CN110128516B - Barley moisture-resistant regulatory gene HvERF2.11, protein and application thereof in breeding - Google Patents

Abstract

The invention discloses a barley moisture-resistant regulatory gene HvERF2.11, protein and application thereof in breeding. Barley moisture-resistant geneHvERF2.11The CDS sequence of (1) is shown in SEQ ID NO. The invention clones the moisture-resistant gene from the moisture-resistant barley for the first timeHvERF2.11And proving that the gene is related to the moisture resistance of barley, adopting a genetic engineering method to performHvERF2.11The gene is over-expressed into arabidopsis, and compared with a control group, the moisture resistance of a transgenic plant is obviously enhanced, which shows thatHvERF2.11The over-expression of the gene improves the moisture resistance of the plant. In the inventionHvERF2.11Cloning of genesAnd the technology of transformation, transgenosis and the like lays a foundation for researching the barley moisture-proof molecular mechanism and breeding application, and has wide breeding application prospect and certain economic value.

Description

Barley moisture-resistant regulatory gene HvERF2.11, protein and application thereof in breeding

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a barley moisture-resistant regulatory geneHvERF2.11And breeding application thereof.

Background

The wet damage (watering) refers to the damage to the normal growth and development of crops caused by an oxygen-deficient environment caused by excessive soil moisture. The damp damage occurs all over the world, and is particularly serious in north america, australia, china, india and other countries. With the deterioration of global ecological environment, the frequency of extreme weather is increasing, drought and waterlogging disasters occur frequently, and the damp damage becomes one of the main abiotic stress factors influencing crop production. According to statistics, about 16% of cultivated lands are harmed by waterlogging with different degrees in the world, the crop yield is reduced by about 20% -30% due to the waterlogging, and even the crop cannot be harvested due to serious wet damage. China is one of countries in the world which are seriously affected by waterlogging, approximately 30% of cultivated land area is affected by floods of different degrees every year, and the influence is increased year by year. In 2013, the disaster-stricken area of the farmland caused by flood in China reaches 1.19 multiplied by 10⁷hm²Resulting in direct economic loss of over 3000 billion yuan.

The plant forms a plurality of gene co-expression regulation and control modes in the long-term evolution process so as to adapt to adversity stress. In recent years, research on plant response to adversity stress has shifted from cloning and functional identification of single functional genes to identification and regulatory function research of transcription factors. A transcription factor related to adversity stress can simultaneously regulate and control the expression change of a series of related downstream functional genes. Therefore, the effect of the transcription factor on the improvement of the stress resistance of crops may be better than that of a single functional gene. The humidity-resistant regulatory genes cloned in plants such as rice, arabidopsis thaliana, kiwi fruit and the like all belong to the subfamily gene of the transcription factor ERF-VIIs, and the humidity-resistant regulatory genes serving as the transcription factors in barley are not reported at present.

Disclosure of Invention

The invention aims to provide a barley moisture-resistant geneHvERF2.11And its breeding application, cloning and constructionHvERF2.11Gene expression vector, using transgenic technologyHvERF2.11Gene transfer into Arabidopsis thaliana, and confirmation of the wet damage stress treatment of transgenic Arabidopsis thalianaHvERF2.11Has the moisture-proof activity, and provides gene resources and theoretical basis for improving barley moisture-proof varieties.

By bioinformatics analysis, we identified 54 genes belonging to the ERF gene family in barley; phylogenetic tree analysis discoveryHvERF2.11Gene related to low oxygen stress in arabidopsis thalianaHRE1、HRE2、RAP2.12AndRAP2.2the evolutionary relationship is recent. Analyzing different organs and different conditions of barley by qRT-PCR technologyHvERF2.11The gene expression level is cloned in moisture-proof barley TF58HvERF2.11A gene, andHvERF2.11the gene is transferred into arabidopsis thaliana, the function of the gene is verified, and a foundation is expected to be laid for barley variety tolerance molecular marker-assisted selective breeding.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a barley moisture resistance regulation related protein is (a) or (b) as follows:

(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2; the specific sequence is MCGGAILAGFIPPSAAAAAAKAAATAKKKQQQRSVTADSLWTGLRKKADEEDFEADFRDFERDSSEEEDDEVEEVPPPPAPATAGFAFAAAAEVALRAPARRDAAVQHDGPAAKQVKRVRKNQYRGIRQRPWGKWAAEIRDPSKGVRVWLGTYDTAEEAARAYDAEARKIRGKKAKVNFPEDAPTVQKSTLKPTAAKSAKLAPPPKACEDQPFNHLSRGDNDLFAMFAFSDKKVPAKPTDSVDSLLPVKHLAPTEAFGMNMLSDQSSNSFGSTDFGWDDEAMTPDYTSVFVPSAAAMPAYGEPAYLQGGAPKRMRNNFGVAVLPQGNGAQDIPAFDNEVKYSLPYVESSSDGSMDNLLLNGAMQDGASSGDLWSLDELFMAAGGY;

(b) a protein which is derived from the amino acid sequence of SEQ ID NO. 2 by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO. 2 and is related to barley moisture resistance regulation.

The invention also provides a gene encoding the protein. The gene is a DNA molecule of any one of the following (a 1) - (a 3);

(a1) 1, DNA molecule shown in SEQ ID NO;

(a2) a DNA molecule which is hybridized with the DNA sequence limited by (a 1) under strict conditions and codes barley humidity-resistant regulation related protein;

(a3) a DNA molecule which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined in (a 1) and encodes a barley moisture-resistance regulation-related protein.

The invention also provides an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene.

It is another object of the present invention to provide the use of (b 1) or (b 2) or (b 3):

(b1) the protein, or the gene, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene is applied to the resistance to barley wet damage stress;

(b2) the protein, or the gene, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene is applied to cultivating a new moisture-resistant variety of barley and arabidopsis thaliana;

(b3) the protein, or the gene, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene is applied to the resistance to arabidopsis thaliana wet damage stress.

The invention also provides a method for cultivating the plant moisture-proof variety, which comprises the steps of transferring the gene into a target plant, or transferring the target plant into the expression cassette and the recombinant vector to obtain a transgenic plant; the transgenic plant has higher wet damage stress resistance than the target plant. The target plants are barley and arabidopsis thaliana. The transgenic plant expresses the barley moisture-resistance related protein.

HvERF2.11The DNA fragment of the gene is shown in a sequence table SEQ ID NO.1, or is basically equivalent to the DNA sequence shown in the SEQ ID NO.1, or is a partial fragment of the sequence shown in the SEQ ID NO.1 in function. The sequence analysis of the gene shows thatHvERF2.11The gene coding region has the full length of 1158bp, codes 385 amino acids, has the molecular weight of 41.38kDa and the isoelectric point of 5.14, and contains a conserved AP2 structural domain. The sequence shown in the over-expression sequence table SEQ ID NO.1 can enhance the tolerance of plants such as arabidopsis thaliana and barley to damp damage.

The gene can be applied to improving the moisture resistance of barley, and the specific operation is as follows:

(1) obtaining of genes: obtaining the extract from barley by bioinformatics analysisHvERF2.11Extracting the total RNA of the moisture-proof strain TF58 root system by using the full-length gene sequence of the gene, and amplifying the total RNA by using RT-PCRHvERF2.11The amplified product is connected to pGEM-Teasy carrier, and the gene sequence is obtained by sequencingHvERF2.11Cloning of the gene.

(2) Construction and genetic transformation of plant expression vectors: construction of barley Using Gateway technology from Invitrogen corporationHvERF2.11A gene overexpression vector. Using a BP Clonase ™ enzyme, willHvERF2.11The gene was ligated to pDONR221 vector to construct an entry vector, which was then ligated to pB2GW7 vector using LR clone ™ enzyme to construct a gene vector containing pDONR221HvERF2.11A gene overexpression vector. Freezing and thawing with liquid nitrogenHvERF2.11The overexpression vector of the gene is introduced into agrobacterium Gv 3101. By utilizing agrobacterium-mediated genetic transformation methodHvERF2.11The coding sequence is introduced into Arabidopsis thaliana for expression. Positive transgenic Arabidopsis plants are screened by antibiotic screening, RT-PCR and the like.

(3) Moisture resistance analysis of transgenic plants: transgenic and wild type Arabidopsis lines that grew normally for 35 days were flooded. After 2 weeks of flooding treatment, the phenotypic differences of transgenic plants and wild plants under the stress of wet injury are observed, thereby proving thatHvERF2.11Gene pair for improving moisture resistance of arabidopsis thalianaCapability.

The invention provides a new method for enhancing the resistance of plants to the wet damage, improves the moisture resistance of crops by means of genetic engineering, not only overcomes the defect of short traditional breeding period, but also has simple operation and is easy to obtain moisture resistant materials. The barley humidity-resistant regulatory gene obtained by first cloningHvERF2.11The gene is transferred into arabidopsis thaliana by an agrobacterium-mediated method, and the moisture damage identification analysis proves that the moisture resistance of a transgenic plant is obviously improved compared with that of a wild plant, so that the gene has wide application prospect in the aspect of improving the moisture resistance of barley.

Drawings

FIG. 1 barleyHvERF2.11The RT-PCR amplification product of the gene sequence,

marker: DL2000DNA Marker (Dalianbao biology) composed of six DNA fragments of 2000bp, 1000bp, 750bp, 500bp, 250bp and 100 bp;

FIG. 2 barleyHvERF2.11The expression difference of the gene in different organs of barley and arabidopsis thaliana,

a: after stress of dampness damage, barleyHvERF2.11The expression difference of the gene in different organs of TF58 includes leaf, adventitious root, seed root, root node and ear seed. B:HvERF2.11the gene is expressed differently in different transgenic Arabidopsis strains and wild type strains;

FIG. 3 shows the difference in moisture resistance phenotype between transgenic Arabidopsis lines and wild type lines at 2 weeks of flooding and under control conditions, (A) plant growth phenotype; (B) the plant height; (C) fresh weight of stem leaves; (D) dry weight of stem leaves; (E) root length; (F) survival rate;

FIG. 4 shows the change of physiological indexes of leaves of transgenic lines and wild lines in different flooding periods,

A-E were SOD, CAT, POD, ADH enzyme activities and proline content, respectively, and the data represent mean. + -. standard deviation, 3 biological replicates of the experiment.*And**respectively representing the difference between the transgenic line and the wild type to a significant and a very significant level (*P < 0.05; * *P < 0.01）；

FIG. 5 shows the expression difference between the transgenic Arabidopsis thaliana and the wild type moisture-resistant related gene in different periods of moisture stress,

a to F each representAtSOD1The gene(s) is (are),AtCAT1the gene(s) is (are),AtPOD1the gene(s) is (are),AtADH1the gene(s) is (are),AtPDC1andAtACO1gene of Arabidopsis thalianaAtActinThe gene is used as an internal reference gene for expressing different genes. Values are expressed as mean ± standard deviation of 3 replicates.*And**respectively representing that the difference between the transgenic line and the wild type reaches a significant level and a very significant level.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

Example 1HvERF2.11Cloning of genes

Designing barleyHvERF2.11Specific primer P for gene₁Forward primers 5'-CCAGTCAGAGATGGTCAAGG-3' (SEQ ID NO. 3) and P₂5'-CATCCATCGTCTTGCTGAG-3' (SEQ ID NO. 4), extracting the total RNA of the wet-proof material TF58 root system by CTAB method, reverse transcribing to synthesize cDNA, taking the reverse transcribed cDNA as a template, and utilizing the primer P₁And P₂The CDS sequence of the barley moisture-resistant gene shown as SEQ ID NO.1 is amplified, and the geneHvERF2.11The full length of the CDS sequence of (1) is 1158 bp.

The method comprises the following specific steps:

(1) adding a CTAB (hexadecyl trimethyl ammonium bromide) extraction buffer solution [ 2% (W/V) CTAB, 1.4 mol/L of NaCl, 20mmol/L of EDTA (ethylene diamine tetraacetic acid), 100mmol/L of Tris-HCl, 2% (W/V) PVP ] and 10% beta-mercaptoethanol into a centrifugal tube, and preheating in a water bath kettle;

(2) cooling and grinding barley root system with liquid nitrogen, adding into the extract, mixing, and water bathing at 65 deg.C for 10 min;

(3) equal volume of chloroform was added: mixing isoamyl alcohol (volume ratio 24: 1), reversing, mixing, standing for 10min, and centrifuging at 4 ℃ at 12000g for 10 min;

(4) taking the supernatant, and repeating the step (3);

(5) taking the supernatant, adding LiCl with the final concentration of 2mol/L, carrying out ice bath for 10-12 hours, centrifuging for 15min at the temperature of 4 ℃ at the rpm of 11000rpm, discarding the supernatant, washing the precipitate twice by using 75% ethanol, and dissolving the precipitate in an appropriate amount of DEPC (diethyl pyrocarbonate) treatment water for later use;

(6) total RNA from root system was extracted from the barley strain TF58 as a template and reverse transcribed to synthesize the first strand cDNA using reverse transcriptase (purchased from Thermo Fisher Scientific Co.) under the following conditions: 5min at 65 ℃, 50min at 42 ℃ and 10min at 70 ℃;

(7) the barley ethylene transcription factor gene was amplified from cDNA reverse transcribed from RNA using the above primers P1 and P2HvERF2.11The CDS sequence of (a);

reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; 30sec at 94 ℃, 30sec at 55 ℃, 1min at 72 ℃ and 33 cycles; extension at 72 ℃ for 10 min. And connecting the PCR product obtained by amplification into a pMD18-T vector (purchased from Takara Bio-engineering Co., Ltd.), transforming escherichia coli competent cells, screening positive clones and sequencing to obtain the required full-length gene. Extracting the carrier from the positive cloneHvERF2.11Plasmid of gene CDS sequence.

Example 2HvERF2.11Construction and genetic transformation of gene overexpression vector

To better analyze the geneHvERF2.11Will further have a biological function ofHvERF2.11Overexpression of the gene in Arabidopsis thaliana was achieved, and barley was constructed using Gateway technology from Invitrogen corporationHvERF2.11A gene overexpression vector. Using a BP Clonase ™ enzyme, willHvERF2.11The gene was ligated to pDONR221 vector to construct an entry vector, which was then ligated to pB2GW7 vector using LR clone ™ enzyme to construct a gene vector containing pDONR221HvERF2.11A gene overexpression vector. Freezing and thawing with liquid nitrogenHvERF2.11The overexpression vector of the gene is introduced into agrobacterium Gv 3101. By utilizing agrobacterium-mediated genetic transformation methodHvERF2.11The coding sequence is introduced into Arabidopsis thaliana. Screening for Positive transformants by antibiotic screening and RT-PCR and the likeHvERF2.11And (4) gene plants.

The practical genetic transformation method of the test is as follows:

(1) cultivation of Agrobacterium

First, solid LB medium (10g/L peptone, 5g/L yeast extract, 10g/L chloride) with selection for corresponding resistance was usedPreculture on sodium, Kan 100mg/L, agar 1.5g/L) and the carrierHvERF2.11Culturing the gene agrobacterium at 28 ℃ for 48 hours; selecting a single colony of the pre-cultured agrobacterium, inoculating the single colony into a liquid LB culture medium (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride and Kan 100mg/L) of corresponding resistance selection, and culturing the single colony overnight in a shaking table at the temperature of 28 ℃ and the rpm of 200 until the OD600 value of the bacterial liquid concentration is about 0.8-1.0.

(2) Inflorescence method for infecting arabidopsis

The arabidopsis thaliana is transformed by adopting an improved inflorescence infection method. Picking up a product containingHvERF2.11And transferring the agrobacterium tumefaciens single colony of the gene plasmid into 100mL of LB liquid culture medium, putting the LB liquid culture medium into a shaking table at 28 ℃ for overnight culture at 200r/min, centrifuging the agrobacterium tumefaciens single colony at 4000r/min for 10min when the concentration of the agrobacterium tumefaciens bacterial liquid reaches OD600 of 0.8-1.0, discarding supernatant, and then re-suspending the agrobacterium tumefaciens bacterial liquid by 100mL of LB liquid culture medium containing 5g of cane sugar and Silwet L-7750 mu L. And (2) inversely buckling an arabidopsis flower pot in a beaker containing bacterial suspension, soaking the inflorescence of the arabidopsis into bacterial liquid for 10-20 s, culturing for 24h in the dark, placing the arabidopsis flower pot in an illumination incubator with the temperature of 25 ℃, the humidity of 60%, the illumination intensity of 3000-4000 lx and the 16h/8h photoperiod for culturing, watering and culturing plants every other day, and harvesting seeds after blooming. Harvested Arabidopsis seeds were sterilized in 8% NaClO alcohol solution (prepared in 95% alcohol) for 5min, and then screened on a selection medium (MS +50mg/L kanamycin +7g/L agar +30g/L sucrose).

Example 3HvERF2.11RT-PCR detection of gene transgenic T3 generation seedling

To verify whether the transgenic Arabidopsis thaliana T3 lines have altered ability to resist moistureHvERF2.11Gene correlation, using RT-PCR method to do partial transgenic arabidopsis plantHvERF2.11The gene expression is detected, the result is shown in figure 2, and the change of the moisture resistance of transgenic arabidopsis T3 strain and the transferred transgenic arabidopsis T3 strain can be knownHvERF2.11Overexpression of the gene is involved.

The method comprises the following specific steps:

total RNA of plants was extracted from 3 lines of T3 generations of transgenic Arabidopsis thaliana by using TRIZOL reagent (purchased from Dalian corporation, Bio-engineering Co., Ltd.) (the extraction method was described in the specification of TRIZOL reagent), and reverse transcriptase was used(purchased from Thermo Fisher Scientific Co.) was reverse transcribed to synthesize the first strand cDNA under conditions of 65 ℃ for 5min, 42 ℃ for 50min, and 70 ℃ for 10 min. Firstly, the cDNA obtained by reverse transcription is detected and the concentration is adjusted by using the reported internal reference gene Actin, and PCR detection is carried out, so that the internal reference genes can be amplified in the plants of contrast wild type arabidopsis thaliana and transgenic arabidopsis thaliana. Then, according toHvERF2.11The sequence of the gene is detected by RT-PCR by using primers P1 and P2, and the reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; 30sec at 94 ℃, 30sec at 55 ℃, 1.5 min at 72 ℃ and 33 cycles; extension at 72 ℃ for 10 min. The agarose gel electrophoresis results of the amplification products show (FIG. 2) that 3 transgenic lines are detectedHvERF2.11Expression of the gene.

Example 4 transferHvERF2.11Identification of moisture resistance of arabidopsis plants

To further analyze the barley moisture resistance geneHvERF2.11The gene is overexpressed in arabidopsis thaliana, and the gene is verified by the phenotypic characteristic of moisture resistance of a transgenic plantHvERF2.11Biological function in the regulation of wet injury stress. The specific experimental steps are as follows: obtained by screening at an earlier stageHvERF2.11In T3 positive transgenic lines obtained by gene transformation of Arabidopsis, 3 lines of seeds are randomly selected, Arabidopsis wild type seeds (WT) are used as a control, the seeds are cultured on 1/2MS culture medium to 4-leaf stage, seedlings are transplanted into pots filled with nutrient soil, and the seedlings are cultured for 35 days in an artificial climate chamber. Putting the transgenic plant line and the wild type into a water tank for flooding treatment, observing the difference of moisture resistance of the transgenic material and the wild type material after 2 weeks of flooding, taking the materials in different flooding time periods for carrying out expression analysis of moisture resistance related genes and measurement of related physiological indexes, and measuring the characters of seedling height, fresh weight of plants, dry weight, root length and the like of different materials after flooding is finished. The result shows that the plant height of the wild type is reduced by 49.09 percent, and the plant heights of the 3 transgenic lines are reduced by 30.54 percent, 27.41 percent and 32.48 percent respectively; the wild chlorophyll content is reduced by 61.56 percent, and the content of 3 transgenic lines is reduced by 29.52 percent, 35.26 percent and 31.37 percent respectively; the fresh weight of wild stems and leaves is reduced by 65.8 percent, and the fresh weight of 3 transgenic lines is reduced by 50 percent, 19.67 percent and 17.86 percent respectively;the dry weight of wild stems and leaves is reduced by 51.01 percent, and the dry weight of 3 transgenic lines is reduced by 46.25 percent, 40.5 percent and 16.1 percent respectively; the wild root length is reduced by 74.87%, and 3 transgenic lines are respectively reduced by 59.69%, 41.47% and 41.88%; after the wet injury treatment, the survival rate of the wild type arabidopsis is only 27.61 percent, while the survival rate of the transgenic arabidopsis is as high as more than 78 percent (figure 3), which shows that the overexpression is performedHvERF2.11The gene obviously enhances the moisture resistance of arabidopsis thaliana.

The changes of antioxidant enzyme activity (SOD, CAT, POD), Alcohol Dehydrogenase (ADH) activity and proline content of wild type and transgenic arabidopsis thaliana under the control and wet damage treatment conditions are shown in figure 4, and the transgenic strains and the wild type have no obvious difference in physiological and biochemical levels under the normal growth conditions; after 3 days of wet damage treatment, the antioxidant enzyme and the alcohol dehydrogenase are obviously increased in both wild type and transgenic lines, the increase reaches the maximum value after 6 days of wet damage treatment, and the increase amplitude of the transgenic lines is obviously higher than that of non-transgenic lines; after 9 days of wet injury treatment, antioxidant enzyme and alcohol dehydrogenase activities began to decrease (fig. 4A, B, C, D). The wet injury resulted in a significant increase in proline content in both wild type and transgenic lines, but the wild type was increased significantly more than the transgenic line (FIG. 4E). Indicating overexpression under wet-injury stressHvERF2.11The gene enhances the efficiency of eliminating harmful substances by an antioxidant enzyme system in arabidopsis thaliana, and improves the resistance of arabidopsis thaliana to the wet damage stress.

To identifyHvERF2.11The regulation and control effect of the gene on the humidity damage stress response related gene, and the genes related to antioxidant enzyme activity, glycolysis and ethylene synthesis in transgenic arabidopsis thaliana and wild type by utilizing qRT-PCR (AtSOD1，AtCAT1，AtPOD1，AtADH1，AtPDC11，AtACO1) Gene expression analysis was performed. As shown in fig. 5, under normal growth conditions,AtPOD1andAtACO1the expression difference of the gene between the wild type and the transgenic arabidopsis thaliana reaches a significant level, and the other genes have no obvious expression difference; after the wet injury stress, the expression levels of the 6 genes are obviously up-regulated in wild arabidopsis and transgenic arabidopsis, and the rise range in the transgenic arabidopsis is larger; the wet damage is treated for 3 days,AtSOD1、AtPOD1andAtACO1gene expression levels reached a maximum and began to decrease gradually after 6 and 9 days of treatment (fig. 5A, C, F); the wet damage is treated for 3 days,AtCAT1、AtADH1andAtPDC1the gene expression level increased slowly, and after 6 days of wet injury treatment, the gene expression level reached the highest level, and after 9 days of wet injury treatment, the expression level began to decrease (fig. 5B, D, E). Show thatHvERF2.11The gene can regulate and control the genes related to the wet damage stress in the ways of antioxidant enzyme, fermentation and ethylene synthesis, and enhance the resistance of arabidopsis thaliana to the wet damage stress.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Claims (5)

1. The barley moisture-resistant regulation related protein shown as SEQ ID NO.1 or the gene of the barley moisture-resistant regulation related protein coded as SEQ ID NO. 2 or the application of an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene in the barley moisture-resistant stress resistance.

2. The barley moisture-resistant regulation related protein shown as SEQ ID NO.1 or the gene of the barley moisture-resistant regulation related protein coded as SEQ ID NO. 2 or the application of an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene in cultivating new barley and arabidopsis thaliana moisture-resistant varieties.

3. The application of the barley moisture-resistant regulation related protein shown as SEQ ID NO.1 or the gene of the barley moisture-resistant regulation related protein coded as SEQ ID NO. 2 or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene in the resistance to arabidopsis thaliana moisture damage stress.

4. The application of any one of claims 1 to 3, wherein the gene is HvERF2.11, the total RNA of the root system of the moisture-resistant strain TF58 is extracted, the gene sequence of the HvERF2.11 is amplified by RT-PCR, the amplified product is connected to a pGEM-Teasy vector, and an overexpression vector containing the HvERF2.11 gene is constructed; introducing the overexpression vector containing the HvERF2.11 gene into agrobacterium Gv3101 by a liquid nitrogen freeze thawing method; introducing the HvERF2.11 coding sequence into arabidopsis thaliana for expression by utilizing an agrobacterium-mediated genetic transformation method; positive transgenic Arabidopsis plants are screened by antibiotic screening, RT-PCR and the like.

5. The use according to claim 4,

the primers for the genes are as follows:

5'-CCAGTCAGAGATGGTCAAGG-3' as a forward primer;

reverse primer 5'-CATCCATCGTCTTGCTGAG-3'.

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