CN110628772A - Gene gammaCOPI and Its Application in Controlling Physalis Ladybug - Google Patents

Abstract

The invention discloses a gene gamma COPI and application thereof in preventing and treating harmonia axyridis. The invention researches and obtains a gene gamma COPI from the harmonia axyridis, and the silencing of the gene expression can efficiently prevent and control the harmonia axyridis, so that a target gene dsRNA with high lethal ability to the harmonia axyridis is designed, namely the dsgamma COPI has high lethal effect on the harmonia axyridis, has the advantages of good sensitivity, high insecticidal efficiency, good prevention and control effect, convenient use and operation, environmental friendliness and the like, and has good application prospect on the prevention and control of the harmonia axyridis.

Description

Gene gammaCOPI and Its Application in Controlling Physalis Ladybug

technical field

The invention belongs to the technical field of pest control. More specifically, it relates to a target gene for controlling Physalis ladybug and its application.

Background technique

Physalis lady beetle Henosepilachnavigintioctopunctata (Fabricius) belongs to Coleoptera and is an important agricultural pest with a wide range of host plants. It mainly damages eggplant, potato and tomato and other solanaceous vegetables. Both the larvae and adults feed on the leaves. They like to cluster on the back of the leaves and feed on the lower epidermis and mesophyll. The damaged leaves usually form irregular transparent spots or perforations. In severe cases, the plants will wilt or even die. Physalis ladybug is widely distributed in my country, especially in the south of the Yangtze River. In recent years, due to climate warming, trade development, and the expansion of vegetable cultivation area in protected areas, its food is available all year round, and the occurrence and damage of Physalis ladybug have become more and more serious. In 2015, my country launched the potato staple food strategy, which will further expand the potato planting area in my country, and the control of Physalis ladybug is urgent.

At present, the control of Physalis ladybug includes artificial capture, trapping with attractants, and chemical pesticides. Among them, artificial capture is not only ineffective, but also has a very heavy labor problem; and the effect of attractant trapping is not satisfactory and incomplete; therefore, it still relies more on chemical pesticides, but chemical pesticides will cause environmental pollution and the quality and safety of agricultural products. and many other issues.

RNA interference (RNA interference, RNAi) is an evolutionarily conserved mechanism that relies on the production of short fragments of RNAs (siRNAs), thereby promoting the degradation of homologous mRNAs or inhibiting their translation. RNAi provides an important tool for functional genomics research in insects and lays the foundation for the development of environmentally friendly pest management methods. Since the use of RNAi technology can specifically inhibit the expression of genes, this technology has been widely used in targeted interference with pest genes to achieve the purpose of pest control. Target gene reporter for insect activity.

The previous research of the inventor's team showed (201710949193.9) that the toxicity to ladybugs can be achieved by directly feeding suitable exogenous dsRNA, so exogenous dsRNA products suitable for preventing and treating ladybug Physalis were developed from the genetic level. The method is convenient, low in cost, precise and excellent control effect can be realized due to the specificity of the gene, it is friendly to the environment, and has a great application prospect in the control of ladybug Physalis. However, the screening of relevant target genes and the design of dsRNA with good control effect, specificity and stability are the biggest difficulties and key issues.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing Physalis ladybug control technology, and provide a highly lethal gene of Physalis ladybug, that is, the gammaCOPI gene. And based on this gene, a technology that can effectively control ladybug Physalis has been developed, that is, direct feeding the target gene dsRNA that has a high lethality to Ladybug Physalis, and using the lethal effect of dsgammaCOPI on Ladybug Physalis to achieve the purpose of prevention and control. The method has many advantages such as convenient operation, good effectiveness and sensitivity, high insecticidal efficiency, and environmental friendliness, and has a good application prospect.

The purpose of the present invention is to provide a physalis beetle gammaCOPI gene and its application in preventing and treating the physalis beetle.

Another object of the present invention is to provide a dsRNA of the gammaCOPI gene that can be used to prevent and treat ladybug Physalis and its application.

Another object of the present invention is to provide a method and a kit for preventing and treating ladybug Physalis.

The above object of the present invention is achieved through the following technical solutions:

The present invention is based on the transcriptome library of ladybug Physalis, obtains a highly lethal gene—gammaCOPI gene by screening, and develops the technology of feeding dsgammaCOPI gene gammaCOPI to control Ladybug Physalis. In the present invention, eggplant leaves are respectively soaked in the solutions of dsgammaCOPI and dsGFP synthesized by the kit, taken out and dried, and then fed to the first instar larvae of Physalis ladybug for 2 days, and then fed with eggplant leaves not treated with dsRNA, and observed and recorded Mortality and developmental status of ladybug Physalis; In addition, the method of expressing dsgammaCOPI in bacterial solution was used to measure its lethal ability to 1st instar, 3rd instar and adults of Physalis ladybug, so as to comprehensively evaluate the effect of exogenous dsgammaCOPI on Physalis ladybug. Insecticidal activity of insects. Finally, the quantitative PCR (qPCR) method was used to detect and analyze the expression level changes of the gammaCOPI gene in ladybugs that fed on dsgammaCOPI and dsGFP Physalis. The results showed that direct feeding of exogenous dsgammaCOPI could significantly inhibit the expression of gammaCOPI gene in ladybug Physalis, and direct feeding of exogenous dsgammaCOPI had obvious lethal effect on ladybug Physalis. Therefore, the following subjects and applications should all fall within the protection scope of the present invention:

A Physalis ladybug gammaCOPI gene, the sequence of which is shown in SEQ ID NO.1.

The application of the gammaCOPI gene in preventing and treating ladybug Physalis or preparing products for preventing and treating ladybug Physalis.

The application of the gammaCOPI gene in inhibiting the growth of the ladybug Physalis or preparing a product for inhibiting the growth of the ladybug Physalis.

The application of the gammaCOPI gene in promoting the death of the ladybug Physalis or preparing a product for promoting the death of the ladybug Physalis.

Application of the inhibitor of the gammaCOPI gene in preventing and treating ladybug Physalis or preparing products for preventing and treating ladybug Physalis.

A dsRNA that can be used to control the Physalis ladybug, the dsRNA can target and silence the gammaCOPI gene. Preferably, the dsRNA sequence is shown in SEQ ID NO.2.

A kit for preventing and controlling ladybug Physalis, containing gammaCOPI gene inhibitor. Preferably, the inhibitor is the aforementioned dsRNA.

Specifically, one of the ways to use the gammaCOPI gene to control Physalis ladybug, that is, a method of controlling Physalis ladybug, directly feeds exogenous dsRNA, so that dsgammaCOPI enters Physalis ladybug, and the dsRNA can silence/inhibit acid The expression of the gammaCOPI gene of the physalis suppresses the growth of the physalis and promotes the death of the physalis, thereby achieving the purpose of preventing and controlling the physalis.

The present invention has the following beneficial effects:

The present invention screens and obtains a highly lethal gene gammaCOPI gene of ladybug Physalis, and develops its efficient silencing dsRNA, and develops a technology capable of efficiently preventing and treating Ladybug Physalis, that is, direct feeding has high lethality to Ladybug Physalis The target gene dsRNA is used to control the lethal effect of dsgammaCOPI on ladybug Physalis. The method has many advantages such as convenient operation, good effectiveness and sensitivity, high insecticidal efficiency, and environmental friendliness, and has a good application prospect.

Description of drawings

Figure 1 is the electrophoresis diagram of dsGFP and dsgammaCOPI expressed in bacterial fluid.

Figure 2 is the effect of different concentrations of dsgammaCOPI on the mortality of ladybug larvae. Using data on larval mortality 10 days after the start of the experiment, survival curves were constructed using a Cox regression procedure. Different letters (such as a, b) indicate significant differences between the control curve and the treatment curve.

Fig. 3 is the effect of dsgammaCOPI expressed in the bacterial liquid on the survival rate of ladybug Physalis (Fig. A: the survival rate of 1st instar larvae; Fig. B: the survival rate of 3rd instar larvae; Fig. C: the survival rate of adults). Using the mortality data of 1st instar larvae, 3rd instar larvae, and adults at 10 days, 10 days, and 14 days respectively, survival curves were constructed using a Cox regression program. Different letters (such as a, b) indicate significant differences between the control curve and the treatment curve.

Figure 4 shows the phenotype difference between the normally developed dsGFP control group (A) and the dead dsgammaCOPI treatment group (B) Physalis ladybug on the 3rd day after feeding dsRNA.

Fig. 5 shows the changes of gammaCOPI gene expression in ladybug Physalis on day 2 and day 4 after feeding on dsgammaCOPI and dsGFP.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

Physalis ladybugs used in the following examples were raised in the Department of Entomology, South China Agricultural University. The eggplants used to raise the Physalis ladybugs are Marshal Wansheng round eggplant seedlings. Put the ladybugs in a petri dish containing filter paper, keep the filter paper moist with cotton balls, and put them in an artificial climate box (temperature 25±1°C, humidity 70%- 80%, photoperiod L:D=14:10) for reproduction.

RNA was extracted using TRIzol extraction method (Invitrogen, USA), reverse transcription reagent (PrimeScript ^TM RTreagent Kit with gDNA Eraser) was purchased from TAKARA Biotechnology Co., Ltd., dsRNA synthesis kit (MEGAscript ^TM T7) was purchased from Thermo Fisher Scientific, PCR reaction The kit (EX Taq ^TM ) used in the system was purchased from TAKARA Biotechnology Co., Ltd., and the DNA purification and recovery kit (Universal DNA Purification Kit) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

The data processing method of following embodiment: For the result analysis that two types of dsRNA carry out bioassay to Ladybug Physalis, use Excel 2010 to carry out statistics to the survival rate of Ladybug Physalis, utilize SPSS 19.0 software to adopt Cox regression analysis to draw a graph , The difference between different concentrations was analyzed by single factor analysis. For the analysis of changes in the expression of target genes after RNA interference, the qPCR data was calculated using the 2 ^-△△Ct method (Ct represents the number of cycles). Data analysis was performed using SPSS 19.0 software using one-way analysis of variance.

Example 1 Obtaining of growth and development-related gene gammaCOPIdsRNA

We constructed a transcriptome library based on the genome of ladybug Physalis, and then based on the constructed transcriptome library, researched and screened genes related to the growth and development of Ladybug Physalis, and screened to obtain the gammaCOPI gene fragment, as shown in SEQ ID NO.1. The dsRNA is then synthesized.

1. Physalis ladybug total RNA extraction and first-strand cDNA synthesis.

Get 10 2nd instar larvae of Ladybug Physalis in a 2ml centrifuge tube, use the TRIzol method to extract the total RNA of Ladybug Physalis, use NanoDropOne ^C to measure the concentration and quality of RNA, and use a reverse transcription kit (PrimeScript ^TM RTreagent Kit with gDNA Eraser, TAKARA) was reverse-transcribed according to the instructions, and the first strand of cDNA was synthesized.

2. Primer design

The gene sequence of gammaCOPI was obtained from the transcriptome library of ladybug Physalis, and the dsRNA primer P1 (Table 1) of the gammaCOPI gene was designed. The green fluorescent protein gene (GFP) was amplified from a plasmid containing GFP preserved in the laboratory. GFP Gene dsRNA primer P2 (Table 1). Homology arms related to restriction sites were added to primer P1 of dsgammaCOPI and primer P2 of dsGFP respectively, and primer P3 related to the construction of expression vector of dsgammaCOPI and primer P4 related to construction of expression vector of dsGFP were designed (Table 1). According to the sequence of the gammaCOPI gene, the qPCR primer P5 of the gammaCOPI gene and the qPCR primer P6 of the internal reference gene GAPDH were designed (Table 1).

Table 1: dsRNA synthesis and qPCR primers

3. The kit synthesizes dsRNAs of gammaCOPI gene and GFP gene

Use the primers P1 and P2 in Table 1 for PCR amplification. The reaction system for PCR amplification is 5 μL of 10x EX Taq Buffer, 0.25 μL of TaKaRa EX Taq, 4 μL of dNTP Mixture, 1 μL of upstream primer (10 μmoL/L), 1 μL of downstream primer (10 μmoL /L) 1 μL, cDNA/GFP plasmid 1 μL, filled to 50 μL with dd H ₂ O. The reaction program of PCR amplification was pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, a total of 30 cycles; extension at 72°C for 5 min. Store the amplified product at 4°C. After the programmed reaction is completed, the amplification result is detected by agarose gel electrophoresis.

Use the DNA Purification Kit (Universal DNA Purification Kit, TIANGEN) to recover and purify the two PCR products obtained above, and use them as templates for in vitro transcription of dsRNA. The in vitro transcription system for dsRNA is 10x ReactionBuffer 5 μL, (ATP, GTP, CTP, UTP) Each solution was 5 μL, Enzyme mix 5 μL, template 20 μL, and ddH ₂ O was added to 50 μL. Place at 37°C for 4h. After the reaction, add 2.5 μL TURBO DNase to remove residual template DNA and single-stranded RNA, then purify the dsRNA, and finally dissolve the dsRNA with 50 μL ddH ₂ O to obtain dsgammaCOPI and dsGFP respectively, and use 1.5% agarose gel electrophoresis to verify the dsRNA Bands.

The gammaCOPIdsRNA of ladybug Physalis is a double-stranded RNA consisting of a sense strand and an antisense strand. The nucleotide sequence of its sense strand is SEQ ID NO.1 in the sequence table, and the nucleotide sequence of its antisense strand is sequence The reverse complementary sequence of SEQ ID NO.1 in the list, the nucleotide sequence of the gene encoding gammaCOPIdsRNA is SEQ ID NO.1 in the sequence list. GFP dsRNA is a double-stranded RNA consisting of a sense strand and an antisense strand. The nucleotide sequence of its sense strand is SEQ ID NO.2 in the sequence listing, and the nucleotide sequence of its antisense strand is SEQ ID NO.2 in the sequence listing. The reverse complementary sequence of NO.2.

4. Acquisition of growth and development-related gene gammaCOPI bacterial liquid expression dsRNA

Two restriction sites were selected on the sequence of L4440, namely BamHI (GGATCC) and SacI (GAGCTC). According to the sequence information of L4440 (this sequence information has been published), the dsgammaCOPI primer P1 and the dsGFP primer P2 respectively add homology arms related to the two restriction sites, and design the primer P3 related to the construction of the expression vector of dsgammaCOPI , Primer P4 (Table 1) related to construction of expression vector for dsGFP. The cDNA template, reaction system, and amplification procedure for PCR amplification were as described above, and the target fragments of dsgammaCOPI and dsGFP were obtained to construct the vector, and the two PCR products obtained above were recovered with a DNA purification and recovery kit (Universal DNA Purification Kit, TIANGEN). product. According to the sequences of the two enzyme cutting sites, the L4440 vector was linearized using QuickCut ^TM SacI and QuickCut ^TM BamHI. For the enzyme digestion reaction system, please refer to the instruction manual. ) to recover the linearized L4440 vector.

Using the Trelief ^TM SoSoo Cloning Kit Ver.2 kit from Guangzhou Qingke Biotechnology Co., Ltd., dsGFP and dsgammaCOPI were reacted with the linearized L4440 vector at 50°C for 20 minutes for recombination. Subsequently, the recombinant expression vector containing dsgammaCOPI and dsGFP was introduced into HT115 competent cells, placed on ice for 30 minutes, then heat-shocked at 37°C for 1 minute; stood on ice for 3 minutes, and then added 700 μL of LB liquid medium without ampicillin , cultured at 37°C and 210 rpm for 1 h, and then cultured overnight on LB plates containing ampicillin and tetracycline. Pick a single colony and place it in 4ml LB liquid medium containing ampicillin (100μg/mL) and tetracycline (10μg/mL), culture at 37°C, 210rpm for 12h, then transfer 50μL to 5mL containing ampicillin (100μg/mL) and tetracycline (10 μg/mL) LB liquid medium, 37 ° C, 210 rpm for 3 h, so that the OD value of the bacterial solution is between 0.5-0.8, add 1 mM IPTG, 37 ° C, 120 rpm for 5 h to induce dsRNA. The mycelia were collected from the two bacterial solutions containing dsGFP and dsgammaCOPI at 4°C and 5000 rpm, RNA was extracted by TRIzol extraction method (Invitrogen, USA), and electrophoresed on 1.5% agarose gel to verify whether dsRNA was successfully induced .

5. Results

Use the P1 primer to amplify to obtain a PCR amplification product with a size of 431bp. After sequencing and deleting the T7 promoter sequence, the nucleotide sequence obtained with a size of 391bp is the target gene gammaCOPI, as shown in SEQ ID NO.2 Show. Using the plasmid carrying GFP as a template and using P2 primers to amplify, a PCR product with a size of 507 bp was obtained. The target bands of dsGFP and dsgammaCOPI were consistent with the sequencing results.

Use bacterial liquid to express the dsRNA of the target gene, extract the RNA of the hyphae, and use agarose gel electrophoresis to verify whether the dsRNA of the target gene is successfully induced. According to the electrophoresis results (Figure 1), you can see the target items that successfully induced dsGFP and dsgammaCOPI bring.

Example 2 Inhibitory effect of dsRNA on ladybug Physalis

1. The application of the dsRNA synthesized by the kit in inhibiting the growth and development of ladybug Physalis

Physalis ladybug dsgamma COPI feeding group: put 10 1st instar larvae of Physalis ladybug in a petri dish with filter paper and humidified cotton balls. Soak circular eggplant leaf discs with a diameter of 12 mm in dsgamma COPI solutions with a concentration of 10 ng/μL, 5 ng/μL, 3 ng/μL, and 2 ng/μL for 1 min, air-dry them at room temperature for 1 h, and then feed the larvae. Replace the leaf discs every 24 h, continuously Two days after feeding dsgammaCOPI-soaked leaf discs, larvae were fed with normal eggplant leaves.

Physalis ladybug dsGFP feeding group: put 10 1st instar larvae of Physalis ladybug in a petri dish with filter paper and humidified cotton balls. Soak circular eggplant leaf disks with a diameter of 12 mm in the dsGFP solution synthesized by the kit for 1 min, air-dry them for 1 hour, and then feed the larvae, replace the leaf disks every 24 hours, and feed the dsGFP-soaked leaf disks continuously for two days. Untreated eggplant leaves feed the larvae.

Every group is set 5 repetitions, counts the death number of ladybug physalis in each petri dish every 24h, and replaces new blade, and petri dish is placed in artificial climate chamber (temperature 25 ± 1 ℃, humidity 70%-80%) , photoperiod L:D=14:10). The number of dead physalis beetles in each petri dish of each group was counted, and the survival rate changes of the control group and dsRNA treatments with different concentrations were calculated.

2. The application of dsgammaCOPI expressed in bacterial liquid to the lethal effect of Physalis ladybug

Physalis ladybug dsgamma COPI feeding group: 10 1st instar larvae, 10 3rd instar larvae, and 5 adults were placed in a petri dish with filter paper and humidified cotton balls, and a total of 3 groups of experiments were set up, with 5 repetitions in each group. Soak a round eggplant leaf disc with a diameter of 12 mm in the bacterial solution expressing dsgammaCOPI for 1 min, air-dry at room temperature for 1 h, and then feed the larvae. In the treatment group, 2 leaf discs were placed in each petri dish for the 1st instar larvae; 5 leaf discs were placed in each petri dish for the 3rd instar larvae; 5 leaf discs were placed in each petri dish for adults. The leaf disks were replaced every 24 hours, and the leaf disks soaked in the dsgammaCOPI bacterial solution were continuously fed for two days, and then fed with normal eggplant leaves.

Physalis ladybug dsGFP feeding group: Put 10 1st instar larvae, 10 3rd instar larvae, and 5 adults in a petri dish with filter paper and humidified cotton balls, set 3 groups of controls in total, and set 5 replicates in each group . Soak round eggplant leaves with a diameter of 12 mm in the bacterial solution expressing dsGFP for 1 min, air-dry them at room temperature for 1 h, and then feed them to larvae. In the control group, 2 leaf discs were placed in each petri dish for 1st instar larvae; 5 leaf discs were placed in each petri dish for 3rd instar larvae; 5 leaf discs were placed in each petri dish for adults. The leaf disks were replaced every 24 hours, and the leaf disks soaked in the dsGFP bacterial solution were continuously fed for two days, and then fed with normal eggplant leaves.

Count the dead number of ladybug physalis in each petri dish every 24h, and replace new blade, petri dish is placed in artificial climate box (temperature 25 ± 1 ℃, humidity 70%-80%, photoperiod L: D= 14:10). Count the number of dead ladybugs in each petri dish in each group, and calculate the changes in the survival rate of ladybugs in the control and different treatment groups.

3. According to the statistical results, after two days of continuous feeding of the 1st instar larvae of Physalis ladybug dsgammaCOPI, the survival rate of the 1st instar larvae of Physalis ladybug showed a downward trend with the increase of time, and the feeding concentrations of the treatment groups were respectively It is 2ng/μL, 3ng/μL, 5ng/μL and 10ng/μL, and the feeding concentration of the control group is 10ng/μL. According to the results in Figure 2, it was found that there were significant differences between the treatment groups of different concentrations and between the control groups (χ2=71.900, df=4, P<0.0001). Treatment group 2ng/μL (P<0.0001, Exp(B)＝5.812) and 5ng/μL (P<0.0001, Exp(B)＝11.603) and 10ng/μL (P<0.0001, Exp(B)＝13.604) There were significant differences between them; there was no significant difference between 2ng/μL (P<0.0001, Exp(B)=5.812) and 3ng/μL (P<0.0001, Exp(B)=8.506); 3ng/μL (P<0.0001, Exp(B)＝8.506), 5ng/μL (P<0.0001, Exp(B)＝11.603), 10ng/μL (P<0.0001, Exp(B)＝13.604) difference. From the statistical results, the following conclusions can be drawn, when the concentration of the treatment group was 2ng/μL, 3ng/μL, 5ng/μL and 10ng/μL, compared with the control group, the mortality rate increased by 5.812 times, 8.506 times, 11.603 times, 13.604 times.

According to the statistical results (as shown in Figure 3), after two days of continuous feeding of dsgammaCOPI expressed by the Physalis ladybug bacterium liquid, the 1st instar larvae (P<0.0001, Exp(B)=26.275), the 3rd instar larvae (P<0.0001, Exp (B) = 24.186) and adult (P = 0.002, Exp (B) = 7.427) survival rate compared with the control group there is a significant difference, the 1st instar larvae of the treatment group, the 3rd instar larvae and adults and the control group Compared with the group, the mortality rate increased by 26.275 times, 24.186 times and 7.427 times respectively.

In addition, the changes in the phenotypic characteristics of ladybug Physalis after feeding dsgammaCOPI for two days were observed with a microscope. It was found that on the third day after feeding dsgammaCOPI, the Physalis ladybugs in the dsGFP control group normally entered the second instar stage, while the larvae in the treatment group could not molt normally and entered the second instar stage and died. The thorns and spots could not be formed, as shown in Figure 4, indicating that feeding on dsgammaCOPI can trigger a strong RNAi effect in the ladybug Physalis, resulting in the death of the ladybug.

Example 3 dsgammaCOPI inhibits the expression of gammaCOPI gene in ladybug Physalis

1. Experimental method

The 1st instar larvae of Physalis physalis treated with 5 ng/μL dsgammaCOPI and dsGFP were collected on the 2nd day and 4th day after starting to feed on dsRNA, respectively, and 3 biological replicates were collected for each treatment. Extract and collect the RNA of ladybug Physalis, then reverse transcribe it into cDNA, and dilute it 10 times as a template for qPCR. qPCR analysis was performed with P5 and P6 as primers. The qPCR system was (15 μL) containing 5.25 μL of ddH ₂ O, 7.5 μL of 2×SYBR Green MasterMix (BIO-RAD Inc, Hercules, CA), 4 μM primers and 1.0 μL of cDNA first-strand template. The reaction instrument for qPCR was Bio-Rad C1000 Real-Time PCR system (BIO-RAD, USA). The reaction conditions were 95°C for 5 min; 95°C for 10 s, 60°C for 30 s, 39 cycles, 3 technical replicates for each sample, and the reaction was carried out in a 96-well plate (BIO-RAD, USA).

2. Experimental results

Taking dsGFP as a control, the changes in the relative expression of gammaCOPI gene in ladybug Physalis on day 2 and day 4 after feeding dsgammaCOPI were counted (as shown in FIG. 5 ). It can be seen from Figure 5 that the expression of gene gammaCOPI in ladybugs fed with dsgammaCOPI showed a clear downward trend compared with the expression of gene gammaCOPI in ladybugs fed with dsGFP. It further shows that feeding dsgammaCOPI can cause a strong RNAi effect in the Physalis ladybug, resulting in a significant decrease in the expression of the gammaCOPI gene in the body, which in turn leads to the death or development of Physalis ladybug. From the 2nd day of feeding dsgammaCOPI, the expression of gammaCOPI had no significant difference compared with the control group (F _1,4 =8.089, P=0.047); from the 4th day of feeding dsgammaCOPI, the expression of gammaCOPI gene Compared with the control group, the amount decreased by 9.87 times (F _1,4 ＝3926.778, P<0.0001), further indicating that feeding dsgammaCOPI can cause a strong RNAi effect in the body of ladybug Physalis, resulting in a significant expression of gammaCOPI gene in vivo. Reduced, which in turn leads to the development inhibition or death of Physalis ladybug.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> South China Agricultural University

<120> Gene gammaCOPI and its application in controlling ladybug Physalis

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Claims (10)

1. A Physalis ladybug gammaCOPI gene, characterized in that its sequence is as shown in SEQ ID NO.1. 2. A dsRNA that can be used to prevent and treat ladybug Physalis, characterized in that, the dsRNA silencing target gene is the gammaCOPI gene described in claim 1. 3. The application of the gammaCOPI gene according to claim 1 or the dsRNA according to claim 2 in preventing and treating ladybug Physalis or preparing products for preventing and treating ladybug Physalis. 4. The application of the gammaCOPI gene of claim 1 or the dsRNA of claim 2 in inhibiting the growth of ladybug Physalis or preparing products for inhibiting the growth of ladybug Physalis. 5. The application of the gammaCOPI gene according to claim 1 or the dsRNA according to claim 2 in promoting the death of the ladybug Physalis or preparing products for promoting the death of the ladybug Physalis. 6. The application of the expression inhibitor of the gammaCOPI gene described in claim 1 in preventing and treating ladybug Physalis or preparing products for preventing and treating ladybug Physalis. 7. The application according to claim 6, wherein the expression inhibitor of the gammaCOPI gene is the dsRNA according to claim 2. 8. A test kit for preventing and treating ladybug Physalis, characterized in that it contains the expression inhibitor of gammaCOPI gene described in claim 1. 9 . The kit according to claim 8 , wherein the expression inhibitor is the dsRNA according to claim 2 . 10. A method for preventing and treating ladybug Physalis, characterized in that, exogenous dsRNA is fed, and the dsRNA can silence/inhibit the expression of the gammaCOPI gene described in claim 1.