Use of camellia sinensis (l.) o. kuntz csmyb1 gene in improving disease resistance of plant and method for improving disease resistance of plant

Abstract

Disclosed are use of the Camellia sinensis (L.) O. Kuntz CsMYB1 gene in improving disease resistance of a plant and a method for improving disease resistance of a plant. The CsMYB1 gene is a transcription factor gene of the MYB family. CsMYB1 is constructed into a plant expression vector, and the vector is transferred into Nicotiana benthamiana. A transgenic tobacco plant is obtained by means of stable genetic transformation, and the plant exhibits relatively strong fungal inhibitory activity against inoculated Botrytis cinerea hyphae. Transient silencing of the CsMYB1 gene of Camellia sinensis (L.) O. Kuntz leaves using a technology of antisense oligonucleotides (AsODNs) significantly reduces the disease resistance of the plant. Therefore, the CsMYB1 gene has the effect of improving the disease resistance of a plant and can be used as a disease-resistant gene. Introducing the gene into plants such as tobacco, Camellia sinensis (L.) O. Kuntz, or vegetables can improve the disease resistance of these plants. The present invention has broad market application prospects.

Description

Application and methods of tea tree CsMYB1 gene in improving plant disease resistance Technical Field

[0001] This invention belongs to the fields of molecular biology and genetic engineering, specifically relating to the application and method of the CsMYB1 gene in tea trees in improving plant disease resistance. Background Technology

[0002] The tea plant (Camellia sinensis (L.) O. Kuntz) is a perennial evergreen shrub or small tree, used as an important economic crop. The tea leaf spot disease caused by *Epicoccum sorghinum* was first isolated and identified by our research group from a tea garden in Duochao Group, Jingqiao Village, Baiquan Town, Dushan County, Guizhou Province (Bao, XT, Dharmasena, DSP, Li, DX, Wang, X., Jiang, SL, Ren, YF, Wang, DL, Song, BA, Chen, Z. First report of *Epicoccum sorghinum* causing leaf spot on tea in China. Plant Dis. 2019, 103:3282). The colonies of *E. sorghinum* are pink or red, with white aerial hyphae. The small conidia are elliptical or oval, with septa preceding the conidia. The dark brown, thick-walled conidia exhibit diverse morphologies, including spherical, subspherical, square, or elliptical shapes. S. Genetic variation of Phoma sorghina isolates from Southern Africa and Texas. Folia Microbiolo. 2009,54:217-229; Bao, XT, Dharmasena, DSP, Li, DX, Wang, Dis.2019,103:3282).

[0003] MYB transcription factors are one of the largest gene families in plants, widely involved in regulating plant growth and development, secondary metabolism, and responses to abiotic stress. MYB transcription factors are ubiquitous in plants, participating in almost every aspect of plant development and metabolism. MYB transcription factors also participate in the regulation of plant responses to external environments, especially stress (Vannini, C., Locatelli, F., Bracale, M., Magnani, E., Marsoni, M., Osnato, M., Mattana, M., Baldoni, E., Coraggio, I. Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants. Plant J. 2004, 37:115-127). In plant disease resistance stress, MYB can also participate in plant disease resistance responses by regulating signal transduction pathways such as abscisic acid (ABA) and gibberellin (GAs). Currently, there is limited research on the function of the MYB gene, particularly its role in disease resistance. Utilizing genetic engineering to cultivate resistant plant varieties and materials has significant advantages and irreplaceable importance. It can facilitate large-scale production of tea, vegetables, tobacco, and other plant varieties, reducing the use of chemical pesticides and environmental pollution. However, to date, there are no reports on the disease resistance function of the MYB gene in tea. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide an application of the tea plant MYB transcription factor CsMYB1 gene, which plays a role in improving the disease resistance of plants and can be applied to improve the disease resistance of plants. The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0005] Preferably, the disease resistance is resistance to diseases caused by pathogens including Botrytis cinerea or Epicoccum sorghinum.

[0006] Preferably, the plant includes tobacco or tea tree.

[0007] A second objective of this invention is to provide a method for improving plant disease resistance, comprising introducing the CsMYB1 gene into a target plant to obtain plants with improved disease resistance; the nucleotide sequence of the CsMYB1 gene is shown in SEQ ID NO.1.

[0008] Preferably, the disease resistance is resistance to diseases caused by pathogens including B. cinerea or E. sorghinum.

[0009] Preferably, the CsMYB1 gene is introduced into the target plant via a plant expression vector.

[0010] Preferably, the plant expression vector includes the pBI121 vector. Other vectors, such as Ti-type plasmid vectors or viral vectors, can also be used.

[0011] Preferably, the import is performed via the leaf disc method.

[0012] Preferably, the plant includes tobacco or tea tree.

[0013] This invention relates to the CsMYB1 gene, a transcription factor gene of the MYB family. CsMYB1 was constructed into a plant expression vector and transformed into Nicotiana benthamiana. Transgenic tobacco plants were obtained through stable genetic transformation, exhibiting strong fungal inhibitory activity against Botrytis cinerea mycelia. Transient silencing of the CsMYB1 gene on tea leaves using antisense oligonucleotides (AsODNs) significantly weakened the disease resistance of the plants. Therefore, the CsMYB1 gene has the function of enhancing plant disease resistance and can be used as a disease resistance gene. Its introduction into plants such as tobacco, tea, or vegetables can improve plant disease resistance and has broad market application prospects. This invention provides a new method for improving plant resistance to fungal diseases. Cultivating disease-resistant plants through genetic engineering overcomes the shortcomings of traditional breeding methods, shortening the breeding cycle, simplifying the operation, and easily obtaining highly resistant materials. Attached Figure Description

[0014] Figure 1 is a clone diagram of the CDS of the CsMYB1 gene in tea plant of the present invention, wherein A is an electrophoresis detection diagram of the CsMYB1 gene and B is a sequencing result diagram.

[0015] Figure 2 is a phylogenetic tree of the CsMYB1 gene in tea plants according to the present invention;

[0016] Figure 3 shows the subcellular localization of the CsMYB1 gene in tea plants observed in tobacco leaves according to the present invention.

[0017] Figure 4 shows the PCR detection of transgenic tobacco after overexpression of gene CsMYB1 in this invention.

[0018] Figure 5 shows the phenotypic diagrams of tobacco overexpressing the CsMYB1 gene and pBI121 control tobacco after inoculation with B. cinerea. In this figure, A is the antibacterial activity phenotypic diagram of tobacco overexpressing the CsMYB1 gene and pBI121 control tobacco after inoculation with B. cinerea, and B is the comparison diagram of lesion area of ​​tobacco overexpressing the CsMYB1 gene and pBI121 control tobacco after inoculation with B. cinerea.

[0019] Figure 6 shows the phenotype of E. sorghinum and the quantitative analysis of the CsMYB1 gene in tea plants inoculated with AsODN to suppress the CsMYB1 gene, according to the present invention. In this figure, A represents the phenotype of E. sorghinum in tea plants inoculated with AsODN to suppress the CsMYB1 gene, B represents the quantitative expression analysis of the CsMYB1 gene in tea plants after AsODN suppression of the CsMYB1 gene, and C represents the lesion area of ​​tea plants in which AsODN suppresses the CsMYB1 gene. Detailed Implementation

[0020] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.

[0021] Preparation before the experiment:

[0022] 1. Data: The CsMYB1 gene sequence was downloaded from the tea plant genome database of Anhui Agricultural University (http: / / tpia.teaplant.org / index.html). The CDS sequence of the CsMYB1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0023] 2. Preparation of some reagents

[0024] 1) Preparation of LB medium:

[0025] 5g of yeast extract;

[0026] Tryptone 10g;

[0027] 10g of sodium chloride (NaCl);

[0028] For liquid culture medium, place the reagent in a 1L glass beaker, add 1L of ddH2O, stir with a glass rod until completely dissolved, and dispense 100mL of liquid culture medium into 250mL Erlenmeyer flasks. For solid LB medium, add 1.5g of agar powder to every 100mL of liquid LB medium.

[0029] 2) Preparation of 1M Morpholine Ethyl Acetic Acid (MES):

[0030] 2.132 g of morpholine ethanesulfonic acid was dissolved in 10 mL of ddH2O, filtered through an aqueous filter membrane (diameter = 0.22 μm), and stored at room temperature.

[0031] 3) Preparation of 200mM acetylsyleugenone (AS):

[0032] Dissolve 0.039 g of acetylsuccinone in 1 mL of dimethyl sulfoxide (DMSO), and store the solution at -20°C.

[0033] 4) Preparation of 1M magnesium chloride (MgCl2):

[0034] Dissolve 2.033g of magnesium chloride in 10mL of ddH2O, autoclave at 121℃ for 20min, and store at 4℃ after preparation.

[0035] 5) Preparation of 50 mg / mL kanamycin (Kana):

[0036] Dissolve 0.5g of kanamycin in 10mL of ddH2O, then filter using an aqueous filter membrane (diameter = 0.22μm). Store the prepared solution at -20℃.

[0037] 6) Preparation of 100 mg / mL rifampin:

[0038] Dissolve 0.5g of rifampicin in 5mL of dimethyl sulfoxide, then filter using an aqueous filter membrane (diameter = 0.22μm), and store the solution at -20℃.

[0039] Experimental Example 1: Cloning and Sequence Analysis of the Full-Length CDS Sequence of the CsMYB1 Gene

[0040] Total RNA was extracted from Fuding Dabaicha tea leaves using TRIzol reagent, and the RNA quantity and purity of each sample were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). First-strand cDNA was generated by reverse transcription and used as a template for PCR amplification of CsMYB1. The upstream and downstream primers used are shown in Table 1. The following PCR program was used: 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). The PCR program consisted of 35 cycles: 94℃ pre-deformation for 2 min, 98℃ denaturation for 10 s, 55℃ annealing for 30 s, and 72℃ extension for 50 s. A final extension at 72℃ for 5 min followed by 50 μL of PCR was then purified using gel extraction. The DNA Gel Extraction Kit (Beijing Qingke Biotechnology) was used for purification. Specific purification steps are detailed in the instruction manual. After purification, 5 μL was subjected to agarose gel electrophoresis. The bands met the expected results (Figure 1A). The sample was sent to Beijing Qingke Biotechnology for sequencing, and the sequencing results were correct, as shown in Figure 1B. The CDS region was completely identical to the downloaded sequence (i.e., the reference sequence), as shown in SEQ ID NO.1. Simultaneously, the concentration of the target product was determined, and the sample was stored for subsequent experiments.

[0041] Table 1 Primers for cloning the full-length CDS sequence of CsMYB1

[0042] The CsMYB1 protein sequence was aligned with the sequences of the Arabidopsis MYB family proteins (http: / / planttfdb.gao-lab.org / ). Amino acid sequences were aligned using Clustal W (http: / / www.clustal.org / clustal2 / ), and phylogenetic analysis was performed using MEGA 11 software with the NJ method, employing 1000 guide repeats. The phylogenetic tree is shown in Figure 2. CsMYB1 is most closely related to the Arabidopsis AtMYB8 gene.

[0043] Experimental Example 2: Subcellular localization of the CsMYB1 gene

[0044] Based on the cDNA sequence of the tea plant MYB transcription factor gene CsMYB1 (as shown in SEQ ID NO.1) and the BamHI and SalI restriction sites of the pCAMBIA2300 vector, homologous recombination primers were designed to contain the homologous arms of the pCAMBIA2300 vector. The primer sequences are shown in Table 2. Then, the subcellular localization vector plasmid of the CsMYB1 gene was constructed using conventional methods.

[0045] The constructed vector plasmid was transformed into Agrobacterium using the freeze-thaw method and cultured at 28°C for 48 hours until colonies appeared. Single colonies were picked and cultured in LB liquid containing Kana resistance for 18–24 hours until OD (Organic Discharge) was achieved. 600 The OD value was around 0.8. The cells were collected by centrifugation at 4000 rpm for 10 min, and resuspended in a suspension of 10 mM MgCl2, 10 mM MES, and 200 μM AS. 600 To a value of approximately 0.8. Select healthy tobacco plants and inject 1 mL of the infection solution into the lower epidermis of the tobacco leaves using a syringe, labeling the injection sites. After incubating the injected tobacco plants in the dark for 48 hours, cut tissue sections near the injection sites and observe the GFP signal using a laser confocal microscope, photographing and saving the images. As shown in Figure 3, the first result shows that the CsMYB1 gene is located in the cell nucleus, while the other result shows that the CsMYB1 gene aggregates into granular substances of different sizes.

[0046] Table 2 Primers for constructing the CsMYB1 subcellular localization vector

[0047] Example 3: Agrobacterium-mediated plant genetic transformation

[0048] 1. Construction of CsMYB1 gene overexpression vector

[0049] The plant expression vector pBI121 plasmid was extracted using the SanPrep column-based plasmid DNA mini-extraction kit (Shanghai Sangon Biotech) (extraction steps are detailed in the Shanghai Sangon Biotech kit instructions). The pBI121 vector was linearized by double digestion of two cloning sites, XbaI and SacI. 1 μL of the extracted plasmid was used for 1.5% agarose gel electrophoresis to check its integrity and concentration. The DNA Gel Extraction Kit (Beijing Qingke Biotechnology) was used for purification (see the instruction manual for specific steps). The size and concentration of the recovered fragments were determined by 1.5% agarose gel electrophoresis. Homologous recombination primers containing the homologous arms of the pBI121 vector were designed, and the primer sequences are shown in Table 3.

[0050] use Following the instructions of the One Step Cloning Kit (Nanjing Novizan) and its accompanying manual, after determining the concentrations of the vector fragment and the target gene, homologous recombination was performed using the kit. The reaction system was as follows: 5×CE II Buffer (2 μL), Exnase II (1 μL), linearized vector (6 μL), and target gene fragment (1 μL). The incubation program was 37℃ for 30 min. After the reaction, the mixture was immediately placed on ice and then transferred to *E. coli* DH5α. After ligation and transformation, the mixture was plated on LB agar containing Kana antibiotic (LB preparation method is attached at the end of the page). The bacterial culture was shaken for 12 h and then analyzed. Positive clones were sent to Qingke Biotechnology Co., Ltd. for sequencing to ensure successful ligation of the target sequence into the pBI121 vector, thus obtaining the overexpression vector.

[0051] Table 3 Primers for overexpression vector construction

[0052] 2. CsMYB1 gene genetic transformation of tobacco

[0053] The constructed recombinant overexpression vector was transformed into Agrobacterium using a freeze-thaw method and then injected into tobacco for transgenic overexpression. The transgenic recipient tobacco in this experiment was Nicotiana benthamiana. The specific steps of transgenic expression are as follows:

[0054] 1) Culture of aseptic tobacco seedlings

[0055] Tobacco seeds were soaked in 75% alcohol for 1 minute, then sterilized with 15% H2O2 for 15 minutes, and then washed three times with water for 3 minutes each time. They were then spread on MS medium in a clean bench and cultured in a light incubator at 28°C for about 15 days.

[0056] 2) Transformation

[0057] In a clean bench, tobacco leaves were cut into 0.5×0.5cm pieces. The tobacco leaves were then transferred into the prepared Agrobacterium tumefaciens solution (OD value around 0.8). After 5 minutes of inoculation, the leaves were blotted dry on sterile filter paper and then inoculated onto MS solid co-medium (1 / 2 MS + 30g / L sucrose + 8g / L agar, pH 5.8) and incubated in a constant temperature incubator at 22℃ in the dark for 2 days.

[0058] 3) Embryo induction

[0059] Transfer the leaves from the plate 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 Kana, pH 5.8), with the wound facing up on the surface of the medium. After transfer, seal the plate with sealing film and place it in a light incubator for cultivation. Transfer the leaves to the same medium once every 15 days.

[0060] 4) Rooting

[0061] When small buds emerged on the induction medium, they were transferred to rooting medium (MS + 0.1 mg / L NAA + 30 g / L sucrose + 8 g / L agar + 100 mg / L Kana, pH 5.8) and cultured under light conditions to induce bud growth and root formation. The control plant was wild-type Nicotiana benthamiana.

[0062] 3. Detection of CsMYB1 gene in genetically modified tobacco

[0063] DNA was extracted from transgenic tobacco using the cetyltrimethylammonium bromide (CTAB) method. PCR was performed using universal primers MYB-F and M13F from the pBI121 vector (see Table 4). The following reagents were used: Taq HS (0.25 μL), dNTP Mixture (4 μL), 10×PCR Buffer (5 μL), DNA template (2 μL), forward and reverse primers (1 μL), and ddH2O (36.75 μL). The PCR program was set to 94℃ pre-deformation for 2 min, 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 50 s, for 35 cycles, followed by a final extension at 72℃ for 5 min. After PCR, 5 μL was subjected to agarose gel electrophoresis. As shown in Figure 4, the bands were correct, indicating successful transgenicity.

[0064] Table 4 Primers for transgenic plant detection

[0065] Experiment Example 4: Disease Resistance Experiment with Transgenic Tobacco

[0066] Transgenic tobacco overexpressing the CsMYB1 gene and wild-type tobacco were inoculated with *Botrytis cinerea*, and the lesion area was recorded 3 days after inoculation for significance analysis. Specifically, *B. cinerea*, preserved in the Key Laboratory of Green Pesticides and Agricultural Bioengineering of the Ministry of Education at Guizhou University, was inoculated onto PDA solid medium and incubated upside down at 25°C. When *B. cinerea* reached 3-4 days of growth, it was inoculated onto leaves. Mycelia were broken into 6 mm diameter mycelial discs, and four small holes were punched in the tobacco leaves. The mycelial discs were then inoculated onto the transgenic tobacco leaves using an inoculation needle, with the mycelial surface in contact with the upper surface of the leaf. The lesion size was recorded 3 days after inoculation. The results showed that the CsMYB1 transgenic tobacco exhibited significant resistance to the growth of *B. cinerea*, as shown in Figure 5. When *B. cinerea* was used to induce disease, the average lesion area of ​​the control wild-type tobacco was 144.34 mm². 2 The average lesion area on CsMYB1 transgenic tobacco was 86.71 mm². 2 The CsMYB1 transgenic tobacco showed significant disease resistance, indicating that the CsMYB1 gene improved the disease resistance of tobacco.

[0067] Experiment Example 5: Gene Expression Repression and Disease Resistance Experiment of CsMYB1 in AsODNs

[0068] The CDS sequence of the CsMYB1 gene was submitted, and an antisense-specific oligonucleotide fragment (OSF) was designed using the Solido online tool (https: / / sfold.wadsworth.org / cgi-bin / soligo.pl / ). The OSF length was typically 20 bp. Olignucleotide fragments with low binding site disruption energy were preferentially selected, and their sequences were reverse-complemented to obtain a sense-specific OSF. The artificially synthesized CsMYB1 OSF sequence (sequences shown in Table 5) was transferred into tea buds. The treatment group consisted of buds containing the antisense OSF of the target gene, while the control group consisted of buds containing the sense OSF. The treated buds were placed in a light incubator at 28℃, 75% humidity, and a 16-hour dark-8-hour light cycle. After 48 hours, leaf samples were collected, and RNA was extracted using a total RNA extraction kit. The RNA was then reverse-transcribed into single-stranded cDNA using a reverse transcription kit. Quantitative primers were designed (sequences shown in Table 6), and qRT-PCR was used to detect the expression level of the target gene CsMYB1 in the treatment and control groups. As shown in Figure 6B, compared with the control group, the expression level of CsMYB1 in the treatment group was significantly reduced, indicating that the CsMYB1 gene was suppressed. The *E. sorghinum* strain (strain number: GZDS2018BXT10, deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC3.20150, address: No. 1, Beichen West Road, Chaoyang District, Beijing), previously identified by our research group, was inoculated onto tea plants in both the treatment and control groups to observe changes in lesion area. The lesion area size was recorded 48 hours after inoculation, as shown in Figures 6A and 6C. The average lesion area of ​​the control plants was 100.43 mm². 2 The average lesion area of ​​silent plants was 126.76 mm. 2 This indicates that the plant's disease resistance was significantly weakened after the CsMYB1 gene was silenced, further proving that the CsMYB1 gene has a disease resistance function.

[0069] Table 5. CsMYB1 oligonucleotide fragments

[0070] Table 6 Quantitative Primers

[0071] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. Application of the CsMYB1 gene in tea trees to improve plant disease resistance, wherein the nucleotide sequence of the CsMYB1 gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The disease resistance refers to resistance to diseases caused by pathogens including Botrytis cinerea or Epicoccum sorghinum.

3. The application as described in claim 1, characterized in that, The plants mentioned include tobacco or tea trees.

4. A method for improving plant disease resistance, characterized in that, This includes introducing the CsMYB1 gene into a target plant to obtain plants with enhanced disease resistance; the nucleotide sequence of the CsMYB1 gene is shown in SEQ ID NO.

1.

5. The method as described in claim 4, characterized in that, The disease resistance refers to resistance to diseases caused by pathogens including B. cinerea or E. sorghinum.

6. The method as described in claim 4, characterized in that, The CsMYB1 gene was introduced into the target plant via a plant expression vector.

7. The method as described in claim 6, characterized in that, The plant expression vector includes the pBI121 vector.

8. The method as described in claim 4, characterized in that, The import is performed using the leaf disc method.

9. The method as described in claim 4, characterized in that, The plants mentioned include tobacco or tea trees.

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