CN113801890B - Application of protein ZmbZIPc3 in regulation and control of salt tolerance and drought resistance of plants - Google Patents

Abstract

The application discloses application of a protein ZmbZIPc3 in regulating and controlling salt tolerance and drought resistance of plants. The application provides application of protein and related biological materials in regulating drought resistance and/or salt tolerance of plants, wherein the protein is a protein with an amino acid sequence shown as SEQ ID NO.2 or a fusion protein obtained by connecting protein tags at N-terminal or/and C-terminal of the amino acid sequence shown as SEQ ID NO. 2. According to the application, a ZmbZIPc3 gene related to drought resistance is cloned from corn, and is transferred into Arabidopsis thaliana and corn, and the ZmbZIPc3 gene is found to improve the osmotic stress resistance and the salt stress tolerance of Arabidopsis thaliana, and improve the drought resistance and the yield of corn. Therefore, the application has important significance for improving the drought tolerance of plants, stabilizing the crop yield, identifying and cultivating new varieties of good crops.

Description

Application of protein ZmbZIPc3 in regulation and control of salt tolerance and drought resistance of plants

Technical Field

The present application relates to the field of biotechnology. In particular to application of a protein ZmbZIPc3 in regulating and controlling salt tolerance and drought resistance of plants.

Background

The arid and semiarid regions of the world occupy more than one third of the total land area, while the cultivated land with irrigation conditions only occupies about three percent of the total land area, thus greatly restricting the global grain production. Drought has become a major factor affecting global grain yield, and now drought areas are enlarged year by year, and natural environment deterioration leads to land desertification and grassland degradation. In the face of the increasing deterioration of plant and crop growth environments, plant biologists and agrologists are increasingly focusing on and researching plant stress resistance, and aim to analyze how plants resist various stress and mechanisms thereof so as to cultivate crop varieties with high yield, good quality and strong stress resistance.

Corn is one of three crops cultivated in the world and is used as feed, food, deep processing and industrial raw material. As an important grain crop in China, the sowing area is up to above 4200 ten thousand hectares, and the annual yield is up to 2.59 hundred million tons. Corn is a crop with large water demand, and water deficiency in critical growth period can seriously affect yield. Corn farmlands in main corn producing areas such as northwest, southwest, north China and northeast China mostly depend on dry lands with natural precipitation, and drought frequently occurs, so that the stability of corn yield is affected. The breeding of drought-resistant corn variety becomes one of the important ways to raise corn yield. The drought-resistant related mechanism of the corn is analyzed from the molecular level, and functional genes or transcription factors related to drought resistance in the corn are cloned, discovered and identified, so that the drought-resistant fine variety is cultivated efficiently by utilizing the genetic engineering means, and the method has practical significance for improving the crop yield.

Disclosure of Invention

The technical problem to be solved by the application is how to improve the stress tolerance, especially the salt tolerance and drought resistance of plants so as to further improve the growth of the plants.

In order to solve the technical problems, the application firstly provides application of a protein in regulating drought resistance and/or salt tolerance of plants, wherein the protein is derived from corn (Zea mays L.), and is named ZmbZIPc3 protein or protein ZmbZIPc3, and the protein is shown in any one of the following A1), A2) or A3):

a1 Protein with the amino acid sequence shown as SEQ ID NO. 2;

a2 Fusion protein obtained by N-terminal or/and C-terminal connexin label of the amino acid sequence shown in SEQ ID NO. 2;

a3 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2, has more than 90 percent of identity with the protein shown in A1) and has the same function.

Wherein SEQ ID NO.2 consists of 410 amino acid residues.

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

In the above application, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed with the target protein by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

In the above applications, identity refers to the identity of amino acid sequences. 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.

In the above applications, the 90% identity or more may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

The application of the biological material related to the protein ZmbZIPc3 in regulating and controlling the drought resistance and/or salt tolerance of plants is also within the protection scope of the application: the biological material is any one of the following C1) -C10):

c1 A nucleic acid molecule encoding the protein zmbzip 3;

c2 An expression cassette comprising C1) said nucleic acid molecule;

c3 A recombinant vector comprising C1) said nucleic acid molecule, or a recombinant vector comprising C2) said expression cassette;

c4 A recombinant microorganism comprising C1) said nucleic acid molecule, or a recombinant microorganism comprising C2) said expression cassette, or a recombinant microorganism comprising C3) said recombinant vector;

c5 A transgenic plant cell line comprising C1) said nucleic acid molecule, or a transgenic plant cell line comprising C2) said expression cassette, or a transgenic plant cell line comprising C3) said recombinant vector;

c6 A) a transgenic plant tissue comprising C1) said nucleic acid molecule, or a transgenic plant tissue comprising C2) said expression cassette, or a transgenic plant tissue comprising C3) said recombinant vector;

c7 A transgenic plant organ comprising C1) said nucleic acid molecule, or a transgenic plant organ comprising C2) said expression cassette, or a transgenic plant organ comprising C3) said recombinant vector;

c8 A transgenic plant containing the nucleic acid molecule of C1), or a transgenic plant containing the expression cassette of C2), or a transgenic plant containing the recombinant vector of C3);

c9 A tissue culture produced by regenerable cells of the transgenic plant of C8);

c10 Protoplasts produced from the tissue culture of C9).

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.

In the above biological material, the nucleic acid molecule of C1) encoding the protein zmbzip 3 may specifically be any one of the following D1), D2) or D3):

d1 51-1283 DNA molecules of SEQ ID NO. 1;

d2 A DNA molecule with the coding sequence shown in the 51 st to 1283 rd positions of SEQ ID NO. 1;

d3 A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in D1) or D2) and which codes for the protein ZmbZIPc3.

Wherein, SEQ ID NO.1 is composed of 1493 nucleotides, the coding sequence is the 51 st to 1283 rd positions from the 5' end of SEQ ID NO.1, and the coding amino acid sequence is the protein shown as SEQ ID NO. 2.

The stringent conditions are hybridization and washing of the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing of the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time.

In the above biological material, the expression cassette of C2) is an expression cassette containing a nucleic acid molecule encoding the protein ZmbZIPc3 (i.e., zmbZIPc3 gene expression cassette), which means that it is capable of being hostedA DNA molecule expressing the protein zmbzip 3 in a host cell, which may include not only a promoter for initiating transcription of the zmbzip 3 gene, but also a terminator for terminating transcription of the zmbzip 3 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 a specific embodiment of the application, the sequence of the ZmbZIPc3 gene expression cassette is shown as SEQ ID NO.4, and the sequence sequentially comprises a promoter 35SS (36-720 th site from the 5' end of SEQ ID NO. 4), a coding sequence of the ZmbZIPc3 gene (1140-2373 th site from the 5' end of SEQ ID NO.4, corresponding to 51-1283 th sites of SEQ ID NO. 1) and a terminator PA35S (2593-2810 th site from the 5' end of SEQ ID NO. 4) on a pLeela vector.

In the above biological material, C3) the recombinant vector may contain a DNA molecule shown in SEQ ID NO.1 for encoding the protein ZmbZIPc3 (i.e., containing the ZmbZIPc3 gene).

The plant expression vector can be used for constructing a recombinant vector containing the ZmbZIPc3 gene expression cassette. The plant expression vector may be a Gateway system vector or a binary agrobacterium vector, etc., such as pweela, pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When ZmbZIPc3 is used to construct recombinant vectors, 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, can be added before the transcription initiation nucleotide thereof, and can 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.

In a specific embodiment of the application, the recombinant vector is a pLeela 35SS: zmbZIPc3 overexpression vector or a pU130 35SS: zmbZIPc3 overexpression vector. ZmbZIPc3 over-expression vector is a vector which is obtained by replacing the sequence between attR1 and attR2 sites of the pLeela vector with a DNA molecule shown in SEQ ID NO.3 and keeping other sequences unchanged; the pU130 35SS is obtained by inserting a ZmbZIPc3 gene expression cassette shown in SEQ ID No.4 between HindIII recognition sites of the pU130 vector and keeping other sequences of the pU130 vector unchanged.

Of the above biological materials, C4) the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.

In the above biological material, the transgenic plant organ of C7) may be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

In the above biological material, C9) the tissue culture may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

In the above biological materials, none of the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs include propagation material.

The application of the protein ZmbZIPc3 or the biological material thereof in plant breeding is also within the protection scope of the application.

Among the above applications, the plant breeding may be specifically performed by crossing a plant containing the protein zmbzip 3 or a biological material related to the protein zmbzip 3 (e.g., the protein zmbzip 3 encoding gene zmbzip 3) with other plants for plant breeding.

In the application, the drought resistance of the regulation plant is improved, and the salt tolerance of the regulation plant is improved.

The application further provides a method for cultivating transgenic plants with high drought resistance and/or salt tolerance.

The method for cultivating transgenic plants with high drought resistance and/or salt tolerance comprises the steps of increasing the expression quantity of a gene of a protein ZmbZIPc3 in a target plant and/or the content of the protein ZmbZIPc3 and/or the activity of the protein ZmbZIPc3 to obtain transgenic plants; the transgenic plants have drought and/or salt tolerance higher than the plants of interest.

In the above method, the method for increasing the expression level of the gene of the protein ZmbZIPc3 in the target plant and/or the content of the protein ZmbZIPc3 and/or the activity of the protein ZmbZIPc3 is to express or overexpress the protein ZmbZIPc3 in the target plant.

In the method, the method of expression or over-expression is to introduce the coding gene of the protein ZmbZIPc3 into a target plant.

In the above method, the gene encoding the protein zmbzip 3 may be introduced into a target plant by a plant expression vector carrying the zmbzip 3 gene of the application. The plant expression vector carrying ZmbZIPc3 of the application may be transformed into plant cells or tissues by using conventional biological methods such as Agrobacterium-mediated, ti-plasmid, ri-plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, etc., and the transformed plant cells or tissues may be cultivated into plants.

In the method, the nucleotide sequence of the coding gene of the protein ZmbZIPc3 is a DNA molecule shown in SEQ ID NO. 1.

In a specific embodiment of the application, the plant expression vector carrying the gene ZmbZIPc3 of the application can be a pLeela 35SS: zmbZIPc3 over-expression vector or a pU130 35SS: zmbZIPc3 over-expression vector. ZmbZIPc3 over-expression vector is a vector which is obtained by replacing the sequence between attR1 and attR2 sites of the pLeela vector with a DNA molecule shown in SEQ ID NO.3 and keeping other sequences unchanged; the pU130 35SS is obtained by inserting a ZmbZIPc3 gene expression cassette shown in SEQ ID No.4 between HindIII recognition sites of the pU130 vector and keeping other sequences of the pU130 vector unchanged.

In the application, the improvement of drought resistance or high drought resistance of plants is particularly characterized by sensitivity to ABA (abscisic acid), high plant survival rate, low plant wilting rate, fast root growth, small aperture opening and the like under drought conditions.

In the present application, the plant is any one of the following E1) to E6):

e1 Dicotyledonous plants;

e2 Monocotyledonous plants;

e3 Cruciferous plants;

e4 Arabidopsis thaliana (Arabidopsis thaliana);

e5 A gramineous plant;

e6 Corn (Zea mays).

The above protein zmbzip 3 and biological materials related to the above protein zmbzip 3 are also within the scope of the application.

The application clones and obtains ZmbZIPc3 gene related to drought resistance from corn. The ZmbZIPc3 gene is transferred into the Arabidopsis thaliana, and the ZmbZIPc3 gene can improve the osmotic stress resistance, improve the salt stress tolerance of the Arabidopsis thaliana, reduce the pore opening of the Arabidopsis thaliana, slow down the water loss rate and facilitate drought resistance and water retention of plants; overexpression of the zmbzip 3 gene in maize has been found to: the ZmbZIPc3 gene affects the growth and development of corn in germination and seedling stage, so as to raise the drought tolerance and yield of corn. Therefore, the application has important significance for improving the drought tolerance of plants, stabilizing the crop yield, identifying and cultivating new varieties of good crops.

Drawings

FIG. 1 shows the expression level identification (A) of ZmZIPc 3-transferred Arabidopsis thaliana and the expression level identification (B) of ZmZIPc 3-transferred corn.

FIG. 2 shows the comparison of growth (a and b) and root length (c) of Arabidopsis thaliana transformed with ZmZIPc 3 under osmotic stress.

FIG. 3 shows the growth of Arabidopsis thaliana transformed with ZmZIPc 3 under salt stress.

FIG. 4 shows germination of Arabidopsis thaliana seeds transformed to ZmZIPc 3 under ABA treatment conditions; wherein A is the germination condition of ZmZIPc 3 Arabidopsis seeds transferred in a 1/2MS culture medium without ABA, B is the germination condition of ZmZIPc 3 Arabidopsis seeds transferred in a 1/2MS culture medium added with 0.25 mu M ABA, C is the germination condition of ZmZIPc 3 Arabidopsis seeds transferred in a 1/2MS culture medium added with 0.5 mu M ABA, and D is the germination condition of ZmZIPc 3 Arabidopsis seeds transferred in a 1/2MS culture medium added with 1 mu M ABA.

FIG. 5 shows the seedling growth of ZmZIPc 3-transformed Arabidopsis under ABA treatment conditions; wherein A is the growth condition of ZmZIPc 3 Arabidopsis seedlings transferred in a 1/2MS culture medium without ABA, B is the growth condition of ZmZIPc 3 Arabidopsis seedlings transferred in a 1/2MS culture medium added with 0.1 mu M ABA, and C is the growth condition of ZmZIPc 3 Arabidopsis seedlings transferred in a 1/2MS culture medium added with 1 mu M ABA.

FIG. 6 shows the growth of Arabidopsis thaliana transformed with ZmZIPc 3 under drought stress.

FIG. 7 is a comparison result of pore opening degrees of Arabidopsis thaliana leaves transformed with ZmbZIPc3 under drought stress.

FIG. 8 shows seed germination (A) and drought resistance index (B) under drought stress during germination of ZmZIPc 3 overexpressing corn.

FIG. 9 shows root observations under drought stress in maize germination over-expressing ZmZIPc 3.

FIG. 10 is an identification of growth under drought stress in maize seedlings over-expressing ZmZIPc 3; a is that seedlings grow to a three-leaf one-heart period; b is the growth condition after 72 hours of first rehydration treatment; c is that the second drought treatment enters a wilting state; d is the growth condition of the second rehydration treatment for 72 h.

In FIGS. 1-7, co1 represents wild type Arabidopsis Col-0,2-1 represents ZmZIPc 3-transferred Arabidopsis strain OE2-1, 5-6 represents ZmZIPc 3-transferred Arabidopsis strain OE 5-6 and 10-4 represents ZmZIPc 3-transferred Arabidopsis strain OE 10-4.

In FIGS. 1, 8-10, WT represents wild-type maize C01, C3-1 represents ZmZIPc 3-transferred Arabidopsis line OE C3-1, C3-3 represents ZmZIPc 3-transferred Arabidopsis line OE C3-3, C3-5 represents ZmZIPc 3-transferred Arabidopsis line OE C3-5 and C3-7 represents ZmZIPc 3-transferred Arabidopsis line OE C3-7.

Detailed Description

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 are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The quantitative tests in the following examples were all performed in triplicate, and the results were averaged.

The pENTR/D-TOPO vector in the following examples is a product of Invitrogen corporation.

The plant expression vector pLeela in the examples described below is described in non-patent document "A novel role for histone methyltransferase KYP/SUVH4 in the control of Arabidopsis primary seed dormancy, new Phytologist (2012) 193:605-616", which is publicly available from plant research at the national academy of sciences, to repeat the experiments of the application, and is not useful for other purposes. .

Maize inbred line B73 in the following examples is described in non-patent document "Zea mays l.; zmbZIP60 mRNA is spliced in maize in response to ER stress, BMC Research Notes (2012) 5: the public was available from the national academy of sciences plant institute to repeat the experiments of the application and was not available for other uses.

pU130 vectors in the examples described below are described in the literature "Development of Novel Glyphosate-Tolerant Japonica Rice Lines: A Step Toward Commercial Release. Cui Y, et al front. Plant Sci. (2016) 7:1218", and are available to the public from national academy of sciences plant research, and this biomaterial is used only for repeated experiments in connection with the application and is not used for other purposes; the public is also available from the national seed group company life science and technology center.

Agrobacterium tumefaciens GV3101 (Agrobacterium tumefaciens strain GV 3101) in the following examples: as described in the document "Binary Agrobacterium vectors for plant transformation, M Bevanin, nucleic Acids Research (1984) 12 (22): 8711-8721", the public is available from the national academy of sciences plant study, and the biomaterial is used only for repeated experiments related to the present application and is not used for other purposes.

Arabidopsis Col-0 in the following examples is described in the literature "Arabidopsis, a use of the strain Meyerowitz EM, cell (1989): 263-270", and is available to the public from plant research institute of national academy of sciences, and this biomaterial is used only for repeated experiments related to the present application and is not used for other purposes.

Example 1 obtaining ZmbZIPc3 Gene

1. cDNA acquisition

Total RNA of the trefoil-heart maize inbred line B73 was extracted and reverse transcribed into cDNA.

2. PCR amplification

And (3) taking the cDNA obtained in the step (1) as a template, and carrying out PCR amplification by adopting a primer F and a primer R to obtain a PCR product. The primer sequences were as follows:

F：5'–CCTCGATCCGGAATGGAGCG-3'

R：5'-CAGTGAGCAGCCAGCACACG-3'

3. sequencing of PCR products

The gene shown in SEQ ID NO.1 was designated as ZmbZIPc3 gene. Sequencing the PCR product obtained in the step 2. Sequencing results showed that: the PCR product is 1382bp DNA fragment, the nucleotide sequence of the DNA fragment is shown as 38 th to 1419 th positions of SEQ ID NO.1, and the DNA fragment comprises the coding sequence of ZmbZIPc3 gene shown as 51 st to 1283 rd positions of SEQ ID NO. 1. The protein coded by the ZmbZIPc3 gene is named as ZmbZIPc3 protein, the ZmbZIPc3 protein consists of 410 amino acid residues, and the amino acid sequence of the ZmbZIPc3 protein is shown as SEQ ID NO. 2.

Example 2 obtaining ZmbZIc 3-transformed Arabidopsis thaliana and phenotypic analysis thereof

1. Obtaining ZmZIPc 3-transformed Arabidopsis thaliana

1. Obtaining recombinant expression vectors

(1) PCR amplification was performed using the cDNA obtained in step 1 of example 1 as a template and the primers F and R of example 1 to obtain a PCR product. The PCR product contains the coding sequence of ZmbZIPc3 gene shown in 51-1283 th position of the 5' end of SEQ ID NO. 1.

(2) And (3) connecting the PCR product obtained in the step (1) into an intermediate vector pENTR/D-TOPO by using a Gateway method, and then recombining the PCR product into a pLeela vector by using a gateway@enzyme Mix of Invitrogen company by using a homologous recombination method to construct the pLeela 35SS: zmZIPc 3 over-expression vector. Specific methods refer to the detailed steps in the gateway@enzyme Mix specification.

(3) The pLeela 35SS is transformed into escherichia coli DH5 alpha by using ZmbZIc3 over-expression vector, cultured overnight at 37 ℃, and monoclonal is selected for colony PCR verification and sequencing.

Sequencing results showed that: zmbZIPc3 is obtained by replacing the sequence between attR1 and attR2 sites of the pLeela vector with the DNA molecule shown in SEQ ID NO.3, and keeping other sequences unchanged. The over-expression vector is a vector obtained by recombining the DNA molecule shown in SEQ ID NO.3 into a pLeela vector. Wherein, the DNA molecule shown in the 51 st to 1283 rd positions of SEQ ID NO.3 is CDS sequence of ZmbZIPc3 gene (corresponding to the 51 st to 1283 rd positions of SEQ ID NO. 1), namely the ZmbZIPc3 gene is successfully connected into the pLeela vector. The pLeela 35SS is that the ZmbZIPc3 over-expression vector expresses the ZmbZIPc3 protein.

2. Acquisition of recombinant Agrobacterium

Transferring the pLeela 35SS:: zmbZIPc3 over-expression vector into agrobacterium tumefaciens GV3101 by using an electric shock method to obtain recombinant agrobacterium tumefaciens GV3101/pLeela 35SS:: zmbZIPc3, and performing amplified culture after colony PCR identification.

3. Obtaining transgenic Arabidopsis thaliana

The Arabidopsis thaliana Col-0 was infected with the recombinant Agrobacterium GV3101/pLeela 35SS:: zmZIPc 3 obtained in step 2 by the Arabidopsis thaliana flower dipping method (specific method is referred to non-patent literature "Clough SJ, bent AF (1998). Flora dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thiana. Plant J16,735-743"), to obtain T ₀ Transgenic arabidopsis seed. Then the obtained T ₀ The transgenic arabidopsis seeds are placed on a 1/2MS solid culture medium containing 10 mug/mL oxadiazon for screening positive seedlings, and the positive seedlings with resistance are transferred to a culture room for culture (the culture condition is that the temperature is 22 ℃ and the light is 16 h/the dark is 8 h) to obtain T ₁ Transgenic arabidopsis seed. And then T is taken ₁ Culturing the transgenic arabidopsis seeds on 1/2MS culture medium solid containing 10 mug/mL oxadiazon, and selecting T with a survival rate of 3/4 (i.e. a separation ratio of 3:1) ₁ Culturing the survival seedling in a culture room, and harvesting T ₂ Transgenic arabidopsis seed. Finally T is set ₂ The transgenic Arabidopsis seeds are placed in a medium containing 10 mug/mL oxadiazonIn 1/2MS solid medium, the total surviving T is added ₂ Transferring the survival seedling to a culture room for culturing to obtain T ₃ The generation transgenic arabidopsis seeds (namely homozygous lines) are cultured to obtain three T ₃ The homozygous lines of the transgenic Arabidopsis thaliana are respectively named as T ₃ The transgenic Arabidopsis homozygous lines OE2-1, OE 5-6 and OE10-4 were used for further experiments.

4. Identification of transgenic Arabidopsis thaliana

Selecting T obtained in step 3 ₃ Leaf total RNA is extracted from homozygous strains OE2-1, OE 5-6 and OE10-4 of the generation transgenic Arabidopsis thaliana and wild Arabidopsis thaliana Col-0 respectively, and RT-PCR amplification is carried out by using primers RT-ZmZIPc 3-F and RT-ZmZIPc 3-R as well as primers 18S-F and 18S-R (18S genes are internal reference genes) as templates, and electrophoresis analysis is carried out, so that the expression quantity of ZmZIPc 3 genes in the transgenic Arabidopsis thaliana is analyzed.

The primer sequences were as follows:

RT-ZmbZIPc3-F：5'-AACTGGAGGCACAGGTAGCACA-3'；

RT-ZmbZIPc3-R：5'-TATCAGAGGCGGAGTCAGAAGG-3'；

18S-F：5’-CTTAGTTGGTGGAGCGATTTG-3’；

18S-R：5’-CCTGTTATTGCCTCAAACTTCC-3。

the results are shown in FIG. 1A, from which it can be seen that: the wild type Arabidopsis thaliana Col-0 (Col) has no target band, the transgenic Arabidopsis thaliana OE2-1, the transgenic Arabidopsis thaliana OE 5-6 and the transgenic Arabidopsis thaliana OE10-4 have target bands, the brightness of the transgenic Arabidopsis thaliana OE is OE2-1 > OE10-4 > OE 5-6, the positive transformation of the transgenic Arabidopsis thaliana OE2-1, the transgenic Arabidopsis thaliana OE 5-6 and the transgenic Arabidopsis thaliana OE10-4 by the positive transformation of the transgenic Arabidopsis thaliana OE2-1, the transgenic Arabidopsis thaliana OE 5-6 and the transgenic Arabidopsis thaliana OE10-4 is OE10-4, and the expression level of the ZmbZIPc3 genes is OE2-1 > OE 5-6 >.

2. Phenotypic analysis of ZmZIPc 3-transformed Arabidopsis thaliana

The plant materials were as follows: wild type Arabidopsis thaliana Col-0 (Col), T ₃ Generation positive ZmZIPc 3 Arabidopsis homozygous strains OE2-1, OE 5-6 and OE 10-4.

1. The plant material seeds are treated with 75% ethanol for 1min, then sterilized with 10% sodium hypochlorite for 15min, then washed with sterilized distilled water for 5 times, and then treated at a low temperature of 4 ℃ for 3 days. Low and lowAfter the temperature treatment, the seeds are respectively sown on 1/2MS solid culture medium, 1/2MS solid culture medium which is permeated by 25 percent PEG overnight, 60mM CaCl is added ₂ 1/2MS solid medium of (C), 150mM NaCl was added ₂ On a 1/2MS solid culture medium, culturing at 22+ -3deg.C under 50% humidity under 16h light/8 h dark condition, observing growth phenotype about 2 weeks, and counting root growth.

Growth phenotype and root length of 1/2MS solid Medium and 25% PEG-infiltrated overnight 1/2MS solid Medium in 1/2MS solid Medium, wild type Arabidopsis thaliana Col-0 (Col) with T is shown in FIG. 2 ₃ The growth phenotype and root length of the generation positive ZmZIPc 3-transformed Arabidopsis homozygous strains OE2-1, OE 5-6 and OE10-4 are equivalent; whereas T was compared to wild-type Arabidopsis thaliana Col-0 (Col) in 1/2MS solid medium infiltrated with 25% PEG overnight ₃ The growth phenotype and root length of the generation positive ZmZIPc 3-transformed Arabidopsis thaliana homozygous strains OE2-1, OE 5-6 and OE10-4 have obvious advantages.

1/2MS solid Medium and 60mM CaCl added thereto ₂ The growth phenotype and root length of the 1/2MS solid medium is shown in FIG. 3, and wild type Arabidopsis thaliana Col-0 (Col) and T in the 1/2MS solid medium ₃ The growth phenotype and root length of the generation positive ZmZIPc 3-transformed Arabidopsis homozygous lines OE2-1, OE 5-6 and OE10-4 are comparable (shown in FIG. 3A); after 60mM CaCl was added ₂ In the 1/2MS solid medium of (2), strain OE2-1 grew slightly weaker than wild-type under stress of 60mM CaCl2 compared to wild-type Arabidopsis thaliana Col-0 (Col), strain OE 5-6 and OE10-4 grew slightly stronger than wild-type (shown in FIG. 3B), and thus it could not be concluded whether ZmbZIPc3 overexpressing transgenic Arabidopsis thaliana was sensitive to CaCl2 stress.

1/2MS solid Medium and 150mM NaCl added ₂ The growth phenotype and root length of the 1/2MS solid medium is shown in FIG. 3, and wild type Arabidopsis thaliana Col-0 (Col) and T in the 1/2MS solid medium ₃ The growth phenotype and root length of the generation positive ZmZIPc 3-transformed Arabidopsis homozygous lines OE2-1, OE 5-6 and OE10-4 are comparable (shown in FIG. 3A); at the time of adding 150mM NaCl ₂ T under stress of 150mM NaCl in 1/2MS solid medium ₃ Generation positive ZmZIPc 3 arabidopsis thaliana homozygous strain OE2-1, OE5-6 and OE10-4 grew stronger than wild-type Arabidopsis thaliana Col-0 (Col) (shown as C in FIG. 3), and therefore ZmZIPc 3 over-expressed transgenic Arabidopsis thaliana was presumed to be resistant to NaCl stress.

The results show that the ZmbZIPc 3-transferred Arabidopsis thaliana strain has a more obvious PEG-resistant phenotype and salt stress-resistant phenotype.

2. The plant material seeds are treated with 75% ethanol for 1min, then sterilized with 10% sodium hypochlorite for 15min, then washed with sterilized distilled water for 5 times, and then treated at a low temperature of 4 ℃ for 3 days. After low temperature treatment, seeds were sown on 1/2MS (CK) and 1/2MS medium supplemented with 0.25. Mu.M, 0.5. Mu.M, and 1. Mu.M ABA, respectively, and cultured for 10-12d, and germination rates were counted daily using a stereoscopic microscope (brand: OPTEC). As a result, as shown in FIG. 4, T was performed on 1/2MS medium without ABA (shown as A in FIG. 4) ₃ The germination rate of the generation positive ZmZIPc 3-transferred arabidopsis thaliana homozygous strain OE2-1 is slightly lower than that of the wild arabidopsis thaliana Col-0 (Col), but the germination rate can reach 100% after 4-5 d; t on 1/2MS medium supplemented with 0.25. Mu.M ABA (shown in FIG. 4B), 2 ₃ The germination rate of the generation positive ZmZIPc 3-transferred arabidopsis thaliana homozygous strain OE2-1 is 60% -70% in 3-5d, which is obviously lower than that of the wild arabidopsis thaliana Col-0 (80% -90%); t on 1/2MS medium (shown as C in FIG. 4) supplemented with 0.5. Mu.M ABA ₃ The germination rate of the generation positive ZmZIPc 3-transferred arabidopsis thaliana homozygous lines OE2-1 and OE 5-6 is only about 45% at the time of 5d, and is obviously lower than that of the wild type arabidopsis thaliana Col-0 (80%); wild-type Arabidopsis Col-0 and T on 1/2MS medium supplemented with 1. Mu.M ABA (shown as D in FIG. 4) ₃ The germination rate of the generation positive ZmZIPc 3-transferred Arabidopsis thaliana strain is inhibited, and the germination rate of the ZmZIPc 3-transferred Arabidopsis thaliana strain OE2-1 in 3-7d is obviously lower than that of the wild type strain. The above results indicate T ₃ The germination rate of the generation positive ZmZIPc 3-transformed Arabidopsis thaliana homozygous lines OE2-1, OE 5-6 and OE10-4 is sensitive to ABA compared with that of wild Arabidopsis thaliana Col-0.

Will T ₃ Seeds of the generation positive ZmbZIPc3 arabidopsis lines OE2-1, OE 5-6 and OE10-4 and wild arabidopsis Col-0 are respectively sown on a 1/2MS culture medium and a 1/2MS culture medium containing 0.1 mu M and 1 mu M ABA after 3d of suction expansion and low-temperature placement, and the seedlings are observed for growth after two weeks of culture, and are photographed. As a result, the result is shown in FIG. 5, at 1/2MS medium and 1/2MS medium containing 0.1. Mu.M ABA, T ₃ The generation positive ZmZIPc 3-transformed Arabidopsis lines OE2-1, OE 5-6 and OE10-4 have no obvious difference from the growth conditions of the wild Arabidopsis Col-0 (shown in A and B in FIG. 5); on 1/2MS medium containing 1. Mu.M ABA, T is compared with wild type Arabidopsis Col-0 ₃ The generation positive ZmZIPc 3-transformed Arabidopsis strain OE2-1 was significantly inhibited (shown as C in FIG. 5). Shows that the seedling stage growth of the ZmZIPc 3-transferred arabidopsis thaliana homozygous strain is sensitive to ABA compared with wild type arabidopsis thaliana Col-0 (Col).

The results show that the ZmbZIPc3 gene plays a positive regulation role in the ABA signal transduction pathway.

3. The plant material seeds are treated with 75% ethanol for 1min, then sterilized with 10% sodium hypochlorite for 15min, then washed with sterilized distilled water for 5 times, and then treated at a low temperature of 4 ℃ for 3 days. The seeds are respectively sown on 1/2MS solid culture medium after low temperature treatment, are placed on a condition of 22+/-3 ℃ and 50% humidity and 16h illumination/8 h darkness for culture, arabidopsis thaliana with consistent size is selected and transferred into soil (nutrient soil: vermiculite=1:2 is filled into eight-hole trays, each hole is filled with 80g of soil, 6L of water is used for soaking soil for 4h, and then the seeds are poured into redundant water, 16 seedlings are transplanted in each hole, each 4 holes are a group, and wild arabidopsis thaliana Col-0 and T are respectively used as a group ₃ The generation positive ZmZIPc 3 arabidopsis homozygous strains OE2-1, OE 5-6 and OE10-4 are respectively provided with an drought group and a control group, and are placed in a constant temperature culture room with 20+/-3 ℃ and 16h illumination/8 h darkness. The result of normal water application of the control group is shown in FIG. 6, and the control group is transformed into ZmZIPc 3 Arabidopsis homozygous strains OE2-1, OE 5-6 and OE10-4, which are consistent with the growth vigor of wild Arabidopsis Col-0. And (3) automatically transferring the drought group into soil, stopping watering, and starting to enter drought when the soil grows normally for 16d, observing and photographing the growth state, and observing and recording when the soil is drought for 7d and the soil is drought for 14d respectively. As shown in FIG. 6, when drought group plants drought to 14d, wild type Arabidopsis thaliana Col-0 leaves have all wilted, and the wilting rate of the over-expressed ZmbZIPc3 transgenic strain is OE2-1<OE 10-4<After rehydration for 3d, the wild arabidopsis thaliana Col-0 plant has a rehydration survival rate of 13%, an OE2-1 survival rate of 82%, an OE 5-6 survival rate of 38% and an OE10-4 survival rate of 69%. The rehydration survival rate is sequentially OE2-1>OE 10-4>OE 2-1>Col, the trend is consistent with the expression level of ZmbZIPc3 genes, which shows that the drought resistance of the transgenic Arabidopsis strain over-expressing ZmbZIPc3 is obviously better than that of wild Arabidopsis Col-0.

4. The plant material seeds are treated with 75% ethanol for 1min, then sterilized with 10% sodium hypochlorite for 15min, and then washed with sterilized distilled water for 5 times, and then treated at a low temperature of 4 ℃ for 3 days. After low temperature treatment, the seeds are respectively sown on 1/2MS solid culture medium and are cultured under the conditions of 22+/-3 ℃, 50% humidity, 16h illumination and 8h darkness. Selecting wild type Arabidopsis Col-0 (WT), T ₃ The generation of positive ZmZIPc 3 arabidopsis homozygous strains OE2-1, OE 5-6 and OE10-4 are arabidopsis with equivalent growth vigor, each plant material is divided into three groups, the isolated leaves are subjected to drought treatment for different time, including drought 0, 0.25, 0.5, 1, 2 and 4 hours, sampling is fixed by FAA and decolored, water-filled tablets are manufactured, and the air hole condition is observed under a positive microscope (Nikco 80 i). The results of the drought 0, 1, 4h stomatal conditions are shown in FIG. 7, T ₃ The leaf pore opening of the generation positive ZmbZIPc 3-transformed arabidopsis homozygous lines OE2-1, OE 5-6 and OE10-4 is small, and the water loss rate of the plant is low, so that the water retention and drought resistance functions of the plant are facilitated due to the over-expression of ZmbZIPc3.

In conclusion, zmbZIPc3 is related to stress resistance such as drought resistance, salt stress and the like.

Example 3 obtaining ZmZIPc 3-transformed maize and phenotypic analysis

1. Obtaining ZmZIPc 3-transformed corn

1. Obtaining recombinant expression vectors

(1) The pLeela 35SS in example 2 is used as a template, and a ZmbZIPc3 overexpression vector is used for PCR amplification by using a primer F2 and a primer R2 to obtain the ZmbZIPc3 gene expression cassette. The primer sequences were as follows:

F：5'-CCCAAGCTTGGGTTCCCGCCTTCGGTTTGG-3'；

R：5'-CCCAAGCTTGGGCCTTGCTCGTCGGTGATGTA-3'。

the sequence of ZmbZIPc3 gene expression cassette is shown as SEQ ID NO.4, and comprises a promoter 35SS (36-720 positions from 5 'end of SEQ ID NO. 4), a coding sequence of ZmbZIPc3 gene (1141-2373 positions from 5' end of SEQ ID NO.4, corresponding 51-1283 positions from 5 'end of SEQ ID NO. 1) and a terminator PA35S (2593-2810 positions from 5' end of SEQ ID NO. 4) on a pLeela carrier in sequence.

(2) The ZmbZIPc3 gene expression cassette is inserted into the pU130 vector by a single digestion method of HindIII (TAKARA) to construct pU130 35SS, wherein the ZmbZIPc3 over-expression vector is obtained.

(3) pU130 35SS, zmbZIPc3 over-expression vector was transformed into E.coli DH 5. Alpha. And cultured overnight at 37℃and the monoclonal was picked for colony PCR verification and sequencing.

Sequencing results showed that: pU130 35SS, zmbZIPc3 over-expression vector is obtained by inserting ZmbZIPc3 gene expression cassette shown in SEQ ID No.4 between HindIII recognition sites of pU130 vector and keeping other sequences of pU130 vector unchanged. pU130 35SS that ZmbZIPc3 protein is expressed by the ZmbZIPc3 over-expression vector.

2. Obtaining transgenic maize

The pU130 35SS with correct sequence is that ZmbZIPc3 over-expression vector entrusts China seed group limited company life science and technology center to carry out corn transformation acceptor corn plant by adopting agrobacterium-mediated method, and the acceptor corn plant is corn C01 (belonging to PB group) (purchased from China seed group limited company life science and technology center) to obtain T0 generation ZmbZIPc3 corn; the corn seeds obtained by the test crossing of the T0 generation ZmbZIPc 3-transformed corn and the recipient corn thereof are T1 generation ZmbZIPc 3-transformed corn, and the genotype of the T1 generation ZmbZIPc 3-transformed corn is Aa: aa=1: 1. through continuous selfing, a homozygous strain with the genotype of T3 generation AA is obtained, and ZmbZIPc 3-transformed corn (namely T3 generation homozygous ZmbZIPc 3-transformed corn) seeds identified as the genotype of T3 generation AA are subjected to the following ZmbZIPc3 gene expression level measurement.

3. Measurement of ZmbZIPc3 Gene expression of transgenic corn

Total RNAs of T3 generation homozygous ZmbZIPc 3-transformed corn and wild corn C01 leaf of three leaves are respectively extracted, and half-quantitative PCR (real-time PCR) amplification is carried out by using a primer RT-ZmbZIPc3-F and a primer RT-ZmbZIPc3-R as well as a primer 18S-F and a primer 18S-R (18S gene is an internal reference gene) by taking the total RNAs as templates, so that the expression amount of the ZmbZIPc3 gene in the ZmbZIPc 3-transformed corn is analyzed (the primer sequence is consistent with that in example 2).

As a result, as shown in FIG. 1B, the expression level of ZmbZIPc3 gene in wild type corn C01 (WT) is used as a reference, the expression level of ZmbZIPc3 gene in ZmbZIPc3 transgenic corn is larger than that of wild type corn C01 (WT), wherein the expression level OEc 3-3> OEc3-1 > OEc3-5 > OEc3-7 > OEc3-2 of the positive T3-generation ZmbZIPc3 transgenic corn strain is higher than that of ZmbZIPc3 gene in ZmbZIPc3 transgenic corn, and the over-expression ZmbZIPc3 homozygous strains C3-1, C3-3, C3-5 and C3-7 are finally selected for subsequent experiments.

2. Phenotypic analysis of ZmbZIPc 3-transformed maize

The plant materials were as follows: wild type maize C01 plants (WT) and over-expressed ZmbZIPc3 homozygous lines C3-1, C3-3, C3-5, C3-7.

1. Wild corn C01 (WT), and the homozygous lines C3-1, C3-3, C3-5 and C3-7 of the over-expression ZmZIPc 3 are selected for germination drought experiments. Reference is made to the local standard of Hebei province in the technical Specification for drought resistance identification of maize. Stress cultures (i.e., treatment groups) were performed separately: each of the replicates was 20, 4, 20ul of polyethylene glycol-6000 solution (distilled water as solvent) was added, and the double-layer filter paper was used as a bud bed. Control culture (i.e., control group): one repetition of 20, 4 repetitions, 20 μl deionized water was added and the double layer filter paper was used as the bud bed. A round culture dish with the diameter of 10cm is used for culturing in a constant temperature incubator (25 ℃ C. Constant temperature care/dark culture for 16h/8 h), and the germination condition and drought resistance index of seeds are observed at 2, 4, 6 and 8 d.

The seed germination drought resistance index is calculated according to the following formula (1):

GDRI＝PIs/PIc×100％………………………………………………………(1)

PI＝1.00×nd2+0.75×nd4+0.50×nd6+0.25×nd8……………………………… (2)

PI _c ＝1.00×nd2 _ck+ 0.75×nd4 _ck +0.50×nd6 _ck +0.25×nd8 _ck ……………(3)

PI _s ＝1.00×nd2 _s +0.75×nd4 _s +0.50×nd6 _s +0.25×nd8 _s …………………………(4)

wherein:

PI-seed germination index, nd2, nd4, nd6, nd8 are seed germination rates at 2d, 4d, 6d, and 8d, respectively;

PI _C -a control seed germination index (ck in formula (3) represents control conditions);

PI _S -seed germination index under stress conditions (s in formula (4) represents stress conditions);

GDRI-seed germination drought resistance index.

The seed germination condition and the seed germination drought resistance index result are shown as A and B in FIG. 8, and the drought resistance indexes of the over-expression ZmZIPc 3 homozygous strains OEc 3-1, OEc 3-5 and OEc 3-7 are obviously higher than that of wild corn C01 (WT); root growth of different corn lines was observed on day 9 of germination, and as shown in FIG. 9, the main roots of the over-expressed ZmZIPc 3 homozygous lines OEc 3-1, OEc 3-5 and OEc 3-7 were significantly longer than that of wild type corn C01 (WT) and the lateral roots were also greater than that of wild type corn C01 (WT) under stress culture conditions.

The above shows that the drought resistance of the strain ZmbZIPc3 over-expressed in germination period is better than that of wild corn C01.

2. Selecting wild corn C01 (WT), over-expression ZmbZIPc3 homozygous strain OEc 3-1, OEc 3-5 and OEc 3-7, and performing seedling drought culture. Soil planting and culturing are adopted, a control group and a treatment group are respectively arranged in each plant line in a basin with the specification of 16cm multiplied by 16cm, the seedling base number of each plant line is 20, and three times of repetition are arranged. Wherein the control group is normally watered in the whole growth period, the seedlings of the treatment group grow to about the three-leaf one-heart period and start drought treatment (A in figure 10), namely, the watering is stopped, the wilting condition of the seedlings is started to be observed and recorded, when the seedlings all show the wilting condition, the first rehydration treatment is carried out, and the survival condition of the seedlings is observed after 72 hours of rehydration (B in figure 10). And after the first rehydration is finished, the second drought treatment is started, and the second rehydration treatment is also carried out after the seedlings are all in a wilting state (C in fig. 10), and the survival rate of the seedlings is observed for 72 hours, wherein the result is that, as shown in D in fig. 10, after two drought rehydration, the survival rate of each strain is OE C3-5 (60%) > OE C3-7 (40%) > WT (10%), the repeated drought rehydration survival rate of the over-expression ZmbZIPc3 homozygous strain is higher than that of wild corn C01 (WT), the trend is consistent with the expression amount trend of each strain, and the drought resistance of the over-expression ZmbZIPc3 strain in the seedling stage is better than that of the wild corn C01 (WT).

The experiment shows that the over-expression of ZmbZIPc3 can improve drought tolerance of corn in germination period and seedling period.

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> institute of plant Material at national academy of sciences

Application of <120> protein ZmbZIPc3 in regulation of drought resistance and/or salt tolerance of plants

<130> GNCFY200882

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1493

<212> DNA

<213> corn (Zea mays)

<400> 1

ccctctccga ggattctgcc cagcagcgcg gcgatccgcc tcgatccgga atggagcgcg 60

tcttctccat ggaggagatc cccaacccct actgggcccc gccgcaccct caaccggcgg 120

ccggcggcgc tgttgctgca ccaggtggag taggaggagc gggggacgcg gcgggcgcga 180

tgaaccggtg cccatctgag tggtacttcc agaagttcct cgaggaggcc gtgctcgaca 240

gtccgggtcc cgtcgccggc gtgggtagaa gcagcggaca agctggagtt gaggcggcgg 300

agagcaagcc gctgggcgcc gcggcgccgg cgtccgtctc gagctcggtc gttgaccccg 360

tcgagtacaa cgcgatgctc aagcagaagc tggagaagga cctcgctgcc atcgctatgt 420

ggagggcttc tggtgcagca cctccagatc tttctgcgac tgctgcttcc ttgccaagtg 480

tcggtgctcc gcatgcagct cctcttaaac ccgtcggagg tactgaaagt ctagttcaaa 540

acatgctagc tggtgctcca gtaggggggt caggtccaca tgtagtacaa attgctgata 600

tccctgttaa gcaaaccact agctcttcct cacgagagca gtcagatgat gatgacatgg 660

aaggcgatgc tgagacaaat ggaaatggaa accctgttca acaaagacaa cagagaagga 720

agcaatccaa ccgggaatca gccaggcgtt cgagaagcag aaaggcggct cacttgaatg 780

aactggaggc acaggtagca caattaagag tcgaaaactc ttcgctgcta aggcggcttg 840

ctgatgttaa ccagaagttc aatgaagctg ctgttgacaa tagggtgcta aaggcagacg 900

tcgaaacctt aagagcaaag gtgaagatgg cagaggactc ggtgaagcgg gtaacaggca 960

tgaacgcatt gtaccctgcc gtgtcggata tgtcttccct cagcatgcca ttcaatggct 1020

ccccttctga ctccgcctct gatagcaccg tcccggtaca agatgacctg aacagttact 1080

tcgccaaccc aagcgaaatc ggatgcaaca acggctacat gccagatata gcctcctcgg 1140

ttcaacagga cgacaatttt gtcaacgggt atcaggctgc tggcaagatg ggcagaacag 1200

actcgctgca gcgggtggca agcctggagc acctccagaa aagaatgtgc ggaggcccgg 1260

cttcatccgg ctcgacctcc taggtttcat atgaaaaagt ccctggtgcc taacggcaag 1320

aatgctaaat taacaatttc aacatgcttt gtactggaca ttgataatcc tgcgatgcgc 1380

attagtgcgt tatggacttg tgggtcaatt gctatttggc gtgtgctggc tgctcactgt 1440

agtgagcaga acaagttcca gtttctatcg aagagcaccc tctccttttc gct 1493

<210> 2

<211> 410

<212> PRT

<213> corn (Zea mays)

<400> 2

Met Glu Arg Val Phe Ser Met Glu Glu Ile Pro Asn Pro Tyr Trp Ala

1 5 10 15

Pro Pro His Pro Gln Pro Ala Ala Gly Gly Ala Val Ala Ala Pro Gly

20 25 30

Gly Val Gly Gly Ala Gly Asp Ala Ala Gly Ala Met Asn Arg Cys Pro

35 40 45

Ser Glu Trp Tyr Phe Gln Lys Phe Leu Glu Glu Ala Val Leu Asp Ser

50 55 60

Pro Gly Pro Val Ala Gly Val Gly Arg Ser Ser Gly Gln Ala Gly Val

65 70 75 80

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

85 90 95

Ser Ser Ser Val Val Asp Pro Val Glu Tyr Asn Ala Met Leu Lys Gln

100 105 110

Lys Leu Glu Lys Asp Leu Ala Ala Ile Ala Met Trp Arg Ala Ser Gly

115 120 125

Ala Ala Pro Pro Asp Leu Ser Ala Thr Ala Ala Ser Leu Pro Ser Val

130 135 140

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

145 150 155 160

Leu Val Gln Asn Met Leu Ala Gly Ala Pro Val Gly Gly Ser Gly Pro

165 170 175

His Val Val Gln Ile Ala Asp Ile Pro Val Lys Gln Thr Thr Ser Ser

180 185 190

Ser Ser Arg Glu Gln Ser Asp Asp Asp Asp Met Glu Gly Asp Ala Glu

195 200 205

Thr Asn Gly Asn Gly Asn Pro Val Gln Gln Arg Gln Gln Arg Arg Lys

210 215 220

Gln Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Ser Arg Lys Ala Ala

225 230 235 240

His Leu Asn Glu Leu Glu Ala Gln Val Ala Gln Leu Arg Val Glu Asn

245 250 255

Ser Ser Leu Leu Arg Arg Leu Ala Asp Val Asn Gln Lys Phe Asn Glu

260 265 270

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

275 280 285

Ala Lys Val Lys Met Ala Glu Asp Ser Val Lys Arg Val Thr Gly Met

290 295 300

Asn Ala Leu Tyr Pro Ala Val Ser Asp Met Ser Ser Leu Ser Met Pro

305 310 315 320

Phe Asn Gly Ser Pro Ser Asp Ser Ala Ser Asp Ser Thr Val Pro Val

325 330 335

Gln Asp Asp Leu Asn Ser Tyr Phe Ala Asn Pro Ser Glu Ile Gly Cys

340 345 350

Asn Asn Gly Tyr Met Pro Asp Ile Ala Ser Ser Val Gln Gln Asp Asp

355 360 365

Asn Phe Val Asn Gly Tyr Gln Ala Ala Gly Lys Met Gly Arg Thr Asp

370 375 380

Ser Leu Gln Arg Val Ala Ser Leu Glu His Leu Gln Lys Arg Met Cys

385 390 395 400

Gly Gly Pro Ala Ser Ser Gly Ser Thr Ser

405 410

<210> 3

<211> 1387

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ggaatggagc gcgtcttctc catggaggag atccccaacc cctactgggc cccgccgcac 60

cctcaaccgg cggccggcgg cgctgttgct gcaccaggtg gagtaggagg agcgggggac 120

gcggcgggcg cgatgaaccg gtgcccatct gagtggtact tccagaagtt cctcgaggag 180

gccgtgctcg acagtccggg tcccgtcgcc ggcgtgggta gaagcagcgg acaagctgga 240

gttgaggcgg cggagagcaa gccgctgggc gccgcggcgc cggcgtccgt ctcgagctcg 300

gtcgttgacc ccgtcgagta caacgcgatg ctcaagcaga agctggagaa ggacctcgct 360

gccatcgcta tgtggagggc ttctggtgca gcacctccag atctttctgc gactgctgct 420

tccttgccaa gtgtcggtgc tccgcatgca gctcctctta aacccgtcgg aggtactgaa 480

agtctagttc aaaacatgct agctggtgct ccagtagggg ggtcaggtcc acatgtagta 540

caaattgctg atatccctgt taagcaaacc actagctctt cctcacgaga gcagtcagat 600

gatgatgaca tggaaggcga tgctgagaca aatggaaatg gaaaccctgt tcaacaaaga 660

caacagagaa ggaagcaatc caaccgggaa tcagccaggc gttcgagaag cagaaaggcg 720

gctcacttga atgaactgga ggcacaggta gcacaattaa gagtcgaaaa ctcttcgctg 780

ctaaggcggc ttgctgatgt taaccagaag ttcaatgaag ctgctgttga caatagggtg 840

ctaaaggcag acgtcgaaac cttaagagca aaggtgaaga tggcagagga ctcggtgaag 900

cgggtaacag gcatgaacgc attgtaccct gccgtgtcgg atatgtcttc cctcagcatg 960

ccattcaatg gctccccttc tgactccgcc tctgatagca ccgtcccggt acaagatgac 1020

ctgaacagtt acttcgccaa cccaagcgaa atcggatgca acaacggcta catgccagat 1080

atagcctcct cggttcaaca ggacgacaat tttgtcaacg ggtatcaggc tgctggcaag 1140

atgggcagaa cagactcgct gcagcgggtg gcaagcctgg agcacctcca gaaaagaatg 1200

tgcggaggcc cggcttcatc cggctcgacc tcctaggttt catatgaaaa agtccctggt 1260

gcctaacggc aagaatgcta aattaacaat ttcaacatgc tttgtactgg acattgataa 1320

tcctgcgatg cgcattagtg cgttatggac ttgtgggtca attgctattt ggcgtgtgct 1380

ggctgct 1387

<210> 4

<211> 3018

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

aagcttgggt tcccgccttc ggtttgggcg cgcccggttc agaagaccag agggctattg 60

agacttttca acaaagggta atatcgggaa acctcctcgg attccattgc ccagctatct 120

gtcacttcat cgaaaggaca gtagaaaagg aagatggctt ctacaaatgc catcattgcg 180

ataaaggaaa ggctatcgtt caagatgcct ctaccgacag tggtcccaaa gatggacccc 240

catccacgag gaacatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg 300

attgatgtga tacatggtgg agcacgacac tctcgtctac tccaagaata tcaaagatac 360

agtctcagaa gaccagaggg ctattgagac ttttcaacaa agggtaatat cgggaaacct 420

cctcggattc cattgcccag ctatctgtca cttcatcgaa aggacagtag aaaaggaaga 480

tggcttctac aaatgccatc attgcgataa aggaaaggct atcgttcaag atgcctctac 540

cgacagtggt cccaaagatg gacccccacc cacgaggaac atcgtggaaa aagaagacgt 600

tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg taagggatga 660

cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt catttcattt 720

ggagaggacc tcgagaaaga ggatccacct gaggcctccg ttccatgggc tagaagcttc 780

tcctcctctg ctaacgtaag cctctctgtt ttttttctct gtttcttttg aaatgaatcc 840

aattagtgat gataatctgt gtttgatgta tcattgattt aacatcttga caatgaatcg 900

tgatcggaag tgataaagtt atgggtcaac ggtttcaaag agagagaaag acttttagag 960

tcaactctcg actctttctt aattatgtta ttgctatttg tctcttttct tgaagtctga 1020

acaattcttg ggattgtttt gcaggttcta gcttctccaa ccacaggaat tcatcgcccg 1080

ggactgggtt atcacaagtt tgtacaaaaa agcaggctcc gcggccgccc ccttcaccgg 1140

aatggagcgc gtcttctcca tggaggagat ccccaacccc tactgggccc cgccgcaccc 1200

tcaaccggcg gccggcggcg ctgttgctgc accaggtgga gtaggaggag cgggggacgc 1260

ggcgggcgcg atgaaccggt gcccatctga gtggtacttc cagaagttcc tcgaggaggc 1320

cgtgctcgac agtccgggtc ccgtcgccgg cgtgggtaga agcagcggac aagctggagt 1380

tgaggcggcg gagagcaagc cgctgggcgc cgcggcgccg gcgtccgtct cgagctcggt 1440

cgttgacccc gtcgagtaca acgcgatgct caagcagaag ctggagaagg acctcgctgc 1500

catcgctatg tggagggctt ctggtgcagc acctccagat ctttctgcga ctgctgcttc 1560

cttgccaagt gtcggtgctc cgcatgcagc tcctcttaaa cccgtcggag gtactgaaag 1620

tctagttcaa aacatgctag ctggtgctcc agtagggggg tcaggtccac atgtagtaca 1680

aattgctgat atccctgtta agcaaaccac tagctcttcc tcacgagagc agtcagatga 1740

tgatgacatg gaaggcgatg ctgagacaaa tggaaatgga aaccctgttc aacaaagaca 1800

acagagaagg aagcaatcca accgggaatc agccaggcgt tcgagaagca gaaaggcggc 1860

tcacttgaat gaactggagg cacaggtagc acaattaaga gtcgaaaact cttcgctgct 1920

aaggcggctt gctgatgtta accagaagtt caatgaagct gctgttgaca atagggtgct 1980

aaaggcagac gtcgaaacct taagagcaaa ggtgaagatg gcagaggact cggtgaagcg 2040

ggtaacaggc atgaacgcat tgtaccctgc cgtgtcggat atgtcttccc tcagcatgcc 2100

attcaatggc tccccttctg actccgcctc tgatagcacc gtcccggtac aagatgacct 2160

gaacagttac ttcgccaacc caagcgaaat cggatgcaac aacggctaca tgccagatat 2220

agcctcctcg gttcaacagg acgacaattt tgtcaacggg tatcaggctg ctggcaagat 2280

gggcagaaca gactcgctgc agcgggtggc aagcctggag cacctccaga aaagaatgtg 2340

cggaggcccg gcttcatccg gctcgacctc ctaggtttca tatgaaaaag tccctggtgc 2400

ctaacggcaa gaatgctaaa ttaacaattt caacatgctt tgtactggac attgataatc 2460

ctgcgatgcg cattagtgcg ttatggactt gtgggtcaat tgctatttgg cgtgtgctgg 2520

ctgctaaggg tgggcgcgcc gacccagctt tcttgtacaa agtggtgata acttccgatc 2580

gattctagac tagttctaga gtccgcaaaa atcaccagtc tctctctaca aatctatctc 2640

tctctatttt tctccagaat aatgtgtgag tagttcccag ataagggaat tagggttctt 2700

atagggtttc gctcatgtgt tgagcatata agaaaccctt agtatgtatt tgtatttgta 2760

aaatacttct atcaataaaa tttctaattc ctaaaaccaa aatccagtga ccgggcggcc 2820

gccaccgcgg tggaggggga tcagattgtc gtttcccgcc ttcagtttaa actatcagtg 2880

tttgacagga tatattggcg ggtaaaccta agagaaaaga gcgtttatta gaataatcgg 2940

atatttaaaa gggcgtgaaa aggtttatcc gttcgtccat ttgtatgtgt acatcaccga 3000

cgagcaaggc ccaagctt 3018

Claims (6)

1. The application of the protein shown in the following A1) or A2) in regulating drought resistance and/or salt tolerance of plants:

a1 Protein with the amino acid sequence shown as SEQ ID NO. 2;

a2 Fusion protein obtained by N-terminal or/and C-terminal connexin label of the amino acid sequence shown in SEQ ID NO. 2;

the regulation of drought resistance and/or salt tolerance of plants is to improve the drought resistance and/or salt tolerance of plants;

the plant is corn or Arabidopsis thaliana.

2. Use of a biological material related to the protein of claim 1 for modulating drought and/or salt tolerance in a plant; the biological material is any one of the following C1) -C10):

c1 A nucleic acid molecule encoding the protein of claim 1;

c2 An expression cassette comprising C1) said nucleic acid molecule;

c3 A recombinant vector comprising C1) said nucleic acid molecule, or a recombinant vector comprising C2) said expression cassette;

c4 A recombinant microorganism comprising C1) said nucleic acid molecule, or a recombinant microorganism comprising C2) said expression cassette, or a recombinant microorganism comprising C3) said recombinant vector;

c5 A transgenic plant cell line comprising C1) said nucleic acid molecule, or a transgenic plant cell line comprising C2) said expression cassette, or a transgenic plant cell line comprising C3) said recombinant vector;

c6 A) a transgenic plant tissue comprising C1) said nucleic acid molecule, or a transgenic plant tissue comprising C2) said expression cassette, or a transgenic plant tissue comprising C3) said recombinant vector;

c7 A transgenic plant organ comprising C1) said nucleic acid molecule, or a transgenic plant organ comprising C2) said expression cassette, or a transgenic plant organ comprising C3) said recombinant vector;

c8 A transgenic plant containing the nucleic acid molecule of C1), or a transgenic plant containing the expression cassette of C2), or a transgenic plant containing the recombinant vector of C3);

c9 A tissue culture produced by regenerable cells of the transgenic plant of C8);

c10 Protoplasts produced from the tissue culture of C9);

the regulation of drought resistance and/or salt tolerance of plants is to improve the drought resistance and/or salt tolerance of plants;

the plant is corn or Arabidopsis thaliana.

3. The use according to claim 2, characterized in that: c1 A nucleic acid molecule encoding the protein according to claim 1 as shown in D1) or D2) below:

d1 A DNA molecule shown in SEQ ID NO. 1;

d2 A DNA molecule with the coding sequence shown as SEQ ID NO. 1.

4. A method of growing transgenic plants with high drought and/or salt tolerance, characterized by: the method comprises increasing the expression level of the gene of the protein of claim 1 and/or the content of the protein and/or the activity of the protein in a plant of interest to obtain a transgenic plant; the transgenic plant has drought and/or alkali tolerance higher than the plant of interest; the plant is corn or Arabidopsis thaliana.

5. The method according to claim 4, wherein: the method for increasing the expression level of the gene of the protein of claim 1 and/or the content of the protein and/or the activity of the protein in a plant of interest is to express or overexpress the protein of claim 1 in a plant of interest.

6. The method according to claim 5, wherein: the method of expression or overexpression is to introduce a gene encoding the protein of claim 1 into a plant of interest.