Application of tomato wrky52 gene in regulating plant resistance to insect stress

Abstract

This invention discloses tomatoes WRKY52 The application of genes in regulating plant resistance to insect pest stress belongs to the field of biotechnology. WRKY52 The nucleotide sequence of the protein-coding region of the gene is shown in SEQ ID NO.1. This invention discloses tomato for the first time. WRKY52 The role of genes in regulating plant resistance to insect pests, especially bollworm, and the use of CRISPR / Cas9 gene editing technology to obtain tomatoes WRKY52 The study found that the mutant with gene deletion exhibited reduced resistance to the cotton bollworm, proving... WRKY52 The application of genes as positive regulators in improving the resistance of tomatoes to insect pest stress can be used for the breeding of insect-resistant tomato varieties. This invention provides more gene resources and new ideas for plant molecular stress resistance genetic breeding and genetic modification of plant stress tolerance.

Description

Technical Field

[0001] This application relates to the field of biotechnology, primarily concerning tomatoes. WRKY52 Application of genes in regulating plant resistance to insect pest stress. Background Technology

[0002] tomato( Solanum lycopersicum Tomatoes, as one of the most popular and widely cultivated vegetable crops globally, serve both fresh consumption and processing purposes, and market demand continues to grow. However, against the backdrop of rapid development in greenhouse agriculture, the year-round production system of tomatoes provides a suitable microenvironment for various plant-eating pests, leading to increasingly prominent pest problems. Among them, the cotton bollworm (… Helicoverpa armigera The cotton bollworm is an omnivorous pest with a very wide host range, and it is most severely affected by tomato plants among vegetable crops. The cotton bollworm primarily feeds on the stems, leaves, and fruits of tomato plants in its larval stage, with the most severe damage occurring to young fruits. Once the interior of the tomato fruit is completely consumed during its tender stage, it quickly rots and falls off, losing its commercial value. Furthermore, wounds on the fruit surface make it susceptible to disease, leading to yield losses exceeding 30%, making it a major limiting factor for late-season tomato cultivation.

[0003] Traditional pest control methods heavily rely on chemical pesticides. Frequent and high-dose application not only accelerates the development of pesticide resistance in pests, making control increasingly difficult year by year, but also brings pesticide residues, environmental pollution, and potential threats to human and animal health. Therefore, exploring and utilizing the plant's own insect-resistant gene resources, elucidating their molecular regulatory mechanisms, and then breeding new insect-resistant varieties is an important direction for achieving green and sustainable agricultural production.

[0004] Plants have developed multi-layered defense mechanisms through long-term evolution, including a systemic signaling regulatory network centered on jasmonic acid, indirect defense mediated by insect-induced volatile organic compounds, and the synergistic effects of various direct insect-resistant substances. Defense-related proteins play a crucial role in these mechanisms. For example, PI-I (Proteinase inhibitor I), an inhibitor of serine proteases, can inhibit trypsin activity in the digestive tract of lepidopteran insect larvae, interfering with protein digestion and absorption, delaying growth and development, and increasing mortality. ARG2 (Arginase 2) participates in plant nitrogen redistribution and metabolic reprogramming by catalyzing arginine hydrolysis, thereby affecting defense responses induced by amino acids.

[0005] Tomatoes, with their small genome, mature genetic transformation system, short life cycle, and sensitivity to abiotic stress, have become an ideal model system for studying plant-pest interaction mechanisms. Transcriptional regulation plays a central role in plant responses to pest stress. Specific transcription factors recognize and bind to cis-acting elements in the promoter regions of target genes, thereby activating or inhibiting the expression of downstream defense-related genes, forming a sophisticated regulatory network. The WRKY transcription factor family is a core regulatory hub unique to plants, and studies have shown its wide involvement in plant growth and development (such as seed dormancy and germination, organ development, flowering, and seed development) and adaptive responses to various abiotic stresses (such as extreme temperatures and salinity). However, research on the functions and mechanisms of WRKY family members in pest stress remains very limited. Specifically, regarding tomatoes… WRKY52 Whether genes are involved in regulating plant defense responses to chewing pests such as bollworms, and the underlying molecular mechanisms, remains unclear.

[0006] In conclusion, a deep analysis of tomatoes WRKY52 The biological functions and regulatory pathways of genes in insect resistance not only contribute to a better understanding of the transcriptional regulatory network of plant insect resistance, but also provide important genetic resources and theoretical basis for improving tomato insect resistance traits and breeding new cotton bollworm-resistant varieties using genetic engineering. Summary of the Invention

[0007] Therefore, this application provides a tomato WRKY52 Application of genes in regulating plant resistance to insect pest stress.

[0008] According to the embodiments of this application, tomatoes are provided. WRKY52 The application of genes in regulating plant resistance to insect pest stress, wherein the application is achieved through... WRKY52 Loss of gene function reduces the number of defense-related genes PI-I , ARG2 The expression level of [certain substances] is reduced, thereby weakening the plant's resistance to insect pest stress; the aforementioned WRKY52 The nucleotide sequence of the protein-coding region of the gene is shown in SEQ ID NO.1. Tomato WRKY52 Gene ID: Solyc03g007380.

[0009] Optionally, the pest is the cotton bollworm.

[0010] Optionally, the WRKY52 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0011] Optionally, the WRKY52 The way to lose gene function is by knockout. WRKY52 Gene or WRKY52 Gene silencing.

[0012] Optionally, the knockout WRKY52 Genes, specifically including: Tomato WRKY52 Sequence analysis was performed to find PAM sequences. The 20 bp sequence before NGG was defined as sgRNA, and sgRNA sequences located in the gene protein coding region with high specificity were selected. Using gene editing and tissue culture technologies, a system was constructed. WRKY52 The gene was vectorized into a CRISPR / Cas9 vector, and genetically stable homozygous mutant lines that did not contain exogenous Cas9 protein were obtained through screening.

[0013] Optionally, the WRKY52 Gene-encoded proteins activate defense-related genes PI-I , ARG2 The promoter promotes PI-I , ARG2 Gene transcription.

[0014] Optionally, the PI-I The nucleotide sequence of the gene promoter is shown in SEQ ID NO.3.

[0015] Optionally, the ARG2 The nucleotide sequence of the gene promoter is shown in SEQ ID NO.4.

[0016] Optionally, the nucleotide sequences of the first 20 bp of the PAM structure of the two target fragments are shown in SEQ ID NO. 5 and SEQ ID NO. 6.

[0017] The WRKY52 The complete DNA sequence of the gene is shown in SEQ ID NO.7, and the nucleotide sequence of the protein-coding region is shown in SEQ ID NO.1, with a protein-coding region length of 1062 bp; WRKY52 The gene encodes a transcription factor containing a WRKY domain, consisting of 353 amino acids, the sequence of which is shown in SEQ ID NO.2.

[0018] Under bollworm stress, compared with wild-type tomato (WT), wrky52 Deletion of defense-related genes in mutants PI-I (Gene ID: Solyc09g084460) , ARG2 The expression level of (gene ID: Solyc01g091160) was significantly decreased. Therefore, in tomatoes... WRKY52 Genes positively regulate the resistance of tomatoes to bollworm.

[0019] The technical solutions provided by the embodiments of this application may include the following beneficial effects: As can be seen from the above embodiments, this invention discloses tomatoes for the first time. WRKY52 The role of genes in regulating plant resistance to insect pest stress, especially bollworm, was explored using CRISPR / Cas9 gene editing technology. WRKY52 Gene function can significantly reduce plant resistance to bollworm, proving that WRKY52 The application of genes in enhancing plant resistance to insect pests can be extended to the breeding of insect-resistant plant varieties. This invention provides more gene resources and new ideas for molecular stress resistance genetic breeding and genetic modification of plant stress tolerance.

[0020] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0021] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0022] Picture 1 Tomato leaves were treated with mechanical wounds and bollworm mouthpart secretions (W+OS) to simulate bollworm feeding. WRKY52 Gene expression status; **, ***, **** represent different time points after treatment. WRKY52 In the T-test, the gene expression level was compared with that of the control group. P The differences were significant at the <0.01, 0.001, and 0.0001 levels.

[0023] Picture 2 CRISPR / Cas9-mediated knockout tomatoes WRKY52 The gene editing sites and changes in encoded amino acids in homozygous mutant plants were analyzed; among them, WT was a non-transgenic wild-type tomato. wrky52 for WRKY52 Gene knockout mutant tomatoes; compared to WT, wrky52 The #1 mutant has a 53 bp deletion at the target 1 position, which leads to premature termination of protein translation; wrky52 The #2 mutant involves a 1 bp base insertion at position 2 of the target protein, which causes premature termination of protein translation.

[0024] Picture 3 For tomatoes WRKY52 Leaf phenotype (A) and leaf weight loss (B) of gene knockout mutant plants 3 days after inoculation with bollworm; more leaf damage indicates more bollworm feeding and more severe infestation of the tomato plant; *** represents differences between different plants in the T-test.P The difference was significant at the <0.001 level.

[0025] Picture 4 For tomatoes WRKY52 The phenotype (A) and weight (B) of cotton bollworms in gene knockout mutant plants 3 days after inoculation; larger cotton bollworm size and weight indicate more feed consumption and more severe pest infestation on tomato plants; **** represents differences between different plants in the T-test. P The difference was significant at the <0.0001 level.

[0026] Picture 5 For tomatoes WRKY52 Defense-related genes in gene knockout mutant plants after simulated bollworm feeding treatment PI-I The expression level (A) and ARG2 The expression level (B); among which, PI-I and ARG2 The higher the expression level, the stronger the plant's insect resistance; lowercase letters a and b represent the differences between different plants in a one-way ANOVA test. P The difference was significant at the <0.05 level.

[0027] Picture 6 For tomatoes WRKY52 Protein regulation PI-I (A) and ARG2 (B) Luciferase reporter gene detection ratio for gene transcription; where a higher relative LUC / REN ratio indicates a stronger ability of the target promoter to drive LUC expression, i.e., higher transcriptional activity; **, *** represent the differences between different treatments in the T-test. P <0.01, the difference at the 0.001 level is significant. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments. The following are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0031] The tomato variety used in the following examples is the conventional tomato variety Condine Red.

[0032] Example 1 Tomato WRKY52 Gene expression mimicking feeding behavior in cotton bollworms; When tomato seedlings reached the six-leaf stage, leaves from the 3rd to 4th nodes (from top to bottom) were selected for W+OS simulated bollworm feeding treatment. Leaf samples were collected at different time points, with three replicates at each time point. Total RNA was extracted from the leaf samples using the Plant RNA Rapid Extract Kit (Coolaber, Beijing, China). 1 μg of total RNA was reverse transcribed into cDNA using HiScript III RTSuperMix for qPCR (Vazyme Biotech Co., Ltd., China). The cDNA was then analyzed using QuantStudio. TM 3. Real-Time PCR System (Applied Biosystems, USA) for detection and analysis WRKY52 Gene expression status.

[0033] The cotton bollworms used were 3rd instar cotton bollworms purchased from Henan Jiyuan Baiyun Industrial Co., Ltd. The method of obtaining the secretions from the mouthparts of the cotton bollworm is as follows: After the cotton bollworm is starved for 12 hours, it is fed tomato leaves, and after 12 hours, the secretions from the mouthparts of the cotton bollworm are extracted as the original solution. The W+OS simulated feeding method of cotton bollworm is as follows: Use hemostatic forceps to clamp the leaf surface on both sides of the main vein of the tomato leaflet to create mechanical wounds, and then apply 10 μl of cotton bollworm mouthpart secretions diluted five times with ddH2O to the wound. analyze WRKY52 The reaction system used to measure gene expression included 10 μl of SYBR Green PCR MasterMix, 1 μl of cDNA, and 0.1 μM of forward and reverse primers. The PCR amplification program was: 95℃, 3 min; 95℃, 30 s; 58℃, 30 s; 72℃, 1 min; 45-50 cycles. Housekeeping genes ACTIN2 and UBI3 were used as internal controls for gene expression. WRKY52 Relative gene expression levels. The primers used are as follows: RT-WRKY52-F: 5'-TCGAGGAAGGCACACTTGTA-3' (SEQ ID NO.8); RT-WRKY52-R: 5'-TAAAGGTGGTGGTGCTGGAT-3' (SEQ ID NO. 9).

[0034] The results are as follows Picture 1 As shown, tomato leaves were treated with simulated bollworm feeding, WRKY52 Gene expression levels were significantly upregulated.

[0035] Example 2 Tomato WRKY52Preparation and identification of deletion mutant plants; 1. Construction of CRISPR / Cas9 vectors containing specific sgRNA; First, find the tomato on the SGN website https: / / solgenomics.net / WRKY52 The full-length DNA sequence of the gene is shown in SEQ ID NO.7. It was entered into the website http: / / crispr.hzau.edu.cn / CRISPR2 / and the 20 bp base sequence before the two PAM structures in the protein coding region with high onscore scores and GC content >40% was identified as the sgRNA specifically targeting the protein coding region of the gene, as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0036] Using tRNA-sgRNA-gRNA scaffold as a template, a fragment containing the selected specific sgRNA sequence was amplified, and its ends were... Bsa I. The restriction endonuclease cleavage site was used to insert the above fragment into the pHEE401 vector. Bsa The plasmid was transformed into E. coli DH5α competent cells at site I, and single clones were selected for sequencing. Plasmids with correct sequencing were electroporated and transformed into GV3101 Agrobacterium competent cells to obtain Agrobacterium strains that can be used to construct CRISPR / Cas9 gene editing materials.

[0037] 2. WRKY52 Preparation and identification of gene mutant materials; Tomato seeds were sterilized and sown in 1 / 2 MS medium. After germination, the seeds were cultured in the dark and then transferred to light. Once the cotyledons emerged, they were cut off and placed in caretaker medium for 12-24 hours of dark culture. Agrobacterium, prepared for constructing CRISPR / Cas9 gene-editing materials, was cultured to the logarithmic growth phase, centrifuged, and the supernatant was removed. The supernatant was resuspended in 0.2 MS medium and used to infect cotyledonary explants. After 2.5 minutes of dark inoculation, the remaining bacterial solution on the surface of the cotyledonary explants was aspirated, and the explants were placed on caretaker medium for two days of co-culture in the dark. After co-culture, the cotyledonary explants were transferred to 2Z medium to induce callus formation. After 2-3 weeks, they were transferred to 0.2Z medium to induce germination. New 0.2Z medium was added every 2 weeks until seedlings emerged. Then, they were transferred to R medium to induce rooting. After approximately 2-3 weeks, when the root system was well developed, the explants were transferred to a substrate and kept moist. The T0 generation gene-editing materials were then validated.

[0038] Genomic DNA was extracted from a small number of leaves of T0 generation plants and used as a template for PCR amplification of DNA fragments containing sgRNA. The PCR products were then sequenced and compared. Plants with base mutations, deletions, or insertions in the sgRNA sequence were selected for self-pollination to obtain T0 generation seeds. The verification primers are as follows: Pre-validation primer: 5'-CTGGATAGATCAAGAAATTTAGTGAGCC-3' (SEQ ID NO.10); Validation primer: 5'-GCTGGCCACCATCTGTTAGAC-3' (SEQ ID NO.11).

[0039] After sowing seeds of generation T0, generation T1 plants were obtained. The sgRNA sequence editing status and the presence or absence of Cas9 in the generation T1 plants were detected using the methods described above. Plants without Cas9 but with mutated sgRNA were selected for propagation. Self-pollination yielded one line of homozygous mutant generation T1 plants that did not contain the exogenous Cas9 gene and exhibited stable inheritance. The gene editing sites are as follows: Picture 2 As shown. wrky52 The #1 mutant has a 53 bp deletion at the target 1 position, which leads to premature termination of protein translation; wrky52 The #2 mutant involves a 1 bp base insertion at position 2 of the target protein, which causes premature termination of protein translation.

[0040] The above-mentioned strains were further self-pollinated to produce T2 generation plants that were stable and genetically inherited and did not contain the exogenous Cas9 gene after seed sowing.

[0041] The following examples all used T2 generation homozygous lines as materials for the experiments.

[0042] Example 3 Tomato WRKY52 Identification of the gene-based resistance phenotype to bollworm; Third-instar bollworms of uniform size were selected and placed in a 24-well plate for overnight starvation. When the tomato seedlings reached six leaves and a central bud, leaves from the 2nd to 4th nodes (from top to bottom) were selected, and leaves of equal weight (1 ± 0.05 g) were placed in a petri dish containing 1% agar to prevent dehydration of the detached leaves. The tomato leaves before inoculation were photographed. Two starved bollworms were then inoculated onto a tomato leaf in a petri dish, the dish was covered, and secured with a rubber band to prevent the bollworms from leaving. After three days of inoculation under low light, the weight of the bollworms was measured. The bollworms were then soaked in 70% methanol until they became inactive, arranged by size, and photographed. The tomato leaves after inoculation were also photographed and weighed accordingly.

[0043] The results are as follows Picture 3 and Picture 4As shown, wrky52 The mutant leaves suffered more damage after insect inoculation treatment compared to wild-type leaves, and the feeding... wrky52 The cotton bollworms fed on mutant leaves were larger and heavier than those fed on wild-type leaves, indicating that... WRKY52 Genes positively regulate bollworm resistance in tomatoes.

[0044] Example 4 Tomato WRKY52 Genes related to defense PI-I , ARG2 The effects on gene expression; When the tomato seedlings have grown to six leaves and one bud, the leaves from the 3rd to 4th nodes from the top are selected for W+OS simulated bollworm feeding treatment, and leaf samples are collected at 0 and 2 hours for analysis. PI-I , ARG2 Gene expression levels were measured in triplicate at each time point.

[0045] Total RNA was extracted from leaf samples using the Plant RNA Rapid Extract Kit (Coolaber, Beijing, China); 1 μg of total RNA was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (Vazyme Biotech Co., Ltd., China); and the RNA was analyzed using QuantStudio. TM 3. Real-Time PCR System (Applied Biosystems, USA) for detection and analysis PI-I , ARG2 Gene expression status.

[0046] analyze PI-I, ARG2 The reaction system used to measure gene expression included 10 μl SYBR Green PCRMaster Mix, 1 μl cDNA, and 0.1 μM of forward and reverse primers. The PCR amplification program was: 95℃, 3 min; 95℃, 30 s; 58℃, 30 s; 72℃, 1 min; 45-50 cycles. Housekeeping genes ACTIN2 and UBI3 were used as internal controls for gene expression. PI-I, ARG2 Relative gene expression levels. The primers used are as follows: RT-PI-IF: 5'-AGTGATGGACCAGAAGTCAT-3' (SEQ ID NO. 12); RT-PI-IR: 5'-TCCATTGTGACTGGAGAACCA-3' (SEQ ID NO. 13); RT-ARG2-F: 5'-TTTTGGTGTGGCAGTACAAAC-3' (SEQ ID NO. 14); RT-ARG2-R: 5'-GGGCGCAATGGATTCTCTTC-3' (SEQ ID NO. 15).

[0047] The results are as follows Picture 5 As shown, 1 hour after W+OS processing, wrky52 mutant plants PI-I, ARG2 The gene expression level was significantly lower than that of the wild-type plant, indicating that the knockout gene... WRKY52 Genes reduced the survival rate of tomato plants after feeding on bollworms. PI-I, ARG2 The expression level of genes is reduced, thereby weakening the bollworm resistance of tomatoes.

[0048] Example 5 Tomato WRKY52 Positive regulation of proteins PI-I , ARG2 Verification of gene transcription; 1. Constructing a system containing WRKY52 The pFGC1008-35S-HA vector of the gene: PCR amplification was performed using tomato cDNA as a template to obtain... WRKY52 The CDS sequence of the gene. Primers are as follows: WRKY52-HA-F: 5'-ttacaattaccatggggcgcgccATGGATAATTACGGAGCTGATAATAACA-3' (SEQ ID NO. 16); WRKY52-HA-R: 5'-aacatcgtatgggtaggtaccAGAGAATCTGTGATAATCATATGGGAA-3' (SEQ ID NO. 17); use Asc I and Kpn I. The vector and PCR product were double-digested with restriction endonucleases and then ligated into E. coli DH5α competent cells.

[0049] 2. Construct separately containing PI-I, ARG2 pGreen II 0800-LUC vector for gene promoter sequence: The method is the same as above, using tomato genomic DNA as a template, with Not I and Nco I restriction enzyme sites. The primers used are as follows: pPI-I-LUC-F: 5'-tccactagttctagagcggccgcATTTTTTACCAAATTCCTTTTTATTTTTT-3' (SEQ ID NO. 18); pPI-I-LUC-R: 5'-tgtttttggcgtcttccatggATTTGTATAAGTTGTATCACATTAATTTATAGGC-3' (SEQ ID NO. 19).

[0050] pARG2-LUC-F: 5'-tccactagttctagagcggccgcGTTACTTGATAAGAAATTAGGTTTTGTGAT-3' (SEQ ID NO. 20); pARG2-LUC-R: 5'-tgtttttggcgtcttccatggCTTATCCAACCAACATACTAAAATAATAATTT-3' (SEQ ID NO. 21).

[0051] 3. Electroporate the correctly sequenced plasmid into Agrobacterium GV3101 (pSoup) competent cells to obtain an Agrobacterium strain that can be used for dual-luciferase reporter gene detection experiments.

[0052] 4. Resuspend the above Agrobacterium bacterial suspension in an infection solution containing 150 μmol / L acetylsyleugenol and adjust to the optimal concentration (OD600=0.75). Activate the suspension at 28℃ for 3 h. Then, co-infect *Fumica benthamiana* with a mixture of WRKY52-HA:pPI-I-LUC (or pARG2-LUC) at a ratio of 10:1. Nicotiana benthamiana Leaves were examined, and three days later, the activities of firefly luciferase and kidney luciferase were detected using a dual-luciferase reporter assay system. The fluorescence ratio of the two was calculated and correlated with the amount of pFGC1008-35S-HA empty vector (EV). PI-I (or ARG2 The relationship between the value of the gene promoter (set to 1).

[0053] The results are as follows Picture 6 As shown, compared to the empty vector, the expression of WRKY52 enables... PI-I, ARG2 Gene promoter activation and a relatively high LUC / REN ratio indicate WRKY52 Activated PI-I, ARG2 Gene transcription.

[0054] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein. The specification and embodiments are to be considered exemplary only.

[0055] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. Tomato WRKY52 The application of genes in regulating plant resistance to insect pest stress is characterized by, The application is carried out through... WRKY52 Loss of gene function reduces the number of defense-related genes PI-I , ARG2 The expression level of [certain substances] is reduced, thereby weakening the plant's resistance to insect pest stress; the aforementioned WRKY52 The nucleotide sequence of the protein-coding region of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The pest in question is the cotton bollworm.

3. The application as described in claim 1, characterized in that, The WRKY52 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

4. The application as described in claim 1, characterized in that, The WRKY52 The way to lose gene function is by knockout. WRKY52 Gene or WRKY52 Gene silencing.

5. The application as described in claim 4, characterized in that, The knockout WRKY52 Genes, specifically including: For tomatoes WRKY52 Sequence analysis was performed to find PAM sequences. The 20 bp sequence before NGG was defined as sgRNA, and sgRNA sequences located in the gene protein coding region with high specificity were selected. Using gene editing and tissue culture technologies, a system was constructed. WRKY52 The gene was vectorized into a CRISPR / Cas9 vector, and genetically stable homozygous mutant lines that did not contain exogenous Cas9 protein were obtained through screening.

6. The application as described in claim 1, characterized in that, The WRKY52 Gene-encoded proteins activate defense-related genes PI-I , ARG2 The promoter promotes PI-I , ARG2 Gene transcription.

7. The application as described in claim 6, characterized in that, The PI-I The nucleotide sequence of the gene promoter is shown in SEQ ID NO.

3.

8. The application as described in claim 6, characterized in that, The ARG2 The nucleotide sequence of the gene promoter is shown in SEQ ID NO.

4.

9. The application as described in claim 5, characterized in that, The first 20 bp nucleotide sequences of the two target fragment PAM structures are shown in SEQ ID NO. 5 and SEQ ID NO. 6.

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