CN114591969B

CN114591969B - Drought-resistant gene CrWRKY57 and application thereof in drought-resistant improvement of plants

Info

Publication number: CN114591969B
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: 2023-08-15
Anticipated expiration: 2042-03-22
Also published as: CN114591969A

Abstract

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

Background

Drought not only severely limits the cultivation range of citrus, but also is a major abiotic factor limiting the development of the citrus industry. Therefore, the cultivation of new varieties of citrus with drought resistance is extremely important for the sustainable, stable and healthy development of the citrus industry. The rapid development of biotechnology provides a new way for plant breeding, can carry out directional genetic improvement on crops through genetic engineering, and has shown important utilization value in cultivating new varieties (materials) of crop stress resistance. The development and identification of stress-resistant genes are the precondition and key for creating stress-resistant gene plants by utilizing genetic engineering, but the screening amount of drought-resistant genes is small at present, and the requirements of scientific research and production cannot be met.

Disclosure of Invention

Therefore, the invention aims to provide a drought-resistant gene CrWRKY57 and application thereof in drought resistance improvement of plants, provide new gene resources for plant stress-resistant molecular design breeding, and provide new genetic resources for green agriculture and water-saving agriculture, and development and utilization of the genetic resources 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, and the nucleotide sequence of the drought-resistant gene CrWRKY57 is shown as SEQ ID NO. 1.

The invention also provides the protein coded by the drought-resistant gene CrWRKY57.

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: performing PCR amplification by using cDNA of the three-lake red orange as a template and using the primer pair to obtain the drought-resistant gene CrWRKY57;

the PCR amplification procedure includes: pre-denaturation at 94℃for 5min; denaturation at 98℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 1min,35 cycles; extending at 72℃for 10min.

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

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

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

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

The beneficial effects are that: the invention provides a drought-resistant gene CrWRKY57, wherein the drought-resistant gene CrWRKY57 is separated from a drought-resistant variety of three-lake red tangerine and cloned, a novel gene CrWRKY57 belonging to a WRKY family is shown in a nucleotide sequence SEQ ID NO.1, the amino acid sequence of the coded protein is shown in SEQ ID NO.2, the coded protein comprises 876bp Open Reading Frame (ORF), 292 coded amino acids, an isoelectric point is 6.19, and the predicted molecular weight is 32.05kDa. 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 using an agrobacterium-mediated genetic transformation method, and is interfered to be expressed in the citrus reticulata, and the obtained transgenic plant is verified by biological functions, so that the drought resistance of the overexpressed plant is obviously improved, and the drought resistance of the interfered expressed plant is obviously reduced, which indicates that the cloned CrWRKY57 gene has the function of improving the drought resistance. The drought resistance gene CrWRKY57 can effectively improve drought resistance of plants, effectively reduce consumption of water in production and reduce production cost; meanwhile, the method can be used for improving the stock material of fruit trees, has no potential safety hazard of transgenic foods, and is easy to accept and accept by the public. The gene is led 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 results of the expression level of the CrWRKY57 gene of the present invention in transcriptome sequencing (RNA-seq) and qRT-PCR analysis; the bar graph is RNA-seq data, expression form 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 representation of the identification of CrWRKY57 transcriptional activation of the present invention;

FIG. 4A is a schematic diagram of the segmentation of CrWRKY57; FIG. 4B is a schematic representation of transcriptional activation assays of CrWRKY57 and various fragments;

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

FIG. 5A is a schematic diagram of the construction of a super-expression vector; FIG. 5B is a schematic diagram of interferometric carrier construction;

FIG. 6 is a schematic diagram of PCR identification of the CrWRKY57 gene overexpression vector and empty transformed tobacco and regenerated plants of the invention; wherein A is a co-cultured leaf, and B is a screening cultured leaf; c is elongation and propagation of the resistant buds on an elongation culture medium, and D is PCR identification of transgenic tobacco;

FIG. 7 is a graph of physiological index measurements of CrWRKY57 overexpressing tobacco (OE 4, OE10, OE 17) of the invention, and of phenotype before and after drought treatment and after dehydration of in vitro leaves of Wild (WT) and empty transgenic tobacco (EV) potting seedlings; wherein A is a phenotype graph of five lines before drought, 14 days after drought and 1 day after rehydration, B is the relative water loss rate after the dehydration of the isolated leaves, C is the malondialdehyde content after the dehydration of the isolated leaves for 80 minutes, and D is the conductivity after the dehydration of the isolated leaves for 80 minutes;

FIG. 8 is a graph of peroxide accumulation in ex vivo dehydrated lamina of CrWRKY57 overexpressing tobacco (OE 4, OE10, OE 17) of the invention with wild-type (WT) and empty transgenic tobacco (EV); wherein A is NBT (the darker the color, O ₂ · ^– The more content) and DAB (the darker the color, H ₂ O ₂ The more the content) of the staining pattern, B is O-resistant ₂ Ability to-the larger the value, the more O is specified ₂ · ^– The lower the content), C is H ₂ O ₂ The content is as follows;

FIG. 9 is a schematic diagram of PCR identification of lemon transformed with the CrWRKY57 gene overexpression vector and regenerated plants of the invention; wherein A is a co-cultured stem segment, and B is a screening cultured stem segment; c, extending and propagating the resistant buds on an extension culture medium, D, carrying out micro-bud grafting on the resistant lemon buds to a stock, and E, carrying out soil culture on the plant; f is the CrWRKY57 gene expression quantity;

FIG. 10 is a schematic diagram of PCR identification of transformed three-lake red orange and regenerated plants by the RNAi vector of the CrWRKY57 gene of the invention; wherein A is a co-cultured stem segment, and B is a screening cultured stem segment; c, elongating and expanding the resistant buds on an elongation culture medium, D, rooting and culturing the resistant buds, and E, detecting RNAi interference conditions by PCR;

FIG. 11 is a graphical representation of the comparison of drought resistance of CrWRKY57 overexpressed lemons (OE-1 and OE-2) of the invention with wild-type (CKL); wherein A is the relative water loss rate after the in-vitro blade is dehydrated, B is the malondialdehyde content after the in-vitro blade is dehydrated for 80 minutes, and C is the conductivity after the in-vitro blade is dehydrated for 80 minutes;

FIG. 12 is a graph showing comparison of drought resistance of CrWRKY57RNAi three-lake red oranges (RNAi-2 and RNAi-19) of the present invention with wild type (CKs); wherein A is the relative water loss rate after the in-vitro blade is dehydrated, B is the malondialdehyde content after the in-vitro blade is dehydrated for 80 minutes, and C is the conductivity after the in-vitro blade is dehydrated for 80 minutes;

FIG. 13 is a Wen diagram and a clustered heat map of a differentially expressed gene, wherein A is an up-regulated differentially expressed gene, B is a down-regulated differentially expressed gene, and C is all differential gene expression patterns;

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

Detailed Description

The invention provides a drought-resistant gene CrWRKY57, and 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 and comprises 876bp, and the drought-resistant gene CrWRKY57 is positioned in a cell nucleus. The drought-resistant gene CrWRKY57 has no transcriptional activation activity, and needs to interact with other transcription factors or promoter elements to form a complex and then regulate and control a target gene, so that the CrWRKY57 can function possibly in the form of the complex.

The drought-resistant gene CrWRKY57 is preferably separated and cloned from a three-lake red orange (Citrus reticulata), and compared with leaf cytology characteristics and drought resistance of the three-lake red orange, the trifoliate orange (Poncirus trifoliata), the trifoliate orange (C.sisensis multiplied by P.trifoliate 'Carrizo') and the chongzhi wild orange (C.reificate), the drought resistance of the three-lake red orange is found to be strongest, so that RNA-seq is sampled after drought treatment of the three-lake red orange, and a drought stress response transcription factor CrWRKY57 is screened from the three-lake red orange.

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

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

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'.

In the invention, when the drought-resistant gene CrWRKY57 is amplified for the first time, partial ESTs screened based on RNA-seq data are optimized, the Primer pair is designed by using the sequence (Cs 7g 03080) of the sweet orange CrWRKY57 gene by using Primer Premier 5.0, and a PCR amplification system is prepared by using the Primer Premier 5.0 and a cDNA template of three-lake red orange, wherein the system is calculated by 50 mu L, and preferably comprises the following steps: taKaRa LA Taq (5U/. Mu.L) 0.5. Mu.L, 10 XLA Taq Buffer II (Mg) ²⁺ Plus) 5. Mu.L, dNTP mix (2.5 mM each) 8. Mu.L, 1. Mu.L each of forward primer and reverse primer (10. Mu.M), 1. Mu.L of template cDNA, and 33.5. Mu.L of sterilized water.

The cDNA template is preferably obtained by reverse transcription of RNA extracted from leaves, and in the embodiment, total RNA in three-lake red orange leaves is extracted by utilizing an RNAiso Plus kit (the kit is purchased from TAKARA company, and the operation method is according to the specification); the extracted three-lake red orange RNA is used for synthesizing the first strand of cDNA by referring to an operation manual of a TOYOBO reverse transcription kit.

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 the over-expression and RNAi technology, and the drought resistance of the transgenic plants after over-expression 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 over-expressing the drought-resistant gene CrWRKY57. The plant of the present invention preferably comprises citrus tangerines, tobacco and/or lemon.

In the construction of the recombinant vector, preferably, xbaI and SmaI are selected as endonucleases according to analysis of a polyclonal site of the pBI121 vector and an ORF cleavage site of the CrWRKY57 gene, and meanwhile, an over-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 are respectively as follows:

forward primer: 5'-GCTCTAGAATGGATGATAGTAGCAAAGAG-3';

reverse primer: 5'-TCCCCCGGGTCATCTATTGCGCATCCCA-3'.

The forward primer and the reverse primer constructed as above are used for amplifying the target fragment containing the cleavage site, and the amplification system is calculated by 50 mu L and preferably comprises: 5X TransStart FastPfu Buffer 10.0.0. Mu.L, dNTP Mix (2.5 mM each) 5.0. Mu. L, cDNA template 1.0. Mu.L, 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 and ddH ₂ O29.0. Mu.L. The prepared amplification system is subjected to PCR amplification, and the PCR amplification process preferably comprises pre-denaturation at 95 ℃ for 1min; denaturation at 95℃for 20s, annealing at 60℃for 20s, elongation at 72℃for 30s,40 cycles; extending at 72℃for 5min. The invention preferably inserts the target fragment into the pBI121 vector based on a double enzyme digestion method, thereby obtaining the pBI121-CrWRKY57 recombinant vector.

The overexpression preferably comprises a method of utilizing genetic transformation, and the recombinant vector is transferred into a genome of a plant 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 accomplished in an agrobacterium tumefaciens-mediated manner in the examples, but is not to be construed as merely limiting the scope of the present invention.

The drought-resistant gene CrWRKY57 and the application thereof in drought resistance improvement of plants are described in detail below with reference to examples, but are not to be construed as limiting the scope of the invention.

Example 1

CrWRKY57 gene separation clone

1. Total RNA from the leaves of three-lake red orange was first extracted using RNAiso Plus kit (kit purchased from TAKARA company, methods of operation according to the instructions). The extracted three-lake red orange RNA is used for synthesizing the first strand of cDNA by referring to an operation manual of a TOYOBO reverse transcription kit.

2. The primers shown in SEQ ID No.3 and SEQ ID No.4 were designed with the sequence of the sweet orange CrWRKY57 gene according to the partial ESTs screened by the RNA-SEQ data using Primer Premier 5.0.

Acquisition of transcriptome sequencing data: the three-lake red orange (Citrus reticulata) is an ancient local special variety of new and dried river and western, and has good anti-biotic stress and abiotic stress characteristics; meanwhile, the drought resistance of the three-lake red orange is superior to that of citrus stocks such as orange, hovenia dulcis, chongyi wild orange and the like, and the three-lake red orange is an excellent drought-resistant germplasm resource.

The RNA-seq is used for analyzing the leaf transcript differences of three-lake red orange drought treatment for 1 day (DroughtAfter Watering, DAW 1), 4 days (DAW 4) and 7 days (DAW 7), performing GO function enrichment, KEGG enrichment, transcription factor mining and other analysis, and performing qRT-PCR verification on 14 DEGs (containing CrWRKY 57).

In DAW4 vs DAW1, the differential gene number was 7519, with a significant up-regulation of gene 3652 and a significant down-regulation of gene 3865; the DAW7 vs DAW1 differential gene number is 6563, the significantly up-regulated gene is 3216, and the significantly down-regulated differential gene is 3348; the DAW7 vs DAW4 differential gene was 7407, 3692 genes significantly up-regulated, 3715 genes significantly down-regulated (table 1). The screened differential genes of the three comparison combinations are subjected to wien diagram analysis, and 2179 differential expression genes which are obvious in the three comparison combinations are found; while the number of genes significantly upregulated in each of the three comparison combinations was 233 and the number of genes significantly downregulated in each of the three comparison combinations was 210. The heat map results showed that DAW4 up-regulated expressed genes were more numerous than DAW1 and DAW7, and that expression of these early response genes was likely to be drought stress induced (fig. 13).

TABLE 1 drought stress differential expression Gene statistics for three-lake red orange

The expression level of 14 DEGs (containing CrWRKY 57) was verified by real-time fluorescent quantitative PCR (qRT-PCR). Real-time quantitative primers were designed with Primer 5.0 (Table 2), and citrus Actin was selected as the reference gene. The correlation coefficient between qRT-PCR verification results and RNA-Seq results reaches 0.72 (FIG. 14), and the reliability of drought stress transcriptome data is proved. qRT-PCR results showed that CrWRKY57 was significantly drought induced.

TABLE 2 qRT-PCR primers for 14 DEGs (CrWRKY 57-containing)

3. The cDNA of the three-lake red orange is used as a template, and the CrWRKY57 of the three-lake red orange is amplified by PCR.

The PCR amplification system comprises: taKaRa LA Taq (5U/. Mu.L) 0.5. Mu.L, 10 XLA Taq Buffer II (Mg2+Plus) 5. Mu.L, dNTP mix (2.5 mM each) 8. Mu.L, forward and reverse primers (10. Mu.M) 1. Mu.L each, template cDNA 1. Mu.L, and sterilized water 33.5. Mu.L.

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

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

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

The connection system is as follows: pMD18-T Vector 0.5. Mu.L, solution I5. Mu.L, and PCR purified product 4.5. Mu.L.

The mixture was then subjected to overnight ligation at 16℃and transformed into E.coli competent DH 5. Alpha. By heat shock (Beijing full gold Biotechnology Co., ltd.) by PCR verification (consistent with the PCR procedure used for the amplification of the above-described genes) using the primers for the target gene sequences and sequencing (completed by Wuhan qingke New Biotechnology Co., ltd.).

This amplified a 904bp fragment. ORF Finder at NCBI predicts the open reading frame and found to contain a 876bp coding region sequence encoding a 292 amino acid protein having a molecular weight of 32.05kDa and a theoretical isoelectric point of 6.19. The gene coding protein and the published WRKY family in Arabidopsis are constructed into a evolutionary tree, and the evolutionary tree and the AtWRKY57 are found to be classified into the II-c subfamily. Comparing it with WRKY57 core amino acid sequence in Arabidopsis thaliana, rice, maize, etc. with DNAMAN 8.0, it was found that they all contained a WRKYGQK heptapeptide structure and a C ₂ H ₂ Zinc finger structure of (a). Therefore, the gene is designated as CrWRKY57, the cDNA sequence is shown as SEQ ID NO.1, and the encoding protein sequence is shown as SEQ ID NO. 2.

Example 2

CrWRKY57 gene expression analysis

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

Designing a real-time quantitative Primer of CrWRKY57 by using Primer 5.0, and selecting citrus Actin as an internal reference gene:

forward primer (SEQ ID No. 7): 5'-AACCTCCTGAGATACCAAGC-3';

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

reference gene:

forward primer (SEQ ID No. 9): 5'-CATCCCTCAGCACCTTCC-3';

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

The quantitative PCR system was 10. Mu.L, including iTaq TM UniversalGreen Supermix (Roche) 5.0. Mu.L, real-time quantitative forward and reverse primers (10 mol/L) 0.2. Mu.L each, cDNA template 0.5. Mu.ddH in example 1 ₂ O 4.1μL。

Instrument use480II (Roche). The reaction procedure was set at 95℃for 30s of pre-denaturation; denaturation at 95℃for 5 s; annealing at 56 ℃ for 10 s; and the temperature is 72 ℃,15s and 40 cycles are extended. Quantitative PCR data was analyzed by the instrument with its own software.

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

Example 3

Subcellular localization of the CrWRKY57 gene

The results of the online ExPASy analysis indicate that CrWRKY57 has a nuclear localization signal, and the subcellular localization of CrWRKY57 is studied by using Arabidopsis leaf protoplasts and a plant subcellular localization vector pL101 YFP.

Subcellular localization primers were designed, the ORF sequence of the CrWRKY57 gene was amplified, and inserted between the KpnI and SmaI cleavage sites on pL101 YFP.

Subcellular localization primers:

forward primer (SEQ ID No. 11): 5'-GGGGTACCATGGATGATAGTAGCAAAGAG-3';

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

The amplification system is as follows: 1-5TM2 XHigh-Fidelity Master Mix (Optimum Biotechnology Co., ltd.) 25. Mu.L, 2. Mu.L each of forward primer and reverse primer, 2. Mu. L, ddH of the three-lake red orange cDNA of example 1 ₂ O 19μL。

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

After completion of PCR amplification, all PCR products were visualized with 1.2% agarose gelThe sample was subjected to electrophoresis, and the Axygen gel recovery kit (Corning Life sciences Co., ltd.) was used to recover the specific target band, and the operations were performed according to instructions. Connecting the recovered productCarrier (full gold, beijing) with a connection system of +.>Blunt Cloning Vector 1 mu L, PCR purified product 4. Mu.L.

After 10min of connection at room temperature, the connection solution was introduced into E.coli competent DH 5. Alpha (Beijing full gold biotechnology Co., ltd.) by thermal excitation, and PCR was performed by selecting positive clones with the target gene sequence primers (consistent with the PCR procedure when constructing the vector described above) and sequencing was completed by Wuhan qingke new industry biotechnology Co., ltd.

The pEASY-CrWRKY57 recombinant vector plasmid and pL101YFP vector plasmid were cut simultaneously with endonucleases KpnI and SmaI, and the double digestion system was 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, qualified strips are detected and cut off by agarose gel electrophoresis, and the target strips are recovered by a gel recovery kit. The T4 DNALigase is used for connecting pEASY-CrWRKY57 recombinant vector plasmid and pL101YFP vector plasmid after double enzyme digestion of KpnI and SmaI to obtain 35S-CrWRKY57-pL101YFP recombinant vector, and agrobacterium competent cells GV3101 (open to Biotechnology Co., shanghai) are transformed, and the transformation steps are shown in the specification.

The positioning condition of CrWRKY57 is detected by adopting an arabidopsis leaf protoplast preparation transformation method. Reagents for extraction of arabidopsis protoplast: arabidopsis leaf protoplast enzymolysis liquid (for preparation and use): 20mM MES, 1.5% cellulase, 0.4% macerating enzyme, 0.4M mannitol, 20mM KCl, and 55℃for 10min, cooling to room temperature, adding 10mM CaCl ₂ 5mM mercaptoethanol, 0.1% BSA, and filtered through a 0.45. Mu.M filter. WI solution: 4mM MES, 0.5M mannitol, 20mM KCl, stored at room temperature. W5 solution: 2mM MES, 154mM NaCl, 125mM CaCl ₂ 5mM KCl. PEG4000 solution: 1g PEG4000, 0.75mL double distilled water, 0.625mL mannitol, 0.25mL CaCl ₂ . MMG solution: 4mM MES,0.4mM mannitol, 15mM MgCl ₂ 。

By transforming the plasmids of 35S-CrWRKY57-pL101YFP and the control empty pL101YFP into protoplasts of Arabidopsis thaliana, the results are shown in FIG. 3, in which the fluorescence of the empty pL101YFP plasmid is distributed throughout the protoplasts, including the cell membrane and the cell nucleus, whereas the fluorescence of the 35S-CrWRKY57-pL101YFP fusion protein is concentrated only in the cell nucleus, indicating that CrWRKY57 is localized in the cell nucleus.

Example 4

CrWRKY57 transcriptional activation Activity assay

Recombinant construction is carried out by adopting pGBKT7 vector, and whether CrWRKY57 has transcriptional activation activity is verified. The WRKY domain of the CrWRKY57 protein is predicted to be distributed in 142th-201th aa on SMART, presumably as the transcriptional activation region. Thus, in addition to full length, crWRKY57 is also divided into three segments bounded by WRKY domains: crWRKY57-1 (1 th-141th aa), crWRKY57-2 (142 th-201th aa), crWRKY57-3 (202 th-291th aa) were ligated to pGBKT7 vector, respectively. After confirming the sequence by sequencing, 4 fusion expression vectors and empty vector (pGBKT 7) were transformed into yeast strain AH109, respectively, and then cultured on different deletion media. The results showed that both the empty vector transformed yeast cells and the 4 fusion vector transformed cells were able to grow only on deletion media SD/-Trp, but not on deletion media SD/-Trp/-ade and SD/-Trp/-ade/-His (FIG. 4), indicating that CrWRKY57 has no transcriptional activation activity and may function as a complex.

Example 5

CrWRKY57 gene overexpression and RNAi vector construction

1. Super expression vector construction

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

Designing the super-expression vector to construct primers SEQ ID NO.5 and SEQ ID NO.6.

Amplification of the mesh using TransStart FastPfu DNA Polymerase (Total gold)Is a fragment of (a). The PCR system is as follows: 5. x TransStart FastPfu Buffer 10.0.0. Mu.L, dNTP Mix (2.5 mM 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 1.0.1.0. Mu. L, ddH ₂ O 29.0μL。

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

The PCR product is subjected to double enzyme digestion after gel cutting recovery, and the system is 100 mu L: 10. Mu.L of 10 XH buffer; xbaI and SmaI (TaKaRa), 5. Mu.L each; recovering 25. Mu.L of the product; ddH ₂ O, 55. Mu.L; and (5) carrying out enzyme digestion at 37 ℃ overnight and then recycling.

The pBI121 vector plasmid was also recovered after cleavage with the same system.

The recovered target fragment and the vector were ligated, and the total ligation volume was 10. Mu.L: 10×T4 ligation buffer, 1 μl; t4 ligase, 1. Mu.L; pBI121 vector, 0.5. Mu.L; 4.5. Mu.L of the fragment of interest; the connection was made at 16℃for 16 hours.

Then the connection product is transformed into escherichia coli DH5 alpha, screening is carried out in an LB solid plate containing 50mg/L kanamycin, monoclonal PCR detection is selected to be positive, and then the obtained product is sent to the Bhan qing laboratory new industry biotechnology Co-Ltd for sequencing, and the sequencing determines that the reading frame is completely correct, namely the pBI121-CrWRKY57 recombinant vector is successfully constructed. Recombinant vectors were introduced into Agrobacterium tumefaciens GV3103 by freeze thawing (see fourth edition, scientific Press, 2017) and the bacterial suspensions were stored at-80℃with 20% glycerol.

RNAi vector construction

In order to obtain interference transgenic plants, pHellgate 2 interference vectors are adopted for recombinant construction. Firstly, selecting a fragment of about 300bp on CrWRKY57, designing a primer, and adding an attB site universal primer in front of a specific primer to serve as a target fragment amplification primer.

Selecting a fragment with CrWRKY57 less than 300bp as a template to design a primer for constructing an RNAi vector:

forward primer (SEQ ID No. 17): 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGAGCGAGTTTGGATGGAA-3';

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

PCR amplification system: 1-5TM2 XHigh-Fidelity Master Mix (Optimum Biotechnology Co., ltd.) 25. Mu.L, 2. Mu.L each of forward primer and reverse primer, 2. Mu. L, ddH of the three-lake red orange cDNA of example 1 ₂ O 19μL。

After the PCR amplification was completed, all the PCR products were subjected to electrophoresis using 1.2% agarose gel, and specific target bands were recovered using Axygen gel recovery kit (Corning Life sciences Co., ltd.) according to instructions.

Ligation of the purified PCR product to RNAi vector pHELLSGATE2 by BP reaction, seeBP/>II Enzyme mix instructions, recovered product 4 u L, pHellsgate plasmid 1 u L, B/P close 1 u L,25 ℃ reaction overnight.

The target fragment was amplified using the three-lake red orange cDNA of example 1 as a template, and the PCR product was recovered and recombined and ligated to pHellgate 2 vector by BP reaction (BP ClonaseTM II Enzyme Mix (Invitrogen) kit) to construct the target vector (FIG. 5B).

All reactants were used to transform E.coli DH 5. Alpha. Competence by heat shock and plated on LB solid plates containing 100mg/L spectinomycin to screen positive clones.

And (3) 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 to carry out PCR detection analysis on positive clones, further carrying out single digestion verification on the positive clones with correct PCR detection by using XbaI and XhoI respectively, and if the two groups of single digestion fragments are consistent in size and larger than the target fragment by about 200bp, correctly connecting the target fragment in the positive clones. The positive monoclonal of correct connection is sent to the Wohangaceae new industry biotechnology Co.Ltd for sequencing, the recombinant plasmid is extracted from the monoclonal of correct sequence, the recombinant vector is led into the Agrobacterium tumefaciens GV3103 by using a freeze thawing method, and the bacterial liquid is preserved at-80 ℃.

Example 6

Application of transcription factor CrWRKY57 in improving drought resistance of tobacco

1. The agrobacterium tumefaciens-mediated genetic transformation of tobacco is as follows:

(1) Culturing agrobacterium tumefaciens: streaking fresh Agrobacterium tumefaciens bacterial liquid on LB solid plate (containing 50mg/L kanamycin), scraping streak bacterial plaque, adding into liquid MS minimal medium, shake culturing at 28deg.C for 200 r/min until bacterial liquid concentration reaches OD ₆₀₀ Dip dyeing is carried out when the color is=0.3 to 0.8;

(2) Dip dyeing: taking healthy tobacco leaves of wild in-vitro seedlings, removing main pulse, cutting into square blocks with the size of 0.5cm multiplied by 0.5cm, immersing in prepared agrobacterium tumefaciens bacterial liquid, immersing for 8-10 minutes, and intermittently oscillating;

(3) Co-cultivation: taking the tobacco leaves after dip dyeing, airing on sterile filter paper, uniformly arranging the leaf backs on a co-culture medium (A in figure 6) downwards, and carrying out dark culture at 25 ℃ for 3 days;

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

(5) Rooting: when the adventitious bud length on the medium to be screened reaches about 1cm, it is cut out and transferred into rooting medium (C in FIG. 6).

TABLE 3 tobacco transformation Medium formulation

Name of the name	Components and contents (30 g/L sucrose; 0.7g/L agar; pH 5.8)
		Co-culture medium	MS minimal medium +2.0 mg/L6-BA +0.3mg/L NAA
Screening media	Co-culture medium +50mg/L hygromycin +500mg/L cephalosporin
		Rooting culture medium	MS minimal medium+0.3 mg/L NAA+50mg/L hygromycin+500 mg/L cephalosporin

2. Positive transgenic tobacco identification

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

The obtained DNA is used as a template, a CrWRKY57 overexpression vector is used for constructing a primer to identify the overexpression positive plants, and a hygromycin gene primer (forward primer SEQ ID NO.13:5'-CTCCATACAAGCCAACCACG-3'; reverse primer SEQ ID NO.14: 5'-AAAAAGCCTGAACTCACCGC-3') is used for identifying the no-load positive plants.

Ubiqutin was amplified as an internal control, forward primer (SEQ ID NO. 15): 5'-AGCTACATGACGCCATTTCC-3', reverse primer (SEQ ID NO. 16): 5'-CCCTGTAAAGCAGCACCTTC-3'.

The fragment of interest was amplified using Fermentas company Taq enzyme. The PCR system is as follows: 10 XPCR Buffer 2.0. Mu.L, dNTP Mix (10 mM 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 each ₂ O15.8. Mu.L. The PCR procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 56℃for 30s, extension at 72℃for 3min,35 cycles; extending at 72℃for 12min.

The results of the partial PCR-validated electrophoresis are shown as D in FIG. 6. Three of the overexpressing strains OE4, OE10 and OE17 were selected as separate transgenic strains and then as female parent plants for seed harvest, respectively.

3. Identification of drought resistance of over-expressed tobacco

Three overexpressing strains OE4, OE10, OE17 and Empty (EV) and wild-type (WT) tobacco seeds of the same batch were harvested for sterile sowing: soaking seeds in 70% ethanol for 1min, washing with sterile double distilled water for three times, sterilizing with 1mL 2.5% NaClO for 8min, oscillating for several times, and sterilizing with sterile ddH ₂ O is washed for 3 times, and finally, seeds are spread and sown on a hygromycin MS basic culture medium containing 50mg/L by a sterile inoculating needle, and the seeds are used for measuring the subsequent drought resistance phenotype and related indexes after sprouting and seedling growth.

(1) Phenotype: the tobacco OE4, OE10 and OE17 which are over-expressed by CrWRKY57 and wild control WT and idle control EV are subjected to potting water control treatment, and when drought treatment is not performed, the growth conditions of the over-expressed CrWRKY57 and control plants are consistent as shown in a figure 7A; after 14 days of drought, WT and EV seriously wilt, most of leaves lose water and dry, and the over-expression plants OE4, OE10 and OE17 have relatively light wilt degree; after rehydration, the control plants had not improved in growth, while the overexpressing plant leaves began to turn green. The result shows that the tobacco with the over-expressed CrWRKY57 has stronger drought resistance and higher recovery speed after drought.

(2) Relative water loss rate: the tobacco leaves of 5 strains are subjected to in-vitro leaf dehydration treatment, and when the tobacco leaves are taken off from the plants, the tobacco leaves are weighed by a ten-thousandth balance, and the reading number is recorded as M ₀ Then the back of the leaf is upwards placed on the dry filter paper, the leaf is weighed once every 30min, and the reading number is recorded as M _n The experiment was set up with 3 replicates, averaged and error calculated, calculation formula:

as shown in FIG. 7B, by the time of 80min dehydration, the relative water loss rates of the over-expressed tobacco OE4, OE10 and OE17 are respectively 27.64%, 28.68% and 26.15%, the relative water loss rates of the WT and EV are respectively 33.72% and 34.24%, and the relative water loss rate of the over-expressed tobacco is significantly lower than that of the control (p < 0.01). The relative water loss rates of the over-expressed tobacco OE4, OE10 and OE17 are smaller than that of the WT and EV, which indicates that the blade water retention capacity is stronger after the over-expression of CrWRKY57.

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

CMDA(μmol/L)＝6.45(A ₅₃₂ -A ₆₀₀ )-0.56A ₄₅₀

as a result, as shown in FIG. 7C, 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, respectively; namely, the MDA content of OE4, OE10 and OE17 is obviously lower than that of a control (p < 0.05), which indicates that the cell membrane of the tobacco leaf blade with the over-expression of CrWRKY57 is damaged to a smaller extent under the condition of in-vitro dehydration.

(4) Conductivity: taking transgenic plant leaves and control plant leaves after 80min of in-vitro dehydration, removing leaf midvein and leaf edge, cutting into small pieces of about 0.5X0.5 cm by scissors, weighing 0.1g, placing into a glass test tube with 10mL deionized water, shaking with shaking table 120r/min at 25deg.C for 3 hr, measuring the conductivity with a conductivity meter (DDS-307), and recording as C ₁ Boiling the test tube in boiling water for 15min, cooling to room temperature, and measuring conductivity to obtain C ₂ Calculation formula

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

(5) Super oxide accumulation conditions:

firstly, carrying out quantitative detection on leaves of transgenic plants and control plants after 80min of in-vitro dehydration.

NBT staining method: the final concentration of the powder of azulene (NBT) was 1mg/mL in 0.01M phosphate buffer, pH=7.8. Subpackaging the prepared NBT staining solution into 50mL centrifuge tubes, completely immersing tobacco leaves, staining for 3h under light, discarding the staining solution, adding absolute ethyl alcohol for decolorizing treatment, changing the greening ethanol until the leaves are completely removed, photographing with a camera, recording, and soaking the leaves in the absolute ethyl alcohol for preservation.

DAB method staining: 3, 3-Diaminobenzidine (DAB) powder was dissolved in 0.01m phosphate buffer, ph=3.8 as solvent, according to V (staining solution) before use: v (H) ₂ O ₂ 30%) =1: 1000 add H ₂ O ₂ Mixing well. And subpackaging the prepared DAB staining solution into 50mL centrifuge tubes, immersing tobacco leaves completely, staining for 8h under light, discarding the staining solution, adding absolute ethyl alcohol, decolorizing and preserving by the same method, and finally photographing and recording by a camera.

As a result, as shown in FIG. 8A, tobacco overexpressing CrWRKY57 under dehydration conditions was lighter in color, indicating less accumulation of superoxide.

H ₂ O ₂ Quantitative determination of content and anti-superoxide anion radical activity using hydrogen peroxide assay kit (A064-1-1) and superoxide anion radical inhibition and generation assay kit (A052-1-1) of Nanjing build company, respectively, the operation process is as described in the specification. anti-O ₂ · ^– The greater the capacity as shown in B in FIG. 8, the greater the value, illustrating O ₂ · ^– The smaller the content is, H ₂ O ₂ The content is shown in fig. 8C, and the results indicate that the accumulation of superoxide in tobacco overexpressing CrWRKY57 under bulk dehydration conditions is less.

Qualitative and quantitative results show that the over-expression CrWRKY57 can effectively enhance the active oxygen scavenging capacity of the transgenic plant, reduce the damage to cells, and further improve the drought resistance of the plant.

Example 7

Application of transcription factor CrWRKY57 in improving drought resistance of lemon and three-lake red orange

In order to provide more abundant and powerful experimental evidence for the effect of CrWRKY57 under drought resistance, the over-expression and RNAi vectors in the embodiment 5 are used for respectively transforming lemon and three-lake red orange by an agrobacterium-mediated citrus epicotyl transformation method, so as to see whether the over-expression lemon drought resistance is enhanced and the RNAi three-lake red orange drought resistance is weakened.

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

(1) Sowing: soaking lemon and three-lake tangerine seeds in 1M NaOH for 20min, washing with clear water, soaking in 2% NaClO on an ultra-clean workbench for 15-20 min, washing with sterile water for 3 times, peeling off seed coats under sterile conditions, seeding on MT solid culture medium, culturing in dark for 3-4 weeks, and culturing in light for 1 week for transformation.

(2) Preparing bacterial liquid: the recombinant vector-containing Agrobacterium solution overexpressed and RNAi in embodiment 5 was streaked onto solid LB medium containing 100mg/L antibiotic (kanamycin for pBI121 vector, spectinomycin for pHellsgate2 vector), and dark cultured at 28℃for 2d; picking single colony, scribing on a new plate again, and culturing in dark at 28 ℃ for 2-3d; scraping off the grown thallus with a scalpel, inoculating into liquid MT culture medium without antibiotics, and shake culturing at 28deg.C and 200rpm to OD ₆₀₀ =0.6 to 0.8, acetosyringone was added to a final concentration of 100 μm for use.

(3) Co-cultivation: taking the epicotyl of aseptic seedlings of lemon and three-lake red tangerine, obliquely cutting the epicotyl into 1-1.5 cm long stem segments in an ultra-clean workbench, soaking the stem segments in prepared agrobacterium tumefaciens bacterial liquid (the bacterial liquid containing the overexpression vector is used for converting lemon, and the bacterial liquid containing the RANi vector is used for converting three-lake red tangerine), and infecting the stem segments for 20min, and continuously shaking the stem segments for several times. After infection, the excess bacterial liquid is absorbed by sterile absorbent paper, the explant is spread on a co-culture medium, and the explant is cultivated in dark at 25 ℃ for 3d (lemon co-cultivation is shown as A in figure 9 and three-lake red orange co-cultivation is shown as A in figure 10).

(4) Screening and rooting: after co-cultivation for 3d, the explants were washed 3-5 times with sterile water, and then surface water was blotted with sterile absorbent paper and transferred to screening medium (lemon screen cultivation see B in FIG. 9, three-lake red orange screen cultivation see B in FIG. 10). Culturing at 25deg.C in dark for 4 weeks, and culturing under illumination. When the resistant bud is >0.5cm, the cut resistant bud is transferred to proliferation medium to promote differentiation (lemon see C in FIG. 9, and three-lake red orange see C in FIG. 10). When the resistant buds are >1.5cm long, lemon resistant buds are grafted onto the rootstock, hovenia dulcis (D in fig. 9), and then earth cultivated (E in fig. 9). The three-lake red orange resistant buds were transferred into rooting medium to induce rooting (D in FIG. 10).

The formula of the common culture medium comprises:

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

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

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

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

three-lake red orange 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 red orange proliferation culture medium: MT+0.5mg/L BA+0.5mg/L IAA+0.5mg/L GA3;

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

during the transformation process, 7.5g/L of agar and 35g/L of sucrose are added into each culture medium, and the pH is adjusted to 5.8.

(5) Positive plant identification:

DNA extraction is the same as that of tobacco.

And (3) PCR detection: the PCR reaction system and the procedure of lemon and three-lake red orange are the same as those of tobacco, except that the internal reference gene is changed into an action in the embodiment 2. The detection result of lemon is shown as E in figure 9, and the detection result of three-lake red orange is shown as D in figure 10. CKL is a lemon control, CKS is a three-lake red orange control, and is obtained by expanding the untransformed epicotyl (i.e., wild type). Two overexpressing lines of lemon were designated OE-1, OE-2, and three-lake red orange picked RNAi-2 and RNAi-19 for subsequent resistance identification.

2. Drought resistance identification of transgenic citrus material

And (3) taking lemon CKL, OE-1, OE-2, three-lake tangerine CKS, RNAi-2 and RNAi-19 leaves for in vitro dehydration. 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 lemon after 80min dehydration was significantly lower than CKL, while malondialdehyde content and conductivity were significantly higher than CKL. In contrast, the relative water loss rate of the three-lake tangerine RNAi line was significantly higher than CKs, while malondialdehyde content and conductivity were significantly lower than CKs (fig. 12). The expression of the CrWRKY57 is proved to be over-expressed, so that the drought resistance of the plant can be obviously enhanced, and the interference of the expression of the CrWRKY57 can obviously reduce the drought resistance of the plant.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> Gannan university of teachers and students

<120> drought-resistant gene CrWRKY57 and application thereof in plant drought resistance improvement

<160> 47

<170> SIPOSequenceListing 1.0

<210> 1

<211> 876

<212> DNA

<213> three-lake red orange (Citrus reticulata)

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atggatgata gtagcaaaga gaaatcggat cgaggccagt cgagctggaa gctaggggag 60

ccaccggacg cgggctgcgt gagttatatt ttgagcgagt ttggatggaa tctgcaagag 120

catgagagtt cgaccagcta cttcgctgct gatcatgaaa gatccgattt ggcgggaaat 180

atcagcagca gttttccggc cgaaactact actgacggtg gcggtttgac aaatcctgga 240

aggtctgctg acgtgtcgac ttcgaatccg tcggtttcgt cgagctccag cgaggatccg 300

acggagaagt ctacgggctc cggcggtaaa cctcctgaga taccaagcaa agcaagaaag 360

aaaggacaaa agcgaattcg gcagccacgt tttgcattta tgaccaagag tgaagttgat 420

caccttgaag atggatacag atggcgaaag tatggtcaga aggctgtaaa aaatagtccg 480

ttccctagga gctactaccg ctgcacaaac agtaaatgta cagtgaagaa gagggtggaa 540

cgatcatctg aagatcccac cattgtaatt actacgtatg aaggtcaaca ctgccatcat 600

accgttggat ttcctcgtgg tggactaatt aatcatgaag cagctgcttt tgctagccat 660

ttgactcacg caatcccacc atattattat catcaaggag ttcagataac ccaagaaact 720

cccggtatca agcagcagtc acatgaagaa gaattaatcc cagttgaagc aagagaacat 780

gaaccaaatg cgttgcccga accaccagcc ttaccacccc ccacggatga aggattacta 840

ggagatattg tgcctcctgg gatgcgcaat agatga 876

<210> 2

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Met Asp Asp Ser Ser Lys Glu Lys Ser Asp Arg Gly Gln Ser Ser Trp

1 5 10 15

Lys Leu Gly Glu Pro Pro Asp Ala Gly Cys Val Ser Tyr Ile Leu Ser

20 25 30

Glu Phe Gly Trp Asn Leu Gln Glu His Glu Ser Ser Thr Ser Tyr Phe

35 40 45

Ala Ala Asp His Glu Arg Ser Asp Leu Ala Gly Asn Ile Ser Ser Ser

50 55 60

Phe Pro Ala Glu Thr Thr Thr Asp Gly Gly Gly Leu Thr Asn Pro Gly

65 70 75 80

Arg Ser Ala Asp Val Ser Thr Ser Asn Pro Ser Val Ser Ser Ser Ser

85 90 95

Ser Glu Asp Pro Thr Glu Lys Ser Thr Gly Ser Gly Gly Lys Pro Pro

100 105 110

Glu Ile Pro Ser Lys Ala Arg Lys Lys Gly Gln Lys Arg Ile Arg Gln

115 120 125

Pro Arg Phe Ala Phe Met Thr Lys Ser Glu Val Asp His Leu Glu Asp

130 135 140

Gly Tyr Arg Trp Arg Lys Tyr Gly Gln Lys Ala Val Lys Asn Ser Pro

145 150 155 160

Phe Pro Arg Ser Tyr Tyr Arg Cys Thr Asn Ser Lys Cys Thr Val Lys

165 170 175

Lys Arg Val Glu Arg Ser Ser Glu Asp Pro Thr Ile Val Ile Thr Thr

180 185 190

Tyr Glu Gly Gln His Cys His His Thr Val Gly Phe Pro Arg Gly Gly

195 200 205

Leu Ile Asn His Glu Ala Ala Ala Phe Ala Ser His Leu Thr His Ala

210 215 220

Ile Pro Pro Tyr Tyr Tyr His Gln Gly Val Gln Ile Thr Gln Glu Thr

225 230 235 240

Pro Gly Ile Lys Gln Gln Ser His Glu Glu Glu Leu Ile Pro Val Glu

245 250 255

Ala Arg Glu His Glu Pro Asn Ala Leu Pro Glu Pro Pro Ala Leu Pro

260 265 270

Pro Pro Thr Asp Glu Gly Leu Leu Gly Asp Ile Val Pro Pro Gly Met

275 280 285

Arg Asn Arg

290

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

attcattgag ctccacggag 20

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<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

actcatctat tgcgcatccc ag 22

<210> 5

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gctctagaat ggatgatagt agcaaagag 29

<210> 6

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

tcccccgggt catctattgc gcatccca 28

<210> 7

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

aacctcctga gataccaagc 20

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tttacagcct tctgaccata c 21

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

catccctcag caccttcc 18

<210> 10

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ccaaccttag cacttctcc 19

<210> 11

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

ggggtaccat ggatgatagt agcaaagag 29

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

tcccccgggt ctattgcgca tccca 25

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

ctccatacaa gccaaccacg 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

aaaaagcctg aactcaccgc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

agctacatga cgccatttcc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

ccctgtaaag cagcaccttc 20

<210> 17

<211> 47

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ggggacaagt ttgtacaaaa aagcaggctg agcgagtttg gatggaa 47

<210> 18

<211> 47

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

ggggaccact ttgtacaaga aagctgggtc gcttttgtcc tttcttt 47

<210> 19

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

ctatccttcg caagaccct 19

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

gtggctattc tgacttgctc g 21

<210> 21

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

tgtactcgta attcctcatc cc 22

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

ctgaagaaca gcccaagtcg 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

ccactgctcc ttatccactc 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

tcatttgcca tcccagttac 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

gagtcaagct cattccctca 20

<210> 26

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

gagtcatcgg agtaatgtcg g 21

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

tgctgttgtc ctgaggctgt 20

<210> 28

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

ttgggtcctg tatgactcct g 21

<210> 29

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

ttcttccgag caagtttctg t 21

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

ttgctgcttc caagaaatgc 20

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

caactcaaag tgctgtccct 20

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

ttctgctgct agataggacg 20

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

aaacatacaa accggacacc 20

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

cgaaaccgaa gaatggagtg 20

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

cgtcaggaac tggagcgaag 20

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

aacctcctga gataccaagc 20

<210> 37

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

tttacagcct tctgaccata c 21

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

tttggacgtg ggagtatgtg 20

<210> 39

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

aatcttgacc aggataagga ga 22

<210> 40

<211> 20

<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. Drought-resistant geneCrWRKY57Application of drought-resistant gene in improving drought resistance of plantsCrWRKY57The nucleotide sequence of (2) is shown as SEQ ID NO. 1; the plant comprises three-lake red orange, tobacco and/or lemon.

2. A method for improving drought resistance of a plant, comprising the steps of: expression or overexpression of drought-resistant genes in plant genomeCrWRKY57The drought-resistant geneCrWRKY57The nucleotide sequence of (2) is shown as SEQ ID NO. 1; the plant comprises three-lake red orange, tobacco and/or lemon.