CN110628773B

CN110628773B - Target gene and method for preventing and treating harmonia axyridis

Info

Publication number: CN110628773B
Application number: CN201910718242.7A
Authority: CN
Inventors: 潘慧鹏; 吕晶; 郭威; 杨春晓; 郭木娟; 陈诗敏; 邱宝利; 刘卓琦
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-08-05
Filing date: 2019-08-05
Publication date: 2021-07-20
Anticipated expiration: 2039-08-05
Also published as: CN110628773A

Abstract

The invention discloses a target gene and a method for preventing and treating ladybug. The gene beta ' COPI is obtained by screening, and the gene is used as a target gene to silence the expression of the gene and can generate high lethal effect on the harmonia axyridis, so that a technology capable of efficiently preventing and treating the harmonia axyridis is developed, namely, the target gene dsRNA (dsbeta ' COPI) with high lethal capability on the harmonia axyridis is directly fed, and the lethal effect of the dsbeta ' COPI on the harmonia axyridis is utilized to achieve the purpose of preventing and treating. The method has the advantages of convenient operation, good effectiveness and sensitivity, high insecticidal efficiency, environmental friendliness and the like, and has good application prospect.

Description

Target gene and method for preventing and treating harmonia axyridis

Technical Field

The invention belongs to the technical field of insect pest prevention and control. More particularly, relates to a target gene for preventing and controlling the harmonia axyridis and application thereof.

Background

The harmonia axyridis (Fabricius) belongs to the family of Coleoptera axyridis, is an important agricultural pest, has wide host plants, and is mainly harmful to solanaceae vegetables such as eggplants, potatoes, tomatoes and the like. The larvae and adults all feed on leaves, prefer to gather on the back of the leaves, and eat down the epidermis and mesophyll, so that the damaged leaves usually form irregular transparent spots or perforations, and the plant wilts or even the whole plant dies when the disease is serious. The distribution range of the harmonia axyridis in China is wide, and particularly the occurrence density of the harmonia axyridis in the south of Yangtze river is high. In recent years, due to the warming of climate, the development of trade and the enlargement of vegetable cultivation area in protected areas, the occurrence and the harm of the ladybug are increasingly serious because of the continuous foodstuff all the year round. In 2015, the potato staple food strategy is started in China, the planting area of potatoes in China must be further enlarged, and the prevention and control of the ladybug are not slow.

At present, the control of the harmonia axyridis comprises artificial capture, attractant trapping and chemical pesticide. Wherein, the manual capture has poor effect and very heavy labor problem; the trapping effect of the attractant is not satisfactory and not thorough; therefore, chemical pesticides are still relied on more, but the chemical pesticides cause environmental pollution and quality safety of agricultural products.

RNA interference (RNAi) is an evolutionarily conserved mechanism of action that relies on the production of short stretches of RNAs (sirnas) to promote degradation or inhibit translation of homologous mrnas. RNAi provides an important tool for functional genomics research in insects, and lays a foundation for developing an environment-friendly pest control method. As RNAi technology can specifically inhibit the expression of genes, the technology is widely applied to target interference of pest genes so as to achieve the purpose of preventing and controlling pests, but the research on the functions of the gene of the harmonia axyridis is less at home and abroad at present, and a target gene report with insecticidal activity is not seen.

The earlier-stage research of the inventor team shows that (201710949193.9), the toxicity to the ladybug can be realized by directly feeding proper exogenous dsRNA, so that the exogenous dsRNA product suitable for preventing and treating the physalis alkekengi is developed from a gene level, the use is convenient, the cost is low, the accurate and excellent prevention and treatment effect can be realized due to the specificity of the gene, the environment is protected, and the application prospect in the prevention and treatment of the physalis alkekengi is great. However, the screening of related target genes and the design of specific and stable dsRNA with good control effect are the biggest difficult problems and key problems.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing wintercherry ladybug control technology and providing a high lethal gene of the wintercherry ladybug, namely a beta' COPI gene. And a technology capable of efficiently preventing and controlling the harmonia axyridis is developed based on the gene, namely, the technology is directly fed with a target gene dsRNA with high lethal capacity to the harmonia axyridis, and the lethal effect of dsbeta' COPI on the harmonia axyridis is utilized to achieve the purpose of preventing and controlling. The method has the advantages of convenient operation, good effectiveness and sensitivity, high insecticidal efficiency, environmental friendliness and the like, and has good application prospect.

The invention aims to provide a coccinella physaloides beta' COPI gene and application thereof in preventing and treating coccinella physaloides.

The invention also aims to provide dsRNA for the beta' COPI gene for preventing and controlling the ladybug and application thereof.

The invention further aims to provide a method and a kit for preventing and treating the harmonia axyridis.

The above purpose of the invention is realized by the following technical scheme:

the invention obtains a high lethal gene, namely beta ' COPI gene, by screening based on a transcriptome library of the ladybug, and develops a technology for preventing and treating the ladybug by dsRNA (dsbeta ' COPI) feeding the beta ' COPI gene. According to the method, eggplant leaves are respectively soaked in dsbeta' COPI and dsGFP solutions synthesized by a kit, the eggplant leaves are taken out and aired, then 1-instar larvae of the ladybug are fed for 2 days, then the eggplant leaves which are not treated by dsRNA are fed, and the mortality rate and the development state of the ladybug are observed and recorded; in addition, the lethality of the strain to 1-year, 3-year and adult ladybug is determined by using a method for expressing dsbeta 'COPI by using a bacterial liquid, so that the insecticidal activity of exogenous dsbeta' COPI to the ladybug is comprehensively evaluated. Finally, the expression quantity change of the beta 'COPI gene in the dsbeta' COPI and dsGFP ladybug eaten is detected and analyzed by a fluorescent quantitative PCR method. The result shows that the direct feeding of exogenous dsbeta ' COPI can obviously inhibit the gene expression of the coccinella physaloides beta ' COPI, and the direct feeding of exogenous dsbeta ' COPI has obvious lethal effect on the coccinella physaloides. Therefore, the following subject matters and applications should be considered to be within the protection scope of the present invention:

a ladybug beta' COPI gene has a sequence shown in SEQ ID NO. 1.

The beta' COPI gene is applied to preventing and treating the harmonia axyridis or preparing products for preventing and treating the harmonia axyridis.

The beta' COPI gene is applied to inhibiting the growth of the harmonia axyridis or preparing products for inhibiting the growth of the harmonia axyridis.

The beta' COPI gene is applied to the promotion of the death of the harmonia axyridis or the preparation of products for promoting the death of the harmonia axyridis.

The beta' COPI gene inhibitor is applied to preventing and treating the harmonia axyridis or preparing products for preventing and treating the harmonia axyridis.

dsRNA can be used for preventing and treating ladybug, and can target and silence beta' COPI gene. Preferably, the dsRNA sequence is shown as SEQ ID NO. 1.

A kit for preventing and treating Laurencia cooperivalis contains beta' COPI gene inhibitor. The inhibitor is the dsRNA.

Specifically, one of the ways of preventing and treating the harmonia axyridis by using the beta ' COPI gene is a method for preventing and treating the harmonia axyridis, exogenous dsRNA is directly fed, so that dsbeta ' COPI enters the body of the harmonia axyridis, the dsRNA can silence/inhibit the beta ' COPI gene expression of the harmonia axyridis, inhibit the growth of the harmonia axyridis and promote the death of the harmonia axyridis, and the aim of preventing and treating the harmonia axyridis is fulfilled.

The invention has the following beneficial effects:

the invention obtains a high lethal gene beta 'COPI gene of the ladybug through screening, develops the high-efficiency silent dsRNA of the gene, and develops a technology capable of efficiently preventing and treating the ladybug, namely directly feeding the target gene dsRNA with high lethal capability to the ladybug, and achieving the purpose of preventing and treating by utilizing the lethal effect of dsbeta' COPI on the ladybug. The method has the advantages of convenient operation, good effectiveness and sensitivity, high insecticidal efficiency, environmental friendliness and the like, and has good application prospect.

Drawings

FIG. 1 is a graph of dsGFP and dsbeta' COPI electrophoreses expressed from bacterial fluid.

FIG. 2 is a graph showing the effect of dsbeta' COPI at various concentrations on mortality of Laimiria serrulata larvae. Survival curves were established using Cox regression procedures using larval mortality data 10 days after the start of the experiment. Different letters (e.g., a, b) indicate significant differences between the control and treatment curves.

FIG. 3 is a graph showing the effect of dsbeta' COPI expressed in the inoculum solution on the survival rate of harmonia axyridis (graph A: survival rate of 1 st larva; graph B: survival rate of 3 rd larva; graph C: survival rate of adult). Survival curves were established using Cox regression programs using mortality data for 1 st, 3 rd and adult larvae at 10, and 14 days, respectively. Different letters (e.g., a, b) indicate significant differences between the control and treatment curves.

FIG. 4 shows the change of beta 'COPI gene expression level in E.paduligera at day 2 and day 4 after feeding dsbeta' COPI and dsGFP.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the present invention are commercially available.

The ladybug, used in the examples below, was bred at the department of insects at southern agricultural university of south China. The eggplants for breeding the harmonia axyridis are Tengsheng Maruashuai round eggplant seedlings, the harmonia axyridis is placed in a culture dish containing filter paper, the filter paper is moisturized by a cotton ball, and the harmonia axyridis is placed in an artificial climate box (the temperature is 25 +/-1 ℃, the humidity is 70-80%, and the photoperiod L: D is 14:10) for propagation.

RNA extraction Using TRIzol extraction (Invitrogen, USA), reverse transcription reagent (PrimeScript)^TMRT reagent Kit with gDNA Eraser) from TAKARA Biotechnology Ltd, dsRNA synthesis Kit (MEGAscript)^TMT7) from Thermo Fisher Scientific, kit for PCR reaction System (EX Taq^TM) Purchased from TAKARA Biotechnology Ltd, and DNA Purification recovery Kit (Universal DNA Purification Kit) purchased from Tiangen Biochemical technology (Beijing) Ltd.

The data processing method of the following example: for the result analysis of the bioassay of the two types of dsRNA on the harmonia axyridis, the survival rate of the harmonia axyridis is counted by using Excel 2010, the SPSS 19.0 software is used for drawing by adopting Cox regression analysis, and the difference analysis among different concentrations is used for single factor analysis. Analyzing the change of target gene expression after RNA interference, wherein qPCR data adopts 2^-△△CtThe method (Ct represents the number of cycles) was performed. Data analysis was performed using single factor analysis of variance using SPSS 19.0 software.

Example 1 acquisition of growth development related Gene beta' COPIPdsRNA

A transcriptome library of the harmonia axyridis is constructed according to the genome of the harmonia axyridis, genes related to growth and development of the harmonia axyridis are researched and screened based on the constructed transcriptome library, and beta 'COPI gene fragments are obtained through screening, wherein the beta' COPI gene fragments are shown as SEQ ID NO. 1. The dsRNA is then synthesized.

1. Extracting total RNA of the harmonia axyridis and synthesizing first strand cDNA.

Taking 10 2-instar larvae of the harmonia axyridis, placing in a 2ml centrifuge tube, extracting total RNA of the harmonia axyridis by using a TRIzol method, wherein the concentration and quality of the RNA are obtained by using NanoDropone^CThe measurement was carried out using a reverse transcription kit (PrimeScript)^TMRT reagent Kit with gDNA Eraser, TAKARA) reverse transcription was performed according to the instruction to synthesize the first strand cDNA.

2. Primer design

The gene sequence of beta 'COPI is obtained from a transcriptome library of the ladybug, a dsRNA primer P1 (table 1) of the beta' COPI gene is designed, a green fluorescent protein Gene (GFP) is obtained by amplifying from a plasmid which contains GFP and is stored in a laboratory, and a dsRNA primer P2 (table 1) of the GFP gene. Homologous arms related to enzyme cutting sites are added to a dsbeta 'COPI primer P1 and a dsGFP primer P2 respectively, a dsbeta' COPI construction expression vector related primer P3 and a dsGFP construction expression vector related primer P4 are designed (Table 1). Based on the sequence of the beta 'COPI gene, a fluorescent quantitative PCR (qPCR) primer P5 of the beta' COPI gene and a qPCR primer P6 of the reference gene GAPDH (Table 1) are designed.

Table 1: dsRNA synthesis and qPCR primers

3. dsRNAs of beta' COPI gene and GFP gene synthesized by kit

PCR amplification was performed using primers P1 and P2 in Table 1, with the reaction system of 10 XEX Taq Buffer 5. mu.L, TaKaRa EX Taq 0.25. mu.L, dNTP mix 4. mu.L, upstream primer (10. mu. moL/L) 1. mu.L, downstream primer (10. mu. moL/L) 1. mu. L, cDNA/GFP plasmid 1. mu.L, dd H₂The content of O is filled to 50 mu L. The reaction program of PCR amplification is pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles; extension at 72 ℃ for 5 min. The amplification product was stored at 4 ℃. And after the program reaction is finished, detecting the amplification result by using an agarose gel electrophoresis method.

Recovering and purifying the two PCR products by using a DNA Purification recovery Kit (TIANGEN) as templates for in vitro transcription of dsRNA, wherein the in vitro transcription system of the dsRNA is 10x Reaction Buffer 5 muL, (ATP, GTP, CTP and UTP) solution respectively 5 muL, Enzyme mix 5 muL, template 20 muL and ddH₂O make up to 50. mu.L. The mixture was left at 37 ℃ for 4 hours. After the reaction, 2.5. mu.L of TURBO DNase was added to remove the residual template DNA and single-stranded RNA, then the dsRNA was purified, and finally 50. mu.L of ddH was used₂O dissolves the dsRNA, resulting in dsbeta' COPI and dsGFP, respectively, and the band of dsRNA was verified by electrophoresis on a 1.5% agarose gel.

The beta 'COPIPdsRNA of the harmonia axyridis is double-stranded RNA and consists of a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is SEQ ID NO.1 in a sequence table, the nucleotide sequence of the antisense strand is a reverse complementary sequence of the SEQ ID NO.1 in the sequence table, and the nucleotide sequence of the beta' COPIPdsRNA encoding gene is SEQ ID NO.1 in the sequence table. The GFP dsRNA is double-stranded RNA and consists of a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is SEQ ID NO.2 in a sequence table, and the nucleotide sequence of the antisense strand is a reverse complementary sequence of the SEQ ID NO.2 in the sequence table.

4. Obtaining of dsRNA expressed by growth development related gene beta' COPI bacterial liquid

Two cleavage sites were selected on the sequence of L4440, BamHI (GGATCC) and SacI (GAGCTC), respectively. According to the sequence information of L4440 (the sequence information is disclosed), homology arms related to two enzyme cutting sites are respectively added to a dsbeta 'COPI primer P1 and a dsGFP primer P2, a dsbeta' COPI construction expression vector related primer P3 and a dsGFP construction expression vector related primer P4 are designed (Table 1). The cDNA template, reaction system and amplification procedure of PCR amplification are shown above, and the target fragments of dsbeta' COPI and dsGFP of the constructed vector are obtained, and the two PCR products obtained above are recovered by using a DNA Purification recovery Kit (Universal DNA Purification Kit, TIANGEN). Utilizing Quickcut according to the sequence of two enzyme cutting sites^TMSacI and Quickcut^TMThe L4440 vector was linearized with BamHI, the reaction system for the enzyme digestion is described in the specification, and after the enzyme digestion reaction was completed, the linearized L4440 vector was recovered with a DNA Purification recovery Kit (Universal DNA Purification Kit, TIANGEN).

Utilizing Trelief of Guangzhou Ongke Biotech Co., Ltd^TMThe SoSoSoSoo Cloning Kit Ver.2 Kit separately reacts dsGFP and dsbeta' COPI with linearized L4440 vector at 50 ℃ for 20min for recombination. Subsequently, the recombinant expression vector containing dsbeta' COPI and dsGFP was introduced into HT115 competent cells, placed on ice for 30min, followed by heat shock at 37 ℃ for 1 min; after standing on ice for 3min, 700. mu.L of LB liquid medium containing no ampicillin was added, and the mixture was incubated at 37 ℃ and 210rpm for 1h, followed by overnight incubation with LB plates containing ampicillin and tetracycline. A single colony was picked and placed in 4mL of LB liquid medium containing ampicillin (100. mu.g/mL) and tetracycline (10. mu.g/mL) and cultured at 37 ℃ and 210rpm for 12 hours, and then 50. mu.L of the single colony was transferred to 5mL of LB liquid medium containing ampicillin (100. mu.g/mL) and tetracycline (10. mu.g/mL) and cultured at 37 ℃ and 210rpm for 3 hours so that the OD of the cell broth was between 0.5 and 0.8, 1mM IPTG was added thereto and cultured at 37 ℃ and 120rpm for 5 hours to induce dsRNA. Will contain dsGFP and dsbetMycelia were collected from the two a' COPI bacterial solutions at 4 ℃ and 5000rpm, RNA was extracted by TRIzol extraction (Invitrogen, USA), and 1.5% agarose gel electrophoresis was performed to verify successful induction of dsRNA.

5. Results

Amplifying by using a P1 primer to obtain a PCR amplification product with the size of 436bp, sequencing and deleting a T7 promoter sequence to obtain a nucleotide sequence with the size of 396bp, namely the target gene beta' COPI, as shown in SEQ ID NO. 1. The plasmid carrying GFP is used as a template, P2 primer is used for amplification, a PCR product with the size of 507bp is obtained, and the target bands of dsGFP and dsbeta' COPI are consistent with the sequencing result.

The dsRNA of the target gene is expressed by using bacterial liquid, the RNA of the hyphae is extracted, agarose gel electrophoresis is used for verifying whether the dsRNA of the target gene is successfully induced, and the target bands for successfully inducing dsGFP and dsbeta' COPI can be seen according to the electrophoresis result (figure 1).

Example 2 inhibition of ladybug by dsRNA

1. Application of dsRNA (double-stranded ribonucleic acid) synthesized by kit in inhibiting growth and development of harmonia axyridis

Wintercherry ladybug dsbeta' COPI feeding group: 10 1 st larvae of the ladybug were placed in a petri dish with filter paper and humidified cotton balls. Soaking round eggplant leaves with the diameter of 12mm in dsbeta 'COPI solutions with the concentrations of 50 ng/mu L, 20 ng/mu L, 10 ng/mu L, 5 ng/mu L and 3 ng/mu L for 1min, air-drying at room temperature for 1h, feeding the larvae, replacing the leaf disc every 24h, continuously feeding the dsbeta' COPI-soaked leaf disc for two days, and feeding the larvae with normal eggplant leaves.

Wintercherry ladybug dsGFP feeding group: 10 1 st larvae of the ladybug were placed in a petri dish with filter paper and humidified cotton balls. Soaking round eggplant leaves with the diameter of 12mm in dsGFP solution with the concentration of 50ng/uL synthesized by the kit for 1min, air-drying for 1h, feeding the larvae, replacing a leaf disc every 24h, continuously feeding the leaf disc soaked by the dsGFP for two days, and feeding the larvae with untreated eggplant leaves.

Each group is set to be 5 times, the death number of the harmonia axyridis in each culture dish is counted every 24 hours, the new leaf is replaced, and the culture dishes are placed in an artificial climate box (the temperature is 25 +/-1 ℃, the humidity is 70-80%, and the light period L: D is 14: 10). And counting the death number of the harmonia axyridis in each culture dish of each group, and calculating the survival rate change of the harmonia axyridis under the treatment of the control group and the dsRNA with different concentrations.

2. Application of dsbeta' COPI expressed by bacterial liquid to killing effect of harmonia axyridis

Wintercherry ladybug dsbeta' COPI feeding group: 10 1-instar larvae, 10 3-instar larvae and 5 adults are placed in a culture dish with filter paper and a humidifying cotton ball, 3 groups of experiments are set in total, and 5 replicates are set in each group. Soaking round eggplant leaves with the diameter of 12mm in a bacterial solution expressing dsbeta' COPI for 1min, and feeding the leaves to larvae after the leaves are air-dried for 1h at room temperature. 2 leaf discs are placed in each culture dish of 1-instar larvae in the treatment group; 5 leaf discs are placed in each culture dish of 3-instar larvae; adult dishes were placed with 5 leaf discs. The leaf disc is replaced every 24 hours, and the leaf disc soaked by the dsbeta' COPI bacterial liquid is continuously fed for two days and then fed by normal eggplant leaves.

Wintercherry ladybug dsGFP feeding group: 10 1-instar larvae, 10 3-instar larvae and 5 adults are placed in a culture dish containing filter paper and a humidifying cotton ball, 3 groups of controls are arranged in total, and 5 replicates are arranged in each group. Round eggplant leaves with the diameter of 12mm are soaked for 1min by using dsGFP-expressing bacterial liquid, and the round eggplant leaves are air-dried for 1h at room temperature and then fed to larvae. 2 leaf discs are placed in each culture dish of 1-instar larvae in the control group; 5 leaf discs are placed in each culture dish of 3-instar larvae; adult dishes were placed with 5 leaf discs. And replacing the leaf disc every 24 hours, continuously feeding the leaf disc soaked by the dsGFP bacterial liquid for two days, and feeding the leaf disc by using normal eggplant leaves.

Counting the death number of the harmonia axyridis in each culture dish every 24h, replacing new leaves, and placing the culture dishes in an artificial climate box (the temperature is 25 +/-1 ℃, the humidity is 70-80%, and the light period L: D is 14: 10). And counting the death number of the harmonia axyridis in each culture dish of each group, and calculating the change of the survival rate of the harmonia axyridis of the control group and the different treatment groups.

3. According to the statistical result, after continuously feeding the 1 st larva dsbeta' COPI of the harmonia axyridis for two days, the survival rate of the 1 st larva of the harmonia axyridis shows a descending trend along with the increase of time, the feeding concentrations of the treatment groups are 3 ng/mu L, 5 ng/mu L, 10 ng/mu L, 20 ng/mu L and 50 ng/mu L respectively, and the feeding concentration of the control group is 50 ng/mu L. From the results of fig. 2, significant differences were found between the treatment groups and the control group at different concentrations (χ 2 ═ 183.816, df ═ 5, P < 0.0001). There was a significant difference between treatment groups of 3ng/μ L (P <0.0001, exp (b) ═ 10.126) and 5ng/μ L (P <0.0001, exp (b) ═ 25.649), 10ng/μ L (P <0.0001, exp (b) ═ 24.315), 20ng/μ L (P <0.0001, exp (b) ═ 46.194), 50ng/μ L (P <0.0001, exp (b) ═ 56.952), and no significant difference between the other 4 concentrations of treatment groups. From the statistical results, it was concluded that the mortality increased 10.126-fold, 11.503-fold, 24.315-fold, 46.194-fold and 56.952-fold, respectively, when the treatment groups were at concentrations of 3 ng/. mu.L, 5 ng/. mu.L, 10 ng/. mu.L, 20 ng/. mu.L and 50 ng/. mu.L, respectively, as compared to the control group

According to the statistical results (as shown in fig. 3), after continuously feeding dsbeta' COPI expressed by the strain liquid of the harmonia axyridis, the survival rates of 1-instar larvae (P <0.0001, exp (B) ═ 20.258), 3-instar larvae (P <0.0001, exp (B) ═ 16.983) and adults (P ═ 0.041, exp (B) ═ 4.940) are significantly different from those of the control group, and the death rates of the 1-instar larvae, the 3-instar larvae and the adults of the treatment group are respectively increased by 20.258 times, 16.983 times and 4.940 times compared with those of the control group.

In addition, changes in phenotypic characteristics of the ladybug were observed microscopically after two days of dsbeta' COPI feeding. On day 3 from the feeding of dsbeta 'COPI, the ladybug of the dsGFP control group normally enters the 2-instar stage, the larvae in the treatment group can not normally molt into the 2-instar stage and die, and the phenotypic characteristic is that branch spines and spots of the anterior dorsal lamella of the larvae can not be formed, which indicates that the feeding of dsbeta' COPI can trigger strong RNAi effect in the body of the ladybug, and the ladybug dies.

Example 3 dsbeta 'COPI inhibits the expression of beta' COPI Gene in Lauremysis serrulata

1. Experimental methods

On

days

2 and 4 after the onset of dsRNA feeding, respectively, 1 st larvae of E.paduligera treated with 5 ng/. mu.L dsbeta' COPI and dsGFP were collected, 3 biological replicates per treatment were collected. Lifting deviceCollecting RNA of the harmonia axyridis, carrying out reverse transcription on the RNA to form cDNA, and diluting by 10 times to obtain a qPCR template. qPCR analysis was performed with P5 and P6 as primers. The qPCR system (15. mu.L) contained 5.25. mu.L of ddH₂O, 7.5. mu.L of 2 XSSYBR Green MasterMix (BIO-RAD Inc, Hercules, Calif.), 4. mu.M primer and 1.0. mu.L of cDNA first strand template. The qPCR reaction apparatus Bio-Rad C1000 Real-Time PCR system (BIO-RAD, USA). The reaction condition is 95 ℃ for 5 min; the reaction was performed in 96 well plates (BIO-RAD, USA) with 95 ℃ for 10s, 60 ℃ for 30s, 39 cycles, and 3 technical replicates per sample.

2. Results of the experiment

Relative expression changes of beta 'COPI gene in E.physaloidis were counted at

day

2 and 4 after dsbeta' COPI feeding, respectively, with dsGFP feeding as a control (as shown in FIG. 4). On day 2 from the start of feeding dsbeta 'COPI, beta' COPI expression decreased 5.26-fold compared to control (F)_1,4588.280, P0.019); on day 4 from the feeding of dsbeta 'COPI, beta' COPI expression was decreased 10.65-fold compared to the control group (F)_1,4＝19956.817,P<0.0001), which shows that the expression level of the gene beta ' COPI in the harmonia axyridis fed with dsbeta ' COPI is obviously reduced compared with the expression level of the gene beta ' COPI in the harmonia axyridis fed with dsGFP. Further shows that the feeding of dsbeta 'COPI can cause strong RNAi effect in the body of the harmonia axyridis, which leads the expression level of beta' COPI gene in the body to be obviously reduced, and further leads the death or the development of the harmonia axyridis to be inhibited.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

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<211> 22

<212> DNA

<213> P6-F

<400> 13

agctcttctc atcatggctt ac 22

<210> 14

<211> 22

<212> DNA

<213> P6-R

<400> 14

gaaagaggtg cagaatgtgt tg 22

Claims

1. A ladybug beta' COPI gene is characterized in that the sequence is shown as SEQ ID NO. 1.

2. A dsRNA for use in the control of ladybug, wherein the dsRNA silencing target gene is the beta' COPI gene of claim 1.

3. The use of the dsRNA of claim 2 for controlling ladybug or for preparing a product for controlling ladybug.

4. Use of the dsRNA of claim 2 for inhibiting the growth of ladybug or for preparing a product for inhibiting the growth of ladybug.

5. Use of the dsRNA of claim 2 for promoting death of or preparing a product for promoting death of E.

6. A kit for controlling ladybug, which contains the dsRNA of claim 2.

7. A method for controlling ladybug, which is characterized in that exogenous dsRNA is fed, and the dsRNA can silence/inhibit the expression of the beta' COPI gene in claim 1.