Application and method of tea tree CsMYB1 gene in improving plant disease resistance

Abstract

The application discloses application and a method of a tea tree CsMYB1 gene in improving plant disease resistance. The CsMYB1 gene is a transcription factor gene of an MYB family, the CsMYB1 is constructed to a plant expression vector, and is introduced into a Nicotiana benthamiana, and a transgenic tobacco plant is obtained through stable genetic transformation, and a strong fungal inhibitory activity is shown to Botrytis cinerea hypha inoculation; the technology of antisense oligonucleotides (AsODNs) is used to carry out transient silencing on the CsMYB1 gene on a tea tree leaf, and the disease resistance of the plant is significantly weakened. Therefore, the CsMYB1 gene has the effect of improving the disease resistance of the plant, can be used as a disease resistance gene, and can be introduced into tobacco, tea trees or vegetables to improve the disease resistance of the plant, and has a wide market application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular biology and genetic engineering, and particularly relates to application and method of tea tree CsMYB1 gene in improving plant disease resistance. BACKGROUND

[0002] Camellia sinensis (L.) O. Kuntz is a perennial evergreen small tree or shrub, which is used as an important economic crop. Tea leaf spot caused by Epicoccum sorghinum is first isolated and identified by the present inventors' research group from the tea leaf disease in Duo Chao group tea garden of Jingqiao village, Dushan county, Guizhou province (Bao, X.T., Dharmasena, D.S.P., Li, D.X., Wang, X., Jiang, S.L., Ren, Y.F., Wang, D.L., Song, B.A., Chen, Z. First report of Epicoccum sorghinum causing leaf spot on tea in China. Plant Dis. 2019, 103: 3282). The colony color of E. sorghinum is pink or red, the aerial hypha is white, the small conidia is oval or ovoid, there is a septum before the conidia, and the chlamydospore morphology is diverse, which can be spherical, subspherical, square or oval (Bao, X.T., Dharmasena, D.S.P., Li, D.X., Wang, X., Jiang, S.L., Ren, Y.F., Wang, D.L., Song, B.A., Chen, Z. First report of Epicoccum sorghinum causing leaf spot on tea in China. Plant Dis. 2019, 103: 3282). S. Genetic variation of Phoma sorghina isolates from Southern Africa and Texas. Folia Microbiolo. 2009, 54: 217-229; Bao, X.T., Dharmasena, D.S.P., Li, D.X., Wang, X., Jiang, S.L., Ren, Y.F., Wang, D.L., Song, B.A., Chen, Z. First report of Epicoccum sorghinum causing leaf spot on tea in China. Plant Dis. 2019, 103: 3282).

[0003] MYB transcription factors are one of the largest gene families in plants, which are widely involved in the regulation of plant growth and development, secondary metabolism and stress response. MYB transcription factors are widely present in plants and almost involved in every aspect of plant development and metabolism. MYB transcription factors are also involved in the regulation of plant response to external environment, especially stress response (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, MYB can also regulate abscisic acid (Abscisic acid, ABA), gibberellic acid (Gibberellic Acid, GAs) and other signal transduction pathways, and participate in plant disease resistance. At present, the research on the function of MYB is less, especially the function of disease resistance. Using genetic engineering technology to cultivate resistant plant varieties and materials has obvious advantages and irreplaceable importance, which can not only provide convenience for large-scale production of tea, vegetables, tobacco and other plant varieties, reduce the use of chemical pesticides, and reduce environmental pollution. However, there is no report on the disease resistance function of tea MYB gene. SUMMARY

[0004] In view of this, one of the purposes of the present application is to provide an application of a tea MYB transcription factor CsMYB1 gene, which has 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 as SEQ ID NO. 1, and the encoded amino acid sequence is shown as 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.

[0007] The second purpose of the present application is to provide a method for improving the disease resistance of plants, which comprises introducing the CsMYB1 gene into the target plant to obtain a plant with improved disease resistance; the nucleotide sequence of the CsMYB1 gene is shown as SEQ ID NO. 1.

[0008] Preferably, the disease resistance is against the disease caused by the pathogen including B. cinerea or E. sorghinum.

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

[0010] Preferably, the plant expression vector comprises a pBI121 vector. Of course, other vectors such as Ti plasmid vectors, viral vectors, etc. can also be used.

[0011] Preferably, the introduction is through a leaf disc method.

[0012] Preferably, the plant comprises tobacco or tea.

[0013] The CsMYB1 gene of the present application is a transcription factor gene of the MYB family. The CsMYB1 gene is constructed into a plant expression vector and introduced into Nicotiana benthamiana to obtain a transgenic tobacco plant through stable genetic transformation. The transgenic tobacco plant shows strong fungal inhibition activity against Botrytis cinerea. The CsMYB1 gene in tea leaves is transiently silenced by using antisense oligonucleotides (AsODNs) technology, and the disease resistance of the plant is significantly weakened. Therefore, the CsMYB1 gene has the effect of improving the disease resistance of plants and can be used as a disease resistance gene to improve the disease resistance of tobacco, tea or vegetables and has a broad market application prospect. The present application provides a new method for improving the resistance of plants to fungal diseases. The disease-resistant plants cultivated through genetic engineering can overcome the shortcomings of traditional breeding, shorten the breeding period, and are easy to operate and obtain high-resistance materials. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Figure 1 is a diagram of the cloning of the tea tree CsMYB1 gene CDS, wherein A is an electrophoresis detection diagram of the CsMYB1 gene, and B is a sequencing result diagram;

[0015] Figure 2 Figure 2 is a phylogenetic tree of the tea tree CsMYB1 gene;

[0016] Figure 3 Figure 3 is a diagram of the subcellular localization of the tea tree CsMYB1 gene in tobacco leaves;

[0017] Figure 4 Figure 4 is a PCR detection diagram of the transgenic tobacco after overexpression of the CsMYB1 gene;

[0018] Figure 5Figure of phenotype of tobacco overexpressing gene CsMYB1 after inoculation of B. cinerea, wherein A is figure of antibacterial activity of tobacco overexpressing gene CsMYB1 after inoculation of B. cinerea, B is figure of lesion area of tobacco overexpressing gene CsMYB1 after inoculation of B. cinerea;

[0019] Figure 6 Figure of phenotype of tea plant with AsODN inhibiting CsMYB1 gene after inoculation of E. sorghinum and quantitative analysis of CsMYB1 gene, wherein A is figure of phenotype of tea plant with AsODN inhibiting CsMYB1 gene after inoculation of E. sorghinum, B is quantitative expression analysis of CsMYB1 gene after using AsODN to inhibit CsMYB1 gene of tea plant, C is lesion area of tea plant with AsODN inhibiting CsMYB1 gene. DETAILED DESCRIPTION

[0020] The application will be described in detail below with reference to the embodiments, which are only illustrative and not limited to the scope of the application. The application is not limited to the following embodiments or examples, and any modification and variation made without departing from the spirit of the application shall be included in the scope of the application. The experimental materials used in the following examples are commercially available unless otherwise specified.

[0021] Preparation before experiment:

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

[0023] 2. Preparation of part of reagents

[0024] 1. Preparation of LB medium:

[0025] Yeast extract 5 g;

[0026] Tryptone 10 g;

[0027] NaCl 10 g;

[0028] Liquid medium: reagents were put in a 1 L glass beaker, 1 L of ddH2O was added, and a glass rod was used to stir and dissolve completely. The solution was then divided into 250 mL conical flasks, each containing 100 mL of liquid medium. Solid LB medium: 1.5 g of agar powder was added to each 100 mL of liquid LB medium.

[0029] 2) Preparation of 1 M morpholine ethanesulfonic acid (MES):

[0030] Morpholine ethanesulfonic acid 2.132 g was dissolved in 10 mL of ddH2O, and then filtered using a water filter membrane (diameter = 0.22 μm). The solution was stored at room temperature.

[0031] 3) Preparation of 200 mM acetyl-syringone (AS):

[0032] Acetyl-syringone 0.039 g was dissolved in 1 mL of dimethyl sulfoxide (DMSO). The solution was stored at -20°C after preparation.

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

[0034] Magnesium chloride 2.033 g was dissolved in 10 mL of ddH2O, and then autoclaved at 121°C for 20 min. The solution was stored at 4°C after preparation.

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

[0036] Kanamycin 0.5 g was dissolved in 10 mL of ddH2O, and then filtered using a water filter membrane (diameter = 0.22 μm). The solution was stored at -20°C after preparation.

[0037] 6) Preparation of 100 mg / mL rifampicin (Rif):

[0038] Rifampicin 0.5 g was dissolved in 5 mL of dimethyl sulfoxide, and then filtered using a water filter membrane (diameter = 0.22 μm). The solution was stored at -20°C after preparation.

[0039] Example 1: Cloning and sequence analysis of the full-length CDS sequence of CsMYB1 gene

[0040] Total RNA from Fuding Dabaicha tea (Camellia sinensis cv. Fuding-dabaicha) 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 generated the first-strand cDNA, which was used as a template for PCR using primers that amplify CsMYB1. The upstream and downstream primers used are shown in Table 1. 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 as 94°C pre-denaturation 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, 50 μL was collected for gel excision and purification. DNA Gel Extraction Kit DNA gel recovery kit (Beijing Qingke Biotechnology) purification specific steps are shown in the instructions, after purification, take 5 μL and perform agarose gel electrophoresis, the bands are consistent with the expected results ( Figure 1 A), and sent it to a biological company (Beijing Qingke Biotechnology) for testing. The sequencing results were correct. Figure 1 As shown in B, the CDS region is exactly the same as the downloaded sequence (ie, the reference sequence), and the CDS sequence is shown in SEQ ID NO. 1; at the same time, the concentration of the target product is detected and stored for subsequent experiments.

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

[0042]

[0043] The CsMYB1 protein sequence was aligned with the Arabidopsis MYB family protein sequence (http: / / planttfdb.gao-lab.org / ). The amino acid sequences were aligned using the Clustal W program (http: / / www.clustal.org / clustal2 / ). Phylogenetic analysis was performed using the Newton-Joint Joint method using MEGA11 software with 1000 bootstrap replicates. The phylogenetic tree is shown in Figure 1. Figure 2 As shown, CsMYB1 is most closely related to the Arabidopsis AtMYB8 gene.

[0044] Experimental Example 2: Subcellular localization of the CsMYB1 gene

[0045] According to the cDNA sequence of the tea tree MYB transcription factor gene CsMYB1 gene (as shown in SEQ ID NO. 1) and the BamHI and SalI enzyme cutting sites of the pCAMBIA2300 vector, the homologous recombination primers are designed to contain the homologous arms of the pCAMBIA2300 vector, and the primer sequences are shown in Table 2. Then the subcellular localization vector plasmid of the CsMYB1 gene is constructed according to the conventional method.

[0046] The constructed vector plasmid is transformed into Agrobacterium by freeze-thaw method, and cultured at 28°C for 48h until colonies grow. Single colonies are picked into liquid LB containing Kana resistance, and cultured at 28°C for 18-24h until the OD 600 is about 0.8. The bacteria are collected by centrifugation at 4000rpm / min for 10min, and resuspended in 10mM MgCl2, 10mM MES, 200μM AS suspension. The OD 600 is adjusted to about 0.8. The tobacco plants with good growth are selected, 1mL of the infection solution is sucked by a syringe and injected from the lower epidermis of the tobacco leaves, and labeled. After the injection is completed, the tobacco plants are cultured in the dark for 48h, and the tissue slices near the injection hole are cut. The laser confocal microscope is used to observe the GFP signal, and the photographs are saved as shown in Figure 3 , the first result shows that the CsMYB1 gene is located in the nucleus, and the other result shows that the CsMYB1 gene can condense into different sized granular materials.

[0047] Table 2 Primers for constructing CsMYB1 subcellular localization vector

[0048]

[0049] Example 3: Agrobacterium-mediated plant genetic transformation

[0050] 1. Construction of CsMYB1 gene overexpression vector

[0051] The SanPrep column plasmid DNA small amount extraction kit (Shanghai Sangon) is used to extract the plant expression vector pBI121 plasmid (the plasmid extraction steps are shown in the Shanghai Sangon kit instruction manual), and the two cloning sites XbaI and SacI on the pBI121 vector are selected for double enzyme digestion to linearize the vector. 1μL is used for 1.5% agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. The Purification was performed using a DNA Gel Extraction Kit (Beijing Qingke Biotechnology Co., Ltd.) (see the instructions for specific steps). The size and concentration of the recovered fragments were determined by 1.5% agarose gel electrophoresis. Homologous recombination primers were designed to contain homology arms to the pBI121 vector. The primer sequences are shown in Table 3.

[0052] use After determining the vector and target gene concentrations using the IIOne Step Cloning Kit (Nanjing Novozymes) and its instructions, 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 completion of the reaction, the cells were placed on ice and transformed into Escherichia coli DH5α. Following ligation and transformation, the cells were plated onto LB solid medium containing Kana antibiotics (LB preparation instructions are attached on the end page). The culture suspension was shaken on a shaker for 12 hours and then tested. Positive clones were selected and sent to Qingke Biotechnology for sequencing to ensure successful ligation of the target sequence into the pBI121 vector, generating an overexpression vector.

[0053] Table 3 Primers for overexpression vector construction

[0054]

[0055] 2. Genetic transformation of CsMYB1 gene into tobacco

[0056] The constructed recombinant overexpression vector was transferred into Agrobacterium by freeze-thaw method and injected into tobacco for transgenic overexpression. The transgenic recipient tobacco in this experiment was N. benthamiana. The specific steps of transgenesis were as follows:

[0057] 1) Tobacco sterile seedling culture

[0058] Tobacco seeds were soaked in 75% alcohol for 1 min, sterilized with 15% H2O2 for 15 min, washed three times with clean water for 3 min each time, spread on MS culture medium in a clean bench, and cultured in a light incubator at 28°C for about 15 days.

[0059] 2) Conversion

[0060] The tobacco leaves were cut into 0.5 x 0.5 cm pieces in a super-clean bench, and then transferred into the treated Agrobacterium solution (OD value was about 0.8), and infected for 5 min, and then dried on a sterile filter paper, and then inoculated on MS solid co-culture medium (1 / 2MS + 30 g / L sucrose + 8 g / L agar, pH 5.8), and placed in a constant temperature box at 22°C for dark culture for 2 d.

[0061] 3) Embryo induction

[0062] The leaves on the plate 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 Kana, pH 5.8), and the wound was adhered to the surface of the medium, and the front was upward, and after the transfer, the sealing film was sealed, and placed in a light incubator for culture, and transferred once in the same medium after about 15 d.

[0063] 4) Rooting

[0064] When the 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 Kana, pH 5.8), and cultured under light conditions to grow the bud body and induce rooting, and the control plants were wild type N. benthamiana.

[0065] 3, Detection of CsMYB1 gene in transgenic tobacco

[0066] The CTAB method was used to extract the DNA of the transgenic tobacco, the universal primers MYB-F and M13F (see Table 4) on the pBI121 vector were used for PCR, Taq HS (0.25 μL), dNTP Mixture (4 μL), 10 x PCR Buffer (5 μL), DNA template (2 μL), upper and lower primers (1 μL), ddH2O (36.75 μL), and the PCR program was set as 94°C pre-denaturation for 2 min, 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 50 s, 35 cycles, 72°C post-extension for 5 min, and 5 μL was taken for agarose gel electrophoresis after the end of PCR, as shown in Figure 4 , the band was correct, indicating that the transgenic was successful.

[0067] Table 4 Primers for detection of transgenic plants

[0068]

[0069] Experimental Example 4: Disease resistance experiment of transgenic tobacco

[0070] The transgenic tobacco overexpressing the CsMYB1 gene and the wild-type tobacco were inoculated with Botrytis cinerea, and the lesion area was recorded 3 days after inoculation, and the significance analysis of the data was performed. Specifically: B. cinerea preserved in the Key Laboratory of Green Pesticides and Agricultural Bioengineering of the Ministry of Education of Guizhou University was inoculated on PDA solid culture medium, inverted and cultured in an incubator at 25°C, and B. cinerea was inoculated on leaves when it grew to 3-4 days. The hyphae were beaten into a 6mm diameter cake, and 4 small holes were punched on the tobacco. The cake was inoculated on the leaves of the transgenic tobacco with an inoculation needle, with the hyphae surface in contact with the front of the leaf, and the size of the lesion was recorded 3 days after inoculation. The experimental results found that CsMYB1 transgenic tobacco has obvious resistance to the growth of B. cinerea, such as Figure 5 As shown, when B. cinerea was used as pathogen, the average lesion area of ​​wild-type tobacco was 144.34 mm 2 , while the average lesion area of ​​CsMYB1 transgenic tobacco was 86.71 mm 2 , CsMYB1 transgenic tobacco has obvious disease resistance, indicating that the CsMYB1 gene improves the disease resistance of tobacco.

[0071] Experimental Example 5: Gene expression inhibition and disease resistance experiment of CsMYB1 in AsODNs

[0072] Submit the CDS sequence of the CsMYB1 gene to the Soligo online tool (https: / / sfold.wadsworth.org / cgi-bin / soligo.pl / ) to design the oligonucleotide antisense chain specific oligonucleotide fragment of the target gene, which is usually 20bp in length. Oligonucleotide fragments with low binding site destruction energy are preferentially selected, and this sequence is reversely complemented to obtain oligonucleotide sense chain specific oligonucleotide fragments. The artificially synthesized oligonucleotide sequence of CsMYB1 (sequence shown in Table 5) was transferred to the tender shoots of the tea tree. The treatment group was the tender shoots containing the target gene oligonucleotide antisense chain, and the control group was the tender shoots containing the oligonucleotide sense chain. The treated young leaves were placed in a light incubator with a temperature of 28°C, a humidity of 75%, and a light cycle of 16h dark and 8h light. After 48h, leaf samples were collected and sample RNA was extracted using a total RNA extraction kit, and reverse transcribed into single-stranded cDNA using a reverse transcription kit. Quantitative primers were designed (sequences are shown in Table 6), and qRT-PCR was used to detect the expression level of the target gene CsMYB1 in the treatment group and control group samples. As Figure 6As shown in B, compared with the control group, the expression amount of CsMYB1 in the treatment group decreased obviously, indicating that the CsMYB1 gene has been inhibited. The E. sorghinum (strain number: GZDS2018BXT10, preservation number: CGMCC3.20150) identified by the inventors' research group in the early stage was inoculated on the tea leaves in the treatment group and the control group to observe the change of lesion area, and the lesion area size of 48h after inoculation was recorded, as shown in Figure 6 As shown in A and 6C, the average lesion area of the control plants was 100.43mm 2 , and the average lesion area of the silencing plants was 126.76mm 2 , indicating that after silencing the CsMYB1 gene, the disease resistance of the plants was significantly weakened, further proving that the CsMYB1 gene has the function of disease resistance.

[0073] Table 5 CsMYB1 oligonucleotide fragment

[0074]

[0075] Table 6 quantitative primer

[0076]

[0077] The conventional techniques and schemes not described in detail in the above examples are well known in the art, and therefore will not be described in detail here. The above examples and / or experimental examples describe the preferred embodiments of the present application in detail, however, the present application is not limited to the specific details in the above embodiments, and within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. Camellia sinensis CsMYB1 application of a gene in improving disease resistance of a plant, wherein the gene has a nucleotide sequence as shown in SEQ ID NO. 1; the disease resistance is against diseases caused by Botrytis cinerea (Bc) or Sphaeropsis balserianus (Sb) pathogen; and the plant is tobacco or tea. CsMYB1 Botrytis cinerea Epicoccum sorghinum application of a gene in improving disease resistance of a plant, wherein the gene has a nucleotide sequence as shown in SEQ ID NO. 1; the disease resistance is against diseases caused by Botrytis cinerea (Bc) or Sphaeropsis balserianus (Sb) pathogen; and the plant is tobacco or tea. CsMYB1 Botrytis cinerea Epicoccum sorghinum application of a gene in improving disease resistance of a plant, wherein the gene has a nucleotide sequence as shown in 2. A method for improving disease resistance in plants, characterized by, comprising CsMYB1 introducing the gene into a plant of interest to obtain a plant with improved disease resistance; the CsMYB1 nucleotide sequence of the gene is shown in SEQ ID NO. 1; the disease resistance is against diseases caused by the pathogen Botrytis cinerea Botrytis cinerea ) or Sphaeropsis fuliginea Epicoccum sorghinum ); and the plant is tobacco or tea.

3. The method of claim 2, wherein, The CsMYB1 Genes are introduced into the plant of interest by means of a plant expression vector.

4. The method of claim 3, wherein, The plant expression vector comprises a pBI121 vector.

5. The method of claim 2, wherein, The introduction is by leaf disc method.

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