Application of CHS2 gene in regulating and controlling resistance of beet armyworm and black cutworm to Vip3Aa protein

Abstract

The invention provides an application of a CHS2 gene in regulating and controlling the resistance of beet armyworm and agrotis ypsilon to Vip3Aa protein, and relates to the technical field of biology. The research of the inventor finds that the CHS2 gene is related to the resistance of beet armyworms and black cutworms to Vip3Aa protein, and the resistance of the beet armyworms and the black cutworms to the Vip3Aa protein can be remarkably improved by knocking out the CHS2 gene; meanwhile, CHS2 can also be used as a marker of the resistance of beet armyworms and black cutworms to the Vip3Aa protein, and is used for detecting the resistance level of the beet armyworms and the black cutworms in the field to the Vip3Aa protein. The research has important theoretical and practical significance for research and development of novel Bt toxins and field monitoring and comprehensive treatment of pest resistance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of a CHS2 gene in regulating the resistance of beet armyworm and black cutworm to Vip3Aa protein. Background Art

[0002] The beet armyworm (Spodoptera exigua) is a major agricultural pest in my country, feeding on crops such as cotton, corn, sesame, peanuts, soybeans, tobacco, tomatoes, cowpeas, and cabbage. The black cutworm (Agrotis ipsilon) is a severe underground pest that harms seedlings in agricultural and forestry plants. Its primary host plants include cotton, corn, wheat, sorghum, tobacco, potatoes, hemp, beans, and vegetables, as well as seedlings of linden, ash, walnut, and Korean pine. Currently, the cultivation of transgenic Bt crops can significantly reduce damage from Lepidoptera insects, including the beet armyworm and black cutworm, but it also poses the risk of pesticide resistance. In response to the widespread resistance to CRY toxins, second-generation VIP-type Bt toxin proteins have garnered increased attention due to their distinct toxicological mechanisms and the rare detection of resistance in the field.

[0003] Currently, the Myb gene and VipR1 gene are involved in Vip3Aa resistance in Lepidoptera insects. Whether other genes are involved in Vip3Aa resistance requires further in-depth research.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The first object of the present invention is to provide an application of the CHS2 (chitin synthase 2) gene in regulating the resistance of beet armyworm and black cutworm to Vip3Aa protein.

[0006] The second object of the present invention is to provide an application of the CHS2 gene in regulating the survival ability of beet armyworm and black cutworm.

[0007] The third object of the present invention is to provide a marker for use in detecting resistance of Spodoptera exigua and Agrotis gambiae to Vip3Aa protein.

[0008] The fourth object of the present invention is to provide a reagent for detecting the resistance of beet armyworm and black cutworm to Vip3Aa protein.

[0009] The fifth object of the present invention is to provide the use of the above reagent in the preparation of a product for detecting the resistance of beet armyworm and cutworm to Vip3Aa protein.

[0010] The sixth object of the present invention is to provide a kit.

[0011] In order to achieve the above objectives, the following technical solutions are adopted:

[0012] In a first aspect, the present invention provides an application of the CHS2 gene in regulating the resistance of Spodoptera exigua and Agrotis diffusa to Vip3Aa protein.

[0013] As a further technical solution, the CHS2 gene was knocked out to improve the resistance of beet armyworm and cutworm to Vip3A protein.

[0014] In a second aspect, the present invention provides the use of the CHS2 gene in regulating the survival ability of beet armyworm and black cutworm.

[0015] As a further technical solution, the CHS2 gene is knocked out to reduce the survival ability of beet armyworm and cutworm.

[0016] In a third aspect, the present invention provides a marker for detecting resistance of beet armyworm and cutworm to Vip3Aa protein, wherein the marker is selected from any one of the following ac:

[0017] a. CHS2 gene;

[0018] b. RNA transcribed from the CHS2 gene;

[0019] c. CHS2 protein.

[0020] In a fourth aspect, the present invention provides a reagent for detecting the resistance of beet armyworm and cutworm to Vip3Aa protein, wherein the reagent comprises a primer for detecting CHS2 gene, a primer for detecting RNA transcribed from CHS2 gene or an antibody for detecting CHS2 protein.

[0021] As a further technical solution, the CDS sequence of the CHS2 gene of Spodoptera exigua is shown as SEQ ID NO.1.

[0022] As a further technical solution, the CDS sequence of the CHS2 gene of the black cutworm is shown as SEQ ID NO.2.

[0023] In a fifth aspect, the present invention provides the use of the above reagent in the preparation of a product for detecting the resistance of beet armyworm and cutworm to Vip3Aa protein.

[0024] In a sixth aspect, the present invention provides a kit comprising the reagent.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The inventors discovered that the CHS2 gene is associated with the resistance of beet armyworm and black cutworm to the Vip3Aa protein, and that knocking out the CHS2 gene significantly increases the resistance of these insects to Vip3Aa. Furthermore, CHS2 can be used as a marker for resistance to Vip3Aa in these insects, allowing detection of resistance levels in field samples. Furthermore, the CHS2 gene is associated with the survival of these insects, with knocking out the CHS2 gene significantly reducing their survival. This research has important theoretical and practical implications for the development of new Bt toxins, as well as for field monitoring and integrated pest control. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 CRISPR / Cas9-mediated knockout of the CHS2 gene in Spodoptera exigua and Agrotis gambiae; the bold sequence is the sgRNA target sequence; the red bases are the protospacer adjacent motif (PAM site); compared with the wild-type (WT) sequence, the red dash indicates a base deletion, and the numbers in brackets are the number of deleted bases;

[0029] Figure 2 The midgut anatomy of wild-type (a and c) and knockout (b and d) Spodoptera exigua and Agrotis minima; PM, peritrophic membrane; MG, midgut;

[0030] Figure 3 This is the survival curve of the larval stage of the beet armyworm knockout strain SeCHS2-KO and the sensitive strain Se-SS;

[0031] Figure 4 Genotype detection of surviving individuals after bioassay of heterozygous offspring of Agrotis miniata; 1-13 represent 13 surviving individuals, and WT represents wild-type individuals. DETAILED DESCRIPTION

[0032] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0033] In a first aspect, the present invention provides an application of the CHS2 gene in regulating the resistance of Spodoptera exigua and Agrotis diffusa to Vip3Aa protein.

[0034] This study discovered for the first time the CHS2-mediated resistance mechanism to Vip3Aa. By knocking out the CHS2 gene, the resistance of beet armyworm and black cutworm to Vip3Aa protein can be significantly improved. This study has important theoretical and practical significance for the development of new Bt toxins, field monitoring and integrated management of pest resistance.

[0035] In a second aspect, the present invention provides the use of the CHS2 gene in regulating the survival ability of beet armyworm and black cutworm.

[0036] The inventors have found that the CHS2 gene is related to the survival ability of beet armyworm and black cutworm. Knocking out the CHS2 gene can significantly reduce the survival ability of beet armyworm and black cutworm. Therefore, the CHS2 gene can be used to regulate the survival ability of beet armyworm and black cutworm.

[0037] In a third aspect, the present invention provides a marker for detecting resistance of beet armyworm and cutworm to Vip3Aa protein, wherein the marker is selected from any one of the following ac:

[0038] a. CHS2 gene;

[0039] b. RNA transcribed from the CHS2 gene;

[0040] c. CHS2 protein.

[0041] The inventors have found that CHS2 is significantly correlated with the resistance of beet armyworm and black cutworm to Vip3Aa protein. Therefore, CHS2 can be used as a marker for the resistance of beet armyworm and black cutworm to Vip3Aa protein, and can be used to detect the resistance level of beet armyworm and black cutworm to Vip3Aa protein in the field.

[0042] In a fourth aspect, the present invention provides a reagent for detecting the resistance of beet armyworm and cutworm to Vip3Aa protein, wherein the reagent comprises a primer for detecting CHS2 gene, a primer for detecting RNA transcribed from CHS2 gene or an antibody for detecting CHS2 protein.

[0043] The reagent provided by the invention can be used for detecting the resistance of beet armyworm and black cutworm to Vip3Aa protein.

[0044] In some optional embodiments, the CDS sequence of the CHS2 gene of Spodoptera exigua is shown as SEQ ID NO.1:

[0045]

[0046] In some optional embodiments, the CDS sequence of the CHS2 gene of the black cutworm is shown in SEQ ID NO.2:

[0047]

[0048] In a fifth aspect, the present invention provides the use of the above reagent in the preparation of a product for detecting the resistance of beet armyworm and cutworm to Vip3Aa protein.

[0049] As a further technical solution, the product includes a PCR kit or an immunochromatography kit.

[0050] For example, primers for detecting CHS2 gene or primers for detecting CHS2 gene RNA are prepared into a PCR kit; antibodies for detecting CHS2 protein are prepared into an immunochromatography kit.

[0051] In a sixth aspect, the present invention provides a kit comprising the reagent.

[0052] The above reagents are used to prepare a kit, which can be used to detect the resistance level of beet armyworm and cutworm to Vip3A protein.

[0053] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0054] Example 1

[0055] In this study, CRISPR / Cas9 technology was used to knock out the CHS2 gene of Spodoptera exigua and Agrotis spp. The Spodoptera exigua knockout strain SeCHS2-KO deleted 5 bases at exon 21 (Exon 21), and the Agrotis spp. knockout strain AiCHS2-KO deleted 146 bases at exon 22 (Exon 22). Figure 1 Screening primers were used to detect the knockout site (Table 1). Ai_CHS2_F and Ai_CHS2_R were used for the black cutworm knockout line, AiCHS2-KO; Se_CHS2_F and Se_CHS2_R were used for the beet armyworm knockout line, SeCHS2-KO. Multiple generations of paired screening were performed to obtain homozygous lines for the CHS2 knockout.

[0056] Table 1 Primer sequences used to detect CHS2 gene knockout mutations

[0057]

[0058] We used the method of coating the feed surface with toxin protein for bioassay. Artificial feed was divided into 24-well plates and different concentrations of Vip3Aa protein (0, 200, 400, 800 μg / cm 2) is evenly applied to the surface of the feed and allowed to dry. One unfed newly hatched larva is placed in each well. After 7 days, the mortality rate is calculated (if the larvae die or remain in the first or second instar stage, they are considered dead). The experiment also tested the wild-type sensitive strain and the knockout strain, and finally calculated the resistance multiple. After the beet armyworm knockout strain SeCHS2-KO and the sensitive strain Se-SS were raised on ordinary feed, their survival rate in the larval stage was tested to determine the fitness cost of knocking out the SeCHS2 gene.

[0059] After knocking out the CHS2 gene in Spodoptera exigua, we found through dissection that the peritrophic membrane of its larvae disappeared ( Figure 2 a and b in the figure). Furthermore, the knockout strain was found to be able to survive on the highest concentration of Vip3Aa in the diet through a coating bioassay. Calculation results confirmed that its resistance multiple was greater than 33,333 times (Table 2). After feeding on ordinary diet, the knockout strain had a significantly higher mortality rate in the larval stage than the sensitive strain (p<0.0001). Figure 3 ).

[0060] Table 2 Resistance multiples of the experimental strains to Vip3Aa

[0061]

[0062]

[0063] LC50, median lethal concentration.

[0064] In cutworms, we found that homozygous larvae of CHS2 knockout also lacked the peritrophic membrane ( Figure 2 Because homozygous knockout lines of Agrotissima cannot produce offspring, we performed bioassays using offspring from heterozygous matings. The results showed that larvae survived on diets treated with Vip3Aa at all concentrations after 7 days, and at ≥200 μg / cm 2 There were 13 larvae surviving in the treatment group with the concentration of 1% (Table 3), and PCR analysis showed that all these larvae were homozygous ( Figure 4 The calculated proportion of surviving individuals was not significantly different from the expected 25% homozygote population (p = 0.174). The results indicate that the homozygote resistance of the cutworm is greater than 11,268 times. Since the number of homozygotes that matured to adulthood and their inability to produce offspring is extremely low, knocking out this gene demonstrates a significant fitness cost (reduced survival).

[0065] Table 3 Bioassay of heterozygous offspring of cutworm

[0066]

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of CHS2 gene in regulating the resistance of beet armyworm and black cutworm to Vip3Aa protein.

2. The use according to claim 1, characterized in that Knocking out the CHS2 gene improves the resistance of beet armyworm and cutworm to Vip3Aa protein.

3. Application of CHS2 gene in regulating the survival ability of beet armyworm and black cutworm.

4. The use according to claim 3, characterized in that Knocking out the CHS2 gene reduces the survivability of beet armyworm and cutworm.

5. Use of a marker in detecting resistance of beet armyworm and cutworm to Vip3Aa protein, characterized in that: The marker is selected from any one of the following ac: a. CHS2 gene; b. RNA transcribed from the CHS2 gene; c. CHS2 protein.

6. A reagent for detecting resistance of beet armyworm and cutworm to Vip3Aa protein, characterized in that: The reagents include primers for detecting CHS2 gene, primers for detecting RNA transcribed from CHS2 gene or antibodies for detecting CHS2 protein.

7. The reagent according to claim 6, characterized in that The CDS sequence of the CHS2 gene of Spodoptera exigua is shown as SEQ ID NO.

1.

8. The reagent according to claim 6, characterized in that The CDS sequence of the CHS2 gene of the black cutworm is shown as SEQ ID NO.

2.

9. Use of the reagent according to any one of claims 6 to 8 in the preparation of a product for detecting resistance of beet armyworm and cutworm to Vip3Aa protein.

10. A kit, characterized in that The invention comprises the reagent according to any one of claims 6 to 8.

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