CN114644691B - EIP1 protein, coding gene thereof and drought resisting application - Google Patents

Abstract

The invention discloses an EIP1 protein, a coding gene and drought resistance application thereof. The EIP1 protein provided by the invention is a fusion protein which has the amino acid sequence of SEQ ID No.1 or is obtained by substituting and/or deleting and/or adding one or more amino acid residues, has the same function with corn or more than 80% of identity with the corn and has the same function with the corn or is obtained by connecting a protein tag at the N end and/or the C end of the protein. Compared with the wild drought resistance, the drought resistance of the positive strain obtained by over-expressing EIP1 in the maize B73 is enhanced, compared with the traditional breeding mode, the time is short, the purpose is strong, and the method has important significance for cultivating and improving new varieties of drought-resistant plants.

Description

EIP1 protein, coding gene thereof and drought resisting application

Technical Field

The invention relates to the technical field of biology, in particular to an EIP1 protein, a coding gene thereof and drought resistance application.

Background

The per capita fresh water resource in China is only 1/4 of the average level in the world, and the spatial-temporal distribution is highly uneven, so that the system is one of 13 countries with serious water shortage in the world. The agricultural water accounts for more than 73% of the total national water consumption, and the paddy water accounts for more than 70% of the total agricultural water consumption. Dry farming accounts for over 52% of the total area of cultivation, with dry farming without irrigation conditions accounting for over 65%. China has 60 hundred million acres of grassland, and 55 hundred million acres of grassland are in a drought state. Corn is one of three major food crops in the world, is the second major food crop except wheat in China, is the first major economic crop, and is widely applied in many aspects. The corn is originally grown in tropical areas with high temperature and high humidity, and the crops in the same period of rain and heat have more water demand, so the drought resistance is not strong. In addition to pest and disease damage in biotic stress, drought stress causes the most severe loss to crops such as corn, and dominates all abiotic stresses. China is in subtropical-temperate regions, and drought is a main abiotic stress factor for limiting the growth yield of corn. The improvement of the stress resistance of plants by editing or over-expressing a certain or some specific genes through a genetic engineering means is one of molecular breeding modes. If the edited or transferred genes can be stably inherited, new varieties can be obtained or new characters can be created, the technology breaks through the limitation of interspecific hybridization, is one of effective ways for improving the stress resistance of crops, can improve the yield of the crops under the adverse circumstances, and has important significance for solving the problem of food shortage caused by environmental stress. Therefore, the method improves the corn variety and improves the drought resistance by means of genetic engineering, and has important significance for yield reduction caused by drought.

Drought can cause osmotic stress in plants, the osmotic pressure in the plant is lower than that in the environment (such as soil solution), and the plant cannot absorb water or even lose water. Osmotic stress has two pathways: ABA-dependent and ABA-independent pathways. When subjected to environmental stress, ABA, PYL, PYR and RCAR are ABA receptors generated in an ABA pathway and can be combined with ABA. The activity of protein phosphatase of PP2C is inhibited, so that SnRK2 has kinase activity and can phosphorylate downstream transcription factors and the like, thereby regulating and controlling the expression of downstream stress response genes, inhibiting stomatal opening, regulating and controlling ABA sensitivity and generating other responses. DREB2A is phosphorylated independent of the ABA pathway, thereby regulating downstream stress response gene expression. Drought stress can induce the generation of abscisic acid (ABA) in plants, which can guide stomata to close in time to reduce the loss of water in plants, and can activate the expression of a large number of drought-related genes to cause various drought stress responses of plants. ABA is an extremely important plant hormone, and plays a key role in stomata closure, expression of stress response genes, and the like.

Most Erwinia are plant pathogenic bacteria, can cause plant necrosis, ulcer, wilting, leaf spot, gummosis and soft rot, and has great harm to agricultural development in China. Erwinia induced protein (Erwinia induced protein) is a protein involved in the plant coping with the Erwinia infection, but the specific disease resistance mechanism is not clear, and meanwhile, the Erwinia induced protein has few reports on the research of the plant coping with abiotic stress such as drought and the like.

Most of the EIP1 protein function researches known in plants at present focus on the aspect of plant disease resistance.

Disclosure of Invention

The invention aims to provide an EIP1 protein, a coding gene thereof and drought resistance application.

In a first aspect, the invention claims a protein.

The protein claimed by the invention is derived from corn (Zea mays L.) and is named EIP1 protein. Specifically, any one of the following may be used:

a1 Protein with the amino acid sequence of SEQ ID No. 1;

a2 The amino acid sequence shown in SEQ ID No.1 is substituted and/or deleted and/or added by one or more amino acid residues and is derived from a protein with the same function of corn;

a3 Protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any of A1) to A2) and derived from corn having the same function;

a4 A fusion protein obtained by attaching a protein tag to the N-and/or C-terminus of a protein as defined in any of A1) to A3).

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

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

In the above protein, the homology of 95% or more may be at least 96%, 97%, 98% identity. The homology of 90% or more may be at least 91%, 92%, 93%, 94% identity. The homology of 85% or more may be at least 86%, 87%, 88%, 89% identity. The homology of 80% or more may be at least 81%, 82%, 83%, 84% identity.

In a second aspect, the invention claims nucleic acid molecules encoding the proteins described in the first aspect above.

Further, 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, and the like.

Further, the nucleic acid molecule (EIP 1 gene) is specifically any one of:

b1 A DNA molecule (complete genome sequence) shown in SEQ ID No. 2;

b2A DNA molecule (T01 transcript) as shown at positions 623-3881 of SEQ ID No. 2;

b3 A DNA molecule (CDS sequence) shown in SEQ ID No. 3;

b4 A DNA molecule hybridizing under stringent conditions with a DNA molecule as defined in any of B1) to B3) and encoding a protein as described in the first aspect above;

b5 A DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity to a DNA sequence as defined in any of B1) to B4) and encoding a protein as defined in the first aspect.

In the above nucleic acid molecule, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na ₃ PO ₄ And 1mM EDTA, in 50 ℃,2 x SSC,0.1% SDS rinsing; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ And 1mM EDTA, and the mixture was subjected to hybridization at 50 ℃ in 1 XSSC, 0.1% SDSRinsing; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ And 1mM EDTA, and rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ And 1mM EDTA, and rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in 6 XSSC, 0.5% SDS solution, hybridization was performed at 65 ℃ and then the SDS and 1 XSSC, 0.1% SDS were used to wash the membranes once each.

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

In the above nucleic acid molecule, the 95% or more homology may be at least 96%, 97%, 98% identity. The homology of 90% or more may be at least 91%, 92%, 93%, 94% identity. The homology of 85% or more may be at least 86%, 87%, 88%, 89% identity. The homology of 80% or more may be at least 81%, 82%, 83%, 84% identity.

In a third aspect, the invention claims an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line comprising a nucleic acid molecule as described in the second aspect above.

The expression cassette refers to a DNA capable of expressing EIP1 in a host cell, and the DNA may include not only a promoter for initiating the transcription of EIP1 gene but also a terminator for terminating the transcription of EIP1. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited toIn the following steps: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: ubiquitin gene ubiqiutin promoter (pUbi); the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120; 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 proteinase inhibitor II promoter (PIN 2) or LAP promoter (both inducible with jasmonic acid ester 26353; heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (Chinese patent 2007 1 0099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., odell et al (I) ⁹⁸⁵ ) Nature 313; rosenberg et al (1987) Gene, 125; guerineau et al (1991) mol.gen.genet, 262; proudfoot (1991) Cell, 64; sanfacon et al Genes dev., 5; mogen et al (1990) Plant Cell, 2; munroe et al (1990) Gene, 91; ballad et al (1989) Nucleic Acids Res.17:7891; joshi et al (1987) Nucleic Acid Res., 15.

Constructing a recombinant expression vector containing the EIP1 gene expression cassette. The plant expression vector used may be a binary Agrobacterium vector or a Gateway system vector, etc., such as pCXUN, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pGWB411, pGWB412, pGWB405, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When ZmEREB167 is used to construct a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi), etc., can be added before the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

In a fourth aspect, the invention claims the use of a protein as described in the first aspect above or a nucleic acid molecule as described in the second aspect above or an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line as described in the third aspect above for modulating drought resistance in a plant.

Further, the method is carried out. In the plant, the expression level and/or activity of the protein is increased, and the drought resistance of the plant is increased; the expression level and/or activity of the aforementioned protein is decreased, and the drought resistance of the plant is decreased.

In a fifth aspect, the invention claims a method of cultivating plants with improved drought resistance.

The method of growing plants with increased drought resistance as claimed in the present invention may comprise the step of increasing the expression level and/or activity of a protein as described in the first aspect hereinbefore in the recipient plant.

The method can be realized by means of hybridization or by means of transgenosis.

In a sixth aspect, the invention claims a method of breeding transgenic plants with improved drought resistance.

The method for cultivating the transgenic plant with improved drought resistance, which is claimed by the invention, can comprise the following steps: introducing into a recipient plant a nucleic acid molecule capable of expressing a protein according to the first aspect of the invention, to obtain a transgenic plant; the transgenic plant has increased drought resistance compared to the recipient plant.

Further, a nucleic acid molecule capable of expressing a protein as described in the first aspect above may be introduced into the recipient plant by means of a recombinant expression vector.

Wherein, the nucleic acid molecule (EIP 1 gene) can be modified as follows and then introduced into the receptor plant to achieve better expression effect:

1) Modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modification is performed using sequences known to be effective in plants;

2) Linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

3) The expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml derived from CaMV, E9 derived from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

4) Enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

In the above aspects, the plant is a monocot or a dicot.

Further, the plant is a gramineous plant.

Further, the plant is a plant of the genus zea, such as corn.

In a specific embodiment of the invention, the plant is maize B73.

Because different transcripts can be generated by the same DNA segment sequence of the corn and different proteins can be translated, the different transcripts generated by the sequence shown in SEQ ID No.2 and the translated different proteins are all in the protection scope of the invention.

More than one EIP1 gene transcript can resist drought sensitivity after the cDNA corresponding to other forms of transcripts is over-expressed, and the invention belongs to the protection scope.

Compared with the wild drought resistance of a positive strain obtained by over-expressing EIP1 in corn B73, the method has the advantages of short time and strong purpose compared with the traditional breeding mode, and has important significance for cultivating and improving new varieties of drought-resistant plants.

Detailed Description

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

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

The corn ecotype is B73; the agrobacterium strain is EHA105. The main reagents comprise: restriction enzymes, DNA polymerases, T4 ligases, etc. from biological companies such as NEB and Toyobo; reverse transcription kit from Thermo corporation; RNA extraction kit from magenta; quantitative PCR reagents of Taraka corporation; the plasmid extraction kit and the DNA recovery kit are products of Tiangen company; MS culture medium, agar powder, agarose, ampicillin, kanamycin, gentamicin sulfate, rifampicin and other antibiotics are sigma products; the various other chemical reagents used in the examples were all imported or domestic analytical reagents; primer synthesis and sequencing was done by Yingjun corporation.

Example 1, application of EIP1 protein and coding gene thereof in regulation and control of plant drought resistance

1. Discovery of EIP1 protein and coding gene thereof

The invention discovers a novel protein named EIP1 protein from corn (Zea mays L.), and the amino acid sequence of the protein is SEQ ID No.1. The EIP1 gene has a sequence of SEQ ID No.2 in a maize B73 genome, consists of 4662 bases, and the reading frame of a T01 transcript is base 623 to base 3881 from the 5' end of the SEQ ID No. 2. The gene consists of 5 exons, wherein a T01 transcript encodes 5 exons, and the 1 st to 664 th bases, the 1452 nd to 1569 th bases, the 1648 th to 1809 th bases, the 1905 th to 1936 th bases, the 3015 th to 3259 th bases and the rest are intron sequences of the exons (namely, the 623 th to 3881 th positions at the 5' end of SEQ ID No. 2). The CDS sequence of EIP1 gene is shown in SEQ ID No. 3.

2. Construction and detection of EIP1 gene overexpression vector

Extracting total RNA from corn (Zea mays L.) B73, carrying out reverse transcription to obtain cDNA, amplifying EIP1 gene by using the cDNA as a template and F and R as primers, wherein the primers are provided with enzyme cutting sites and are connected to an over-expression vector after enzyme cutting.

The vector construction method comprises the following steps:

(1) The total RNA of the corn B73 is extracted by using an RNA extraction kit of magenta company, and the specific steps refer to the kit instruction.

(2) The RNA was reverse transcribed to give cDNA using a reverse transcription kit from thermo, and the detailed procedures were as described in the kit's instructions.

(3) Using cDNA as a template and F and R as primers to amplify EIP1 gene cDNA, running electrophoresis on an amplification product, cutting gel and recovering, wherein the recovery method refers to a reagent kit of Tiangen corporation.

The primers used were:

an upstream primer F:5 'ATGGCGACGCCGCCGACTG-3';

a downstream primer R:5 'CTACACACAGAGGAATCCCACCCG-3'.

(4) The recovered EIP1 gene cDNA (SEQ ID No. 3) was cloned into pBCXUN vector, 5. Mu.L of ligation product was taken, and E.coli competence was transformed. Screening was performed on LB plates containing 50. Mu.g/mL kanamycin. And (5) identifying the single clone by colony PCR, and selecting a positive clone for sequencing. The obtained recombinant expression vector with correct sequencing is named pBCXUN-EIP1. Colony PCR and sequencing universal primers were as follows:

UbiP-seq：5’-TTTTAGCCCTGCCTTCATACGC-3’；

NosR-seq：5’-AGACCGGCAACAGGATTCAATC-3’。

in the recombinant plasmid pBCXUN-EIP1, the EIP1 gene shown in SEQ ID No.3 is transcribed by a Ubi promoter and is terminated by a Nos terminator, so that the target protein EIP1 (SEQ ID No. 1) is expressed.

The pBCXUN vector is an expression vector obtained by replacing the HYG gene (hptII, hygromycin resistance gene) of the pCXUN vector (GenBank: FJ905215.1, 06-JUL-2009) with a Bar gene (encoding phosphinothricin acetyltransferase) (GenBank: 284-835 th nucleotide in MG719235.1, 02-OCT-2018) and keeping the other nucleotides of the pCXUN unchanged.

3. Construction and detection of EIP1 gene overexpression plant

And (3) transforming the pBCXUN-EIP1 overexpression plasmid constructed in the step two into a competent agrobacterium EHA105 strain by a heat shock method, and identifying a positive clone by colony PCR. Inoculating single colony of Agrobacterium identified correctly in 2-3mL liquid culture medium containing 100 μ g/mL kanamycin and 50 μ g/mL rifampicin, shake culturing at 28 deg.C overnight, inoculating to liquid culture medium containing large amount of antibiotics the next day, shake culturing, collecting thallus after several times of inoculation, and resuspending to OD ₆₀₀ Between 0.8 and 1.0. Infecting the B73 maize immature embryo which is scraped out under the aseptic condition by using the obtained recombinant agrobacterium tumefaciens suspensionThen, callus is induced to grow seedlings. The transgenic plants are subjected to selfing and seed reproduction to obtain T3 generation for subsequent experiments.

Extracting genome DNA of plant leaves, carrying out PCR amplification by adopting a primer pair (the specific sequence is the same as the above) consisting of Ubi P-seq (corresponding to a Ubi promoter) and NosR-seq (corresponding to a Nos terminator), and if a specific amplification product is obtained, the plant is a transgenic plant.

4. Statistics of dry treatment survival rate of corn with EIP1 gene overexpression

1. Homozygous T identified as positive by step three ₃ Transgenic maize seeds (OE 1 and OE 2) of the generation EIP1 and Wild Type (WT) maize seeds of B73 were sown in small pots with nutrient soil, 6 per plant line and 10 per pot, respectively.

2. After growing for 7 days in a culture room at 25 ℃, selecting seedlings with consistent growth vigor, transplanting the seedlings into large rectangular pots filled with 2500g of nutrient soil, planting Wild Type (WT) in one half area of each pot, planting EIP1 transgenic lines in the other half area of each pot, and planting 5 seedlings in each row, namely 15 seedlings of the wild type and the transgenic lines in each large rectangular pot. Three replicates were set, 5 pots per replicate.

3. After 7 days of growth under normal watering conditions, the watering was stopped for drought treatment.

4. After 20 days, significant differences in phenotype between Wild Type (WT) and EIP1 transgenic plants were observed.

5. After being rehydrated for 7 days, the survival of each strain is observed and the survival rate is counted. Survival rate is the percentage of the number of surviving plants in each line to the total number of plants. And taking the average value of multiple repetitions for statistical analysis.

The results show that: the survival rate of maize B73 plants (WT) was 40% ± 4%. The survival rate of plants of the OE1 line is 85% + -5%, and the survival rate of plants of the OE2 line is 78 + -4%.

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

<110> university of agriculture in China

<120> EIP1 protein, coding gene and drought-resistant application thereof

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ctttaaactt taaaattaaa gtaccacttg aaatggctga tctatgatta ggtgggacaa 1740

gccaagcttg cagcggctag gttgtgaaag attttctttt ctgtgtttgt gtggtatatg 1800

tgaacaattc tatttgctat ggttcatgaa ttttagcaag ggttgctttg agtgaatata 1860

gtattcagtg cattagctaa agataactta taaaaatgtg cagtgacagg ttttcatttt 1920

ttatggaagt gtctttcact ttcatggagg catatctttt gttttgctga ttgattcaat 1980

aatgatcgtt gagaaggata ctttctaatt gtgccgttct ctctgctgat cagtacttcc 2040

tttctaacga caaccatttg tttctttcat tagcttgcgc ctcagcattt cctaactctg 2100

cttcggacta tggattgctt gtggcaaatg gaacctatgc actgactgct ggtaactgtg 2160

tggagtgcag ttgtgggcca gcgaatctca agtaaactta cccttctgtt caatgtagtt 2220

ttcttgcctt ttatttgttt tggactgtaa cacccttatt tctttgtagt ttgtattgca 2280

caccgtcttc actatcagca tcatgttcaa gtatgcaatg ctctaatagc agcttaattc 2340

ttggtaatgt gactgcacaa cccaccactg gtggctgcag tgtctcatct tgcaactatg 2400

atggctatgt caatggaacc attgcaacat cgtaagcaaa aaatcccaaa taattttcgt 2460

tttactgatt tactctatct aaactgtgga actcaaagat tatcacatct tacaatgcat 2520

gtgcaggcta tcctcaggtc ttcaacccat gtgtccaggt acttgtgcaa tcttcagttc 2580

tagtgtcctt catatttagc tactggagtg ctgatgcagt aaatttcagt gttttactct 2640

tttgttcaaa gttccttttc aacaccatca ttgacttgtc caaagctgca tttttgaaac 2700

attcgtcaat aaatgtattg tctagtcaca acctataaga ttttttaggt gtgtgcctcc 2760

tttttagaag agagaataaa ggacggcccc gatgtataat taagtgtaaa gaaaaatatt 2820

catgtcctag aatattaata tagaagcaaa catagttctg atcttcttat gtcaaatcta 2880

tgatggtatt tccttttttt atattggtag ttgccctcaa tcttgtttta gttagtatca 2940

gattatccct gcctaaccct atctaccttg ctttaaaggt tctatatact tctttatttg 3000

aggtagtatc aaaggaaacc ctgttaaata aatgaagtga tggttattta actaaattat 3060

caagggcacc tttgaaacat tcgtcaatat ttgtattgtc tagtcacaac ctataatatt 3120

ttttaggtat gtgcctcctt tttagaagag agaataaagg atgaacctga tgtatgattt 3180

caagtgtaaa aaaaagatat tcatgttcca gaatattatt ctagcccttt ggtttggatt 3240

taaagcgaac atagttctga actccttgta tcaaatttat gatggtattt ccttttttta 3300

ttggtaggtt gccctcaatc ttgtttcagt tagtatcaga ttatccctgc ctaacactac 3360

ttaccttgct ttaaaggttc tgtatagttt ctggaacatc attgacatta taatacgtgg 3420

attaaggcat taaccaaata aacatgatgt ctagaaatgt gaagctttca ctttgttctc 3480

aatgtatttc ggtatagcaa acaataggaa ctgtaccgac agaattttgt tagatggatg 3540

ctgttgtttc atttgcccgg tgctattcat catgagttcc tctgtttcga tctttactgg 3600

tgtctgttct ttcaatcact ttgaccatga ttacaggacc gcatcaattt cccccactca 3660

cggcggtgcc aactgcggca aaccatgggt cttattctcc atcacctgca ccaggacctg 3720

gagatgctgg tggtgccacc ccaggaggtt ccagcctttc tccatcaaat gagcctgctg 3780

ggaacagctc acaagcggct gcgataaatc aaccatgccg tttcctcctc attttcattc 3840

tttctgtgac attgtccttg cgggtgtgga ttcctgtgta gagtattgtt ttttttaaaa 3900

aaataactat gtagagtatt tgttggctgt atttgtattg agcgtggaag gccttattgg 3960

ggccaggtgg aacctgaatg aagaatgcag tagcctgtct tcgttagtgt ccggtgaaac 4020

tttggtatga ttttggtttc tccagaattt gtatatgaat aagcctatct ccagaatcta 4080

tccatcaagg cagtagcacg agatagagat ggtaaggttc atggtcccca tgggggcacc 4140

aagcttattg cctagagctt attgcctagt gcggctgcta gtatgcatct cccattagtc 4200

ctgcaaaaac gaaaaaaaaa tcaggtgaag aataccccca ccatgtatcc gcgggagctc 4260

accgtggatt tatgctactt gtctgtatct ctactactaa ttataaacct aacgtaggcg 4320

tgcacacccc gcggtgttct gccgcccgcg cgtcccgccc gcggctgcac cttgccccgc 4380

atcacgcaat ccagcgcaat tccccggccc acacgctcga gcacgcccct gccccacacg 4440

ctcgagcacg cgccgccccg ggggatccgc ctccgcctct tcctcagccg tggcggtgag 4500

cgcagcgccg ccccctagtg tagatcttga gacggcgacg aagcttcaag cccacgcgcc 4560

cctcgaggta tggctacgac gcagtcccca ggtcgcaact gatttagacg cagatggagg 4620

cgtcggcggc ctccgacgaa accctagccg acgtgctcga cc 4662

<210> 3

<211> 1221

<212> DNA

<213> Zea mays L.

<400> 3

atggcgacgc cgccgactgc gctgctgttg ctcctcgcgg cggcagcggc attccacggc 60

gcgacggcga agacgacgat cgagccgtgc tctggcgcgg acgcgtgccc ggctcttctg 120

ggctacaagc tctacgcgga catgaaggtg tccgaggtgg cggcgctctt cggcgccgac 180

ccggcagcgg tactggctgc caacgcgctg gacttcgcct ctccaggcgc cgccaaccgc 240

atcctccccg cgggaacacc cctccgcgtg cccacccgct gcgcctgcgc cgacggcgtc 300

cgcaagtccg tcgccatccg ctacgccgcc cggccttccg acacgctcgg ctccatctcc 360

gaggtcgtct tcgccggcct cccctcggcc gaccagatcc gcaccgccaa cgggctcgcc 420

gccgaggacc ccgacgcgcc gctcaacccg ggccaggagc tcgtcatccc gctcccctgc 480

gtctgcttca actccaccga caacaacctc cccgctgtgt acctctccta tgtcgtgcag 540

gtcggggaca ccgtggaatc catcgccgcc agccacacaa ccaccgtcac tgatatcagc 600

aatgtgaacg ccatggggag ccccattgtc gcaccaggcg acatccttgc cataccattg 660

tctgcttgcg cctcagcatt tcctaactct gcttcggact atggattgct tgtggcaaat 720

ggaacctatg cactgactgc tggtaactgt gtggagtgca gttgtgggcc agcgaatctc 780

aatttgtatt gcacaccgtc ttcactatca gcatcatgtt caagtatgca atgctctaat 840

agcagcttaa ttcttggtaa tgtgactgca caacccacca ctggtggctg cagtgtctca 900

tcttgcaact atgatggcta tgtcaatgga accattgcaa catcgctatc ctcaggtctt 960

caacccatgt gtccaggacc gcatcaattt cccccactca cggcggtgcc aactgcggca 1020

aaccatgggt cttattctcc atcacctgca ccaggacctg gagatgctgg tggtgccacc 1080

ccaggaggtt ccagcctttc tccatcaaat gagcctgctg ggaacagctc acaagcggct 1140

gcgataaatc aaccatgccg tttcctcctc attttcattc tttctgtgac attgtccttg 1200

cgggtgtgga ttcctgtgta g 1221

Claims (11)

1. The application of protein or nucleic acid molecule or expression cassette or recombinant vector or recombinant bacterium or transgenic cell line in improving plant drought resistance;

the protein is any one of the following proteins:

a1 Protein with the amino acid sequence of SEQ ID No. 1;

a2 A fusion protein obtained by connecting a protein tag to the N-terminal and/or C-terminal of the protein defined in A1);

the nucleic acid molecule is a nucleic acid molecule encoding the protein;

the expression cassette is an expression cassette containing the nucleic acid molecule;

the recombinant vector is a recombinant vector containing the nucleic acid molecule;

the recombinant bacterium is a recombinant bacterium containing the nucleic acid molecule;

the transgenic cell line is a transgenic cell line containing the nucleic acid molecule;

the plant is a monocot.

2. Use according to claim 1, characterized in that: the nucleic acid molecule is any one of the following:

b1 A DNA molecule shown as SEQ ID No. 2;

b2A DNA molecule shown at positions 623-3881 of SEQ ID No. 2;

b3 A DNA molecule shown by SEQ ID No. 3.

3. Use according to claim 1, characterized in that: in the plant, the expression level of the protein is increased, and the drought resistance of the plant is improved.

4. Use according to any one of claims 1 to 3, characterized in that: the monocotyledon is a gramineous plant.

5. Use according to claim 4, characterized in that: the gramineous plant is corn.

6. A method of growing plants with increased drought resistance comprising the steps of increasing the expression of a protein in a recipient plant;

the protein is any one of the following proteins:

a1 Protein with the amino acid sequence of SEQ ID No. 1;

a2 A fusion protein obtained by connecting a protein tag to the N-terminal and/or C-terminal of the protein defined in A1);

the plant is a monocot.

7. A method of breeding a transgenic plant with improved drought resistance comprising the steps of: introducing a nucleic acid molecule capable of expressing a protein into a recipient plant to obtain a transgenic plant; the transgenic plant has increased drought resistance compared to the recipient plant;

the protein is any one of the following proteins:

a1 Protein with the amino acid sequence of SEQ ID No. 1;

a2 A fusion protein obtained by connecting a protein tag to the N-terminal and/or C-terminal of the protein defined in A1);

the plant is a monocot.

8. The method of claim 7, wherein: the nucleic acid molecule is any one of the following:

b1 A DNA molecule shown as SEQ ID No. 2;

b2A DNA molecule shown at positions 623-3881 of SEQ ID No. 2;

b3 A DNA molecule shown by SEQ ID No. 3.

9. The method of claim 7, wherein: the nucleic acid molecule capable of expressing the protein is introduced into the recipient plant by means of a recombinant expression vector.

10. The method according to any one of claims 6-9, wherein: the monocotyledon is a gramineous plant.

11. The method of claim 10, wherein: the gramineous plant is corn.