Application and method of tea plant CsETR2 gene in improving plant disease resistance

Abstract

The present invention discloses the application and method of the tea plant CsETR2 (ethylene receptor 2) gene in improving plant disease resistance. The CsETR2 gene is an important gene in the ethylene receptor family. CsETR2 is constructed into a plant expression vector and introduced into Nicotiana benthamiana. Transgenic tobacco plants are obtained through stable genetic transformation, and exhibit strong fungal inhibitory activity against Botrytis cinerea mycelium. Antisense oligonucleotides (AsODNs) technology is used to transiently silence the CsETR2 gene in tea leaves, and the plants are inoculated with Lasiodiplodia theobromae, significantly weakening their disease resistance. Therefore, the CsETR2 gene has the function of improving plant disease resistance and can be used as a disease-resistant gene to improve plant disease resistance by introducing it into plants such as tobacco, tea, or vegetables. The gene has broad market application prospects.

Description

Technical Field

[0001] The invention belongs to the technical fields related to molecular biology and genetic engineering, and particularly relates to an application and method of a tea plant CsETR2 gene in improving plant disease resistance. Background Art

[0002] Tea (Camellia sinensis (L.) O. Kuntz) is a perennial, evergreen, small tree or shrub used as an important economic crop. Tea leaf spot disease, caused by Lasiodiplodia theobromae, was first isolated and identified by the present inventors' research group from a tea plantation in Huishui County, Guizhou Province (Li DX, Bao XT, Ren YF, et al. First report of Lasiodiplodia theobromae causing leaf spot on tea plants in Guizhou Province of China [J]. Plant Disease, 2019, 103(2): 374). Lasiodiplodia theobromae is the asexual form of the Botryosphaeria pathogen, and its sexual form is Botryosphaeria rhodian (Berk. & M.A. Curtis) Arx. Although it is found almost worldwide, it occurs more frequently in tropical and subtropical regions. It is a non-host-specific plant pathogen or endophytic fungus (Phillips AJ., Alves A., Abdollahzadeh J., et al. The Botryosphaeriaceae: genera and species known from culture [J]. Studies in Mycology. 2013, 76(1): 51-167; Mehl J., Wingfield MJ, Roux J., Slippers B. Invasive everywhere? Phylogeographic analysis of the globally distributed tree pathogen Lasiodiplodia theobromae [J]. Forests. 2017, 8: 145.).

[0003] Plant defense response hormones usually include salicylic acid, jasmonic acid and ethylene. Ethylene is generally considered to be related to plant resistance (Dong X.SA,JA,ethylene,and disease resistance in plants[J].Currentopinion in plant biology,1998,1(4):316-323.). Ethylene can regulate various physiological and biochemical reactions in plants, such as seed germination, seedling growth, leaf and petal abscission, organ aging, maturation, stress response and pathogen response (Abeles FB,Morgan PW,Saltveit Jr ME.Ethylene in plant biology[M].Academic press,2012.). In the early stages of pathogen infection, plants respond to pathogen attack by increasing ethylene content. Ethylene is related to the induction of defense responses in plants (Boller T.Ethylene in pathogenesis and disease resistance[M].The plant hormone ethylene.CRC press,2018:293-314.). Studies have shown that when plants are under stress, increasing ethylene content helps plants alleviate stress. On the other hand, some plant pathogens can enhance their colonization ability in plant tissues by increasing the ethylene content in their bodies (Chagué V, Danit LV, Siewers V, et al. Ethylenesensing and gene activation in Botrytis cinerea: amissing link in ethylene regulation of fungus-plant interactions? [J]. Molecular plant-microbe interactions, 2006, 19(1): 33-42.).

[0004] Currently, little research has been conducted on the functions of the tea plant ethylene receptor family gene, CsETR2 (ethylene receptor 2), particularly its role in disease resistance. Using genetic engineering techniques to cultivate resistant plant varieties and materials has significant advantages and irreplaceable importance. It not only facilitates the large-scale production of tea, vegetables, tobacco, and other plant varieties, but also reduces the use of chemical pesticides and environmental pollution. However, to date, no reports have been found on the disease resistance function of the tea plant CsETR2 gene. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide an application of the tea plant ethylene receptor gene CsETR2 gene, which plays a role in improving the disease resistance of plants and can be used 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.

[0006] Preferably, the disease resistance is resistance to diseases caused by pathogens including Botrytis cinerea and Lasiodiplodia theobromin.

[0007] Preferably, the plant comprises Nicotiana benthamiana or Camellia sinensis.

[0008] A second object of the present invention is to provide a method for improving plant disease resistance, comprising introducing the CsETR2 gene into a target plant to obtain a plant with improved disease resistance; the nucleotide sequence of the CsETR2 gene is shown in SEQ ID NO.1.

[0009] Preferably, the disease resistance is resistance to diseases caused by pathogens including B. cinerea or L. theobromae.

[0010] Preferably, the CsETR2 gene is introduced into the target plant via a plant expression vector.

[0011] Preferably, the plant expression vector comprises a pBI121 vector. Of course, it can also be other vectors, such as Ti-type plasmid vectors, viral vectors, etc.

[0012] Preferably, the introduction is carried out by a blade disk method.

[0013] Preferably, the plant comprises Nicotiana benthamiana or Camellia sinensis.

[0014] The CsETR2 gene of the present invention is an ethylene receptor family gene. CsETR2 is constructed into a plant expression vector and introduced into Nicotiana benthamiana. Transgenic tobacco plants are obtained through stable genetic transformation, and exhibit strong fungal inhibitory activity against inoculated Botrytis cinerea hyphae. The CsETR2 gene is transiently silenced on tea leaves using antisense oligonucleotides (AsODNs) technology, and the plant's disease resistance is significantly weakened by inoculation with Diplodia theobromin. Therefore, the CsETR2 gene has the function of improving plant disease resistance and can be used as a disease-resistant gene to improve plant disease resistance by introducing it into plants such as tobacco, tea, or vegetables, thus having broad market application prospects. The present invention provides a new method for improving plant resistance to fungal diseases. Cultivating disease-resistant plants through genetic engineering can overcome the shortcomings of traditional breeding, shorten the breeding cycle, and simplify the operation, making it easy to obtain highly resistant materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the gel electrophoresis diagram of the tea plant CsETR2 gene clone of the present invention;

[0016] Figure 2 is the evolutionary tree of the tea plant CsETR2 gene of the present invention;

[0017] Figure 3 This is an observation diagram of Nicotiana benthamiana leaves showing the subcellular localization of the CsETR2 gene in tea plants of the present invention;

[0018] Figure 4 This is a PCR detection diagram of transgenic tobacco after overexpressing the gene CsETR2 of the present invention;

[0019] Figure 5 Figures 1 and 2 are phenotypic images of Nicotiana benthamiana overexpressing the CsETR2 gene and Nicotiana benthamiana control with pBI121 after inoculation with B. cinerea; Figure A shows the antibacterial activity phenotype of Nicotiana benthamiana overexpressing the CsETR2 gene and Nicotiana benthamiana control with pBI121 after inoculation with B. cinerea; Figure B shows the comparison of lesion area of ​​Nicotiana benthamiana overexpressing the CsETR2 gene and Nicotiana benthamiana control with pBI121 after inoculation with B. cinerea.

[0020] Figure 6 The present invention shows the phenotype of L. theobromae inoculated on tea leaves with CsETR2 gene suppressed by AsODN and the quantitative analysis of CsETR2 gene; wherein A is the phenotype of L. theobromae inoculated on tea plants with CsETR2 gene suppressed by AsODN; B is the quantitative expression analysis of CsETR2 gene after tea plants with CsETR2 gene suppressed by AsODN; C is the lesion area of ​​tea plants with CsETR2 gene suppressed by AsODN. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below in conjunction with the examples, which are merely illustrative 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, and any modifications and variations made without violating the spirit of the present invention are intended to be included within the scope of the present invention. Unless otherwise specified, the experimental materials used in the following examples are commercially available.

[0022] Preparation before the experiment:

[0023] 1. Data: The CsETR2 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 CsETR2 gene is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2.

[0024] 2. Configuration of some reagents

[0025] 1) Preparation of LB medium:

[0026] Yeast extract 5g;

[0027] Tryptone 10g;

[0028] Sodium chloride (NaCl) 10g;

[0029] For liquid culture, place the reagent in a 1L glass beaker, add 1L of ddH2O, stir with a glass rod until completely dissolved, and dispense into 250mL Erlenmeyer flasks, 100mL of liquid culture medium per flask. For solid LB medium, add 1.5g of agar powder per 100mL of liquid LB medium.

[0030] 2) Preparation of 1M morpholineethanesulfonic acid (MES):

[0031] 2.132 g of morpholineethanesulfonic acid was dissolved in 10 mL of ddH2O, filtered using a water filter membrane (diameter = 0.22 μm), and stored at room temperature.

[0032] 3) Preparation of 200 mM acetosyringone (AS):

[0033] 0.039 g of acetosyringone was dissolved in 1 mL of dimethyl sulfoxide (DMSO) and stored in a -20°C refrigerator after preparation.

[0034] 4) Preparation of 1M magnesium chloride (MgCl2):

[0035] Dissolve 2.033 g of magnesium chloride in 10 mL of ddH2O, sterilize by autoclaving at 121°C for 20 min, and store in a refrigerator at 4°C after preparation.

[0036] 5) Preparation of 50 mg / mL Kanamycin (Kana):

[0037] 0.5 g of kanamycin was dissolved in 10 mL of ddH2O, and then filtered using a water filter membrane (diameter = 0.22 μm). After preparation, the solution was stored in a refrigerator at -20°C.

[0038] 6) Preparation of 100 mg / mL Rifampicin (Rif):

[0039] 0.5 g of rifampicin was dissolved in 5 mL of dimethyl sulfoxide, and then filtered using a water filter membrane (diameter = 0.22 μm). After preparation, the mixture was stored in a refrigerator at -20°C.

[0040] Experimental Example 1: Cloning and sequence analysis of the full-length CDS sequence of the CsETR2 gene

[0041] 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 CsETR2. 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 ) and sent it to a biological company (Beijing Qingke Biotechnology) for testing. The sequencing results were correct, and the CDS sequence was shown as SEQ ID NO.1. At the same time, the concentration of the target product was detected and stored for subsequent experiments.

[0042] Table 1 Primers for cloning the full-length CDS sequence of CsETR2

[0043]

[0044] The CsETR2 protein sequence was aligned with the ethylene receptor family protein sequences from tea plant, Arabidopsis thaliana, tomato, and wheat (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 with MEGA 11 software, using 1000 bootstrap replicates. The phylogenetic tree is shown in Figure 1. Figure 2 As shown, CsETR2 is most closely related to the Arabidopsis LeETR4 gene.

[0045] Experimental Example 2: Subcellular localization of CsETR2 gene

[0046] Based on the cDNA sequence of the tea plant CsETR2 gene (as shown in SEQ ID NO.1) and the BamHI and XbaI restriction sites of the pCAMBIA2300 vector, homologous recombination primers were designed to contain homology arms of the pCAMBIA2300 vector. The primer sequences are shown in Table 2. Then, the subcellular localization vector plasmid of the CsETR2 gene was constructed according to conventional methods.

[0047] The constructed vector plasmid was transferred into Agrobacterium by freeze-thaw method and cultured at 28℃ for 48h until colonies were grown. Single clones were picked and transferred to liquid LB containing Kana resistance and cultured at 28℃ for 18-24h until OD 600 At about 0.8, centrifuge at 4000 rpm / min for 10 min to collect the cells, resuspend the cells in 10 mM MgCl2, 10 mM MES, 200 μM AS suspension, and adjust the OD 600 To about 0.8. Select tobacco plants with good growth conditions, use a syringe to draw 1mL of infection solution and inject it from the lower epidermis of tobacco leaves, and mark it. After the injected tobacco plants are cultured in the dark for 48 hours, cut tissue sections near the injection hole, observe the GFP signal using a laser confocal microscope, and take pictures for preservation. Figure 3 As shown, the CsETR gene was observed to be localized in the cell membrane, cytoplasm, and nucleus.

[0048] Table 2 Primers for constructing CsETR2 subcellular localization vector

[0049]

[0050] Example 3: Agrobacterium-mediated plant genetic transformation

[0051] 1. Construction of CsETR2 gene overexpression vector

[0052] 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. 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.

[0053] 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.

[0054] Table 3 Primers for overexpression vector construction

[0055]

[0056] 2. Genetic transformation of tobacco with CsETR2 gene

[0057] 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:

[0058] 1) Sterile seedling culture of Nicotiana benthamiana

[0059] Nicotiana benthamiana seeds were soaked in 75% alcohol for 1 minute, sterilized with 15% H2O2 for 15 minutes, washed three times with clean water for 3 minutes each time, spread on MS culture medium in a clean bench, and cultured in a light incubator at 28°C for about 15 days.

[0060] 2) Conversion

[0061] In a clean bench, Nicotiana benthamian leaves were cut into small pieces of 0.5×0.5 cm, and the leaves were transferred to the treated Agrobacterium solution (OD value was measured at about 0.8). After infection for 5 minutes, the leaves were blotted dry on 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 cultured in the dark at 22°C in a constant temperature box for 2 days.

[0062] 3) Embryo induction

[0063] Transfer the leaves on the plate to the induction medium (MS+0.5mg / L BA+30g / L sucrose+8g / L agar+500mg / L cef+100mg / L Kana, pH5.8), let the wound stick to the surface of the culture medium with the front side facing up. After the transfer, seal it with sealing film and place it in a light incubator for culture. Transfer it once in the same culture medium after about 15 days.

[0064] 4) Rooting

[0065] When small shoots grew 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 allow the shoots to grow and induce rooting. The control plant was wild-type Nicotiana benthamiana (N. benthamiana).

[0066] 3. Detection of CsETR2 gene in transgenic Nicotiana benthamiana

[0067] DNA of transgenic Nicotiana benthamiana was extracted using the cetyltrimethylammonium bromide (CTAB) method. PCR was performed using the universal primers CsETR2-DNA and M13F (see Table 4) on the pBI121 vector. 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, 5 μL was collected for agarose gel electrophoresis. Figure 4 As shown, the bands are correct, indicating that the transgenesis is successful.

[0068] Table 4 Primers for transgenic plant detection

[0069]

[0070] Experimental Example 4: Disease resistance experiment of transgenic tobacco

[0071] The transgenic Nicotiana benthamiana and wild-type Nicotiana benthamiana were inoculated with Botrytis cinerea, and the area of ​​lesions 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 in an incubator and cultured at 25°C. B. cinerea was inoculated on leaves when it grew to 3-4 days old, and the mycelium was beaten into a 6mm diameter cake. Four small holes were punched on the tobacco, and the cake was inoculated on the leaves of the transgenic Nicotiana benthamiana with an inoculation needle, with the mycelium surface in contact with the front of the leaf. The size of the lesions was recorded 3 days after inoculation. The experimental results found that CsETR2 transgenic Nicotiana benthamiana had 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 ​​the wild-type Nicotiana benthamiana was 216.5 mm 2 The average lesion area of ​​CsETR2 transgenic Nicotiana benthamiana was 50.8 mm 2 The results showed that CsETR2 transgenic Nicotiana benthamiana had obvious disease resistance, indicating that the CsETR2 gene improved the disease resistance of Nicotiana benthamiana.

[0072] Experimental Example 5: Gene expression inhibition and disease resistance experiment of CsETR2 in AsODNs

[0073] Submit the CDS sequence of the CsETR2 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 reverse complemented to obtain oligonucleotide positive chain specific oligonucleotide fragments. The artificially synthesized oligonucleotide sequence of CsETR2 (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 positive 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 shown in Table 6), and qRT-PCR was used to detect the expression level of the target gene CsETR2 in the treatment group and control group samples. As Figure 6As shown in B, compared with the control group, the expression level of CsETR2 in the treatment group decreased significantly, indicating that the CsETR2 gene has been inhibited. Lasiodiplodia theobromae (strain number: GZHS-2017-010, deposit number: CGMCC3.20151) identified by the inventors' research group in the early stage was inoculated into the tea leaves of the treatment group and the control group to observe the changes in the lesion area. The size of the lesion area was recorded 48 hours after inoculation. Figure 6 As shown in A and 6C, the average lesion area of ​​the control plants was 27.7 mm 2 , while the average lesion area of ​​silent plants was 38.2 mm 2 , indicating that after silencing the CsETR2 gene, the plant's disease resistance was significantly weakened, further proving that the CsETR2 gene has disease resistance function.

[0074] Table 5 CsETR2 oligonucleotide fragments

[0075]

[0076] Table 6 Quantitative primers

[0077]

[0078] The conventional techniques and schemes not described in detail in the above embodiments are well known in the art and will not be described in detail here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple variations of the technical scheme of the present invention can be made, and these simple variations all fall within the scope of protection of the present invention.

Claims

1. Tea Tree CsETR2 Application of genes in improving plant disease resistance, the CsETR2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the disease resistance is resistance to Botrytis cinerea ( Botrytis cinerea ) or Lactodiplodia theobrominata ( Lasiodiplodia theobromae ) diseases caused by pathogens; the plant is tobacco or tea.

2. A method for improving plant disease resistance, characterized in that: Including CsETR2 The gene is introduced into the target plant to obtain a plant with improved disease resistance; CsETR2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the disease resistance is caused by B. cinerea or L. theobromae Diseases caused by pathogens; the plant is tobacco or tea.

3. The method according to claim 2, wherein described CsETR2 The gene is introduced into the target plant via a plant expression vector.

4. The method according to claim 3, wherein The plant expression vector includes the pBI121 vector.

5. The method according to claim 2, wherein The introduction is carried out by the leaf disk method.

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