAAAAACTCCATA (SEQ ID NO: 22), using the cassava genomic DNA as a template, the target fragment was amplified by PCR, ligated into the pMDT18-vector vector (purchased from TAKARA), and sequence correctness was verified by sequencing. Thereafter, it was digested with EcoRI and Sail into the pGAD424 vector (Clontech) to obtain a recombinant vector pGAD424-MeCB ⁇ , as shown in Fig. 13A.
  • the recombinant vectors pHISi-3wDRE-HS/3wDRE-HA and pHISi-3mDRE-HS2/3mDRE-HA2 were first transferred to a yeast strain (strain YM4271, purchased from Clontech) by applying different concentrations of 3 -AT screening pressure verified the background resistance of the transformants to 3-AT.
  • strain YM4271 purchased from Clontech
  • 3 -AT screening pressure verified the background resistance of the transformants to 3-AT.
  • transformants can be grown on His-deficient medium, but the addition of 15 mM 3-AT inhibits the growth of the bacteria, indicating that the obtained transformants have lower background resistance and can be used for further experiments.
  • the present inventors transformed the pGAD424 vector containing the gene of interest and the empty pGAD424 vector into two transformants containing the correct DNA sequence (normal DRE/CRT sequence) and the mutated DNA sequence (mutated DRE/CRT sequence), respectively.
  • the SD/-Le U and -Hi S medium were cultured for 2-3 days, and all clones were found to have clones, but if 15 mM of 3-AT was applied, only the pGAD424 vector containing the gene of interest and the pHISi vector of the correct DNA sequence were contained. Clones can be grown, and no other clones have grown clones, as shown in Figure 13B.
  • the above results indicate that MeCBF1 can interact with the correct DRE/CRT motif and cannot interact with the mutated DRE/CRT motif, which also demonstrates the specificity of this interaction.
  • MeCBF1 has transcriptional activation activity
  • the inventors first amplified full-length MeC ⁇ F7 (SEQ ID NO: 1 , full CDS), 5 '-end-MeCB ⁇ sequence (SEQ ID NO: 1, 1-l- 354 bp, MeCBF1-N) and 3'-MeC ⁇ FV sequence (SEQ ID NO: 1, 355-660 bp, MeCBF1-C), ligated into pMDT18-vector vector (purchased from TAKARA), correctly sequenced Thereafter, it was digested with EcoRI and B Sail into the pGBKT7 vector (purchased from Clontech), and then transformed into a yeast strain (; yeast strain: AH109, purchased from Clontech), respectively, and a pGBKT7 empty vector was used as a negative control.
  • the pCAMBIAlSOU. MeC ⁇ FV transgenic Arabidopsis seeds were disinfected and seeded on NaCl-containing medium. After 2 weeks of culture, the leaves of transgenic Arabidopsis plants were greenish and the number of leaves was high. Under high salt stress conditions, the seedlings were under high salt stress. The survival rate is high, therefore, the transgenic material is significantly resistant to salt than the wild type, as shown in Figure 15.
  • the inventors examined the Na + and K + concentrations of the aerial parts of the transgenic and wild type materials under salt stress conditions. As shown in FIG. 16 , it was found that the Na + concentration of the transgenic lines was lower than that of the wild type, and the K+ concentration was higher than that of the wild. High type. This result indicates that transgenic vaccines resist salt stress by lowering the above-ground Na + concentration and increasing the K+ concentration. Polyethylene glycol PEG treatment can induce osmotic stress and simulate drought stress. The present inventors disinfected pCAMBIABOl ⁇ .
  • MeC ⁇ FJ transgenic Arabidopsis thaliana and wild-type seeds on a normal medium and transferred to a medium containing 20% PEG two days after germination, and detected the overexpressed plants for osmotic stress. Whether it is resistant.
  • MeCBF1 overexpresses Arabidopsis plants Has a strong ability to resist osmotic stress.
  • DAB dyes are often used to detect the accumulation of 3 ⁇ 40 2 . Therefore, in the present example, the present inventors used DAB staining to detect the ability of MeCBF1 to overexpress Arabidopsis thaliana plants against oxidative stress.
  • the present inventors performed DAB staining after treating the seedlings grown on a normal medium for about 14 days at a low temperature (10 ° C) for 7 days.
  • DAB staining method is as follows: Add 10ml DAB staining solution to 50ml centrifuge tube, take a certain amount for dyeing leaves, vacuum for 10min; immerse the leaves in DAB liquid, place under light, observe color change at any time, dye 6-8h ; When coloring, pour off the staining solution, add 95% ethanol, and decolorize at 80 ° C for 10 min.
  • the H 2 0 2 content was determined as follows: l) lg leaf + 10 ml 0.1% pre-cooled TCA solution, ground on ice bath, lump slurry 12000 g, 15 min ; 2) 1 ml supernatant, add 1 ml lOOmM phosphate buffer (PBS) (pH 7.0), 2 ml KI (lmol/L) ; shake the hook, let stand for a while (10 min), measure the OD value at 390 nm (ultraviolet spectrophotometer); 3) Determine the content of 3 ⁇ 40 2 according to the standard curve.
  • the CAT enzyme and SOD enzyme activity assays are as follows:
  • Test agent enzyme dosage (ml) final concentration (colorimetric)
  • AO light absorption value of the light control tube
  • AS light absorption value of the sample tube
  • VT total volume of the sample liquid (ml)
  • VI sample amount (ml) when measured
  • F W sample fresh weight (g).
  • DAB staining result found that the wild-type and transgenic seedlings have been dyed brown, but compared with the wild type transgene staining shallow, FIG. 19, described at low temperature, the amount of accumulation of transgenic material 3 ⁇ 40 2 may be less than the wild type.
  • Plants have a mechanism of ROS clearance, in which superoxide dismutase (SOD) and catalase (CAT) play an important role. Since the transgenic vaccine has a lower content of 3 ⁇ 40 2 under low temperature treatment conditions, the inventors further examined the enzymatic activities of the two enzymes, and found that under normal conditions, wild type and transgenic plants have the same enzymatic activity, but at low temperatures. Under the treatment conditions, the enzyme activities of SOD and CAT in transgenic plants were higher than those in wild type, as shown in Figure 21. This result suggests that, due to the increased activity of these two enzymes, the H 2 0 2 content is lowered, and the transgenic seedlings have a higher ability to resist oxidative stress.
  • Example 11 Acquisition of MeCBF1 Overexpressing Cassava Plants
  • MeCBF1 To further verify the function of MeCBF1, the inventors transformed the same overexpression vector (pCAMBIAl 301 -35S:: MeCBFl, SMI for short) into cassava, and obtained three independent transgenic lines to verify MeCBF1 overexpression of transgenic cassava. Copy number, two single-copy lines (SM1-1, SM1-2) and one multi-copy line (SM1-3) were identified by Southern blotting, as shown in Figure 22.
  • CBF cold stress signaling pathway
  • FAD7 fatty acid desaturase
  • COR47 dehydrin protein
  • KIN1 antifreeze protein
  • COR15a localized to chloroplast, protective chloroplast
  • GolS3 inositol galactosidase 3
  • Rd29a COR6.6 (co Id regulated gene).
  • MeFAD7-F CCATGATTGTGGTCATGGGAGC (SEQ ID NO: 23);
  • MeFAD7-R GGTACCAGGGAAGTTTGTCCTC (SEQ ID NO: 24);
  • MeCOR47-F CTGAGGAGCACCACAACAAGG (SEQ ID NO: 25);
  • MeCOR47-R GGCGTAGCTACCTCTTCAGGC (SEQ ID NO: 26);
  • MeGolS3-F GGCTTACGTGACTTTCTTGGC (SEQ ID NO: 27);
  • MeGolS3-R CACTGTTGGCAGTAGCCGATC (SEQ ID NO: 28).
  • the present inventors prepared a polyclonal antibody against MeCBF1 by a conventional method using MeCBFl full-length protein as an antigen.
  • MeCBF1 protein in over-expressed cassava nuclear proteins in transgenic plants and wild-type controls were extracted.
  • Western blotting results showed that MeCBF1 protein was increased in transgenic cassava plants, and the number of single-copy plants increased more than multiple copies, with histone H3 as an internal reference, as shown in Figure 24.
  • the cassava tissue culture seedlings of the T1 generation for about 10 days were placed in a 4 °C light incubator for 10 days, and the culture was resumed. It was found that some of the leaves of the transgenic seedlings remained green, and the buds remained viable, while the control C3 leaves were all whitish, buds. The point loses vitality, and in subsequent cultures, the transgenic seedlings can continue to grow, while C3 does not, as shown in Figure 25. This result indicates that MeCBF1 overexpressed cassava material has high cold resistance, which also proves that MeCBF1 protein also has low temperature resistance to other species. To further verify the cold resistance of the transgenic material, the inventors planted the transgenic and control materials in the field to observe the effects of low temperature weather in nature.
  • 95% ethanol The chlorophyll (Chi) content was determined by overnight extraction and UV spectrophotometer. The chlorophyll content was determined as follows: Leaf soaking 95% alcohol was extracted in the dark for 12-24 h, until the leaves were white, and OD663 (nm) and OD646 (nm) were determined by spectrophotometry;
  • Proline extraction Take 0.5g of the differently processed shredded hooks (the dry sample is reduced according to the moisture content), place them in a large test tube, and add 5ml of 3% sulfosalicylic acid solution. The glass ball was capped and immersed in a boiling water bath for 10 min. After cooling to room temperature, centrifugation at 3000 rpm for 10 min;
  • C the concentration of proline in the extract g), obtained from the standard curve
  • Methyl viologen can induce plant oxidation, causing the chlorophyll content of leaves to decrease, showing whitening of leaves, which is a quantitative indicator.
  • This example used MV to treat cassava overexpressing plants to observe their ability to resist oxidative stress.
  • the MV staining method was as follows: Two sheets of filter paper were placed on a glass dish, 25 ml of lOOuM MV working solution was added, the leaves were placed in a liquid, and 20 leaves were placed under continuous light conditions to observe changes in the leaves.
  • the inventors measured the chlorophyll content of the leaves before and after MV treatment. The results showed that the chlorophyll content of the wild type was significantly reduced after MV treatment, while the chlorophyll content of the transgenic plants was significantly higher than that of the wild type (p ⁇ 0.01), as shown in Fig. 29.
  • Example 14 MeCBF1 overexpressing cassava plants have obvious water loss resistance
  • the leaves of the tissue culture seedlings were placed on a dry filter paper, and the water loss of the leaves was photographed at intervals. It was found that the water loss rate of the transgenic cassava was slower than that of the wild type. Under the same treatment time, the wild type was more likely to lose water. Wilting 30.
  • MeCB ⁇ overexpressing cassava was planted in the transgenic test field to observe the phenotype and root yield of the transgenic plants. After about 6 months of cultivation, over-expression of cassava plant height and root yield was similar to wild type, as shown in Figure 34.
  • MeCBF1 and MeCBF2 are involved in plant stress resistance signaling pathways.
  • Cell localization and yeast experiments demonstrated that MeCBF1 may function as a transcription factor.
  • Transgenic experiments have also demonstrated that MeCBF1 overexpresses plants with strong resistance to stress, such as: low temperature, salt, permeation and oxidation, further validating the function of this protein.
  • the inventors also overexpressed MeCBF1 in cassava, and the stress treatment and physiological experiments showed that the mesocarpic plants overexpressed by MeCBFl had higher resistance to cold, oxidation and water loss.
  • the invention proves that the MeCBF1 protein has the ability to resist various stresses such as cold and oxidation in both the Arabidopsis and cassava systems, and provides a new strategy and possibility for obtaining the genetically modified cassava with improved cold resistance.
  • the study of the molecular mechanism of MeCBFl has laid a solid foundation for studying the molecular mechanism of low temperature response of cassava and tropical crops.

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Abstract

本发明涉及一种提高植物抗逆境能力的基因及其用途。本发明人首次分离到一种新的对于调节植物抗逆境能力有用的基因，其可极好地应用于植物品种的改良，提高植物对于逆境的抵抗力。本发明还提供了所述基因的启动子，其具有诱导目的基因在逆境下表达的功能。本发明对延长植物种植时间、扩大植物种植面积、提高植物产量有重要意义。

Description

一种提高植物抗逆境能力的基因及其用途
技术领域
本发明属于生物技术和植物学领域； 更具体地， 本发明涉及一种提高植物抗逆境能 力的基因及其用途。 背景技术
植物抗逆是植物对抗不良环境， 比如抗旱、 抗冷、 抗盐碱的能力。 在自然界条件下， 由于不同的地理位置和气候条件以及人类活动等多方面原因， 造成了各种不良环境， 超 出了植物正常生长、 发育所能忍受的范围， 致使植物受到伤害甚至死亡。 这些对植物产 生伤害的环境称为逆境或胁迫。 而植物对不良环境的适应性和抵抗力为抗逆性或抗性。
逆境的种类多种多样， 包括物理的、 化学的、 生物因素等， 可分为生物逆境和非生 物逆境两大类。 对植物产生重要影响的非生物逆境主要有水分 (干旱和淹涝)、 温度 (高、 低温)、 盐碱等理化逆境。
低温胁迫是影响植物正常生长的主要障碍因子之一， 植物尤其是经济作物的抗冷性 强弱直接影响作物产量。 近年来， 本领域技术人员已对植物抗冷性进行了较多研究， 探 讨出植物抗冷性的鉴定方法， 对植物抗冷性生理生化机理及各生理生化指标与植物抗冷 性的关系有了更深入的了解； 通过分子标记技术， 在分子水平上对植物抗冷性基因进行 分析， 获得了部分与抗冷性有关的基因。
然而， 还需要在此基础上进一步地鉴定和分离抗逆境相关的基因， 并深入研究其抗 逆境机制， 更重要地是切实地将之应用于生产实践。 发明内容
本发明的目的在于提供一种提高植物抗逆境能力的基因及其用途。
本发明的目的还在于提供一种具有诱导目的基因在逆境下表达功能的启动子。 在本发明的第一方面， 提供一种分离的多肽， 所述多肽选自下组-
(a) 如 SEQ ID NO: 3所示氨基酸序列的多肽；
(b) 如 SEQ ID NO: 4所示氨基酸序列的多肽； 或
(c) 将 SEQ ID NO: 3或 SEQ ID NO: 4所示氨基酸序列经过一个或多个 (如 1-50个， 较佳地 1-20个， 更佳地 1-10， 更佳地 1-5个， 如 2个、 3个)氨基酸残基的取代、 缺失或 添加而形成的， 且具有提高植物抗逆境能力功能的由 (a)或 (b)衍生的多肽。
在一个优选例中， 该多肽是如 SEQ ID NO: 3或 SEQ ID NO: 4所示氨基酸序列的多 肽； 或
该多肽是相对于 SEQ ID NO: 3氨基酸序列， 第 40-53位、 第 56-1 12位、 第 114-1 17 位氨基酸序列不变而其它位置经过一个或多个 (如 1-50个，较佳地 1-20个，更佳地 1-10， 更佳地 1-5个， 如 2个、 3个)氨基酸残基的取代、 缺失或添加而形成的， 且具有提高植 物抗逆境能力功能的由 (a)衍生的多肽； 或
该多肽是相对于 SEQ ID NO: 4， 第 52-65位、 第 68-124位、 第 126-129位氨基酸序 列不变而其它位置经过一个或多个 (如 1-50个，较佳地 1-20个，更佳地 1-10，更佳地 1-5 个， 如 2个、 3个)氨基酸残基的取代、 缺失或添加而形成的， 且具有提高植物抗逆境能 力功能的由 (b)衍生的多肽。
在另一优选例中， 所述的植物选自 (但不限于)： 大戟科植物、旋花科植物、茄科植物、 十字花科植物、 禾本科植物、 木本科植物。
在另一优选例中， 所述植物选自大戟科的木薯、 橡胶、 蓖麻、 乌桕； 或旋花科放入 甘薯； 或茄科的马铃薯； 或禾本科的水稻、 小麦、 大麦、 玉米、 高粱等； 或十字花科的 拟南芥。
在本发明的另一方面， 提供一种分离的多核苷酸， 它包含一核苷酸序列， 该核苷酸 序列选自下组：
(1) 编码所述多肽的多核苷酸； 或
(2) 与多核苷酸 （1) 互补的多核苷酸。
在一个优选例中， 该多核苷酸编码如 SEQ ID NO: 3或 SEQ ID NO: 4所示氨基酸序 列的多肽。
在一个优选例中， 该多核苷酸：
(i) 核苷酸序列如 SEQ ID NO: 1所示； 或
(ϋ) 核苷酸序列如 SEQ ID NO: 2所示。
在本发明的另一方面， 提供一种载体， 它含有所述的多核苷酸。
在本发明的另一方面， 提供一种遗传工程化的宿主细胞， 它含有所述的载体。
在本发明的另一方面， 提供一种制备植物的方法， 其包括将所述的多核苷酸转入植 物中。
在一个优选例中， 所述方法包括：
(1) 提供携带表达载体的农杆菌， 所述的表达载体含有所述的多核苷酸；
(2) 将植物细胞或组织或器官与步骤 (1)中的农杆菌接触， 从而使所述的多核苷酸转 入植物细胞， 并且整合到植物细胞的染色体上；
(3) 选择出转入了所述的多核苷酸的植物细胞或组织或器官； 和
(4) 将步骤 (3)中的植物细胞或组织或器官再生成植物。 在本发明的另一方面， 提供一种植物， 其包含前面任一项所述的多核苷酸。
在本发明的另一方面， 提供一种所述的多肽的制备方法， 该方法包含- (a) 在适合表达的条件下， 培养所述的宿主细胞； (b) 从培养物中分离出所述的多肽。
在本发明的另一方面， 提供所述的多肽或编码该多肽的多核苷酸的用途， 用于提高 植物抗逆境能力或制备提高植物抗逆境能力的物质。
在一个优选例中， 所述的逆境是冷 (胁迫)环境、 高盐 (胁迫)环境、 干旱 (胁迫)环境、 氧化 (胁迫)环境、 渗透 (胁迫)环境。
在另一优选例中， 所述的多肽或编码该多肽的多核苷酸还用于：
降低植物 (较佳地为逆境或胁迫环境下的植物)地上部分的 Na+浓度， 增加 K+浓度； 降低植物 (较佳地为逆境或胁迫环境下的植物)中积累的 ¾02量；
提高植物 (较佳地为逆境或胁迫环境下的植物）中超氧化物歧化酶 (SOD)或过氧化氢 酶 (CAT)的酶活性；
提高植物 (较佳地为逆境或胁迫环境下的植物）中 CBF信号通路基因 (如 FAD7、COR47 和 GolS3)的表达；
提高植物 (较佳地为逆境或胁迫环境下的植物)中叶绿素含量；
提高植物 (较佳地为逆境或胁迫环境下的植物)中脯氨酸含量；
提高植物 (较佳地为逆境或胁迫环境下的植物)的抗失水能力；
改善植物在冷环境 (如 10°C左右的低温环境)下的生长状态；
提高植物在冷环境 (如 10°C左右的低温环境)下的生物量 (如鲜重或干重)；
提高植物在冷环境 (如 10°C左右的低温环境)下的叶长叶宽。
在本发明的另一方面， 提供一种提高植物抗逆境能力的方法， 所述方法包括： 提高 植物中所述的多肽的表达或活性。
在本发明的另一方面， 提供一种所述的多肽或其编码基因的激动剂。
在本发明的另一方面， 提供一种受逆境诱导表达的启动子， 所述的启动子选自下组：
(1) 如 SEQ ID NO: 5或 SEQ ID NO: 6所示的核苷酸序列的多核苷酸；
(2) 在严格条件下能够与 (1)限定的多核苷酸序列杂交且具有诱导目的基因在逆境下 表达功能的多核苷酸； 或
(3) 与 SEQ ID NO: 5或 SEQ ID NO: 6所示的核苷酸序列具有 70% (；优选 80%以上，更优 选 90%以上， 最优选 95%以上， 如 98%， 99%) 以上相同性且具有诱导目的基因在逆境下 表达功能的多核苷酸。
在一个优选例中， 所述的启动子选自下组：
相对于 SEQ ID NO: 5的核苷酸序列， 第 574-579位、 第 716-720位、 第 130-133位、 第 417-420位、 284-289、第 649-654位碱基序列不变而其它位置经过一个或多个 (；如 1-50 个， 较佳地 1-20个， 更佳地 1-10， 更佳地 1-5个， 如 2个、 3个)碱基的取代、 缺失或添 加而形成的， 且具有诱导目的基因在逆境下表达功能的多核苷酸； 或
相对于 SEQ ID NO: 6的核苷酸序列， 第 745-749位、 第 1340-1345位、 第 533-536 位、 第 878-881位、 第 302-307位、 第 482-487位、 第 510-515位、 第 744-749位、 第 1 155-1 160位、 第 1278-1283位、 第 1306-131 1位、 第 1375-1380位碱基序列不变而其它 位置经过一个或多个 (如 1-50个， 较佳地 1-20个， 更佳地 1-10， 更佳地 1-5个， 如 2个、
3个)碱基的取代、 缺失或添加而形成的， 且具有诱导目的基因在逆境下表达功能的多核 苷酸。
在本发明的另一方面， 提供所述的启动子的用途， 用于诱导目的基因在逆境下表达。 在本发明的另一方面， 提供一种构建物， 所述的构建物含有所述的受逆境诱导表达 的启动子。
在另一优选例中， 所述构建物中， 受逆境诱导表达的启动子的下游含有至少一个多 克隆位点 (如酶切位点)， 其与所述的受逆境诱导表达的启动子可操作性连接， 用于插入 目的基因。
在另一优选例中， 所述的构建物是表达载体。
在另一优选例中， 所述的构建物含有以下可操作性连接的元件：
所述的启动子； 和目的基因。
在另一优选例中， 所述的目的基因是外源基因。
在另一优选例中， 所述的目的基因是结构基因。
在另一优选例中， 所述的目的基因可编码具有特定功能的蛋白。
在另一优选例中， 所述的目的基因位于所述启动子的下游， 且与所述启动子的间隔 小于 2000bp (优选的， 小于 l OOObp; 更优选的， 小于 500bp ; 最优选的， 小于 300bp)。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的。 附图说明
图 1显示了 Southern印迹的结果， 显示木薯 CBF基因可能存在两个拷贝。
图 2显示了 MeCBFl和 MeCBF2核苷酸序列比对 (A)和 MeCBFl禾 B MeCBF2蛋白序 列比对。 NLS、 AP2和 DSW为 CBF保守序列。
图 3显示了 MeCBFl在受冷胁迫 (A)及其它逆境胁迫条件下 (B)表达水平的变化， 以 及 MeCBFl的组织特异性表达情况 (C)。 其中， " GA-冷"指： GA预处理 3h后再 4 °C处 理 3h后的基因表达情况。
图 4显示了 MeCBF2在受冷胁迫 (A)及其它逆境胁迫条件下 (B)表达水平的变化， 以 及 MeCBF2的组织特异性表达情况 (C)。 其中， " GA-冷"指： GA预处理 3h后再 4 °C处 理 3h后的基因表达情况。
图 5A显示了 MeCBFl和 MeCBF2洋葱表皮细胞定位试验。
图 5B显示了 MeCBFl和 MeCBF2烟草原生质体定位。
图 6显示 MeCBFl过表达拟南芥抗冷性增强。将野生型 Col-0和 3个单拷贝过 表达纯合株系（SMl-13-3、 SM1-14-19, SMl-19-10)的种子， 经表面消毒后铺于基 本培养基上。 4°C春化 3天后， 将其分别放于 22°C和 10°C光照培养箱中水平和 垂直培养。 培养一段时间后观察幼苗生长状态。
图 Ί显示了低温条件下 MeCBFl过表达拟南芥具有较好的长势。分别取水平培 养 30 d的 Col-0与过表达株系叶片，称量其鲜重和干重， n=20 o并测量低温 (10°C) 水平培养 30 d幼苗子叶的叶长与叶宽。 **代表 p<0.01。
图 8显示了低温冷处理比较过量表达 MeCBFl的拟南芥与野生型拟南芥的抗冷能力。 SM1-19、 SM1-17为两种 T2代植株。
图 9显示了平皿实验比较过量表达 MeCBFl的拟南芥与野生型拟南芥的抗冷能力。 图 10显示了正常萌发的 SM1转基因拟南芥较野生型明显抗盐。
图 1 1显示了 MeCBFl启动子受冷诱导表达。
图 12显示了 BL21(pET32a-MeCBFl)和 BL21(pET32a) 重组原核表达菌株在抗性筛选 培养基上的生长状态。
图 13 显示 MeCBFl 具有专一的 DNA 结合活性。 其中， A 中上图为重组载体 pGAO424-MeCBFl 的部分结构示意图。 A 中中图为重组载体 pHISi-3wDRE-HS/ 3wDRE-HA的部分结构示意图。 A中下图为重组载体 pHISi-3mDRE-HS2/3mDRE-HA2 的部分结构示意图。 B显示转入不同的重组载体后， 酵母细胞的生长状况。
图 14显示 MeCBFl的 C端具有转录激活活性。 A图为用于酵母杂交系统的各重组载 体的部分元件示意图。 B图为各酵母转化子的显色结果。
图 15显示 MeCBFl过表达拟南芥植株具有较高抗盐能力。图中所示两次独立重复实 验 的 结果 ， Col-0 为 野生型 ， 其余三个株系（14-19 、 19-10 、 13-3)为 pCAMBIA 1301-355:： MeCBFl独立的纯合株系。
图 16显示 MeCBFl过表达拟南芥植株 (SM1- 14-19、 SM1-19-10 SM1-13-3)具有较低 的 Na+浓度和较高的 K+浓度。
图 17显示 MeCBFl过表达拟南芥植株 (SM1- 14-19、 SM1-19-10 SM1-13-3)具有明显 的抗渗透胁迫能力。
图 18显示 MeCBFl过表达拟南芥植株具有较强的抗干旱能力。
图 19显示 MeCBFl过表达拟南芥植株 (SM1- 14-19、 SM1-19-10 SM1-13-3)具有明显 的抗氧化胁迫能力。
图 20显示 MeCBFl过表达拟南芥植株 (SM1- 14-19、 SM1-19-10 SM1-13-3)积累较低 的 ¾02含量。
图 21显示 MeCBFl过表达拟南芥植株 (SM1- 14-19、 SM1-19-10 SM1-13-3)在低温处 理条件下具有较高的 SOD(A)和 CAT酶活性 (B)。
图 22显示 Southern Blotting检测 MeCBFl 过表达转基因木薯的拷贝数。 Lanel和 Lane2证明相应植株为单拷贝株系 (SM1-1， SM1-2), Lane3证明相应植株为为多拷贝株 系（SMl-3)。
图 23显示 MeCBFl过表达木薯植株诱导 CBF-靶基因的表达情况。
图 24显示 MeCBFl过表达木薯植株中 MeCBFl蛋白的表达量增加。
图 25显示 MeCBFl过表达木薯植株在培养基下培育时， 具有较高的抗低温能力。 图 26显示 MeCBFl过表达木薯植株在大田培育时， 表现出较高的抗低温能力。 图 27显示 MeCBFl过表达木薯大田苗具有较高的叶绿素 (A)和脯氨酸 (B)含量。
图 28显示 MeCBFl过表达木薯植株具有较高的抗氧化胁迫的能力。
图 29显示 MeCBFl过表达木薯植株具有较高的叶绿素含量。
图 30显示 MeCBFl过表达木薯植株具有较强的抗失水能力。
图 31显示 MeCBFl过表达木薯植株具有较强的复水能力。
图 32显示 MeCBFl过表达木薯植株具有较强的抗失水能力。
图 33显示 MeCBFl过表达木薯植株具有较强的抗干旱能力。
图 34显示 MeCBFl过表达木薯田间表型。 具体实施方式
本发明人经过广泛的研究， 首次分离到一种新的对于调节植物抗逆境能力有用的基 因。本发明的基因可极好地应用于植物品种的改良， 提高植物对于逆境的抵抗力。并且， 本发明对延长植物种植时间、 扩大植物种植面积 (如种植北移)、 提高植物产量有重要意 义。 在此基础上完成了本发明。 本发明中， 对于适用于本发明的植物 (或作物)没有特别的限制， 只要其适合进行基 因的转化操作， 或适合于进行基因敲除的操作， 如各种农作物、 花卉植物、 或林业植物 等。 所述的植物比如可以是 （不限于)： 双子叶植物、 单子叶植物、 或裸子植物。 更具体 地， 所述的植物包括 (但不限于)： 小麦、 大麦、 黑麦、 水稻、 玉米、 高梁、 甜菜、 苹果、 梨、 李、 桃、 杏、 樱桃、 草莓、 木莓、 黑莓、 豆、 扁豆、 豌豆、 大豆、 油菜、 芥、 罂粟、 齐墩果、 向日葵、 椰子、 蓖麻油植物、 可可豆、 花生、 葫芦、 黄瓜、 西瓜、 棉花、 亚麻、 大麻、 黄麻、 柑桔、 柠檬、 葡萄柚、 菠菜、 苘苣、 芦笋、 洋白菜、 大白菜、 小白菜、 胡 萝卜、 洋葱、 土豆、 西红柿、 青椒、 鳄梨、 桂皮、 樟脑、 烟叶、 坚果、 咖啡、 茄子、 甘 蔗、 茶叶、 胡椒、 葡萄树、 蚝麻草、 香蕉、 天然橡胶树和观赏植物等。
较佳地， 所述的 "植物" 或 "作物"包括但不限于： 大戟科 (E^/wr aceae植物、 旋花科植物、 茄科植物、 十字花科植物、 禾本科植物、 木本科植物等。 更优选的， 所述 的大戟科植物包括但不限于：
Figure imgf000007_0001
esculenta)、 橡胶、 蓖麻、 乌桕等； 所述的旋 花科植物包括但不限于： 甘薯； 所述的茄科植物包括但不限于： 马铃薯； 所述的禾本科植 物包括但不限于： 水稻、 小麦、 大麦、 玉米、 高粱等； 或所述的十字花科植物包括但不 限于： 拟南芥。
如本文所用， "分离的" 是指物质从其原始环境中分离出来 (如果是天然的物质， 原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯 化的， 但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开， 则为分离 纯化的。
如本文所用， "分离的提高植物抗逆境能力的多肽" 、 "分离的 MeCBF蛋白"或 "分离的 MeCBF多肽" 是指 MeCBF蛋白基本上不含天然与其相关的其它蛋白、 脂类、 糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯化技术纯化 MeCBF蛋白。 基 本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主带。本发明中，所述的 MeCBF 蛋白可以是 MeCBFl蛋白和 MeCBF2蛋白。
如本文所用， 所述的 "含有"， "具有 "或"包括"包括了 "包含"、 "主要由 ...... 构成" 、 "基本上由 ......构成" 、 禾 B "由 ... ...构成" ； "主要由 ......构成" 、 "基本上 由 ......构成" 和 "由 ......构成"属于 "含有" 、 "具有" 或 "包括" 的下位概念。 本发明的多肽可以是重组多肽、 天然多肽、 合成多肽， 优选的是重组多肽。 本发明 的多肽可以是天然纯化的产物， 或是化学合成的产物， 或使用重组技术从原核或真核宿 主 (例如， 细菌、 酵母、 高等植物、 昆虫和哺乳动物细胞)中产生。 根据重组生产方案所 用的宿主， 本发明的多肽可以是糖基化的， 或可以是非糖基化的。 本发明的多肽还可包 括或不包括起始的甲硫氨酸残基。
本发明还包括 MeCBF蛋白的片段、 衍生物和类似物。 如本文所用， 术语 "片段" 、 "衍生物" 和 "类似物" 是指基本上保持本发明的 MeCBF蛋白相同的生物学功能或活 性的多肽。 本发明的多肽片段、 衍生物或类似物可以是 (i)有一个或多个保守或非保守性 氨基酸残基 (优选保守性氨基酸残基)被取代的多肽， 而这样的取代的氨基酸残基可以是 也可以不是由遗传密码编码的， 或 (ii)在一个或多个氨基酸残基中具有取代基团的多肽， 或 (iii)成熟多肽与另一个化合物（比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形 成的多肽， 或 O)附加的氨基酸序列融合到此多肽序列而形成的多肽 (如前导序列或分泌 序列或用来纯化此多肽的序列或蛋白原序列， 或融合蛋白）。 根据本文的定义这些片段、 衍生物和类似物属于本领域熟练技术人员公知的范围。
在本发明中， 术语 " MeCBF蛋白"指具有提高植物抗逆境能力或提高植物对盐的敏 感性功能的 SEQ ID NO: 3或 SEQ ID NO: 4序列的多肽。 该术语还包括具有提高植物抗 逆境能力提高植物对盐的敏感性功能的、 SEQ ID NO:3或 SEQ ID NO: 4序列的变异形 式。 这些变异形式包括 (但并不限于)： 若干个 (通常为 1-50个， 较佳地 1-30个， 更佳地 1-20个， 最佳地 1-10个， 还更佳如 1-8个或 1-5个)氨基酸的缺失、 插入和 /或取代， 以 及在 C末端和 /或 N末端添加一个或数个 (通常为 20个以内， 较佳地为 10个以内， 更佳 地为 5个以内)氨基酸。 例如， 在本领域中， 用性能相近或相似的氨基酸进行取代时， 通 常不会改变蛋白质的功能。 又比如， 在 C末端和 /或 N末端添加一个或数个氨基酸通常 也不会改变蛋白质的功能。 该术语还包括 MeCBF蛋白的活性片段和活性衍生物。
多肽的变异形式包括： 同源序列、 保守性变异体、 等位变异体、 天然突变体、 诱导 突变体、 在高或低的严紧度条件下能与 MeCBF蛋白 DNA杂交的 DNA所编码的蛋白、 以及利用抗 MeCBF蛋白的抗血清获得的多肽或蛋白。 本发明还提供了其他多肽， 如包 含 MeCBF蛋白或其片段的融合蛋白。 除了几乎全长的多肽外， 本发明还包括了 MeCBF 蛋白的可溶性片段。 通常， 该片段具有 MeCBF蛋白序列的至少约 20个连续氨基酸， 通 常至少约 30个连续氨基酸， 较佳地至少约 50个连续氨基酸， 更佳地至少约 80个连续 氨基酸， 最佳地至少约 100个连续氨基酸。
本发明还提供 MeCBF蛋白或多肽的类似物。 这些类似物与天然 MeCBF蛋白的差别 可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。 这些多肽包括天然或诱导的遗传变异体。 诱导变异体可以通过各种技术得到， 如通过辐 射或暴露于诱变剂而产生随机诱变， 还可通过定点诱变法或其他已知分子生物学的技 术。 类似物还包括具有不同于天然 L-氨基酸的残基 (如 D-氨基酸)的类似物， 以及具有非 天然存在的或合成的氨基酸 (如 β、 Υ -氨基酸)的类似物。 应理解， 本发明的多肽并不限 于上述例举的代表性的多肽。
修饰 (通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或 羧基化。 修饰还包括糖基化。修饰形式还包括具有磷酸化氨基酸残基 (如磷酸酪氨酸， 磷 酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶 解性能的多肽。
在本发明中， " MeCBF蛋白保守性变异多肽"指与 SEQ ID NO: 3或 SEQ ID NO: 4 的氨基酸序列相比， 有至多 20个， 较佳地至多 10个， 更佳地至多 5个， 最佳地至多 3 个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。 这些保守性变异多肽最好根据 表 1进行氨基酸替换而产生。
表 1
Figure imgf000009_0001
Figure imgf000010_0001
本发明还提供了编码本发明 MeCBF蛋白或其保守性变异多肽的多核苷酸序列。 本发明的多核苷酸可以是 DNA形式或 RNA形式。 DNA形式包括 cDNA、 基因组
DNA或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编码链或非编 码链。 编码成熟多肽的编码区序列可以与 SEQ ID NO: 1或 SEQ ID NO: 2所示的编码区 序列相同或者是简并的变异体。 如本文所用， "简并的变异体"在本发明中是指编码具 有 SEQ ID NO: 3或 SEQ ID NO: 4的蛋白质， 但与 SEQ ID NO: 1或 SEQ ID NO: 2所示 的编码区序列有差别的核酸序列。
编码 SEQ ID NO: 3或 SEQ ID NO: 4的成熟多肽的多核苷酸包括： 只编码成熟多肽 的编码序列； 成熟多肽的编码序列和各种附加编码序列； 成熟多肽的编码序列 (和任选的 附加编码序列）以及非编码序列。
术语 "编码多肽的多核苷酸"可以是包括编码此多肽的多核苷酸， 也可以是还包括 附加编码和 /或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽 或多肽的片段、 类似物和衍生物。 此多核苷酸的变异体可以是天然发生的等位变异体或 非天然发生的变异体。 这些核苷酸变异体包括取代变异体、 缺失变异体和插入变异体。 如本领域所知的， 等位变异体是一个多核苷酸的替换形式， 它可能是一个或多个核苷酸 的取代、 缺失或插入， 但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少 50%， 较佳地至少 70%， 更佳地至少 80%相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷 酸可杂交的多核苷酸。 在本发明中， "严格条件" 是指: (1)在较低离子强度和较高温度 下的杂交和洗脱， 如 0.2 X SSC, 0.1%SDS, 60 °C ; 或 (2)杂交时加有变性剂， 如 50%(ν/ν) 甲酰胺， 0.1%小牛血清 /0.1%Ficoll， 42°C等；或 (3)仅在两条序列之间的相同性至少在 80% 以上， 较好至少 90%以上， 更好是 95%以上时才发生杂交。 并且， 可杂交的多核苷酸编 码的多肽与 SEQ ID NO: 3所示的成熟多肽有相同的生物学功能和活性。
本发明还涉及与上述的序列杂交的核酸片段。 如本文所用， "核酸片段" 的长度至 少含 15个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50个核苷酸， 最好是至少 100 个核苷酸以上。 核酸片段可用于核酸的扩增技术 (如 PCR)以确定和 /或分离编码 MeCBF 蛋白的多聚核苷酸。
本发明的 MeCBF蛋白核苷酸全长序列或其片段通常可以用 PCR扩增法、 重组法或 人工合成的方法获得。 对于 PCR扩增法， 可根据本发明所公开的有关核苷酸序列， 尤其 是开放阅读框序列来设计引物， 并用市售的 cDNA库或按本领域技术人员已知的常规方 法所制备的 cDNA库作为模板， 扩增而得有关序列。 当序列较长时， 常常需要进行两次 或多次 PCR扩增， 然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通常是将其 克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外， 还可用人工合成的方法来合成有关序列， 尤其是片段长度较短时。 通常， 通 过先合成多个小片段， 然后再进行连接可获得序列很长的片段。
目前， 已经可以完全通过化学合成来得到编码本发明蛋白（或其片段， 或其衍生物) 的 DNA序列。然后可将该 DNA序列引入本领域中已知的各种现有的 DNA分子 (或如载 体)和细胞中。 此外， 还可通过化学合成将突变引入本发明蛋白序列中。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或 MeCBF蛋白编 码序列经基因工程产生的宿主细胞， 以及经重组技术产生本发明所述多肽的方法。
通过常规的重组 DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组 的 MeCBF蛋白。 一般来说有以下步骤：
(1).用本发明的编码 MeCBF 蛋白的多核苷酸 (或变异体)， 或用含有该多核苷酸的重 组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养宿主细胞；
(3).从培养基或细胞中分离、 纯化蛋白质。
本发明中， MeCBF蛋白多核苷酸序列可插入到重组表达载体中。 术语 "重组表达载 体"指本领域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒 或其他载体。 总之， 只要能在宿主体内复制和稳定， 任何质粒和载体都可以用。 表达载 体的一个重要特征是通常含有复制起点、 启动子、 标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含 MeCBF蛋白编码 DNA序列和合适的转 录 /翻译控制信号的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成技术、 体内 重组技术等。所述的 DNA序列可有效连接到表达载体中的适当启动子上，以指导 mRNA 合成。 表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转化的宿 主细胞的表型性状， 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗性以及绿色荧光蛋 白 (GFP)， 或用于大肠杆菌的卡那霉素或氨苄青霉素抗性。
包含上述的适当 DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当 的宿主细胞， 以使其能够表达蛋白质。 宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高 等真核细胞， 如植物细胞。 代表性例子有： 大肠杆菌， 链霉菌属、 农杆菌； 真菌细胞如 酵母； 植物细胞等。
本发明的多核苷酸在高等真核细胞中表达时， 如果在载体中插入增强子序列时将会 使转录得到增强。 增强子是 DNA的顺式作用因子， 通常大约有 10到 300个碱基对， 作 用于启动子以增强基因的转录。
本领域一般技术人员都清楚如何选择适当的载体、 启动子、 增强子和宿主细胞。 用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核 生物如大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获， 用 CaCl2法处 理， 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl2。 如果需要， 转化也可用 电穿孔的方法进行。 当宿主是真核生物， 可选用如下的 DNA转染方法： 磷酸钙共沉淀 法， 常规机械方法如显微注射、 电穿孔、 脂质体包装等。 转化植物也可使用农杆菌转化 或基因枪转化等方法， 例如叶盘法、 水稻幼胚转化法等。 对于转化的植物细胞、 组织或 器官可以用常规方法再生成植株， 从而获得性状发生改变的植物。
获得的转化子可以用常规方法培养， 表达本发明的基因所编码的多肽。 根据所用的 宿主细胞， 培养中所用的培养基可选自各种常规培养基。 在适于宿主细胞生长的条件下 进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法 (如温度转换或化学诱导) 诱导选择的启动子， 将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、 或在细胞膜上表达、 或分泌到细胞外。 如 果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。 这些方法是本领域技术人员所熟知的。 这些方法的例子包括但并不限于： 常规的复性处 理、 用蛋白沉淀剂处理 (盐析方法)、 离心、 渗透破菌、 超处理、 超离心、 分子筛层析 (凝 胶过滤)、 吸附层析、 离子交换层析、 高效液相层析 (HPLC)和其它各种液相层析技术及 这些方法的结合。
重组的 MeCBF蛋白有多方面的用途。 例如用于筛选促进或对抗 MeCBF蛋白功能的 抗体、 多肽或其它配体。 用表达的重组 MeCBF蛋白筛选多肽库可用于寻找有价值的能 抑制或剌激 MeCBF蛋白功能的多肽分子。
本发明的多核苷酸的一部分或全部可作为探针固定在微阵列 (microarray)或 DNA 芯 片 (又称为 "基因芯片"）上， 用于分析组织中基因的差异表达分析。 用 MeCBF蛋白特异 的引物进行 RNA-聚合酶链反应 (RT-PCR)体外扩增也可检测 MeCBF蛋白的转录产物。
本发明还涉及一种改良作物的方法，该方法包括提高所述植物中 MeCBF基因的表达 或蛋白活性。 从而使得所述植物具有更优良的抗逆境 （包括： 冷环境、 高盐环境或干旱 环境） 能力。
增加 MeCBF基因表达的方法是本领域周知的。 例如， 可通过转入携带 MeCBF编码 基因的表达构建物使植株过表达 MeCBF; 或可通过用强启动子驱动从而增强 MeCBF基 因的表达； 或者通过增强子 (如水稻 waxy基因第一内含子、 Actin基因第一内含子等)来 增强该 MeCBF基因的表达。 适用于本发明方法的强启动子包括但不限于： 35S启动子、 水稻、 玉米的 Ubi启动子等。
抑制 MeC^P基因表达的方法也是本领域周知的。 例如， 可通过 RNA干扰 (RNAi)或 基因沉默 (敲除)的技术来实现。
作为本发明的一种优选方式， 获得 MeCBF高表达的植株的方法如下：
(1) 提供携带表达载体的农杆菌， 所述的表达载体含有 MeCBF蛋白的 DNA编码序 列；
(2) 将植物细胞或组织或器官与步骤 (1)中的农杆菌接触，从而使该 MeCBF蛋白 DNA 编码序列转入植物细胞， 并且整合到植物细胞的染色体上；
(3) 选择出转入所述 MeCBF蛋白 DNA编码序列的植物细胞或组织； 和
(4) 将步骤 (3)中的植物细胞或组织再生成植株。
其中， 可采用任何适当的常规手段， 包括试剂、 温度、 压力条件等来实施此方法。 本发明还包括 MeCBF 蛋白或其编码基因的激动剂。 由于 MeCBF 的激动剂可调节
MeCBF的活性或表达， 因此， 所述的 MeCBF的激动剂也可通过对 MeCBF的影响来提 高植物的抗逆境能力， 从而达到性状改良的目的。
所述的 MeCBF的激动剂是指任何可提高 MeCBF的活性、 维持 MeCBF的稳定性、 促进 MeCBF表达、 延长 MeCBF有效作用时间、 或促进 MeC^P的转录和翻译的物质， 这些物质均可用于本发明， 作为对于提高植物的抗逆境能力有用的物质。
在本发明的一个实例中， 提供了一种 MeC^Pl基因， 其基因组序列如 SEQ ID NO: 1 所示， 编码一个含有 219个氨基酸的蛋白质 （SEQ ID NO: 3)。所述的 MeCBFl基因可以 为植物的抗逆境改良提供新的途径， 因而具有巨大的应用前景。
在本发明的另一个实例中， 提供了一种 MeCBF2基因， 其基因组序列如 SEQ ID NO: 2所示， 编码一个含有 231个氨基酸的蛋白质 （SEQ ID NO: 4)。 所述的 MeCAF2基因可 以为植物的抗逆境改良提供新的途径， 因而具有巨大的应用前景。 启动子及其应用
如本文所用， 所述的 "启动子"或 "启动子区 (域) "是指一种核酸序列， 其通常存在 于目的基因编码序列的上游 (5 ')， 能够引导核酸序列转录为 mRNA。 一般地， 启动子或 启动子区提供 RNA聚合酶和正确起始转录所必需的其它因子的识别位点。 在本文中， 所述的启动子或启动子区包括启动子的变异体， 该变异体可以是天然发生的等位变异体 或非天然发生的变异体。 所述变异体包括取代变异体、 缺失变异体和插入变异体。
如本文所用， 所述的 "可操作性连接" 是指两个或多个核酸区域或核酸序列的功能 性的空间排列。 例如： 启动子区被置于相对于目的基因核酸序列的特定位置， 使得核酸 序列的转录受到该启动子区域的引导， 从而， 启动子区域被 "可操作地连接" 到该核酸 序列上。
本发明提供一种启动子， 所述的启动子选自下组：
(1) 具有 SEQ ID NO: 5或 SEQ ID NO: 6所示的核苷酸序列的多核苷酸； 或
(2) 在严格条件下能够与 (1)限定的多核苷酸序列杂交且具有诱导目的基因在逆境下 表达功能的多核苷酸。
在本发明中， "严格条件" 是指： （1)在较低离子强度和较高温度下的杂交和洗脱， 如 0.2 X SSC, 0.1%SDS , 60 °C ; 或 (2)杂交时加有变性剂， 如 50%(v/v)甲酰胺， 0.1%小 牛血清 /0.1%Ficoll， 42°C等； 或 (3)仅在两条序列之间的相同性至少在 80%以上， 较好至 少 90%以上， 更好是 95%以上时才发生杂交。 并且， 可杂交的多核苷酸也具有诱导目的 基因在逆境下表达的功能。
多核苷酸的杂交是本领域技术人员熟知的技术， 特定的一对核酸的杂交特性指示它 们的相似性或同一性。 因此， 本发明还涉及与 SEQ ID NO: 5或 SEQ ID NO: 6所示的核 苷酸序列杂交且两个序列之间具有至少 50%， 较佳地至少 60%， 更佳地至少 70%， 更 佳地至少 80%， 更佳地至少 85%， 更佳地至少 90% (例如 95%、 96%、 97%、 98%、 或 99%) 相同性的多核苷酸。
本发明的启动子是诱导目的基因在逆境下表达的， 例如诱导目的基因在冷环境下表 达。 在本发明的实例中， 本发明人发现， 在本发明的启动子的指导下， 可以使 Me ^ 或 基因特异地在逆境诱导下表达。
本发明的启动子可以被可操作地连接到目的基因上， 该目的基因相对于启动子可以 是外源 (异源)的。所述的目的基因通常可以是任何核酸序列 (优选结构性核酸序列)， 所述 的目的基因优选编码具有特定功能的蛋白。
本发明的启动子还可以被可操作地连接到被改进的目的基因序列上， 该目的基因相 对于启动子是外源 (异源)的。所述的目的基因可以被改进来产生各种期望的特性。例如， 目的基因可以被改进来增加表达量， 改变翻译后的修饰 (如磷酸化位点）， 将翻译产物转 运到细胞外， 改善蛋白的稳定性， 插入或删除细胞信号等。
此外， 启动子和目的基因可以设计成下调特定基因。 这一般是通过将启动子连接到 目的基因序列上来实现， 该序列以反义反向被引导。 本领域的普通技术人员熟悉这种反 义技术。 任何核酸序列可以以这种方式被调节。
任何一种前述的启动子和目的基因序列可被包含在构建物中， 更具体地如重组载体 中。
所述的重组载体一般包括可操作性连接的 (通常从 5 '到 3 '方向)： 引导目的基因转录 的启动子， 和目的基因。 如果需要， 所述的重组载体还可以包括 3 '转录终止子， 3 '多聚 核苷酸化信号， 其它非翻译核酸序列， 转运和靶向核酸序列、 抗性选择标记、 增强子或 操作子。
通常， 所述的目的基因位于所述受逆境诱导表达的启动子的下游， 且与所述启动子 的间隔小于 2000bp (；优选的，小于 l OOObp ;更优选的，小于 500bp ;最优选的，小于 300bp)。
重组载体中除了含有本发明的启动子， 还可含有一种或多种其它启动子。 所述的其 它启动子例如是： 组织特异性的、 组成型的或诱导型的。
包含上述适当的启动子和目的基因的载体， 可以用于转化适当的宿主细胞， 以使其 能够表达蛋白质。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件如 J.萨姆布鲁克等编著， 分子克隆实验指南， 科学出版社， 2002中所述的条件， 或 按照制造厂商所建议的条件。 除非另外说明， 否则百分比和份数按重量计算。
除非另行定义， 文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义 相同。 此外， 任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。 文中所 述的较佳实施方法与材料仅作示范之用。 实施例 1、 木薯抗逆基因的获得
木薯为热带和亚热带作物， 对低温非常敏感。 为了研究木薯 cassava (Manihot esculenta Crantz) 非生物胁迫调控机制， 本发明人首先根据 CBF保守序列 -AP2 结构域， 试图克隆木薯类似序列， 再通过 5 '和 3 '-RACE获得全长序列， 但此方法未获成功。
接着，本发明人通过筛选木薯一个干旱处理后叶片和块根混合组织的 cDNA文库 (全 长 cDNA RIKEN 文库序列参考文献 Sakurai T, Plata G, Rodriguez-Zapata F, Seki M, Salcedo A, Toyoda A, Ishiwata A, Tohme J, Sakaki Y, Shinozaki K et ah Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response. Bmc Plant Biology 2007, 7:)， 得至 Ll一条类 4以 DREB1A/CBF序列， 通过设计引物 (；序列： 正义引物： atggatgttttctctcagtactc, 反义引物： ttatacagaaaaactccataatgg) ,本发明人分离到一个基因，命名为 MeCBFl , 其序列如 SEQ ID NO: 1所示。
由于 CBF主要以基因家族形式存在于各物种中， 因此以此序列为探针，经三种限制 性内切酶 （EcoRI、 HindllK Xball) 分别酶解木薯 TMS60444(瑞士联邦理工大学)基因组， 通过 Southern印迹实验检测该基因的拷贝数， 结果显示该基因可能有两个拷贝（图 1)。 根据同源序列， 本发明人也克隆到了另一个 MeCBF基因， 命名为 MeC¾F (克隆过程： 我们用克隆 MeCBFl 的引物， PCR得到序列后连接到 T- 18载体后测序， 在测序结果中 发现主要是 MeCBFl序列， 但同时也有另一种序列， 通过序列比对发现， 这两条序列在 引物位置只有几个碱基的差别， 并且与 MeCBFl同源性很高， 因此命名为 MeCBF2。 正 义引物： atggatgttttctctcagtactc , 反义引物： ttatacagaaaaactccataatgg)。 核苷酸和蛋白序列 比对得知， 这两个基因同源性很高， 特别是在保守区域 NLS (相对于 SEQ ID NO: 3氨基 酸序列， 第 40-53位； 相对于 SEQ ID NO: 4氨基酸序列， 第 52-65位）、 AP2/EREBP (相 对于 SEQ ID NO: 3氨基酸序列， 第 56-1 12位； 相对于 SEQ ID NO: 4氨基酸序列， 第 68-124位）、 DSW(相对于 SEQ ID NO: 3氨基酸序列，第 1 14- 1 17位； 相对于 SEQ ID NO: 4氨基酸序列， 第 126-129位)， 见图 2。 实施例 2、 验证 MeCBF蛋白是否参与木薯抗逆信号途径
为验证 MeCBF蛋白是否参与了木薯抗逆信号途径。 本发明人在 4 °C冷处理扦插一个 月的木薯 (TMS60444)苗 0hr、 2hr、 4hr、 9hr、 25 hr, 通过 Realtime-PCR检测 MeCBFl的 表达情况（引物序列为： 正义引物： TTCTTGGTCGCCTGATACCTG， 反义引物： GCTTCTTGGGATAGCTGGAAG) , 发现 MeCB ^在 2hr时明显上调， 之后 4hr略微下降， 9hr后又上升直至 25hr(本实验已重复 3次)， 结果预示 MeCBF受冷诱导表达， 提示其参 与冷信号途径， 见图 3A。
同时， 本发明人观察到 MeCBFl在 4hr出现下调， 提示这是该基因被某种机制反馈 调控， 也就是说， 如果基因被诱导达到一定的量后， 植物会启动反馈机制抑制该基因继 续上调， 然而冷胁迫仍然存在， 所以为发挥抗冷的作用该基因会继续上调， 而这时它被 上调的程度往往会高于第一次。本发明人推测， 该反馈机制的存在是由于 CBF的高表达 会对植株正常生长起负调控作用， 因此出于保护自身的原因， 此反馈机制会抑制 CBF 基因的过高表达。
然而， 进一步还要验证 MeC¾F7是否也受其它胁迫调控。 实验结果显示 MeC¾F7特 异性受冷诱导表达， 而对其它胁迫条件不响应。 具体实验如下： 本发明人先将 20天的 木薯 (TMS60444)组培苗根部的培养基洗净， 再用 MS营养液预处理 4-5天， 之后， 分别 用 250mM NaCl、 25%PEG、 Ι ΟΟμΜ ABA 4 °C处理 6hr (本实验重复 2次）， Realtime-PCR 结果显示 MeCBFl在盐胁迫和干旱胁迫和 ABA条件下略微诱导， 但是非常强烈的受冷 诱导表达， 这说明 MeCB ^可能在冷胁迫条件下起主要作用（图 3B)。 同时， 本发明人发 现预先用 80μΜ ΟΑ处理组培苗， 之后再 4°C处理， Me ^的表达量明显受到抑制， 降 至 50%左右， 这说明 MeC^FV与 GA信号途径存在一定的相互作用 （crosstalk)。
GA预处理明显抑制 Me ^基因的表达，这可能是植物生长与抵抗胁迫需要一定的 平衡： 如果植物需要茁壮生长， 就会降低对恶劣环境的耐受力， 而为了提高抗逆境能力， 就必须以影响正常的生长为代价； 换句话说， 在适宜的环境条件下， 植物会完成正常的 生理周期， 而在逆境环境下， 植物会以牺牲生长为代价来抵抗周边不适环境以求生存。 为进一步研究该基因的组织特异表达情况， 本发明人分别提取了温室 4-5株木薯苗的不 同组织的 RNA，这些组织包括非常幼嫩的顶芽 (AP : apical buds)、幼叶 (YL： young leaves) 成熟叶 (第一片伸展叶 XML： mature leaves) 老叶 (第 10或 1 1片伸展叶) (OL :old leaves) 茎 (ST ： stem) 周边形成层 (CA: cambia)及须根 (RT: fibrous root)等组织， 通过定量 PCR 验证， 该基因主要在茎中高表达， 在成熟叶片及须根中也有微弱的表达， 结果见图 3C。 此结果同样说明： 在幼嫩组织如顶芽、 幼叶， 主要表达与生长相关基因， 而与胁迫响应 相关基因主要在成熟组织如茎、 成熟叶等组织中表达。 这也同样解释了为什么在胁迫条 件下， 生长能力强的幼嫩组织最先受到伤害， 而成熟组织受胁迫表型却不明显。
同样的， 本发明人也检测了 MeC¾F2在冷胁迫、 多种逆境胁迫条件下的表达及组织 特异性表达 (引物：正义引物： atggatgttttctctcactaccc ,反义弓 |物： gcaaccgtgatcagataaagaaga) , 发现 MeC¾F2有类似的表达谱特征， 见图 4。 实施例 3、 MeCSF基因的定位
在分析了 MeCBF 基因在多种胁迫条件及组织表达后， 本发明人通过 Genomewalking (试齐 [J盒： Genome Walker Universal Kit User Manual, 货号为： Cat. No. 638904)禾 B TAIL-PCR(弓 I 物 ： 扩增 MeCBF 1 启 动 子 引 物 ： MeCBFLl-1 ： ttgcagcactgcaggtatcagg ， MeCBFLl-2 ： agggagtgagtcagagtactgagag ， MeCBFLl-3 ： agaagtagagagtgtgagtgtgtaag； 扩增 MeCBF2 启 动子 引 物（两次扩增 结果）： MeCBF2-GSPl-l ： CGAGGAGCACTGCAACCGTGATCAGAT ， MeCBF2-GSPl-2 ： GGGTC AGGGT AGTGAGAGAAAAC ATCC； MeCBF2-GSP2-l： ATATACTCTATCTCTT TGGGCAGCTGT, MeCBF2-GSP2-2: ATGGAGGTTGGTCTATGAAGTAGAGGT)的方法 克隆了 MeCBF 1的启动子 (SEQ ID NO: 5)和 MeCBF2的启动子 (SEQ ID NO: 6)， 分别约 为 lkb 禾 B 1.5kb。 通过分析（软件： PLACE, http：〃 www.dna.affrc.go.jp/PLACE/ signalscan.html)得知， 这两个基因的启动子区域存在多个与胁迫相关的顺式作用元件， 包括与干旱胁迫 (ACGT)、 盐胁迫 (ACGTG)、 冷胁迫 (CANNTG)等相关的基序， 见表 2， 也预示着 MeCBF 1和 MeCBF2参与植物胁迫响应。
表 2、 MeCBFl和 MeC^F 启动子顺式作用元件分析 顺式作用
MeCBFl 启动子 MeCBF2 启动子中起 元件教量
顺式作用
中起始位置 始位置
元件名称
(基于 SEQ ID NO: 5) (基于 SEQ ID NO: 6) MeCBFl MeCBF2
AKR 1 41 0 1 ABA诱 ¾^勺
744, 753,
A K相关 ACGYCiK 574， 583 2 3 Ca++相应的
1340
ABRE 类似
ACGTG 575, 716 745, 1341 2 2 黄化诱导的表达， 十 ， 盐 /erdl
130， 417， 533, 745,
ACGTATERDl ACGT 4 4 脱水胁迫和黑暗诱导的 sensescence
575, 716 878, !341
AIMYB2 TAACTG 658 1 0 水胁迫响应
284, 574， 302, 482, 510, 744, 1155
AtMYC2 CANNTG 3 9 脱水响、 V：， 冷
649 ， 1278, 1306， 1340, !375
754, 501,
CCGC 丽 VCGCGR 584, 592 2 4 Ca++/钙调蛋 信号响应
762， 829
Dcpbl" ACAC NG 1339 0 1 L A型 ¾因， A13A响应
284, 574, 302, 482, 510, 744, 1155 riapA; 储存蛋白; A匿; R-box； 种
ΕΒΟΧΒΝΝΛΡΛ CANNTG 3 9
649 ， 1278, 1306, 1340, 1375 子
(；一 box CACGT (； 574 744， 1340 1 2 防御相关¾因
S0RIJP1 GCCAC 631, 720 817 2 1 Phy oc romc A诱 的：; ¾序
121， 128， 167, 172 29， 266， 800， 876， 1275,
WR Y710S TGAC 5 8 WR Y; GA; WR; W box; PR蛋 [Ί;
， 687 1282, 1632
693, 1106, 1113, 1460， 1
Tat box ΤΛΤΛΛ 494， 538, 844， S6S 4 6
496， 1535
上表序列中， K表示 T或 G; M表示 A或 C; B表示 T或 C或 G; Y表示 C或 T; N表示任意核苷酸； V表示 A或〔或0。
为验证木薯 MeCBF蛋白是否也位于细胞核， 本发明人构建了 pA7-MeC^F7::GFP和 pA7-MeC¾F ::GFP载体， 同时以 pA7-GFP空载体 (购自 TAKARA)为阴性对照， 以拟南 芥 基因为阳性对照， 构建了 pA7-AC^P4::GFP阳性克隆。 载体构建方法如下： 设计 5'端含有 Xhol和 3'端含有 Spel酶切位点的正向和反向引物， 通过 PCR方法， 分 别以木薯和拟南芥基因组为模板， 分别得到目的条带， 再分别与 pMD 18 T vector (购自 TAKARA公司）连接， 通过 Xhol和 Spel双酶切验证目的片段已插入到 T载体中。 选取 测序正确的克隆， 测序过程中发现一个克隆插入的不是 MeCBFl, 而是 MeCBF2， 因此 同时构建了 MeCBF2 瞬时表达载体）， 通过 Xhol 和 Spel 双酶切得到目的带后与 pA7-GFP (购自 Invitrogen公司， 已经 Xhol和 Spel酶切)经 T4连接酶连接， 连接液转化 Top 10大肠杆菌 (购自 Invitrogen公司）感受态后， 挑单克隆， 再经 Xhol和 Spel酶切验证 正确即为最终载体， 可以用于洋葱定位实验。
引物序列如下：
MeCBF 1/2-XhoI cactcgqgATGGATGTTTTCTCTC (SEQ ID NO: 7)
MeCBFl/2-SpeI gcoctog ACAGAAAAACTCCATAATG (SEQ ID NO: 8) AtCBF4-XhoI cactcgqgATGAATCCATTTTACTCTAC (SEQ ID NO: 9) AtCBF4-SpeI tc ctqgtacCTCGTCAAAACTCCAGAG (SEQ ID NO: 10) 通过常规的基因枪轰击洋葱细胞 (分离自商购的普通洋葱)表皮实验， 将目标载体 (即 pA7-MeCBFl : :GF? , pA7-MeCBF2: :GF?载体和 pA7- tCB ^: :GFP)转入细胞内。 激光共 聚焦显微镜观察到： MeCBFl和 MeCBF2绿色 GFP荧光信号除了位于细胞膜外， 主要 存在于细胞核， 与拟南芥的 AtCBF4具有类似的细胞定位， 而 pA7-GFP空载体的荧光信 号则位于整个细胞内（图 5A)。 因此， 确信 MeCBFl和 MeCBF2为核定位蛋白。
另外， 将上述载体通过烟草原生质体瞬时转化实验 (；方法参见 Sheen , J. 2002， A transient expression assay using Arabidopsis mesophyll protoplasts) , 激光共聚焦显微镜观 察至 Ij : MeCBFl和 MeCBF2绿色 GFP荧光信号存在于细胞核， 与拟南芥的 AtCBF4具有 类似的细胞定位， 而 pA7-GFP空载体的荧光信号则位于整个细胞内（图 5B)， 因此可以 确信 MeCBF l和 MeCBF2为核定位蛋白。 同时， 木薯 MeCBF细胞核定位预示其功能是 转录因子。 实施例 4、 MeCBF的生物学功能验证
为了进一步研究 MeCBF的生物学功能，本发明人借助模式生物拟南芥简单快捷的遗 传转化体系， 在拟南芥中过量表达 MeCBF l (pCAMBIA1301 -35S: : MeCBFl , 简称 SM1)。 pCAMBIA1301 -35S: :MeCBFl 的构建以及转基因拟南芥的制备方法如下： 根据已知的 MeCBFl 核苷酸信息， 设计引物（atggatgttttctctcactaccc (SEQ ID NO: 1 1)和 ttatacagaaaaactccataatgg (SEQ ID NO: 12))，以冷处理 TMS60444木薯苗 cDNA为模板 PCR 扩增得到目的带 (实施例 1方法)， 割胶回收后与 pMD 18 T vector载体连接， 连接液转化 Top 10大肠杆菌感受态后， 挑单克隆再经 EcoRI和 Hindlll酶切验证目的带已插入 T载 体后送去测序， 选取测序正确， 同时插入方向是 Sall-ATG-MeCBF l -TAA-Xbal的克隆。 将该克隆通过 Sail和 Xbal双酶切后与改造的 pCAMBIA1301 -35S (改造方法： 在商业化 的 pCAMBIA1301载体 (；购自 Invitrogen公司）的多克隆位点的一端接入 35 S启动子， 另 一端接入 NOS终止子 X已经 Sail和 Xbal双酶切)连接，连接液转化 Top l O大肠杆菌感受 态后， 挑单克隆， 再经 Xhol 和 Spel 酶切验证正确即为最终载体， 将载体转化农杆菌 GV3101 (购自 Invitrogen) , 验证正确后即可用于拟南芥转化。
除非另外说明， 拟南芥转化采用经典的 flower dip方法， 该方法已广泛应用， 可参 考文献： Agrobacterium- mediated transformation of Arabidopsis thaliana using the floral dip method (Nature Protocols 1， 641 - 646 (2006) Xiuren Zhang , Rossana Henriquesl , Shih-Shun Linl， Qi-Wen Niu & Nam-Hai Chua)。
将野生型 Col-0和过表达株系 (SMl-13-3、 SM1-14-19、 SM1-19-10)的种子， 经表面消毒 后铺于基本培养基上。 4°C春化 3天后， 水平或垂直置于 22°C和 10°C条件下培养， 发现 SM1 株系生长状态明显好于野生型， 见图 6。测量 20株 30天 (d)的幼苗鲜重、干重、 叶长和叶宽， 发现在正常培养条件下， 转基因株系的鲜重和干重与野生型无明显差异； 但在低温培养下， 转基因株系的鲜重和干重都明显高于野生型， 见图 7。 同时， 水平低温培养条件下， 转基因 株系的叶长和叶宽都明显大于野生型， 见图 7。
经 -6°C处理两天恢复培养 5天后观察， 发现野生型 Col-0全部死亡， 而转基因 T2代 植株则保持较高的存活率： SM1-19为 86.7%， SM1- 17为 64.7%， 见图 8。
为进一步验证上述结果， 本发明人用平皿实验重复此实验： 在 MS基本培养基上萌发 约 14天的小苗经 -5 °C处理 l lhr， 恢复 5天， 发现 Col-0大部分苗叶片发白， 只有最中央 叶片保留绿色； 相比之下， 转基因苗则大部分叶片呈绿色， 保持良好生存活力， 见图 9。
本发明人还注意到， 在正常培养条件下， 转基因苗生长较野生型矮化， 提示这与组 成型表达 MeCBFl有关。 从这两个实验结果看， MeCBFl转基因苗表现出较野生型明显 抗冷， 说明 MeCBFl为具有抗冷功能的蛋白， 这在拟南芥体系中得到了较好的验证。 本发明人分析了 MeCBFl蛋白的结构域， MeCBFl的主要结构域为 NLS (相对于 SEQ ID NO: 3氨基酸序列， 第 40-53位）、 AP2/EREBP(相对于 SEQ ID NO: 3氨基酸序列， 第 56-1 12位）、 DSW(相对于 SEQ ID NO: 3氨基酸序列， 第 1 14-1 17位）， 见图 2B， 这些结 构域是蛋白发挥抗逆境作用的关键性位点。基于以上分析，本发明人建立了多个 MeCBFl 蛋白的变异体序列， 依次如下：
基于 MeCBFl蛋白 （SEQ ID NO: 3) 序列， 其中第 1 1位氨基酸由 L变异为 I， 获得 MeCBFl -Ml变体。
基于 MeCBFl蛋白 （SEQ ID NO: 3) 序列， 其中第 210位氨基酸由 A变异为 V， 获 得 MeCBFl -M2变体。
基于 MeCBFl 蛋白 （SEQ ID NO: 3) 序列， 其中第 1-3 位氨基酸缺失， 获得 MeCBFl -M3变体。
基于 MeCBFl蛋白 （SEQ ID NO: 3) 序列， 其中第 210-214位氨基酸缺失， 获得 MeCBFl -M4变体。
基于 MeCBFl蛋白 CSEQ ID NO: 3) 序列， 其中 C末端添加 4个氨基酸 ΑΤΑΑ, 获 得 MeCBFl -Μ5变体。
首先将 SEQ ID NO: 1所示的 MeCBFl基因的 CDS序列克隆入 pCAMBIA1301-35S 载体的多克隆位点中， 获得含有该 CDS 的重组载体。 然后根据以上设计的蛋白变体序 列， 利用常规的定点突变技术， 分别对重组载体中编码相应位点氨基酸的碱基进行碱基 突变或碱基缺失或碱基添加， 获得含有相应的变体的重组载体。
将以上构建的重组载体转入农杆菌中， 并转化拟南芥， 分别获得拟南芥转基因植株 Ml-Linel Ml-Line2; M2-Linel、 M2-Line2; M3-Linel M3-Line2; M4-Linel、 M4-Line2; M5-Linel M5-Une2。 用平皿实验进行验证： 在 MS基本培养基上萌发约 14天的小苗经 -5 °C处理 l lhr， 恢复 5天， 观察植株性状。 结果发现， 与野生型 (叶片发白)相比， 转基因苗绝大部分叶 片呈绿色， 保持良好生存活力。 实施例 5、 MeCBFl的抗盐性
在拟南芥体系中， 除了发现 Me ^转基因植株有较明显的抗冷表型外， 本发明人 还观察了其在盐胁迫条件下的萌发率。
为验证 SM1转基因苗在 NaCl培养基上生长是否受影响， 本发明人将转基因和野生 型拟南芥种子先在正常培养基上萌发 4天后转移至 100 mM NaCl培养基上， 培养 16天 后发现， 转基因苗叶片较伸展， 生长状态也较对照好 (图 10)。 以上结果证明， 已正常萌 发的 SM1转基因苗较野生型有更好的抗盐性。 实施例 6、 MeCBFl和 MeCSF2启动子在逆境下诱导表达
MeCBFJ启动子含有多个胁迫诱导元件， 同时 MeC^FV mRNA受逆境诱导表达。 为 进一步验证 MeCBFl 启动子是否受逆境诱导表达， 本发明人构建了 MeCBFl promoter: :GUS载体。 载体构建方法如下： 设计引物并两端加 Pstl和 Ncol酶切位点， 以 TMS60444木薯苗基因组为模板 PCR， 获得目的带后割胶回收连接 pMD 18T-vector (；购自 TAKARA), 之后转化大肠杆菌感受态， 得到阳性克隆， 经测序正确， 通过 Pstl和 Ncol 酶切连入 PCAMBIA1303载体 （获自 CAMBIA公司） 中， 连接液转化大肠杆菌， 挑取阳 性克隆， 经验证正确后转化 GV3101农杆菌， 得到阳性克隆后按照拟南芥转化方法转化 拟南芥。 引物如下：
MeCBFl -P-P: GCCTGCAGTCAAAGTATATTTCGACTTC (SEQ ID NO: 13)；
MeC5 7-P-N: TACCATGGAGAAGTAGAGAGTGTGAGTG (SEQ ID NO: 14)。
将上述构建的载体进行拟南芥转化， 拿到了至少 3个 T3代纯合系。 转基因拟南芥苗 经 4°C处理不同时间后用于 GUS染色， 同时取未经冷处理的拟南芥苗 GUS染色作为对 八眧1 *、。°
结果显示， MeCB ^启动子是受冷诱导表达的， 在冷处理 9-10h时表达最强， 并且这 种表达有组织特异性， 在顶芽， 幼叶和成熟叶的叶脉中高表达， 而在根中未发现 GUS 信号（图 1 1)。 基于类似实验， Me ^启动子也是受冷诱导表达的。
启动子变体：
根据前述实施例 3中的分析， MeCBFl的启动子 (SEQ ID NO: 5)和 MeCBF2的启动子 (SEQ ID NO: 6)存在多个与胁迫相关的顺式作用元件。 经分析其中与干旱胁迫 (ACGT)、 盐胁迫 (ACGTG)、 冷胁迫 (CANNTG)等相关的基序是决定其功能所关键的序列。 因此， 本发明人根据这些关键序列， 在这些序列保守的前体下改变所述启动子其它位点的序 列， 获得一些启动子变体如下：
基于 MeCBFl的启动子 (SEQ ID NO: 5) 序列， 其中第 6位碱基由 G变异为 C， 第 25位碱基由 C变异为 G， 第 318位碱基由 T变异为 A， 获得 MeCS ^7-P l变体。
基于 MeCBFl的启动子 (SEQ ID NO: 5) 序列， 其中第 857位碱基由 T变异为 A， 第 495-497位 ATA缺失， 获得 MeCB ^-P2变体。
基于 Me(¾F7的启动子 (SEQ ID NO: 5) 序列， 其中 5 '端加上碱基 AGTCC , 3 '端缺 失 4个碱基获得 MeCBFl -?3变体。
基于 MeCS 的启动子 CSEQ ID NO: 6) 序列， 其中第 40位碱基由 A变异为 T， 第 243-246碱基缺失， 第 1465位由 C变异为 G， 获得 MeCBF2-?4变体。
基于 MeCBF2的启动子 （SEQ ID NO: 6) 序列， 其中 5 '端添加 4个碱基 ATGG， 第
1576-1583位碱基缺失， 获得 MeCBF2-?5变体。
将上述启动子变体构建入 MeCSW promoter::GUS载体， 构建方法如下： 设计引物 并两端加 Pstl 和 Ncol 酶切位点， 以基因组为模板 PCR， 获得目的带后割胶回收连接 pMD 18T-vector (购自 TAKARA) , 之后转化大肠杆菌感受态， 得到阳性克隆， 经测序正 确， 通过 Pstl和 Ncol酶切连入 pCAMBIA1303载体 （获自 CAMBIA公司） 中， 连接液 转化大肠杆菌， 挑取阳性克隆， 经验证正确后转化 GV3101农杆菌， 得到阳性克隆后按 照拟南芥转化方法转化拟南芥。 将以上构建的重组载体转入农杆菌中， 并转化拟南芥， 分别获得拟南芥转基因植株 P I -Line 1、P1-Line2; P2-Line 1 P2-Line2； M3-Line 1、P3-Line2; P4-Linel P4-Line2; P5-Linel P5-Line2。 转基因拟南芥苗经 4 °C处理不同时间后用于 GUS染色， 同时取未经冷处理的拟南芥苗 GUS染色作为对照。 结果显示， MeCB ^启 动子是受冷诱导表达的， 在冷处理 9-10h时表达最强， 并且这种表达有组织特异性。 实施例 7、 在原核体系中 MeCBFl有明显的抗盐和抗旱功能
利用原核重组蛋白高效表达体系， 本发明人将 MeCBFl基因构建到 pET32a载体中。 构建 pET32a-MeC¾F7载体所需引物如下：
MeCBFl-EcoRl : cggaattcATGGATGTTTTCTCTC (SEQ ID NO: 15)
MeCBFl -Hindlll : ccaagcTTcTACAGAAAAACTCCAT (SEQ ID NO: 16)
通过以上引物克隆到目的带后经测序正确，通过 EcoRl和 Hindlll酶切，连入 pET32a 载体（Novagen 公司）， 连接液转化 BL21 感受态， 经筛选得到阳性克隆 BL21 (pET32a-MeCBFl) 0
通过 IPTG诱导重组蛋白的表达， 分析重组菌 (BL21)在含有不同浓度的 NaCl和甘露 醇（matmitol)的 LB 培养基上的生长情况。 具体方法如下： 分别挑取新活化的 BL21(pET32a-MeCBi^)和对照 BL21(pET32a)单克隆， 接种于 5mlLB(Kan)中， 37 °C振荡 培养过夜。 分别取过夜培养物按 1%比例接菌到 LB(Kan)培养基中（10ml)， 直至 OD 为 0.5-0.8(约培养 2hr)， 加诱导剂 0.5mmol/L IPTG继续培养 4h后用于胁迫实验。 取含有不 同浓度的 NaCl和 mannitol的抗性 LB平板， 将两种菌分别在平板上划线， 倒置培养 12h 左右， 观察这两种菌的生长情况。
结果发现， 在对照 LB培养基上， 两种菌的生长状态类似， 然而在 0.4 M NaCl、 0.6 M NaCl、 0.4 M mannitol和 0.6 M mannitol培养基上， BL21(pET32a- MeC^FV)生长明显好 于对照 BL21(pET32a)(图 12)。
从该结果可以看出， 在原核表达体系中， MeCBFl有明显的抗盐和抗旱功能。
本发明人同时验证了 MeCBFl 的变体 MeCBFl-Ml、 MeCBFl-M2、 MeCBFl -M3
MeCBFl-M4、 MeCBFl -M5的抗盐和抗旱功能。 将它们的编码基因构建到 pET32a载体 中， 转化 BL21， 经过上述同样培养后， 将菌在不同浓度的 NaCl和 mannitol的抗性 LB 平板上划线， 倒置培养， 观察菌的生长情况。 结果发现， 这些菌也能够正常生长， 状态 显著好于对照 BL21(pET32a)。 实施例 8、 MeCBFl具有专一的 DNA结合活性和转录激活活性
为了验证 MeCBFl是否具有 DNA结合活性， 本发明人首先引物合成正常的和突变 的 DRE/CRT序列， 分别命名为 3wDRE-HS/3wDRE-HA禾口 3mDRE-HS2/3mDRE-HA2。 引物序列分别为-
(SEQ ID NO: 17)；
(SEQ ID NO: 18)；
3mDRE-HS2： GTGAATTCTAaatcaATTAaatcaATTAaatcaATTCTAGACT (SEQ ID NO: 19)；
3mDRE-HA2: AGTctagAATtgattTAATtgattTAATtgattTAGAATTCAC (SEQ ID NO: 20)。
通过 PCR方法将正反义引物序列退火成双链，分别命名为野生型 DRE/CRT和突变 型 DRE/CRT, 之后通过 EcoRI, Xbal酶切连入 pHISi载体 （Clontech) 中， 测序验证重 组载 体 构 建 正 确 ， 分 别 获 得 重 组载 体 pHISi-3wDRE-HS/3wDRE-HA 和 pHISi-3mDRE-HS2/3mDRE-HA2 , 如图 13A。
同样，设计引物 MeCBFl-EcoRl : cggaattcATGGATGTTTTCTCTC (SEQ ID NO: 21)，
MeCBFl -Sail: gagtcgaccTACAGAAAAACTCCATA (SEQ ID NO: 22)，以木薯基因组 DNA 为模板， 通过 PCR扩增到目的片段， 连入 pMDT18-vector载体 (；购自 TAKARA)中， 测 序验证片段序列正确性。之后通过 EcoRI和 Sail酶切连入 pGAD424载体 （Clontech) 中， 获得重组载体 pGAD424-MeCB ^， 如图 13A。 按照常规酵母转化方法， 首先分别将重组载体 pHISi-3wDRE-HS/3wDRE-HA 和 pHISi-3mDRE-HS2/3mDRE-HA2转入酵母菌株 （菌株 YM4271 , 购自 Clontech) 中，通过 施加不同浓度的 3-AT筛选压验证转化子对 3 -AT的本底抗性。实验证明， 转化子可以在 缺失 His的培养基上生长， 但添加 15mM的 3-AT即能抑制该菌的生长， 说明获得的转 化子有较低的本底抗性， 可以用于进一步实验。
之后， 本发明人将含有目的基因的 pGAD424载体和空 pGAD424载体分别转化含 正确 DNA序列 (正常的 DRE/CRT序列)和突变 DNA序列 (突变的 DRE/CRT序列）的两种 转化子中， 在 SD/-LeU,-HiS培养基培养 2-3天， 发现所有转化都长出克隆， 但如果施加 15mM的 3-AT, 则只有同时含有目的基因的 pGAD424载体与正确 DNA序列的 pHISi 载体可以长出克隆， 其它的转化都未长出克隆， 如图 13B。 上述结果说明， MeCBFl可 以和正确的 DRE/CRT基序相互作用， 不能和突变的 DRE/CRT基序相互作用， 同样证明 了这种互作的专一性。
为了验证 MeCBFl是否具有转录激活活性，本发明人首先扩增了全长 MeC^F7(SEQ ID NO: 1 , full CDS), 5 '端 -MeCB ^序列（SEQ ID NO: 1中， 第 l-354bp， MeCBFl-N)禾口 3 '端 -MeC^FV序列（SEQ ID NO: 1中， 第 355-660bp， MeCBFl-C), 连入 pMDT18-vector 载体 (；购自 TAKARA)中， 测序正确后通过 EcoRI禾 B Sail 酶切连入 pGBKT7载体 (；购自 Clontech)中， 之后分别将其转化酵母菌株 (；酵母菌株： AH109, 购自 Clontech) , pGBKT7 空载体为阴性对照。 所有转化子都可以在 SD/-Trp生长， 但只有阳性转化子可以在三缺 的培养板上 (SD/-Trp， -His, -Ade)生长， 同时在培养基上加 X-Gal， 阳性转化子可以显 示蓝色。结果显示， 全长 MeCBFl及 3 '端 -MeCBFl具有转录激活活性， 而 5 '端 -MeCBFl 是没有激活活性的， 如图 14。 实施例 9、 MeCBFl过表达拟南芥植株具有较强的抗盐和干旱能力
将 pCAMBIAlSOU . MeC^FV转基因拟南芥种子消毒后点播在含 NaCl的培养基 上， 培养 2周后， 发现转基因拟南芥植株叶片颜色偏绿， 叶片数目多， 在高度的盐胁迫 条件下苗的存活率高， 因此， 转基因材料较野生型明显抗盐， 如图 15。
之后，本发明人检测了盐胁迫条件下转基因和野生型材料地上部分的 Na+和 K+浓度， 结果如图 16所示， 发现转基因株系的 Na+浓度较野生型低， 而 K+浓度较野生型高。 这 个结果说明转基因苗通过降低地上部分 Na+浓度， 增加 K+浓度来对抗盐胁迫。 聚乙二醇 PEG处理可以引发渗透胁迫， 模拟干旱胁迫。 本发明人将 pCAMBIABOl ^. MeC^FJ 转基因拟南芥和野生型种子消毒后点播在正常培养基上， 待其萌发两天后转移至含 20%PEG的培养基上， 检测过表达植株对于渗透胁迫是否具有抗性。
经过 20天的培养， 发现大多数转基因苗可以形成明显的子叶， 而野生型生长受到 明显的抑制， 基本不能形成明显的子叶， 如图 17。 因此， MeCBFl过表达拟南芥植株具 有较强的抗渗透胁迫能力。
为进一步验证转基因植株的抗干旱能力， 将温室生长约 10天的野生型和转基因拟 南芥植株进行干旱处理。 种植前倒入相同质量的土壤， 干旱处理开始阶段， 选择生长一 致的盆栽， 测定土壤相对含水量， 保持每个盆中土壤湿度一致后停止浇水。 结果发现野 生型和转基因株系虽然都可以抽苔完成生长周期， 但是可以看到野生型干枯死亡， 而转 基因苗仍然存活， 见图 18。 实施例 10、 MeCBFl过表达拟南芥植株具有较强的抗氧化胁迫能力
DAB染液常用于检测 ¾02的积累量。 因此， 本实施例中， 本发明人采用 DAB染 色法检测 MeCBFl过表达拟南芥植株的抗氧化胁迫能力。
本发明人将在正常培养基上生长 14天左右的幼苗低温 (10°C)处理 7天后进行 DAB 染色。
DAB染色方法如下： 在 50ml离心管中加入 10ml DAB染色液， 取一定量用于染叶片， 抽真空 lOmin; 将叶片浸没于 DAB液体中， 放置在光下， 随时观察颜色变化， 染色 6-8h; 显色时， 倒掉染色液， 加入 95%乙醇， 80°C脱色 10min。
H202含量的测定如下： l)lg叶片 +10ml 0.1%预冷的 TCA溶液， 冰浴上研磨， 勾浆液 12000g, 15min; 2)lml上清， 添加 lml lOOmM磷酸盐缓冲液 (PBS)(pH7.0)， 2ml KI(lmol/L); 摇勾，静置片刻 (10min)， 390nm测 OD值 (紫外分光光度计)； 3)根据标准曲线求得 ¾02含量。
CAT酶和 SOD酶活测定方法如下：
1) lg的样品加入 5ml酶提取缓冲液 C50mM磷酸缓冲液， 1% PVP， ImM EDTA)研磨，
4°C， lOOOOrpm离心 20min， 取上清， 将酶液转移至 EP管中 (约 3-4ml)， 置冰上；
2) CAT 酶活测定： 2ml 反应体系 =1.6ml酶提取缓冲液 +0.2ml 0.1mol/L 酶液 +0.2ml O.lmol/L的 ¾02。加完 ¾02立即计时，试管上下颠倒混勾， 240nm下测定吸光度，每隔 lmin 读数 1次， 共测 5min。 以 lmin内 A240减少 0.1的酶量为 1个酶活单位 (u);
3) SOD酶活测定： 显色反应： 取透明度、 质地相同玻璃试管 5支， 3支为测定、 2支为 对照， 按表 3加入试剂。
表 3
试 剂 (酶） 用量 (ml) 终浓度 (比色时）
酶提取缓冲液 1.8
130mmol/L Met溶液 0.3 13mmol
750μηιο1/ί ΝΒΤ溶液 0.3 75μηιο1
20μηιο1/ί核黄素 0.3 2.0μηιο1
酶 液 0.3 空白和对照管加
总体积 3 缓冲液代替酶液 按照表 3混勾后，给 1支对照管罩上比试管稍长的双层黑色硬纸套遮光，与其他各管同 时置于 4000 lx日光灯下反应 lOmin (要求各管照光情况一致，反应温度控制在 25〜35 °C之间， 视酶活性高低适当调整反应时间)。 当对照管变蓝， 而样品管仍为黄色时， 结束反应， 测定 各管的吸光度。
SOD活性 = (A0-AS)xVT/A0x0.5xFWxVl
AO: 照光对照光管的光吸收值； AS: 样品管的光吸收值； VT: 样液总体积 (ml); VI： 测定时样品用量 (ml); F W: 样品鲜重 (g)。
DAB 染色检测结果， 发现转基因苗和野生型都被染成褐色， 但是转基因染色较野 生型浅， 如图 19， 说明在低温处理下， 转基因材料积累的 ¾02量可能少于野生型。 本 发明人检测了低温处理条件下， 转基因和野生型材料的 ¾02的积累量， 发现无论在正 常条件下还是低温胁迫条件下， 转基因苗中积累的 ¾02量明显少于野生型材料， 如图 20， 从而验证上面的推测。
植物具有 ROS清除机制， 其中超氧化物歧化酶 (SOD)和过氧化氢酶 (Catalase, CAT) 发挥重要作用。 由于在低温处理条件下， 转基因苗具有较低的 ¾02含量， 本发明人进 一步检测了这两种酶的酶活性， 发现在正常条件下野生型和转基因苗有相同的酶活性， 但在低温处理条件下， 转基因苗中 SOD和 CAT的酶活性高于野生型， 如图 21。 该结果 提示， 由于这两种酶活性的提高， 降低了 H202含量， 使转基因苗具有较高的抗氧化胁 迫的能力。 实施例 11、 MeCBFl过表达木薯植株的获得
为 了进一步验证 MeCBFl 的功能， 本发明人将同样的过表达载体 (pCAMBIAl 301 -35S:: MeCBFl ,简称 SMI) 转化了木薯，得到了 3个独立的转基因株系， 为验证 MeCBFl过表达转基因木薯的拷贝数， 通过 Southern blotting方法鉴定出两个单 拷贝株系（SM1-1， SM1-2)和一个多拷贝株系（SM1-3), 如图 22。
在模式植物拟南芥中 CBF介导的冷胁迫信号通路已研究的比较透彻， CBF作为早 期冷胁迫响应的转录因子， 能激活下游 COR等基因的表达， 这其中包括 FAD7(脂肪酸 去不饱和酶)、 COR47(脱水素蛋白）、 KIN1(抗冻蛋白）、 COR15a (定位于叶绿体， 保护叶 绿体）、 GolS3(肌醇半乳糖苷转移酶 3)、 Rd29a、 COR6.6(co Id regulated gene)。 以这些基 因的 DNA和 cDNA序列在木薯基因组中 Blast比对， 仅 FAD7、 COR47和 GolS3找到木 薯同源基因， 分别命名为 MeFAD7、 MeCOR47和 MeGolS3。
首先， 本发明人检测了 MeC^FV过表达木薯中这三个基因的表达情况， 以 Meactin 为内参。 从图 23中可以看出， 在木薯体系中同样存在着 CBF信号通路， 且 MeCB ^过 表达可以使这些下游基因的表达量上调。 但是， 从另一个方面可以看出， 木薯中的 CBF 信号通路并不完善， 因为在木薯基因组中， 未找到许多在拟南芥中广为报道的下游基因 的同源序列。 这提示木薯不完善或者不强大的抗逆信号通路使木薯对低温非常敏感。 用于测定各基因表达情况的引物序列如下：
MeFAD7-F: CCATGATTGTGGTCATGGGAGC (SEQ ID NO: 23)；
MeFAD7-R: GGTACCAGGGAAGTTTGTCCTC (SEQ ID NO: 24)；
MeCOR47-F: CTGAGGAGCACCACAACAAGG (SEQ ID NO: 25)；
MeCOR47-R: GGCGTAGCTACCTCTTCAGGC (SEQ ID NO: 26)；
MeGolS3-F: GGCTTACGTGACTTTCTTGGC (SEQ ID NO: 27)；
MeGolS3-R: CACTGTTGGCAGTAGCCGATC (SEQ ID NO: 28)。
同时， 本发明人以 MeCBFl全长蛋白为抗原常规方法制备了抗 MeCBFl 的多克隆 抗体。 为验证过表达木薯中 MeCBFl蛋白含量是否增加， 提取了转基因植株和野生型对 照中的核蛋白。 Western blotting结果表明， 在转基因木薯植株中 MeCBFl蛋白有增加， 并且在单拷贝植株的增加量多于多拷贝的株系， 以组蛋白 H3为内参， 如图 24。
实施例 12、 MeCBFl过表达木薯植株具有具有明显的抗冷能力
将继代 T1代约 10天的木薯组培苗放 4°C光照培养箱中处理 10天后恢复培养， 发 现转基因苗部分叶片保持绿色， 芽点仍有活力， 而对照 C3 叶片全部发白， 芽点失去活 力， 在后续的培养中， 转基因苗仍可以继续生长， 而 C3却不能， 如图 25。 此结果说明 MeCBFl过表达的木薯材料具有较高的抗冷能力， 同样证明了 MeCBFl蛋白也有同其他 物种保守的抗低温功能。 为进一步验证转基因材料的抗冷性， 本发明人将转基因和对照 材料种于田间， 观察自然界的低温天气对其影响。 由于今年入秋早， 并且多次寒流袭击 过实验所在地上海，实验从 1 1月中旬开始，本发明人观察到转基因材料到 11月底为止， 叶片完整， 顶芽很少焦化或完全健壮。相比之下， 野生型对照材料 (C3)则叶子枯黄焦化， 顶芽大面积焦化， 如图 26。 从大田试验看， 木薯转基因材料也明显好于野生型， 表现出 较好的抗冷性。
本发明人观察田间木薯表型， 发现野生型 C3的顶部叶片变黄， 而转基因植株的叶 片保持较好的绿色， 为量化叶片颜色的改变， 取相同位置的转基因和野生型叶片， 95% 乙醇过夜抽提， 紫外分光光度计测定叶绿素 (Chi)含量。 叶绿素含量测定方法如下： 叶片浸泡 95%酒精在黑暗中 12-24h提取色素， 至叶片白色为止， 分光光度计法测 定 OD663(nm)和 OD646(nm); 计算公式如下：
Chi a=12.21 *A663-2.81A646;
Chi b=20.13*A646-5.03*A663 ;
Chi a+ Chi b =8.02*A663+20.2A646。
从测量结果可以看出， 转基因木薯叶片的叶绿素含量明显高于野生型， 如图 27A。 同时， 由于转基因材料较野生型抗冷， 本发明人检测了叶片中的脯氨酸含量， 脯氨 酸含量的测定方法如下：
(1) 脯氨酸提取： 取不同处理的剪碎混勾待测叶片 0.5g (干样根据水分含量酌减)， 分别置于大试管中， 加入 5ml 3%磺基水杨酸溶液， 管口加盖玻璃球， 于沸水浴中浸提 10min。 冷却至室温后， 以 3000rpm离心 lOmin;
(2) 吸取上清液 2ml， 加 2ml冰乙酸和 4ml显色液， 于沸水浴中加热 60min， 下一 步操作按标准曲线制做方法进行甲苯萃取和比色。
(3) 结果计算： 从标准曲线中查出测定液中脯氨酸浓度， 按下式计算样品中脯氨酸 含量的百分数。 脯氨酸 Oig/g FW或 DW)= W
式中， C: 提取液中脯氨酸浓度 g)， 由标准曲线求得；
V： 提取液总体积 (ml);
A: 测定时所吸取的体积 (ml);
W: 样品重 (g)。
计算： 脯氨酸 (microg*g-l *FW)=(C*V/a)/W=C*2.5/W
C: 浓度， a:2ml， V： 5ml, W: 0.5000g。
脯氨酸含量的测定结果显示， 转基因材料的叶片含有较高的脯氨酸， 如图 27B。 这 提示转基因材料的细胞质中有较高的渗透调节物质 (如脯氨酸等)， 从而使其具有较强的 抗冷胁迫的能力。 实施例 13、 MeCBFl过表达木薯植株具有具有明显的抗氧化胁迫的能力
甲基紫精 (MV)可以诱导植物氧化， 引起叶片叶绿素含量降低， 表现出叶片发白， 为一种量化指标。本实施例采用 MV处理木薯过表达植株， 以观察其抗氧化胁迫的能力。
MV染色方法如下： 玻璃平皿放上 2张滤纸， 加入 25ml lOOuM的 MV工作液， 将叶片 放置在液体中， 20持续光照条件放置， 观察叶片变化。
100 uM MV处理 MeCBFl过表达木薯组培叶片 24 h后， 本发明人发现转基因苗可 以更强的抵抗 MV处理， 表现出叶片偏绿， 而野生型 C3则明显变白， 如图 28。
本发明人测量了 MV处理前和 MV处理后叶片的叶绿素含量。 结果显示， MV处理 后， 野生型叶绿素含量明显降低， 而转基因植株的叶绿素含量明显高于野生型 (p<0.01)， 如图 29。 实施例 14、 MeCBFl过表达木薯植株具有具有明显的抗失水能力
将离体的组培苗叶片置于干燥的滤纸上， 每隔一段时间拍照记录叶片失水情况， 发 现转基因木薯失水速率较野生型慢， 在同样的处理时间下， 野生型更容易失水萎蔫如图 30。
经过 1小时的离体干旱处理， 转基因与野生型的叶片都明显失水萎蔫， 之后在平皿 中加等量的水， 观察叶片的复水能力。 结果发现， 转基因植株的叶片基本恢复伸展， 如 脱水处理前的表型， 而 C3仍然萎蔫， 复水效果较慢， 如图 31。
为量化叶片的失水速率， 每 15分钟对叶片称重， 按照 (FW0-FW1)/FW0公式计算特 定时间点的失水速率， 绘制曲线， 如图 32。 可以看出， 转基因苗失水速率较野生型低。 实施例 15、 MeCBFl过表达木薯植株具有具有较强的抗干旱能力
温室生长约 3个月的木薯扦插苗， WT和转基因材料生长状态良好。 停止浇水 3周 后， WT木薯叶片多变白干枯， 并且大部分已脱落； 而过表达株系底部仅有部分叶片干 枯， 顶部叶片仍呈现绿色， 如图 33。
同时我们发现，温室生长 3个月木薯扦插苗，每 3天浇等量且过量的含 200 mM NaCl 的盐水， 3周后， 本发明人发现， 过表达木薯的生长状态明显好于野生型， 表明其有较 强的抗盐能力。
2011-2012年度将 MeCB ^过表达木薯种于转基因试验田，观察转基因植株表型及块 根产量。 经过约 6个月的栽培， 过表达木薯株高与块根产量与野生型相似， 见图 34。
综上实施例， 本发明人从木薯基因组中分离得到了两个新的 MeCBF蛋白， 分别为 MeCBFl和 MeCBF2， 通过冷胁迫处理下的 MeC^P基因表达量变化及启动子顺式作用 元件分析，说明 MeCBFl和 MeCBF2参与了植物抗逆境信号途径。细胞定位和酵母实验 证明了 MeCBFl可能以转录因子的活性发挥功能。转基因实验也证明了 MeCBFl过表达 植株较强的抗胁迫能力， 如： 低温， 盐， 渗透和氧化等， 进一步验证了此蛋白的功能。 同样的， 本发明人也在木薯中过表达了 MeCBFl , 胁迫处理及生理实验证明 MeCBFl过 表达的木薯植株具有较高的抗冷， 氧化和失水能力。 本发明通过在拟南芥和木薯两套体 系中证明了 MeCBFl蛋白具有抗冷， 氧化等多种胁迫的能力， 为获得抗冷能力提高的转 基因木薯提供了新的策略与可能。同时对 MeCBFl分子机制的研究也为研究木薯及热带 作物低温响应的分子机制奠定了坚实的基础。 在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引 用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员 可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所附权利要求书所限定 的范围。

Claims

权 利 要 求
1. 一种分离的多肽， 其特征在于， 所述多肽选自下组- (a) 如 SEQ ID NO: 3所示氨基酸序列的多肽；
(b) 如 SEQ ID NO: 4所示氨基酸序列的多肽； 或
(c) 将 SEQ ID NO: 3或 SEQ ID NO: 4所示氨基酸序列经过一个或多个氨基酸残基的 取代、 缺失或添加而形成的， 且具有提高植物抗逆境能力功能的由 (a)或 (b)衍生的多肽。
2. 如权利要求 1所述的多肽，其特征在于，该多肽是如 SEQ ID NO: 3或 SEQ ID NO: 4所示氨基酸序列的多肽； 或
该多肽是相对于 SEQ ID NO: 3氨基酸序列， 第 40-53位、 第 56-112位、 第 1 14-117 位氨基酸序列不变而其它位置经过一个或多个氨基酸残基的取代、 缺失或添加而形成 的， 且具有提高植物抗逆境能力功能的由 (a)衍生的多肽； 或
该多肽是相对于 SEQ ID NO: 4， 第 52-65位、 第 68-124位、 第 126-129位氨基酸序 列不变而其它位置经过一个或多个氨基酸残基的取代、 缺失或添加而形成的， 且具有提 高植物抗逆境能力功能的由 (b)衍生的多肽。
3. 一种分离的多核苷酸， 其特征在于， 它包含一核苷酸序列， 该核苷酸序列选自下 组：
(1) 编码如权利要求 1或 2所述多肽的多核苷酸； 或
(2) 与多核苷酸 （1) 互补的多核苷酸。
4. 如权利要求 3所述的多核苷酸， 其特征在于， 该多核苷酸编码如 SEQ ID NO: 3 或 SEQ ID NO: 4所示氨基酸序列的多肽。
5. 如权利要求 3所述的多核苷酸， 其特征在于， 该多核苷酸：
(i) 核苷酸序列如 SEQ ID NO: 1所示； 或
(ϋ) 核苷酸序列如 SEQ ID NO: 2所示。
6. 一种载体， 其特征在于， 它含有权利要求 3-5中任一项所述的多核苷酸。
7. 一种遗传工程化的宿主细胞， 其特征在于， 它含有权利要求 6所述的载体。
8. 一种制备植物的方法， 其包括将权利要求 3-5中任一项所述的多核苷酸转入植物 中。
9. 如权利要求 8所述的方法， 其特征在于， 所述方法包括：
(1) 提供携带表达载体的农杆菌， 所述的表达载体含有权利要求 3-5任一所述的多核 苷酸；
(2) 将植物细胞或组织或器官与步骤 (1)中的农杆菌接触，从而使权利要求 3-5任一所 述的多核苷酸转入植物细胞。
10. 一种权利要求 1所述的多肽的制备方法， 其特征在于， 该方法包含：
(a) 在适合表达的条件下， 培养权利要求 7所述的宿主细胞；
(b) 从培养物中分离出权利要求 1所述的多肽。
11. 权利要求 1或 2所述的多肽或编码该多肽的多核苷酸的用途，用于提高植物抗逆 境能力或制备提高植物抗逆境能力的物质。
12. 如权利要求 11所述的用途， 其特征在于， 所述的逆境是冷环境、 高盐环境、 干 旱环境、 氧化环境、 渗透环境。
13. 如权利要求 11所述的用途， 其特征在于， 还用于：
降低植物地上部分的 Na+浓度， 增加 K+浓度；
降低植物中积累的 ¾02量；
提高植物中超氧化物歧化酶或过氧化氢酶的酶活性；
提高植物中 CBF信号通路基因的表达；
提高植物中叶绿素含量；
提高植物中脯氨酸含量；
提高植物的抗失水能力；
改善植物在冷环境下的生长状态；
提高植物在冷环境下的生物量；
提高植物在冷环境下的叶长叶宽。
14. 一种提高植物抗逆境能力的方法， 其特征在于， 所述方法包括： 提高植物中的权 利要求 1所述的多肽的表达或活性。
15. 一种权利要求 1所述的多肽或其编码基因的激动剂。
16. 一种受逆境诱导表达的启动子， 其特征在于， 所述的启动子选自下组：
(1) 如 SEQ ID NO: 5或 SEQ ID NO: 6所示的核苷酸序列的多核苷酸；
(2) 在严格条件下能够与 (1)限定的多核苷酸序列杂交且具有诱导目的基因在逆境下 表达功能的多核苷酸； 或
(3) 与 SEQ ID NO: 5或 SEQ ID NO: 6所示的核苷酸序列具有 70%以上相同性且具有 诱导目的基因在逆境下表达功能的多核苷酸。
17. 如权利要求 16所述的启动子， 其特征在于， 所述的启动子选自下组： 相对于 SEQ ID NO: 5的核苷酸序列， 第 574-579位、 第 716-720位、 第 130-133位、 第 417-420位、 284-289、 第 649-654位碱基序列不变而其它位置经过一个或多个碱基的 取代、 缺失或添加而形成的， 且具有诱导目的基因在逆境下表达功能的多核苷酸； 或 相对于 SEQ ID NO: 6的核苷酸序列， 第 745-749位、 第 1340-1345位、 第 533-536 位、 第 878-881位、 第 302-307位、 第 482-487位、 第 510-515位、 第 744-749位、 第 1155-1160位、 第 1278-1283位、 第 1306-131 1位、 第 1375-1380位碱基序列不变而其它 位置经过一个或多个碱基的取代、 缺失或添加而形成的， 且具有诱导目的基因在逆境下 表达功能的多核苷酸。
18. 权利要求 16或 17所述的启动子的用途， 其特征在于， 所述的启动子用于诱导目 的基因在逆境下表达。
19. 一种构建物， 其特征在于， 所述的构建物含有权利要求 15或 16所述的受逆境诱 导表达的启动子。
20. 如权利要求 19所述的构建物， 其特征在于， 所述的构建物含有以下可操作性连 接的元件：
权利要求 16或 17所述的启动子； 和
目的基因。
PCT/CN2013/075532 2012-05-11 2013-05-13 一种提高植物抗逆境能力的基因及其用途 WO2013166996A1 (zh)

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BR102013006244A2 (pt) &#34;mêtodo para aprimorar uma ou mais características relacionadas ao rendimento em plantas em relação a plantas de controle, ácido nucléico isolado, polipepíideo isolado, construção de super-expressão, planta transgênica, cêlula hospedeira, uso, método para a produção de uma planta transgênica, parte colheitável de uma planta, produto derivado, dna cromossômico recombinante, construção de expressão, grão de pólen transgênico e composição&#34;
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