Application and method of tea tree CsETR2 gene in improvement of plant disease resistance

Abstract

The invention discloses an application of a tea tree CsETR2 (ethene receptor 2) gene in improving the disease resistance of a plant and a method of the tea tree CsETR2 gene in improving the disease resistance of the plant. The CsETR2 gene is an important gene of an ethylene receptor family, the CsETR2 is constructed to a plant expression vector and is transferred into nicotiana benthamiana, a transgenic tobacco plant is obtained through stable genetic transformation, and the CsETR2 gene shows relatively strong fungal inhibitory activity on botrytis cinerea inoculated hyphae; according to the present invention, the CsETR2 gene on the tea tree leaf is subjected to instantaneous silencing by using the antisense oligonucleotides (AsODNs) technology, and the lasiodiplodia theobroma is inoculated, such that the disease resistance of the plant is significantly weakened; therefore, the CsETR2 gene has the effect of improving the disease resistance of the plants, can be used as a disease-resistant gene, can improve the disease resistance of the plants by being introduced into the plants such as tobacco, tea trees or vegetables, and has a wide market application prospect.

Description

Technical Field

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

[0002] Tea tree (Camellia sinensis (L.) O. Kuntz) is a perennial evergreen small tree or shrub used as an important economic crop. Tea leaf spot caused by Lasiodiplodia theobromae was first isolated and identified by the inventor's research group from tea leaf diseases in Huishui County, Guizhou Province (Li DX, Bao XT, Ren YF, et al. First report of Lasiodiplodia theobromae causing leaf spot on tea plant in Guizhou Province of China [J]. Plant Disease, 2019, 103 (2): 374.). Lasiodiplodia theobromae is an asexual form of the Botryosphaeria pathogen, and its sexual form is Botryosphaeria rhodian (Berk. & M.A. Curtis) Arx. Although it is found almost all over the world, 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 shedding, organ aging, maturity, stress response and pathogen response (Abeles FB, Morgan PW, Saltveit Jr M E. Ethylene in plant biology [M]. Academic press, 2012.). In the early stage 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: a missing link in ethylene regulation of fungus-plant interactions? [J]. Molecular plant-microbe interactions, 2006, 19(1): 33-42.).

[0004] At present, there are few studies on the function of tea tree ethylene receptor family gene CsETR2 (ethylene receptor 2), especially the function of disease resistance. The use of genetic engineering technology to cultivate resistant plant varieties and materials has obvious advantages and irreplaceable importance. It can not only facilitate the large-scale production of tea trees, vegetables, tobacco and other plant varieties, but also reduce the use of chemical pesticides and reduce environmental pollution. However, so far, there has been no report on the disease resistance function of tea tree CsETR2 gene. Summary of the invention

[0005] In view of this, one of the purposes of the present invention is to provide an application of a tea tree ethylene receptor gene CsETR2 gene, which plays a role in improving plant disease resistance and can be used to improve plant disease resistance. 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 Lactodiplodia 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 pBI121 vector. Of course, it can also be other vectors, such as Ti-type plasmid vectors, viral vectors, etc.

[0012] Preferably, the introducing is carried out by a leaf 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 transferred into Nicotiana benthamiana. A transgenic tobacco plant is obtained by stable genetic transformation, and a strong fungal inhibitory activity is shown on the mycelium inoculated with Botrytis cinerea. The CsETR2 gene on tea leaves is transiently silenced by using the technology of antisense oligonucleotides (AsODNs), and the disease resistance of the plant is significantly weakened by inoculating Diplodia theobromin. Therefore, the CsETR2 gene has the function of improving the disease resistance of plants, can be used as a disease resistance gene, and can be introduced into plants such as tobacco, tea trees or vegetables to improve the disease resistance of plants, and has broad market application prospects. The present invention provides a new method for improving the resistance of plants to fungal diseases. Cultivating disease-resistant plants by genetic engineering means can overcome the shortcomings of traditional breeding, not only shorten the breeding cycle, but also simplify the operation and easily obtain highly resistant materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the gel electrophoresis diagram of the tea plant CsETR2 gene cloning 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 for the subcellular localization of the tea plant CsETR2 gene of the present invention;

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

[0019] Figure 5 The phenotype diagrams of the Nicotiana benthamiana overexpressing the gene CsETR2 of the present invention and the Nicotiana benthamiana control pBI121 after inoculation with B. cinerea; wherein A is a phenotype diagram of the antibacterial activity of Nicotiana benthamiana overexpressing the gene CsETR2 and the Nicotiana benthamiana control pBI121 after inoculation with B. cinerea; B is a comparison diagram of the lesion area of ​​Nicotiana benthamiana overexpressing the gene CsETR2 and the Nicotiana benthamiana control 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 inhibited by AsODN and the quantitative analysis of CsETR2 gene; wherein A is the phenotype of L. theobromae inoculated on tea plants with CsETR2 gene inhibited by AsODN; B is the quantitative expression analysis of CsETR2 gene after the tea plants with CsETR2 gene inhibited by AsODN; C is the lesion area of ​​the tea plants with CsETR2 gene inhibited by AsODN. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below in conjunction with 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 should be included within the scope of the present invention. The experimental materials used in the following examples are commercially available unless otherwise specified.

[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 10 g;

[0028] Sodium chloride (NaCl) 10 g;

[0029] For liquid culture medium, place the reagent in a 1L glass beaker, add 1L of ddH2O to the glass beaker, stir with a glass rod to dissolve completely, and dispense into 250mL conical flasks, 100mL of liquid culture medium in each conical flask. For solid LB culture medium, add 1.5g of agar powder to every 100mL of liquid LB culture 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] 2.033 g of magnesium chloride was dissolved in 10 mL of ddH2O, sterilized by high pressure at 121°C for 20 min, and stored 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, it was stored in a -20°C refrigerator.

[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, it was stored in a -20°C refrigerator.

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

[0041] Total RNA from Camellia sinensis cv. Fuding-dabaicha tea leaves was extracted using TRIzol reagent, and the RNA quantity and purity of each sample were quantified using NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). The first strand of cDNA was generated by reverse transcription and used as a template. PCR was performed using primers for amplifying 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 to 94°C pre-deformation for 2 min, 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 50 s, 35 cycles, and 72°C post-extension for 5 min. After the PCR, 50 μL was taken for gel excision purification. Gel excision purification was used. DNA Gel Extraction Kit DNA Gel Extraction Kit (Beijing Qingke Biotechnology) Purification specific steps are shown in the instructions. After purification, take 5 μL and perform agarose gel electrophoresis. The bands meet the expected results ( Figure 1 ) and sent it to a biological company (Beijing Qingke Biotechnology) for testing. The sequencing result was correct, wherein 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 in tea, Arabidopsis, tomato, and wheat (http: / / planttfdb.gao-lab.org / ). The amino acid sequences were aligned using the Clustal W program (http: / / www.clustal.org / clustal2 / ), and phylogenetic analysis was performed using the NJ method using MEGA 11 software with 1000 bootstrap replicates. The evolutionary tree is shown in Figure 2 As shown, CsETR2 is most closely related to the Arabidopsis LeETR4 gene.

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

[0046] According to 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 the homologous arms of the pCAMBIA2300 vector. The primer sequences are shown in Table 2, and 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 grew. Single clone colonies were picked and placed in 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, it was observed that the CsETR gene was 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 plasmid DNA small-scale extraction kit (Shanghai Biotech) (see the instructions for the Shanghai Biotech kit for the plasmid extraction steps). The two cloning sites XbaI and SacI on the pBI121 vector were selected for double restriction digestion to linearize the vector. 1 μL was taken for 1.5% agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. DNA Gel Extraction Kit DNA gel recovery kit (Beijing Qingke Biotechnology) was used for purification (see the instruction manual for specific steps), and the size of the recovered fragments and the concentration were detected by 1.5% agarose gel electrophoresis. Homologous recombination primers were designed to contain homology arms of the pBI121 vector. The primer sequences are shown in Table 3.

[0053] use IIOne Step Cloning Kit homologous recombination kit (Nanjing Novozyme) and its instructions, after determining the concentration of vector fragment and target gene concentration, homologous recombination was performed using the homologous recombination kit reagents, the system is as follows: 5×CEⅡBuffer (2μL), ExnaseⅡ (1μL), linearized vector (6μL), target gene fragment (1μL), the program is 37℃, 30min. After the reaction is completed, it is immediately placed on ice and then transferred into Escherichia coli DH5α. After ligation and transformation, it is plated on LB solid medium containing Kana antibiotics (LB preparation method attached to the end page), and the bacterial solution is shaken in a shaker for 12h before the bacterial solution is tested. The bacterial solution of the positive clone is picked and sent to Qingke Biological Company for sequencing to ensure that the target sequence is successfully connected to the pBI121 vector to obtain the overexpression vector.

[0054] Table 3 Primers for overexpression vector construction

[0055]

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

[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, and the specific steps of transgenic were as follows:

[0058] 1) Cultivation of sterile seedlings of Nicotiana benthamiana

[0059] The seeds of Nicotiana benthamiana 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.

[0060] 2) Conversion

[0061] In a clean bench, cut Nicotiana benthamian leaves into small pieces of 0.5×0.5 cm, transfer the Nicotiana benthamian leaves into the treated Agrobacterium solution (OD value measured at about 0.8), infect for 5 minutes, blot dry on sterile filter paper, and then inoculate on MS solid co-culture medium (1 / 2MS+30g / L sucrose+8g / L agar, pH 5.8) and culture in a constant temperature incubator at 22°C in the dark 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 culture it in a light incubator. Transfer it once in the same culture medium after about 15 days.

[0064] 4) Rooting

[0065] When small shoots grow on the induction medium, they are 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 is wild-type Nicotiana benthamiana (N. benthamiana).

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

[0067] The DNA of transgenic Nicotiana benthamiana was extracted using the cetyltrimethylammonium bromide (CTAB) method, and 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-deformation for 2 min, 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 50 s, 35 cycles, and 72°C post-extension for 5 min. After the PCR, 5 μL was taken for agarose gel electrophoresis, as shown in FIG. Figure 4 As shown, the bands are correct, indicating that the transgenic was 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 lesion area was recorded 3 days after inoculation, and the data significance analysis 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 hyphae were 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 hyphae surface in contact with the front of the leaf. The size of the lesion 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 ​​wild-type Nicotiana benthamiana was 216.5 mm 2 The average lesion area of ​​CsETR2 transgenic N. 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 fragment specific for the antisense strand of the target gene oligonucleotide, which is usually 20 bp in length. Oligonucleotide fragments with low binding site destruction energy are preferentially selected, and this sequence is reversely complemented to obtain oligonucleotide fragments specific for the sense strand of the oligonucleotide. The artificially synthesized oligonucleotide sequence of CsETR2 (sequence as shown in Table 5) was transferred to the young buds of the tea tree. The treatment group was the young buds containing the antisense strand of the oligonucleotide of the target gene, and the control group was the young buds containing the sense strand of the oligonucleotide. 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 CsETR2 in the samples of the treatment group and the control group. Figure 6As shown in B, compared with the control group, the expression of CsETR2 in the treatment was significantly reduced, indicating that the CsETR2 gene had 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 on the tea leaves of the treatment group and the control group to observe the changes in the lesion area, and the size of the lesion area after 48 hours of inoculation was recorded, as shown in FIG. 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, so they 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 in the above embodiments. Within the scope of the technical concept of the present invention, the technical scheme of the present invention can be subjected to a variety of simple modifications, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. Application of tea plant CsETR2 gene in improving plant disease resistance, the nucleotide sequence of the CsETR2 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The disease resistance is resistance to diseases caused by pathogens including Botrytis cinerea or Lasiodiplodia theobromae.

3. The use according to claim 1, characterized in that The plant includes tobacco or tea.

4. A method for improving plant disease resistance, characterized in that: The method comprises 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.

5. The method according to claim 4, characterized in that The disease resistance is resistance to diseases caused by pathogens including B. cinerea or L. theobromae.

6. The method according to claim 4, characterized in that The CsETR2 gene is introduced into the target plant via a plant expression vector.

7. The method according to claim 6, characterized in that The plant expression vector includes pBI121 vector.

8. The method according to claim 4, characterized in that The introduction is carried out by a leaf disk method.

9. The method according to claim 4, characterized in that The plant includes tobacco or tea.

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