Application of CsNAC2 gene in improving resistance of tea trees to tea geometrid

Abstract

The invention discloses a tea tree CsNAC2 gene and application thereof, and belongs to the technical field of tea tree gene utilization. The CDS nucleotide sequence of the tea tree CsNAC2 gene is as shown in SEQ ID No. 1. The amino acid sequence of the protein coded by the CsNAC2 gene is as shown in SEQ ID No. 2. A transcription factor gene CsNAC2 from a tea tree is cloned, it is revealed for the first time that the tea tree CsNAC2 gene participates in regulation and control of insect pest response of the tea tree, and the tea geometrid defense capacity of the tea tree can be reduced by reducing the expression quantity of the tea tree CsNAC2 gene. The tea tree CsNAC2 gene is remarkably expressed under the induction of leaf acetate, the tea geometrid defending capability of the tea tree is enhanced, a good gene resource is provided for cultivating a new insect-resistant tea tree variety, and potential application value is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of tea tree gene utilization, and particularly relates to an application of a CsNAC2 gene in improving the resistance of tea trees to tea geometrids. Background Art

[0002] Tea (Camellia sinensis) is one of the most widely consumed non-alcoholic beverages in the world, enjoyed by billions of people worldwide. As an important economic crop, its young leaves are the primary raw material for tea production. However, the defoliating pest, the tea geometrex, poses a serious threat to both tea yield and quality. While chemical pesticides help control tea geometrex infestations, their widespread use raises concerns about the environment and human health. Therefore, understanding the natural defense mechanisms of tea plants and developing environmentally friendly biopesticides are crucial for reducing pesticide dependence and enhancing resistance to herbivorous pests.

[0003] (Z) -3-hexene-acetate (leaves acetate) is a key component in green leaf volatiles and is synthesized by acetyl CoA and cis-3-hexen-1-ol under the catalysis of acetyltransferase. The biosynthesis and release of leaves acetate remain at baseline levels under normal physiological conditions, but are rapidly increased under the treatment of herbivory and abiotic stress. Recent studies have shown that leaves acetate plays a vital role in plant-insect interactions. For example, exogenous application of leaves acetate significantly increases the expression of defense-related genes in hybrid poplar leaves, thereby enhancing their resistance to gypsy moth larvae (Frost CJ, et al. (2008) Priming defense genes and metabolites in hybrid poplar by the greenleaf volatile cis-3-hexenyl acetate. New Phytologist 180: 722–734). Jasmonic acid, as an important plant hormone, is essential for plants to resist necrotic pathogens and chewing herbivores. In tea plants, phytyl acetate acts as a signal molecule to induce the accumulation of jasmonic acid, thereby enhancing the plant's resistance to tea looper (Gu H, et al. (2024) A defensive pathway from NAC and TCP transcription factors activates a BAHDacyltransferase for (Z)-3-hexenyl acetate biosynthesis to resist herbivore in tea plant (Camellia sinensis). New Phytologist 245:1232–1248).

[0004] Transcription factors play an important regulatory role in plant defense responses. The NAC (NAM, ATAF, and CUC) transcription factor family is one of the largest transcription factor families in plants, with a large number of genes and a wide range of functions. In addition to participating in multiple processes of growth and development, NAC transcription factors also play a vital role in mediating plant defense mechanisms. Studies have shown that NAC transcription factors can enhance plant stress resistance by regulating the expression of defense-related genes. NAC2, as a key regulator in the tobacco airborne immune signaling pathway, confers tobacco resistance to aphid and virus infection (Gong Q, et al. (2023) Molecular basis of methyl-salicylate-mediated plant airborne defense. Nature 622: 139–148). In Arabidopsis, AtNAC2 / ANAC092 is involved in the salt stress response pathway (He XJ, et al. (2005) AtNAC2, a transcription factor downstream of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development. Plant J 44:903–916). Downregulation of OsNAC2 in rice enhances resistance to bacterial blight without affecting growth performance or yield components (Zhong Q, et al. (2024) Rice transcription factor OsNAC2 maintains the homeostasis of immune responses to bacterial blight. Plant Physiol 195:785–798). The NAC transcription factor GmNAC12 enhances drought stress tolerance in soybean (Yang C, et al. (2022) NAC Transcription Factor GmNAC12 Improved Drought Stress Tolerance in Soybean. Int J Mol Sci 23:12029). Currently, the CsNAC2 gene has not been used to improve insect resistance in tea plants.

[0005] Therefore, exploring the role of the CsNAC2 gene in improving tea plant resistance to tea loopers and how to utilize phytosterol acetate in sustainable management strategies for tea plant defense against herbivores remain unresolved. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to apply the gene CsNAC2 and the protein encoded by it to improve the ability of tea plants to defend against tea geometrids and how to provide a method for improving and breeding tea plants' resistance to tea geometrids.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] The first aspect of the present invention proposes the use of the tea plant transcription factor CsNAC2 gene in improving the resistance of tea plants to the tea geometrid. The CDS sequence of the CsNAC2 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2. By promoting the expression of the CsNAC2 gene, the resistance of tea plants to the tea geometrid is improved.

[0009] The improvement of tea tree's resistance to tea geometrids is reflected in: promoting the expression of tea tree's CsNAC2 gene, increasing the jasmonic acid (JA) content in tea leaves, and thus reducing the feeding ability of tea geometrids.

[0010] The second aspect of the present invention proposes the use of CsNAC2 as a drug target in the preparation of a drug for defending tea plants against tea geometrids.

[0011] Preferably, the CsNAC2 includes a CsNAC2 gene and a CsNAC2 protein.

[0012] Preferably, the drug is a CsNAC2 overexpression agent or an agent that promotes CsNAC2 overexpression.

[0013] Preferably, the active ingredient of the drug includes phytyl acetate.

[0014] A third aspect of the present invention provides a drug for controlling tea geometrids, the active ingredient of which includes phytol acetate.

[0015] A fourth aspect of the present invention provides the use of the CsNAC2 gene in identifying or breeding tea varieties resistant to tea geometrids.

[0016] A fifth aspect of the present invention provides a method for controlling tea loopers on tea trees, comprising the following steps: spraying a leaf acetate solution onto tea leaves of the tea tree.

[0017] Preferably, the concentration of phytol acetate is 0.3-0.8 ng / μL, more preferably 0.5 ng / μL.

[0018] The beneficial effects of the present invention are:

[0019] 1. The present invention cloned the transcription factor gene CsNAC2 from tea plants. By exposing tea plants to exogenous phytyl acetate, its expression level was significantly increased compared with the control group, and the content of tea leaf feeding by tea loopers was reduced, indicating that it plays an important role in tea plants' defense against tea loopers.

[0020] 2. The CsNAC2 gene and the protein it encodes provided by the present invention can provide a new strategy for breeding new insect-resistant tea varieties, and at the same time provide new theoretical insights for studying the defense mechanism of tea trees; promoting the expression of the CsNAC2 gene in tea trees, increasing the jasmonic acid (JA) content in tea leaves, and thus affecting the feeding ability of tea loopers.

[0021] 3. The present invention reveals for the first time that the CsNAC2 gene of tea plants is involved in regulating the response of tea plants to insect pests. Reducing its expression level will reduce the insect resistance of tea plants, indicating that CsNAC2 can improve the insect resistance of tea plants.

[0022] 4. The CsNAC2 gene in tea trees is significantly expressed under the induction of phytol acetate, which enhances the tea tree's ability to defend against tea loopers. This provides a good genetic resource for breeding new insect-resistant tea varieties and has potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure 1 shows the phylogenetic tree cluster analysis of the CsNAC2 gene and the Arabidopsis NAC family in Example 1 of the present invention. Figure A shows the amino acid sequence alignment of the conserved motifs of CsNAC2 and homologous proteins from other plant species, with subdomains A, B, C, D, and E marked with green boxes. Figure B shows the phylogenetic tree of NAC transcription factors in tea plants and Arabidopsis.

[0024] Figure 2 The agarose gel electrophoresis diagram of the CsNAC2 CDS amplification product in Example 1 of the present invention is shown. Wherein: M, 2000 bp DNA marker;

[0025] Figure 3 This is a graph showing the expression levels of the CsNAC2 gene in Example 1 of the present invention when tea plants are exposed to leaf acetate at different time points;

[0026] Figure 4 Figure 1 is a graph showing the expression of the CsNAC2 gene at different times of silencing in tea plants in Example 1 of the present invention, wherein A is a graph showing the leaf phenotype of tea trees after CsNAC2 inhibition, B is a graph showing the expression levels of CsLOX2 at different time points after CsNAC2 inhibition, and C is a graph showing the expression of CsNAC2 in tea trees after inhibition and in the control;

[0027] Figure 5Graph showing the effect of silencing the CsNAC2 gene in tea plants on the feeding amount of tea geometrids in Example 1 of the present invention, wherein A is a phenotypic graph of tea leaves fed by tea geometrids for 10 hours, B is a graph showing the water loss rate of tea leaves during the feeding stage, and C is a statistical graph showing the feeding weight of tea leaves by tea geometrids;

[0028] Figure 6 Figure 1 is a graph showing the JA and JA-Ile content in tea plants after the CsNAC2 gene was silenced in Example 1 of the present invention, wherein A is a graph showing the JA content in tea plants after being inhibited by CsNAC2 and in a control, and B is a graph showing the JA-Ile content in tea plants after being inhibited by CsNAC2 and in a control;

[0029] Figure 7 This is a graph showing the expression of CsNAC2 in tea plant leaves exposed to phytol acetate after silencing the CsNAC2 gene for 12 hours in Example 1 of the present invention;

[0030] Figure 8 Graph showing the effect of silencing the CsNAC2 gene in tea plants for 12 hours and then exposing them to leaf acetate on the feeding amount of tea geometrids in Example 1 of the present invention, wherein A is a phenotypic graph of tea leaves exposed to leaf acetate after silencing the CsNAC2 gene in tea plants for 12 hours and then exposing them to leaf acetate, after being fed by tea geometrids for 10 hours; B is a graph showing the water loss rate of tea leaves during the feeding stage; and C is a statistical graph showing the weight of tea leaves fed by tea geometrids;

[0031] Figure 9 This is a graph showing the JA and JA-Ile content in the leaves of the tea plant after the CsNAC2 gene was silenced for 12 hours and then exposed to phytol acetate in Example 1 of the present invention, wherein A is a graph showing the JA content in the tea leaves after the CsNAC2 gene was silenced for 12 hours and then exposed to phytol acetate, and B is a graph showing the JA-Ile content in the tea leaves after the CsNAC2 gene was silenced for 12 hours and then exposed to phytol acetate. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below have the same meaning as those understood by professional and technical personnel in this field.

[0033] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources or prepared by known methods.

[0034] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples were repeated three times and the results were averaged.

[0035] The main reagents and formulations used in the following examples are:

[0036] (1) Leaf acetate (98%, Aladdin) standard

[0037] (2) Jasmonic acid and jasmonic acid-isoleucine standards

[0038] (3) Polysaccharide and polyphenol plant total RNA extraction kit

[0039] (4) RNA reverse transcription kit

[0040] (5) Gel recovery kit

[0041] (6) LB medium: 5g yeast powder, 10g peptone, and 10g sodium chloride are dissolved in 1L pure water. 4-5g agar powder is added to every 250ml of solid LB medium and sterilized at high temperature and high pressure for 20min.

[0042] (7) 1.5% agarose gel formula: Weigh 0.3 g agarose in 20 mL 0.5× TBE buffer, mix well, and heat to dissolve until clear.

[0043] Example 1:

[0044] 1. CsNAC2 gene cloning

[0045] (1) RNA extraction: Add 50 mg of sample to a 2 mL RNA extraction tube, add steel balls, and grind thoroughly using a ball mill. The extraction was performed according to the instructions of the Universal Plant Total RNA Isolation Kit (Novozymes).

[0046] After RNA extraction, the quality of RNA extraction was tested by 1.5% agarose gel electrophoresis at a voltage of 130 V. The quality of extracted nucleic acid was determined using a nucleic acid quantification instrument Nanodrop-2000 (Thermo Corporation) to check whether OD260 / OD280 was between 1.8 and 2.1.

[0047] (2) cDNA reverse transcription: according to PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara) instructions: RNA was reverse transcribed into cDNA as a template for PCR amplification of the gene.

[0048] (3) PCR amplification: The CDS sequence of CsNAC2 was amplified using upstream and downstream primers in the CDS region. The required reagents were added in sequence according to the reaction system (1.0 μL cDNA template, 1.0 μL upstream primer, 1.0 μL downstream primer, 12.5 μL LA Taq premix, 9.5 μL ddH2O) and PCR amplification was performed. The cloning procedure was 94°C (3 min), 94°C (30 s), 56°C (30 s), 72°C (1 min), 30 cycles, 72°C (10 min), and the reaction product was stored at 4°C.

[0049] (4) Ligation, transformation, and sequencing: ① After electrophoresis of the PCR product on a 1.5% agarose gel, the agarose gel containing the target fragment was quickly excised under UV detection conditions. Subsequently, the target gene fragment was recovered according to the instructions of the gel recovery kit. Finally, the concentration and purity of the target fragment were determined using a Nanodrop-2000 nucleic acid quantifier (Thermo).

[0050] ②According to -T1 cloning vector instructions to ligate the recovered product into After the reaction, add the ligation product to 100 μL of Trans1-T1 competent cells, incubate on ice for 30 minutes, then heat shock in a 42°C water bath for 30 seconds, and immediately cool on ice for 2 minutes.

[0051] ③ Then, add 400 μL of resistance-free LB medium to the centrifuge tube in a clean bench and culture on a 37°C horizontal shaker at 180 rpm for 1 hour.

[0052] ④ On the clean bench, take 200 μL of bacterial solution from the cultured cells and apply it to 100 ng / mL ampicillin (Amp + ) on solid LB medium and cultured in an inverted position in a 37°C incubator overnight.

[0053] ⑤Pick a single clone and perform PCR verification using specific primers. After screening positive clones, send them to a sequencing company for analysis.

[0054] 2. Tea trees exposed to leaf acetate

[0055] (1) Strictly select 8 one-year-old and pest-free 'Shucha Zao' plug seedlings and place them in a transparent cylindrical glass jar (20 cm high, 15 cm in diameter), with a 1 cm -3A cotton ball was placed on the surface of the device. A leaf acetate solution (dissolved in 100 μL of methanol) was added dropwise to the cotton ball to a final concentration of 0.5 ng / μL after the glass cover was placed. An equal volume of methanol was added to the control group. The gaps in the device were frosted and sealed with plastic wrap to ensure airtightness.

[0056] (2) The entire device was then moved into a greenhouse, where the temperature was maintained at 25±2°C, the photoperiod was 12 hours light / 12 hours dark, and the relative humidity was maintained at 70%.

[0057] (3) After 0.5 h, 1 h, 3 h, 6 h, 12 h, and 24 h of treatment, the second leaf of the tea plant was picked as a sample. Five biological replicates were set for each treatment. The experimental samples were immediately placed in liquid nitrogen after picking and then transferred to a -80 °C freezer for storage.

[0058] 3. Analysis of CsNAC2 Expression Pattern

[0059] cDNA reverse transcription: According to the instructions of HiScript II One Step RT-PCR Kit (Vazyme), RNA was quantitatively reverse transcribed into cDNA as a template for gene qPCR amplification.

[0060] Fluorescence quantitative PCR: Amplification was performed using the upstream and downstream primers of qPCR. The reaction system (1.2 μL cDNA template, 0.3 μL upstream primer, 0.3 μL downstream primer, 5 μL TB Green Premix Ex Taq II, 3.2 μL ddH2O) was used and detected using a CFX fluorescence quantitative PCR instrument (Bio-Rad). Five biological replicates were set for each sample, and each biological replicate had three technical replicates. CsGAPDH was used as the internal reference. The results were analyzed using Bio-Rad CFX Manager software.

[0061] The analysis showed that compared with the control group without phytol acetate exposure, the expression of CsNAC2 in tea leaves exposed to phytol acetate increased significantly, and the highest expression was at 1h ( Figure 3 ). This indicates that phytyl acetate can induce the expression of CsNAC2.

[0062] 4. Tea Tree Gene Transient Antisense Inhibition Experiment

[0063] (1) Based on the CsNAC2 gene sequence, antisense probes were designed and selected using the Soligo online website (https: / / sfold.wadsworth.org / cgi-bin / soligo.pl) and sent to Sangon Biotech (https: / / www.sangon.com) for synthesis.

[0064] (2) The complementary sequence of AsODNs is named as sense oligonucleotide (sODN). Take three young branches with one bud and two leaves and place them in a 1.5 mL test tube, add 1 mL of 100 μmol·L -1 AsODN. The control group was added with an equal volume of 100 μmol·L -1 sODN or sterile deionized water. After 8, 10, and 12 h of treatment, the second leaf of the treated and control groups was removed for analysis.

[0065] After analysis, it was found that the silencing effect of CsNAC2 gene was the best after 12 hours in tea plants. Compared with the control group (sODN and H2O), the expression level was significantly decreased ( Figure 4 ).

[0066] 5. Statistical analysis of tea looper feeding amount

[0067] After the tea trees were treated, the second leaf of each tea tree was removed and weighed (initial mass: M1). The third-instar tea geometrid larvae were pre-starved for 8 hours and then placed in a culture dish with the second tea leaf for 10 hours. Afterwards, the larvae were removed and the remaining leaves were reweighed (M2). To calculate the water loss rate, the center of the leaf in a control culture dish without tea geometrid larvae was weighed 10 hours later (M3). The water loss rate (W1) was calculated as: W1 = (M1-M3) / M1. The average water loss rate (W) was then determined. A The calculation formula of tea geometrid's leaf feeding amount (C1) is: C1=M1-M2-(W A ×M1). Feeding behavior was assessed based on leaf mass consumption. Each treatment included 20–30 biological replicates.

[0068] After analysis, the leaves of tea plants that had been silenced for 12 hours after the CsNAC2 gene was silenced were fed to tea geometrids for 10 hours. Compared with the control group (sODN and H2O), the amount of tea geometrids feeding on the leaves after CsNAC2 silence was significantly reduced ( Figure 5 This indicates that CsNAC2 can respond to the tea plant's defense against the tea looper and is one of the factors affecting the tea plant's insect resistance.

[0069] 6. Determination of hormone content

[0070] The contents of jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile) were determined according to a previously established method (Wu J, et al. (2007) Herbivory rapidly activates MAPK signaling in attacked and unattacked leaf regions but not between leaves of Nicotiana attenuata. The Plant Cell, 19(3): 1096-1122). 25 mg of freeze-dried tea leaves were ground in liquid nitrogen, and 1 mL of the internal standard (D6-JA and JA-Ile) was added to each sample. 13 C6-JA-Ile) in ethyl acetate. Centrifuge at 13,000 × g for 10 min at 4°C. The supernatant was transferred to a 2 ml tube and evaporated to dryness using a vacuum concentrator (Eppendorf, Germany). The residue was re-extracted with 1 mL of 50% methanol (v / v), and the supernatant was filtered through a 0.22 μm organic membrane. Liquid chromatography-mass spectrometry-triple quadrupole (LC-MS-QQQ; Agilent, USA) equipped with C 18 Chromatographic column (100 mm × 4.6 mm, 2.6 μm). Column temperature was maintained at 40°C, flow rate was 0.4 mL min -1 .

[0071] After analysis, the hormone content of leaves was measured 12 hours after the CsNAC2 gene was silenced in tea plants. It was found that the content of JA and JA-Ile in leaves after CsNAC2 silencing was significantly decreased compared with the control group (sODN and H2O). Figure 6 This indicates that CsNAC2 can respond to the JA signaling pathway in tea plants and may affect the insect resistance of tea plants by changing the JA content.

[0072] 7. Analysis of CsNAC2 gene expression pattern, hormone content, and insect resistance after silencing CsNAC2 in tea plants by exogenous leaf acetate

[0073] To further explore whether phytyl acetate (CAS No. 3681-71-8) is the main mediator that may induce CsNAC2 to regulate JA signaling in tea plant defense against tea loopers, we exogenously exposed phytyl acetate to tea leaves for 1 h in the control group (H2O and sODN) and after 12 h of CsNAC2 silencing treatment.

[0074] After analysis, the expression level of CsNAC2 was measured after 1 hour of phytol acetate treatment. It was found that the expression level of CsNAC2 in the control group (H2O and sODN) was significantly higher than that in the group not exposed to phytol acetate after 1 hour of phytol acetate exposure, increasing by 93.47% and 159.13% respectively. However, the expression level of CsNAC2 in the CsNAC2 silenced tea tree group was not significantly different from that in the group not exposed to phytol acetate after 1 hour of phytol acetate exposure, and the degree of increase was lower than that in the control group (H2O and sODN), increasing by only 14.55% ( Figure 7 Statistical analysis of the weight of leaves fed by tea geometrids showed that the weight of leaves in the control group (H2O and sODN) not exposed to phytol acetate for 1 hour was 0.0483g and 0.0469g, respectively, while the weight of leaves in the control group (H2O and sODN) exposed to phytol acetate for 1 hour was 0.0290g and 0.0299g, respectively, which were 39.89% and 36.22% lower than those in the unexposed group. However, the leaf mass of the tea looper feeding on the CsNAC2 silenced tea plant group after 1 hour of exposure to phytol acetate was not significantly different from that of the group not exposed to phytol acetate. The leaf mass of the tea looper feeding on the unexposed phytol acetate was 0.0560 g, while the leaf mass of the tea looper feeding on the exposed phytol acetate was 0.0410 g. The degree of decrease was lower than that of the control group (H2O and sODN), which was only reduced by 26.80% ( Figure 8 ). Finally, the results of hormone determination showed that the JA content in the control group (H2O and sODN) was significantly higher than that in the group not exposed to phytol acetate for 1 hour, increasing by 94.56% and 144.06% respectively; although the JA content in the CsNAC2 and CsMYB306 silenced tea tree group was also significantly higher than that in the group not exposed to phytol acetate for 1 hour, the degree of increase was lower than that in the control group (H2O and sODN), increasing by only 60.25% and 62.40% respectively. JA-Ile also showed similar results. This shows that phytol acetate can induce CsNAC2 to increase the jasmonic acid content and enhance the resistance of tea trees to tea loopers ( Figure 9 ).

[0075] CDS sequence of the CsNAC2 gene from tea plant (SEQ ID NO.1)

[0076] ATGACGAGCAGTAGCAGTCAGTTGGAGTTACCTCCTGGATTCAGATTCCATCCGACGGATGAGGAGCTCGTGATGCACTACCTGTGCCGTAAATGCGCAGCTCAGCCGATTTCTGTCCCGATCATCGCCGAGATCGATCTGTACAAGTTCGATCCATGGCAACTTCCAGGAATGGCGATGTATGGTGAAAAGGAGTGGTATTTTTTCTCTCCGAGAGATCGGAAGTATCCGAACGGTTCGAGGCCGAACAGGGCGGCGGGGACGGGGTACTGGAAGGCGACCGGAGCCGATAAGCCAATAGGGCGGCCGAAGGCGGTGGGAATCAAGAAGGCTCTGGTTTTTTACGCCGGAAAAGCACCCAAAGGGGTGAAGACCAATTGGATTATGCATGAATATCGGTTAGCCAATGTGGATAGGTCCGCCGGCAAGAAAAACAGTTTAAGGCTTGATGATTGGGTGTTATGTCGAATATACAACAAGAAGGGTGCTATTGAGAAGCATTTAAATTCGACAGGTCCAAAGCCAACTCAATATTTAGAAATTGAAGAGAGAAAACCCGAAATTTTATCGGGATACGAGGCCATCTCAATGCCGCCGCCGCGGCCACCACCATCACAACCACTAATGTCTAATGACTTGCTGCATTTTGAGACATCGGATTCGGTGCCGTGGTTGCATACGGACTCGAGTGGCTCCGAGCATGCGGGGTCACCCGACTTCATGTGTGACAAAGAAGTGCAGAGCGAGCCGAGATGGAATAATGACTTGGAAAATGCCCTCGATTTTCAGTTTAATTACATGGATGCCTTCCTAGATGACTCTTTTGCCTCTCAAATGCAGGAGTTTCAGCAGGACCAGCTCTCCCCATTGCAGGACATGTTCATGTTCATGCCGAAGCCATTCTAG

[0077] Amino acid sequence of the CsNAC2 gene of Camellia sinensis (SEQ ID NO.2)

[0078] MTSSSSQLELPPGFRFHPTDEELVMHYLCRKCAAQPISVPIIAEIDLYKFDPWQLPGMAMYGEKEWYFFSPRDRKYPNGSRPNRAAGTGYWKATGADKPIGRPKAVGIKKALVFYAGKAPKGVKTNWIMHEYRLANVDRSAGKKNSLRLD DWVLCRIYNKKGAIEKHLNSTGPKPTQYLEIEERKPEILSGYEAISMPPPPRPPPSQPLMSNDLLHFETSDSVPWLHTDSSGSEHAGSPDFMCDKEVQSEPRWNDLENALDFQFNYMDAFLDDSFASQMQEFQQDQLSPLQDMFMFMPKPF

[0079] CDS region amplification upstream primer: ATGACGAGCAGTAGCAGTCA (SEQ ID NO. 3)

[0080] CDS region amplification downstream primer: CTAGAATGGCTTCGGCATGA (SEQ ID NO.4)

[0081] qPCR upstream primer: AGGCTTGATGATTGGGTGTT (SEQ ID NO. 5)

[0082] qPCR downstream primer: GCATTGAGATGGCCTCGTAT (SEQ ID NO. 6)

[0083] Antisense oligonucleotide chain probe: TTAGTGGTTGTGATGGTGGT (SEQ ID NO.7)

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Application of the CsNAC2 gene in improving resistance of tea plants to tea geometrids, characterized in that: The CDS sequence of the CsNAC2 gene is shown in SEQ ID NO. 1; by promoting the expression of the CsNAC2 gene, the resistance of tea plants to tea geometrids is improved.

2. Application of CsNAC2 as a drug target in the preparation of drugs to defend tea trees against tea loopers.

3. The use according to claim 2, characterized in that The CsNAC2 includes the CsNAC2 gene and the CsNAC2 protein.

4. The use according to claim 2, characterized in that The drug is a CsNAC2 overexpression reagent or a reagent that promotes CsNAC2 overexpression.

5. The use according to claim 2, characterized in that The active ingredient of the medicine includes phytol acetate.

6. A drug for preventing and treating tea geometrid, characterized in that: Its active ingredients include leaf acetate.

7. Application of CsNAC2 gene in identifying or breeding tea varieties resistant to tea looper.

8. A method for controlling tea loopers on tea trees, characterized in that: The following steps are involved: Just spray the leaf acetate solution on the tea leaves of the tea plant.

9. The method for controlling tea looper on tea trees according to claim 8, wherein: The concentration of the phytol acetate is 0.3-0.8 ng / μL.

10. The method for controlling tea looper on tea trees according to claim 9, characterized in that: The concentration of the phytyl acetate was 0.5 ng / μL.

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