阅读说明：本技术 植物衰老相关蛋白GhWRKY91及其编码基因和应用 (Plant senescence-associated protein GhWRKY91, and coding gene and application thereof ) 是由 喻树迅 顾丽姣 魏恒玲 王寒涛 王聪聪 马亮 苏政政 于 2018-08-14 设计创作，主要内容包括：本发明公开了一种植物衰老相关蛋白GhWRKY91及其编码基因和应用。本发明提供的蛋白质为序列表中序列1所示的蛋白质。编码所述蛋白质的核酸分子也属于本发明的保护范围。本发明还保护所述蛋白的应用：调控植物衰老进程；调控植物叶片衰老进程；抑制植物衰老进程；抑制植物叶片衰老进程；调控植物干旱胁迫环境中的衰老进程；调控植物叶片干旱胁迫环境中的衰老进程；调控植物的抗旱性；提高植物的抗旱性。本发明还保护一种制备转基因植物的方法,包括如下步骤：在出发植物中导入所述基因,得到衰老进程延缓的转基因植物。本发明对于植物衰老机制的研究具有重大的理论价值。对于培育抗早衰植物,特别是培育抗早衰棉花具有重大的应用价值。(The invention discloses a plant senescence-associated protein GhWRKY91, and a coding gene and application thereof. The protein provided by the invention is the protein shown in a sequence 1 in a sequence table. Nucleic acid molecules encoding such proteins are also within the scope of the invention. The invention also protects the application of the protein: regulating the senescence process of plants; regulating and controlling the aging process of plant leaves; inhibiting the senescence process of plants; inhibiting the aging process of plant leaves; regulating and controlling the aging process in the drought stress environment of the plant; regulating and controlling the aging process of plant leaves in drought stress environment; regulating and controlling the drought resistance of the plant; improve the drought resistance of the plants. The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the gene is introduced into the original plant to obtain the transgenic plant with the senescence process being delayed. The invention has great theoretical value for the research of plant senescence mechanism. Has great application value for cultivating premature senility resistant plants, in particular to premature senility resistant cotton.)

1. A protein which is (a1) or (a2) or (a3) or (a4) as follows:

(a1) protein shown as a sequence 1 in a sequence table;

(a2) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);

(a3) a plant senescence-associated protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a 1);

(a4) a protein derived from upland cotton, having 98% or more identity to (a1) and associated with plant senescence.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is (b1) or (b2) or (b3) as follows:

(b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(b2) a DNA molecule derived from gossypium hirsutum and having 95% or more identity to (b1) and encoding said protein;

(b3) a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in (b1) and encodes said protein.

4. An expression cassette, recombinant vector or recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3.

5. The use of the protein of claim 1, wherein the protein is (c1), (c2), (c3), (c4), (c5), (c6), (c7), (c8), (c9) or (c 10):

(c1) regulating the senescence process of plants;

(c2) regulating and controlling the aging process of plant leaves;

(c3) inhibiting the senescence process of plants;

(c4) inhibiting the aging process of plant leaves;

(c5) regulating and controlling the aging process in the drought stress environment of the plant;

(c6) regulating and controlling the aging process of plant leaves in drought stress environment;

(c7) inhibiting the senescence process in plant drought stress environments;

(c8) inhibiting the aging process of plant leaves in drought stress environment;

(c9) regulating and controlling the drought resistance of the plant;

(c10) improve the drought resistance of the plants.

6. The use of the nucleic acid molecule of claim 2 or 3, which is (d1), (d2), (d3), (d4), (d5), (d6), (d7), (d8), (d9) or (d 10):

(d1) cultivating a transgenic plant with the changed senescence trait;

(d2) cultivating transgenic plants with the changed leaf senescence traits;

(d3) cultivating transgenic plants with delayed senescence process;

(d4) cultivating transgenic plants with delayed leaf senescence process;

(d5) cultivating a transgenic plant with improved premature senility;

(d6) cultivating transgenic plants with improved leaf senilism;

(d7) cultivating a transgenic plant with a delayed senescence process in a drought stress environment;

(d8) cultivating a transgenic plant with a delayed leaf senescence process in a drought stress environment;

(d9) cultivating a transgenic plant with changed drought resistance;

(d10) cultivating the transgenic plant with enhanced drought resistance.

7. A method of making a transgenic plant comprising the steps of: a transgenic plant with delayed senescence process, obtained by introducing the nucleic acid molecule of claim 2 or 3 into a starting plant.

8. A method of plant breeding comprising the steps of: increasing the content and/or activity of the protein according to claim 1 in the plant of interest, thereby delaying the senescence process in the plant of interest.

9. A method of making a transgenic plant comprising the steps of: introducing the nucleic acid molecule of claim 2 or 3 into a starting plant to obtain a transgenic plant with enhanced drought resistance.

10. A method of plant breeding comprising the steps of: increasing the content and/or activity of the protein of claim 1 in a plant of interest, thereby enhancing the drought resistance of the plant of interest.

Technical Field

The invention relates to a plant senescence-associated protein GhWRKY91, and a coding gene and application thereof.

Background

Senescence is an integral part of plant development and is the final stage of development. During senescence, leaf cells undergo highly coordinated changes in structure, metabolism, and gene expression. The earliest, most obvious change in cell structure is chloroplast disintegration. Metabolically, carbon metabolism (photosynthesis) is replaced by the catabolism of chloroplasts and macromolecular substances such as proteins, membrane lipids, RNA, etc. At the molecular level, the above changes are accompanied by changes in gene expression.

Senescence is programmed cell death. Leaf senescence plays an important role in physiological activities such as the circulation and reuse of nutrient elements. The leaf assimilation function reduction caused by aging limits the crop yield.

Cotton is an important economic crop and textile raw material in China, and has no substitutable effect on the development of the national economy of China. The contradiction of land competition of grain and cotton in China is prominent, the contradiction of land competition of grain and cotton can be relieved by breeding and popularizing the short-season cotton variety, but the precocity of the short-season cotton is often accompanied with premature senility. In the production of cotton, the premature senility of cotton causes the reduction of boll weight, the reduction of clothes content and the reduction of fiber strength and maturity, thus reducing the yield and quality of cotton.

Disclosure of Invention

The invention aims to provide a plant senescence-associated protein GhWRKY91, and a coding gene and application thereof.

The protein provided by the invention is obtained from upland cotton (Gossypium hirsutum) and is named GhWRKY91 protein, and is (a1) or (a2) or (a3) or (a4) as follows:

(a1) protein shown as a sequence 1 in a sequence table;

(a2) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);

(a3) a plant senescence-associated protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a 1);

(a4) a protein derived from upland cotton, having 98% or more identity to (a1) and associated with plant senescence.

The labels are specifically shown in table 1.

TABLE 1 sequences of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL
HA	9	YPYDVPDYA

The protein can be synthesized artificially, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

Nucleic acid molecules encoding such proteins are also within the scope of the invention. The nucleic acid molecule is a DNA molecule or an RNA molecule.

The DNA molecule (named GhWRKY91 gene) for coding the protein is (b1) or (b2) or (b3) as follows:

(b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(b2) a DNA molecule derived from gossypium hirsutum and having 95% or more identity to (b1) and encoding said protein;

(b3) a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in (b1) and encodes said protein.

An expression cassette, a recombinant vector or a recombinant microorganism containing the GhWRKY91 gene all belong to the protection scope of the invention.

The recombinant expression vector containing the gene can be constructed by using the existing expression vector. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added in front of the transcription initiation nucleotide, and can be used alone or combined with other plant promoters; in addition, when the gene is used to construct a recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or adjacent regions initiation codons, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and screening of the transgenic plant or the transgenic microorganism, an expression vector to be used may be processed, for example, a gene for expressing an enzyme or a luminescent compound which produces a color change in the plant or the microorganism, a gene for an antibiotic marker having resistance or a chemical-resistant agent marker, etc. From the viewpoint of safety of transgenes, the transformed plants or microorganisms can be directly screened phenotypically without adding any selectable marker gene.

The recombinant expression vector may specifically be: the recombinant plasmid is obtained by inserting a DNA molecule shown in a sequence 2 in a sequence table between XbaI enzyme cutting sites and SmaI enzyme cutting sites of the pBI121 plasmid.

The invention also protects the application of GhWRKY91 protein, which is (c1), (c2), (c3), (c4), (c5), (c6), (c7), (c8), (c9) or (c 10):

(c1) regulating the senescence process of plants;

(c2) regulating and controlling the aging process of plant leaves;

(c3) inhibiting the senescence process of plants;

(c4) inhibiting the aging process of plant leaves;

(c5) regulating and controlling the aging process in the drought stress environment of the plant;

(c6) regulating and controlling the aging process of plant leaves in drought stress environment;

(c7) inhibiting the senescence process in plant drought stress environments;

(c8) inhibiting the aging process of plant leaves in drought stress environment;

(c9) regulating and controlling the drought resistance of the plant;

(c10) improve the drought resistance of the plants.

The plant is a dicotyledonous plant or a monocotyledonous plant. The dicot may be a cotton plant or an arabidopsis plant. The cotton plant may be upland cotton. The arabidopsis plant may specifically be colombian ecotype arabidopsis thaliana.

The invention also protects the application of the GhWRKY91 gene, which is (d1), (d2), (d3), (d4), (d5), (d6), (d7), (d8), (d9) or (d 10):

(d1) cultivating a transgenic plant with the changed senescence trait;

(d2) cultivating transgenic plants with the changed leaf senescence traits;

(d3) cultivating transgenic plants with delayed senescence process;

(d4) cultivating transgenic plants with delayed leaf senescence process;

(d5) cultivating a transgenic plant with improved premature senility;

(d6) cultivating transgenic plants with improved leaf senilism;

(d7) cultivating a transgenic plant with a delayed senescence process in a drought stress environment;

(d8) cultivating a transgenic plant with a delayed leaf senescence process in a drought stress environment;

(d9) cultivating a transgenic plant with changed drought resistance;

(d10) cultivating the transgenic plant with enhanced drought resistance.

The plant is a dicotyledonous plant or a monocotyledonous plant. The dicot may be a cotton plant or an arabidopsis plant. The cotton plant may be upland cotton. The arabidopsis plant may specifically be colombian ecotype arabidopsis thaliana.

The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the GhWRKY91 gene is introduced into the original plant to obtain the transgenic plant with delayed senescence process. The gene can be specifically introduced into the starting plant by any of the above recombinant expression vectors. The recombinant expression vector carrying the gene can be transformed into a starting plant by a conventional biological method such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation and the like. The starting plant is a dicotyledonous plant or a monocotyledonous plant. The dicot may be a cotton plant or an arabidopsis plant. The cotton plant may be upland cotton. The arabidopsis plant may specifically be colombian ecotype arabidopsis thaliana. The senescence process delaying may specifically be leaf senescence process delaying.

The invention also provides a plant breeding method, which comprises the following steps: increasing the content and/or activity of GhWRKY91 protein in the target plant, thereby delaying the aging process of the target plant. The target plant is a dicotyledonous plant or a monocotyledonous plant. The dicot may be a cotton plant or an arabidopsis plant. The cotton plant may be upland cotton. The arabidopsis plant may specifically be colombian ecotype arabidopsis thaliana. The process for delaying the senescence of the target plant can be specifically the process for delaying the senescence of the target plant leaves.

The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the GhWRKY91 gene is introduced into the original plant to obtain the transgenic plant with enhanced drought resistance. The gene can be specifically introduced into the starting plant by any of the above recombinant expression vectors. The recombinant expression vector carrying the gene can be transformed into a starting plant by a conventional biological method such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation and the like. The starting plant is a dicotyledonous plant or a monocotyledonous plant. The dicot may be a cotton plant or an arabidopsis plant. The cotton plant may be upland cotton. The arabidopsis plant may specifically be colombian ecotype arabidopsis thaliana. The drought resistance enhancement is embodied as delaying of the senescence process in drought stress environment. The senescence process delaying may specifically be leaf senescence process delaying.

The invention also provides a plant breeding method, which comprises the following steps: the content and/or activity of GhWRKY91 protein in the target plant is increased, so that the drought resistance of the target plant is enhanced. The target plant is a dicotyledonous plant or a monocotyledonous plant. The dicot may be a cotton plant or an arabidopsis plant. The cotton plant may be upland cotton. The arabidopsis plant may specifically be colombian ecotype arabidopsis thaliana. The method for enhancing the drought resistance of the target plant is specifically embodied in the process of delaying the aging process of the target plant in the drought stress environment. The senescence process delaying may specifically be leaf senescence process delaying.

The invention has great theoretical value for the research of plant senescence mechanism. Has great application value for cultivating premature senility resistant plants, in particular to premature senility resistant cotton.

Drawings

FIG. 1 shows the relative expression level of the GhWRKY91 gene.

FIG. 2 is a photograph of the natural senescence phenotype in step four.

FIG. 3 shows the relative expression amounts of AtSAG12 gene and AtSAG13 gene in step four.

Fig. 4 is a schematic diagram of the division of zones in the nutrition bowl.

FIG. 5 is a photograph of the drought treatment phenotype in step five.

FIG. 6 shows the relative expression amounts of step five AtSAG12 gene and AtSAG13 gene.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

A new protein is found from Gossypium hirsutum Miq 74, which is shown as a sequence 1 in a sequence table (273aa) and named as GhWRKY91 protein. The GhWRKY91 protein has a relative molecular weight of 29.82kDa and an isoelectric point of 5.13. The gene encoding the GhWRKY91 protein is named as GhWRKY91 gene. In the cDNA of No. 74 of Gossypium hirsutum, the coding frame of the GhWRKY91 gene is shown as sequence 2 (822bp) in the sequence table.