CN111424039A - Cymbidium CgWRKY65 gene and application thereof - Google Patents

Abstract

The invention discloses a Chinese cymbidiumCgWRKY65A coding gene and an application thereof,CgWRKY65the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2. The invention relates to a cymbidium goeringii cultivated variety 'Songmei'CgWRKY65Cloning and identification of genes, analysis of gene expression, and verification of their function, discoveryCgWRKY65At the seedling stage, compared with the wild arabidopsis WT,CgWRKY65the transgenic plant is short and small as a whole, has yellow leaves and has the tendency of premature senility, so that the gene can be widely used in the aspect of orchid nutrition and reproduction improvement。

Description

Cymbidium CgWRKY65 gene and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to cymbidium goeringiiCgWRKY65Genes and their use.

Background

The orchid family (Orchidaceae) is one of the largest of the flowering plants, and contains 25000 or more species worldwide, accounting for about 10% of all flowering plants. Goering cymbidium (A. fern)Cymbidium goeringii) Belongs to the species of floret type Geshenlan in orchid family, and has peculiar flower type, light flower color, delicate flower fragrance, beautiful leaf appearance and high ornamental value and economic value. The cymbidium goeringii has high requirements on the growth environment, is very easily influenced by severe environments such as high temperature, low temperature, drought and the like in the growth process, and can cause the reduction of the ornamental quality of gardening and even the death of plants in severe cases. Therefore, the research on the molecular mechanism of plants for coping with abiotic stress and the identification of genes with stress resistance function are of great significance to the breeding and production of cymbidium goeringii. In a plant cell signal transduction pathway, the WRKY transcription factor is considered as a key pivot of plant growth and various stress responses, and provides an important basis for genetic improvement of plants.

WRKY transcription factors are one of the largest families of regulatory proteins in plants, involved in a variety of physiological processes, the most prominent of which are stress responses to biotic and abiotic stresses. The WRKY gene is reported to enhance the tolerance of plants to adversity stress. The sunflower HaWRKY76 transgenic plant shows stronger stress resistance to waterlogging stress, and the yield is also obviously increased.AtWRKY25The overexpression of (a) enhances the salt tolerance of Arabidopsis thaliana.AtWRKY57The over-expression in rice not only improves the drought resistance of rice, but also enhances the tolerance of the rice to salt and PEG. Therefore, it is obtained by cloning from cymbidium goeringii by using genetic engineering technologyCgWRKY65The gene has obvious expression difference under ABA stress, so the gene will be expressedCgWRKY65The gene is transferred into plants, and has great application prospect.

Different plants have different gene sequences even if they are of the same gene family, and even if more than 80% of the gene sequences are identical, the functions of transferring some genes into plants are different because of different insertion sites. When a foreign gene enters a chromosome, a foreign protein encoded by the gene is generated, the foreign protein or enzyme causes the activity of other enzymes or proteins to be enhanced or reduced through a series of reactions, and when the foreign gene is embedded in a certain segment of the chromosome, the whole chromosome is influenced, so that the activity or the regulation mechanism of other genes is influenced.

Even if the same gene is inserted, the same gene is inevitably influenced on the chromosome due to different insertion sites or different chromosome groups, and further influences the gene on the chromosome, and slight changes of the gene can influence the regulation mechanism, so that the changes of gene inactivation, activity reduction, activity enhancement and the like can be caused, and the gene action is different, and the gene action can be obtained only by verification and can not be obtained by inference.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the defects in the prior breeding technology, the invention aims to provide the cymbidium goeringiiCgWRKY65A gene. Another object of the present invention is to provide cymbidium goeringiiCgWRKY65The application of the gene in orchid breeding.

The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a kind of Chinese cymbidiumCgWRKY65The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

The cymbidium goeringiiCgWRKY65The amino acid sequence of the gene expression protein is shown in SEQ ID NO. 2.

The cymbidium goeringiiCgWRKY65The use of genes in plant production and breeding.

Contains the cymbidium goeringiiCgWRKY65A vector for the gene.

Contains the cymbidium goeringiiCgWRKY65A host cell for the gene.

Has the advantages that: compared with the prior art, the invention has the advantages that the cymbidium goeringii is treatedCgWRKY65Cloning and identification of gene, expression analysis of gene, verification of its function, finding over-expressionCgWRKY65Compared with wild Arabidopsis WT, the transgenic Arabidopsis has smaller whole leaf, slightly yellow leaf and premature senility trend, and prolonged vegetative growth period, so that the transgenic Arabidopsis has vegetative growth in orchid and other plantsWill have wide application.

Drawings

FIG. 1 is cymbidium goeringiiCgWRKY65Gene cloning and constructed over-expression vector map;

in FIG. 2, a isCgWRKY65Expression in various tissues of cymbidium goeringii, wherein R represents root, P represents pseudobulb, L represents leaf, F represents flower, and b represents cymbidium goeringii under ABA stressCgWRKY65The expression of the gene;

FIG. 3a is a diagram showing the results of the cleavage, wherein M is D L2000 Marker;CgWRKY65b, performing double enzyme digestion with XbaI and SnaBI after being connected with pBI121, wherein the picture b is a screening picture of a positive recombinant, M is D L2000 Marker, and the size of a target band is 780 bp;

FIG. 4 is a diagram showing the PCR results of transgenic Arabidopsis plants, wherein M is D L2000 Marker, 1 is vector plasmid DNA as positive control, and 2 is wild type DNA as negative control;

FIG. 5 is a view of a rotary tableCgWRKY65The plant types of the gene plant and the wild type arabidopsis thaliana plant are compared, wherein WT and the wild type arabidopsis thaliana are shown in the figure; 65, turn toCgWRKY65Different strains of genes;

FIG. 6 is a view of a rotary tableCgWRKY65The expression quantity of the gene plant and the wild type arabidopsis thaliana under the stress of ABA;

FIG. 7 is a view of a rotary drumCgWRKY65The root length variation graph of the gene plant; WT: common wild type Arabidopsis thaliana; the abscissa represents the gene number; the ordinate represents the root length (cm); concentration units of ABA (. mu.M).

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1

The material used in this example was the "songmei" leaf of cymbidium goeringii, which was snap frozen in liquid nitrogen after harvesting and stored in an ultra-low temperature freezer (-80 ℃).

1) Extraction of total RNA from cymbidium goeringii leaves

The method is carried out according to the instruction of a TaKaRa plant total RNA extraction kit, and comprises the following specific operations:

quickly transferring the frozen cymbidium goeringii leaves at ultralow temperature to a mortar precooled by liquid nitrogen for grindingGrinding the tissue with pestle while adding liquid nitrogen continuously until it is ground into powder, adding the ground sample to 1.5m L sterilized Tube containing 450. mu.l Buffer PE, repeatedly pipetting until there is no significant precipitate in the lysate, centrifuging the lysate at 12,000 rpm and 4 ℃ for 5min, carefully pipetting the supernatant into a new 1.5m L sterilized Tube, adding 1/10 volumes of Buffer NB to the supernatant, Vortex mixing with agitation, centrifuging at 12,000 rpm and 4 ℃ for 5min, carefully pipetting the supernatant into a new 1.5m L0 sterilized Tube, adding 450. mu. L. mu.1 of Buffer R L, mixing the solution uniformly with a pipetting gun, adding 1/2 volumes of absolute ethanol to the mixture, mixing the solution uniformly with a pipetting gun, immediately transferring the total amount of RNA into SpinColinColinumn, 12,000 rpm, centrifuging 1min, discarding the filtrate, disposing the RNA in Buffer 2min, disposing the filtrate back into the Buffer 2, adding RWA 2. mu.20 rpm of Buffer NB to the Buffer NB, centrifuging the RNA in a Buffer 2, adding the RNA into a Buffer RNA in a Buffer 12. mu.5,000 rpm, centrifuging RNA in a Buffer DNA, adding the RNA in a Buffer 2, discarding the RNA in a Buffer 2, centrifuging the RNA in a Buffer DNA, adding the RNA in a Buffer 2, adding RNA in a Buffer 2, centrifuging a Buffer DNA of Buffer 2, adding RNA in a Buffer 2, centrifuging a Buffer to a Buffer of a Buffer, adding RNA in a Buffer of a Buffer 2, adding RNA in a Buffer of a Buffer to a Buffer of a Buffer, adding RNA of a Buffer of a₂O is left to stand at room temperature for 5min and centrifuged at 12,000 rpm for 2min to elute RNA. The obtained RNA is stored in a refrigerator at minus 80 ℃ for later use after concentration and purity detection.

2 mu L RNA is absorbed and detected by 1% agarose gel electrophoresis, the result shows that 28S and 18S bands are clearer, the brightness of the 28S band is about twice of 18S, the RNA quality is better, and the RNA purity and OD are detected by a micro-computerised protein analyzer₂₆₀/OD₂₈₀Is 2.02, OD₂₆₀/OD₂₃₀2.01, better integrity, can be used for reverse transcription.

2) Synthesis of first Strand cDNA

The total RNA obtained was used as a template, reverse transcription was performed using a TaKaRa reverse transcription kit, and oligo (dT) was used as an anchor primer, and first strand cDNA was synthesized by reverse transcription. The specific operation is as follows:

the mix was prepared in the order of template RNA/Primer in a centrifuge tube at a total of 10. mu. L for template 1. mu.g, oligo (dT) Primer (50. mu.M) 1. mu. L mix (10mM reach) 1. mu. L, and the remaining volume was treated with RNase-freeddH₂After completion of the O addition, the mixture was kept at 65 ℃ for 5min and rapidly cooled on ice, the tube was centrifuged to deposit the mixture in the tube at the bottom, and a reverse transcription reaction solution (20. mu. L) was prepared in a new tube, which was denatured, 10. mu. L, 5 × PrimeScriptBuffer 4. mu. L Inhibitor (40U/. mu.l) 0.5. mu. L RTase (200U/. mu.l) 1. mu. L FreedH₂And O is supplemented by 20 mu L, the mixture is slowly shaken up, and is put on a PCR instrument for heat preservation for 10min at the temperature of 30 ℃, for heat preservation for 30min at the temperature of 42 ℃ and for heat preservation for 5min at the temperature of 95 ℃ to inactivate the enzyme, and the cDNA solution is obtained after the mixture is placed on ice.

3) Design and cloning of target gene primer

Based on existing sequencing data for the cymbidium transcriptome, using other speciesWRKYBlast homology alignment is carried out on related gene sequences. Corresponding primers are designed by utilizing Oligo6.0 and Prime5.0, and the sequences of the primers are as follows:

CgWRKY65-F：5'-ATGAACGCCAAGGAAGAACC-3'，

CgWRKY65R：5'-TCATCCTGTGCTGCCGCACG-3'。

performing cymbidium goeringii by using PrimerStar Max high fidelity enzyme by using cDNA first strand as templateCgWRKY65The PCR amplification system (50. mu. L) was 25. mu.l L PrimerStar Max, 2. mu. L Forward Primer, 2. mu. L Reverseprimer, 2. mu. L Template DNA, 19. mu. L ddH₂And O. The PCR procedure was: the reaction conditions are pre-denaturation at 94 ℃ for 3min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 5s, extension at 72 ℃ for 30s, 32 cycles, total extension at 72 ℃ for 5min and heat preservation at 16 ℃.

After the PCR reaction is finished, taking all PCR products, detecting by 1.5% agarose gel electrophoresis, cutting target fragments, carrying out gel recovery and purification on the PCR target amplification products, adopting a DNA gel recovery kit of Tiangen company to carry out purification and recovery on the target fragments, and specifically, adding 500 mu L equilibrium liquid B L into an adsorption column CA2 (the adsorption column is put into a collecting pipe), centrifuging at 12000rpm for 1min, pouring off waste liquid in the collecting pipePutting the adsorption column back into the collection tube, cutting off a single target band from agarose gel, putting the cut single target band into a clean centrifuge tube, weighing, adding an equal volume of solution PN (if the gel is 0.1g and the volume can be regarded as 100 mu L, adding 100 mu L PN solution), putting the gel block in a 50 ℃ water bath, turning the centrifuge tube up and down continuously and gently until the gel block is completely dissolved, adding the solution obtained in the previous step into an adsorption column CA2, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, pouring off the waste liquid in the collection tube, putting the adsorption column CA2 into the collection tube, adding 600 mu L rinsing liquid W into the adsorption column CA2, centrifuging at 12000rpm for 1min, pouring off the waste liquid in the collection tube, putting the adsorption column back into the collection tube, centrifuging at 12000rpm for 2min, removing the rinsing liquid as much as possible, putting the adsorption column into the collection tube, standing at room temperature for 5min, airing completely, putting the adsorption column into a clean centrifuge tube, hanging 30 mu L ddH in the middle position of the adsorption₂O, standing at room temperature for 2min, centrifuging at 12000rpm for 2min to collect DNA solution, collecting purified product of 2 μ L, and detecting by gel electrophoresis using 1% agarose.

4) Ligation of the fragment of interest to the vector

The cloning Vector was pEASY-Blunt Vector from the entire gold company, and the ligation reaction was carried out in a ligation system (5. mu. L) of 4. mu. L PCR purified product and 1. mu. L pEASY-Blunt Vector, which were gently pipetted and mixed, and then the mixture was left at room temperature for 5min, and the tube was placed on ice.

5) Conversion of ligation products

The competent cell Trans5 α strain was taken out from the ultra-low temperature refrigerator, placed on ice to melt, 5. mu. L of overnight ligation product was taken up and added to 100. mu. L of competent cells, the centrifuge tube was placed on ice for 30min, water bath was carried out in a 42 ℃ water bath kettle with heat shock for 90s without shaking, immediately placed on ice for 2min, 800. mu. L of antibiotic-free liquid medium was added to the super clean bench, thawed by shaking at 37 ℃ and 180 rpm for 1h, centrifuged at 4000rpm for 3min, 800. mu. L supernatant was aspirated, and the precipitated cells were resuspended and smeared on L B plates (Amp concentration 100 mg/L) and cultured overnight at 37 ℃.

6) Screening and validation of recombinant plasmids

Single colonies grown overnight on L B solid medium containing antibiotic (Amp) were picked and inoculated into L B liquid medium containing 750. mu. L of the same antibiotic at 200 rpm and 37 ℃ overnight.

The PCR amplification system comprises 10u L Green TaqMix, 1 mu l M13-F/R, 1 mu L bacterial liquid and 7 mu L ddH₂O was replenished to 20. mu. L.

The PCR procedure was: 10min at 94 ℃; 30 cycles at 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 1 min; 5min at 72 ℃; at 16 ℃ forever.

And absorbing a PCR product of 5 mu L for agarose gel electrophoresis detection analysis, verifying, entrusting the bacterial liquid sample with correct strip size to sequencing by Nanjing Kingsry Biotechnology Co., Ltd, and performing comparative analysis on the sequencing result on NCBI by using a sequencing primer which is a universal primer M13F/R.

According to the analysis of the sequencing result, 1 cymbidium can be finally obtained by determining the cloneWRKYThe coding gene is namedCgWRKY65The nucleotide sequence of the gene is shown as SEQ ID NO.1,CgWRKY65the gene coding length is 780bp, the ATG start codon and the TGA stop codon are contained, wherein the ORF total length is 780bp, 259 amino acid proteins are coded, and the amino acid sequence of the proteins is shown as SEQ ID NO. 2.

Example 2

The research result shows thatWRKY65The gene is expressed in various tissues and organs in cymbidium (FIG. 2 a), but the expression level of the gene is the highest in cymbidium, which indicates that the gene is active in flower function. Expression analysis of cymbidium ABA stress treated leaves (FIG. 2 b) demonstratedCgWRKY65The gene plays an important regulation role in the ABA stress response of the cymbidium goeringii leaves.

The plant material used in this example was Arabidopsis thaliana (Arabidopsis thaliana) Col (Columbia) wild type seeds.

The E.coli strain used in this example was Trans5 α, the Agrobacterium strain was GV3101, which was used to transform Arabidopsis thaliana, respectively, and the plant expression vector used in the experiment was pBI121, which were purchased from holotype gold and prism, respectively.

1）CgWRKY65Construction of Gene overexpression vectors

Obtained in example 1CgWRKY65The full-length sequence of the gene ORF is connected with a plant expression vector pBI121, and the constructed vector is shown in figure 1.

2) And (3) plasmid extraction:

extracting plasmids according to the specification of the small-extraction medium-volume kit of the Tiangen plasmids, and specifically comprising the following steps:

centrifuging 10m L bacteria liquid cultured overnight at 12000rpm for 1min, removing supernatant, adding 500 μ L P1 solution (containing RNaseA) into centrifuge tube with thallus precipitate, suspending thallus precipitate completely with vortex apparatus, adding 500 μ L P2 solution into centrifuge tube, cracking thallus fully when turning up and down gently, adding 700 μ L P3 solution into centrifuge tube, turning up and down gently, mixing well, centrifuging at 12000rpm for 10min after white flocculent precipitate appears, adding 500 μ L equilibrium solution B L into adsorption column CP4, centrifuging at 12000rpm for 1min, discarding waste liquid in collection tube, returning adsorption column to collection tube, adding collected supernatant into filtration column CS in batches, centrifuging at 12000rpm for 2min, adding collected solution into adsorption column CP4 in batches, discarding waste liquid in collection tube at 12000rpm for 4 min, returning PD of adsorption column to L min, adding collected supernatant to adsorption column PD of collection tube, discarding supernatant to centrifugation column, adding collected supernatant to PW column 4, removing supernatant, adding collected supernatant to adsorption column PW column 4, adding collected supernatant to centrifugation column PW column, discarding supernatant, adding collected supernatant to centrifugation column PW column 4, adding collected supernatant to centrifugation column, removing waste liquid, centrifuging, removing waste liquid in centrifugation column, adding supernatant to centrifugation column PW column 4, removing medium PW column, centrifuging, removing waste liquid in centrifugation column, removing medium for 12000rpm, removing medium, adding supernatant, centrifuging, removing waste liquid, removing medium, removing waste liquid in centrifugation column, centrifuging, removing medium column 638, removing medium column, removing medium column, removing medium adsorbing column, removing medium column₂O; standing at room temperature for 2min, centrifuging at 12000rpm for 1min, and collecting the solution in the centrifuge tube as plasmid. Finally, the plasmid concentration was determined and prepared for the next experiment.

3) Addition of specific cleavage sites

cDNA is taken as a template, and specific enzyme cutting sites are added on two sides of a target gene by a PCR method. Chinese cymbidiumCgWRKY65SmaI and SnaBI enzyme cutting sites are added on both sides of the gene. The PCR reaction system, procedure and primers used were as follows:

PCR amplification System (50. mu. L): 25μL PrimerStar Max，2μL Forward Primer，2μL ReversePrimer，2μL Template DNA，19μL ddH₂And O. The PCR procedure was: the reaction conditions are pre-denaturation at 94 ℃ for 3min, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 5s, extension at 72 ℃ for 30s, 32 cycles, total extension at 72 ℃ for 5min and heat preservation at 16 ℃.

The primer sequences used were:

CgWRKY65-Xbal-F：5'-GAGAACACGGGGGACTCTAGAATGAACGCCAAGGAAGAACCC -3'，

CgWRKY65-SnaBI-R：5'-TCACACAAACGGTGATACGTATCCTGTGCTGCCGCACGC-3'

the obtained PCR product was separated by 1.5% agarose gel electrophoresis, recovered and purified using a Tiangen agarose gel DNA recovery kit, and the recovered product was ligated with the pBI121 vector to construct an expression vector.

4) Double enzyme digestion reaction

Digesting the extracted pBI121 plasmid with Xbal and SnaBI at 37 deg.C for 15min, recovering linear vector by electrophoresis, and storing at-20 deg.C for use, wherein the double digestion reaction system is 50 μ L, pBI121 plasmid 20 μ L, 5 × buffer 5 μ L1 μ L1 μ L, ddH₂O23 mu L, the enzyme cutting result is shown in figure 3a, wherein M is D L2000 Marker, 1:CgWRKY65after ligation with pBI121, the DNA fragment was digested simultaneously with XbaI and SnaBI.

5) Ligation reaction

Agarose gel electrophoresis is used for detecting a target gene and a vector pBI121 recovered after enzyme digestion, and according to the detected purity and concentration, all reagents are added according to a connection system, wherein the number of target fragment molecules is =3:1-5:1, the connection reaction system is a linearized pBI121 vector 7 mu L, an insert fragment 3 mu L, a 5 × CE II buffer 4 mu l, an Exnase II 2 mu l and a ddH2OUp to 20 mu L, the reaction is carried out for 30min at 37 ℃, and the reaction is placed on ice for cooling.

6) Transfer of the ligation product into E.coli

The product of the ligation of the desired fragment with the vector pBI121 was transferred into competent cells of E.coli Trans5 α in the same manner as in example 1.

7) Identification of recombinants

The single colony on the plate is selected and inoculated into L B liquid culture medium containing antibiotic (kanamycin), shaking culture is carried out at 37 ℃ and 200 rpm for overnight, bacterial liquid PCR is carried out by using a target gene full-length primer, positive clones are screened, the screened positive clones are sent to Nanjing Kingsry company for sequencing, plasmid is extracted by using a Tiangen plasmid extraction kit, enzyme digestion verification is carried out, whether the sizes of fragments after enzyme digestion are consistent or not is judged, the result is shown in figure 3B, the target primer PCR result is the target primer, and the size of a strip is 780 bp.

8) Preparation and transformation of Agrobacterium-infected competent cells

The agrobacterium is used for preparing agrobacterium-induced competence for carrying out an infection experiment of arabidopsis thaliana by using agrobacterium GV3101, and the agrobacterium-induced competence preparation process comprises the steps of selecting an activated agrobacterium single colony, inoculating the agrobacterium-induced competence in a 5m L liquid L B culture medium, carrying out shake culture at 28 ℃ and 250rpm for 20-24 h, absorbing 2m L bacterial liquid, inoculating the agrobacterium-induced competence in a triangular flask containing a 50m L liquid L B culture medium, carrying out shake culture at 28 ℃ and 250rpm to OD₆₀₀The value is about 0.8, the bacterial liquid after the propagation is put on ice and iced for 30min, the temperature is 4 ℃, the centrifugal speed is 5000 rpm for 5min, the supernatant is discarded, 10m L precooled 0.1 mo 1/L CaCl is added₂Suspending the precipitated thallus thoroughly, centrifuging at 4 deg.C and 5000 rpm for 5min, discarding supernatant, adding 1m L precooled 20 mm O1/L CaCl₂The solution fully suspends the thalli to obtain GV3101 competent cells to be prepared, the competent cells are subpackaged into 100 mu L/tube by a centrifuge tube, 20% of sterile glycerol is rapidly added, and the competent cells are placed and stored at minus 80 ℃.

Agrobacterium transformation of recon, namely, thawing agrobacterium competent cells by ice bath, adding 1 to 5 mul of recovered and purified plasmid into 200 mul of agrobacterium competent cells, lightly mixing uniformly, performing ice bath for 30min, quickly freezing for l min by using liquid nitrogen, thermally shocking in a water bath kettle at 37 ℃ for 1 to 5min, quickly placing on ice for 1 to 2min, adding 800 mul of L B culture medium without any antibiotic, recovering for 2 to 4h at 28 ℃ and 100 rpm, centrifuging for 3min at 4000rpm, sucking off part of the culture medium, fully mixing the rest of bacteria liquid by using a liquid transfer gun, smearing on a solid L B culture medium added with 50 mg/L kanamycin and 50 mg/L streptomycin (EHA 105) or 100 mg/L gentamycin (GV 3101), and performing inverted culture for 30 to 48 h at 28 ℃.

Identification of Agrobacterium recombinants: picking out single colony from the plate culture medium, and inoculating the single colony in a liquid culture medium containing corresponding antibiotics; culturing at 28 deg.C and 220 rpm overnight; carrying out PCR on the bacterial liquid by respectively matching 35S-F with the following primers, wherein the sequences of the primers are as follows:

35S-F：5'-GATAGTGGAAAAGGAAGGTG-3'，

35S-CgWRKY65-R：5'-TCACACAAACGGTGATACGTATCCTGTGCTGCCGCACGC -3'。

detecting the PCR product by 1% agarose gel electrophoresis to identify whether the PCR product contains a target fragment; identifying positive clones, performing amplification culture, extracting plasmids by an alkaline lysis method, and performing double enzyme digestion verification; and adding a proper amount of sterile glycerol into the identified positive clone, and storing at-80 ℃ for later use.

9) Agrobacterium-mediated transformation of Arabidopsis thaliana

The method comprises the specific operation steps of maintaining healthy growth state of arabidopsis (col wild type) until the arabidopsis flowers, activating agrobacterium EHA105 strain carrying the target gene, selecting a single colony, inoculating the single colony on L B culture medium containing 5m L kanamycin and streptomycin, shaking the bacteria at 28 ℃ and 250rpm until the bacteria become turbid just and the bacteria is about 8-10 hours, sucking 1m L bacteria liquid, inoculating the bacteria liquid into a triangular flask (50m L) for shaking the bacteria for 24 hours until the OD value is about 0.8, centrifuging the bacteria liquid at 5000 rpm for 5min at room temperature, removing supernatant, collecting the bacteria, suspending the bacteria with 5% sucrose solution, adding Silwet L-77 at the concentration of 0.05% (500 mu l/L) before soaking, shaking out foams, soaking the overground part of arabidopsis in agrobacterium suspension solution for 15-30 seconds, slightly shaking, laying the soaked arabidopsis thaliana in a tray covered with a foil, sealing the foil, placing the arabidopsis thaliana in a sealed box, placing the box for 24 hours, and culturing the arabidopsis thaliana in a light-shielding condition, and watering the foil to grow.

The 5% sucrose solution resuspension comprises the following components of MS culture medium, sucrose 50 g/L0.5.5 g/L and Silwet-77500 mu l/L (note: after preparation, pH is adjusted to 5.8, after bacterial liquid is centrifugally resuspended, Silwet L-77 is added, and the conversion relation between resuspension liquid and bacterial liquid is that the dosage of resuspension liquid is bacterial liquid OD bacterial liquid volume =0.8 bacterial liquid resuspension).

10) Screening of transgenic plants

The collected seeds of transgenic Arabidopsis thaliana of T1 generation are sterilized by alcohol and mercuric chloride, and the method comprises placing appropriate amount of the obtained transgenic seeds in a centrifuge tube of 1.5m L, soaking in 75% alcohol for 30s, sterilizing with 10% sodium hypochlorite for 2min for 30s, washing with sterile water for 3-4 times, replacing the sterilized new centrifuge tube after the first washing, and suspending with 0.1% agarose solution.

The sterilized transgenic Arabidopsis seeds were sown on 1/2MS solid medium containing antibiotics (kanamycin 50 mg/L and cefamycin 100 mg/L), cultured at 22 ℃ under light, and after about one week, Arabidopsis seeds which can normally grow on the medium were transplanted into soil and continued to grow.

11) Detection of transgenic plants

Taking a proper amount of young leaves of arabidopsis thaliana and transgenic plants, and extracting DNA by adopting a CTAB method, wherein the specific operation steps comprise placing a proper amount of leaves in a sterilized 2m L centrifuge tube, adding 700 mu l of CTAB solution, thoroughly grinding by using a ball mill, standing for 10min at 65 ℃, adding chloroform isoamylol with the same volume, evenly mixing by reversing for several times, centrifuging for 10min at 14000rpm, transferring supernatant into a new sterile centrifuge tube, adding isopropanol with the same volume, evenly mixing by reversing for several times, standing for 2min at room temperature, centrifuging for 10min at 14000rpm, pouring off supernatant, adding 70% absolute ethyl alcohol, blowing and washing twice by using a liquid transfer gun, centrifuging for 1min at 14000rpm, discarding supernatant, drying surface liquid, adding 20 mu L ddH₂And dissolving the O. Taking the DNA of the above-mentioned extracted transgenic and wild type Arabidopsis thaliana, and usingCgWRKY65PCR detection is carried out by specific primers of the gene.

Chinese cymbidiumCgWRKY65After transgenic Arabidopsis thaliana, a total of 5 over-expressions were obtainedCgWRKY65A transgenic Arabidopsis line. The PCR results are shown in FIG. 4 with the recombinant plasmid as positive control, wild type as negative control, and water as blank control, and the positive control asCgWRKY65As for the result of gene vector PCR, water was used as a template for negative control, and wild type Arabidopsis DNA was used as a template for WT.

12) Phenotypic observations

Obtaining transgenic plants of different generations: the harvested transgenic T1 generation seeds are sterilized, screened and cultured, and then transplanted into nutrient soil to be cultured at 22 ℃ for 16 h in light/8 h in darkness; after detection, retaining the preliminarily confirmed transgenic plants, harvesting seeds of T1 generations after the plants are mature, and numbering to obtain T2 generations; like the T1 generation, seeds of the T2 generation are sterilized and then coated on a screening culture medium containing antibiotics, and the culture medium is placed at 22 ℃ for continuous illumination; performing survival rate statistics on T2 generation seeds with different numbers for about 10 days, selecting plants with 75% survival rate, transplanting the plants into nutrient soil, culturing the plants in the nutrient soil at 22 ℃ for 16 h in light/8 h in dark, and taking leaves for positive detection; continuously numbering positive T2 generation plants, and collecting seeds to obtain T3 generation seeds; sterilizing the seeds, screening by using a screening culture medium, and placing under the light for continuous illumination culture; around 10d, different numbered T3 generation plants were observed, all survived and no segregating homozygous plants for the T3 generation appeared.

The obtained transgenic lines were observed in batches.

(1) Selecting 3 transgenic plants with obvious phenotype for observation, as shown in figure 5, finding that the transgenic arabidopsis plants grow slowly compared with wild arabidopsis,CgWRKY65the transgenic plants had smaller overall leaf size at the seedling stage compared to wild type Arabidopsis WT.

(2) Carrying out ABA stress treatment on transgenic Arabidopsis, sowing the disinfected transgenic Arabidopsis seeds and wild Arabidopsis seeds on 1/2MS culture medium to grow for about 4d, transferring the seeds to 1/2MS culture medium added with 100 MuM ABA, photographing for about 10d to observe the root length of Arabidopsis, recording data, and finding out the result shown in figure 6,CgWRKY65the expression level is adjusted to be the highest in 1h and is about 3.2 times of that of a control, the expression level in a common wild type arabidopsis plant (WT) is almost 0, and the expression level in the transgenic arabidopsis plant is different in different degrees at different times, and experimental results prove that the expression level is the highest in 1h and is about 3.2 times of that of the controlCgWRKY65The gene has been successfully transferred into arabidopsis; as shown in FIG. 7, at different ABA concentrationsCgWRKY65The root length reduction of transgenic Arabidopsis is obviously larger than that of WT, which shows thatCgWRKY65Is sensitive to ABA and can promote the inhibiting effect of ABA on root development.

This example shows cymbidium goeringii to be overexpressedCgWRKY6535S:CgWRKY65the transgenic plants were transformed into Arabidopsis thaliana, and phenotypic observation and analysis were performed. As can be seen from the results, 35S was overexpressed:CgWRKY65the arabidopsis T2 generation plants have slow growth and development, and the leaves of the plants become small;CgWRKY65overexpression can enhance the inhibiting effect of ABA on root development by regulating the expression of ABA signal pathway related genes.

Sequence listing

<110> Nanjing university of forestry

<120> cymbidium CgWRKY65 gene and application thereof

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>780

<212>DNA

<213> Cymbidium goeringii

<400>1

atgaacgcca aggaagaacc cgggacctcg ccggagctca gcccatatcc tgaaccggag 60

ccttctccta agaaaagccg gcggtcggtg cagaagaggg ttgtgtcgtt accggtgccg 120

gaagcggagg ggccaaggct gaaggccggc ggtgaggcgg cgccgccgcc ggattcgtgg 180

gcctggcgaa agtatgggca gaagccgatc aagggatcgc cttatcccag gggttattac 240

agatgtagca gttcgaaggg atgcccggcg agaaaacaag tagagagaag ccgtgcggac 300

cctaccatgt tcattgtcac ctactcctcc gaacacaacc atccatggcc gccggcccac 360

aagaaaagca agccggcgga agacccaaaa ccggtcgaga ccagcaccgc cgaaccagcc 420

tcggatcccg aagagaagtt ctccgatcta atcggagaag aaccgtccct gttgattcat480

gacgacttcc gctggccgtc caatgcctcc gccgccgccg acgacgatac cgggctccta 540

tacggcccgc tgttcgttgg cgcttctctg ggtgatgagt gtgataagtt gagtggcagg 600

ggaagcgggg cggcgcttga cgaggaggaa gacgcgctct tcgccggact cggagagctg 660

ccggaatatt ctgttatttt ccggcgtgga tttctggacc gtcaggtgcg tggcggagag 720

gacgggaaac gatgtggatt ggaggcgacg gctgcggcgg cgtgcggcag cacaggatga 780

<210>2

<211>259

<212>PRT

<213> Cymbidium goeringii

<400>2

Met Asn Ala Lys Glu Glu Pro Gly Thr Ser Pro Glu Leu Ser Pro Tyr

1 5 10 15

Pro Glu Pro Glu Pro Ser Pro Lys Lys Ser Arg Arg Ser Val Gln Lys

20 25 30

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

35 40 45

Ala Gly Gly Glu Ala Ala Pro Pro Pro Asp Ser Trp Ala Trp Arg Lys

50 55 60

Tyr Gly Gln Lys Pro Ile Lys Gly Ser Pro Tyr Pro Arg Gly Tyr Tyr

65 70 75 80

Arg Cys Ser Ser Ser Lys Gly Cys Pro Ala Arg Lys Gln Val Glu Arg

85 90 95

Ser Arg Ala Asp Pro Thr Met Phe Ile Val Thr Tyr Ser Ser Glu His

100 105 110

Asn His Pro Trp Pro Pro Ala His Lys Lys Ser Lys Pro Ala Glu Asp

115 120 125

Pro Lys Pro Val Glu Thr Ser Thr Ala Glu Pro Ala Ser Asp Pro Glu

130 135 140

Glu Lys Phe Ser Asp Leu Ile Gly Glu Glu Pro Ser Leu Leu Ile His

145 150 155 160

Asp Asp Phe Arg Trp Pro Ser Asn Ala Ser Ala Ala Ala Asp Asp Asp

165 170 175

Thr Gly Leu Leu Tyr Gly Pro Leu Phe Val Gly Ala Ser Leu Gly Asp

180 185 190

Glu Cys Asp Lys Leu Ser Gly Arg Gly Ser Gly Ala Ala Leu Asp Glu

195 200 205

Glu Glu Asp Ala Leu Phe Ala Gly Leu Gly Glu Leu Pro Glu Tyr Ser

210 215 220

Val Ile Phe Arg Arg Gly Phe Leu Asp Arg Gln Val Arg Gly Gly Glu

225 230 235 240

Asp Gly Lys Arg Cys Gly Leu Glu Ala Thr Ala Ala Ala Ala Cys Gly

245 250 255

Ser Thr Gly

<210>3

<211>20

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>3

atgaacgcca aggaagaacc 20

<210>4

<211>20

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>4

tcatcctgtg ctgccgcacg 20

<210>5

<211>42

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>5

gagaacacgg gggactctag aatgaacgcc aaggaagaac cc 42

<210>6

<211>39

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>6

tcacacaaac ggtgatacgt atcctgtgct gccgcacgc 39

<210>7

<211>20

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>7

gatagtggaa aaggaaggtg 20

<210>8

<211>39

<212>DNA

<213> Artificial sequence (artiartiartifical sequence)

<400>8

tcacacaaac ggtgatacgt atcctgtgct gccgcacgc 39

Claims (5)

1. A kind of Chinese cymbidiumCgWRKY65The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

2. The cymbidium goeringii of claim 1CgWRKY65The amino acid sequence of the gene expression protein is shown in SEQ ID NO. 2.

3. The cymbidium goeringii of claim 1CgWRKY65The use of genes in plant production and breeding.

4. Contains the cymbidium goeringii of claim 1CgWRKY65A vector for the gene.

5. Contains the cymbidium goeringii of claim 1CgWRKY65A host cell for the gene.

Images