CN110592117B

CN110592117B - Highly lethal gene vATPase B and its application against ladybugs

Info

Publication number: CN110592117B
Application number: CN201910718244.6A
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: CN110592117A

Abstract

The invention discloses a highly lethal gene vATPase B and its application in preventing ladybugs. In the present invention, a gene vATPase B is obtained from Coccinella solani, and silencing the expression of the gene can effectively prevent and control Coccinella solani, so a target with high lethal ability to Coccinell solani is designed. Gene dsRNA, namely dsvATPase B, has a high lethal effect on Coccinella 28 stars, good sensitivity, high insecticidal efficiency, good control effect, and is easy to use and operate, and also has many advantages such as environmental friendliness. The control of the eight-star ladybug has a good application prospect.

Description

High-lethal gene vATPase B and ladybug-proof application thereof

Technical Field

The invention belongs to the technical field of insect pest prevention and control. More particularly, relates to a high lethal gene vATPase B and an application thereof in preventing ladybug.

Background

The coccinella solanacearum (Fabricius) belongs to the family of Coleoptera axyridoides, 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 harmonia axyridis has wide distribution range in China, and particularly has higher occurrence density in the south of the Yangtze river. In recent years, due to the warming of climate, the development of trade and the enlargement of vegetable cultivation area in protected areas, the food materials are continuously fed all the year round, and the occurrence and harm of the ladybug with twenty-eight star are increasingly serious. In 2015, the strategy of potato staple food production is started in China, the planting area of potatoes in China must be further enlarged, and the prevention and control of the harmonia axyridis is slow.

At present, the control of the ladybug with the eggplant and the dioctadecylate comprises artificial capture, attractant trapping and killing 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 at present, the research on the gene function of the ladybug twenty-eight star does not exist at home and abroad, and the target gene report of insecticidal activity does not exist.

The earlier-stage research of the inventor team shows (201710949193.9) that 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 ladybug with twenty-eight stars is developed from a gene level, is convenient to use and low in cost, can realize accurate and excellent prevention and treatment effects due to the specificity of the gene, is environment-friendly, and has a great application prospect in the prevention and treatment aspect of the ladybug with twenty-eight stars. 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 prevention and control technology of the ladybug twenty-eight star and provides a high lethal gene of the ladybug twenty-eight star, namely vATPase B gene. And a technology capable of efficiently preventing and treating the axyridis solaris is developed based on the gene, namely, the target gene dsRNA with high lethal capacity to the axyridis solaris is directly fed, and the lethal effect of dsvATPase B on the axyridis solaris 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.

The invention aims to provide a ladybug VATPase B gene and application thereof in preventing and treating ladybug dioctadecylate.

The invention also aims to provide dsRNA of vATPase B gene for preventing and treating ladybug twenty-eight and application thereof.

The invention further aims to provide a method and a kit for preventing and treating the ladybug with dioctadecylate.

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

the invention screens and obtains a high lethal gene, namely vATPase B gene based on a transcriptome library of the harmonia axyridis, and develops a technology for preventing and treating the harmonia axyridis by feeding dsRNA (dsvATPase B) of the vATPase B gene. The invention soaks the eggplant leaves in dsvATPase B and dsGFP solution that the kit synthesizes respectively, take out and air-dry, feed 1-year-old larva of the ladybug with twenty-eight stars for 2 days, then feed with the eggplant leaves that are not treated with dsRNA, observe and record the death rate and development state of the ladybug with twenty-eight stars; in addition, the lethality of the dsvATPase B to 1-year, 3-year and adult ladybug of twenty eight star ladybug is determined by a method for expressing the dsvATPase B by using bacterial liquid, so that the insecticidal activity of the exogenous dsvATPase B to dioctadecylladybug of eggplant is comprehensively evaluated. Finally, the method of fluorescent quantitative PCR (qPCR) is used for detecting and analyzing the expression quantity change of vATPase B gene in dsvATPase B and dsGFP ladybug eggplant. The result shows that the direct feeding of exogenous dsvATPase B can obviously inhibit the gene expression of the ladybug VATPase B, and the direct feeding of exogenous dsvATPase B has high lethal effect on the ladybug dioctadecylate. Therefore, the following subject matters and applications should be considered to be within the protection scope of the present invention:

a gene VATPase B of ladybug Eicosarum and the sequence is shown in SEQ ID NO. 1.

The vATPase B gene is applied to the prevention and treatment of the harmonia axyridis or the preparation of products for preventing and treating the harmonia axyridis.

The application of the vATPase B gene in inhibiting the growth of the axyridis solanacearum or preparing products for inhibiting the growth of the axyridis solanacearum.

The application of the vATPase B gene in promoting death of the harmonia axyridis or preparing products for promoting death of the harmonia axyridis.

The application of the vATPase B gene inhibitor in preventing and treating the ladybug with twenty eight stars or in preparing products for preventing and treating the ladybug with twenty eight stars.

The dsRNA can be used for preventing and controlling the ladybug Eicosarum solani, and the dsRNA silences a target gene as the vATPase B gene. Preferably, the dsRNA sequence is shown as SEQ ID NO. 1.

A kit for preventing and treating ladybug Eicosarum comprises vATPase B gene inhibitor. Preferably, the inhibitor is the above-described dsRNA.

Specifically, one way of preventing and controlling the axyridis solanacearum by using the vATPase B gene is a method for preventing and controlling the axyridis solanacearum, exogenous dsRNA is directly fed, so that the dsvATPase B enters the body of the axyriasis solanacearum, the dsRNA can silence/inhibit the vATPase B gene expression of the axyriasis solanacearum, inhibit the growth of the axyriasis solanacearum and promote the death of the axyriasis solanacearum, and the purpose of preventing and controlling the axyriasis solanacearum is achieved.

The invention has the following beneficial effects:

the invention obtains a high lethal gene vATPase B gene of the ladybug, develops the high-efficiency silencing dsRNA of the gene, develops a technology capable of efficiently preventing and treating the ladybug, namely directly feeding a target gene dsRNA with high lethal capability to the ladybug, and achieves the prevention and treatment purpose by utilizing the lethal effect of dsvATPase B 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 diagram of the electrophoresis of dsGFP and dsvATPase B synthesized by the kit.

FIG. 2 is an electrophoretogram of dsGFP and dsvATPase B expressed from the bacterial suspension.

FIG. 3 is a graph showing the change in survival of E.solani following feeding dsGFP and dsvATPase B; 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. 4 is a graph showing the phenotypic changes of dsGFP control group (A) and dsvATPase B-treated group (B) at day 6 from dsRNA feeding.

FIG. 5 shows the effect of silencing vATPase B gene expression on the growth and development of E.solariciressa; the development of ladybug larvae was significantly inhibited in the treated group compared to the control group after feeding dsvATPase B.

FIG. 6 is a graph showing the effect of dsvATPase B expressed in the inoculum solution on the survival rate of E.solasoni (panel A: survival rate of 1 st larva; panel B: survival rate of 3 rd larva; panel C: survival rate of adult). Survival curves were established using Cox regression procedures using 12 day larval mortality data. Different letters (e.g., a, b) indicate significant differences between the control and treatment curves.

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 E.solani used in the examples below was bred at the department of insects of southern agricultural university of south China. The eggplants for breeding the ladybug twenty-eight-star are Tengsheng Maruashuai round eggplant seedlings, the ladybug is placed in a culture dish containing filter paper, the filter paper is moisturized by a cotton ball, and the culture dish is placed in an artificial climatic chamber (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.

EXAMPLE 1 acquisition of dsRNA synthesized by Gene vATPase B kit

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

1. Extracting the total RNA of the ladybug with twenty eight stars and synthesizing the first strand cDNA.

Placing 10 2-instar larvae of E.solaricius in 2ml centrifuge tube, extracting total RNA of E.solaricius by TRIzol method, and reverse transcription with 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 vATPase B was obtained from the transcriptome library of E.solariciressa, and dsRNA primer P1 (Table 1) of vATPase B gene was designed and synthesized by Hipponica Biotech, Guangzhou. The green fluorescent protein Gene (GFP) was amplified from a plasmid containing GFP stored in the laboratory, and the dsRNA primer P2 for the GFP gene (Table 1).

Table 1: dsRNA synthesis and qPCR primers

The reaction system for PCR amplification is 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) 1. mu.L, downstream primer (10. mu. moL. L-1) 1. mu. L, cDNA/GFP plasmid 1. mu.L, dd H2O is supplemented 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.

The synthesized cDNA is used as a template, a P1 primer is utilized to carry out PCR amplification reaction to obtain a PCR amplification product with the size of 473bp, and after sequencing and deleting a T7 promoter sequence, a nucleotide sequence with the size of 433bp is obtained and is the target gene vATPase B, as shown in SEQ ID NO. 2.

The plasmid carrying GFP is used as a template, and P2 primer is used for amplification to obtain a PCR product with the size of 507 bp.

3. dsRNAs preparation of vATPase B Gene and GFP Gene

Recovering and purifying the two PCR products obtained in step 2 by using a DNA Purification recovery Kit (TIANGEN) as templates for in vitro transcription of dsRNA, wherein the in vitro transcription system of dsRNA is 10 × Reaction Buffer 5 μ L, (ATP, GTP, CTP, UTP) solution is 5 μ L each, Enzyme mix 5 μ L, template 20 μ L, 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, yielding dsvATPase B and dsGFP, respectively.

The vATPaseBdsRNA sequencing results of the harmonia solani are as follows:

the vATPaseBdsRNA of the ladybug solani 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, and the nucleotide sequence of the antisense strand is a reverse complementary sequence of 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.

Example 2 obtaining of dsRNA expressed by vATPase B bacterial liquid

1. Construction of dsvATPase B and L4440 expression vectors

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 primer P1 of dsvATPase B and a primer P2 of dsGFP, a primer P3 related to dsvATPase B construction expression vectors and a primer P4 related to dsGFP construction expression vectors are designed (Table 1). The target fragments of dsvATPase B and dsGFP for constructing the vector were obtained as shown in example 2 using the cDNA in example 2 as a template and the reaction system and amplification procedure for PCR amplification, and the two PCR products obtained above were recovered 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 dsvATPase B with linearized L4440 vector for 20min at 50 ℃ for recombination. Subsequently, the recombinant expression vector containing dsvATPase B 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 thereto, and the mixture was incubated at 37 ℃ and 210rpm for 1h, followed by overnight incubation with LB plates containing ampicillin and tetracyclineAnd (5) culturing. 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.

Both of the culture solutions containing dsGFP and dsvATPase B were subjected to hyphal collection at 4 ℃ and 5000rpm, RNA extraction using TRIzol extraction (Invitrogen, USA), and 1.5% agarose gel electrophoresis to confirm successful induction of dsRNA, as shown in FIG. 1.

Example 3 application of dsRNA to inhibition of growth and development of harmonia axyridis

1. Preparation of ladybug host plant and artificial incubator

The eggplant variety for feeding the harmonia axyridis is a marshally round eggplant seedling, and the artificial incubator is a 90mm culture dish containing filter paper and a humidifying cotton ball.

2. Application of dsvATPase B synthesized by kit in inhibiting growth and development of harmonia solaricellica

Ladybug dsvATPase B feeding group: 101 st larvae of E.solaricius are placed in a petri dish with filter paper and a humidified cotton ball. Soaking a round eggplant leaf disc with the diameter of 12mm in dsvATPase B solutions with the concentrations of 50 ng/mu L, 100 ng/mu L, 250 ng/mu L and 500 ng/mu L, which are synthesized by the kit, for 1min, air-drying for 1h, feeding larvae, replacing the leaf disc every 24h, continuously feeding the leaf disc soaked in dsvATPase B for two days, and feeding the larvae with normal eggplant leaves.

Ladybug dsGFP feeding group: 101 st larvae of E.solaricius are placed in a petri dish with filter paper and a humidified cotton ball. Soaking a round eggplant leaf disc with the diameter of 12mm in dsGFP solution with the concentration of 500ng/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 provided with 5 replicates, the death number of the E.solariciresis in each culture dish is counted every 24 hours, and new leaves are replaced, and the culture dishes are placed in an artificial climate box (the temperature is 25 +/-1 ℃, the humidity is 70-80%, and the photoperiod L: D is 14: 10). Counting the number of deaths of the E.solani in each culture dish of each group, calculating the survival rate change of the E.solani under the treatment of the control group and different concentrations of dsRNA, counting the survival rate of the E.solani by using Excel2010, and mapping by using SPSS 19.0 software and a Cox regression analysis method (as shown in figure 2).

From the results, after continuously feeding dsRNA for 1-instar ladybug of twenty eight star, the survival rate of the 1-instar ladybug of twenty eight star shows a trend of decreasing along with the increase of time, the feeding concentrations of the treatment groups are 10 ng/mu L, 50 ng/mu L, 250 ng/mu L and 500 ng/mu L respectively, and the feeding concentration of the control group is 500 ng/mu L. There were significant differences between the treatment and control groups at different concentrations (χ 2 ═ 101.448, df ═ 4, P < 0.0001). When the treatment group was fed at a concentration of 10ng/μ L (P <0.0001, exp (b) ═ 6.540), the difference was significant from each of the groups at concentrations of 50ng/μ L (P <0.0001, exp (b) ═ 15.496), 250ng/μ L (P <0.0001, exp (b) ═ 18.179) and 500ng/μ L (P <0.0001, exp (b) ═ 19.436), and there was no significant difference between the treatment groups at the other concentrations. From the statistical results, it can be concluded that the mortality increased 19.436-fold, 18.179-fold, 15.496-fold and 6.540-fold, respectively, when the concentrations of the treatment groups were 500 ng/. mu.L, 250 ng/. mu.L, 50 ng/. mu.L and 10 ng/. mu.L, respectively, compared to the control group.

Changes in phenotypic characteristics of E.solani following two days of feeding dsvATPase B and dsGFP were observed microscopically. It was found that day 6 from the feeding of dsvATPase B, the ladybug of dsGFP control group normally entered the 3 rd instar stage, but the larvae in the treatment group failed to normally molt into the 3 rd instar stage and died, and the phenotypic characteristics were shown that the systemic sticks were bent and in a blackened state as shown in FIG. 3, indicating that feeding of dsvATPase B could induce a strong RNAi effect in vivo of ladybug, resulting in the death of ladybug. Wherein, at a concentration of 10 ng/. mu.L in the treated group, a lower mortality rate (50% mortality rate at day 10) was caused, at which concentration feeding dsvATPase B was observed to affect the growth and development of E.solani dissonii as shown in FIG. 5, the control groups at day 3, day 6 and day 9 from dsRNA feeding entered 2, 3 and 4 years, respectively, while the E.solani dissonii of the treated group was in the 2-year state after 2-year entry at day 3 until death (as shown in FIG. 4).

3. Application of dsvATPase B expressed by bacterial liquid in killing harmonia solaricellica

Ladybug dsvATPase B feeding group: 10 larvae of 1 year, 10 larvae of 3 years and 5 adults are respectively placed in a culture dish with filter paper and a humidifying cotton ball, 3 groups of experiments are set in total, and 5 experiments are set in each group. And soaking a circular eggplant leaf disc with the diameter of 12mm for 1min by using a bacterial liquid expressing dsvATPase B, air-drying at room temperature for 1h, and feeding larvae. 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. And replacing the leaf disc every 24 hours, continuously feeding the leaf disc soaked by the dsvATPase B bacterial solution for two days, and feeding the leaf disc by using normal eggplant leaves.

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.

And 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). The number of deaths of the E.solaris in each culture dish of each group was counted, the change of the survival rate of the E.solaris in the control and different treatment groups was calculated, the survival rate of the E.solaris was counted by using Excel2010, and the SPSS 19.0 software was used to plot by Cox regression analysis (see FIG. 5).

According to the statistical results (as shown in fig. 6), after two days of continuously taking dsva atpase B expressed by the liquid extract of the eggplanta glaber, the survival rates of 1-instar larvae (P <0.0001, exp (B) ═ 13.068), 3-instar larvae (P <0.0001, exp (B) ═ 15.258) and adults (P ═ 0.021, exp (B) ═ 6.001) 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 treated group were increased by 13.068 times, 15.258 times and 6.001 times, respectively, compared with those of the control group.

Example 4 dsvATPase B inhibition of vATPase B Gene expression in E.solasonia

Based on the sequence of vATPase B gene, qPCR primer P5 for vATPase B gene and qPCR primer P6 for reference gene GAPDH were designed (Table 1).

Ladybug 1 st larvae treated with 10 ng/. mu.L dsvATPase B and dsGFP were collected on

days

2 and 4, respectively, after initiation of dsRNA feeding, and 3 biological replicates were collected for each treatment. Extracting the collected RNA of the harmonia axyridis, carrying out reverse transcription to form cDNA, and diluting by 10 times to be used as 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 primers 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. Final result calculation using 2^-ΔΔCtThe method (Ct represents the number of cycles) was performed. Data statistics were performed using one-way anova.

Relative expression changes of vATPase B gene in E.solani after 2 days and 4 days of dsvATPase B feeding were counted respectively with dsGFP feeding as a control (see FIG. 6). As can be seen from FIG. 6, the expression level of vATPase B gene in E.solaris feeding dsvATPase B shows a significant decrease trend compared with the expression level of vATPase B gene in E.solaris feeding dsGFP. And on day 2, expression of vATPase B gene in the treated group compared to the control groupThe amount decreased by 9.8 times (F)_1,4＝1727.253,P<0.0001); on day 4, the expression level of vATPase B gene was 13.1-fold lower in the treated group than in the control group (F)_1,4＝9762.102,P<0.0001), further indicates that the feeding of dsvATPase B can cause strong RNAi effect in vivo of the ladybug, so that the expression level of vATPase B gene in vivo is obviously reduced, and further the development of the ladybug is inhibited or the ladybug is dead.

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> 20

<212> DNA

<213> P5-F

<400> 11

agcatccttc gctcgtttag 20

<210> 12

<211> 20

<212> DNA

<213> P5-R

<400> 12

ttcgacaacc tgccattagg 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 Coccinella solani vATPase B 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 Coccinella solani, characterized in that the dsRNA silencing target gene is the vATPase B gene of claim 1.

3. the application of dsRNA described in claim 2 in preventing and treating Coccinella solani or preparing a product for preventing and treating Coccinella solani.

4. the application of dsRNA described in claim 2 in suppressing the growth of Coccinella solani or preparing the product of inhibiting the growth of Coccinella solani.

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

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

7. A method for preventing and treating Coccinella solani, characterized in that, by feeding exogenous dsRNA, the dsRNA can silence/inhibit the expression of the vATPase B gene of claim 1.