Application of tea tree-derived transcription factor CsWRKY71 in improving plant insect resistance and preparing insect-resistant plant varieties

Abstract

The present invention provides an application of tea tree-derived transcription factor CsWRKY71 in improving plant insect resistance and preparing plant insect-resistant varieties, belonging to the field of biological control technology. The nucleotide sequence of the tea tree-derived transcription factor CsWRKY71 is shown in SEQ ID No. 1, and the gene can play a key role in improving plant insect resistance. Studies have shown that the feeding preference of diamondback moth in Arabidopsis overexpressing the CsWRKY71 gene is significantly reduced; compared with the control, the weight gain of tea geometrid on tea trees overexpressing the CsWRKY71 gene is significantly reduced. That is, the insect resistance of plants overexpressing the CsWRKY71 gene is significantly enhanced. The present invention can provide genetic resources and technical means for the breeding and application of new insect-resistant plant varieties and the green prevention and control of pests, and has potential application prospects and value.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and in particular relates to application of tea tree-derived transcription factor CsWRKY71 in improving plant insect resistance and preparing plant insect-resistant varieties. Background Art

[0002] Tea tree (Camellia sinensis) is a perennial evergreen woody plant. Its tender buds and leaves are often used to make different types of tea and sold all over the world. It is an important economic crop with great characteristics and value. However, during its growth process, especially in the summer and autumn when the temperature is high, tea trees are often harmed by various pests, and the occurrence of pests often presents the characteristics of many types and great harm, which not only significantly reduces the yield and quality of tea in the current year, but also causes the growth of tea trees to weaken in severe cases, which has a serious impact on tea production in the next year. At present, the prevention and control of important tea pests mainly relies on chemical pesticides. The increasingly serious environmental and ecological safety and human life and health problems caused by the widespread use of pesticides make it urgent to explore and study new insect-resistant genes in tea trees and cultivate new insect-resistant tea varieties.

[0003] Insect pests often occur during the cultivation of Arabidopsis thaliana, which will hinder the normal growth and development of Arabidopsis thaliana, affecting subsequent experiments, phenotypic observations and long-term preservation of material germplasm.

[0004] Studies in recent years have shown that transcription factor genes play an important role in various life activities of plants, such as growth and development, and response to adversity. Among them, WRKY transcription factors, as one of the largest transcription factor families unique to plants, have been proven to play a very critical role in the process of plants responding to biotic and abiotic stresses. For example, the WRKY29 gene of tea trees can significantly improve the cold resistance of plants. However, so far, there have been no studies and reports on the research of tea tree-derived WRKY transcription factor genes and their encoded products in plant insect resistance. As a perennial woody plant, tea trees have many problems such as long traditional breeding cycles, low efficiency, and the lack of a sound transgenic technology system.

[0005] Therefore, studying the insect-resistant function of tea tree-derived WRKY transcription factor genes and applying them to the molecular breeding of insect-resistant tea trees has important practical significance for the green prevention and control of tea tree pests and ensuring the green and healthy development of the tea industry. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide an application of the tea tree-derived transcription factor CsWRKY71 in improving plant insect resistance and preparing plant insect-resistant varieties. The tea tree-derived transcription factor CsWRKY71 can improve the resistance of herbaceous plants and woody plants to pests, and provides genetic resources and technical means for the breeding and application of new insect-resistant plant varieties and the green prevention and control of pests, and has good application prospects and value.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides application of tea tree-derived transcription factor CsWRKY71 in improving plant insect resistance. The nucleotide sequence of the tea tree-derived transcription factor CsWRKY71 is shown in SEQ ID No.1.

[0009] Preferably, the insect resistance of the plant is improved by overexpressing the tea tree-derived transcription factor CsWRKY71.

[0010] Preferably, the plant is a herbaceous plant or a woody plant.

[0011] Preferably, the herbaceous plant is Arabidopsis thaliana, and the woody plant is tea tree.

[0012] The present invention also provides a recombinant vector for overexpressing the tea tree-derived transcription factor CsWRKY71, comprising the tea tree-derived transcription factor CsWRKY71 and a pBWA(V)HS vector.

[0013] The present invention also provides a method for constructing the recombinant vector for overexpressing the tea tree-derived transcription factor CsWRKY71, comprising the following steps:

[0014] (1) connecting the tea tree-derived transcription factor CsWRKY71 to the pEASY-blunt zero vector to obtain pEASY-CsWRKY71;

[0015] (2) PCR amplification of pEASY-CsWRKY71 to obtain the amplified product rDNA;

[0016] (3) The amplified rDNA and the expression vector pBWA(V)HS were subjected to BsaI / Eco31I double restriction digestion reaction to obtain linearized rDNA and linearized vector pBWA(V)HS;

[0017] (4) The linearized rDNA obtained in step (3) and the linearized vector pBWA(V)HS were subjected to homologous recombination and GoldenGate ligation reaction to obtain the overexpression recombinant vector pBWA(V)HS-CsWRKY71.

[0018] The present invention also provides a recombinant bacterium over-expressing the tea tree-derived transcription factor CsWRKY71, which is obtained by transferring the over-expression recombinant vector into Agrobacterium GV3101.

[0019] The present invention also provides the use of overexpressing tea tree-derived transcription factor CsWRKY71 in preparing insect-resistant Arabidopsis varieties.

[0020] Preferably, when preparing the insect-resistant Arabidopsis thaliana variety, the recombinant bacteria overexpressing the tea tree-derived transcription factor CsWRKY71 are transformed into Arabidopsis thaliana by Agrobacterium-mediated floral infection.

[0021] The present invention also provides the use of overexpressing tea tree-derived transcription factor CsWRKY71 in preparing insect-resistant tea tree varieties.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses RT-PCR and RACE technology to isolate and clone a tea tree-derived transcription factor gene CsWRKY71 for the first time, and verifies the biological function of the gene for the first time through homologous and heterologous overexpression experiments, proving that it can improve the resistance of plants to pests, providing valuable gene resources and technical means for breeding new varieties of insect-resistant plants and green pest control, and has good potential application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the map of the overexpression recombinant vector pBWA(V)HS-CsWRKY71;

[0025] Figure 2 This is the gel electrophoresis image of the PCR amplification product of the full length of CsWRKY71 gene;

[0026] Figure 3 The relative expression of CsWRKY71 gene in overexpressed plants; the left figure shows the relative expression of CsWRKY71 gene in control plants, Arabidopsis transgenic lines 1 and 2, and the right figure shows the relative expression of CsWRKY71 gene in control tea leaves and CsWRKY71 overexpressed tea leaves after transient overexpression for 24h and 48h;

[0027] Figure 4 Schematic diagram of the experimental setup for feeding preference of CsWRKY71-overexpressing Arabidopsis;

[0028] Figure 5 The results of feeding preference analysis of the Arabidopsis pest Plutella xylostella on control plants and CsWRKY71 overexpressing Arabidopsis strains 1 and 2;

[0029] Figure 6This is a diagram of the experimental setup for identifying the insect resistance function of Arabidopsis thaliana overexpressing the CsWRKY71 gene in Example 8;

[0030] Figure 7 The analysis results of the biological assay on Plutella xylostella in Example 8, wherein: Figure 7 Each circle in the figure represents one biological replicate;

[0031] Figure 8 The results of the insect resistance analysis of tea plants transiently overexpressing CsWRKY71 are shown in Figure 2. Figure 8 Each circle in the figure represents one biological replicate; the 25 replicates of the control and treatment were averaged and statistically analyzed. DETAILED DESCRIPTION

[0032] The present invention provides application of tea tree-derived transcription factor CsWRKY71 in improving plant insect resistance. The nucleotide sequence of the tea tree-derived transcription factor CsWRKY71 is shown in SEQ ID No.1.

[0033]

[0034] The amino acid sequence of the protein encoded by the tea tree-derived transcription factor CsWRKY71 is: MSDEPGGFYYHDPFHDDRYATGATAFSFSTSTNSSLYNSPSHDLHGLGSSHMSFTESLHGSTDYNTIETAFGLSPPSSQLFSVVKEEQKPLVMDGGDTVVGVGETAVTPNSSISSSSTEAGGDEDTSKSKKDRLQKEVEDGGGESSKKVNKSKKKVEKIKREPRFAFMTKSEVDHLEDGYRWRKYGQKAVKNSPFPRSYYRCTTQKCTVKKRVERSFQDPSTVITTYEGQHNHQIPATLRGNAAGVMLPPSMLTMTPPLIAGTGFPQEFIVQMAPMYNHGGMSSFYPQSSNLTPLHHQQLQHPDDYGLLQDMFPSTFFKQEP* (SEQ ID No. 2).

[0035] The present invention improves the insect resistance of plants by overexpressing the tea tree-derived transcription factor CsWRKY71; the plants are preferably herbaceous plants or woody plants; the herbaceous plants are preferably Arabidopsis thaliana, and the woody plants are preferably tea trees.

[0036] The present invention also provides a recombinant vector for overexpressing the tea tree-derived transcription factor CsWRKY71, comprising the tea tree-derived transcription factor CsWRKY71 and a pBWA(V)HS vector.

[0037] The present invention also provides a method for constructing the recombinant vector for overexpressing the tea tree-derived transcription factor CsWRKY71, comprising the following steps:

[0038] (1) connecting the tea tree-derived transcription factor CsWRKY71 to the pEASY-blunt zero vector to obtain pEASY-CsWRKY71;

[0039] (2) PCR amplification of pEASY-CsWRKY71 to obtain the amplified product rDNA;

[0040] (3) The amplified rDNA and the expression vector pBWA(V)HS were subjected to BsaI / Eco31I double restriction digestion reaction to obtain linearized rDNA and linearized vector pBWA(V)HS;

[0041] (4) The linearized rDNA obtained in step (3) and the linearized vector pBWA(V)HS were subjected to homologous recombination and GoldenGate ligation reaction to obtain the overexpression recombinant vector pBWA(V)HS-CsWRKY71.

[0042] In the present invention, the tea tree-derived transcription factor CsWRKY71 is connected to the pEASY-blunt zero vector to obtain pEASY-CsWRKY71.

[0043] In the present invention, the tea tree-derived transcription factor CsWRKY71 is preferably obtained after purification, and the purification method is preferably PCR amplification. The primers used in the PCR amplification are preferably composed of an upstream primer with a nucleotide sequence of 5'-ATGTCTGATGAACCAGGAGGC-3' (SEQ ID No.3) and a downstream primer with a nucleotide sequence of 5'-CGTCGTTAAGTCAAGCAGCC-3' (SEQ ID No.4); the template for the PCR amplification is preferably cDNA obtained by reverse transcription of the tea tree-derived transcription factor CsWRKY71 gene; the connection method is preferably carried out according to the instructions of the pEASYblunt zero vector purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0044] In the present invention, pEASY-CsWRKY71 is PCR amplified to obtain the amplified product rDNA. In the present invention, the primers used in the PCR amplification are preferably composed of an upstream primer with a nucleotide sequence of 5'-CAGTGGTCTCACAACATGTCTGATGAACCAGGAGG-3' (SEQ ID No. 5) and a downstream primer with a nucleotide sequence of 5'-CAGTGGTCTCATACATCATGGCTCTTGTTTAAAAA-3' (SEQ ID No. 6).

[0045] In the present invention, the amplified product rDNA and the expression vector pBWA(V)HS are subjected to BsaI / Eco31I double restriction enzyme digestion reaction respectively to obtain linearized rDNA and linearized vector pBWA(V)HS.

[0046] In the present invention, the enzyme digestion reaction system is preferably: based on 20 μL, 3 μL of rDNA or pBWA(V)HS plasmid, 2 μL of 10× buffer, 1 μL of BsaI, 1 μL of Eco31I, and 13 μL of water.

[0047] In the present invention, homologous recombination and Golden Gate ligation reaction are performed on the obtained linearized rDNA and the linearized vector pBWA(V)HS to obtain the overexpression recombinant vector pBWA(V)HS-CsWRKY71.

[0048] In the present invention, the homologous recombination conditions are preferably 20-25°C for 1-2h, more preferably 21-24°C for 1.2-1.8h, and more preferably 23°C for 1.5h; the Golden Gate ligation reaction conditions are preferably 15-20°C for 1-1.5h, more preferably 16-18°C for 1.2-1.4h, and more preferably 17°C for 1.3h; the overexpression recombinant vector pBWA(V)HS-CsWRKY71 has a map as shown in Figure 1 shown.

[0049] The present invention also provides a recombinant bacterium over-expressing the tea tree-derived transcription factor CsWRKY71, which is obtained by transferring the over-expression recombinant vector into Agrobacterium GV3101.

[0050] In the present invention, the transfer method is preferably a chemical conversion method.

[0051] The present invention also provides the use of overexpressing tea tree-derived transcription factor CsWRKY71 in preparing insect-resistant Arabidopsis varieties.

[0052] In the present invention, when preparing insect-resistant Arabidopsis varieties, the recombinant bacteria overexpressing the tea tree-derived transcription factor CsWRKY71 are preferably transformed into Arabidopsis by Agrobacterium-mediated floral infection, and the Arabidopsis is preferably wild-type Arabidopsis.

[0053] The present invention also provides the use of overexpressing tea tree-derived transcription factor CsWRKY71 in preparing insect-resistant tea tree varieties.

[0054] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] Example 1 Acquisition and sequence analysis of tea tree derived transcription factor gene CsWRKY71

[0056] The total RNA from the leaves of tea plant 'Longjing 43' (two-year-old potted seedlings, obtained by cuttings and propagation in the laboratory) was extracted using RNAPlant Plus Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The concentration and quality of the extracted total RNA were tested using Nanodrop. RNA samples with OD260 / 280>2.0 and OD260 / 230>1.8 were considered qualified.TM The qualified biological samples were reverse transcribed into cDNA using RT reagent Kit (purchased from TaKaRa). The detailed operation method was carried out according to the manufacturer's instructions.

[0057] Primers capable of amplifying the full length of the CsWRKY71 gene were designed using the NCBI PrimerBlast website. The primers consisted of an upstream primer with a nucleotide sequence of 5'-ATGTCTGATGAACCAGGAGGC-3' (SEQ ID No. 3) and a downstream primer with a nucleotide sequence of 5'-CGTCGTTAAGTCAAGCAGCC-3' (SEQ ID No. 4).

[0058] The cDNA obtained by reverse transcription was used as a template to perform PCR amplification of the full length of the CsWRKY71 gene using KOD high-fidelity enzyme (purchased from Toyobo Co., Ltd.) to obtain a PCR purified product containing the complete CsWRKY71 gene coding region sequence. The bands obtained by the reaction were as follows: Figure 2 shown.

[0059] The PCR amplification system is: 50 μL, cDNA template 2 μL, 2×PCR buffer 25 μL, 2 mM dNTP 10 μL, 10 pmol / μL upstream primer 1.5 μL, 10 pmol / μL downstream primer 1.5 μL, KOD high-fidelity enzyme 1 μL, water 9 μL. The amplification conditions are: 98°C×4min→(98°C×20sec→62°C×20sec→68°C×60sec)×35 cycles→68°C×3min.

[0060] The PCR purified product obtained by the above amplification was connected to the pEASYblunt zero vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to obtain pEASY-CsWRKY71.

[0061] Plasmid PCR identification was performed using universal primers M13 (M13F: 5'-GTAAAACGACGGCCAGT-3' (SEQ ID No. 7), M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID No. 8)).

[0062] The specific connection method and PCR identification method were carried out according to the manufacturer's instructions. The positive plasmid obtained by identification was sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing, and the CsWRKY71 gene sequence shown in SEQ ID NO.1 was obtained after sequencing.

[0063] Example 2 Construction of a recombinant vector for overexpressing tea tree-derived transcription factor CsWRKY71

[0064] The pEASY-CsWRKY71 plasmid obtained in Example 1 was used as a template, and a primer pair consisting of an upstream primer: 5'-CAGTGGTCTCACAACATGTCTGATGAACCAGGAGG-3' (SEQ ID No. 5) and a downstream primer: 5'-CAGTGGTCTCATACATCATGGCTCTTGTTTAAAA-3' (SEQ ID No. 6)) was used for PCR amplification to obtain the PCR product rDNA. The PCR amplification system is: based on 50 μl, 2 μl of plasmid template, 25 μl of 2×PCR buffer, 10 μl of 2mM dNTP, 1.5 μl of 10 pmol / μl upstream primer, 1.5 μl of 10 pmol / μl downstream primer, 1 μl of KOD high-fidelity enzyme (purchased from Toyobo Co., Ltd.), and 9 μl of water. The conditions for PCR amplification were: 98°C×4 min→(98°C×20 sec→62°C×20 sec→68°C×60 sec)×35 cycles→68°C×3 min.

[0065] The PCR product rDNA and the expression vector pBWA(V)HS (purchased from Wuhan Boyuan Biotechnology Co., Ltd.) were subjected to BsaI / Eco31I double restriction enzyme digestion reaction to obtain linearized rDNA and linearized vector pBWA(V)HS.

[0066] The enzyme digestion reaction system is: based on 20 μL, 3 μL of rDNA or pBWA(V)HS plasmid, 2 μL of 10× buffer, 1 μL of BsaI, 1 μL of Eco31I, and 13 μL of water.

[0067] The obtained linearized rDNA and linearized vector pBWA(V)HS were purified using a PCR purification kit (purchased from Axygen), and then the two were subjected to homologous recombination and Golden Gate ligation to obtain the overexpression recombinant vector pBWA(V)HS-CsWRKY71. The map of the overexpression recombinant vector is shown in Figure 1 shown.

[0068] Sequencing analysis showed that the overexpression recombinant vector pBWA(V)HS-CsWRKY71 was obtained by cloning the CsWRKY71 gene shown in SEQ ID No.1 into the plasmid pBWA(V)HS, and the vector expressed the CsWRKY71 protein shown in SEQ ID No.2.

[0069] Example 3 Construction of recombinant bacteria overexpressing tea tree-derived transcription factor CsWRKY71

[0070] The overexpression recombinant vector pBWA(V)HS-CsWRKY71 was transferred into Agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) by chemical transformation to obtain the overexpression recombinant bacteria named GV3101 / pBWA(V)HS-CsWRKY71.

[0071] The empty vector pBWA(V)HS was introduced into Agrobacterium GV3101 to obtain the recombinant bacteria GV3101 / pBWA(V)HS as an empty vector control.

[0072] Example 4 Construction of transgenic Arabidopsis thaliana overexpressing CsWRKY71 gene

[0073] The recombinant bacterium GV3101 / pBWA(V)HS-CsWRKY71 constructed in Example 3 was transformed into wild-type Arabidopsis thaliana (Colombia ecotype variety, a commonly used Arabidopsis thaliana variety in the laboratory) by Agrobacterium-mediated floral infection to obtain transgenic Arabidopsis thaliana T0 generation seeds.

[0074] Since the pBWA(V)HS vector carries a hygromycin resistance selection gene, the transgenic Arabidopsis T0 seeds were screened on MS medium plates containing 15 mg / L hygromycin. Arabidopsis that can grow normally on the hygromycin medium plates are transgenic T1 positive seedlings, and those that cannot grow normally are negative seedlings. The seeds harvested from the positive seedlings are T1 seeds. The T1 seeds were screened for hygromycin resistance, and the segregation ratio was identified. The strain with a positive seedling: negative seedling ratio of about 3:1 was selected, indicating that in this strain, the vector carrying the CsWRKY71 gene was inserted into the Arabidopsis genome in a single copy form. The T2 positive seedlings in these strains were removed, and single-plant planting and harvesting were performed to obtain T2 seeds, which were then screened for hygromycin resistance. The T3 seedlings that were all positive seedlings were single-copy T3 CsWRKY71 overexpressing transgenic lines. Two lines were randomly selected and named Line 1 and Line 2, and they were used as Line 1 and Line 2 for subsequent experiments.

[0075] The recombinant bacteria GV3101 / pBWA(V)HS were transformed into wild-type Arabidopsis thaliana using the same method as above to obtain control plants with empty vectors.

[0076] Example 5 Construction of tea plants with transient overexpression of CsWRKY71 gene

[0077] The recombinant bacteria GV3101 / pBWA(V)HS-CsWRKY71 constructed in Example 3 was injected into the third leaf of three-year-old Longjing 43 tea tree potted seedlings (Longjing 43 tea tree potted seedlings were obtained by cutting propagation in the laboratory) by Agrobacterium infiltration to obtain CsWRKY71-OE overexpression samples.

[0078] The same method was used to transform the recombinant bacteria GV3101 / pBWA(V)HS into the leaves at the same position of tea plants to obtain a control sample with an empty vector.

[0079] Example 6 Detection of relative expression level of CsWRKY71 gene in overexpressed plants

[0080] The relative expression levels of CsWRKY71 in Arabidopsis overexpressing the CsWRKY71 gene and in tea plants transiently overexpressing CsWRKY71 in Examples 4 and 5 were detected. Total RNA from Arabidopsis and tea leaves was extracted using the RNAPlant Plus Kit, and PrimeScript TM The total RNA was reverse transcribed using the RT reagent Kit to synthesize cDNA. The detailed operation method was carried out according to the manufacturer's instructions.

[0081] The Light Cycler480 fluorescence quantitative PCR system (qRT-PCR) was used to detect the relative expression of CsWRKY71 in Arabidopsis and tea plants, respectively. The detection primer pair used consisted of an upstream primer: 5'-TTCCATGATGATCGCTACGC-3' (SEQ ID No. 9) and a downstream primer: 5'-AGCTCATGTGTGAAGAGCCT-3' (SEQ ID No. 10). The detection results are shown in Figure 2. Figure 3 shown.

[0082] Depend on Figure 3 It can be seen that the overexpression effect of the CsWRKY71 gene can be detected in transgenic Arabidopsis lines 1 and 2 and tea plants transiently expressed for 24h and 48h.

[0083] Example 7 Determination of feeding preference of Plutella xylostella on Arabidopsis thaliana overexpressing CsWRKY71 gene

[0084] The control plants in Example 4 and the seeds of Arabidopsis strains 1 and 2 overexpressing the CsWRKY71 gene were sterilized and grown on MS medium (purchased from Beijing Solebow Technology Co., Ltd.) for 10 days, and then moved into cylindrical seedling blocks with a diameter and height of 4 cm and 5 cm, respectively, and placed in a light incubator for further cultivation. The incubator conditions were set as follows: the photoperiod was 16 h of light and 8 h of darkness; the light intensity was 10,000 lux; the temperature was 22°C under light conditions and 20°C under dark conditions. After 25 days, the control plants and Arabidopsis strains 1 and 2 overexpressing the CsWRKY71 gene with consistent growth were selected for the experiment.

[0085] Select 5 leaves from the same leaf position, cut them and place them in a 10 cm × 10 cm square culture dish, and cover the base of the petiole with filter paper to keep it moist. After the leaves are placed, five third-instar diamondback moth larvae (purchased from Keyun Biopesticide Technology Research and Development Center, an important leaf-feeding pest on Arabidopsis) are placed in the upper middle of the culture dish. Figure 4 shown.

[0086] After 3, 6, 18, and 30 hours of access, the number of larvae feeding on the control plants and the CsWRKY71 overexpression lines was investigated and statistically analyzed. Figure 5 shown.

[0087] Depend on Figure 5 It can be seen that compared with overexpression lines 1 and 2, the larvae of the diamondback moth are more inclined to feed on the control plants, that is, the insect resistance of Arabidopsis thaliana overexpressing the CsWRKY71 gene is significantly improved.

[0088] Example 8 Identification of the insect resistance function of Arabidopsis thaliana overexpressing the CsWRKY71 gene

[0089] The 30-day-old control plants and the CsWRKY71 gene overexpressing Arabidopsis strains 1 and 2 plants of Example 4 were selected and placed in cylindrical plastic cups (upper diameter 9.5 cm, lower diameter 5.5 cm, height 10.5 cm), and a second-instar diamondback moth larva was inoculated in the central leaf position of each plant. Figure 6 After the diamondback moth had been feeding for 3 days, its weight was taken out and statistically calculated. Figure 7 shown.

[0090] Depend on Figure 7 It can be seen that compared with the control, the weight of the diamondback moth larvae fed on the overexpression lines 1 and 2 was significantly reduced, indicating that the insect resistance of Arabidopsis thaliana overexpressing the CsWRKY71 gene was significantly improved.

[0091] Example 9 Identification of the insect resistance function of tea plants transiently overexpressing the CsWRKY71 gene

[0092] One second-instar tea geometrid larva was inoculated on each tea leaf transiently expressing CsWRKY71-OE obtained in Example 5 (tea geometrids were collected from the experimental tea garden of the Tea Research Institute of the Chinese Academy of Agricultural Sciences, and placed in an environment with a temperature of 26±2°C, a relative humidity of 70-80%, and a photoperiod of 12h light and 12h darkness, and then reared on tea branches for successive generations. Second-instar larvae of the same size after hatching from eggs were used in the experiment), and the larvae were taken out and weighed after feeding for 5 days. The experiment was repeated 25 times in total, i.e., one tea geometrid larva was inoculated on each of 25 tea leaves transiently expressing CsWRKY71-OE. Leaves transiently expressing an empty vector were used as a control. The experimental results are shown in the figure. Figure 8 shown.

[0093] Depend on Figure 8 It can be seen that compared with the control, the weight of tea geometrid larvae feeding on CsWRKY71-OE overexpressing tea plants was significantly reduced, indicating that the insect resistance of tea plants overexpressing the CsWRKY71 gene was significantly improved.

[0094] The above research results prove that the CsWRKY71 gene and its encoded product play a key role in improving plant insect resistance, and its good effect has been verified in both the herbaceous plant Arabidopsis and the woody plant tea. This result will provide genetic resources and basis for the selection and application of new insect-resistant plant varieties and the green prevention and control of pests.

[0095] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Tea tree-derived transcription factors CsWRKY71 The application of the invention in improving the insect resistance of plants is characterized in that: Tea tree-derived transcription factor CsWRKY71 The nucleotide sequence is shown in SEQ ID No. 1; The plant is a herbaceous plant or a woody plant; The herbaceous plant is Arabidopsis thaliana, and the woody plant is tea tree; The pest species covered by the insect resistance are Plutella xylostella or Tea Geometridae.

2. The use according to claim 1, characterized in that: Overexpression of tea tree-derived transcription factors CsWRKY71 To improve the insect resistance of plants.

3. Overexpression of tea tree-derived transcription factors CsWRKY71 Application in the preparation of insect-resistant varieties of Arabidopsis; The insect-resistant variety is resistant to the diamondback moth.

4. The use according to claim 3, characterized in that: Overexpression of tea tree-derived transcription factors in the preparation of insect-resistant Arabidopsis varieties CsWRKY71 The recombinant bacteria were transformed into Arabidopsis thaliana by Agrobacterium-mediated floral infection.

5. The use according to claim 4, characterized in that: The overexpression of tea tree-derived transcription factor CsWRKY71 The preparation method of the recombinant bacteria comprises the following steps: (1) The tea tree-derived transcription factor according to claim 1 or 2 CsWRKY71 Ligated to pEASY-blunt zero vector to obtain pEASY- CsWRKY71 ; (2) PCR amplification of pEASY- CsWRKY71 , and obtain the amplified product rDNA; (3) The amplified product rDNA and the expression vector pBWA(V)HS were subjected to BsaI / Eco31I double restriction digestion reaction to obtain linearized rDNA and linearized vector pBWA(V)HS; (4) Perform homologous recombination and GoldenGate ligation on the linearized rDNA obtained in step (3) and the linearized vector pBWA(V)HS to obtain the overexpression recombinant vector pBWA(V)HS- CsWRKY71; (5) Overexpression recombinant vector pBWA(V)HS- CsWRKY71 Overexpression of tea tree-derived transcription factors by transformation into Agrobacterium tumefaciens GV3101 CsWRKY71 of recombinant bacteria.

6. Overexpression of tea tree-derived transcription factors CsWRKY71 Application in the preparation of insect-resistant varieties of tea trees; The insect-resistant variety is resistant to the tea geometrid.

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