CN113563443B

CN113563443B - Salt tolerance related protein IbWRKY32, and coding gene and application thereof

Info

Publication number: CN113563443B
Application number: CN202110980074.6A
Authority: CN
Inventors: 翟红; 刘庆昌; 何绍贞; 高少培; 张欢; 孙思凡; 李思语
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2023-10-31
Anticipated expiration: 2041-08-25
Also published as: CN113563443A

Abstract

The application discloses application of a protein IbWRKY32 in regulating and controlling salt tolerance of plants. The application firstly discloses application of a protein in improving salt tolerance of plants; the protein is a protein with an amino acid sequence shown as SEQ ID NO.1 or a fusion protein obtained by connecting protein tags at the N end or/and the C end of the amino acid sequence shown as SEQ ID NO. 1. The application further discloses the protein-related biological material and application thereof. The application discovers IbWRKY32 protein and a coding gene thereof, and introduces the coding gene of the IbWRKY protein into tobacco to obtain a transgenic tobacco plant. The transgenic plant is subjected to salt stress treatment, so that 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.

Description

Salt tolerance related protein IbWRKY32, and coding gene and application thereof

Technical Field

The present application relates to the field of biotechnology. In particular to a salt-tolerant related protein IbWRKY32, and a coding gene and application thereof.

Background

In the natural world, adverse conditions such as high temperature, high salt, low temperature, waterlogging, drought, diseases, weeds and the like can influence the morphological structure and physiological and biochemical processes of plants, so that the normal growth and development of the plants are stopped, the quality is reduced, and the yield is reduced. Wherein drought and salt and alkali are stress factors which harm plant growth and have the highest influence on the global crop yield. At present, chinese saline-alkali soil and global saline-alkali soilThe products reach 0.27×108hm respectively ² And 9.5×108hm ² The latter occupies almost 10% of the world's surface area. The yield of crops planted on medium saline soil is reduced by about 95%, for example, the average yield reduction caused by saline alkali in various places of Huang-Huai-Hai plain is about 25%, and the serious yield reduction is even 50%. Therefore, it is important to explore stress-resistance mechanism of plants to dig stress-resistance genes and to improve stress resistance of plants by genetic engineering. Although sweet potato is drought-tolerant, in the absence of water, the growth, development, physiology and yield are affected as in other crops. Through the deep research on the salt-tolerant drought-resistant mechanism of plants, salt-tolerant drought-resistant gene resources are excavated, and the cultivation of new varieties of salt-tolerant drought-resistant sweet potatoes is one of the most economical and effective measures for utilizing saline-alkali soil and arid and semiarid resources.

Disclosure of Invention

The application aims to solve the technical problem of improving the salt tolerance of plants.

To solve the above technical problems, the present application provides first an application of a protein or a substance regulating the expression of a gene encoding the protein or a substance regulating the activity or content of the protein, wherein the application may be an application of a protein or a substance regulating the expression of a gene encoding the protein or a substance regulating the activity or content of the protein in regulating salt tolerance of plants, the protein may be IbWRKY32 protein, and the IbWRKY32 protein may be any 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 the A1), has more than 80 percent of identity with the protein shown in the A1) and has the function of regulating and controlling the salt tolerance of plants;

a3 Fusion proteins obtained by ligating protein tags at the N-terminus or/and the C-terminus of A1) or A2).

Further, the protein may be derived from sweet potato.

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

The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.

As used herein, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed with a protein of interest using DNA in vitro recombinant techniques to facilitate expression, detection, tracking and/or purification of the protein of interest. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

Herein, the identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

Herein, the 80% identity or more may 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 that regulates the activity or content of the protein may be a substance that regulates the expression of a gene encoding the protein.

In the above application, the substance for regulating gene expression may be a substance for performing at least one of the following 6 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 the 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 the protein translated by the gene).

In the above applications, the modulating gene expression may be increasing or augmenting the gene expression.

The substance that increases or increases the expression of the gene 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 comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2).

Wherein, the nucleic acid molecule of B1) can 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 as shown in the following G1) or G2):

g1 A cDNA molecule or a DNA molecule of SEQ ID No. 2;

g2 A cDNA molecule or a DNA molecule of SEQ ID No. 2;

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

In the above related biological materials, the expression cassette of B2) isRefers to DNA capable of expressing the protein IbWRKY32 in a host cell, which may include not only a promoter for promoting transcription of the IbWRKY32 gene, but also a terminator for terminating transcription of the IbWRKY32 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present application 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: a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed specific promoters such as the millet seed specific promoter pF128 (CN 101063139B (China patent 2007 1 0099169.7)), seed storage protein specific promoters (e.g., phaseolin, napin, oleosin and soybean beta-glucose promoters (Beachy et al (1985) EMBO J.4:3047-3053) which may 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, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, tml terminator, the pea rbcS E9 terminator and the 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; proudroot (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 above related biological material, the recombinant vector of B3) may contain a DNA molecule shown in SEQ ID No.2 for encoding the protein IbWRKY32.

The plant expression vector can be used for constructing a recombinant vector containing the IbWRKY32 coding gene expression cassette. The plant expression vector may be a Gateway system vector or a binary agrobacterium vector, etc., such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300-35S, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When IbWRKY32 is used to construct a recombinant vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like may be added before the transcription initiation nucleotide thereof, and they may be used alone or in combination with other plant promoters; in addition, when the gene of the present application is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants.

Of the above-mentioned related biological materials, the recombinant microorganism of B4) may be specifically yeasts, bacteria, algae and fungi.

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

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

Among the above-mentioned related biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may or may not include propagation material.

The application 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 target plants with the salt tolerance higher than that of the receptor plant.

Further, the coding gene in the above method is G1) or G2) as follows:

g1 A coding sequence of the coding strand is a cDNA molecule or a DNA molecule as shown in 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 plant of interest by a plant expression vector carrying the protein-encoding gene.

The plant expression vector carrying the protein-encoding gene of the present application may be obtained by transforming plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium-mediated transformation, etc., and cultivating the transformed plant cells or tissues into plants.

In a specific embodiment of the application, the recombinant vector is recombinant plasmid pCB-IbWRKY32. 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 cleavage recognition sites of the vector pCAMBIA1300-35S by utilizing restriction enzymes KpnI and XbaI and keeping other sequences of the vector pCAMBIA1300-35S unchanged.

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

The plant may be a dicotyledonous plant, which may be a plant of the order Solanaceae. The plant of Solanaceae is plant of Solanaceae. The plant of the Solanaceae family may be a plant of the Nicotiana genus. The nicotiana plant can be tobacco.

The proteins described above, and/or biological materials associated with proteins, are also within the scope of the present application.

The application provides IbWRKY32 protein and a coding gene thereof, and introduces the coding gene of the IbWRKY32 protein into tobacco W38 to obtain a transgenic tobacco plant of IbWRKY32. Salt stress treatment is carried out on the transgenic plant, compared with the control, the salt tolerance of the transgenic plant is enhanced, and the specific embodiment is that H is reduced ₂ O ₂ The content is as follows. Therefore, the IbWRKY32 gene and the protein encoded by the gene play an important role in the plant salt tolerance process, have important application value in the research of improving the plant salt tolerance, and have wide application space and market prospect in the agricultural field.

Drawings

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

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

FIG. 3A shows plant growth in a salt tolerance test of transgenic tobacco plants over-expressing IbWRKY32 and wild type W38 plants; wherein, WT is a wild W38 plant; l1, L2 and L3 are transgenic tobacco plants over-expressing IbWRKY 32; controls represent growth vigor and fresh weight for 4 weeks in normal medium, salt stress for 4 weeks in stress medium.

FIG. 3B shows plant fresh weight in salt tolerance assay of transgenic tobacco plants overexpressing IbWRKY32 and wild type W38 plants; wherein, WT is a wild W38 plant; l1, L2 and L3 are transgenic tobacco plants over-expressing IbWRKY 32; controls represent growth vigor and fresh weight for 4 weeks in normal medium, salt stress for 4 weeks in stress medium.

FIG. 4 is a H over-expressing IbWRKY32 transgenic tobacco plant and wild type control plant ₂ O ₂ Measuring the content; wherein, WT is a wild type W38 plant; l1, L2 and L3 are transgenic tobacco plants over-expressing IbWRKY32.

Detailed Description

The bradyctolagus chinensis 55-2 is disclosed in the literature: zhu Hong cloning and functional identification of drought-resistance related genes IbWRKY2, ibGATA24 and IbSDT are disclosed in doctor's thesis, chinese university of agriculture, 2018, and the public, after approval by the authors, can be obtained from the national university of agriculture, sweet potato genetic breeding laboratory to repeat the experiment and cannot be used for other purposes.

Tobacco variety W38 is disclosed in the literature "Jiang T, zhai H, wang FB, zhou HN, si ZZ, he SZ, liu QC. Cloning and haracterization of a Salt Tolerance-Associated Gene Encoding Trehalose-6-Phosphate Synthase in Sweetpotato, journal of Integrative Agriculture 2014,13 (8): 1651-1661", and is publicly available from the national agricultural university sweet potato genetic breeding laboratory after approval by the authors for repeat of the experiment and is not available for other uses.

Vector pCAMBIA1300-35S was purchased from wuhan transduction biology laboratory limited under the product catalog number VT4004.

Agrobacterium tumefaciens EHA105 was purchased from Beijing bayer Di Biotechnology Co.

Coli DH5a (from Beijing full gold biotechnology Co., ltd., catalog number CD 201-01)

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

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 production of protein IbWRKY32 related to improving salt tolerance of sweet Potato and Gene encoding the same

Cloning of IbWRKY32 Gene cDNA of Ipomoea batatas

1.1.1 Total RNA extraction from sweet Potato

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

Grinding 0.1g of tender leaves of sweet potato into powder in liquid nitrogen, adding into a 2mL centrifuge tube, and extracting total RNA of the sweet potato by using an RNAprep pure plant total RNA extraction kit (catalog number: DP 432) of TIANGEN, wherein the kit comprises: lysate RL, deproteinized solution RW1, rinsing solution RW, RNase-Free ddH ₂ O, RNase-Free adsorption column CR3, RNase-Free filtration column CS, DNase I, buffer RDD, RNase-Free centrifuge tube, and RNase-Free collection tube. 1. Mu.L of the sample was subjected to 1.2% agarose gel electrophoresis to determine the integrity, 2. Mu.L of the sample was diluted to 500. Mu.L, and the mass (OD) was measured by an ultraviolet spectrophotometer _260nm ) And purity (OD) _260nm /OD _280nm ) The extracted total RNA of the yam 55-2 is detected by non-denaturing gel agarose gel electrophoresis, 28S and 18S bands are clear, and the brightness ratio of the two bands is 1.5-2:1, so that the total RNA is not degraded, the mRNA meets the experimental requirements, and the mRNA can be used for cloning the full length of the IbWRKY32 protein cDNA.

1.1.2 cloning of cDNA sequence of IbWRKY32 Gene

Primers were designed to clone the IbWRKY32 cDNA sequence.

The primer sequences were as follows:

primer 1:5'-ATGGATGACACAGGAGAAGCG-3'

Primer 2:5'-TCAACAAGGCTTGATTTCGAAT-3'

The total RNA extracted in the step 1 was reverse transcribed into a template of QuantScript RT Kit (TIANGEN, beijing) and amplified by a high-fidelity LA enzyme. And detecting the PCR amplification product by agarose gel electrophoresis to obtain the amplified fragment with the length of 1488 bp.

Through sequencing, the PCR product has a nucleotide sequence shown in SEQ ID No.2, a gene shown in the sequence is named as IbWRKY32 gene, the coding region of the gene is 1 st-1488 th nucleotides from the 5' end of the SEQ ID No.2, the protein coded by the gene is named as IbWRKY32 protein or protein IbWRKY32, the amino acid sequence is named as 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 sweet potato IbWRKY32 gene cDNA, designing a primer sequence for amplifying the complete coding sequence, and respectively introducing Kpn I and XbaI enzyme cutting sites into forward and reverse primers (primer 3 and primer 4), wherein the primer sequence is as follows:

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

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

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

The vector pCAMBIA1300-35S (available from Whan transduction biological laboratory under the product catalog number VT 4001) was digested with Kpn I and XbaI, and the vector large fragment was recovered, and at the same time, the above PCR product was digested with Kpn I and XbaI, and an intermediate fragment of about 1.5kb was recovered, and the recovered vector large fragment was ligated with the intermediate fragment of about 1.5kb to obtain the objective plasmid. The target plasmid was transformed into E.coli DH5a (purchased from Beijing full-size gold biotechnology Co., ltd., catalog number of CD 201-01), cultured at 37℃for 20 hours, subjected to PCR analysis and enzyme digestion identification of the recombinant vector, and subjected to sequencing verification. Sequencing results show that the sequence shown in the 1 st to 1488 th positions of SEQ ID No.2 from the 5' end is inserted between KpnI and XbaI cleavage recognition sites of the vector pCAMBIA1300-35S, and other nucleotide sequences of the vector pCAMBIA1300-35S remain unchanged, which indicates that the recombinant vector is constructed correctly, and the recombinant vector is named pCB-IbWRKY32.

1.3 transformation of Agrobacterium with plant expression vectors

(1) 200. Mu.L of EHA105 competent cells (purchased from Beijing Bayer Di Biotechnology Co., ltd.) were taken out from a-80℃low temperature refrigerator, thawed on ice, and 1. Mu.g of the plant expression vector pCAMBIA1300-IbWRKY32 obtained in the above step 1 was added and mixed well.

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

(3) 800. Mu.L of LB liquid medium was added thereto, and the mixture was cultured at 28℃for 2-6 hours.

(4) mu.L of the bacterial liquid was spread uniformly on LB solid medium (containing 100. Mu.g/mL rifampicin (Rif), 25. Mu.g/mL kanamycin (Kan)), and the dish was sealed. The dish was inverted and incubated at 28℃for 2d.

(5) And (3) taking a single colony positive to PCR identification, inoculating the single colony into an LB liquid culture medium containing 100 mug/mL of Rif and 25 mug/mL of Kan, culturing at 28 ℃ for 30 hours to logarithmic phase, and diluting a proper amount of agrobacterium with a liquid MS culture medium for 30 times for later use to obtain an agrobacterium liquid (agrobacterium liquid containing a target gene) introduced into pCAMBIA1300-IbWRKY 32.

EXAMPLE 2 genetic transformation and regeneration of tobacco

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

the W38 tobacco leaves grown in the medium for about 4 weeks were selected and placed in sterilized glass dishes. The main vein and leaf edge were cut off, and a tobacco leaf disk of 1X 1cm was cut. The tobacco leaf discs were placed face up on pre-culture medium (MS of 1.0mg/L6-BA, 0.1mg/L NAA), grown for 2-3d at 28℃in the dark. Soaking the tobacco leaf disc after preculture in the OD prepared in the step 2 _600nm Infection soaking in 0.4-0.6 of Agrobacterium for 5-10min. The excessive bacterial liquid on the tobacco leaf disc is sucked, and the tobacco leaf disc is placed on a co-culture medium at 28 ℃ for co-culture for 2-3d under the dark condition. 150 mg/L CS (fully called or Chinese name) is added into a liquid MS culture medium, and the tobacco leaf discs after 3 times of co-culture are cleaned according to the concentration from high to low. Excess liquid on leaf discs was blotted with clean filter paper sterilized at high temperature and high pressure, placed on regeneration medium (MS of 15mg/L hygromycin, 400mg/L CS, 1.0mg/L6-BA and 0.1mg/L NAA), illuminated with 3000lux light, incubated at 28℃for 30d until shoots had differentiated. Cutting off 1cm regeneration bud, transferring onto rooting culture medium (1.0 mg/L6-BA, 0.1mg/L NAA, 400mg/L cefalexin, 15mg/L hygromycin 1/2MS solid culture medium) until complete plant is grown to obtain quasi-transfer mediumDue to the plant.

Identification of transgenic plants uses a combination of PCR detection and qRT-PCR detection.

A. PCR detection

Genomic DNA of the quasi-transgenic plants and wild tobacco plants was extracted by CTAB method. PCR was performed using conventional methods using the IbWRKY32 gene primers:

primer 5:5'-TCCTTCGCAAGACCCTTCCTC-3'

Primer 6:5'-TCAACAAGGCTTGATTTCGAAT-3'.

10 XPCR buffer 2. Mu.l, 4dNTP (10 mol/L) 1. Mu.l, 1. Mu.l primer (10. Mu. Mol/L), 2. Mu.l template DNA (50 ng/ul), 1ul Taq DNA polymerase and H were added to a 0.2ml Eppendorf centrifuge tube ₂ O to a total volume of 20. Mu.l. The reaction procedure was 94℃denaturation for 4min,57℃renaturation for 1.5min,72℃extension for 1min30s for 35 cycles. The pCAMBIA1300-IbWRKY32 vector plasmid was used as positive control, water and wild tobacco plants as negative control, and then electrophoretically detected.

The result is shown in FIG. 1, and it can be seen from the graph that the target bands of 1500bp are amplified by the quasi-transgenic plants L1-L6 and the positive control, which shows that the IbWRKY32 gene is integrated into the genome of tobacco, and the plants are proved to be transgenic plants; the water and wild type tobacco plants did not amplify the target band.

B、qRT-PCR

Extracting RNA of the transgenic tobacco positive plant, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR by taking a wild W38 plant as a control.

Taking a tobacco Actin (action) gene as an internal reference, the primer sequences are as follows:

NtActin-F：5'-GAGGAATGCAGATCTTCGTG-3'

NtActin-R：5'-TCCTTGTCCTGGATCTTAGC-3'

the IbWRKY32 primer sequence is as follows:

primer 7:5'-GATGAGCCTAGTGGAGCGAC-3'

Primer 8:5'-GTATGGAAGGCGAGTCCACC-3'

The qRT-PCR results are shown in FIG. 2, and the results show that the IbWRKY32 gene is expressed in transgenic plants to different degrees. And selecting transgenic tobacco plants L1, L2 and L3 for propagation and carrying out subsequent experiments.

Example 3 salt tolerance identification of transgenic plants

3.1 phenotypic identification

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

As a result, as shown in FIGS. 3A and 3B, wild-type W38 tobacco plants (indicated by "WT" in the figures) grew poorly on MS medium with NaCl (200 mM concentration), and rooting was difficult; the growth state of the 3 transgenic tobacco lines L1, L2 and L3 with the over-expression IbWRKY32 is better than that of wild type W38 tobacco to different degrees. Under the condition of MS culture medium culture (control), the fresh weights of the transgenic tobacco plants and the wild tobacco plants are not significantly different, and the fresh weights of 3 over-expressed IbWRKY32 transgenic tobacco lines L1, L2 and L3 under the condition of salt stress are significantly higher than that of the wild type W38 tobacco.

The above results demonstrate that over-expression of IbWRKY32 increases the salt tolerance of transgenic tobacco plants.

3.2、H ₂ O ₂ Content determination

H due to enhanced in vivo active oxygen metabolism in plants under adverse conditions or aging ₂ O ₂ Accumulation occurs. H ₂ O ₂ Can oxidize biomacromolecules such as nucleic acid, protein and the like in cells directly or indirectly and damage cell membranes, thereby accelerating aging and disintegration of cells. Thus H ₂ O ₂ The higher the content of (c) the greater the extent to which the plant suffers from stress injury.

Hydrogen peroxide (H) ₂ O ₂ ) Kit (su zhou koku Ming biology, catalog number: h ₂ O ₂ -2-Y) to detect H of tobacco plants ₂ O ₂ Accumulation amount. The tobacco plants were tobacco plants treated for 4 weeks with the control group in step 3 above and tobacco plants treated for 4 weeks with salt stress in step 3 above. Tobacco plantStrains include those that are over-expressing IbWRKY32 transgenic tobacco plants L1, L2, L3 and wild-type tobacco plants (WTs). The experiment was repeated three times and the results averaged.

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

Transgenic tobacco plant H as described above ₂ O ₂ Compared with wild tobacco plants, the salt tolerance of transgenic plants over-expressing IbWRKY32 is obviously improved, which indicates that the protein IbWRKY32 and the coding gene thereof can be used for regulating and controlling plant stress tolerance, in particular to improving plant salt tolerance.

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

Sequence listing

<110> Chinese university of agriculture

<120> salt tolerance related 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)

<400> 1

Met Asp Asp Thr Gly Glu Ala Ser Lys Pro Ser Leu Gln Leu Gln Asn

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

195 200 205

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

<210> 2

<211> 1488

<212> DNA

<213> sweet potato (Ipomoea batatas)

<400> 2

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

Claims

1. The use of a protein or a biological material related to said protein for increasing the salt tolerance of a plant,

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 Fusion proteins obtained by ligating protein tags at the N-terminus or/and the C-terminus of A1);

the biomaterial is any one of the following B1) to B7):

b1 A nucleic acid molecule encoding said protein;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2);

the plant is tobacco.

2. The use according to claim 1, wherein the nucleic acid molecule is a DNA molecule whose coding sequence of the coding strand is SEQ ID No. 2.

3. A method for improving salt tolerance of a plant, comprising increasing the expression level of a gene encoding the protein of claim 1 in a plant of interest, thereby improving salt tolerance of the plant of interest;

the plant is tobacco.

4. The method of claim 3, wherein the coding gene of the protein is a DNA molecule having a coding sequence of SEQ ID No. 2.