阅读说明：本技术 耐盐相关蛋白IbWRKY32及其编码基因与应用 (Salt tolerance related protein IbWRKY32 and coding gene and application thereof ) 是由 翟红 刘庆昌 何绍贞 高少培 张欢 孙思凡 李思语 于 2021-08-25 设计创作，主要内容包括：本发明公开了一种蛋白质IbWRKY32在调控植物耐盐性中的应用。本发明首先公开了一种蛋白质在提高植物耐盐性中的应用；所述蛋白质为氨基酸序列如SEQ IDNO.1所示的蛋白质或SEQ ID NO.1所示的氨基酸序列的N端或/和C端连接蛋白标签得到的融合蛋白。本发明进一步公开了上述蛋白相关生物材料及其应用。本发明发现了IbWRKY32蛋白及其编码基因,并将IbWRKY蛋白的编码基因导入烟草中,得到转基因烟草植株。将转基因植株进行盐胁迫处理,其耐盐性增强,在植物耐盐的过程中起着重要的作用,在农业领域具有广阔的应用空间和市场前景。(The invention discloses an application of protein IbWRKY32 in regulation and control of plant salt tolerance. The invention firstly discloses the application of protein in improving the salt tolerance of plants; the protein is a protein with an amino acid sequence shown in SEQ ID NO.1 or a fusion protein obtained by connecting protein labels to the N end or/and the C end of the amino acid sequence shown in SEQ ID NO. 1. The invention further discloses the protein-related biomaterial and application thereof. The invention discovers IbWRKY32 protein and an encoding gene thereof, and introduces the encoding gene of the IbWRKY protein into tobacco to obtain a transgenic tobacco plant. The transgenic plant is subjected to salt stress treatment, the salt tolerance of the transgenic plant is enhanced, the transgenic plant plays an important role in the salt tolerance process of the plant, and the transgenic plant has wide application space and market prospect in the agricultural field.)

1. Use of a protein or a substance which regulates the expression of a gene encoding said protein or a substance which regulates the activity or content of said protein, characterized in that: the application is the application of protein or an expression substance for regulating and controlling the protein coding gene or a substance for regulating and controlling the activity or the content of the protein in regulating and controlling the salt tolerance of plants,

the protein is IbWRKY32 protein, and the IbWRKY32 protein is any one of the following proteins:

A1) a protein having an amino acid sequence of SEQ ID No. 1;

A2) a protein which is obtained by substituting and/or deleting and/or adding more than one amino acid residue in the amino acid sequence shown in A1), has more than 80% of identity with the protein shown in A1), and has the function of regulating and controlling the salt tolerance of plants;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

2. Use according to claim 1, characterized in that: the protein is derived from sweet potato.

3. Use according to claim 1 or 2, characterized in that: the substance regulating the expression of the gene encoding the protein or the substance regulating the activity or content of the protein is a biological material related to the protein, and the biological material is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing B2) the expression cassette.

4. The use according to claim 3, wherein the nucleic acid molecule is a gene as shown in G1) or G2):

G1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule of SEQ ID No. 2;

G2) the nucleotides of the coding strand are cDNA molecules or DNA molecules of SEQ ID No. 2.

5. Use according to claim 1, wherein the recipient plant is any one of:

1) a dicotyledonous plant;

2) plants of the order Solanales;

3) a plant of the Solanaceae family;

4) a plant of the genus nicotiana;

5) tobacco.

6. A method for improving salt tolerance of a plant, comprising introducing a gene encoding the protein of claim 1 into a recipient plant to obtain a plant of interest having higher salt tolerance than the recipient plant.

7. The method according to claim 6, wherein the gene encoding the protein is a gene represented by G1) or G2) below:

G1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule of SEQ ID No. 2;

G2) the nucleotides of the coding strand are cDNA molecules or DNA molecules of SEQ ID No. 2.

8. The method of claim 7, wherein the recipient plant is a dicot.

9. The method according to claim 8, wherein the dicotyledonous plant is any one of:

1) a plant belonging to the order of Solanales,

2) a plant of the family Solanaceae,

3) a plant of the genus Nicotiana,

4) tobacco.

10. The IbWRKY32 protein as described in claim 1 or 2, and/or the biomaterial as described in claim 3 or 4.

Technical Field

The invention relates to the field of biotechnology. In particular to a salt tolerance related protein IbWRKY32 and an encoding gene and application thereof.

Background

In nature, adverse conditions such as high temperature, high salinity, low temperature, waterlogging, drought, diseases, pests, weeds and the like can influence the morphological structure and physiological and biochemical processes of plants, so that the plants can stop normal growth and development, reduce quality and reduce yield. Wherein, drought and saline-alkali are the stress factors which harm the growth of plants and have the highest influence on the yield of crops all over the world. At present, the areas of the Chinese saline-alkali soil and the global saline-alkali soil respectively reach 0.27 multiplied by 108hm²And 9.5 x 108hm²The latter occupies almost 10% of the world's surface area. When crops are planted on medium saline soil, the yield can be reduced by about 95 percent, for example, the yield reduction of various places in Huang-Huai-Hai plain caused by saline and alkaline is about 25 percent on average, and even reaches 50 percent seriously. Therefore, exploring the stress resistance mechanism of plants has important significance for exploring the stress resistance genes and improving the stress resistance of the plants by genetic engineering. Although sweet potatoes are drought-enduring crops, the growth, development, physiology and yield of sweet potatoes are affected in the absence of water, as in other crops. By deeply researching the salt and drought resistance mechanism of plants, the salt and drought resistance gene resources are excavated, and the cultivation of new salt and drought resistant sweet potato varieties is one of the most economic and effective measures for utilizing saline-alkali soil and arid and semi-arid resources.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the salt tolerance of plants.

In order to solve the technical problems, the invention firstly provides an application of a protein or an expression substance for regulating a gene encoding the protein or a substance for regulating the activity or content of the protein, wherein the application can be the application of the protein or the expression substance for regulating the gene encoding the protein or the substance for regulating the activity or content of the protein in regulating and controlling the salt tolerance of plants, the protein can be IbWRKY32 protein, and the IbWRKY32 protein can be any one of the following proteins:

A1) a protein having an amino acid sequence of SEQ ID No. 1;

A2) a protein which is obtained by substituting and/or deleting and/or adding more than one amino acid residue in the amino acid sequence shown in A1), has more than 80% of identity with the protein shown in A1), and has the function of regulating and controlling the salt tolerance of plants;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

Further, the protein may be derived from sweetpotato.

Wherein SEQ ID No.1 consists of 495 amino acid residues.

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

Herein, the protein-tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a protein of interest using in vitro recombinant DNA technology, so as to facilitate the expression, detection, tracking and/or purification of the protein of interest. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

Herein, the identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

Herein, the 80% or greater identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In the above application, the substance for regulating the activity or content of the protein may be a substance for regulating the expression of a gene encoding the protein.

In the above application, the substance for regulating gene expression may be a substance for regulating at least one of the following 6 kinds of regulation: 1) regulation at the level of transcription of said gene; 2) regulation after transcription of the gene (i.e., regulation of splicing or processing of a primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; 6) post-translational regulation of the gene (i.e., regulation of the activity of a protein translated from the gene).

In the above application, the regulation of gene expression may be increasing or increasing the gene expression.

The substance that increases or increases the gene expression may be a biological material related to the protein, and the biological material may be any one of the following B1) to B7):

B1) nucleic acid molecules encoding the above proteins;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing B2) the expression cassette.

Wherein, the nucleic acid molecule of B1) 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.

Further, the nucleic acid molecule of B1) may be a gene represented by G1) or G2) as follows:

G1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule of SEQ ID No. 2;

G2) the nucleotide sequence of the coding strand is a cDNA molecule or a DNA molecule of SEQ ID No. 2;

wherein, SEQ ID No.2 consists of 1488 nucleotides, the Open Reading Frame (ORF) thereof is from 1 st to 1488 th from the 5' end, and the encoded amino acid sequence is the protein shown in SEQ ID No. 1.

In the related biological materials, the expression cassette B2) is DNA capable of expressing protein IbWRKY32 in host cells, and the DNA not only can comprise a promoter for starting the transcription of IbWRKY32 gene, but also can comprise a terminator for terminating the transcription of IbWRKY32 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053.) they can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

In the related biological material, B3) the recombinant vector can contain a DNA molecule shown in SEQ ID No.2 and used for encoding the protein IbWRKY 32.

A plant expression vector can be used for constructing a recombinant vector containing the IbWRKY32 coding gene expression cassette. The plant expression vector can be a Gateway system vector or a binary agrobacterium vector and the like, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300-35S, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa or pCAMBIA 1391-Xb. When the IbWRKY32 is used for constructing a recombinant vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added in front of the transcription initiation nucleotide, such as a cauliflower mosaic virus (CAMV)35S promoter, a ubiquitin gene Ubiqutin promoter (pUbi) and the like, and the promoters can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, 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 the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

In the related biological material, the recombinant microorganism B4) can be yeast, bacteria, algae and fungi.

In the above-mentioned related biological materials, B6) the plant tissue may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

In the above-mentioned related biological materials, B7) the transgenic plant organ may be a root, a stem, a leaf, a flower, a fruit and a seed of the transgenic plant.

In the related biological materials, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ may or may not include propagation material.

The invention also provides a method for improving the salt tolerance of plants, which comprises the step of introducing the protein coding gene into a receptor plant to obtain a target plant with higher salt tolerance than the receptor plant.

Further, the encoding gene in the above method is the following G1) or G2):

G1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule shown as SEQ ID No. 2;

G2) the nucleotide sequence of the coding strand is a cDNA molecule or a DNA molecule as shown in SEQ ID No. 2.

In the above method, the protein-encoding gene may be introduced into a target plant via a plant expression vector carrying the protein-encoding gene.

The plant expression vector carrying the protein-encoding gene of the present invention can be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and culture the transformed plant cells or tissues into plants.

In a specific embodiment of the invention, the recombinant vector is a recombinant plasmid pCB-IbWRKY 32. The recombinant plasmid pCB-IbWRKY32 is a recombinant vector obtained by inserting a DNA molecule shown in SEQ ID No.2 between KpnI and XbaI restriction recognition sites of the vector pCAMBIA1300-35S by using restriction enzymes KpnI and XbaI and keeping other sequences of the vector pCAMBIA1300-35S unchanged.

In the recombinant plasmid pCB-IbWRKY32, the promoter for starting IbWRKY32 gene transcription is 35S promoter located in the vector pCAMBIA 1300-35S.

The above plant may be a dicot, and the dicot may be a plant of the order solanales. The plant of Solanales can be plant of Solanaceae. The Solanaceae plant can be plant of Nicotiana. The nicotiana species plant can be tobacco.

The protein and/or the biological material related to the protein also belong to the protection scope of the invention.

The invention provides IbWRKY32 protein and an encoding gene thereof, and the encoding gene of the IbWRKY32 protein is introduced into tobacco W38 to obtain a transgenic tobacco plant of IbWRKY 32. The salt stress treatment is carried out on the transgenic plant, and compared with the contrast, the salt tolerance of the transgenic plant is enhanced, particularly embodied in that H is reduced₂O₂And (4) content. Therefore, the IbWRKY32 gene and the protein coded by the same play an important role in the plant salt tolerance process, have important application values in the research of improving plant salt tolerance, and have wide application space and market prospects in the agricultural field.

Drawings

FIG. 1 shows the PCR detection result of transgenic tobacco plants; wherein M is marker; w is water; p is positive plasmid; WT is wild type tobacco W38 plant; L1-L6 is a transgenic plant.

FIG. 2 is the expression analysis of IbWRKY32 in transgenic tobacco; wherein, WT is wild tobacco W38 plant; L1-L6 is a transgenic plant.

FIG. 3A shows the plant growth conditions in the salt tolerance identification experiment of the transgenic tobacco plant overexpressing IbWRKY32 and the wild W38 plant; wherein, WT is wild type W38 plant; l1, L2 and L3 are transgenic tobacco plants overexpressing IbWRKY 32; controls indicate growth and fresh weight in normal medium for 4 weeks, and salt stress indicates 4 weeks in stress medium.

FIG. 3B shows the fresh weight of plants in the salt tolerance identification experiment of the transgenic tobacco plant overexpressing IbWRKY32 and the wild W38 plant; wherein, WT is wild type W38 plant; l1, L2 and L3 are transgenic tobacco plants overexpressing IbWRKY 32; controls indicate growth and fresh weight in normal medium for 4 weeks, and salt stress indicates 4 weeks in stress medium.

FIG. 4 is H of transgenic tobacco plants overexpressing IbWRKY32 and wild type control plants₂O₂Measuring the content; wherein, WT is a wild type W38 plant; l1, L2 and L3 are transgenic tobacco plants overexpressing IbWRKY 32.

Detailed Description

Xushu 55-2 in literature: the method comprises the steps of analyzing a dry transcriptome of the Zhuhong sweet potato and cloning and functional identification of drought-resistant related genes IbWRKY2, IbGATA24 and IbSDT, a doctor academic paper, China agriculture university, 2018, wherein the method can be obtained from a sweet potato genetic breeding research laboratory of Chinese agriculture university after the consent of the authors of the public, so that the experiment can be repeated, and the method cannot be used for other purposes.

The tobacco variety W38 is disclosed in the literature "Jiang T, ZHai H, Wang FB, Zhou HN, Si ZZ, He SZ, Liu QC. cloning and characterization of a Salt Tolerance-Associated Gene Encoding Trehase-6-Phosphate Synthase in Sweetpotato, Journal of Integrated agricultural culture 2014,13(8): 1651-1661", and is available from the sweet potato genetic Breeding research laboratory of Chinese Agriculture after the approval of the university of the author, to repeat the experiment, and is not useful for other purposes.

The vector pCAMBIA1300-35S was purchased from Wuhan transduction Biolabs, Inc., and has catalog number VT 4004.

Agrobacterium tumefaciens EHA105 was purchased from Beijing Bylendi Biotechnology, Inc.

Escherichia coli DH5a (from Beijing Quanjin Biotechnology Co., Ltd., catalog No. CD201-01)

The following examples were processed using SPSS19.0 statistical software and the results were expressed as mean ± standard deviation, with P < 0.05 (x) indicating significant differences, P < 0.01 (x) indicating very significant differences and P < 0.001 (x) indicating very significant differences.

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1, IbWRKY32 related to improving salt tolerance of sweet potato and obtaining of coding gene thereof

1.1 cloning of IbWRKY32 Gene cDNA of Ipomoea batatas

1.1.1 extraction of Total RNA from sweet Potato

Experimental materials: the sweet potato strain 'xu potato 55-2'.

Grinding 0.1g of young and tender leaves of sweet potato into powder in liquid nitrogen, adding into a 2mL centrifuge tube, and extracting the total RNA of the sweet potato by using a TIANGEN RNAprep pure plant total RNA extraction kit (catalog number: DP432), wherein the kit comprises: lysis solution RL, deproteinization solution RW1, rinsing solution RW, RNase-Free ddH₂O, RNase-Free adsorption column CR3, RNase-Free filtration column CS, DNase I, buffer RDD, RNase-Free centrifuge tube, RNase-Free collection tube. Collecting 1 μ L, performing 1.2% agarose gel electrophoresis to detect its integrity, diluting 2 μ L to 500 μ L, and detecting its quality (OD) with ultraviolet spectrophotometer_260nm) And purity (OD)_260nm/OD_280nm) The extracted Xushu 55-2 total RNA is detected by non-denaturing gel agarose gel electrophoresis, 28S and 18S bands are clear, the brightness ratio of the two is 1.5-2: 1, the total RNA is not degraded, the obtained mRNA meets the experimental requirements, and the method can be used for the full-length cDNA of IbWRKY32 proteinCloning of (4).

1.1.2 cloning of cDNA sequence of IbWRKY32 Gene

Primers were designed for cloning of the IbWRKY32 cDNA sequence.

The primer sequences are as follows:

primer 1: 5'-ATGGATGACACAGGAGAAGCG-3'

Primer 2: 5'-TCAACAAGGCTTGATTTCGAAT-3'

The total RNA extracted in the step 1 is subjected to reverse transcription by a QuantScript RT Kit (TIANGEN, Beijing) to be used as a template, and high-fidelity LA enzyme is used for PCR amplification. Detecting the PCR amplification product by agarose gel electrophoresis to obtain an amplified fragment 1488bp in length.

After sequencing, the PCR product has a nucleotide sequence shown in SEQ ID No.2, a gene shown in the sequence is named as an IbWRKY32 gene, a coding region of the gene is nucleotides 1-1488 from the 5' end of the SEQ ID No.2, a protein coded by the gene is named as IbWRKY32 protein or protein IbWRKY32, an amino acid sequence is SEQ ID No.1, and the gene consists of 495 amino acid residues.

1.2 construction of plant expression vectors

According to the coding sequence of the cDNA of IbWRKY32 gene of sweet potato, designing and amplifying a primer sequence of the complete coding sequence, respectively introducing Kpn I and XbaI enzyme cutting sites into forward and reverse primers (primer 3 and primer 4), wherein the primer sequences are as follows:

primer 3: 5' -ACGGGGGACGAGCTCGGTACCATGGATGACACAGGAGAAGCG-3' (the underlined part is the Kpn I cleavage site sequence),

primer 4: 5' -GCTCACCATGTCGACTCTAGAACAAGGCTTGATTTCGAATCC-3' (the XbaI cleavage site is underlined).

And (3) carrying out PCR amplification by using the artificially synthesized SEQ ID No.2 as a template, and harvesting a PCR product for later use.

The vector pCAMBIA1300-35S (purchased from Wuhan transduction biology laboratory, product catalog number VT4001) was digested with Kpn I and XbaI, the large vector fragment was recovered, and at the same time, the PCR product was digested with Kpn I and XbaI, the intermediate fragment of about 1.5kb was recovered, and the recovered large vector fragment was ligated with the intermediate fragment of about 1.5kb to obtain the desired plasmid. The target plasmid is transformed into escherichia coli DH5a (purchased from Beijing Quanyujin biotechnology limited, product catalog number is CD201-01), cultured for 20h at 37 ℃, subjected to PCR analysis and enzyme digestion identification of the recombinant vector, and subjected to sequencing verification. Sequencing results show that a sequence shown in the 1 st-1488 th from the 5' end of SEQ ID No.2 is inserted between KpnI and XbaI restriction enzyme recognition sites of the vector pCAMBIA1300-35S, and other nucleotide sequences of the vector pCAMBIA1300-35S are kept unchanged, so that the construction of the recombinant vector is correct, and the recombinant vector is named as pCB-IbWRKY 32.

1.3 plant expression vector transformation of Agrobacterium

(1) 200 μ L of EHA105 competent cells (purchased from Beijing Byerdi Biotechnology Co., Ltd.) were taken out from a low temperature refrigerator of-80 deg.C, thawed on ice, added with 1 μ g of the plant expression vector pCAMBIA1300-IbWRKY32 obtained in the above step 1, and mixed well.

(2) Freezing with liquid nitrogen for 1min, and incubating at 37 deg.C for 5 min.

(3) Adding 800 μ L LB liquid culture medium, and culturing at 28 deg.C for 2-6 h.

(4) mu.L of the resulting suspension was applied to LB solid medium (containing 100. mu.g/mL rifampicin (Rif) and 25. mu.g/mL kanamycin (Kan)), and the applied solution was spread uniformly, followed by sealing the petri dish. The plates were inverted and incubated at 28 ℃ for 2 d.

(5) Taking a single colony which is positive in PCR identification, inoculating the single colony into LB liquid culture medium containing Rif of 100 mu g/mL and Kan of 25 mu g/mL, culturing at 28 ℃ for 30h to logarithmic phase, taking a proper amount of agrobacterium, and diluting the agrobacterium with the liquid MS culture medium by 30 times for later use to obtain agrobacterium liquid (agrobacterium liquid containing target genes) introduced into pCAMBIA1300-IbWRKY 32.

Example 2 genetic transformation and regeneration of tobacco

The coding sequence of IbWRKY32 cDNA was introduced into tobacco variety Wisconsin38(W38) using Agrobacterium-mediated method. The specific method comprises the following steps:

w38 tobacco leaves growing for about 4 weeks in culture medium were selected and placed in sterilized glass petri dishes. The main vein and the leaf edge are cut off, and a tobacco leaf disc of 1X 1cm is cut. The tobacco leaf discs were placed right side up on preculture medium (1.0mg/L6-BA, 0.1mg/L NAA in MS) and grown for 2-3d at 28 ℃ in the dark. Pre-cultured tobaccoThe leaf disk is soaked in the OD prepared in the step 2_600nmSoaking in 0.4-0.6 of Agrobacterium for 5-10 min. Sucking off the redundant bacterial liquid on the tobacco leaf disc, placing the tobacco leaf disc on the co-culture medium at the front side, and co-culturing for 2-3d under the dark condition at 28 ℃. 150, 300 and 600mg/L CS (full name or Chinese name) are respectively added into the liquid MS culture medium, and the co-cultured tobacco leaf discs are washed for 3 times according to the concentration from high to low. The excess liquid on the leaf disks was blotted with clean filter paper sterilized at high temperature and high pressure, and placed on a regeneration medium (MS of 15mg/L hygromycin, 400mg/L CS, 1.0mg/L6-BA and 0.1mg/L NAA) and cultured for 30 days at 28 ℃ under 3000lux of light until the shoots differentiated. 1cm of the regenerated bud is cut off and subcultured on a rooting medium (1/2 MS solid medium of 1.0mg/L6-BA, 0.1mg/L NAA, 400mg/L cephalexin and 15mg/L hygromycin) until a complete plant is grown to obtain a pseudotransgenic plant.

The identification of the transgenic plants uses a method combining PCR detection and qRT-PCR detection.

A. PCR detection

Extracting genome DNA of a pseudotransgenic plant and a wild tobacco plant by a CTAB method. PCR detection is carried out by a conventional method, and the used IbWRKY32 gene primers are as follows:

primer 5: 5'-TCCTTCGCAAGACCCTTCCTC-3'

Primer 6: 5'-TCAACAAGGCTTGATTTCGAAT-3' are provided.

To a 0.2ml Eppendorf centrifuge tube were added 2. mu.l of 10 XPCR buffer, 1. mu.l of 4dNTP (10mol/L), 1. mu.l of each primer (10. mu. mol/L), 2. mu.l of template DNA (50ng/ul), 1ul of Taq DNA polymerase, and H₂O to a total volume of 20. mu.l. The reaction program is denaturation at 94 ℃ for 4min, renaturation at 57 ℃ for 1.5min, and extension at 72 ℃ for 1min30s, for 35 cycles. The pCAMBIA1300-IbWRKY32 vector plasmid is used as a positive control, water and wild tobacco plants are used as negative controls, and then electrophoresis detection is carried out.

The result is shown in figure 1, and it can be seen from the figure that a 1500bp target band is amplified by the pseudotransgenic plant L1-L6 and the positive control, which indicates that the IbWRKY32 gene has been integrated into the genome of tobacco, and proves that the plants are transgenic plants; water and wild type tobacco plants did not amplify the target bands.

B、qRT-PCR

And extracting RNA of the positive plant of the transgenic tobacco, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR (quantitative reverse transcription-polymerase chain reaction) by taking the wild W38 plant as a control.

Taking a tobacco Actin (Actin) gene as an internal reference, wherein the primer sequence is as follows:

NtActin-F：5'-GAGGAATGCAGATCTTCGTG-3'

NtActin-R：5'-TCCTTGTCCTGGATCTTAGC-3'

the sequence of the IbWRKY32 primer is as follows:

primer 7: 5'-GATGAGCCTAGTGGAGCGAC-3'

Primer 8: 5'-GTATGGAAGGCGAGTCCACC-3'

The qRT-PCR result is shown in figure 2, and the result shows that the IbWRKY32 gene is expressed in different degrees in transgenic plants. Transgenic tobacco plants L1, L2 and L3 are selected for expanding propagation for subsequent experiments.

Example 3 identification of salt tolerance of transgenic plants

3.1 phenotypic characterization

The IbWRKY32 transgenic tobacco strains L1, L2 and L3 and wild type W38 were respectively cultured in MS culture medium (control culture medium) and MS culture medium (salt stress culture medium) containing NaCl (concentration 200mM), and each strain had 3 plants per culture medium. The culture conditions are 27 +/-1 ℃, the culture is carried out for 13h and 3000lux illumination every day, and the growth condition of the strain is observed after the strain culture is carried out for 4 weeks.

As shown in FIGS. 3A and 3B, the wild type W38 tobacco plant (denoted by "WT" in the figure) was less strongly grown on the MS medium containing NaCl (200 mM) and was difficult to root; the growth states of 3 IbWRKY32 overexpression transgenic tobacco strains L1, L2 and L3 are more superior to those of wild type W38 tobacco in different degrees. Under the culture condition of the MS culture medium (contrast), the fresh weights of the transgenic tobacco plants and the wild-type tobacco plants have no significant difference, and the fresh weights of 3 IbWRKY32 transgenic tobacco strains L1, L2 and L3 under the salt stress condition are significantly higher than those of wild-type W38 tobacco.

The results show that the salt tolerance of the transgenic tobacco plants is improved by over-expressing IbWRKY 32.

3.2、H₂O₂Determination of content

When plants are in stress or aging, the metabolism of active oxygen in vivo is enhanced to increase H₂O₂Accumulation occurs. H₂O₂Can directly or indirectly oxidize intracellular biomacromolecules such as nucleic acid, protein and the like, and damage cell membranes, thereby accelerating the aging and disintegration of cells. Thus, H₂O₂The higher the content of (a), the greater the degree to which the plant suffers stress injury.

Hydrogen peroxide (H)₂O₂) Kit (sumac gming biology, catalog No.: h₂O₂-2-Y) to detect H in tobacco plants₂O₂The accumulated amount. The tobacco plants are the tobacco plants which are treated for 4 weeks by adopting the control group in the step 3 and the tobacco plants which are treated for 4 weeks by adopting salt stress in the step 3. The tobacco plants include strains of transgenic tobacco plants L1, L2 and L3 and wild type tobacco plants (WT) which overexpress IbWRKY 32. The experiment was repeated three times and the results averaged.

H of plants over-expressing IbWRKY32 transgenic tobacco lines L1, L2 and L3 and wild type control plants₂O₂The results of the content determination are shown in FIG. 4, which shows the H of 3 transgenic lines on MS medium with sodium chloride (concentration 200mM)₂O₂The content is obviously lower than that of wild sweet potato plants.

The transgenic tobacco plant H₂O₂The content determination result shows that compared with a wild tobacco plant, the salt tolerance of a transgenic plant over-expressing IbWRKY32 is remarkably improved, which indicates that the protein IbWRKY32 and the coding gene thereof can be used for regulating and controlling the stress resistance of the plant, and particularly improving the salt tolerance of the plant.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> university of agriculture in China

<120> salt tolerance associated protein IbWRKY32, and coding gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 495

<212> PRT

<213> sweet potato (Ipomoea batatas)

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Thr Cys Ala Asp Ile Gly Gly Gly Gly Gly Gly Gly Asp Glu Pro Ser

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Gly Ala Thr Thr Gly Thr Glu Thr Ser Glu Glu Ala Gln Val Gly Gly

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Ser Asp Ser Glu Glu Thr Leu Asp Thr Val Asp Ser Pro Ser Ile Gln

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Leu Asp Lys Ser Ala Ser Arg Pro Asp Ser Leu Ala Thr Ser Ser Ser

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

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Phe Leu Lys Glu Ile Lys Asp Glu Val Gly Ile Ser Asn Gln Lys Ala

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Ser Thr Val Gln Ala Gln Arg Arg Asn Gln Leu Gln Ser Ala Asp Asp

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Pro Ser Val Leu Glu Leu Ser Pro Thr Ser Val Thr Gln Ser Ile Ser

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Ser Ile Pro Ser Pro Thr Pro Gly Glu Arg Arg Leu Ser Pro Leu Glu

145 150 155 160

Asn Arg Asn Gly Ala Cys Ile Gln Glu Val Asp Asn Gln Asn Ser Ser

165 170 175

Asn Ser Lys Ala Leu Ser Leu Val Pro Val Leu Lys Ile Gln Ala Pro

180 185 190

Asp Gly Tyr Asn Trp Arg Lys Tyr Gly Gln Lys Gln Val Lys Ser Pro

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Gln Gly Ser Arg Ser Tyr Tyr Arg Cys Thr Tyr Ser Asp Cys Cys Ala

210 215 220

Lys Lys Ile Glu Cys Ser Asp His Thr Asn Arg Val Thr Glu Ile Val

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Tyr Arg Ser Pro His Asn His Glu Pro Pro Arg Lys Val Asn Thr Pro

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Lys Val Asn Lys Leu Ala Ile Ser Ser Met Pro Arg Ser Gln Asp Ser

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Lys Val Ala Arg Leu Asn Ser Asn Ala Asp Glu Thr Val Pro Ser Thr

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Ser Lys Lys His Val Lys Glu Thr Ile Pro Ile Ser Glu Thr Lys Gln

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Gln Asp Phe Phe Gly Leu Asp Asp Asn Ala Glu Thr Asn Val Lys Arg

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Glu Asp Cys Asp Glu Pro Thr Gln Lys Lys Arg Leu Lys Lys Cys Ser

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Ser Ser Pro Glu Ser Leu Pro Lys Pro Gly Lys Lys Ala Lys Leu Val

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Val His Ala Gly Gly Asp Val Gly Ile Ser Ser Asp Gly Tyr Arg Trp

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Arg Lys Tyr Gly Gln Lys Met Val Lys Gly Asn Pro His Pro Arg Asn

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Tyr Tyr Arg Cys Thr Ser Ala Gly Cys Pro Val Arg Lys His Ile Glu

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Arg Ala Val Asp Asn Thr Thr Ala Val Ile Ile Thr Tyr Lys Gly Val

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His Asp His Gly Met Pro Val Pro Lys Lys Arg Tyr Gly Gln Pro Ser

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Ala Pro Leu Val Ala Ala Thr Ala Ser Ala Ser Met Thr Asp Ser Gln

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Thr Lys Lys Ser Glu Pro Thr Thr Gln Trp Ser Val Asp Lys Glu Gly

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Ala Leu Thr Gly Glu Thr Leu Glu His Glu Gly Glu Lys Thr Val Glu

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Ser Ala Lys Thr Leu Leu Ser Ile Gly Phe Glu Ile Lys Pro Cys

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atggatgaca caggagaagc gtctaagcca tctctgcagc tccaaaatac atgcgccgac 60

atcggcggcg gaggtggcgg tggtgatgag cctagtggag cgacgactgg aactgagact 120

tcagaagaag ctcaagtggg aggttctgac tccgaagaaa ccctagatac ggtggactcg 180

ccttccatac agttggataa gagtgctagc cgaccggatt cccttgctac ctcctcctcg 240

cacgtgctct ctgaagttcc aatcgagtat agcttgcacc cgtccgaatt cctgaaggaa 300

atcaaggatg aggttggcat ttctaaccag aaagcctcaa ctgttcaagc tcaaaggcgg 360

aaccaactgc agtctgctga tgatccatct gtgttggaat tatctccaac ttctgttaca 420

cagtccatat catccattcc cagcccaact ccaggagagc gaagattgtc tccattagag 480

aacagaaatg gcgcatgcat ccaagaagta gataaccaga attcttccaa ctccaaagct 540

ttatctcttg ttcctgtctt aaagatacaa gcacctgatg ggtacaattg gcggaaatat 600

ggtcaaaagc aagtgaagag tcctcaaggt tctcggagct attacagatg cacatattct 660

gactgctgtg caaaaaagat tgagtgttct gaccacacta accgtgttac agagattgtt 720

tatagaagtc ctcacaatca cgagccaccc cgaaaagtaa atacccctaa agtaaacaag 780

cttgcaatct catctatgcc tcgtagtcag gatagcaaag tagctcgcct aaatagtaat 840

gctgatgaga cagtgccatc cacttcaaag aaacatgtta aagaaacaat accaatatca 900

gagacaaagc agcaggattt ctttggattg gatgacaatg ctgaaactaa tgttaaacgg 960

gaggattgtg atgaacctac acagaagaaa agattgaaga aatgttcctc aagtcctgag 1020

tctcttccta aacctggcaa gaaagcaaaa ttggttgttc acgctggtgg tgatgtggga 1080

atctccagtg atggctatag gtggcgcaag tatggacaaa aaatggtgaa gggtaacccc 1140

catcccagga actattatcg gtgcacttcg gctggatgtc ctgttcggaa acacattgag 1200

agggctgtag acaacacgac tgctgtcatt ataacctata agggggttca tgatcatggc 1260

atgccagtac ctaagaaacg ttatggccaa cctagtgctc ccctagttgc cgcgactgcc 1320

tccgcttcca tgactgattc gcagactaag aaatctgaac caaccaccca gtggtcagta 1380

gacaaagaag gtgcattaac aggcgagaca ttggagcatg aaggagagaa aactgtggaa 1440

tcagctaaaa ctctattgag tattggattc gaaatcaagc cttgttga 1488