CN114591969A

CN114591969A - Drought-resistant gene CrWRKY57 and application thereof in drought-resistant improvement of plants

Info

Publication number: CN114591969A
Application number: CN202210284601.4A
Authority: CN
Inventors: 彭婷; 熊欣婷; 王敏; 钟灶发; 程方婷; 莫锦夏
Original assignee: Gannan Normal University
Current assignee: Gannan Normal University
Priority date: 2022-03-22
Filing date: 2022-03-22
Publication date: 2022-06-07
Anticipated expiration: 2042-03-22
Also published as: CN114591969B

Abstract

The invention provides a drought-resistant gene CrWRKY57 and application thereof in plant drought-resistant improvement, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the drought-resistant gene CrWRKY57 is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2. The gene is overexpressed in lemon and tobacco by an agricultural genetic transformation method and is subjected to interference expression in the citrus reticulata blanco, and the drought resistance of the overexpressed plant is remarkably improved and the drought resistance of the plant subjected to interference expression is remarkably reduced by biological function verification of the obtained transgenic plant, so that the cloned CrWRKY57 gene has the function of improving the drought resistance.

Description

Drought-resistant gene CrWRKY57 and application thereof in drought-resistant improvement of plants

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a drought-resistant gene CrWRKY57 and application thereof in plant drought-resistant improvement.

Background

Drought not only severely limits the scope of citrus cultivation, but also is a major abiotic factor that restricts the development of the citrus industry. Therefore, the cultivation of new varieties of citrus with drought resistance is extremely important for the continuous, stable and healthy development of the citrus industry. The rapid development of biotechnology provides a new approach for plant breeding, can perform directional genetic improvement on crops through genetic engineering, and has shown important utilization value in the cultivation of new stress-resistant varieties (materials) of crops. The discovery and identification of the stress-resistant gene are the premise and key of creating a stress-resistant transgenic plant by using genetic engineering, but the screening amount of the drought-resistant gene is very small at present, and the requirements of scientific research and production cannot be met.

Disclosure of Invention

In view of the above, the invention aims to provide a drought-resistant gene CrWRKY57 and application thereof in plant drought resistance improvement, provide a new gene resource for plant stress-resistant molecule design breeding, provide a new genetic resource for implementing green agriculture and water-saving agriculture, and the development and utilization of the genetic resource are beneficial to reducing agricultural production cost and realizing environmental friendliness.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a drought-resistant gene CrWRKY57, wherein the nucleotide sequence of the drought-resistant gene CrWRKY57 is shown in SEQ ID No. 1.

The invention also provides a protein coded by the drought-resistant gene CrWRKY 57.

Preferably, the amino acid sequence of the protein is shown as SEQ ID NO. 2.

The invention also provides a primer pair for amplifying the drought-resistant gene CrWRKY57, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4.

The invention also provides a method for amplifying the drought-resistant gene CrWRKY57, which comprises the following steps: carrying out PCR amplification by using cDNA of the citrus reticulata blanco as a template and the primer pair to obtain the drought-resistant gene CrWRKY 57;

the PCR amplification program comprises: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extension for 10min at 72 ℃.

The invention also provides application of the drought-resistant gene CrWRKY57, the protein or the drought-resistant gene CrWRKY57 obtained by amplification by the method in improving drought resistance of plants.

Preferably, the plant comprises citrus reticulata blanco, tobacco and/or lemon.

The invention also provides a recombinant vector for over-expressing the drought-resistant gene CrWRKY57, wherein the basic vector of the recombinant vector comprises a pBI121 vector, and the drought-resistant gene CrWRKY57 is inserted between XbaI and SmaI sites of the basic vector.

The invention also provides a method for improving the drought resistance of plants, which comprises the following steps: the drought-resistant gene CrWRKY57 is expressed or over-expressed in a plant genome.

Preferably, the overexpression comprises the step of transferring the recombinant vector into the genome of a plant by using a genetic transformation method to obtain the plant with improved drought resistance.

Has the advantages that: the invention provides a drought-resistant gene CrWRKY57 which is obtained by separating and cloning a drought-resistant variety CrWRKY57 from a super-drought-resistant variety of red tangerines, belongs to a new gene CrWRKY57 of a WRKY family, and has a nucleotide sequence shown in SEQ ID No.1, wherein an amino acid sequence of encoded protein is shown in SEQ ID No.2, the encoded protein comprises an 876bp Open Reading Frame (ORF), 292 amino acids are encoded, the isoelectric point is 6.19, and the predicted molecular weight is 32.05 kDa. In the embodiment of the invention, after the full length of the drought-resistant gene CrWRKY57 is obtained, the gene is overexpressed in lemon and tobacco by utilizing an agrobacterium-mediated genetic transformation method and is subjected to interference expression in the citrus reticulata blanco Hay, the drought resistance of an overexpressed plant is obviously improved and the drought resistance of an interference-expressed plant is obviously reduced through biological function verification, which shows that the cloned CrWRKY57 gene has the function of improving the drought resistance. By means of the drought-resistant gene CrWRKY57, the drought-resistant capability of plants can be effectively improved, the water consumption can be effectively reduced in production, and the production cost is reduced; meanwhile, the method can also be used for improving the stock material of fruit trees, does not have potential safety hazards of transgenic food, and is easy to accept and accept by the public. The gene is introduced into plants through agrobacterium mediation, the functions of the gene under drought or in vitro dehydration conditions are identified, new gene resources are provided for plant drought-resistant molecular breeding, and new genetic resources are provided for green agriculture and water-saving agriculture.

Drawings

FIG. 1 is a technical flow chart of the present invention;

FIG. 2 is a graph showing the result of expression level of CrWRKY57 gene in transcriptome sequencing (RNA-seq) and qRT-PCR analysis; histogram is RNA-seq data, expressed as FPKM; the line graph is qRT-PCR data, and the expression form is relative expression quantity;

FIG. 3 is a schematic diagram of subcellular localization fluorescence of the CrWRKY57 gene of the invention;

FIG. 4 is a schematic diagram of the identification of CrWRKY57 transcriptional activation of the present invention;

a in FIG. 4 is a schematic diagram of a segmentation case of CrWRKY 57; b in FIG. 4 is a schematic representation of the transcriptional activation identification of CrWRKY57 and different fragments;

FIG. 5 is a schematic diagram of the construction of a CrWRKY57 gene vector of the invention;

FIG. 5A is a schematic diagram of the construction of the overexpression vector; FIG. 5B is a schematic representation of the interference vector construction;

FIG. 6 is a schematic diagram of PCR identification of a CrWRKY57 gene overexpression vector, no-load transformation tobacco and a regeneration plant; wherein A is co-cultured leaf blade, B is screened cultured leaf blade; c is the elongation and propagation of the resistant bud on an elongation culture medium, and D is the PCR identification of the transgenic tobacco;

FIG. 7 is a graph of measurement of phenotype and physiological index after dehydration of leaves in vitro before and after drought treatment of CrWRKY57 overexpression tobacco (OE4, OE10 and OE17), Wild Type (WT) and no-load transgenic tobacco (EV) potted seedlings; wherein A is a table diagram of five lines before drought, 14 days after drought and 1 day after rehydration, B is the relative water loss rate of the in vitro leaves after dehydration, C is the malondialdehyde content of the in vitro leaves after 80 minutes of dehydration, and D is the conductivity of the in vitro leaves after 80 minutes of dehydration;

FIG. 8 is a graph of peroxide accumulation in vitro dehydrated leaves of CrWRKY57 overexpressing tobacco (OE4, OE10, OE17) and Wild Type (WT) and unloaded transgenic tobacco (EV) according to the invention; wherein A is NBT (the darker the color, O)₂·^–Higher content) and DAB (darker colour, H₂O₂More content) staining pattern, B is anti-O₂Ability (the larger the value, the more the indication of O₂·^–The smaller the content), C is H₂O₂Content (c);

FIG. 9 is a schematic diagram of PCR identification of CrWRKY57 gene overexpression vector transformed lemon and regenerated plant; wherein A is a stem section of co-culture, and B is a stem section of screening culture; c, extending and propagating the resistant buds on an extending culture medium, D, carrying out micro-bud grafting on the resistant lemon buds onto rootstock trifoliate orange, and E, obtaining soil-culture plants; f is CrWRKY57 gene expression quantity;

FIG. 10 is a schematic diagram of PCR identification of a CrWRKY57 gene RNAi vector transformed Sanhu red oranges and regeneration plants; wherein A is a stem section of co-culture, and B is a stem section of screening culture; c, elongation and propagation of the resistant bud on an elongation culture medium, D, rooting culture of the resistant bud, and E, detection of RNAi interference condition by PCR;

FIG. 11 is a schematic diagram showing the comparison of drought resistance between CrWRKY57 overexpressed lemons (OE-1 and OE-2) and wild type (CKL) according to the present invention; wherein A is the relative water loss rate of the in vitro leaves after dehydration, B is the malondialdehyde content of the in vitro leaves after 80 minutes of dehydration, and C is the conductivity of the in vitro leaves after 80 minutes of dehydration;

FIG. 12 is a schematic diagram showing the comparison between CrWRKY57RNAi three-lake red oranges (RNAi-2 and RNAi-19) and wild type (CKs) drought resistance according to the present invention; wherein A is the relative water loss rate of the in vitro leaves after dehydration, B is the malondialdehyde content of the in vitro leaves after 80 minutes of dehydration, and C is the conductivity of the in vitro leaves after 80 minutes of dehydration;

FIG. 13 is a Wen plot and cluster heatmap of differentially expressed genes, where A is an up-regulated differentially expressed gene, B is a down-regulated differentially expressed gene, and C is the expression pattern of all differentially expressed genes;

FIG. 14 is a graph of qRT-PCR validation results, wherein the lower right graph is a correlation analysis of qRT-PCR data and RNA-Seq data.

Detailed Description

The invention provides a drought-resistant gene CrWRKY57, wherein the nucleotide sequence of the drought-resistant gene CrWRKY57 is shown as SEQ ID No. 1. The sequence shown in SEQ ID NO.1 is an open reading frame which comprises 876bp, and the drought-resistant gene CrWRKY57 is positioned in a cell nucleus. The drought-resistant gene CrWRKY57 has no transcription activation activity, and needs to be interacted with other transcription factors or promoter elements to form a complex and then regulate and control a target gene, so that CrWRKY57 can possibly function in the form of the complex.

The drought-resistant gene CrWRKY57 is preferably separated and cloned from a Citrus reticulata blanco (Citrus reticulata), and the cytology characteristics and the drought resistance of the Citrus reticulata blanco, a Citrus aurantium (Poncirus trifoliata), a Citrus aurantium (C.sesensis x P.trifoliate 'Carrizo'), and a Citrus maxima (C.reticulata) under drought stress are compared to find that the drought resistance of the Citrus reticulata is the strongest, so that the Citrus reticulata blanco is sampled after being subjected to drought treatment to carry out RNA-seq, and a drought stress response transcription factor CrWRKY57 is screened from the RNA-seq.

The amino acid sequence of the protein is preferably shown as SEQ ID NO. 2: MDDSSKEKSDRGQSSWKLGEPPDAGCVSYILSEFGWNLQEHESSTSYFAADHERSDLA GNISSSFPAETTTDGGGLTNPGRSADVSTSNPSVSSSSSEDPTEKSTGSGGKPPEIPSKAR KKGQKRIRQPRFAFMTKSEVDHLEDGYRWRKYGQKAVKNSPFPRSYYRCTNSKCTVK KRVERSSEDPTIVITTYEGQHCHH

TVGFPRGGLINHEAAAFASHLTHAIPPYYYHQGVQITQETPGIKQQSHEEELIPVEAREH EPNALPEPPALPPPTDEGLLGDIVPPGMRNR, which codes for 292 amino acids. The protein CrWRKY57 coded by the drought-resistant gene CrWRKY57 has the isoelectric point of 6.19 and the predicted molecular weight of 32.05 kDa.

The invention also provides a primer pair for amplifying the drought-resistant gene CrWRKY57, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 3: 5'-ATTCATTGAGCTCCACGGAG-3', the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 4: 5'-ACTCATCTATTGCGCATCCCAG-3' are provided.

In the invention, when the drought-resistant gene CrWRKY57 is amplified for the first time, part of ESTs screened based on RNA-seq data are preferably selected, a sequence (Cs7g03080) of orange CrWRKY57 gene is used, a Primer Premier 5.0 is used for designing a Primer pair, and a PCR amplification system is prepared by the Primer pair and a cDNA template of the orange of Sanhu red orange, wherein the system is calculated by 50 mu L, and preferably comprises: TaKaRa LA Taq (5U/. mu.L) 0.5. mu.L, 10 × LA Taq Buffer II (Mg²⁺Plus) 5. mu.L, dNTP mix (2.5mM each) 8. mu.L, forward and reverse primers (10. mu.M) each 1. mu.L, template cDNA 1. mu.L and sterile water 33.5. mu.L.

The cDNA template is preferably obtained by reverse transcription of RNA extracted from leaves, in the embodiment, total RNA in leaves of tangerine of three lakes is extracted by an RNAioso Plus kit (the kit is purchased from TAKARA company, and the operation method is according to the instruction); the RNA of the three-lake tangerine orange obtained by extraction refers to an operation manual of a TOYOBO reverse transcription kit to synthesize a first strand of cDNA.

The invention also provides application of the drought-resistant gene CrWRKY57, the protein or the drought-resistant gene CrWRKY57 obtained by amplification by the method to improvement of plant drought resistance.

In the invention, drought stress can induce the expression of the drought-resistant gene CrWRKY57, which indicates that the drought-resistant gene CrWRKY57 is a drought response gene. In the invention, the drought resistance of the drought-resistant gene CrWRKY57 in transgenic plants is compared based on overexpression and RNAi technology, and the drought resistance of the transgenic plants after overexpression is obviously improved, and the drought resistance of the transgenic plants after RNAi inhibition expression is obviously reduced, so that the cold resistance of the plants can be improved by overexpressing the drought-resistant gene CrWRKY 57. The plants of the present invention preferably include citrus tristimulus, tobacco and/or lemon.

When the recombinant vector is constructed, XbaI and SmaI are preferably selected as endonucleases according to the analysis of a pBI121 vector multiple cloning site and a CrWRKY57 gene ORF enzyme digestion site, and simultaneously, a super-expression vector construction primer is designed, wherein the primer comprises a forward primer (SEQ ID NO.5) and a reverse primer (SEQ ID NO.6), and the sequences of the primers are respectively as follows:

a forward primer: 5'-GCTCTAGAATGGATGATAGTAGCAAAGAG-3', respectively;

reverse primer: 5'-TCCCCCGGGTCATCTATTGCGCATCCCA-3' are provided.

The invention utilizes the forward primer and the reverse primer which are obtained by the construction to amplify the target fragment containing the enzyme cutting site, and an amplification system is calculated by 50 mu L, and preferably comprises the following components: 5 × TransStart Fastpfu Buffer 10.0 μ L, dNTP Mix (2.5mM each)5.0 μ L, cDNA template 1.0 μ L, forward primer (10 μ M)2.0 μ L, reverse primer (10 μ M)2.0 μ L, TransStart Fastpfu DNA Polymerase 1.0 μ L and ddH₂O29.0. mu.L. The PCR amplification is carried out on the amplification system obtained by preparation, and the PCR amplification program preferably comprises pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 30s, and 40 cycles; extension at 72 ℃ for 5 min. The target fragment is preferably inserted into a pBI121 vector based on a double-enzyme digestion method, so that a pBI121-CrWRKY57 recombinant vector is obtained.

The overexpression of the invention preferably comprises the step of transferring the recombinant vector into the genome of a plant by using a genetic transformation method to obtain the plant with improved drought resistance. The method of genetic transformation is not particularly limited in the present invention, and the genetic transformation is performed in an Agrobacterium tumefaciens-mediated manner in the examples, but it cannot be considered as the full scope of the present invention.

The drought-resistant gene CrWRKY57 and its application in improving drought resistance of plants provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

Example 1

Isolated clone of CrWRKY57 gene

1. Total RNA was first extracted from leaves of Trifolium pratense using RNAioso Plus kit (kit available from TAKARA, Inc., procedures according to the instructions). The RNA of the three-lake tangerine orange obtained by extraction refers to an operation manual of a TOYOBO reverse transcription kit to synthesize a first strand of cDNA.

2. Primers shown in SEQ ID NO.3 and SEQ ID NO.4 were designed by using the sequence of orange CrWRKY57 gene and the Primer Premier 5.0 based on a part of ESTs screened from RNA-SEQ data.

Obtaining of transcriptome sequencing data: the Citrus reticulata blanco (Citrus reticulata) is an ancient local characteristic variety of new stems in Jiangxi, and has good characteristics of resisting biotic stress and abiotic stress; meanwhile, the drought resistance of the three-lake red tangerine is superior to that of citrus stocks such as poncirus trifoliata, chongyi wild tangerine and the like, and the three-lake red tangerine is an excellent drought-resistant germplasm resource.

RNA-seq is utilized to analyze the differences of leaf transcripts of the three-lake tangerine after drought treatment for 1 day (DroughtAfter Watering, DAW1), 4 days (DAW4) and 7 days (DAW7), GO function enrichment, KEGG enrichment, transcription factor mining and other analyses are carried out, and qRT-PCR verification is carried out on 14 DEGs (containing CrWRKY 57).

In DAW4 vs DAW1, the number of differential genes was 7519, with gene 3652 significantly up-regulated and gene 3865 significantly down-regulated; the number of DAW7 vs DAW1 differential genes is 6563, the significant up-regulation gene is 3216, and the significant down-regulation differential gene is 3348; the DAW7 vs DAW4 differential gene was 7407, 3692 genes were significantly up-regulated, and 3715 genes were significantly down-regulated (table 1). The screened differential genes of the three comparative combinations are analyzed by a Wein diagram, and 2179 significant differential expression genes are found in the three comparative combinations; whereas the number of genes that were all significantly up-regulated in the three comparative combinations was 233 and the number of genes that were all significantly down-regulated was 210. The heat map results showed that the number of up-regulated expression genes of DAW4 was greater than that of DAW1 and DAW7, and that the expression of these early response genes was likely to be induced by drought stress (fig. 13).

TABLE 1 statistics of drought stress differential expression genes of citrus tangerina sanguinea

The expression level of 14 DEGs (containing CrWRKY57) is verified by adopting real-time fluorescent quantitative PCR (qRT-PCR). Real-time quantitative primers (table 2) were designed using Primer 5.0, and citrus Actin was selected as an internal reference gene. The correlation coefficient of the qRT-PCR verification result and the RNA-Seq result reaches 0.72 (figure 14), and the drought stress transcriptome data is proved to be reliable. The qRT-PCR result shows that CrWRKY57 is significantly induced by drought.

qRT-PCR primers for 214 DEGs (CrWRKY 57-containing) in Table

3. The cDNA of the orange of the third lake is taken as a template, and the CrWRKY57 of the orange of the third lake is amplified by PCR.

The PCR amplification system is as follows: TaKaRa LA Taq (5U/. mu.L) 0.5. mu.L, 10 × LA Taq Buffer II (Mg2+ Plus) 5. mu.L, dNTP mix (2.5mM each) 8. mu.L, forward and reverse primers (10. mu.M) each 1. mu.L, template cDNA 1. mu.L, and sterilized water 33.5. mu.L.

PCR was performed as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, 35 cycles, and extension at 72 ℃ for 10min after the cycle is completed.

4. After PCR amplification was completed, the mixture was electrophoresed on a 1.2% agarose gel for 30min in a TAE buffer on an DYY-6C electrophoresis apparatus (Liuyi, Beijing) with the parameters set to 120V and 150 mA. The band of interest was excised under an ultraviolet lamp, and the specific band of interest was recovered using an Axygen gel recovery kit (Corning Life sciences Co., Ltd.) and the operation was performed according to the instructions.

5. The recovered and purified product was ligated with pMD18-T vector (Takara, Japan).

The connecting system is as follows: pMD18-T Vector 0.5. mu.L, Solution I5. mu.L, PCR purified product 4.5. mu.L.

Connecting overnight at 16 ℃, transforming into escherichia coli competence DH5 alpha (Beijing hologold biotechnology limited) by adopting a heat shock method, selecting positive clone by using a target gene sequence primer, carrying out PCR verification (consistent with the PCR program in the gene amplification), and sequencing (completed by Wuhan Kyok encyclopedia biotechnology limited).

The above procedure amplified a 904bp fragment. ORF Finder at NCBI predicts the open reading frame and finds that this sequence contains a 876bp long coding region sequence which codes for a 292 amino acid protein with a molecular weight of 32.05kDa and a theoretical isoelectric point of 6.19. The gene coding protein and a published WRKY family in Arabidopsis are used for constructing an evolutionary tree, and the evolutionary tree and AtWRKY57 are found to belong to the II-c subfamily. The DNA is compared with the WRKY57 core amino acid sequence in arabidopsis, rice, corn and other species by using DNAMAN 8.0, and the WRKYGQK heptapeptide structure and C are found to be contained₂H₂The zinc finger structure of (1). Therefore, the gene is named as CrWRKY57, the cDNA sequence is shown as SEQ ID NO.1, and the sequence of the encoded protein is shown as SEQ ID NO. 2.

Example 2

CrWRKY57 Gene expression analysis

The expression of the gene in leaves after 1d, 4d and 7d of drought was analyzed by real-time quantitative PCR using the same samples as the RNA-seq in example 1. The RNA extraction and reverse transcription process are as in example 1.

The Primer 5.0 is used for designing a CrWRKY57 real-time quantitative Primer, and citrus Actin is selected as an internal reference gene:

forward primer (SEQ ID NO. 7): 5'-AACCTCCTGAGATACCAAGC-3', respectively;

reverse primer (SEQ ID NO. 8): 5'-TTTACAGCCTTCTGACCATAC-3', respectively;

internal reference genes:

forward primer (SEQ ID NO. 9): 5'-CATCCCTCAGCACCTTCC-3', respectively;

reverse primer (SEQ ID NO. 10): 5'-CCAACCTTAGCACTTCTCC-3' are provided.

The quantitative PCR system was 10. mu.L, including iTaq TM Universal

Green Supermix (Roche) 5.0. mu.L, real-time quantitative forward and reverse primers (10mol/L) 0.2. mu.L each, cDNA template 0.5. mu.in example 1, ddH₂O 4.1μL。

Use of the apparatus

480II (Roche). The reaction program is set to be 95 ℃ and pre-denatured for 30 s; denaturation at 95 ℃ for 5 s; annealing at 56 ℃ for 10 s; 72 ℃ for 15s, 40 cycles of extension. Quantitative PCR data were analyzed by the instrument with its own software.

The results of RNA-seq and qRT-PCR are shown in FIG. 2, and drought can induce the gene expression (see FIG. 2), which indicates that CrWRKY57 is a drought response gene.

Example 3

Subcellular localization of CrWRKY57 Gene

The ExPASy online analysis result shows that CrWRKY57 has a nuclear localization signal, and the subcellular localization of CrWRKY57 is researched by using arabidopsis leaf protoplast and plant subcellular localization vector pL101YFP in the embodiment.

And (3) designing a subcellular localization primer, amplifying an ORF sequence of the CrWRKY57 gene, and inserting the ORF sequence between KpnI and SmaI enzyme cutting sites on pL101 YFP.

Subcellular localization primers:

forward primer (SEQ ID NO. 11): 5'-GGGGTACCATGGATGATAGTAGCAAAGAG-3', respectively;

reverse primer (SEQ ID NO. 12): 5'-TCCCCCGGGTCTATTGCGCATCCCA-3' are provided.

The amplification system is as follows: 1-5TM2 Xhigh-Fidelity Master Mix (Pongke New Biotechnology Co., Ltd.) 25. mu.L, each of the forward and reverse primers 2. mu.L, and the cDNA of Tri-huh red orange in example 1 2. mu. L, ddH₂O 19μL。

The amplification PCR program is: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 1min, 35 cycles, and extension at 72 ℃ for 10min after the cycle is completed.

After PCR amplification, all PCR products were electrophoresed on 1.2% agarose gel, and specific target bands were recovered by Axygen gel recovery kit (Corning Life sciences, Ltd.), and the operation was performed according to the instruction. Connecting the recovered product

The carrier (all-purpose gold, Beijing) and the connecting system are

-Blunt Cloning Vector 1. mu. L, PCR purification of 4. mu.L of product.

After 10min of ligation at room temperature, the ligation solution was introduced into E.coli competent DH5 α (Beijing holotype gold Biotechnology Co., Ltd.) by thermal excitation, positive clones were selected with the target gene sequence primers for PCR verification (identical to the PCR procedure described above in constructing the vector) and sequencing was completed by Wuhan Pongku Seiko Biotechnology Co., Ltd.

The pEASY-CrWRKY57 recombinant vector plasmid and the pL101YFP vector plasmid were simultaneously cleaved with the enzymes KpnI and SmaI as follows in a double cleavage system of KpnI 1. mu. L, SmaI 1. mu.L, 10 XTbuffer 2. mu.L, 0.1% BSA 2. mu.L, and plasmid DNA 14. mu.L.

The double enzyme digestion system is subjected to enzyme digestion at 37 ℃ overnight, agarose gel electrophoresis is used for detection, qualified bands are cut off, and a gel recovery kit is used for recovering target bands. T4 DNALigase is used for connecting pEASY-CrWRKY57 recombinant vector plasmid and pL101YFP vector plasmid after KpnI and SmaI double digestion to obtain 35S-CrWRKY57-pL101YFP recombinant vector, agrobacterium-infected cell GV3101 (only biotechnology, Inc., Shanghai) is transformed, and the transformation steps are shown in the specification.

The CrWRKY57 positioning condition is detected by adopting an arabidopsis leaf protoplast preparation and transformation method. Reagents used for extraction of arabidopsis protoplasts: arabidopsis thaliana leaf protoplast enzymolysis liquid (used in present preparation): 20mM MES, 1.5% cellulase, 0.4% macerating enzyme, 0.4M mannitol, 20mM KCl, warm bathing at 55 deg.C for 10min, cooling to room temperature, adding 10mM CaCl₂5mM mercaptoethanol, 0.1% BSA, filtered through a 0.45. mu.M filter and used. WI solution: 4mM MES, 0.5M mannitol, 20mM KCl, stored at room temperature. W5 solution: 2mM MES, 154mM NaCl, 125mM CaCl₂5mM KCl. PEG4000 solution: 1g PEG4000, 0.75mL double distilled water, 0.625mL mannitol, 0.25mL CaCl₂. MMG solution: 4mM MES, 0.4mM mannitol, 15mM MgCl₂。

By transforming 35S-CrWRKY57-pL101YFP and control pL101 YFP-unloaded plasmids into Arabidopsis protoplasts, the results are shown in FIG. 3, the fluorescence of the pL101 YFP-unloaded plasmid is distributed in the whole protoplast including cell membrane and nucleus, while the fluorescence of the 35S-CrWRKY57-pL101YFP fusion protein is only concentrated in the nucleus, indicating that CrWRKY57 is located in the nucleus.

Example 4

CrWRKY57 transcriptional activation Activity assay

pGBKT7 vector is adopted for recombinant construction, and whether CrWRKY57 has transcription activation activity or not is verified. The WRKY domain of the predicted CrWRKY57 protein on SMART is distributed in 142th-201th aa, and is presumed to be a transcription activation region. Thus, in addition to full length, CrWRKY57 is further divided into three segments bounded by the WRKY domain: CrWRKY57-1(1th-141th aa), CrWRKY57-2(142th-201th aa) and CrWRKY57-3(202th-291th aa) are respectively connected to pGBKT7 vectors. After the sequence is confirmed to be correct by sequencing, 4 fusion expression vectors and an empty vector (pGBKT7) are respectively transformed into a yeast strain AH109, and then the yeast strain is cultured on different deletion media. The results showed that both the yeast cells transformed with the empty vector and the cells transformed with 4 fusion vectors could only grow on the deletion medium SD/-Trp, but not on the deletion media SD/-Trp/-ade and SD/-Trp/-ade/-His (FIG. 4), indicating that CrWRKY57 has no transcriptional activation activity and may function by the complex.

Example 5

Overexpression of CrWRKY57 gene and construction of RNAi vector

1. Overexpression vector construction

XbaI and SmaI were selected as endonucleases (A in FIG. 5) based on the analysis of the pBI121 vector multiple cloning site and the ORF cleavage site of the CrWRKY57 gene.

Designing an over-expression vector to construct primers SEQ ID NO.5 and SEQ ID NO. 6.

The fragment of interest was amplified using TransStart FastPfu DNA Polymerase (all-type gold). The PCR system is as follows: 5 XTransStart FastPfu Buffer 10.0. mu.L, dNTP Mix (2.5mM each) 5.0. mu.L, cDNA template 1.0. mu.L in example 1, forward primer (10. mu.M) 2.0. mu.L, reverse primer (10. mu.M) 2.0. mu.L, TransStart FastPfu DNA Polymerase 1.0. mu. L, ddH₂O 29.0μL。

The PCR procedure was: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 30s, and 40 cycles; extension at 72 ℃ for 5 min.

And performing double enzyme digestion after the PCR product is subjected to gel cutting recovery, wherein the system is 100 mu L: 10 XH buffer, 10 μ L; XbaI and SmaI (TaKaRa), each 5. mu.L; the product was recovered, 25 μ L; ddH₂O, 55 μ L; the enzyme was cleaved at 37 ℃ overnight and recovered.

The pBI121 vector plasmid was also digested with the same system and recovered.

The recovered target fragment and the vector were ligated, and the total ligation reaction volume was 10. mu.L: 10 XT 4 ligation buffer, 1 μ L; t4 ligase, 1. mu.L; pBI121 vector, 0.5. mu.L; 4.5 μ L of the target fragment; ligation was carried out at 16 ℃ for 16 hours.

Then the connecting product is transformed into escherichia coli DH5 alpha, screening is carried out in an LB solid plate containing 50mg/L kanamycin, monoclonal PCR is selected to be detected to be positive, then sequencing is carried out by Wuhan engine new industry biotechnology limited company, and the sequencing determines that the reading frame is completely correct, namely the pBI121-CrWRKY57 recombinant vector is constructed successfully. The recombinant vector was introduced into Agrobacterium tumefaciens GV3103 by a freeze-thaw method (see molecular cloning, A laboratory Manual, fourth edition, science Press, 2017) and the cell suspension was stored at-80 ℃ with 20% glycerol.

RNAi vector construction

In order to obtain an interference transgenic plant, a pHellsgate2 interference vector is adopted for recombination construction. Firstly, selecting a fragment design primer of about 300bp on CrWRKY57, and adding an attB site universal primer in front of a specific primer to serve as a target fragment amplification primer.

Selecting a fragment of CrWRKY57 smaller than 300bp as a template to design and construct a primer of an RNAi vector:

forward primer (SEQ ID NO. 17): 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGAGCGAGTTTGGATGGAA-3', respectively;

reverse primer (SEQ ID NO. 18): 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCGCTTTTGTCCTTTCTTT-3', respectively;

PCR amplification System: 1-5TM2 Xhigh-Fidelity Master Mix (Pongke New Biotechnology Co., Ltd.) 25. mu.L, 2. mu.L each of forward and reverse primers, and 2. mu. L, ddH of Saururi red orange cDNA in example 1₂O 19μL。

After PCR amplification, all PCR products were electrophoresed on 1.2% agarose gel, and specific target bands were recovered by Axygen gel recovery kit (Corning Life sciences, Ltd.), and the operation was performed according to the instruction.

The purified PCR product was ligated to the RNAi vector pHELLSGATE2 by BP reaction, as described in

BP

II Enzyme mix instructions, i.e., recovered product 4. mu. L, pHellsgate2 plasmid 1. mu. L, B/P clone 1. mu.L, reacted at 25 ℃ overnight.

The target fragment was amplified using the tricuspid tangerine cDNA in example 1 as a template, and the PCR product was recovered and recombinantly linked to the pHellsgate2 vector by BP reaction (BP ClonaseTM II Enzyme Mix (Invitrogen) kit) to construct the target vector (B in fig. 5).

All the reactants are taken to be transformed into the competence of Escherichia coli DH5 alpha by a heat shock method, and the competence is smeared on an LB solid plate containing 100mg/L spectinomycin to screen positive clones.

Carrying out PCR detection analysis on the positive clone by taking a 35S promoter sequence primer (SEQ ID NO. 19: 5'-CTATCCTTCGCAAGACCCT-3') and a reverse specific primer (SEQ ID NO.18) as amplification primers, further carrying out single enzyme digestion verification on the positive clone with correct PCR detection by using XbaI and XhoI respectively, and if the sizes of two groups of single enzyme digestion fragments are consistent and are about 200bp larger than that of a target fragment, correctly connecting the target fragment in the positive clone. The positive single clone with correct connection is sent to Wuhan engine new industry biotechnology Limited company for sequencing, the single clone with correct sequence extracts recombinant plasmid, the recombinant vector is introduced into the agrobacterium tumefaciens GV3103 by using a freeze-thaw method, and the bacterial liquid is preserved at the temperature of minus 80 ℃ (containing 20% of glycerol).

Example 6

Application of transcription factor CrWRKY57 in improving drought resistance of tobacco

1. The agrobacterium tumefaciens-mediated tobacco genetic transformation steps are as follows:

(1) culturing agrobacterium tumefaciens: taking fresh Agrobacterium tumefaciens bacterial liquid, streaking on an LB solid plate (containing 50mg/L kanamycin), scraping streaked bacterial plaque, adding into a liquid MS minimal medium, performing shaking culture at 28 ℃ at 200rpm until the bacterial liquid concentration reaches OD₆₀₀Carrying out dip dyeing when the dyeing rate is 0.3-0.8;

(2) dip dyeing: taking healthy wild type in-vitro seedling tobacco leaves, removing main veins, cutting into 0.5cm × 0.5cm square blocks, soaking in the prepared agrobacterium tumefaciens bacterial solution for 8-10 minutes, and intermittently oscillating;

(3) co-cultivation: taking the impregnated tobacco leaves, air drying on sterile filter paper, uniformly arranging the back of the leaves downwards on a co-culture medium (A in figure 6), and carrying out dark culture at 25 ℃ for 3 days;

(4) screening and culturing: after co-cultivation, the cells were washed once with a cefuroxime solution (500mg/L), then washed 3-5 times with sterile water, and transferred to a screening medium (B in FIG. 6).

(5) Rooting: when the adventitious bud grows to about 1cm on the screening medium, it is excised and transferred to a rooting medium (C in FIG. 6).

TABLE 3 tobacco transformation Medium formulation

Name (R)	Components and content (30g/L sucrose; 0.7g/L agar; pH5.8)
		Co-cultivation medium	MS minimal medium +2.0 mg/L6-BA +0.3mg/L NAA
Screening media	Co-culture medium +50mg/L hygromycin +500mg/L cefuroxime
		Rooting culture medium	MS minimal medium +0.3mg/L NAA +50mg/L hygromycin +500mg/L cephamycin

2. Positive transgenic tobacco identification

When 2 to 3 leaves grow on the rooted seedling, DNA is extracted by using a CTAB method.

The obtained DNA is used as a template, a primer is constructed by a CrWRKY57 overexpression vector to identify an overexpression positive plant, and a hygromycin gene primer (a forward primer SEQ ID NO. 13: 5'-CTCCATACAAGCCAACCACG-3'; a reverse primer SEQ ID NO. 14: 5'-AAAAAGCCTGAACTCACCGC-3') is used to identify an unloaded positive plant.

Ubiqutin as an internal reference for amplification, forward primer (SEQ ID NO. 15): 5'-AGCTACATGACGCCATTTCC-3', reverse primer (SEQ ID NO. 16): 5'-CCCTGTAAAGCAGCACCTTC-3' are provided.

The target fragment was amplified using Taq enzyme from Fermentas. The PCR system is as follows: 10 XPCR Buffer 2.0. mu.L, dNTP Mix (10mM each)) 0.4. mu.L, tobacco DNA 1.0. mu.L, forward and reverse primers (10. mu.M) 0.3. mu. L, Polymerase 0.2.2. mu. L, ddH each₂O15.8. mu.L. The PCR procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 3min, and 35 cycles; extension at 72 ℃ for 12 min.

The partial PCR validation electrophoresis results are shown in FIG. 6D. Three of these overexpression lines OE4, OE10 and OE17 were selected as individual transgenic lines and then as the female parent plants for seed harvest, respectively.

3. Identification of drought resistance of over-expression tobacco

Three over-expression lines OE4, OE10, OE17 and Empty (EV) and Wild Type (WT) tobacco seeds of the same batch were harvested and aseptically sown: soaking the seeds in 70% ethanol for 1min, washing with sterile double distilled water for three times, sterilizing with 1mL of 2.5% NaClO for 8min, shaking for several times, and sterilizing with sterile ddH₂And cleaning for 3 times, spreading and sowing the seeds on a hygromycin MS basic culture medium containing 50mg/L by using an aseptic inoculating needle, and after the seeds grow into seedlings after germination, using the seedlings for subsequent determination of drought-resistant phenotype and related indexes.

(1) Phenotype: carrying out potted plant water control treatment on over-expressed CrWRKY57 tobacco OE4, OE10 and OE17 and wild type control WT and no-load control EV thereof, wherein as shown in A in figure 7, when drought treatment is not carried out, the growth conditions of over-expressed CrWRKY57 and control plants are consistent; after 14 days of drought, WT and EV are wilted severely, most leaves are dried up due to water loss, and the wilting degree of over-expression plants OE4, OE10 and OE17 is relatively light; after rehydration, the growth of the control plants was not improved, and the leaves of the over-expressed plants began to turn green. The result shows that the tobacco over-expressing CrWRKY57 has stronger drought resistance and faster recovery speed after drought.

(2) Relative water loss rate: carrying out in-vitro leaf dehydration treatment on 5 strains of tobacco leaves, weighing the leaves by a ten-thousandth balance when the leaves are taken off from the plants, and recording the reading as M₀OfPlacing the back of the rear leaf upward on dry filter paper, weighing every 30min, and recording the reading as M_nThe experiment was set to 3 replicates, the average was taken and the error calculated, the formula:

as shown in B in FIG. 7, the relative water loss rates of over-expressed tobacco OE4, OE10 and OE17 were 27.64%, 28.68% and 26.15% respectively, and the relative water loss rates of WT and EV were 33.72% and 34.24% respectively, at 80min of dehydration, and the relative water loss rate of over-expressed tobacco was significantly lower than that of the control (p < 0.01). The relative water loss rates of the over-expressed tobaccos OE4, OE10 and OE17 are all smaller than those of WT and EV, which shows that the water retention of the leaf blade is stronger after the over-expressed CrWRKY 57.

(3) Content of malonaldehyde: the thiobarbituric acid method is adopted. Weighing in vitro dehydrated 80min leaf 0.5g, adding precooled 5% trichloroacetic acid solution and a little quartz sand, grinding into homogenate, transferring into centrifuge tube, washing with 5% TCA, adding cleaning solution into centrifuge tube, metering volume to 10mL, centrifuging at 4 deg.C and 7000rpm for 10min, collecting supernatant 2mL (V)₁) Adding 0.67% thiobarbituric acid (TBA)2mL into test tube, mixing, boiling in boiling water for 20min, cooling, centrifuging at 25 deg.C and 7000rpm for 10min, and collecting supernatant as solution to be tested (V)₂) Taking the supernatant to be subjected to color comparison at 450nm, 532nm and 600nm, and calculating the formula:

CMDA(μmol/L)＝6.45(A₅₃₂-A₆₀₀)-0.56A₄₅₀

as shown in C in FIG. 7, at 80min, the MDA contents of OE4, OE10 and OE17 were 0.21. mu. mol/L, 0.20. mu. mol/L and 0.21. mu. mol/L, respectively, and the MDA contents of WT and EV were 0.34. mu. mol/L and 0.29. mu. mol/L; namely, the MDA content of OE4, OE10 and OE17 is obviously lower than that of a control (p is less than 0.05), which indicates that the cell membrane of the tobacco leaf overexpressing CrWRKY57 is less damaged under the in vitro dehydration condition.

(4) Conductivity: taking leaves of transgenic and control plants dehydrated 80min in vitro, removing midrib and leaf edge, cutting into small pieces of about 0.5 × 0.5cm with scissors, weighing 0.1g, and adding glass containing 10mL deionized waterIn the test tube, after shaking at 25 ℃ for 3h at 120r/min by a shaking table, measuring the conductivity by a conductivity meter (DDS-307) and recording the conductivity as C₁Boiling the test tube in boiling water for 15min, cooling to room temperature, measuring the electric conductivity and recording as C₂Calculating the formula

As shown in D in fig. 7, at 80min, the conductivities of OE4, OE10, OE17 were significantly lower than the control (p <0.05), indicating that the cell membrane of the tobacco leaf overexpressing CrWRKY57 was less damaged under the in vitro dehydration conditions.

(5) Superoxide accumulation:

firstly, transgenic and control plant leaves dehydrated for 80min in vitro are used for dyeing qualitative detection.

NBT staining method: nitroblue tetrazolium (NBT) powder was dissolved in 0.01M phosphate buffer at pH 7.8 to a final concentration of 1 mg/mL. And (3) subpackaging the prepared NBT staining solution into 50mL centrifuge tubes, completely immersing the tobacco leaves in the NBT staining solution, removing the staining solution after 3 hours of under-light staining, adding absolute ethyl alcohol for decoloring, replacing the greening ethyl alcohol until the leaves are completely green, taking pictures by a camera for recording, and immersing the leaves in the absolute ethyl alcohol for storage.

Dyeing by using a DAB method: 3, 3-Diaminobenzidine (DAB) powder was dissolved in 0.01M phosphate buffer at pH 3.8 as a solvent, and the mixture was mixed with V (staining solution): v (H)₂

O

₂30%): 1000 addition of H₂O₂And (5) uniformly mixing. And (3) subpackaging the prepared DAB staining solution into a 50mL centrifuge tube, completely immersing the tobacco leaves, dyeing for 8h under the light, discarding the staining solution, adding absolute ethyl alcohol, decoloring and storing in the same way, and finally photographing and recording by using a camera.

The results are shown in FIG. 8, A, where the color of tobacco overexpressing CrWRKY57 under dehydration conditions is lighter, indicating less superoxide accumulation.

H₂O₂The content and the activity of the superoxide anion free radical are quantitatively determined by using the peroxide of Nanjing KangjiThe hydrogen hydride determination kit (A064-1-1) and the determination kit (A052-1-1) for inhibiting and generating superoxide anion free radicals, and the operation processes are as the specification. anti-O₂·^–The ability is shown as B in FIG. 8, with larger values indicating O₂·^–The lower the content, H₂O₂The content is shown as C in figure 8, and the result shows that the over-expression of CrWRKY57 tobacco under the condition of dehydration has less superoxide accumulation.

Qualitative and quantitative results show that the over-expression CrWRKY57 can effectively enhance the active oxygen scavenging capability of transgenic plants and reduce the damage of the transgenic plants to cells, thereby improving the drought resistance of the plants.

Example 7

Application of transcription factor CrWRKY57 in improving drought resistance of lemon and citrus tristimulus

In order to provide more powerful experimental evidence to show the effect of CrWRKY57 under drought resistance, the overexpression and RNAi vectors in embodiment 5 are used for respectively transforming lemon and Sanhu red tangerine through an agrobacterium-mediated transformation method of the epicotyl of the citrus, so that whether the drought resistance of the overexpressed lemon is enhanced or not and the drought resistance of the RNAi Sanhu red tangerine is weakened can be judged.

1. The steps of agrobacterium tumefaciens-mediated transformation of the epicotyl of citrus and identification of a positive transgenic plant are as follows:

(1) sowing: taking lemon and sanhu tangerine seeds, soaking for 20min by using 1M NaOH, cleaning by using clean water, soaking for 15-20 min by using 2% NaClO on an ultra-clean workbench, washing for 3 times by using sterile water, peeling off seed coats under the sterile condition, sowing on an MT solid culture medium, culturing for 3-4 weeks in dark, culturing for 1 week by using light again, and then using for conversion.

(2) Preparing a bacterial liquid: taking the agrobacterium liquid containing the overexpression and RNAi recombinant vectors in the embodiment 5, streaking the liquid on a solid LB culture medium containing 100mg/L antibiotics (the pBI121 vector is kanamycin, and the pHellsgate2 vector is spectinomycin), and carrying out dark culture at 28 ℃ for 2 d; selecting single colony, streaking on new plate, dark culturing at 28 deg.C for 2-3 days; scraping the well grown thallus with scalpel, inoculating in liquid MT culture medium without antibiotic, and shake culturing at 28 deg.C and 200rpm to OD₆₀₀And (5) adding acetosyringone to the concentration of 0.6-0.8 to 100 mu M for later use.

(3) Co-cultivation: taking epicotyls of the aseptic seedlings of the lemons and the sanhu tangerine, obliquely cutting the epicotyls into 1-1.5 cm long stems in a super clean workbench, soaking the stems in prepared agrobacterium liquid (the lemon is converted from the liquid containing a super expression vector, and the sanhu tangerine is converted from the liquid containing a RANi vector) for 20min of infection, and continuously shaking for several times. After infection, the excess bacteria solution is sucked dry by using sterile absorbent paper, the explants are paved and aged on a co-culture medium, and dark culture is carried out for 3d at 25 ℃ (the lemon co-culture is shown as A in figure 9, and the three-lake tangerine co-culture is shown as A in figure 10).

(4) Screening and culturing and rooting: after co-culturing for 3 days, washing the explant with sterile water for 3-5 times, then, absorbing the surface moisture with sterile absorbent paper, and transferring to a screening medium (the lemon screening is shown as B in figure 9, and the three-lake tangerine screening is shown as B in figure 10). Culturing at 25 deg.C in dark for 4 weeks, and culturing under light. When the resistant shoots are >0.5cm, the cut resistant shoots are transferred to a proliferation medium to promote differentiation (C in FIG. 9 for lemon and C in FIG. 10 for three-lake tangerine). When the resistant bud is >1.5cm long, the lemon resistant bud is grafted onto the rootstock trifoliate (D in fig. 9), then soil-cultured (E in fig. 9). The resistant shoots of sanhu tangerine were transferred to rooting medium to induce rooting (D in fig. 10).

Common media formulations:

LB solid Medium: 10g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl and 15g/L of agar;

lemon co-culture medium: MT +0.5mg/L BA +0.1mg/L NAA +0.5mg/L KT +50mg/L AS;

three-lake tangerine co-culture medium: MT +1.0mg/L BA +20mg/L AS;

lemon screening culture medium: MT +0.5mg/L BA +0.1mg/L NAA +0.5mg/L KT +50mg/L AS +400mg/L Cef +50mg/L Km;

three-lake tangerine screening culture medium: MT +1.0mg/L BA +400mg/L Cef +50mg/L Km;

lemon proliferation medium: MT +0.1mg/L BA +0.5mg/L GA3+0.5mg/L IAA +400mg/L Cef;

three-lake tangerine proliferation culture medium: MT +0.5mg/L BA +0.5mg/L IAA +0.5mg/L GA 3;

three-lake tangerine rooting culture medium: 1/2MT +0.5mg/L NAA +0.1mg/L IBA +0.5g/L activated carbon;

agar 7.5g/L and sucrose 35g/L were added to each medium during transformation, and the pH was adjusted to 5.8.

(5) And (3) positive plant identification:

DNA extraction is performed in the same manner as in tobacco.

And (3) PCR detection: the PCR reaction system and procedure of lemon and three-lake tangerine are the same as those of tobacco, except that the internal reference gene is changed into Actin in the embodiment 2. The detection results of lemon are shown in E in FIG. 9, and the detection results of sanhu tangerine are shown in D in FIG. 10. CKL is a lemon control and CKS is a sanhu tangerine control, both obtained from untransformed epicotyl propagation (i.e. wild type). The two overexpression lines of the lemon are named as OE-1 and OE-2, and RNAi-2 and RNAi-19 are selected from the citrus reticulata blanco for subsequent resistance identification.

2. Drought resistance identification of transgenic citrus material

Dehydrating leaves of lemon CKL, OE-1, OE-2, Sanhu red orange CKS, RNAi-2 and RNAi-19 in vitro. The relative water loss, malondialdehyde content and conductivity were determined in the same manner as in example 6. As shown in fig. 11, the relative water loss rate of OE lemons after 80min dehydration was significantly lower than CKL, while the malondialdehyde content and conductivity were significantly higher than CKL. In contrast, the relative water loss rate of the RNAi line of the citrus tristimulus glauca was significantly higher than that of CKs, while the malondialdehyde content and conductivity were significantly lower than that of CKs (fig. 12). The drought resistance of the plants can be obviously enhanced by over-expression of CrWRKY57, and the drought resistance of the plants can be obviously reduced by interfering with the expression of CrWRKY 57.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of Jiangxian teachers

<120> drought-resistant gene CrWRKY57 and application thereof in drought-resistant improvement of plants

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Arg Asn Arg

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<400> 19

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

<400> 33

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

<400> 37

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<213> Artificial Sequence (Artificial Sequence)

<400> 38

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

aatcttgacc aggataagga ga 22

<210> 40

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

gcagcaaccc ttcaaactaa 20

<210> 41

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

cacaagcgaa caaataacag aa 22

<210> 42

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

cgccaccgag accaaataca 20

<210> 43

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

aaatccctta cgggcaaacc 20

<210> 44

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

tctttactgg ctgcctgttt 20

<210> 45

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

tagacgagca tctggtccct 20

<210> 46

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

tctgtcggga atgtttggtt 20

<210> 47

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

ctcctcggta atctcgccta 20

Claims

1. A drought-resistant gene CrWRKY57 is characterized in that the nucleotide sequence of the drought-resistant gene CrWRKY57 is shown as SEQ ID No. 1.

2. The protein encoded by the drought resistant gene CrWRKY57 of claim 1.

3. The protein of claim 2, wherein the amino acid sequence of the protein is represented by SEQ ID No. 2.

4. A primer pair for amplifying the drought resistant gene CrWRKY57 in claim 1, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4.

5. A method for amplifying the drought resistant gene CrWRKY57 of claim 1, comprising the following steps: carrying out PCR amplification by using cDNA of the citrus reticulata blanco as a template and the primer pair of claim 4 to obtain the drought-resistant gene CrWRKY 57;

6. The application of the drought-resistant gene CrWRKY57 as defined in claim 1, the protein as defined in claim 2 or 3 or the drought-resistant gene CrWRKY57 obtained by amplification by the method as defined in claim 5 in improving the drought resistance of plants.

7. Use according to claim 6, wherein the plants comprise Citrus trizandra, tobacco and/or lemon.

8. A recombinant vector for over-expressing the drought resistant gene CrWRKY57 as claimed in claim 1, wherein the basic vector of the recombinant vector comprises a pBI121 vector, and the drought resistant gene CrWRKY57 is inserted between XbaI and SmaI sites of the basic vector.

9. A method for improving drought resistance of a plant, comprising the steps of: the drought resistant gene CrWRKY57 of claim 1 is expressed or over-expressed in a plant genome.

10. The method of claim 9, wherein the overexpression comprises transforming the genome of the plant with the recombinant vector of claim 8 by genetic transformation to obtain a plant with increased drought resistance.