CN110551730B

CN110551730B - Ladybug RPS18 gene and application thereof in pest control

Info

Publication number: CN110551730B
Application number: CN201910718865.4A
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: CN110551730A

Abstract

The invention discloses a ladybug RPS18 gene and application thereof in insect pest prevention. The invention screens a high lethal gene RPS18 of the ladybug with twenty-eight stars, designs high-efficiency dsRNA aiming at the gene, has high lethal capability to the ladybug with twenty-eight stars, can efficiently prevent and control the ladybug with twenty-eight stars, namely directly feeds the dsRNA, and can achieve the purpose of preventing and controlling by utilizing the lethal effect of dsRPS18 on the ladybug with twenty-eight stars. The method has the advantages of convenient operation, good effectiveness and sensitivity, high insecticidal efficiency, environmental friendliness and the like, and has good application prospect.

Description

Ladybug RPS18 gene and application thereof in pest control

Technical Field

The invention belongs to the technical field of insect pest prevention and control. More particularly, relates to an RPS18 gene of the harmonia axyridis and application thereof in preventing and treating the harmonia axyridis.

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 solanacearum and provides a high lethal gene of the ladybug solanacearum, namely an RPS18 gene. And a technology capable of efficiently preventing and treating the ladybug twenty-eight star is developed based on the gene, namely, the target gene dsRNA with high lethal capability to the ladybug twenty-eight star is directly fed, and the lethal effect of dsRPS18 on the ladybug twenty-eight star is utilized to achieve the prevention and treatment purpose. 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 RPS18 gene and application thereof in preventing and treating ladybug.

The invention also aims to provide dsRNA for the RPS18 gene for preventing and treating the 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-RPS 18 gene based on the transcriptome library of the ladybug with twenty-eight eggplant, and develops the technology for preventing and treating the ladybug with the dsRNA (dsRPS18) feeding the RPS18 gene. According to the invention, eggplant leaves are respectively soaked in dsRPS18 and dsGFP solutions synthesized by a kit, the eggplant leaves are taken out and aired, and then 1-instar larvae of the ladybug with twenty-eight delicacies are fed for 2 days, then the eggplant leaves which are not treated by dsRNA are fed, and the death rate and the development state of the ladybug with twenty-eight delicacies are observed and recorded; in addition, the lethal ability of the dsRPS18 on 1-year, 3-year and adult ladybug of the ladybug with a bacterium liquid expression method is determined, and the insecticidal activity of the exogenous dsRPS18 on the ladybug of the twenty-eight. Finally, the change of the expression level of the RPS18 gene in the ladybug eating dsRPS18 and dsGFP was analyzed by the method of fluorescent quantitative PCR (qPCR). The result shows that the direct feeding of the exogenous dsRPS18 can obviously inhibit the expression of the RPS18 gene of the ladybug with twenty eight stars, and the direct feeding of the exogenous dsRPS18 has high lethal effect on the ladybug with twenty eight stars. Therefore, the following subject matters and applications should be considered to be within the protection scope of the present invention:

a ladybug RPS18 gene with sequence shown in SEQ ID NO. 1.

The RPS18 gene is applied to the prevention and treatment of the ladybug with twenty eight stars or the preparation of products for the prevention and treatment of the ladybug with twenty eight stars.

The RPS18 gene is applied to inhibiting the growth of the ladybug with dioctadecylate or preparing products for inhibiting the growth of the ladybug with dioctadecylate.

The RPS18 gene is applied to the promotion of the death of the harmonia axyridis or the preparation of products for promoting the death of the harmonia axyridis.

The inhibitor of the RPS18 gene is applied to the prevention and treatment of the ladybug with twenty eight stars or the preparation of products for the prevention and treatment of the ladybug with twenty eight stars.

The dsRNA can be used for preventing and controlling the ladybug with twenty-eight stars, and the dsRNA silences a target gene as the RPS18 gene. Preferably, the dsRNA sequence is shown as SEQ ID NO. 1.

A kit for preventing and treating ladybug with twenty-eight stars contains RPS18 gene inhibitor. Preferably, the inhibitor is the above-described dsRNA.

Specifically, one way of preventing and controlling the axyridis solaris by using the RPS18 gene is a method for preventing and controlling the axyridis solaris, exogenous dsRNA is directly fed, so that the dsRPS18 enters the body of the axyridis solaris, the dsRNA can silence/inhibit the expression of the RPS18 gene of the axyridis solaris, inhibit the growth of the axyridis solaris and promote the death of the axyridis solaris, and the aim of preventing and controlling the axyridis solaris is fulfilled.

The invention has the following beneficial effects:

the invention obtains a high lethal gene RPS18 gene of the ladybug with twenty-eight stars through screening, develops the high-efficiency silent dsRNA of the gene, and develops a technology capable of efficiently preventing and treating the ladybug with twenty-eight stars, namely, directly feeding a target gene dsRNA with high lethal capability to the ladybug with twenty-eight stars, and achieving the purpose of preventing and treating by utilizing the lethal effect of dsRPS18 on the ladybug with twenty-eight stars. 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 photograph of the electrophoresis of dsGFP and dsRPS18 synthesized by the kit.

FIG. 2 is an electrophoretogram of dsGFP and dsRPS18 expressed from the bacterial suspension.

FIG. 3 is a graph showing the survival rate of E.solariciressa after feeding dsGFP and dsRPS 18; 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 E.solaricius larvae after feeding dsGFP and dsRPS 18; a is a control group; b, C, D and E are treatment groups.

FIG. 5 is a graph showing the effect of feeding dsGFP and dsRPS18 on the growth and development of E.coli.

FIG. 6 shows the change of 1 st, 3 rd and adult survival rate of E.coli Tsakayasu after taking dsGFP and dsRPS18 expressed by the inoculum solution; establishing a survival curve using Cox regression using 12 day larval mortality data; different letters (e.g., a, b) indicate significant differences between the control and treatment curves; FIG. A: survival rate of 1 instar larvae; and B: survival rate of 3 instar larvae; and (C) figure: survival rate of adults.

FIG. 7 shows the relative expression of the RPS18 gene at 2 and 4 days after feeding dsRPS 18.

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

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 an RPS18 gene fragment is obtained by screening, wherein the 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 RPS18 gene fragment was obtained from the ladybug transcriptome results, and the dsRNA primer P1 (Table 1) of the RPS18 gene was designed and synthesized by Hippocastellate Hipponica technologies, Inc., Guangzhou. The green fluorescent protein Gene (GFP) was amplified from a GFP plasmid stored in the laboratory, and the dsRNA primer P2 for the GFP gene is shown in 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, ddH₂The 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. Taking the ladybug cDNA as a template, carrying out PCR amplification by using a P1 primer to obtain a 453bp PCR product, namely a dsRPS18 amplified fragment, and obtaining the nucleotide shown by the 413bp SEQ ID NO.1 after sequencing and deleting a T7 promoter sequence.

The plasmid carrying GFP was used as a template, and amplification was carried out with P2 primer to obtain 507bp PCR product (the result of the PCR electrophoresis is shown in FIG. 1).

3. Preparation of dsRNAs synthesized by RPS18 gene and GFP gene kit

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 was completed, 2.5. mu.L of TURBO DNase was added to remove the remaining template DNA and single-stranded RNA, then the dsRNA was purified, and finally the dsRNA was dissolved with 50. mu.L of ddH2O to obtain dsRPS18 and dsGFP, respectively.

The results of sequencing the RPS18dsRNA of E.solaricius are as follows:

the RPS18dsRNA of the ladybug solanacearum is double-stranded RNA and consists of a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is SEQ ID NO.1 in a sequence table, the nucleotide sequence of the antisense strand is a reverse complementary sequence of the SEQ ID NO.1 in the sequence table, and the nucleotide sequence of the coding gene of the RPS18dsRNA is SEQ ID NO.1 in the sequence table.

The GFP dsRNA is double-stranded RNA and consists of a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is SEQ ID NO.2 in a sequence table, and the nucleotide sequence of the antisense strand is a reverse complementary sequence of the SEQ ID NO.2 in the sequence table.

EXAMPLE 2 obtaining of dsRNA expressed in bacterial liquid of RPS18 Gene

1. Construction of dsRPS18 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 added to the primer P1 of dsRPS18 and the primer P2 of dsGFP respectively, and a primer P3 related to the dsRPS18 construction expression vector and a primer P4 related to the dsGFP construction expression vector are designed (Table 1). Using the cDNA in example 2 as a template, the reaction system and the amplification procedure for PCR amplification were as described in example 2, and the target fragments dsRPS18 and dsGFP of the constructed vector were obtained, and the two PCR products obtained above were recovered using a DNA Purification and 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 dsRPS18 with linearized L4440 vector for 20min at 50 ℃ for recombination. Subsequently, the recombinant expression vector containing dsRPS18 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, after which time ampicillin and tetracycline were applied to LB platesThe culture was carried out overnight. Single colonies were 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, then 50. mu.L of the single colonies were 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 to bring the OD of the cells to 0.5-0.8, and then 1mM of IPTG was added thereto and cultured at 37 ℃ and 120rpm for 5 hours to induce dsRNA.

Both of the culture solutions containing dsGFP and dsRPS18 were subjected to hyphal collection at 4 ℃ and 5000rpm, RNA was extracted by TRIzol extraction (Invitrogen, USA), and 1.5% agarose gel electrophoresis was performed to confirm successful induction of dsRNA.

The electrophoretograms of dsGFP and dsRPS18 synthesized by the kit are shown in FIG. 1, and the electrophoretograms of dsGFP and dsRPS18 expressed in bacterial liquid are shown in FIG. 2.

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 dsRPS18 synthesized by kit in inhibiting growth and development of ladybug twenty-eight star

Ladybug RPS18dsRNA feeding group (dsRPS 18): 10 1 st larvae of E.solaricius are placed in a petri dish with filter paper and a humidified cotton ball. Soaking round eggplant leaves with the diameter of 12mm in dsRPS18 solutions with the concentrations of 10 ng/mu L, 50 ng/mu L, 100 ng/mu L, 250 ng/mu L and 500 ng/mu L for 1min, air-drying for 1h, feeding the larvae, replacing leaf disks every 24h, continuously feeding the leaf disks soaked in dsRPS18 for two days, and feeding the larvae with normal eggplant leaves.

Sabdaria solanacearum GFPdsRNA feeding group (dsGFP): 10 1 st larvae of E.solaricius are placed in a petri dish with filter paper and a humidified cotton ball. Soaking round eggplant leaves 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 the larvae, replacing a leaf disc every 24h, continuously feeding the leaf disc soaked by the dsGFP for two days, and feeding the larvae with untreated eggplant leaves. Each group is 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.solaris in each culture dish of each group, calculating the survival rate change of the E.solaris under the treatment of the control group and dsRNA with different concentrations, counting the survival rate of the E.solaris by using Excel 2010, and mapping by using SPSS 19.0 software and adopting a Cox regression analysis method.

The effect of different concentrations of dsRPS18 on mortality of E.solani larvae is shown in FIG. 3.

As can be seen from fig. 3, after feeding dsRPS18 for two days for 1 st larvae of axyridis solani, the survival rate of 1 st larvae of axyridis solani decreased with time, and significant differences were found between the treatment groups and the control group at different concentrations (χ 2 ═ 103.095, df ═ 5, P < 0.0001). There was a significant difference analysis between dsRPS18 fed 50ng/μ L (P <0.0001, exp (b) ═ 6.920) and 10ng/μ L (P <0.0001, exp (b) ═ 4.436) and dsRPS18 fed 100ng/μ L (P <0.0001, exp (b) ═ 10.657), 250ng/μ L (P <0.0001, exp (b) ═ 14.025) and 500ng/μ L (P <0.0001, exp (b) ═ 14.494), whereas there was no significant difference between the treatment groups fed 50ng/μ L and 10ng/μ L dsRPS18 and between the treatment groups fed 100ng/μ L, 250ng/μ L and 500ng/μ L dsRPS 18. From the statistical results, it can be concluded that the mortality increased 14.494-fold, 14.025-fold, 10.657-fold, 6.920-fold and 4.436-fold, respectively, when the concentrations of the treatment groups were 500 ng/. mu.L, 250 ng/. mu.L, 100 ng/. mu.L, 50 ng/. mu.L and 10 ng/. mu.L, respectively, as compared to the control group.

Changes in phenotypic characteristics of the E.solani dioctadecylate were observed microscopically after two days of dsRPS18 feeding. It was found that day 3 from the feeding of dsRPS18, the ladybug of dsGFP control group normally entered the 2 nd stage, and the larvae in the treatment group failed to normally molt into the 2 nd stage and died, with phenotypic features showing failure of branch spines and spots at the forebreast backplate as shown in fig. 4, suggesting that feeding dsRPS18 could induce strong RNAi effect in vivo of ladybug, resulting in ladybug death. At a concentration of 10 ng/. mu.L in the treated group, a lower mortality rate (50% mortality at day 10) was caused, at which concentration feeding dsRPS18 was observed to affect the growth and development of E.solaris axyridis at 3d, 6d, 9d control groups, which started with dsRNA feeding, entered 2, 3, 4 years, respectively, whereas E.solaris octacosyridis in the treated group, which entered 2 years at 3d, remained in the 2-year state until death, as shown in FIG. 5. Development of ladybug larvae was significantly inhibited in the treated group compared to the control group after feeding dsRPS 18.

3. Application of dsRPS18 expressed by bacterial liquid in killing harmonia solaricoides

Ladybug RPS18dsRNA feeding group (dsRPS 18): 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 (3) soaking a circular eggplant leaf disc with the diameter of 12mm for 1min by using a bacterial liquid expressing dsRPS18, and feeding larvae after the disc is air-dried for 1h at room temperature. 2 leaf discs are placed in each culture dish of 1-instar larvae in the treatment group; 5 leaf discs are placed in each culture dish of 3-instar larvae; adult dishes were placed with 5 leaf discs. Changing the leaf disc every 24h, continuously feeding the leaf disc soaked by the dsRPS18 bacterial liquid for two days, and feeding the leaf disc with normal eggplant leaves.

Sabdaria solanacearum GFPdsRNA feeding group (dsGFP): 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 Excel 2010, and the SPSS 19.0 software was used to plot by Cox regression analysis (see FIG. 6).

According to the statistical results (as shown in fig. 6), after the dsRPS18 expressed by the liquid of the ladybug is continuously fed for two days, the survival rates of 1-instar larvae (P <0.0001, exp (b) ═ 39.114), 3-instar larvae (P <0.0001, exp (b) ═ 25.399) and adults (P ═ 0.021, exp (b) ═ 48.445) are significantly different from those of the control group, and the death rates of the 1-instar larvae, the 3-instar larvae and the adults of the treatment group are increased by 39.114 times, 25.399 times and 48.445 times respectively compared with those of the control group.

Example 4dsRPS18 inhibition of expression of the RPS18 Gene in E.solasoni

Based on the sequence of the RPS18 gene, qPCR primer P5 for the RPS18 gene and qPCR primer P6 for the reference gene GAPDH were designed (Table 1).

Ladybug 1 st larvae treated with 10 ng/. mu.LdsRPS 18 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 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. Final result calculation using 2^-ΔΔCtThe method (Ct represents the number of cycles) was performed. Data statistics were performed using one-way anova.

The relative expression amount change of the RPS18 gene in the ladybug with dioctadecylate is counted after feeding dsRPS for 182 days and 4 days respectively by taking the dsGFP feeding as a control. FromIn FIG. 7, it can be seen that the expression level of the RPS18 gene in E.solasoni fed with dsRPS18 showed a significantly decreased tendency compared with the expression level of the RPS18 gene in E.solasoni fed with dsGFP. Further shows that the feeding of dsRPS18 can cause strong RNAi effect in the ladybug with twenty-eight star, which leads to the obvious reduction of the expression level of RPS18 in vivo, and further leads to the death or the development inhibition of the ladybug with twenty-eight star. The expression of the RPS18 gene in the ladybug with dioctadecylate was significantly different between day 2 and day 4 from the feeding of dsRPS18, and the expression of the RPS18 gene was reduced 6.8 times in the treatment group on day 2 compared with the control group (F)_1,4＝78646.985,P<0.0001), the expression level of RPS18 gene was reduced by 26.5 times (F) in the treatment group on day 4 compared with the control group_1,4＝34807.770,P<0.0001), the expression of the RPS18 gene in E.solasoni was significantly different in comparison with day 2 and day 4 from the feeding of dsRPS 18.

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

<110> southern China university of agriculture

<120> ladybug RPS18 gene and application thereof in pest control

<130>

<160> 14

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<213> P3-F

<400> 7

ctgatatcat cgatgaattc ttctccgtat cctcggtac 39

<210> 8

<211> 38

<212> DNA

<213> P3-R

<400> 8

cgaattcctg cagcccggga gacacaccca cagttcta 38

<210> 9

<211> 41

<212> DNA

<213> P4-F

<400> 9

ctgatatcat cgatgaattc aagttcagcg tgtccggcga g 41

<210> 10

<211> 40

<212> DNA

<213> P4-R

<400> 10

cgaattcctg cagcccgggt tcacgttgat gccgttcttc 40

<210> 11

<211> 21

<212> DNA

<213> P5-F

<400> 11

cgcaatcaaa ggtgttggaa g 21

<210> 12

<211> 21

<212> DNA

<213> P5-R

<400> 12

gcctagggtt ggccataata g 21

<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 ladybug RPS18 gene is characterized in that the sequence is shown in SEQ ID NO. 1.

2. A dsRNA for controlling ladybug twenty-eight, wherein the dsRNA silences a target gene as the RPS18 gene of claim 1.

3. The dsRNA of claim 2 for use in controlling ladybug eggplant eight star or in preparing products for controlling ladybug eggplant twenty-eight star.

4. The use of the dsRNA of claim 2 for inhibiting the growth of E.

5. Use of the dsRNA of claim 2 for promoting death of E.

6. A kit for controlling E.solariciressa, characterized in that it contains the dsRNA of claim 2.

7. A method of controlling ladybug dioctadecylate fed with exogenous dsRNA which silences/inhibits the expression of the RPS18 gene of claim 1.