CN110195064B - Chimonanthus praecox WRKY transcription factor gene CpWRKY71 and cloning and application of promoter thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a chimonanthus nitens WRKY transcription factor gene CpWRKY71 and cloning and application of a promoter thereof. The invention aims to provide a new choice for the researches on the adversity stress of Chimonanthus praecox. The invention provides a chimonanthus nitens WRKY transcription factor gene CpWRKY71, wherein the coding protein of the gene has an amino acid sequence shown as SEQ ID No. 3. The invention clones and obtains the chimonanthus nitens CpWRKY71 gene for the first time, detects the expression characteristic of the CpWRKY71 gene by a real-time fluorescent quantitative PCR technology, and verifies the function of the gene promoter in arabidopsis thaliana by a transgenic technology.

Description

Chimonanthus praecox WRKY transcription factor gene CpWRKY71 and cloning and application of promoter thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a chimonanthus nitens WRKY transcription factor gene CpWRKY71 and cloning and application of a promoter thereof.

Background

The deciduous shrubs of Chimonanthus praecox of Chimonanthus family are native to China and widely distributed in the southwest and southwest regions of China. Chimonanthus praecox is a rare winter flowering plant, with a flowering period of 12 months to 2 months in the next year. The wintersweet has unique color and pleasant fragrance, and can be potted to be used as a stake scene, a cut flower for appreciation and a landscaping plant. Currently, some progress has been made in the research on the abiotic stress and flower development of Chimonanthus fragrans.

The WRKY transcription factor is a specific superfamily transcription factor of a plant, and is named because the N-ends in the WRKY structural domain contain highly conserved WRKYGQK amino acid sequences. It can be specifically combined with (T) (T) TGAC (C/T) sequence (W box) in downstream gene promoter so as to regulate expression of downstream gene, and can be involved in various growth and development processes of plant, such as seed dormancy, leaf senescence and abiotic stress, etc. However, reports about the cloning and function research of the chimonanthus nitens WRKY transcription factor gene are not found at present.

Disclosure of Invention

The invention aims to provide a new choice for the researches on the adversity stress of Chimonanthus praecox.

The invention provides a chimonanthus nitens WRKY transcription factor gene CpWRKY71, wherein the coding protein of the gene has an amino acid sequence shown as SEQ ID No. 3.

Further, the chimonanthus praecox WRKY transcription factor gene CpWRKY71 has a nucleotide sequence shown as SEQ ID No. 1.

The invention also provides a promoter of the chimonanthus nitens WRKY transcription factor gene CpWRKY71, which has a nucleotide sequence shown as SEQ ID No. 2.

The promoter of the chimonanthus nitens WRKY transcription factor gene CpWRKY71 and/or the chimonanthus nitens WRKY transcription factor gene CpWRKY71 is used for regulating and controlling the stress resistance or senescence of chimonanthus nitens.

Specifically, the adversity stress is high temperature, low temperature, drought, salt stress, abscisic acid ABA, salicylic acid SA, gibberellin GA or methyl jasmonate MeJA treatment.

Specifically, the high temperature is 42 ℃.

Specifically, the low temperature is 2 ℃.

Specifically, the drought is 15% PEG6000 treatment.

In particular, the salt stress is 200mM NaCl treatment.

Specifically, the abscisic acid treatment is 50 mu M ABA.

Specifically, the salicylic acid is treated to 100 mu M SA.

Specifically, the gibberellin treatment is 10. mu.M GA/50. mu.M GA.

Specifically, the methyl jasmonate is treated into 100 mu M MeJA.

The invention has the beneficial effects that: the invention provides a chimonanthus nitens CpWRKY71 protein, a gene for coding the protein and a clone of a promoter, and application thereof. The invention clones and obtains the chimonanthus nitens CpWRKY71 gene for the first time, detects the expression characteristic of the CpWRKY71 gene by a real-time fluorescent quantitative PCR technology, and verifies the function of the gene promoter in a plant (arabidopsis thaliana) by a transgenic technology. Provides reference and basis for the research on senescence, stress response and regulation mechanism of the wintersweet flowers and leaves, and provides possibility for further improving ornamental plants, such as prolonging the flowering phase of the wintersweet flowers.

Drawings

FIG. 1 alignment of the deduced amino acid sequence of CpWRKY71 with WRKY protein sequences from other species.

At is Arabidopsis thaliana; vv is grape; gh is poplar; nt: tobacco

FIG. 2 analysis of CpWRKY71 and Arabidopsis WRKY protein evolutionary tree

FIG. 3 shows the expression analysis of CpWRKY71 in Chimonanthus praecox, and relative expression is relative expression. A, analyzing the expression of CpWRKY71 in each tissue of Chimonanthus praecox; r: a root; s: a stem; c, cotyledon; YY is young leaf; OL is old leaf; f: and (4) flower. B: expression analysis of CpWRKY71 in the plum blossom development process; SDS (sodium dodecyl sulfate): sepal primordia differentiation period; PDS, petal primordium differentiation period; StDS, differentiation period of stamen primordium; PiDS: pistil primordial differentiation period; s1, sprouting; s2 bud device; s3, the phase of valve emergence; s4, initial startup; s5, full bloom period; s6: early stage of failure

FIG. 4 expression analysis of CpWRKY71 gene under different stresses and hormone treatments. A: 4 ℃; b: 42 ℃; c: 15% PEG 6000; d: 200mM NaCl; e: 50 μ M ABA (abscisic acid); f: 100 μ M SA (salicylic acid); g: 10 μ M GA (gibberellin); h: 100 μ M MeJA (methyl jasmonate)

FIG. 5 promoter sequence and homeopathic elements of chimonanthus nitens CpWRKY71 gene. CpWRKY71 promoter sequence, the shaded part corresponds to each homeopathic element in the B picture in turn; b: schematic representation of the homeopathic elements of the CpWRKY71 promoter.

Promoter core elements: TATA-BOX CAAT-BOX;

the light-responsive element: LAMP-element, Box I, TGG-motif, G-Box;

abiotic stress-like elements: MBS (drought response element), HSE (high temperature response element), LTR (low temperature response element), MYB1AT (dehydration response element);

hormone-responsive elements: CGTCA-motif and TGACG-motif (methyl jasmonate responsive element), TCA-element (salicylic acid responsive element), ABRE, CE3 and ABRELATED 1 (abscisic acid responsive element), P-box and GAREAT (gibberellin responsive element);

other elements: skn-1_ motif, circadian, polen 1lelat52

FIG. 6pCpWRKY71 GUS transgenic Arabidopsis GUS histochemical staining results. A: vernalized seed; b: germinating the seeds for 2 days; C-G: 3, 5, 7, 11, 15 days old arabidopsis seedlings; h: inflorescence; i: single flower; j: old leaves; k: young leaves; l: young leaves; m: young leaves.

FIG. 7 Effect of abiotic stress and hormone treatment on the activity of GUS in pCpWRKY71 GUS transgenic Arabidopsis.

FIG. 8 shows the relative expression level of CpWRKY71 gene in Arabidopsis thaliana transformed with CpWRKY71 gene. OE1 and OE2 are transgenic lines, EV is a 1300-transgenic empty vector plant, and WT is a wild type plant.

FIG. 9 CpWRKY71 transgenic Arabidopsis thaliana leaf senescence

FIG. 10 statistics of time spent in apical meristems of OE1, OE2, EV and WT plants

Detailed Description

Example 1 isolation of the Chimonanthus nitens CpWRKY71 Gene

According to the sequence fragment known in a Chimonanthus fragrans transcriptome database, a software Primer 5.0 is used for designing a specific Primer for PCR amplification, and the sequence of the Primer is as follows:

CpWRKY71-F：5‘-aagctaaacctcttccctct-3’(SEQ ID No.4)

CpWRKY71-R：5‘-ccacactaggatgttggttc-3’(SEQ ID No.5)

amplification of the chimonanthus nitens CpWRKY71 gene by using chimonanthus nitens cDNA as a template, wherein a PCR reaction system is as follows: 10 × Taq PCR Buffer 2.5 μ L, dNTP (10mM)1.5 μ L, CpWRKY71-F (10 μ M)1 μ L, CpWRKY71-R (10 μ M)1 μ L, TaKaRa Ex TaqTM 0.2 μ L, template 2 μ L, plus ddH₂O to 25. mu.L. The reaction conditions were as follows: 5min at 95 ℃; 30s at 95 ℃ and 30s at52 ℃, and 26 cycles; 1min at 72 ℃; 10min at 72 ℃.

And recovering the PCR product, connecting the PCR product to a T vector, transforming escherichia coli competent cells, selecting recombinants for sequencing (engine, Chengdu), analyzing the sequencing result, and displaying that the sequence obtained by cloning is consistent with the sequence in a transcriptome database. The cDNA sequence of the chimonanthus nitens CpWRKY71 gene is shown as SEQ ID No. 1.

The full length of cDNA of the chimonanthus nitens CpWRKY71 gene is 1137bp, and comprises a 951bp complete open reading frame which encodes 316 amino acids. The 5 'end has a 93bp long untranslated region, and the 3' end contains a 93bp non-coding sequence. The deduced amino acid sequence has a WRKYGQK 7 peptide at the N-terminal of the WRKY conserved domain, and a zinc finger structure of C2H2 type (C-X4-5-C-X22-23-H-X1-H) at the C-terminal (FIG. 1), and the gene belongs to the structural subgroup II (II subfamily members) of the WRKY family according to the existing literature report. Based on phylogenetic analysis of Arabidopsis thaliana WRKY family II subfamily members, the gene is found to belong to group IIc subgroup and has close evolutionary relationship with AtWRKY71 (FIG. 2).

Example 2 analysis of expression characteristics of chimonanthus nitens CpWRKY71 Gene

1 RNA extraction

(1) Wrapping the instruments for extracting RNA, such as a mortar, a mortar bar, a spoon, a pair of scissors, and the like with tinfoil paper, placing the wrapped products in an oven at 180 ℃ for 4h, and cooling the wrapped products for later use.

(2) Taking Chimonanthus praecox leaf, grinding with liquid nitrogen in a mortar, pouring into a RNase-free centrifuge tube rapidly, adding 600 μ L Trizol (plant RNA extraction reagent, Thermo, USA), vortex-suspending, mixing well, and standing at room temperature for 10 min.

(3) Adding 120 μ L chloroform, mixing by vortex, and standing at room temperature for 10 min.

(4) Centrifuge at 12000rpm for 10min at 4 ℃ and aspirate 200. mu.L of supernatant into another centrifuge tube.

(5) Adding 2 times volume of anhydrous ethanol, mixing, centrifuging at 12000rpm and 4 deg.C for 30min, removing supernatant, and retaining precipitate.

(6) The precipitate was washed 3 times with 75% ethanol and then air-dried.

(7) The precipitate was dissolved in an appropriate amount of RNase-free water.

(8) Total RNA was analyzed for quality by electrophoresis and concentration and purity by using NanoDrop ND-2000 (ultramicro nucleic acid protein analyzer, Thermo, USA).

2 Synthesis of first Strand of cDNA

Removing the genome DNA reaction system: 5 XgDNA Eraser Buffer 2. mu.L, gDNA Eraser 1. mu.L, mRNA 1. mu.g, RNase Free H₂O up to 10 mL; denaturation at 42 deg.C for 5min, taking out and placing on ice. Adding to the initial reaction system: 5 XPrimeScript Buffer 4. mu.L, RNase Free H₂O4. mu.L, RT Primer Mix 1. mu.L, PrimeScript RT Enzyme Mix 1. mu.L; and (3) PCR reaction conditions: synthesizing a first chain of cDNA at 37 ℃ for 15 min; denaturation at 85 ℃ for 5s inactivated reverse transcriptase. The reverse transcription product was stored at-20 ℃.

3 real-time fluorescent quantitative PCR

The expression patterns of the CpWRKY71 gene under different tissues, flower development periods and stress treatment conditions are analyzed by a fluorescent quantitative PCR method. RNA extraction and cDNA synthesis of each sample were performed as described above. The reaction was completed on a Real-time quantitative PCR instrument Bio-Rad CFX96Real-time system (USA) using the Ssofast EvaGreen Supermix (Bio-Rad, CA) reaction kit. Chimonanthus praecox CpActin and CpTublin are used as internal reference genes. The primers used were as follows:

CpActin-F：5‘-aggctaagattcaagacaagg-3’(SEQ ID No.6)

CpActin-R：5‘-ttggtcgcagctgattgctgtg-3’(SEQ ID No.7)

CpTublin-F：5‘-gtgcatctctatccacatcg-3’(SEQ ID No.8)

CpTublin-R：5‘-caagcttccttatgcgatcc-3’(SEQ ID No.9)

RT-CpWRKY71-F：5‘-tgaccactaccacgatgacaaca-3’(SEQ ID No.10)

RT-CpWRKY71-R：5‘-ggattgccactgggtcctcta-3’(SEQ ID No.11)

the PCR reaction system is as follows: 2 XSsofast EvaGreen Supermix 5. mu.L, dNTP (10mM) 1.5. mu.L, Primer 1 (10. mu.M) 0.5. mu.L, Primer 2 (10. mu.M) 0.5. mu.L, cDNA template 0.5. mu.L, RNase Free dH₂O to 10. mu.L. The reaction conditions were as follows: 5min at 95 ℃; 5s at 95 ℃, 5s at 60 ℃, 5s at 72 ℃ and 40 cycles; melt cycle from 65to 95 ℃ at 0.5 ℃/s.

First, the expression levels of genes in roots, stems, leaves and flowers of the waxberry were analyzed using a fluorescent quantitative PCR technique (FIG. 3). The CpWRKY71 gene is mainly and predominantly expressed in flowers and senescent leaves, and the expression level in roots and stems is low. Meanwhile, the gene expression levels of 10 different flowering stages from the differentiation stage to the senescence stage of the calyx canthus flower primordium are detected, and the result shows that the CpWRKY71 gene is expressed in each stage of the calyx canthus flower development, wherein the expression level is relatively high in the differentiation stage of the calyx canthus flower primordium, and the expression level is highest in the senescence stage.

Secondly, the hexalobal stage chimonanthus nitens seedlings with consistent growth are used for detecting the expression mode of the CpWRKY71 gene under abiotic stress and hormone treatment. High-temperature and low-temperature treatment, namely respectively putting the Chimonanthus praecox seedlings into a 42 ℃ or 4 ℃ artificial climate box; drought and salt stress treatment, irrigating wintersweet seedlings with 15% PEG6000 and 200mM NaCl respectively; and (3) hormone treatment, namely spraying wintersweet seedlings with 50 mu M ABA, 10 mu M GA, 100 mu M SA and 100 mu M MeJA respectively, and sampling after treatment for 0h, 2h, 6h, 12h and 24h, wherein two top leaves of each plant are taken as a biological repetition, and each treatment comprises three biological repetitions. The results show that the expression of the CpWRKY71 gene can be induced by low temperature, high temperature, drought, salt stress, abscisic acid, salicylic acid, gibberellin and jasmonic acid treatment (figure 4). After low-temperature treatment, the expression level of CpWRKY71 gradually increases, and the expression level is highest at 24 h. After high temperature treatment for 2h, the expression level of CpWRKY71 is remarkably reduced and then gradually increased to reach the highest level at 24 h. Under the conditions of PEG6000, ABA, SA and GA treatment, the expression patterns of CpWRKY71 are similar and are increased firstly and then reduced to the original expression level. After NaCl treatment, the expression level of CpWRKY71 gradually increased, and at 6h, the expression level was the highest, and then rapidly decreased. After MeJA treatment, the expression level of CpWRKY71 is rapidly reduced, a lower expression level is maintained in the stage of 2-12 h, and the original level is recovered at 24 h.

Example 3 cloning of the chimonanthus nitens CpWRKY71 promoter

1 obtaining of chimonanthus nitens CpWRKY71 gene promoter fragment

The promoter of the chimonanthus nitens CpWRKY71 gene was amplified using the Clontech chromosome walking kit. Based on the cloned chimonanthus nitens CpWRKY71 gene sequence, a specific Primer is designed by using software Primer 5.0 for PCR amplification, and the Primer sequence is as follows:

pCpWRKY71-GSP1：5‘-gccaacactccagaagcactccc-3’(SEQ ID No.12)

pCpWRKY71-GSP2：5‘-catcgccagagccgaacacg-3’(SEQ ID No.13)

according to the steps of the chromosome walking kit, the promoter of the chimonanthus nitens CpWRKY71 gene is amplified, and a PCR reaction system and reaction conditions are as follows:

a first round: 10 × Advantage 2PCR Buffer 2.5 μ L, dNTP (10mM)0.5 μ L, AP1 (supplied with kit) (10mM)0.5 μ L, GSP1(10mM)0.5 μ L, Advantage 2Polymerase Mix (50X) 0.5 μ L, template 2 μ L, plus ddH₂O to 20 μ L; at 94 ℃ for 25s and 72 ℃ for 3min, and 7 cycles; 25s at 94 ℃, 3min at 67 ℃ and 32 cycles; 7min at 67 ℃.

The PCR products obtained in the first round were diluted 50-fold each and used as templates in the second round. The second round of PCR reaction system and reaction conditions were as follows: 10 × Advantage 2PCR Buffer 2.5 μ L, dNTP (10mM)0.5 μ L, AP2 (supplied with kit) (10mM)0.5 μ L, GSP2(10mM)0.5 μ L, Advantage 2Polymerase Mix (50X) 0.5 μ L, template 2 μ L, plus ddH₂O to 20 μ L; 25s at 94 ℃, 3min at 72 ℃ and 5 cycles; 25s at 94 ℃, 3min at 67 ℃ and 20 cycles; 7min at 67 ℃.

And recovering the PCR product, connecting the PCR product to a T vector, transforming escherichia coli competent cells, and selecting a recombinant for sequencing.

The obtained promoter fragments were submitted to the online databases PlantCARE (http:// bioinformatics. psb. element. be/webtools/plantaCARE/html /) and PLACE (http:// www.dna.affrc.go.jp/PLACE) for regulatory element prediction.

The result shows that the upstream start codon ATG of the CpWRKY71 gene is cloned into 4170bp upstream by a chromosome walking method. The cis-acting elements comprised by the CpWRKY71 promoter sequence may be classified into 5 types: promoter core elements, abiotic stress-like elements, hormone response-like elements, light-inducing elements and other elements (fig. 5). Core elements include TATA Box and CAAT Box. The abiotic stress elements comprise a low temperature response element LTR, a high temperature response element HSE, a drought response element MBS, a dehydration response element MYB1AT and a W-box. Hormone-responsive elements include methyl jasmonate-responsive elements CGTCA-motif and TGACG-motif, salicylic acid-responsive element TCA-element, abscisic acid-responsive elements ABRE, CE3 and ABRELATITED 1, gibberellin-responsive elements P-box and GAREAT. The light induction element comprises LAMP, TGG, G-Box, Box I and ATCT-motif. Other elements include Skn-1_ motif, circadian, pollen1lelat52, and the like. The result shows that the expression of the CpWRKY71 gene is possibly induced by factors such as abiotic stress, hormone and the like, and is consistent with the expression mode of CpWRKY71 in Chimonanthus praecox.

Example 4 analysis of spatio-temporal expression characteristics and inducible expression characteristics of chimonanthus nitens CpWRKY71 promoter development

1 construction of expression vector

According to experimental requirements, the pBI121 vector (present at gardening school of gardening and forestry, southwest university) is used in the present example, and the cloned promoter fragment is used to replace 35S promoter on the vector pBI121 by using a double enzyme digestion method.

2 chimonanthus nitens CpWRKY71 gene promoter fusion plant expression vector transferred into agrobacterium tumefaciens

(1) Adding 0.5 μ g plasmid into 50 μ L GV3101 Agrobacterium competent cells, mixing, and placing on ice for 30 min;

(2) taking out the mixture of the cells and the plasmids, transferring the mixture into an electric shock cup (the electric shock cup is precooled at-20 ℃), and carrying out electric shock at the high voltage of 2500V;

(3) taking out the electric shock cup, adding 500 mu L of precooled YEB culture solution (containing no antibiotics), gently blowing and uniformly mixing, transferring the sucked bacterial solution into a 1.5mL centrifuge tube, and carrying out shaking culture at 28 ℃ and 200rpm for 3-5 h;

(4) appropriate amount of cells were plated on screening YEB solid medium plates and cultured at 28 ℃ for 36 h.

3 inflorescence infection method for transforming arabidopsis thaliana

(1) Single-line activation of Agrobacterium GV3101 containing pCpWRKY71: GUS plasmid on YEB solid medium containing antibiotic (Gen:50mg/L, Kan:50mg/L), dark culture at 28 deg.C for 24 h;

(2) picking single colony with aseptic toothpick, inoculating into 50mL YEB liquid culture medium containing antibiotic (Gen:50mg/L, Kan:50mg/L), culturing overnight at 28 deg.C under shaking at 200 rpm;

(3) inoculating 1mL of the bacterial liquid into 100mL of non-antibiotic YEB liquid culture medium, and shaking the bacterial liquid at 28 ℃ and 200rpm until the OD600 is between 1.0 and 2.0 for later use;

(4) collecting the bacterial liquid, centrifuging at 5000rpm for 15min, discarding the supernatant, collecting the precipitate, re-suspending the precipitate with infection liquid (100mL sterile water +5g sucrose +50 μ L surfactant Silwet L-77), and diluting until OD600 is approximately equal to 0.8;

(5) cutting off the opened flowers, and only keeping the flowers with white flowers; the inner wall of the incubator is sprayed with water (humidity is maintained for the infection process) by a water spraying kettle. Soaking the scape head into the dye liquor (0.5cm), taking out after 3-5 seconds, putting into an incubator, covering a cover, covering the outer surface of the incubator with black cloth (shading), taking out after about 20 hours, keeping overnight in a dark environment, and then transferring to a normal growth environment.

4 identification of transgenic Arabidopsis thaliana

(1) The T0 generation transgenic seeds are disinfected by 20 percent sodium hypochlorite for 10min, washed by clean water for 5 times, sowed on a culture medium plate containing Kan 50mg/L for screening, and the successfully transformed arabidopsis thaliana can normally grow on a culture medium containing antibiotics.

(2) And (3) carrying out PCR (polymerase chain reaction) inspection by taking the DNA of the positive plant as a template to confirm whether the transgenic plant has the inserted target segment.

5 transgenic Arabidopsis GUS tissue staining analysis and enzyme activity determination

And (3) carrying out GUS histochemical staining identification on the screened T3 generation homozygous Arabidopsis plants, taking 1-15 days after germination of the Arabidopsis seedlings and inflorescences, leaves, stems and siliques at the reproductive growth stage, adding prepared histochemical GUS staining solution to enable the Arabidopsis seedlings and the inflorescences, leaves, stems and siliques to submerge materials, and placing the Arabidopsis seedlings and the inflorescences in a 37 ℃ thermostat for overnight staining. Decolorizing with 70% ethanol until the solution is colorless, eliminating color interference, and observing under microscope.

GUS plants, namely 10-day-old pCpWRKY71, are used for detecting the inducible expression characteristic of the CpWRKY71 promoter. Seedlings were treated with high temperature, low temperature for 6h, 15% PEG6000, 200mM NaCl for 12h, 50. mu.M ABA, 50. mu.M GA, 100. mu.M SA, 100. mu.M MeJA for 6h, and untreated seedlings were used as controls. The treated Arabidopsis plants were ground into powder in liquid nitrogen, and then put into a 1.5mL centrifuge tube, and 600. mu.L of GUS enzyme extract was added. Centrifuge at 12000rpm for 10min at 4 deg.C, and take the supernatant to a new centrifuge tube. Protein concentration was determined using the Bradford method protein concentration assay kit. GUS activity of each sample was measured with a microplate reader under conditions of excitation light 365nm, emission light 455nm, and slit 10 nm.

The results show that the vernalized seeds are not stained blue. Subsequently, GUS staining was detected in Arabidopsis at different developmental stages. The embryonic root after the seed germination and the overground part of the 3-15-day-old arabidopsis seedling can be dyed with dark blue, and the GUS activity of the root is weaker. After entering the reproductive growth stage, GUS activity gradually increased in inflorescences with flower opening, and was the most active in mature and senescent flowers. Further analyzing GUS activity in mature flowers, GUS expression activity of stamens, pistils and abscissas is strong. GUS staining was stronger in senescent leaves than in young leaves. GUS staining was weak in the stem and was barely detectable. In mature siliques, GUS staining was mainly concentrated at the top and abscission of the siliques. The tissue-specific expression pattern of GUS gene driven by CpWRKY71 promoter is identical to the expression pattern of CpWRKY71 in different tissues of Chimonanthus praecox, and the expression levels of CpWRKY71 in the aged flower and leaf are high, which indicates that CpWRKY71 may be involved in the regulation of flower senescence and leaf senescence of Chimonanthus praecox (FIG. 6).

The CpWRKY71 promoter contains cis-acting elements associated with abiotic stress and hormones. To further investigate the transcriptional regulatory properties of these cis-acting elements in the CpWRKY71 promoter, pCpWRKY71 was analyzed for GUS activity in GUS transgenic Arabidopsis seedlings under hormonal and abiotic stress treatment (FIG. 7). The results show that GUS activity is obviously improved after the seedlings are treated by low temperature, salt stress, salicylic acid SA and abscisic acid ABA. After high temperature and methyl jasmonate MeJA treatment, GUS activity is obviously reduced. The results are consistent with the expression pattern of the CpWRKY71 gene after abiotic stress and hormone treatment. However, GUS activity was not altered after gibberellin GA treatment.

Example 5 Agrobacterium mediated transformation of the Chimonanthus Nitns CpWRKY71 Gene (Arabidopsis)

1 construction of expression vector

According to experimental requirements, the modified 1300 vector (present in Yangxian university gardening garden school Yang Jianfeng) was used in this example, and the cDNA fragment of CpWRKY71 was ligated into the expression vector by using the double enzyme digestion method.

2 chimonanthus nitens CpWRKY71 gene promoter fusion plant expression vector transferred into agrobacterium tumefaciens

See example 4 for specific procedures.

3 inflorescence infection method for transforming arabidopsis thaliana

See example 4 for specific procedures.

4 identification of transgenic Arabidopsis thaliana

(1) The T0 generation transgenic seeds are disinfected by 20% sodium hypochlorite for 10min, washed by clean water for 5 times, sowed on a culture medium plate containing Hyg 25mg/L for screening, and the successfully transformed arabidopsis thaliana can grow normally on a culture medium containing antibiotics.

(2) And (3) carrying out PCR (polymerase chain reaction) inspection by taking the DNA of the positive plant as a template to confirm whether the transgenic plant has the inserted target segment.

5 real-time quantitative RT-PCR

The transgenic plant leaves were used as material, RNA extraction, reverse transcription of cDNA and quantitative PCR were performed as described in example 2.

6 results of transgene expression

The expression levels of exogenous genes in different transgenic strains are detected by an RT-PCR method, a strain OE-1 with the highest expression quantity and a strain OE-2 with the medium expression quantity are selected from the strains (figure 8), after T3 generation homozygotes are obtained by screening, Wild Type (WT) and empty vector transfer plants (EV) are used as controls, and phenotype observation and subsequent tests are carried out.

Under normal growth conditions, leaves of plants OE1 and OE2, overexpressed by CpWRKY71, exhibited a pre-senescent phenotype (fig. 9). In addition to the leaf senescence observed in OE1 and OE2, the senescence time of the entire plants was also significantly advanced. The arrest of apical meristem growth is a marker for whole plant senescence, therefore, we counted apical meristem growth for transgenic plants, wild type and blank control plants. The results showed that OE1 and OE2 had no flower buds on days 48.37 and 49.18, respectively, whereas WT and EV plants had no flower buds on days 53.62 and 54.01, respectively (fig. 10). The above results indicate that CpWRKY71 plays an important role in the senescence process of leaves and whole plants.

Sequence listing

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<120> chimonanthus nitens WRKY transcription factor gene CpWRKY71 and cloning and application of promoter thereof

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tattcatatt aggtgggtag tacacaaccc attaccaaac gcttggccta cccattggcc 180

aaactataag gagcaaattt aatttggttg ccaaatacca tttgacaaga actggtcagg 240

ctttgtagca cacatcagta catgcattca caccatagcc catatgggcc atgaaaagta 300

gagcacgatg cttgagccat gtgggtaaag ttcaccaata tggtgtgcac ataactggtg 360

gtcggctgca acccttctct cctccaaacc agcaacctat tcgtacatat ccgtagctgc 420

atacatggat ggatatgtgt gaatgcggat acaccagcac atttttcgaa aagggatgtg 480

ctctatatga agtagaaatg agagtttttt attagtttct ttgatgcaat ttagttgatc 540

cggactttta aaattatatt aaaagtttta ggttcaacta tttctgaaat tatctgatac 600

atgagactta acagatggtt ttatacccat tttatatata aaaaaaaccc tcatttttat 660

aacaaaatac aaatcaaaac atctaagaag tgttttccaa ctctttcttg ggcctcttgc 720

ctttttttta tattcacatg tgtctgtgtc agcaagattc cttcttgtgc atagacctaa 780

caaaaacctg cccacccctt cccttgattg ggacgtttga aggcccaaat gccatacgta 840

taatacaaac acctacaagg tcaacaatct catgctccta ttttagtctt ctcccccatc 900

aaaagtcgtc tctctctctc tctctctctc tgctttgaac aacaccagag actcgacacc 960

gtcatctctt caaaacttgt actgtagttg gtcatacaaa tgttcttcta gaccctcttt 1020

ctttccacta ttctattttt tttattcaac tactataaca tctattatca ttcatttgct 1080

attgttttat attaaacatg tacccaatac tcagttttct cttgtgggca tcatttttct 1140

ctatttgact cttatcaaag agaatgagaa gacgggcaat acgcttctaa tatgtacaaa 1200

tctgatgaca aagactgaga aattgtgccg atgactcagg catgacaagc tgcatacctc 1260

cccttgttta tgagaaatgg agcaatcttg agagtattaa tacctactgt tgtgcgaaat 1320

ctcaataagt caaacaacgg aagcgattca aacataaaca caattcaaac aattatccac 1380

aatagagagg acatagattt acatggttcg gccgaaacct acatccactt gcagcacagg 1440

aaaaaaaatt cactaatatc aaagtggaga tacaaattta caaatgttct taaactcatt 1500

ttctcacatt cgtctctctc actcaaatta atctctcgat tgctcgagag aggctacaat 1560

gccaaagcca aaccacaaca ctctcaccgt ctctcactat atatagtttt catataaaat 1620

gagaaagaaa ttgagtcgga ccataaacga gccaagtcga gacaatattc taacacgtac 1680

cacttgtatc aggggccatc acctgatgca aaagtttgat gtatgagttg atgatgaagt 1740

gatgtgaatc ttgaccaaga aaaaaataat gtacagcatc ttcaatgttt agggttcgag 1800

acaacttagc gcaattgtca aactttgatc gaaatgacag tcccaccggg aagtgactgc 1860

cgatggtgga cccggtgcct gaaaagcatc tgatgaacgt caaacagcca tgaataagcc 1920

ccacacctcc atctcttgaa gatgaaagtc accattgtgt catcacaagc gagcacttca 1980

tatatgagac tttttttttt tctcaatttc aatggtgaat gaatcgtgca attcttacta 2040

aaaattgtct atcacacagt agctgattaa attcataaca ttctgactta ggttataaaa 2100

atatgaaaca tgtttcctaa acttcattca atctagaagc atatgtaaca tgaattatta 2160

atgtagaaac acatgcaaca cgaaatagaa agactgggtt gcttctttct gcacatttct 2220

ttatttttcc ctctttccat tttctaaagt ttaaaaaaga cttcttcaaa gatactaaaa 2280

gcaatttgta aaaaggcagt caaaaatgtg gctcaaacga tattattgaa tgttgaatat 2340

ctcaagcagc gtgctttcat ggcagtgtca ttattgtaat ttgcgtgcaa acaggtaatt 2400

cggtgcagct caggaccgtt ggtagaaagc ctgctccaat cccctttgtg ggacagcttc 2460

atttaattta aagaataata ttgaatgttt atggaattaa atcaagcagg gtgtctgtat 2520

ggattttcat gaaaatcaag ttttctaata taattcaaaa aaataaggtc tctacattta 2580

ataatgattg atttgtgtgt aagttttctc atattacagt atttttataa aattcagttt 2640

ttttttacaa attatagtag aaaatacacg ttactatagg atattaaaga atatgtatat 2700

tatattatga gtttcttcgt atttgtttcc tattttctaa aattttatat ttttttaaaa 2760

tttgagaaca tctttattcg tatcaaaatt ttgactctaa gggacttgat acttcataaa 2820

aaatgacact cttttaagaa aaatatgctt agactaggtt ttcaattttt ttctaatttt 2880

ctaatatttt tatctacttt ttcaaaaaaa aattaaaaaa caaaaaaata tctaataaaa 2940

agagaaagaa tgcatatcta aattataatg ttgatgtact acttagcgcc gatactccta 3000

tattattagt aataagtaac aatatgtatg gaccgattct tttcttaacc cctgatacac 3060

attagtcacg cttacttatc aaatcatagt tggattgact agatttaaac tcacattaaa 3120

ataattgaat attgaattta aatacatcta aatttaaaat ttaacatttc tcaaatctgc 3180

ttcaaactta gtacagtcag atttttaaat ctttggtact attaagttcc atgcacatct 3240

aaattatttt tcttttttta tgcctacgtt gtctagatct tttagaacat ctgattaatt 3300

ctagtggcat atataagcct ctttacctat tcacactatg taattctacg aagtggccct 3360

ttgacattaa gtgagaatcg aatccatgat ttttaccacc taaattgagt ttttgacaat 3420

cactctaatc attttgaata ttctatagat gcgaattcat tagactaacg ttataaaaga 3480

acagtaatga ttcatcacat tttttagaac ggttttgtct ctaccgttga tctagtgtga 3540

tgataaagaa aaattcggcg acgcccaccg aaaaagtagg gcactaatta gaccgtccag 3600

atccatcaca atcctcacca atcaaagggc gggtaccatt tagtacatag gtagctgacg 3660

tgcaccgact cccaacctac gtctttgacg cttcattgac accggaggcg tcacccattt 3720

gtttgtcatt ctagcccaca ctcatattat atgtgacgtc cacgccccat cagcaacccc 3780

cacgtatcat ctaggctgta ccttcccacg tgccaaacga cagaatccag ctacagtaaa 3840

aagccggtgc taaaacggac gcgtgtcggt gtgatctccc caggcccaaa tctttgaaac 3900

attgaccgat ccccgtcacg ggaccggcac tagccggtgg tcgttgctga ctcataatat 3960

attgcgaccg tagactattc aaacatcaaa aggagaagaa aactcacaca agaaagagag 4020

tgatatgaag ggattcccac acacacactt cttttctttt ccatacaaaa aaaaccaaag 4080

ctaaacctct tccctctctc ctcttatctc taaccctttc tttcttttca cgctctcctt 4140

ctctctgctc ctctctcccg caccagaccc 4170

<210> 3

<211> 316

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ser Glu Asn Arg Asp Ser Ser His His Asp His Tyr His Asp Asp

1 5 10 15

Asn Met Gly Ile Gly Ser Met Gly Gly Phe Gly Phe Ser Phe Ser Asn

20 25 30

His Pro Ser Thr Ile Tyr Ser Asn Met Ser Pro Met Ala Pro Glu Ser

35 40 45

Gln Pro Met Arg Asp Gln His Gly Phe Asp Pro Ser Pro Tyr Met Ser

50 55 60

Phe Thr Glu Cys Leu Gln Ala Pro Met Asp Tyr Gly Ser Leu Ser Arg

65 70 75 80

Ala Phe Asp Phe Ser Pro Ser Ser Ser Asp Val Phe Gly Ser Gly Asp

85 90 95

Gly Gly Gly Val Val Asn Glu Leu Val Asp Ser Arg Gly Pro Ser Gly

100 105 110

Asn Pro Ala Thr Pro Asn Ser Ser Val Ser Ser Ser Ser Thr Glu Ala

115 120 125

Ala Ala Glu Glu Glu Ser Ser Arg Cys Lys Lys Asp Gln Gln Pro Lys

130 135 140

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

145 150 155 160

Lys Lys Gly Glu Lys Arg Pro Arg Glu Pro Arg Phe Ala Phe Met Thr

165 170 175

Lys Ser Val Val Asp His Leu Glu Asp Gly Tyr Arg Trp Arg Lys Tyr

180 185 190

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

195 200 205

Cys Thr Ser Gln Lys Cys Ser Val Lys Lys Arg Val Glu Arg Ser Phe

210 215 220

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

225 230 235 240

Pro Ser Pro Ala Ser Ile Arg Gly Ser Ala Ser Gly Val Leu Ala Pro

245 250 255

Ser Leu Leu Thr Ser Ala Pro Gln Met Pro Ser Phe His His Glu Phe

260 265 270

Met Met Pro Thr Asn Ile Gln Gly Asp Pro Ser Ser Met Tyr Leu Gln

275 280 285

Asn Phe Ala Thr Gln Gln Gln Phe Gln Thr Pro Asp Tyr Gly Leu Leu

290 295 300

Gln Asp Ile Val Pro Ser Phe Ile Pro Arg Gln Pro

305 310 315

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aagctaaacc tcttccctct 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccacactagg atgttggttc 20

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aggctaagat tcaagacaag g 21

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttggtcgcag ctgattgctg tg 22

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtgcatctct atccacatcg 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

caagcttcct tatgcgatcc 20

<210> 10

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgaccactac cacgatgaca aca 23

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggattgccac tgggtcctct a 21

<210> 12

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gccaacactc cagaagcact ccc 23

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

catcgccaga gccgaacacg 20

Claims (13)

1. The Chimonanthus praecox WRKY transcription factor gene CpWRKY71 is characterized in that: the coded protein has an amino acid sequence shown as SEQ ID No. 3.

2. The Chimonanthus praecox WRKY transcription factor gene CpWRKY71 as claimed in claim 1, wherein: has a nucleotide sequence shown as SEQ ID No. 1.

3. The promoter of the Chimonanthus praecox WRKY transcription factor gene CpWRKY71 is characterized in that: has a nucleotide sequence shown as SEQ ID No. 2.

4. Use of the chimonanthus nitens WRKY transcription factor gene CpWRKY71 in regulating chimonanthus nitens senescence according to claim 1 or 2, and/or use of the chimonanthus nitens WRKY transcription factor gene CpWRKY71 promoter in regulating chimonanthus nites stress tolerance or senescence according to claim 3.

5. Use according to claim 4, characterized in that: the adversity stress is high temperature, low temperature, drought, salt stress, abscisic acid ABA, salicylic acid SA, gibberellin GA or methyl jasmonate MeJA treatment.

6. Use according to claim 5, characterized in that: the high temperature is 42 ℃.

7. Use according to claim 5, characterized in that: the low temperature is 2 ℃.

8. Use according to claim 5, characterized in that: the drought is 15% PEG6000 treatment.

9. Use according to claim 5, characterized in that: the salt stress was 200mM NaCl treatment.

10. Use according to claim 5, characterized in that: the abscisic acid treatment is 50 mu M ABA.

11. Use according to claim 5, characterized in that: the salicylic acid treatment was 100 μ M SA.

12. Use according to claim 5, characterized in that: the gibberellin treatment was 10. mu.M GA/50. mu.M GA.

13. Use according to claim 5, characterized in that: the methyl jasmonate treatment was 100. mu.M MeJA.

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