阅读说明：本技术 蛋白质CkWRKY33及其编码基因与应用 (Protein CkWRKY33 and coding gene and application thereof ) 是由 龙艳 裴新梧 梁凤萍 于 2019-11-13 设计创作，主要内容包括：本发明公开了蛋白质CkWRKY33及其编码基因与应用。蛋白质CkWRKY33的氨基酸序列如SEQ ID NO：2所示。实验证明,向野生型拟南芥Columbia-0亚型中导入CkWRKY33基因,得到转基因拟南芥；与野生型拟南芥Columbia-0亚型相比,转基因拟南芥的抗旱性提高,抗旱性提高体现在丙二醛含量降低、可溶性糖含量增加、脯氨酸含量增加、过氧化物酶活性增加、失水率降低和存活率提高。由此可见,蛋白质CkWRKY33可以提高植物抗逆性。本发明具有重要的应用价值。(The invention discloses a protein CkWRKY33 and an encoding gene and application thereof. The amino acid sequence of the protein CkWRKY33 is shown as SEQ ID NO: 2, respectively. Experiments prove that CkWRKY33 gene is introduced into a wild type Arabidopsis thaliana Columbia-0 subtype to obtain transgenic Arabidopsis thaliana; compared with the wild type Arabidopsis thaliana Columbia-0 subtype, the drought resistance of the transgenic Arabidopsis thaliana is improved, and the improvement of the drought resistance is reflected in that the malonaldehyde content is reduced, the soluble sugar content is increased, the proline content is increased, the peroxidase activity is increased, the water loss rate is reduced and the survival rate is improved. Therefore, the protein CkWRKY33 can improve the stress resistance of plants. The invention has important application value.)

1. Protein CkWRKY33, as follows a1) or a 2):

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2 and the N end or/and the C end of the protein shown in the figure is connected with a label to obtain the fusion protein.

2. A nucleic acid molecule encoding the protein CkWRKY33 of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a DNA molecule shown as b1) or b 2):

b1) the coding region is SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1.

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

5. Use of the protein CkWRKY33 of claim 1, or the nucleic acid molecule of claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 2 or 3, as a1) or a 2):

A1) regulating and controlling the stress resistance of the plants;

A2) cultivating the transgenic plant with the changed stress resistance.

6. A method of breeding a transgenic plant comprising the steps of: increasing the expression quantity and/or activity of the protein CkWRKY33 in the starting plant to obtain a transgenic plant; the transgenic plants have improved stress resistance compared to the starting plants.

7. The method of claim 6, wherein: the expression level and/or activity of the protein CkWRKY33 in the starting plant are/is improved by introducing a nucleic acid molecule encoding the protein CkWRKY33 into the starting plant.

8. A method of plant breeding comprising the steps of: increasing the content and/or activity of the protein CkWRKY33 as defined in claim 1 in plants, thereby improving the stress resistance of plants.

9. The protein CkWRKY33 of claim 1, the use of claim 5, or the method of any one of claims 6 to 8, wherein: the plant is any one of the following c1) to c 5): c1) a dicotyledonous plant; c2) a monocot plant; c3) a cruciferous plant; c4) arabidopsis thaliana; c5) the wild type Arabidopsis thaliana Columbia-0 subtype.

10. The protein CkWRKY33 as defined in claim 1 or 9 or the use as defined in claim 5 or 9 or the method as defined in any one of claims 6 to 9 wherein: the stress resistance is drought resistance.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a protein CkWRKY33, and a coding gene and application thereof.

Background

Caragana microphylla is a leguminous shrub mainly growing in sandy land of desert or semi-desert regions, and has a highly developed root system and extremely strong drought and cold resistance. Caragana microphylla is not only important pasture and industrial raw material, but also can maintain water and soil, prevent soil desertification and have important economic and Ecological value (Fang X W, Li J H, Xiong Y C, Xu D H, Fan X W, Li F M. Responses of Caraganaakorshiki Kom. to shoot removal: mechanisms undersiding growth [ J ]. Ecological Research, 2007, 23 (5): 863-871.).

At present, the caragana korshinskii researches mainly focus on the aspects of biological characteristics, physiological changes, anatomical structures and the like, and the researches on stress resistance related genes are very little.

Disclosure of Invention

The invention aims to improve the stress resistance of plants.

The invention provides a protein, which is obtained from caragana korshinskii and is named as protein CkWRKY33, and can be a1) or a2) as follows:

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2 and the N end or/and the C end of the protein shown in the figure is connected with a label to obtain the fusion protein.

Wherein, SEQ ID NO: 2 consists of 537 amino acid residues.

To facilitate purification and detection of the protein, the protein may be identified in the sequence set forth by SEQ ID NO: 2, to the amino terminus or the carboxy terminus of the protein consisting of the amino acid sequence shown in table 1.

TABLE 1 sequence 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

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

The coding gene of the protein can be obtained by converting SEQ ID NO: 1 is linked at its 5 'end and/or 3' end to the coding sequence of the tag shown in table 1 above.

Nucleic acid molecules encoding the protein CkWRKY33 are also within the scope of the invention.

The nucleic acid molecule encoding the protein CkWRKY33 can be a DNA molecule shown as b1) or b2) as follows:

b1) the coding region is SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID NO: 1 consists of 1614 nucleotides, SEQ ID NO: 1 encodes the nucleotide sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

The nucleotide sequence encoding the protein CkWRKY33 of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the protein CkWRKY33 isolated in the present invention, as long as the nucleotide sequence encoding the protein CkWRKY33 is derived from the nucleotide sequence of the present invention and is identical to the sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes the identity to the nucleotide sequence of the present invention encoding SEQ ID NO: 2, or 80% or more, or 85% or more, or 90% or more, or 95% or more, of the nucleotide sequence of protein CkWRKY 33. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The nucleic acid molecule encoding the protein CkWRKY33 can be specifically a gene encoding the protein CkWRKY33 and is named as a CkWRKY33 gene.

Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines comprising any of the above-described nucleic acid molecules are also within the scope of the present invention.

The recombinant vector containing any one of the nucleic acid molecules can be obtained by inserting the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.

The recombinant vector containing any one of the nucleic acid molecules can be specifically a recombinant plasmid pBinGlyRed3-CkWRKY 33. The recombinant plasmid pBinGlyRed3-CkWRKY33 can replace a small DNA fragment between restriction enzymes EcoRI and XmaI recognition sequences of a pBinGlyRed3 vector with a nucleotide sequence shown as SEQ ID NO: 1 to obtain the recombinant plasmid.

The recombinant microorganism containing any of the above-described nucleic acid molecules may be a recombinant bacterium obtained by introducing a recombinant vector containing any of the above-described nucleic acid molecules into a starting microorganism.

The starting microorganism can be agrobacterium or escherichia coli. The agrobacterium may specifically be agrobacterium tumefaciens EHA 105.

The recombinant microorganism containing any one of the nucleic acid molecules can be specifically EHA105/pBinGlyRed3-CkWRKY 33. The EHA105/pBinGlyRed3-CkWRKY33 is used for introducing a recombinant plasmid pBinGlyRed3-CkWRKY33 into the agrobacterium tumefaciens EHA105 to obtain the recombinant agrobacterium tumefaciens.

The transgenic cell line does not include propagation material.

The invention also protects the application of any one of the protein CkWRKY33, or any one of the nucleic acid molecules, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing any one of the nucleic acid molecules, which can be A1) or A2):

A1) regulating and controlling the stress resistance of the plants;

A2) cultivating the transgenic plant with the changed stress resistance.

In the above application, the regulation of the plant stress resistance may be an increase in the plant stress resistance.

In the application, the cultivation of the transgenic plant with the changed stress resistance can be the cultivation of the transgenic plant with the increased stress resistance.

The invention also provides a method for cultivating transgenic plants, which comprises the following steps: improving the expression quantity and/or activity of any one of the proteins CkWRKY33 in the starting plant to obtain a transgenic plant; the transgenic plants have improved stress resistance compared to the starting plants.

In the method, the effect of improving the expression quantity and/or activity of the protein CkWRKY33 can be achieved by using methods known in the field, such as multiple copies, promoter change, regulatory factor change, transgenosis and the like, for improving the expression quantity and/or activity of any one of the protein CkWRKY33 in the starting plant.

In the above method, the "improvement in the expression level and/or activity of any of the above-mentioned proteins CkWRKY33 in the starting plant" may be achieved by introducing a nucleic acid molecule encoding the protein CkWRKY33 into the starting plant.

In any of the above methods, the nucleic acid molecule encoding the protein CkWRKY33 may be a DNA molecule represented by b1) or b2) as follows:

b1) the coding region is SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID NO: 1 consists of 1614 nucleotides, SEQ ID NO: 1 encodes the nucleotide sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

The "introduction of a nucleic acid molecule encoding the protein CkWRKY33 into a starting plant" can be specifically achieved by introducing a recombinant vector containing a nucleic acid molecule encoding the protein CkWRKY33 into a starting plant. The "recombinant vector containing a nucleic acid molecule encoding the protein CkWRKY 33" can be specifically the recombinant plasmid pBinGlyRed3-CkWRKY 33.

The invention also provides a plant breeding method, which comprises the following steps: increasing the content and/or activity of the protein CkWRKY33 in the plant, thereby improving the stress resistance of the plant.

Any of the plants or starting plants described above may be any of the following c1) to c 5): c1) a dicotyledonous plant; c2) a monocot plant; c3) a cruciferous plant; c4) arabidopsis thaliana; c5) the wild type Arabidopsis thaliana Columbia-0 subtype.

Any of the above stress resistance may be drought resistance.

Any of the above described plant drought resistance enhancements is manifested in at least one of reduced malondialdehyde content, increased soluble sugar content, increased proline content, increased peroxidase activity, reduced water loss rate, and increased survival rate.

Experiments prove that CkWRKY33 gene is introduced into a wild type Arabidopsis thaliana Columbia-0 subtype to obtain transgenic Arabidopsis thaliana; compared with the wild type Arabidopsis thaliana Columbia-0 subtype, the drought resistance of the transgenic Arabidopsis thaliana is improved, and the improvement of the drought resistance is reflected in that the malonaldehyde content is reduced, the soluble sugar content is increased, the proline content is increased, the peroxidase activity is increased, the water loss rate is reduced and the survival rate is improved. Therefore, the protein CkWRKY33 can improve the stress resistance of plants. The invention has important application value.

Drawings

FIG. 1 shows the growth state of Arabidopsis plants to be tested before and after natural drought and 3 days after rehydration.

FIG. 2 shows the statistics of the survival rate of Arabidopsis plants.

FIG. 3 shows the statistical result of the water loss rate of the isolated leaf of Arabidopsis thaliana.

FIG. 4 is a statistical result of the soluble sugar content in Arabidopsis thaliana leaves.

FIG. 5 is a statistical result of the malondialdehyde content in Arabidopsis thaliana leaves.

FIG. 6 shows the statistics of proline content in Arabidopsis leaves.

FIG. 7 is a statistical result of peroxidase activity in Arabidopsis thaliana leaves.

FIG. 8 shows the growth state of Arabidopsis thaliana on 1/2MS solid medium and 1/2MS solid medium containing mannitol.

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.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Caragana microphylla seeds were collected from a Denseian desert botanical garden in Gansu, by inventor (010-.

Wild type Arabidopsis thaliana (Arabidopsis thaliana) (Columbia-0 subtype) is described in the following references: kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J.A geneticin between column responses and flowing time through FVE in Arabidopsis thaliana. Nature genetics.2004, 36: 167-.