CN110628772B

CN110628772B - Gene gammaCOPI and its application in the control of Physalis lady beetle

Info

Publication number: CN110628772B
Application number: CN201910718240.8A
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: CN110628772A

Abstract

The invention discloses a gene gammaCOPI and its application in controlling Physalis ladybird. In the present invention, a gene gammaCOPI is obtained from C. Physalis, and silencing the expression of this gene can effectively prevent C. Physalis. Therefore, a target gene dsRNA with high lethality to C. Physalis is designed, namely dsgammaCOPI, which is effective against C. Physalis. It has many advantages such as high lethal effect, good sensitivity, high insecticidal efficiency, good control effect, convenient use and operation, and environmental friendliness.

Description

Gene gamma COPI and application thereof in 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 no target gene report with insecticidal activity exists.

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 technology for preventing and controlling the harmonia axyridis and provides a high lethal gene of the harmonia axyridis, namely a gamma COPI gene. And a technology capable of efficiently preventing and treating the ladybug is developed based on the gene, namely, the target gene dsRNA with high lethal capacity to the ladybug is directly fed, and the lethal effect of dsgamma COPI on the ladybug is utilized to achieve the purpose of prevention and treatment. 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 harmonia axyridis gamma COPI gene and application thereof in preventing and treating harmonia axyridis.

The invention also aims to provide dsRNA of the gamma COPI gene for preventing and treating 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 screens a high lethal gene, i.e. gamma COPI gene, based on a transcriptome library of the ladybug, and develops a technology for preventing and treating the ladybug by dsRNA (dsgamma COPI) feeding the gamma COPI gene. The method comprises the steps of respectively soaking eggplant leaves in dsgammaCOPI and dsGFP solutions synthesized by a kit, taking out the eggplant leaves, airing the eggplant leaves, feeding 1-instar larvae of the ladybug for 2 days, feeding the 1-instar larvae with the eggplant leaves which are not treated by dsRNA, and observing and recording the mortality rate and the development state of the ladybug; in addition, the lethality of the strain to 1-year-old and 3-year-old ladybug and adults is determined by using a method for expressing dsgamma COPI by using a bacterial liquid, so that the insecticidal activity of exogenous dsgamma COPI to the ladybug is comprehensively evaluated. Finally, the expression quantity change of the gamma COPI gene in the dsgamma COPI and dsGFP ladybug eaten is detected and analyzed by a fluorescent quantitative PCR (qPCR) method. The result shows that the direct feeding of exogenous dsgamma COPI can obviously inhibit the gene expression of the harmonia axyridis gamma COPI, and the direct feeding of exogenous dsgamma COPI has obvious lethal effect on the harmonia axyridis. Therefore, the following subject matters and applications should be considered to be within the protection scope of the present invention:

a gene of the ladybug gammaCOPI has a sequence shown in SEQ ID NO. 1.

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

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

The application of the gamma COPI gene in promoting the death of the harmonia axyridis or preparing products for promoting the death of the harmonia axyridis.

The inhibitor of the gamma COPI gene 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 be used for targeted silencing of gamma COPI gene. Preferably, the dsRNA sequence is shown as SEQ ID NO. 2.

A kit for preventing and treating Laurencia cooperivalis contains gamma COPI gene inhibitor. Preferably, the inhibitor is the above-described dsRNA.

Specifically, one of the ways of preventing and treating the harmonia axyridis by using the gamma COPI gene is a method for preventing and treating the harmonia axyridis, exogenous dsRNA is directly fed, so that dsgamma COPI enters the body of the harmonia axyridis, the dsRNA can silence/inhibit the gamma 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 gamma COPI gene of the ladybug through screening, develops the high-efficiency silencing 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 dsgamma 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 dsgamma COPI electrophoreses expressed from the bacterial suspension.

FIG. 2 is a graph showing the effect of dsgamma COPI at various concentrations on mortality of E.varivestis 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 dsgammaCOPI expressed in the inoculum solution on the survival rate of E.varivestis (FIG. A: survival rate of 1 st larva; FIG. B: survival rate of 3 rd larva; FIG. 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 phenotypic differences between a normally developed dsGFP control group (A) and a dead dsgammacCOPI-treated group (B) of E.salmonides at day 3 from the start of dsRNA feeding.

FIG. 5 shows the change in the expression level of gamma-COPI gene in E.physaloidis at

days

2 and 4 after feeding dsgamma-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 TAKAdsRNA synthesis kit (MEGAscript) from RA Biotechnology Ltd^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 gamma COPIPIIRNA

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 a gamma COPI gene fragment is obtained through screening, wherein the gamma COPI gene fragment is 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 gamma COPI was obtained from the transcriptome library of E.variegatus, and dsRNA primer P1 (Table 1) of gamma COPI gene was designed, and the green fluorescent protein Gene (GFP) was amplified from a plasmid containing GFP stored in the laboratory, and dsRNA primer P2 (Table 1) of GFP gene was designed. Homologous arms related to the enzyme cutting sites are added to a dsgamma COPI primer P1 and a dsGFP primer P2 respectively, a dsgamma COPI primer P3 related to the construction of an expression vector and a dsGFP primer P4 related to the construction of the expression vector are designed (Table 1). Based on the sequence of the gamma COPI gene, qPCR primer P5 for the gamma COPI gene and qPCR primer P6 for the reference gene GAPDH were designed (Table 1).

Table 1: dsRNA synthesis and qPCR primers

3. dsRNAs of gamma 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-solubilizing the dsRNA to obtain dsgamma COPI and dsGFP, and verifying the band of dsRNA by 1.5% agarose gel electrophoresis.

The gamma COPIPIL dsRNA 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 a gamma COPIPIL dsRNA encoding gene is the 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 gamma 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 added to the dsgamma COPI primer P1 and the dsGFP primer P2, respectively, so as to design a dsgamma COPI expression vector-related primer P3 and a dsGFP expression vector-related primer P4 (Table 1). The cDNA template, reaction system and amplification procedure of PCR amplification are shown above, and the target fragments of dsgamma 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 SoSoSoSoSoo Cloning Kit Ver.2 Kit separately reacts dsGFP and dsgamma COPI with linearized L4440 vector at 50 ℃ for 20min for recombination. Subsequently, the recombinant expression vector containing dsgamma 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), cultured at 37 ℃ and 210rpm for 12 hours, 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), cultured at 37 ℃ and 210rpm for 3 hours to bring the OD of the cell broth to 0.5-0.8, 1mM of IPTG was added, and cultured at 37 ℃ and 120rpmRaising for 5h, and inducing dsRNA. Both of the bacterial solutions containing dsGFP and dsgamma COPI were subjected to mycelia collection at 4 ℃ and 5000rpm, RNA was extracted by TRIzol extraction (Invitrogen, USA), and 1.5% agarose gel electrophoresis was performed to verify the successful induction of dsRNA.

5. Results

Amplifying by using a P1 primer to obtain a PCR amplification product with the size of 431bp, sequencing and deleting a T7 promoter sequence to obtain a nucleotide sequence with the size of 391bp, namely the target gene gamma COPI, which is shown as SEQ ID NO. 2. 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 dsgamma 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 dsgamma 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

The harmonia axyridis dsgammaCOPI 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 leaf disks with the diameter of 12mm in dsgamma COPI solutions with the concentration of 10 ng/mu L, 5 ng/mu L, 3 ng/mu L and 2 ng/mu L for 1min, air-drying at room temperature for 1h, feeding larvae, replacing the leaf disks every 24h, continuously feeding the leaf disks soaked by the dsgamma COPI 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 a round eggplant leaf disc with the diameter of 12mm in a dsGFP solution with the concentration of 10ng/uL synthesized by the kit for 1min, air-drying for 1h, feeding larvae, replacing the leaf disc every 24h, continuously feeding the leaf disc soaked with 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 dsgammaCOPI expressed by bacterial liquid to lethal effect of harmonia axyridis

The harmonia axyridis dsgammaCOPI feeding group: in a culture dish containing filter paper and a humidifying cotton ball, 10 1-instar larvae, 10 3-instar larvae and 5 adults are arranged, 3 groups of experiments are arranged in total, and each group is provided with 5 replicates. Soaking a round eggplant leaf disc with the diameter of 12mm for 1min by using a dsgammacOPI-expressing bacterial liquid, and feeding larvae after air-drying 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. Changing the leaf disc every 24h, continuously feeding the leaf disc soaked by the dsgammaCOPI bacterial liquid for two days, and feeding the leaf disc by using 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 of the harmonia axyridis for dsgamma COPI for two days, the survival rate of the 1 st larva of the harmonia axyridis shows a trend of decreasing along with the increase of time, the feeding concentrations of the treatment groups are respectively 2 ng/mu L, 3 ng/mu L, 5 ng/mu L and 10 ng/mu L, and the feeding concentration of the control group is 10 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 2-71.900, df-4, P < 0.0001). There were significant differences between treatment groups of 2ng/μ L (P <0.0001, exp (b) ═ 5.812) and 5ng/μ L (P <0.0001, exp (b) ═ 11.603) and 10ng/μ L (P <0.0001, exp (b) ═ 13.604); there was no significant difference between 2ng/μ L (P <0.0001, exp (b) ═ 5.812) and 3ng/μ L (P <0.0001, exp (b) ═ 8.506); there were no significant differences between 3ng/μ L (P <0.0001, exp (b) ═ 8.506), 5ng/μ L (P <0.0001, exp (b) ═ 11.603), and 10ng/μ L (P <0.0001, exp (b) ═ 13.604). From the statistical results, it was concluded that the mortality increased 5.812-fold, 8.506-fold, 11.603-fold, 13.604-fold, respectively, when the concentrations of the treatment groups were 2 ng/. mu.L, 3 ng/. mu.L, 5 ng/. mu.L, and 10 ng/. mu.L, respectively, compared to the control group.

According to the statistical results (as shown in fig. 3), after continuously feeding dsgammacopii expressed by the strain liquid of the ladybug for two days, the survival rates of 1-instar larvae (P <0.0001, exp (b) ═ 26.275), 3-instar larvae (P <0.0001, exp (b) ═ 24.186) and adults (P ═ 0.002, exp (b) ═ 7.427) were significantly different from those of the control group, and the mortality rates of the 1-instar larvae, the 3-instar larvae and the adults of the treatment group were increased by 26.275 times, 24.186 times and 7.427 times, respectively, compared with those of the control group.

In addition, changes in phenotypic characteristics of E.varivestis were observed microscopically after two days of feeding dsgamma COPI. It was found that day 3 from the feeding of dsgamma COPI, the ladybug in the dsGFP control group normally entered the 2-instar stage, and the larvae in the treatment group failed to normally molt into the 2-instar stage and died, with phenotypic characteristics showing that branches and spots of the anterior dorsal lamella of the larvae failed to form as shown in FIG. 4, indicating that feeding of dsgamma COPI could induce a strong RNAi effect in vivo in the ladybug, resulting in the death of the ladybug.

Example 3 inhibition of expression of the gamma COPI Gene in L.variegates by dsgamma COPI

1. Experimental methods

Day 2 and day 2 after the start of dsRNA feeding,

respectivelyOn day

4, 1 st larvae of E.varivestis treated with 5 ng/. mu.L of dsgammacopiI and dsGFP were collected, and 3 biological replicates were collected per treatment. And extracting and collecting RNA of the harmonia axyridis, then carrying out reverse transcription on the RNA into cDNA, and diluting the cDNA 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 C1000Real-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 the gamma-COPI gene in the ladybug at

day

2 and 4 after feeding dsgamma-COPI were counted, respectively, using dsGFP feeding as a control (as shown in FIG. 5). As can be seen from FIG. 5, the expression level of the gene gamma COPI in E.varivestis fed with dsgamma COPI showed a significantly decreased tendency as compared with the expression level of the gene gamma COPI in E.varivestis fed with dsGFP. Further shows that the feed of dsgammaCOPI can cause strong RNAi effect in the body of the harmonia axyridis, which leads to the obvious reduction of the expression level of the gammaCOPI gene in the body and further leads to the death or the development of the harmonia axyridis to be inhibited. No significant difference was observed in the amount of gamma COPI expressed on day 2 from the time of feeding dsgamma COPI (F)_1,48.089, P0.047); on day 4 from the feeding of dsgammacopiI, the expression level of gammacopiI gene was decreased by 9.87 times as compared with the control group (F)_1,4＝3926.778,P<0.0001), further indicates that the feed dsgamma COPI can cause strong RNAi effect in the body of the harmonia axyridis, which leads to the obvious reduction of the expression level of the gamma COPI gene in the body and further leads to the inhibition or death of the development of the harmonia axyridis.

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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<130>

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<400> 10

cgaattcctg cagcccgggt tcacgttgat gccgttcttc 40

<210> 11

<211> 20

<212> DNA

<213> P5-F

<400> 11

ggattgccag acacagatga 20

<210> 12

<211> 20

<212> DNA

<213> P5-R

<400> 12

ggcttcttcc catgcagtat 20

<210> 13

<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 Physalis ladybird gammaCOPI gene, is characterized in that, its sequence is as shown in SEQ ID NO.1.

2. a kind of dsRNA that can be used for preventing and treating Physalis ladybird, it is characterized in that, described dsRNA silencing target gene is the described gammaCOPI gene of claim 1.

3. the application of dsRNA described in claim 2 in preventing and controlling Physalis Physalis or preparing the product of controlling Physalis Physalis.

4. the application of dsRNA described in claim 2 in suppressing the growth of Physalis Physalis or preparing the product that suppresses the growth of Physalis Physalis.

5. the application of the described dsRNA of claim 2 in promoting the death of Physalis Physalis or preparing the product that promotes the death of Physalis Physalis.

6. A test kit for preventing and treating Physalis ladybird, characterized in that, containing the described dsRNA of claim 2.

7. A method for preventing and treating Physalis ladybird, characterized in that, by feeding exogenous dsRNA, the dsRNA can silence/inhibit the expression of the gammaCOPI gene described in claim 1.