Camellia sinensis MYB transcription factor gene CsMYB4 and application thereof

Abstract

The application discloses a tea tree MYB transcription factor gene CsMYB4 and application thereof, and belongs to the technical field of biotechnology.The CDS sequence of the gene is shown as SEQ ID NO:1, and the gene encodes a MYB transcription factor.The CsMYB4 gene has the function of improving the fungal resistance of plants, the fungal resistance gene CsMYB4 is constructed on a plant expression vector and is introduced into Nicotiana benthamiana to perform transient overexpression, or is stably genetically transformed into tobacco plants, and all have strong in-vitro fungal resistance activity, the transgenic tobacco with overexpression of CsMYB4 has strong bacteriostatic activity on Botrytis cinerea and Epicoccum sorghinum, after the CsMYB4 gene is silenced by using a virus induced gene silencing (VIGS) technology, the growth of the plants is hindered, and the disease resistance is greatly weakened.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology and genetic engineering, and relates to a tea tree MYB transcription factor gene CsMYB4 and application thereof. BACKGROUND

[0002] Tea tree is one of the most important economic crops in the world, widely planted in tropical and subtropical regions. Tea leaf diseases have a serious impact on tea yield, and there is currently a lack of effective prevention and control measures. Among them, tea leaf spot caused by Didymella bellidis is isolated from the organization of tea leaf spot in Guizhou Province, Yujing County, Songyan Town, Erlong tea area (Wang, X., Yin, Q. X., Jiang, S. L., Wu, X., Wang, D. L., Song, B. A., Chen, Z. First report of Didymella bellidis causing tea leaf spot in China. Plant Dis. 2020, 104: 1254.). The pathogen can infect tea shoots, young leaves and mature leaves. Due to the lack of effective control measures for leaf spot caused by D. bellidis, the disease seriously affects the quality and yield of tea. D. bellidis can also cause leaf spot in daisy (Bellis perennis), chrysanthemum (Chrysanthemum x morifolium), water chestnut (Eleocharis dulcis), white peony (Angelica dahurica), angelica (Angelica gigas), kiwi fruit, etc. (Chen, Q., Jiang, J. R., Zhang, G. Z., Cai, L., Crous, P. W. Resolving the Phoma enigma. Stud. Mycol. 2015, 82: 137-217; Lv, R., Zheng, L., Zhu, Z., Pan, L., Huang, J., Hsiang, T. First report of stem blight of Eleocharis dulcis caused by Phoma bellidis in China. Plant Dis. 2011, 95 (9): 1190; Xu, H. J., Cui, J. C., Zhou, R. J., Fu, J. F., Hao, N. First report of leaf spot disease in Angelica dahurica caused by Phoma bellidis in China. J. Phytopathol. 2016, 164 (7-8): 448-454; Liu, Y. H., Zhang, C. Q., Dai, D. J.First report of leaf black spot on white chrysanthemum (Chrysanthemum morifolium) caused by Phoma bellidis in China. Plant Dis. 2019, 103(9): 2475.; Lee, D.H., Choi, K.M., Jung, C.R., Lee, S.H. First report of Didymella bellidis causing leaf spots on Angelica gigas in South Korea. J. Plant Pathol. 2020, 102: 1297; Zou, M.F., Wang, Y.X., Yan, M.F., Zhou, Y., Xiong, G.H., Jiang, J.X. First report of leaf spot on kiwifruit caused by Didymella bellidis in China. Plant Dis. 2020, 104(1): 287). Traditional disease control methods mainly include pesticide application and breeding of elite varieties. Pesticide application has risks such as human and livestock safety and environmental pollution. Breeding of elite varieties has long time, slow effect, large investment, and difficulty in exploring disease-resistant genetic resources. Transcriptomics, gene silencing, and gene overexpression technologies can quickly find disease-resistant genes and are a method for quickly exploring disease-resistant resources.

[0003] During growth and development, plants have evolved various defense mechanisms to resist the attack of pathogens and pests. Plant hormones can regulate the growth and development of plants, and can also induce plants to produce disease-resistant substances, such as pathogen-related proteins (PR). Transcription factors (TF) play an important role in regulating gene expression and inducing disease-resistant substances (Wani, S. H., Anand, S., Singh, B., Bohra, A., Joshi, R. WRKY transcription factors and plant defense responses: latest discoveries and future prospects. Plant Cell Rep. 2021, 40(7): 1071-1085). Studies have shown that the regulation mechanism is divided into three levels, namely transcriptional level regulation, post-transcriptional level regulation and translational level regulation. Transcription factors are a class of proteins that can bind to specific gene sequences, which can bind to the upstream of the gene and regulate gene transcription. There are many transcription factors in plants, such as bHLH (basic helix-loop-helix), bZIP (basic leucine zipper), Zinc-finger and MYB (v-myb avian myeloblastosis viral oncogene homolog); among them, MYB is the largest class of transcription factors.

[0004] MYB transcription factors, as the largest family of transcription factors in plants, play an important role in plant stress resistance. In plant stress response, the regulation of functional gene expression by transcription factors is a key link in plant stress response (Zhu, T., Zhou, X., Zhang, J. L., Zhang, W. H., Zhang L. P., You C. X., Jameson P. E., Ma P. T., Guo S. L. Ethylene-induced NbMYB4L is involved in resistance against tobacco mosaic virus in Nicotiana benthamiana. Mol. Plant Pathol. 2022, 23(1): 16-31.). Transcription factors directly regulate the expression of target genes by binding to the cis-acting elements of the promoter region of downstream genes, or form homodimers or heterodimers, or interact with other proteins to form an activated form, thereby participating in the signal transduction pathways of jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), etc., forming a regulatory network of gene expression. For example, AtMyb30 in Arabidopsis is a transcription factor expressed in the early stage of hypersensitive response (HR). In Arabidopsis and tobacco, through the overexpression and gene inhibition experiments of this gene, it is shown that AtMyb30 is a positive regulator of HR response when plants are infected by pathogens, and its expression depends on the content and accumulation of SA in plants, but not on NPR1 (nonexpressor of pathogenesis-related genes 1). In addition, Myb1 gene in tobacco can regulate PR genes downstream of salicylic acid signaling pathway and participate in the activation of PR genes and plant defense response.In addition, MYB-type transcription factors such as Arabidopsis AS1 (asymmetric leaves 1), Antirrhinum majus PHAN (phantastica), the Nicotiana tabacum ortholog of PHANTASTICA, and Zea mays RS2 (rough sheath 2) can be involved in the jasmonic acid signaling pathway and plant defense response, promote overexpression of disease resistance functional genes, and reduce the damage caused by pathogenic bacteria to plants (Li J., Han G., Sun C., Sui N. Research advances of MYB transcription factors in plant stress resistance and breeding. Plant Signal Behav. 2019, 14(8): 1613131. doi: 10.1080 / 15592324.2019.1613131).

[0005] MYB72 gene-deficient Arabidopsis thaliana is constructed by gene knockout method, and the disease resistance of mutant plants to Pseudomonas adaceae, Peronospora brassicae gaumann, Alternaria alternata and Botrytis cinerea is reduced, which indicates that MYB72 of Arabidopsis thaliana plays an important role in plant disease resistance (Van Wees, S.C., Van der Ent, S., Pieterse, C.M. Plant immune responses triggered by beneficial microbes. Curr. Opin. Plant Biol. 2008, 11(4): 443-448.); by constructing SSH library (suppression subtractive hybridization cDNA library) of grapevine induced by Erysiphales, it is found that the expression of MYB transcription factor and multiple disease resistance-related genes (Fekete, C., Fung, R.W., Szabo, Z., Qiu, W., Chang, L., Schachtman, D.P., Kovacs, L.G. Up-regulated transcripts in a compatible powdery mildew-grapevine interaction. Plant Physiol Biochem. 2009, 47(8): 732-728). This indicates that MYB may be involved in the defense of grapevine against Erysiphales. Therefore, application of MYB transcription factor to improve the disease resistance of plants has developed into an important technology for plant disease resistance. SUMMARY

[0006] The technical problem to be solved by the present application is to provide disease resistance genetic engineering application of tea tree MYB transcription factor gene CsMYB4, which is from tea tree and can be introduced into plants as a target gene to improve the disease resistance of tobacco, tea tree or vegetables against Botrytis cinerea and Sphaerodes sorghi, and to improve plant varieties.

[0007] The present application clones cDNA of MYB transcription factor gene CsMYB4 with antifungal activity from tea tree, and the nucleotide sequence of CsMYB4 is shown as SEQ ID NO:1, the full length of the gene is 810 bp, and the amino acid sequence is shown as SEQ ID NO:2.

[0008] The present application clones cDNA of MYB transcription factor gene CsMYB4 with antifungal activity from tea tree, and the nucleotide sequence of CsMYB4 is shown as SEQ ID NO:1, the full length of the gene is 810 bp, and the amino acid sequence is shown as SEQ ID NO:2.

[0009] The expression vector described in the present application is obtained by inserting the tea tree MYB transcription factor gene CsMYB4 into the XbaI and SacI enzyme cutting sites of the expression vector pBI121. The target gene is introduced into tobacco for overexpression by Agrobacterium tumefaciens mediation, and whether the gene has the function of resisting fungi is verified by the transgenic plants. In the later stage, the gene can be used to improve tobacco, tea tree or vegetable plants and the like to make them obtain the ability to resist diseases; the inventor names the gene as CsMYB4.

[0010] The CsMYB4 gene described above can be applied to improve the anti-fungal effect of tobacco, and the specific technical scheme is as follows:

[0011] Specific primers for amplifying CsMYB4 are used to extract total RNA from mycelium after inoculation of D. bellidis, and the full-length coding region of CsMYB4 is amplified by RT-PCR, and then the full-length coding region is connected to the vector, and the cloning with the target gene is obtained by sequencing.

[0012] The pBI121 vector and the plant expression vector are cut by restriction endonuclease XbaI and SacI, and the target gene fragment and the vector large fragment are recovered; then the obtained CsMYB4 gene fragment is connected with the pBI121 vector fragment to construct a plant overexpression vector; then the constructed recombinant vector is transferred into Agrobacterium by freeze-thaw method, and is injected into tobacco for transient overexpression; 6 days later, Botrytis cinerea and Clavibacter michiganensis are inoculated, the lesion area after inoculation for 3 days is recorded, and the data is subjected to significance analysis. When Botrytis cinerea is used, the average lesion area of the wild-type tobacco is 2.31 cm 2 , and the average lesion area of the overexpressed tobacco is 3.38 cm 2 ; when Clavibacter michiganensis is used, the average lesion area of the wild-type tobacco is 1.54 cm 2 , and the average lesion area of the overexpressed tobacco is 1.01 cm 2 , which has obvious disease resistance effect.

[0013] The technical method for obtaining the transgenic plants is as follows:

[0014] 1) Construction of overexpression vector

[0015] According to the cDNA sequence SEQ ID NO:1 of the tea tree MYB transcription factor gene CsMYB4 and the XbaI and SacI enzyme cutting sites of the vector, homologous recombination primers containing the homologous arms of the overexpression vector are designed, and the primer sequences of P3 and P4 are SEQ ID NO:3 and SEQ ID NO:4 respectively;

[0016] The cDNA of CsMYB4 was used as a template for polymerase chain reaction (PCR) amplification. The purified product was introduced into the linearized vector pBI121 digested by restriction endonuclease sites XbaI and SacI, and sequencing was performed to ensure that the reading frame sequence of the coding region in the expression vector was correct.

[0017] 2) Construction of VIGS expression vector

[0018] According to the CDS sequence of the tea tree MYB transcription factor gene CsMYB4, the homologous gene ID in Nicotiana benthamiana is Niben101Scf00448g02011.1. The gene is MYB4-Like in NCBI (National Center for Biotechnology Information) alignment. The target sequence required for silencing is designed using the Solanaceae database (https: / / solgenomics.net / ). The CDS sequence and the target sequence are shown in SEQ ID NO: 5 and SEQ ID NO: 6. According to the PstI restriction enzyme site of the VIGS vector Pash18 (TRV2), the VIGS primers P7 and P8 are designed. The primer sequences of P7 and P8 are SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0019] The homologous gene of CsMYB4 in tobacco was used as a template for PCR amplification and purification. The target gene was connected to the linearized vector pBI121 digested by restriction endonuclease site PstI, and sequencing was performed to ensure that the reading frame sequence of the coding region in the expression vector was correct.

[0020] 3) Obtaining of transgenic plants

[0021] The expression vector pBI121-CsMYB4 obtained in step 1) was transformed into Agrobacterium (GV3101), which was further introduced into Nicotiana benthamiana. The transgenic plants were verified by PCR and RT-qPCR to be positive and expressed in large quantities in the plants. The transgenic T2 generation plants growing to 3-4 leaf stage were inoculated with Botrytis cinerea, and the lesion area of the diseased leaves of the plants was recorded 3 days after inoculation. Compared with the control, the transgenic T2 generation plants had stronger disease resistance.

[0022] 4) Obtaining of VIGS silenced plants

[0023] The expression vector TRV2 obtained in step 2) is transferred into Agrobacterium, and the obtained silencing plants are obtained by injecting the TRV1 into Nicotiana benthamiana, the expression amount of the CsMYB4 homologous gene in the plants is verified by using reverse transcription quantitative-PCR (RT-qPCR), the plants subjected to gene silencing are selected to perform disease resistance analysis, the silencing plants grown for 14 days are inoculated with Botrytis cinerea, the disease incidence of the plants is recorded when the plants are inoculated with the pathogenic bacteria for 3 days, the size of the disease spot area of the leaves is compared with that of the control, it is found that the growth of the plants subjected to gene silencing is hindered, and the disease spot is large, which indicates that the gene has the disease resistance function.

[0024] The application provides a new method for improving the resistance of plants to fungal diseases, and the disease-resistant plants can be cultivated by using genetic engineering, so that the disadvantages of traditional breeding can be overcome, the breeding cycle is shortened, the operation is simple, and high-resistance materials can be easily obtained; the CsMYB4 gene from the tea tree in the application can enhance the resistance of plants to several pathogenic fungi, and the gene can be introduced into tobacco to produce new varieties and new materials with fungal resistance. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 PCR detection results of the CDS of the CsMYB4 gene from the tea tree in the application;

[0026] Figure 2 Expression amount analysis results of CsMYB4 in the tobacco with transient overexpression of the CsMYB4 gene in the application, wherein CK is the PCR product with the wild-type tobacco total RNA reverse transcription cDNA as a template; and the positive control is the PCR product with pBI121-CsMYB4 as a template;

[0027] Figure 3 Expression amount analysis results of the transcription level of CsMYB4 in the tobacco with the transgenic CsMYB4 in the application, wherein WT is the relative expression amount of the non-transgenic tobacco; and CsMYB4 is the relative expression amount of CsMYB4 in the transgenic tobacco;

[0028] Figure 4 Inhibition activity phenotype diagrams of the tobacco with overexpressed CsMYB4 and the pBI121 control tobacco after inoculation with pathogenic bacteria, wherein A is the front side of the Botrytis cinerea pathogenic leaf; B is the front side of the Claviceps fusiformis pathogenic leaf; and C is the back side of the Claviceps fusiformis pathogenic leaf;

[0029] Figure 5Comparison of lesion area and gene expression levels between tobacco and pBI121 after transient overexpression of CsMYB4, where A is the comparison of lesion area; B is the comparison of gene expression levels;

[0030] Figure 6 This is a phenotypic comparison diagram of tobacco plants after silencing MYB4-Like using VIGS in the present invention and control tobacco plants;

[0031] Figure 7 This is a comparison of the lesion area and gene expression levels of the TRV2 negative control and CsMYB4 silenced tobacco plants after inoculation with pathogens using VIGS in the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described below by way of examples, but the scope of protection of the present invention is not limited to the contents described. In the examples, the methods are operated according to conventional methods unless otherwise specified, and the reagents used are conventional reagents or reagents prepared according to conventional methods unless otherwise specified.

[0033] Example 1: CsMYB4 full-length cDNA cloning and sequence analysis

[0034] The CDS sequence of the pathogen-like interaction pathway CsMYB4 gene in tea plants was downloaded from the Tea Plant Genome Database of Anhui Agricultural University (http: / / tpia.teaplant.org / index.html). Total RNA from healthy and infected tea leaves was extracted using TRIzol reagent, and the RNA quantity and purity of each sample were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). Reverse transcription was performed to generate the first-strand cDNA, which was used as a template. PCR was performed using homologous primers for amplifying CsMYB4. The upstream and downstream homologous primers used were SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Taq HS (0.25 μL), dNTP Mixture (4 μL), 10× PCR Buffer (5 μL), DNA template (2 μL), upstream and downstream primers (1 μL), and ddH2O (36.75 μL) were used. The PCR program was set to 94°C pre-deformation for 2 min, 98°C denaturation for 10 s, 55°C annealing for 30 s, and 72°C extension for 50 s for 35 cycles, followed by 72°C post-extension for 5 min. After the PCR was completed, 50 μL was taken for gel excision and purification. The gel excision and purification were used. DNA Gel Extraction Kit (Beijing Qingke Biotechnology) was used for purification. The specific steps are shown in the instructions. After purification, 5 μL of the product was taken for agarose gel electrophoresis, and the concentration of the target product was detected and stored for later use.

[0035] Case Study 2: Application of Overexpression of CsMYB4 Gene for Disease Resistance in Plants

[0036] The plant expression vector pBI121 plasmid was extracted using the SanPrep column-based plasmid DNA miniprep kit (Shanghai Biotech). The extraction steps were described in the kit instructions. The two cloning sites on the pBI121 vector, XbaI and SacI, were double-digested to linearize the vector. 1 μL of the extracted plasmid was used for 1.5% agarose gel electrophoresis to check the integrity and concentration of the extracted plasmid. DNA Gel Extraction Kit (Beijing Qingke Biotechnology) was used for purification (see the instructions for specific steps), and the size of the recovered fragments and the concentration were determined by 1.5% agarose gel electrophoresis. The IIOne Step Cloning Kit (Nanjing Novozymes) and its instructions were used to determine the concentrations of the vector fragment and the target gene (the gene fragment in Case 1). Homologous recombination was performed using the following reagents: 5×CEⅡ Buffer (2μL), ExnaseⅡ (1μL), linearized vector (6μL), and target gene fragment (1μL). The reaction was incubated at 37°C for 30 min. Immediately after the reaction, the cells were placed on ice and transformed into Escherichia coli DH5α. After ligation and transformation, the cells were plated onto LB solid medium containing Kana antibiotics (LB preparation method is attached on the end page). The culture suspension was shaken on a shaker for 12 hours and then tested. The culture suspension of positive clones was sent to Qingke Biotechnology for sequencing to ensure that the target sequence was successfully ligated into the pBI121 vector, thereby obtaining an overexpression vector.

[0037] The constructed recombinant vector was transferred into Agrobacterium by freeze-thaw method and injected into tobacco for transient overexpression. Six days later, the plate of Botrytis cinerea and E. sorghum were inoculated respectively. The lesion area was recorded 3 days after inoculation and the significance of the data was analyzed. When Botrytis cinerea was used for pathogenicity, the average lesion area of ​​wild-type tobacco was 2.31 cm 2 , while the average lesion area on the overexpressed tobacco was 3.38 cm 2 When E. sorghum was used as the pathogen, the average lesion area of ​​wild-type tobacco was 1.54 cm 2 The average lesion area of ​​overexpressing tobacco was 1.01 cm 2 , has obvious disease-resistant effect.

[0038] Implementation Case 3: Construction of VIGS Vector

[0039] The present invention also discloses silencing CsMYB4 using VIGS technology, and determining the function of the CsMYB4 gene after silencing it. The specific implementation scheme is as follows:

[0040] The CDS sequence of CsMYB4 gene was downloaded from the tea tree (http: / / tpdb.shengxin.ren / index.html) database, Niben101Scf00448g02011.1 on Nicotiana benthamiana was retrieved as the homologous gene of CsMYB4 gene in the Solanaceae database (https: / / solgenomics.net / ), and was matched as MYB4-Like on NCBI, and a silent target sequence was designed on the website, and the CDS sequence of MYB4-Like gene was constructed into the TRV2 (Pash18) vector using the Link technology. The constructed vector was transformed into Agrobacterium (GV3101). The Agrobacterium solution containing TRV2-MYB4-Like, PDS, TRV2, and TRV1 was mixed at a ratio of 1:1, and the Agrobacterium suspension was adjusted using a buffer (buffer preparation method attached at the end), and the OD 600 was 1.0, and was injected into 3-week-old Nicotiana benthamiana, and was placed in a plant climate chamber, and was kept at 25°C and 20°C under day and night conditions, respectively, and was cycled for 16h / d light and 8h / d darkness, and the relative humidity was 70-80%. At 14d after inoculation, the positive control plants showed whitening, and the negative control plants grew more robustly than the silenced plants, the expression amount of the gene was analyzed by RT-qPCR, the efficiency of the silenced plants was about 70%, and the pathogenic phenotype was further observed. The Botrytis cinerea was inoculated on the systemic leaves to observe the change of the lesion area, and the lesion area size was recorded continuously for 3d, the average lesion area of the negative control plants TRV2 was 2.24cm 2 , and the average lesion area of the silenced plants was 2.49cm 2 , indicating that after silencing the homologous gene of the gene of the application, the growth condition of the plants was poor and the disease resistance was weakened, further proving that the homologous gene of the gene of the application has similar disease resistance function.

[0041] Example 4: Agrobacterium GV3101 mediated genetic transformation of plants

[0042] The transgenic receptor of the experiment was Nicotiana benthamiana, and the specific steps of transgenics were as follows:

[0043] 1) Tobacco sterile seedling culture

[0044] The tobacco seeds were immersed with 75% alcohol for 1min, then sterilized with 15% H2O2 for 15min, and then washed with water three times, each time for 3min, and were placed on MS medium in a clean bench, and were cultured in a light incubator at 28°C for about 15d.

[0045] 2) Transformation

[0046] Tobacco leaves were cut into 0.5 x 0.5 cm pieces in a clean bench, and then transferred into the prepared Agrobacterium solution (OD value was about 0.5) for 5 min. After the leaves were dried on sterile filter paper, they were inoculated on MS solid co-culture medium (1 / 2MS + 30 g / L sucrose + 8 g / L agar, pH 5.8) and cultured in a constant temperature box at 22°C in the dark for 2 d.

[0047] 3) Bud induction

[0048] The leaves on the plates were transferred to the induction medium (MS + 0.5 mg / L BA + 30 g / L sucrose + 8 g / L agar + 500 mg / L cef + 100 mg / L Kan, pH 5.8), with the wound surface adhering to the medium, and the front side facing up. After the transfer, the plates were sealed with sealing film and placed in a light incubator for culture. The plates were transferred to the same medium again after about 15 d.

[0049] 4) Rooting

[0050] When small buds grew on the induction medium, they were transferred to the rooting medium (MS + 0.1 mg / L NAA + 30 g / L sucrose + 8 g / L agar + 100 mg / L Kan, pH 5.8) for culture under light conditions to allow the buds to grow and induce rooting. The control plants were wild-type N. benthamiana.

[0051] Example 5: Expression analysis of CsMYB4 in transgenic tobacco and analysis of disease resistance of transgenic plants

[0052] Genomic RNA was extracted from transgenic and wild-type tobacco leaves using a high-purity RNA extraction kit (Beijing Zomanjin). The extraction method is described in the Zomanjin kit manual. The RNA was reverse transcribed using the EasyScript One-Step gDNA Rempvaland cDNA Synthesis SuperMix kit (Beijing Zomanjin). The reagents and their amounts were as follows: RNA (3 μL), Anchored Oligo(dT)18 Primer (1 μL), 2 x TS Reaction Mix (10 μL), TransScript RT / RI Enzyme MIX (1 μL), gDNA Remover (1 μL), RNase-free Water (4 μL). The program was set to 42°C for 30 min and 85°C for 5 s. After obtaining the reverse-transcribed cDNA, RT-qPCR was performed using the Zomanjin kit, and the system was as follows Green qPCR SuperMix (1 μL), P9 (0.2 μL), P10 (0.2 μL), cDNA (1 μL), Nuclease-free Water (3.6 μL) (the primer sequences of P9 and P10 are SEQ ID NO: 9 and SEQ ID NO: 10, respectively), the PCR program is set as 94°C pre-denaturation for 30 s, 94°C denaturation for 5 s, 55°C annealing for 15 s, 72°C extension for 10 s, 40 cycles, and the obtained relative expression result is analyzed by the method of 2 -ΔΔct .

[0053] The laboratory preserved Botrytis cinerea and Sphacelotheca reiliana were inoculated on PDA solid medium and cultured in an incubator at 25°C. Botrytis cinerea was inoculated on leaves when it grew to 3-4 days, and Sphacelotheca reiliana was inoculated on leaves when it grew to 6-7 days. The mycelium was beaten into 6 mm diameter fungus cake and inoculated on the leaves of transgenic tobacco. The mycelium was in contact with the front of the leaves. After continuously recording the lesion size for 3 days, it was found that the transgenic tobacco of CsMYB4 had obvious resistance to the growth of Botrytis cinerea and Sphacelotheca reiliana.

[0054] Reagent preparation method:

[0055] 1) Preparation of LB medium:

[0056] Yeast extract 5 g

[0057] Tryptone 10 g

[0058] Sodium chloride (NaCl) 10 g

[0059] Liquid medium is to place the reagent in a 1L glass beaker, add 1L of ddH2O to the glass beaker, stir with a glass rod until completely dissolved, and then pour into a 250mL capacity conical flask, 100mL of liquid medium per conical flask. Solid LB medium, add 1.5g of agar powder to every 100mL of liquid LB medium.

[0060] 2) Preparation of 1M MES:

[0061] Morpholine ethanesulfonic acid 2.132 g is dissolved in ddH2O 10 mL, and then filtered using a 0.22 μm bacterial filter and stored at room temperature.

[0062] 3) Preparation of 200mM AS:

[0063] Acetyl-syringone 0.039 g is dissolved in dimethyl sulfoxide (DMSO) 1 mL, and after preparation, it is stored in a refrigerator at -20°C.

[0064] 4) Preparation of 1M Magnesium Chloride (MgCl2):

[0065] Magnesium chloride 2.033g was dissolved in ddH2O 10mL, autoclaved at 121°C for 20min, and stored at 4°C after preparation.

[0066] 5) Preparation of 50mg / mL Kanamycin (Kana):

[0067] Kanamycin 0.5g was dissolved in ddH2O 10mL, filtered using 0.22pm bacterial filter, and stored at -20°C after preparation.

[0068] 6) Preparation of 100mg / mL Rifampicin (Rif):

[0069] Rifampicin 0.5g was dissolved in dimethyl sulfoxide 5mL, filtered using 0.22pm bacterial filter, and stored at -20°C after preparation.

Claims

1. The tea tree MYB transcription factor CsMYB4 improves tobacco resistance to Botrytis cinerea ( Botrytis cinerea ) and E. sorghumensis ( Epicoccum sorghinum ) resistance; the nucleotide sequence of the tea tree MYB transcription factor gene CsMYB4 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: Overexpression of the tea plant MYB transcription factor CsMYB4 enhances tobacco resistance to Botrytis cinerea and Ephemerococcus kaoliang.

3. The use according to claim 2, characterized in that: The overexpression vector is obtained by inserting the tea tree transcription factor gene CsMYB4 into the XbaI and SacI restriction sites of the plant expression vector pBI121.

4. Application of the tea tree MYB transcription factor gene CsMYB4 in breeding tobacco resistant varieties resistant to Botrytis cinerea and Echinococcus kaoliang by genetic engineering means. The nucleotide sequence of the tea tree MYB transcription factor gene CsMYB4 is shown in SEQ ID NO:

1.

5. The use according to claim 4, characterized in that: The following steps are involved: 1) Construction of overexpression vector Based on the cDNA sequence of the tea plant MYB transcription factor gene CsMYB4 (SEQ ID NO: 1) and the vector's XbaI and SacI restriction sites, homologous recombination primers were designed. These primers contained homology arms of the overexpression vector. The primer sequences for P3 and P4 were SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Using CsMYB4 cDNA as a template, the amplified product was amplified by polymerase chain reaction and introduced into the linearized vector pBI121, which had been digested with restriction endonucleases XbaI and SacI and purified. The coding region reading frame sequence in the expression vector was then sequenced to ensure that it was correct. 2) Obtaining transgenic plants The expression vector pBI121-CsMYB4 obtained in step 1) was transformed into Agrobacterium GV3101 and further transferred into Nicotiana benthamiana. The transgenic plants were verified as positive by PCR and real-time fluorescence quantitative analysis. The transgenic T2 generation plants grown to the 3-4 leaf stage were inoculated with Botrytis cinerea, and the size of the lesions on the diseased leaves of the plants was recorded 3 days after inoculation. Compared with the control, the transgenic T2 generation plants had stronger disease resistance.

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