CN112111508A

CN112111508A - Cotton stress-tolerant gene GhCBF and coding protein and application thereof

Info

Publication number: CN112111508A
Application number: CN201910539228.0A
Authority: CN
Inventors: 李建平; 黄全生; 郝晓燕; 足木热木·吐尔逊; 高升旗; 常晓春; 胡文冉; 陈果
Original assignee: Xinjiang Academy Of Agricultural Sciences Institute Of Nuclear Technology Biotechnology (xinjiang Uygur Autonomous Region Biotechnology Research Center)
Current assignee: Xinjiang Academy Of Agricultural Sciences Institute Of Nuclear Technology Biotechnology (xinjiang Uygur Autonomous Region Biotechnology Research Center)
Priority date: 2019-06-20
Filing date: 2019-06-20
Publication date: 2020-12-22

Abstract

The invention provides a cotton stress tolerance gene GhCBF, and a coding protein and application thereof, and particularly can remarkably enhance the stress tolerance of plants (such as cotton plants) by improving the expression or activity of the GhCBF gene or the coding protein thereof.

Description

Cotton stress-tolerant gene GhCBF and coding protein and application thereof

Technical Field

The invention relates to the field of botany, in particular to a cotton stress tolerance gene GhCBF, and a coding protein and application thereof.

Background

Plants are affected by low temperatures and their photosynthetic capacity, ability to synthesize energy and substances are reduced, eventually leading to the possibility of death. However, plants improve their tolerance in low temperature environments in order to adapt to the environment, and have a complex set of low temperature signal transmission and regulation mechanisms.

CBFs regulate the expression of a number of genes, the products of which are involved in phosphoinositide metabolism, transcription, biosynthesis of permeating substances, ROS detoxification, membrane transport, hormone metabolism, signaling, and the synthesis of many other substances known or presumed to have a cytoprotective function.

Cotton including upland cotton and island cotton is a kind of economic crop sensitive to low temperature, and in most cotton planting areas in China, the cotton seedlings are in a low-temperature stress environment due to unstable temperature and sudden cold flow in spring, so that the cotton planting and the cotton growth are greatly damaged, and the cotton yield is reduced.

Therefore, there is an urgent need in the art to develop methods for improving the low temperature tolerance of plants and to study the correlation between the improvement of the low temperature tolerance of plants and the yield and quality of plants (such as cotton).

Disclosure of Invention

The invention aims to provide a method for improving the low-temperature tolerance of a plant and research the correlation between the improvement of the low-temperature tolerance of the plant and the yield and the quality of the plant (such as cotton).

The invention provides a use of GhCBF gene or its coding protein, which is used for one or more of the following:

(a) for preparing agents or compositions for improving stress resistance in plants;

(b) is used for improving the stress resistance of plants.

In another preferred embodiment, the stress resistance comprises cold resistance.

In another preferred embodiment, the cold resistance comprises one or more properties selected from the group consisting of:

(a) enhancing the tolerance of the plant to low-temperature environment;

(b) improving the survival ability of the plant in low temperature environment.

In another preferred embodiment, the plant comprises a malvaceae plant.

In another preferred embodiment, the plant comprises a cotton plant.

In another preferred embodiment, the plant comprises cotton.

In another preferred example, the GhCBF gene includes a GhCBF protein encoding gene and a GhCBF gene conserved region sequence.

In another preferred example, the GhCBF gene includes a wild-type GhCBF gene and a mutant GhCBF gene.

In another preferred embodiment, the mutant form comprises a mutant form in which the function of the encoded protein is not altered after mutation (i.e., the function is the same or substantially the same as the wild-type encoded protein).

In another preferred embodiment, the polypeptide encoded by the mutant type GhCBF gene is the same as or substantially the same as the polypeptide encoded by the wild type GhCBF gene.

In another preferred embodiment, the mutant GhCBF gene comprises a polynucleotide having a homology of 80% or more (preferably 90% or more, more preferably 95% or more) with respect to the wild-type GhCBF gene.

In another preferred example, the mutant type GhCBF gene includes a polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or 3' end of the wild type GhCBF gene.

In another preferred embodiment, the GhCBF gene is selected from the group consisting of: a cDNA sequence, a genomic sequence, or a combination thereof.

In another preferred embodiment, the amino acid sequence of the GhCBF protein is selected from the group consisting of:

(i) a polypeptide having an amino acid sequence as set forth in SEQ ID No. 2;

(ii) (ii) a polypeptide which is formed by substituting, deleting or adding one or more (such as 1-10) amino acid residues of the amino acid sequence shown in SEQ ID NO.2, has the function of improving the stress resistance of plants and is derived from (i); or

(iii) The homology of the amino acid sequence and the amino acid sequence shown in SEQ ID NO.2 is more than or equal to 80 percent (preferably more than or equal to 90 percent, more preferably more than or equal to 95 percent or more than or equal to 98 percent), and the polypeptide has the GhCBF activity.

In another preferred embodiment, the nucleotide sequence of the GhCBF gene is selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID No. 2;

(b) a polynucleotide having a sequence as shown in SEQ ID No. 1;

(c) polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95%) with the sequence shown in SEQ ID No. 1;

(d) a polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or the 3' end of the polynucleotide shown in SEQ ID No. 1;

(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).

In another preferred example, the nucleotide sequence of the GhCBF gene is shown in SEQ ID No. 1.

In another preferred example, the coding protein of the GhCBF gene is shown in SEQ ID No. 2.

In another preferred embodiment, the GhCBF gene is derived from a plant, preferably from a plant of the family malvaceae, more preferably from cotton, most preferably from gossypium hirsutum.

In another preferred embodiment, the composition comprises an agricultural composition.

In a second aspect, the present invention provides a method for improving stress resistance of a plant, comprising the steps of:

(a) introducing an exogenous construct into a plant cell, wherein the construct comprises an exogenous GhCBF gene sequence, an exogenous nucleotide sequence that promotes expression of the GhCBF gene, thereby obtaining a plant cell into which the exogenous construct is introduced;

(b) regenerating the plant cell into which the exogenous construct is introduced, obtained in the previous step, into a plant: and

(c) optionally identifying said regenerated plants, thereby obtaining plants having increased plant stress resistance.

In another preferred embodiment, the exogenous GhCBF gene sequence further comprises a promoter and/or a terminator operably linked to the ORF sequence.

In another preferred embodiment, the promoter is selected from the group consisting of: constitutive promoters, tissue specific promoters, inducible promoters, and strong promoters.

In another preferred embodiment, the constitutive promoter comprises a 35S promoter.

In a third aspect, the present invention provides a method for improving stress resistance of a plant, the method comprising the steps of: in the plant, the expression of a GhCBF gene is promoted or the activity of a GhCBF protein is promoted.

In another preferred embodiment, the method comprises administering to the plant an enhancer of the GhCBF gene or polypeptide encoded thereby.

In another preferred embodiment, the method comprises introducing an exogenous GhCBF gene into the plant.

In another preferred example, the method comprises the steps of:

(i) providing a plant or plant cell; and

(ii) introducing a GhCBF gene sequence into said plant or plant cell, thereby obtaining a transgenic plant or plant cell.

In another preferred example, the method comprises the steps of:

(a) providing agrobacterium carrying an expression vector of a GhCBF gene sequence;

(b) contacting a plant cell or tissue or organ with the agrobacterium of step (a) such that the gene sequence for GhCBF is transferred into the plant cell and integrated into the chromosome of the plant cell;

(c) selecting plant cells or tissues or organs which are transferred with the GhCBF gene sequence; and

(d) regenerating the plant cell or tissue or organ of step (c) into a plant.

The fourth aspect of the invention provides an application of a regulator of a GhCBF gene or a coding protein thereof in regulating and controlling the stress resistance of plants or preparing a reagent or a composition for regulating and controlling the stress resistance of plants.

In another preferred embodiment, the regulating agent comprises an accelerating agent and an inhibiting agent.

In another preferred embodiment, the promoter is a substance which promotes the expression of the GhCBF gene or the protein coded by the GhCBF gene.

In another preferred embodiment, the inhibitor is selected from the group consisting of: antisense nucleic acids, antibodies, small molecule compounds, criprpr reagents, siRNA, shRNA, miRNA, small molecule ligands, or combinations thereof.

In another preferred embodiment, the accelerator is selected from the group consisting of: a small molecule compound, a nucleic acid molecule, or a combination thereof.

In another preferred embodiment, the regulator is an accelerant, and the regulation refers to enhancing the stress resistance of the plant.

In another preferred embodiment, the modulator is an inhibitor, and the modulation is the attenuation of stress resistance in a plant.

In another preferred embodiment, the modulator comprises a small molecule compound, or a nucleic acid.

In another preferred embodiment, the nucleic acid is selected from the group consisting of: miRNA, shRNA, siRNA, or a combination thereof.

The fifth aspect of the present invention provides a method for screening a substance that modulates stress resistance of a plant, the method comprising the steps of:

a) administering a test substance to a plant;

b) detecting the activity or expression condition of the GhCBF gene or the coding protein thereof in the plant;

if the activity or expression of the GhCBF gene or the protein coded by the GhCBF gene is up-regulated compared with that of a control plant not administered with a test substance, the test substance is a candidate substance for enhancing the stress resistance of the plant; and/or

The test substance is a candidate substance for reducing the stress resistance of a plant if the activity or expression of the GhCBF gene or its encoded protein is down-regulated compared to a control plant to which the test substance has not been administered.

In a sixth aspect, the present invention provides a method for preparing a genetically engineered plant tissue or plant cell, comprising the steps of:

regulating and controlling the expression and/or activity of GhCBF gene or its coded protein in plant tissue or plant cell so as to obtain the plant tissue or plant cell with gene engineering.

In another preferred example, the modulation comprises increasing the expression and/or activity of the GhCBF gene or protein encoding it in plant tissue or plant cells; and/or reducing the expression and/or activity of the GhCBF gene or its encoded protein in plant tissues or plant cells.

In another preferred embodiment, the regulation further comprises introducing an promoter or inhibitor of the GhCBF gene or its encoded protein into plant tissue or plant cells.

In another preferred embodiment, the expression level or activity of the GhCBF gene or the protein encoded by the GhCBF gene in the plant tissue or the plant cells is reduced by more than or equal to 50 percent, preferably more than or equal to 70 percent, and more preferably more than or equal to 90 percent or 100 percent.

In another preferred embodiment, the "decrease" means that the decrease in expression or activity of the GhCBF gene or its encoded protein satisfies the following condition:

the ratio of A1/A0 is less than or equal to 80 percent, preferably less than or equal to 50 percent, more preferably less than or equal to 20 percent, and most preferably 0 to 10 percent; wherein, A1 is the expression or activity of GhCBF gene or its coding protein; a0 is the expression or activity of the same GhCBF gene or its coded protein in wild-type plant tissue or plant cells of the same type.

In another preferred embodiment, said reduction means that the expression level of the GhCBF gene or protein encoding it in said plant tissue or plant cells E1 is 0-80%, preferably 0-60%, more preferably 0-40% of the wild type, compared to the expression level of the wild type GhCBF gene or protein encoding it E0.

In another preferred embodiment, said reducing the expression or activity of the GhCBF gene or its encoded protein in plant tissues or plant cells is effected by a means selected from the group consisting of: gene mutation, gene knockout, gene disruption, RNA interference technology, criprpr technology, ZFN (zinc finger endonuclease technology), TALEN (transcription activator-like effector nuclease), VIGS technology, or a combination thereof.

In another preferred embodiment, the method further comprises introducing an promoter or inhibitor of the GhCBF gene or its encoded protein into the plant tissue or plant cells.

The seventh aspect of the present invention provides a method for preparing a genetically engineered plant, comprising the steps of:

the genetically engineered plant tissue or plant cell prepared by the method of the sixth aspect of the present invention is regenerated into a plant body, thereby obtaining a genetically engineered plant.

In an eighth aspect, the present invention provides a genetically engineered plant prepared by the method of the seventh aspect.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the survival rate of upland cotton TM-1 at different temperatures.

FIG. 2 shows the results of analysis of the transcription levels of the GhCBF gene after the induction treatment at a low temperature of 10 ℃ for various periods of time.

FIG. 3A shows the growth phenotype of cotton after silencing of the VIGS gene of CBF gene at low temperature of 10 ℃; FIG. 3B is the survival rate statistics of cotton after silencing CBF gene VIGS at low temperature of 10 ℃.

Detailed Description

After extensive and intensive research, the inventor finds that the stress resistance (such as plant cold resistance) of a plant (such as a cotton plant) can be obviously improved by improving the expression or activity of the GhCBBF F gene or the coded protein thereof. On this basis, the present inventors have completed the present invention.

GhCBF gene

As used herein, the terms "GhCBF gene", "stress tolerance gene" and "gene of the invention" are used interchangeably and refer to a gene of the invention that increases stress tolerance in plants (e.g., cotton), particularly low temperature tolerance.

CBF protein C-repeat binding factors, also known as dehydration response element binding proteins, belong to the group of low temperature stress-induced response element transcription factors which can bind to cis-acting elements such as the COR gene promoter, activate the expression of these genes and the expression products are involved in phosphoinositide metabolism, transcription, biosynthesis of osmotic agents, ROS detoxification, membrane transport, hormone metabolism, signaling and the synthesis of many other substances known or assumed to have a cytoprotective function.

The invention separates and clones a gene cDNA segment related to stress tolerance from upland cotton TM-1, selects a gene conserved region sequence to construct a VIGS vector through sequence analysis, triggers gene silencing of upland cotton endogenous GhCBF gene after converting upland cotton through a VIGS technical system, and obviously reduces the tolerance capability of gene silencing upland cotton seedlings to low temperature compared with a contrast. The inventors named this gene as the GhCBF gene.

The inventors have conducted the following studies on the cotton GhCBF gene:

cloning of the GhCBF gene: the full-length nucleotide sequence of the GhCBF gene or a partial fragment thereof can be obtained by various methods such as an RT-PCR amplification method, an RACE-PCR method or a probe method. For example, polynucleotide primers designed based on conserved sequences of CBF genes of other species are directly amplified from cDNA or genome by RACE-PCR method, and the amplified cDNA sequence is connected to a cloning vector and sequenced to obtain the correct GhCBF gene.

Virus-Induced gene silencing (VIGS) is the inhibition of the expression of a gene of interest by a specific nucleic acid sequence. When the virus vector carries the target gene segment and enters mRNA of the target gene in the plant body, the mRNA is degraded, and the plant can show the symptom of function deficiency, so that the research and the understanding of the function of the gene are facilitated.

The recombinant vector obtained by the invention can be used as a genetic engineering resource in the fields of scientific research, agriculture and commerce to assist other genetic resources in exploring or cultivating new varieties of crops with excellent agronomic characters.

The GhCBF gene of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA of the present invention may be single-stranded or double-stranded, and the DNA may be a coding strand or a non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in SEQ ID NO.1 or may be a degenerate variant.

As used herein, "degenerate variant" means in the present invention a nucleic acid sequence which encodes a protein having SEQ ID NO.2, but differs from the sequence of the coding region shown in SEQ ID NO. 1.

The polynucleotide encoding the mature polypeptide of SEQ ID No.2 comprises: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention.

The invention also relates to nucleic acid fragments which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR) to determine and/or isolate polynucleotides encoding polypeptides associated with cotton fiber/plant cell length.

Polypeptide coded by GhCBF gene

As used herein, the terms "GhCBF polypeptide", "GhCBF protein", "protein of the invention", "protein encoded by the GhCBF gene" are used interchangeably and refer to a protein of the invention that has increased stress resistance, particularly low temperature tolerance, in plants.

In a preferred embodiment, the protein of the invention is derived from cotton (e.g., upland cotton).

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the invention can be naturally purified products, or chemically synthesized products, or using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptides of the invention may or may not also include an initial methionine residue.

The invention also includes fragments, derivatives and analogs of the GhCBF polypeptide. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as a native GhCBF polypeptide of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues 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 polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

In a preferred embodiment, the polypeptide of the present invention refers to a polypeptide having the sequence of SEQ ID NO.2 which has the function of regulating the length of cotton fibers/plant cells. Also included are variants of the sequence of SEQ ID NO.2 having the same function as the GhCBF polypeptide. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of GhCBF polypeptides.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to DNA of a GhCBF polypeptide under high or low stringency conditions, and polypeptides or proteins obtained using antisera directed against a GhCBF polypeptide. The invention also provides other polypeptides, such as fusion proteins comprising a GhCBF polypeptide or fragment thereof. In addition to nearly full-length polypeptides, the invention also encompasses soluble fragments of GhCBF polypeptides. Typically, the fragment has at least about 10 contiguous amino acids, 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 contiguous amino acids of the GhCBF polypeptide sequence.

The invention also provides a GhCBF polypeptide or analogue thereof. The analogs may differ from the native GhCBF polypeptide by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized 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 (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, the "conservative variant polypeptide of GhCBF polypeptide" refers to that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties as compared with the amino acid sequence of SEQ ID NO.2 to form a polypeptide. In such proteins, substitutions with amino acids of similar or analogous nature will not generally alter the function of the protein, nor will the addition of one or more amino acids at the C-terminus and/or \ terminus. These conservative variants are preferably produced by amino acid substitutions according to the following table.

Cotton

Cotton is seed fiber of Malvaceae (Malvaceae) cotton (Gossypium) plants of the order Malvales (Malvales) and native to the subtropics. The plant is shrubbery and can grow to 6 meters in height, generally 1 to 2 meters, when cultivated in tropical regions. Flowers are milky white and turn deep red shortly after flowering and then wither, leaving a small green capsule called boll. The cotton bolls are filled with cotton seeds, and the fuzz on the cotton seeds grows out of the cotton seed skins and fills the cotton bolls.

Upland cotton (Gossypium hirsutum L.) is the most important cotton cultivar in the world, since it was first planted in the continental americas, and accounts for more than 90% of the global cotton planting area. Upland cotton is an allotetraploid, comprising two subgenomic groups, subgroup a and subgroup D.

Recombinant techniques and plant improvements

The full-length sequence of the gene of the present invention or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising a polynucleotide of the invention, as well as genetically engineered host cells transformed with a vector of the invention or a sequence encoding a polypeptide of the invention, and methods for producing a polypeptide of the invention by recombinant techniques.

The polynucleotide sequence of the present invention may be used to express or produce a recombinant polypeptide of the present invention by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) separating and purifying protein from culture medium or cell.

The polynucleotide sequences of the present invention may be inserted into a recombinant expression vector. The term "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. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the polynucleotides of the present invention 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 may be operably linked to a suitable 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.

Furthermore, 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, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells (e.g., cells of crops and forestry plants). Representative examples are: escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, electricity may also be used for the conversionThe perforation method is carried out. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformed plant may also be transformed by Agrobacterium transformation or gene gun transformation, such as leaf disk method. The transformed plant cell, tissue or organ can be regenerated into a plant by a conventional method, so that the plant with the changed low-temperature stress character can be obtained.

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 culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. 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 (such as salt precipitation), centrifugation, cell disruption by osmosis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Recombinant polypeptides of the invention have a variety of uses. For example, for screening compounds, polypeptides or other ligands that have the ability to increase the tolerance of plants to low temperatures. Screening polypeptide libraries using expressed recombinant polypeptides of the invention can be used to find valuable polypeptide molecules that can improve the ability of plants to tolerate low temperatures.

In another aspect, the invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the polypeptides of the invention. The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, or chimeric antibodies.

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, a purified gene product of a polypeptide of the present invention, or an antigenic fragment thereof, can be administered to an animal to induce the production of polyclonal antibodies. The antibodies of the invention can be obtained by conventional immunization techniques using fragments or functional regions of gene products related to cotton fiber length. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of cotton fiber length-related gene products can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (e.g., e.coli); antibodies that bind to post-translationally modified forms (e.g., glycosylated or phosphorylated proteins or polypeptides) can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell). Antibodies to the polypeptides of the invention may be used to detect the relevant polypeptides in a sample.

The invention also relates to assays for quantifying and localizing the level of polypeptides associated with an optical signaling pathway. These assays are well known in the art. The level of the polypeptide related to stress tolerance detected in the test can be used for explaining the function of improving the low-temperature tolerance of the plant.

One method for detecting the presence of a stress tolerance-related polypeptide in a sample is to use an antibody specific for the polypeptide of the invention to detect it, which comprises: contacting the sample with an antibody specific for a polypeptide of the invention; observing whether an antibody complex is formed, the formation of an antibody complex is indicative of the presence of the polypeptide of interest in the sample.

A part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also called a "gene chip") for analyzing the differential expression analysis of genes in tissues. The transcription product of the polypeptide of the present invention may also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using primers specific for the polypeptide of the present invention.

VIGS technology

Virus-Induced gene silencing (VIGS) belongs to post-transcriptional gene silencing (PTGS). VIGS, also known as reverse genetics technology, silences plant endogenous genes by recombinant viruses. VIGS is a phenomenon that viruses carrying target gene segments infect plants, and the endogenous genes of the plants are induced to be silenced, so that the phenotype of the plants is changed.

The basic principle of VIGS is that when VIGS is started, a target gene segment connected to a viral vector is synthesized into a large amount of double-stranded RNA (dsRNA) by RNA-guided RNA polymerase. When the dsRNA is accumulated in a plant body in a large amount, the dsRNA can be recognized by Dicer enzyme similar to RNase-III enzyme and degraded into small molecular RNA (siRNA) of approximately 21-24 nt. siRNA binds to Argonaute (AGO) protein and other RNAs in a single stranded form to form an RNA Induced Silencing Complex (RISC). RISC has recognition and cutting effects on target mRNA, and can be specifically combined with homologous target mRNA, so that the target mRNA is degraded, and finally, the gene silencing effect is achieved. The VIGS technology is an important method for researching plant gene functions in recent years, and compared with other analysis methods for identifying gene functions, the VIGS technology has the advantages of simplicity, rapidness, high flux, no influence of genetic background and the like.

In the invention, the VIGS technical system is widely applied to the aspects of high-throughput research on cotton gene functions, particularly rapid verification of important quality trait genes with obvious phenotype and large effect, such as genes closely related to cotton growth and development, stress resistance and the like, and key metabolic pathway gene functions.

Polypeptide of the invention and application of its coding sequence

The present invention provides the application of stress tolerance related polypeptide and its coding sequence in raising the stress tolerance of plant, especially low temperature tolerance. In the invention, the stress tolerance related polypeptide and the coding sequence thereof can also be used for preparing a composition for improving the stress resistance of plants, particularly the low-temperature tolerance capability. The invention also relates to the application of the anti-adversity related polypeptide and the antagonist (or inhibitor) of the coding sequence thereof.

Any substance that can decrease the activity of the GhCBF protein and decrease the expression of the GhCBF protein can be used in the present invention as an inhibitor of GhCBF. Methods for making interfering molecules that interfere with the expression of a particular gene, once the target sequence is known, are well known to those skilled in the art.

The main advantages of the invention include:

(a) the invention screens a stress tolerance related protein and a coding gene thereof for the first time.

(b) The invention discovers for the first time that the expression or activity of stress tolerance related protein GhCBF is improved, and the stress resistance of plants (such as cotton plants) can be obviously improved, especially the low-temperature tolerance.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Unless otherwise specified, the reagents and materials in the examples are all commercially available products.

Example 1 evaluation of preferred Low temperature stress conditions

Cotton seedlings which have developed 2-3 true leaves and grown for 3 weeks under a Light cycle of 16hr Light/8h Dark at 26 ℃ are respectively treated for 72 hours at 22 ℃, 18 ℃, 16 ℃,12 ℃, 10 ℃ and 4 ℃, then moved to 26 ℃ for treatment for one week, and the survival rate of the seedlings is counted. The survival rate of cotton seedlings gradually decreases with the decrease of temperature. The survival rate after treatment at 22 ℃, 18 ℃ and 16 ℃ is not obvious, but after treatment at 12 ℃, 10 ℃ and 4 ℃ for 72 hours, cotton seedlings wither, cotton leaves begin to shrink and dehydrate, and after the cotton leaves are turned to 26 ℃ to recover normal growth for one week, the survival rate is obviously reduced compared with that at 22 ℃, 18 ℃ and 16 ℃. Considering that the low temperature resistance difference between different cotton materials can be better distinguished in future research, the phenotype analysis difference after low temperature stress is possibly not obvious by adopting the low temperature stress of 22 ℃, 18 ℃, 16 ℃ and 4 ℃, so that the temperature of 12-10 ℃ is suitable for the low temperature stress of cotton seedlings (as shown in figure 1).

EXAMPLE 2 cloning and sequence identification of Gene silencing fragments of interest

According to the sequence of the upland cotton GhCBF gene, a 500bp sequence in a conserved region is selected to design a primer, an EcoRI enzyme cutting site is introduced into the 5 'end of a forward primer, a KpnI enzyme cutting site is introduced into the 5' end of a reverse primer, and the primer sequence is as follows:

GhCBFVi-F：5`-GGAATTCTTGATTCTGGGTCGGTTTCT-3' (SEQ ID No.:3) (EcoRI cleavage site in the underlined section), GhCBBFVi-R: 5-GGGGTACCCCTTTCTCCATCTCCGTGTTT-3' (SEQ ID NO.:4) (the KpnI cleavage site is underlined). And (3) performing PCR amplification on the VIGS fragment by using the constructed GhCBF cloning plasmid as a template. The PCR reaction system is 50 μ L: 1 μ L of plasmid, 1 μ L of each primer, 25 μ L of 2 XPrimTAR PCR Mix (Dalibobao), plus ddH₂And O is supplemented to 50 mu L. And (3) amplification procedure: pre-denaturation at 98 ℃ for 1 min; 15s at 98 ℃, 15s at 56 ℃, 30s at 72 ℃ and 30 cycles; extending for 10min at 72 ℃; storing at 4 ℃. And (3) observing the PCR product by agarose gel electrophoresis, and recovering the target fragment by using a DNA gel recovery kit. The target fragment was ligated into a pEASY blunt cloning vector, the DH 5. alpha. strain was transformed, positive clones were selected based on the antibiotic carried on the plasmid and sequenced.

Example 3 Gene expression analysis induced by Low temperature

And when the cotton seedlings grow to 3 weeks, transferring the cotton seedlings to a low-temperature plant incubator at 10 ℃ for low-temperature treatment. And taking a sample before low-temperature treatment as a control, sampling at 3h, 6h, 12h, 24h and 48h of treatment, and analyzing the gene expression condition of the candidate gene under low-temperature induction through the steps of total RNA extraction, cDNA synthesis, real-time fluorescence quantitative PCR and the like. The GhCBF gene was induced to vary gene expression to varying degrees in response to low temperature (figure 2). The expression level of the GhCBF gene gradually increases to a higher level within 6 hours of the initial induction along with the increase of the low-temperature induction time, but the transcription level begins to be gradually reduced along with the increase of the low-temperature induction time, but the expression level is still higher than that of a control.

EXAMPLE 3 construction of VIGS vector for Gene of interest

The clone plasmid with completely correct sequencing result is double-digested by restriction enzymes EcoRI and KpnI, and is connected with pYL156 vector which is also digested by EcoRI and KpnI, DH5 alpha strain is transformed, according to the antibiotic carried on the plasmid, the positive clone is double-digested and identified by restriction enzymes EcoRI and KpnI, and the recombinant plasmid is named as TRV: GhCBF. The identified recombinant plasmid is transformed into agrobacterium strain GV3101 by a freeze-thaw method.

Example 4 creation of GhCBF Gene-silenced upland Cotton plants Using the VIGS technology

(1) Three days prior to inoculation, Agrobacterium TRV: RNA1, TRV: pYL156 empty vector, TRV: GhCL 1 and TRV: GhCBF cryopreserved with glycerol were streaked on LB solid medium containing 50. mu.g/ml kanamycin and 25. mu.g/ml gentamicin. The plate was incubated at 28 ℃ for 24 hours. (2) Two days before infection with VIGS, a single colony was selected from each plate, inoculated into LB liquid medium containing 50. mu.g/ml kanamycin and 25. mu.g/ml gentamicin, and cultured overnight at 28 ℃ on a shaker at 50 rpm. (3) The Agrobacterium containing the vector of interest was cultured overnight at 28 ℃ and 50rpm in 50ml LB medium containing 50. mu.g/ml kanamycin, 25. mu.g/ml gentamicin, 10mM EMS, 20. mu.M acetosyringone. (4) The next day, Agrobacterium cells were collected by centrifugation at 4000rpm for 5 minutes, the supernatant was discarded, and the pellet was suspended in a medium containing 10mM MgCl₂OD was adjusted in 200. mu.M acetosyringone liquid at 10mM MES₆₀₀To 1.5. (5) And left at room temperature for 3 hours. (6) Mixing TRV: RNA1 with bacterial suspension of TRV: pYL156 empty carrier, TRV: GhCLA1 and TRV: GhCBF respectively according to the proportion of 1:1, puncturing one or two small holes below cotton cotyledons by using a needle, removing the needle, and permeating the bacterial liquid into the cotton cotyledons by using a syringe. (7) And culturing the cotton after injection in a dark environment at room temperature overnight. (8) The cotton was transferred to 23 ℃ at 120. mu. E m^-2S^-1The cultivation room with light intensity takes 12h light and 12h dark as the period. (9) And 7-8 days after infiltration, the blooming phenomenon of the true leaf phenotype of the cotton injected with TRV: GhCLA1 is used as a reference, and the cotton injected with TRV: pYL156 empty vector is used as a negative Control (CK). (10) Taking the control and gene silencing plant leaves, extracting total RNA by a Trizol method, and carrying out mRNA transcription level expression analysis, wherein the result is shown in figure 3A, the transcription level of the GhCBF gene in the TRV/GhCBF gene silencing plant is obviously reduced, and the expression of the GhCBF gene is inhibited. Cotton co-infected with TRV: pYL156 empty vector and TRV: RNA1 was used as a negative Control (CK) for observationThe difference between the growth phenotype of cotton subjected to TRV and GhCBF interference under low temperature (10 ℃) and a negative control is shown in figures 3A and 3B, and the result shows that after the GhCBF gene in the cotton is subjected to virus induction and silenced, the cotton subjected to gene silencing shows a low-temperature sensitive phenotype compared with the control under the low-temperature stress condition, and the survival rate is lower than that of the control.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> institute of Nuclear technology and Biotechnology of academy of agricultural sciences in Xinjiang (center for Biotechnology research in autonomous region of Uygur autonomous region in Xinjiang)

<120> cotton stress tolerance gene GhCBF, and coding protein and application thereof

<130> P2019-0856

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gaatatgtga tgttagcttc gagttatcca aagaggcgag ctgggaggaa gaagttccgg 120

gagactcgac acccggtgta ccgtggagtt cgccggagga atcccgggaa gtgggtttct 180

gaagtgaggg agcctaataa gaagtcgagg atttggcttg gaactttccc gacggcggat 240

atggcggcgc gtgctcacga cgtggcagct atagcactga gagggaagtc agcttgtttg 300

aacttcgctg actcagcttg gaatcttccg gtcccggctt cttccgaccc aaaggatatt 360

caaaagacgg cggcggaggt ggcggagact ttcagaacgg ctgagtgttc gtgcgggaat 420

tctagaaacg attcaaagag aagtgaaaac acggagatgg agaaagggtt ttatttggac 480

gaagaagcgt tgtttgggac acaaagattt tgggcaaata tggctgccgg tatgatgatg 540

tcacctcctc gttccggtca tgacggagga tgggaagaac atgaagtaga tgattatgta 600

cctttatgga gttattctat ttaa 624

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<212> PRT

<213> upland cotton (Gossypium hirsutum)

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Asn Phe Ser Asp Glu Tyr Val Met Leu Ala Ser Ser Tyr Pro Lys Arg

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Arg Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Val Tyr Arg

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Gly Val Arg Arg Arg Asn Pro Gly Lys Trp Val Ser Glu Val Arg Glu

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Pro Asn Lys Lys Ser Arg Ile Trp Leu Gly Thr Phe Pro Thr Ala Asp

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Met Ala Ala Arg Ala His Asp Val Ala Ala Ile Ala Leu Arg Gly Lys

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Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Asn Leu Pro Val Pro

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Glu Glu Ala Leu Phe Gly Thr Gln Arg Phe Trp Ala Asn Met Ala Ala

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Gly Met Met Met Ser Pro Pro Arg Ser Gly His Asp Gly Gly Trp Glu

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Glu His Glu Val Asp Asp Tyr Val Pro Leu Trp Ser Tyr Ser Ile

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ggaattcttg attctgggtc ggtttct 27

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<212> DNA

<213> Artificial sequence ()

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ggggtacccc tttctccatc tccgtgttt 29

Claims

1. Use of the GhCBF gene or its encoded protein for one or more uses selected from the group consisting of:

(b) is used for improving the stress resistance of plants.

2. Use according to claim 1, wherein the stress resistance comprises cold resistance.

3. Use according to claim 2, wherein the cold resistance comprises one or more properties selected from the group consisting of:

(a) enhancing the tolerance of the plant to low-temperature environment;

(b) improving the survival ability of the plant in low temperature environment.

4. The use according to claim 1, wherein the amino acid sequence of the GhCBF protein is selected from the group consisting of:

(i) a polypeptide having an amino acid sequence as set forth in SEQ ID No. 2;

5. A method for improving the stress resistance of plants is characterized by comprising the following steps:

6. A method for improving stress resistance of a plant, comprising the steps of: in the plant, the expression of a GhCBF gene is promoted or the activity of a GhCBF protein is promoted.

7. The application of a regulator of GhCBF gene or its coded protein is characterized by that it is used for regulating the stress resistance of plant or preparing the reagent or composition for regulating the stress resistance of plant.

8. A method of screening for a substance that modulates stress resistance in a plant, the method comprising the steps of:

a) administering a test substance to a plant;

9. A method of producing genetically engineered plant tissue or plant cells comprising the steps of:

10. A method of producing a genetically engineered plant comprising the steps of:

regenerating the genetically engineered plant tissue or plant cell prepared by the method of claim 9 into a plant body, thereby obtaining a genetically engineered plant.