CN110759983B

CN110759983B - Recombinant fungus expressed by targeted silent pest pattern recognition protein GNBP3 gene and application thereof in pest control

Info

Publication number: CN110759983B
Application number: CN201910855074.6A
Authority: CN
Inventors: 许小霞; 金丰良; 洪莹莹; 曹苗苗; 李树忠; 曾路; 张雨欣; 花艳艳
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-09-10
Filing date: 2019-09-10
Publication date: 2021-07-09
Anticipated expiration: 2039-09-10
Also published as: CN110759983A

Abstract

The invention discloses a recombinant fungus expressed by a targeted silent pest pattern recognition protein GNBP3 gene and application thereof in pest control, and the invention constructs a recombinant beauveria bassiana capable of inhibiting or blocking the transcription of a GNBP3 gene. Therefore, when the recombinant beauveria bassiana infects pests, the expression of the antibacterial peptide in the pests is obviously reduced, the signal conduction of the pests for identifying the immune entomogenous fungi is weakened or interrupted, and the pests are more easily infected with pathogenic microorganisms such as the entomogenous fungi and the like to cause death. The recombinant beauveria bassiana remarkably improves the death rate of pests, shortens the death time and has concentration dependence. The microbial source pesticide is combined with a microbial source pesticide bacillus thuringiensis Bt or serratia marcescens, the mortality and death time of the recombinant beauveria bassiana to pests can be obviously improved, the microbial source pesticide is a good synergist of the microbial pesticide, the killing effect is not improved, the insecticidal spectrum is widened, the microbial pesticide resistance is reduced, and the microbial source pesticide is an environment-friendly microbial source pesticide.

Description

Recombinant fungus expressed by targeted silent pest pattern recognition protein GNBP3 gene and application thereof in pest control

Technical Field

The invention relates to the technical field of biology, in particular to a recombinant fungus expressed by a targeted silent pest pattern recognition protein GNBP3 gene and application thereof in pest control.

Background

There are about 150 thousands of scientifically recorded insects all over the world, which account for about half of the species of terrestrial organisms, and about 45% of insects harm plants by taking leaves of green plants as food, and insects feeding on crops can seriously increase the variety yield of crops, thereby affecting the income of farmers.

At present, agricultural pest control mainly depends on chemical pesticides, but the use of a large amount of chemical pesticides causes eye-surprise environmental pollution, causes serious harm to human health, and improves the drug resistance of insects, so that the selection of effective and pollution-free pest biological control is increasingly emphasized.

Entomogenous fungi play an important role in the aspects of insect population density regulation and biological control of harmful insects, and more than 750 species are identified and mainly distributed in ascomycetes and zygomycota. Insect entomogenous fungi are very suitable for being used as biological insecticides, wherein metarhizium anisopliae, metarhizium locustum, beauveria bassiana and the like of ascomycetes are developed into important biological pest control agents. The entomogenous fungi can be cultured in a large scale and can be stored for a long time, the fungal spores are suitable for common agriculture and forestry spraying equipment, compared with chemical insecticides, the entomopathogenic fungi have small toxicity to mammals and have negligible influence on the environment, and the entomopathogenic fungi have the characteristic of sustainable control of pests and are widely used for prevention and treatment of agriculture and forestry pests. The commercial fungal insecticides registered and used in the world can reach more than 170 varieties, and have good ecological, economic and social benefits in biological control of pests in agriculture, forestry, grassland and the like.

But the immune defense reaction of pests greatly delays the insecticidal efficiency of the currently used fungal insecticides, and the innate immune system of insects consists of cellular immunity and humoral immunity. When the entomogenous fungi infects insects and enters the blood cavity of the insects through the body wall, the insects are defended by the innate immune system of the insects, the entomogenous fungi are inhibited or killed, the continuous propagation of the fungi is hindered, and the insecticidal efficiency of the entomogenous fungi is directly delayed.

Cellular immunity is primarily the phagocytosis and coating of blood cells (primarily plasma cells and granulocytes) against foreign bodies; the main organ of humoral immunity is fat body, which induces and generates antibacterial peptide and releases it to haemolymph, and at the same time, including lysozyme action and prophenoloxidase (PPO) cascade reaction, etc., which cooperates with cellular immunity to carry out defense reaction. When infected by a foreign pathogen, natural immunity of the insect is initiated by recognizing the foreign body, then initiating, activating serine protease and releasing the extracellular cascade of serine protease inhibitors, initiating Toll or Imd signal transduction pathways to induce the production of different antimicrobial peptides and phenol oxidase cascade, and killing/inhibiting the infected foreign microorganism. Insect immunity research using Drosophila melanogaster as a model has been greatly developed, and it is clear that the recognition of foreign substances is mainly realized by different Pattern Recognition Proteins (PRPs). On the other hand, in response to insect immune recognition, insect pathogens develop different immune evasion mechanisms through long-term evolutionary adaptation, evade or overcome host cell immune recognition and immune defense, causing insect morbidity or ultimately killing of host insects. The interaction between insect immunity and its pathogens is a dynamic process, which is reflected in different levels of molecules, cells, individuals to populations. The switch button for the initiation of any immune response is the recognition by the insect of the invasion of foreign substances or foreign bodies. Insects have a complete set of systems that clearly identify "self" and "non-self" components. Insect immune switches rely on the recognition and binding of Pattern Recognition Receptors (PRRs) to pathogen-associated molecular patterns (PAMPs) or microorganism-associated molecular patterns (MAMPs) to activate relevant immune signaling pathways. PAMPs or MAMPs include microbial nucleic acids, proteins, lipids, lipoproteins, generally located in conserved structures essential for pathogen survival, and are ligands that specifically activate the innate immune pathway. Pattern recognition receptors for insects mainly include peptidoglycan recognition proteins (PGRPs), gram-negative bacteria binding proteins (GNBPs), β -1, 3-glucan recognition proteins (β GRPs), C-type lectins (CTLs), scavenger receptors (scavenger receptors), hemolymph immunoglobulins (hemolins), and thiolipoproteins (TEs Ps). After the pattern recognition protein recognizes the heterosis, the organism can start the self immune mechanism to amplify the danger signal. After intracellular signal transduction, Toll, Imd and STAT signal pathways in the insect body are activated, a danger signal is transmitted to the downstream, and finally, the expression of an effector is activated, so that the insect is protected.

Diamondback moth (Plutella xylostella L.) belongs to worldwide crucifer pests, and due to the characteristics of wide feeding property, short generation period and the like, the diamondback moth brings great loss to crop production, leads the diamondback moth to generate serious drug resistance due to abuse of chemical drugs for a long time, and increases the difficulty of prevention and control. Alternatives to high efficacy and low toxicity, especially microbial pesticides, are continually being sought. However, there is currently no microbial pesticide that can escape its recognition of invading microorganisms in vivo.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a recombinant fungus for targeted silencing of the expression of a pest pattern recognition protein GNBP3 gene and application of the recombinant fungus in pest control.

The first purpose of the invention is to provide a diamondback moth pattern recognition receptor protein GNBP3 gene.

The second purpose of the invention is to provide a diamondback moth pattern recognition receptor protein GNBP 3.

The third purpose of the invention is to provide the application of the diamondback moth pattern recognition receptor protein GNBP3 gene, the fragment of the diamondback moth pattern recognition receptor protein GNBP3 gene, the diamondback moth pattern recognition receptor protein GNBP3or the fragment of the diamondback moth pattern recognition receptor protein GNBP3 as a pest control target.

The fourth purpose of the invention is to use the inhibitor of any one of the diamondback moth pattern recognition receptor protein gene GNBP3, the fragment of the diamondback moth pattern recognition receptor protein gene GNBP3, the diamondback moth pattern recognition receptor protein GNB P3or the fragment of the diamondback moth pattern recognition receptor protein GNBP3 in preparing pesticides.

The fifth object of the present invention is to provide a recombinant expression vector.

It is a sixth object of the present invention to provide a recombinant fungus.

It is a seventh object of the present invention to provide a method for controlling pests.

The eighth purpose of the invention is to provide the application of the recombinant bacterium as a synergist in pest control.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the mode recognition receptor gene selected by the patent is a switch button-gram negative bacteria binding protein gene GNBP3 for recognizing an invading pathogen, and a recombinant vector which takes a plutella xylostella GNBP3 gene as a target is constructed, so that a recombinant beauveria bassiana strain containing GNBP3 is obtained. The process of infecting the plutella xylostella by using the fungi integrates the exogenous plasmid into the plutella xylostella genome, the lethal effect of the recombinant beauveria bassiana on the plutella xylostella is obvious, the mortality rate is obviously improved, and the death time is shortened. Further, the recombinant beauveria bassiana is mixed with a microbial insecticide bacillus thuringiensis (Bt) and Serratia marcescens (Sm) for use, and the insecticidal efficiency can be greatly improved.

The invention therefore claims the following:

a diamondback moth pattern recognition receptor protein gene GNBP3 has a nucleotide sequence shown as SEQ ID NO: 1 is shown.

A diamondback moth pattern recognition receptor protein GNBP3, the amino acid sequence of which is shown in SEQ ID NO: 2, respectively.

The application of any one of the diamondback moth pattern recognition receptor protein gene GNBP3, the fragment of the diamondback moth pattern recognition receptor protein gene GNBP3, the diamondback moth pattern recognition receptor protein GNBP3or the fragment of the diamondback moth pattern recognition receptor protein GNBP3 as a pest control target.

The application of any inhibitor of the diamondback moth pattern recognition receptor protein gene GNBP3, the fragment of the diamondback moth pattern recognition receptor protein gene GNBP3, the diamondback moth pattern recognition receptor protein GNBP3or the fragment of the diamondback moth pattern recognition receptor protein GNBP3 in preparing pesticides.

Preferably, the nucleotide sequence of the fragment of the diamondback moth pattern recognition receptor protein gene GNBP3 is shown in SEQ ID NO: 3, respectively.

Preferably, the amino acid sequence of the fragment of the diamondback moth pattern recognition receptor protein GNBP3 is shown in SEQ ID NO: 4, respectively.

The invention further claims a recombinant expression vector which is characterized in that the plutella xylostella pattern recognition receptor protein gene GNBP3 is silenced and/or the expression of the plutella xylostella pattern recognition receptor protein GNBP3 is inhibited by targeting the plutella xylostella pattern recognition receptor protein gene GNBP3or a fragment thereof.

The invention does not limit the type of the recombinant expression vector, and the RNAi vector or CRISPR vector which is commonly used at present can be used.

Preferably, the recombinant vector is an RNAi vector, and the recombinant expression vector contains the diamondback moth pattern recognition receptor protein gene GNBP3or a fragment thereof, or a reverse complementary sequence of the diamondback moth pattern recognition receptor protein gene GNBP3or a fragment thereof.

Preferably, the RNAi vector is a psilient vector.

The invention further claims a recombinant bacterium carrying the recombinant expression vector.

Preferably, the expression vector is beauveria bassiana.

More preferably, the recombinant bacterium is a bacterium which carries a polypeptide containing a nucleotide sequence shown as SEQ ID NO: 3, and (3) white muscardine fungus of pSilent vector of fragment of Plutella xylostella pattern recognition receptor protein gene GNBP 3.

The application of the recombinant expression vector or the recombinant bacterium in preventing and controlling pests also belongs to the protection scope of the invention.

The invention also claims a method for preventing and controlling pests, which silences the diamondback moth pattern recognition receptor protein gene GNBP3 and/or inhibits the expression of the diamondback moth pattern recognition receptor protein GNBP 3.

Preferably, the pests include, but are not limited to, agricultural pests such as beet armyworm, prodenia litura, Asiatic corn borer, and the like.

The method is also suitable for controlling other pests, such as agricultural pests of beet armyworm, prodenia litura, Asiatic corn borer and the like. As long as the cDNA sequences of the pattern recognition protein GNBP3 genes of the pests are screened, a series of recombinant beauveria bassiana are constructed and applied to the biological control of the pests.

Preferably, the recombinant bacteria or the recombinant expression vector is used for silencing the diamondback moth pattern recognition receptor protein gene GNBP3 and/or inhibiting the expression of the diamondback moth pattern recognition receptor protein GNBP 3.

Furthermore, the invention also protects the application of the recombinant bacterium as a synergist in pest control.

The recombinant beauveria bassiana enhances the pathogenic effect of the bacillus thuringiensis Bt and the serratia marcescens Sm on the insect pest diamondback moth, and the recombinant beauveria bassiana is 10⁴At the concentration, the recombinant beauveria bassiana with the death rate of the diamondback moth is 10 by combining with the bacillus thuringiensis Bt⁷The results were consistent. In the case of recombinant Beauveria bassiana of 10⁴At the concentration, the recombinant beauveria bassiana is 10 when combined with the serratia marcescens Sm and the death rate of the diamondback moth⁶Compared with the prior art, the insecticidal time is shortened by half a day.

Compared with the prior art, the invention has the following beneficial effects:

the invention is based on RNA interference technology, uses a pattern recognition protein gene GNBP3 as a target, and utilizes a genetic engineering means to construct recombinant beauveria bassiana capable of inhibiting or blocking the expression of the GNBP3 gene. Therefore, when the beauveria bassiana infects pests, the expression of the antibacterial peptide in the pests is obviously reduced, the signal conduction of identifying the immune entomogenous fungi by the pests is weakened or interrupted, and the pests are more likely to infect pathogenic microorganisms such as the entomogenous fungi and the like. The death rate of pests is improved, and the death time is shortened. In addition, the compound can also be used as a synergist, so that the use efficiency and the use range of the microbial insecticide are improved, the insecticidal spectrum is expanded, the occurrence of drug resistance of the microbial insecticide is reduced, and the compound is an IPM advocating comprehensive biological control method. The invention takes the pest immune system as a target to research and develop the fungal active pesticide which can inhibit the pest immune system, is safe to human and livestock and is environment-friendly, effectively improves the natural control effect of pathogenic microorganisms existing in the nature on pests and delays the occurrence of microbial drug resistance.

Drawings

FIG. 1 is the nucleotide and deduced amino acid sequence diagram of the Plutella xylostella GNBP3 gene.

FIG. 2 is a spatiotemporal expression pattern of the plutella xylostella GNBP3 gene.

FIG. 3 is the expression pattern detection of GNBP3 after three fungi infect plutella xylostella.

FIG. 4 shows PCR amplification of GNBP3 gene and double restriction enzyme identification of recombinant plasmid; 1: PCR amplification of GNBP 3; 2: double enzyme digestion identification of the recombinant plasmid T-GNBP 3; 3: carrying out double enzyme digestion identification on BglII/KpnI of the recombinant plasmid pSilent-GNBP 3; 4: xho I/Hind III double restriction of the recombinant plasmid pSilent-GNBP 3.

Fig. 5 is a picture of recombinant beauveria bassiana.

FIG. 6 shows the expression of Plutella xylostella GNBP3 gene after RNAi.

FIG. 7 shows the expression of the plutella xylostella antimicrobial peptides Cecropin1 and Cecropin2 after RNAi.

FIG. 8 shows the expression of the plutella xylostella antimicrobial peptide Cecropin3 and Moricin genes after RNAi.

FIG. 9 shows the expression of the Lysozyme2 and Lysozyme2 genes of Plutella xylostella antimicrobial peptides Lysozyme1 after RNAi.

FIG. 10 is a graph of the mortality statistics of plutella xylostella after RNAi.

FIG. 11 is a statistical chart of mortality of recombinant Beauveria bassiana and microorganisms cooperatively infecting plutella xylostella.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Table 1 primer sequences in the examples of the invention:

example 1 cloning of the Plutella xylostella gram-negative binding protein Gene GNBP3 cDNA sequence

First, experiment method

1. Unigene amplification of the GNBP3 Gene

The sequence of the GNBP3 Unigene was obtained by bioinformatics analysis and screening based on the Plutella xylostella transcriptome database determined in the laboratory, and in order to ensure the correctness of the sequence GNBP3, a pair of primers (GNBP3FUni and GNBP3RUni) of the Unigene were designed, and the sequences of the primers are shown in Table 1.

The method comprises the steps of using diamondback moth cDNA as a template, carrying out PCR amplification on a Unigene sequence, recovering a product, carrying out T cloning, extracting a recombinant plasmid, carrying out double enzyme digestion identification on a pMD18-T-GNBP3, PstI and EcoR I to obtain a recombinant plasmid pMD18-T-GNBP3, and sending the recombinant plasmid pMD18-T-GNBP3 which is subjected to the enzyme digestion identification to the Correct sequence determination of Guangzhou Elegano biotechnology Limited. The sequencing results were compared by DNAMAN software, and sequence analysis was performed by using the online biology website http:// www.ncbi.nlm.nih.GOv/blast, confirming that the Unigene sequence is a partial sequence of GNBP 3.

2. Amplification of full-Length cDNA of GNBP3 Gene

According to the RACE kit, template 5 'RACE cDNA and 3' RACE cDNA are synthesized, two 5 'and 3' RACE primers are respectively designed according to a GNBP3 Unigene sequence with correct sequencing, the 3 'RACE primer is GNBP3-3F1 and GNBP3-3F2, the 5' RACE primer is GNBP3-5R1 and GNBP3-5R2, the primer sequences are shown in a table 1, the 3 'RACE/5' RACE primer is matched with a universal primer, and semi-nested PCR amplification is carried out. T clone 5 'and 3' RACE PCR products, and finally sequencing to identify 5 'and 3' RACE PCR products. And splicing the 5 'RACE product and the 3' RACE product with correct sequencing by using DNAstar software to obtain a cDNA full-length sequence.

In order to ensure the accurate sequence splicing, a pair of specific primers of the full-length open reading frame of the GNBP3 gene, namely GNBP3ORFF and GNBP3ORFR are redesigned, the sequences of the primers are shown in Table 1, the diamondback moth cDNA is taken as a template, RT-PCR amplification is carried out on the full-length cDNA, and after enzyme digestion and PCR identification, the sequence is sequenced to finally confirm the sequence of the GNBP3 cDNA.

Second, experimental results

The full-length cDNA sequence cloned to the diamondback moth GNBP3 by using RT-PCR combined with RACE technology and the deduced amino acid sequence of the GNBP3 are shown in figure 1. As can be seen from FIG. 1, the full-length cDNA sequence of the plutella xylostella GNBP3 gene is 1485bp (the nucleotide sequence is shown as SEQ ID NO: 1), the 5 'UTR is 126bp, the 3' UTR is 60bp, the open reading frame is 1299bp, and 432 amino acid residues are coded (the amino acid sequence is shown as SEQ ID NO: 2). The molecular weight of the protein is 48.36kDa, and the isoelectric point is 5.92. The GNBP3 gene sequence contains a beta-1, 3-glucan recognition site, an N-terminal carbohydrate binding domain, a glycosyl hydrolase region and a laminin G protein region.

Example 2 spatiotemporal expression Pattern of the Plutella xylostella GNBP3 Gene

First, experiment method

Selecting samples of the healthy diamondback moth at different development stages including eggs, larvae, prepupa, pupae and imagoes. Healthy 4-instar plutella xylostella larvae are selected, and 6 tissue samples of the head, the hemolymph, the midgut, the mahalanobis tube, the fat body and the epidermis are collected through ice dissection.

All samples were extracted for total RNA and first strand of synthetic cDNA. Two pairs of fluorescent quantitative PCR primers are respectively designed according to the full-length cDNA sequence of the diamondback moth GNBP3 and the cDNA sequence of the diamondback moth RPS13 gene. The GNBP3 quantitative primers are GNBP3 primers GNBP3-qF and GNBP3-qR, the Rps13 primers are Rps13-qF and Rps13-qR, the primer sequences are shown in Table 1, the RT-qPCR reaction system is 25 mu L: ddH2O 8.5.5 μ L, SYBR Premix ExTaq II 12.5 μ L, upstream primer 1 μ L, downstream primer 1 μ L, template 1 μ L. After the reaction system is prepared, the mixture is mixed evenly and is quickly centrifuged in a short time. The reaction is carried out in a Bio-CFX 96TM instrument, the reaction program is set as a two-step method, and the reaction temperature is set as follows: pre-denaturation at 95 deg.C for 3 min; circulating the temperature at 95 ℃, 10s and 60 ℃, and collecting fluorescence signals; 95 ℃ for 10 s; the melting temperature is 65-95 ℃ for 5s, and signals are collected. After the reaction is finished, the amplification curve and the melting curve of the reaction are firstly analyzed, and whether the data are available is judged. Quantitative result processing adopts

The relative gene expression was calculated and the results were plotted as a bar Graph using the software Graph PadPrism 7. Data variability was analyzed using IBM SPSS Statistics20 software. And detecting the expression quantity of the plutella xylostella GNBP3 in different developmental stages and different tissues by using real-time fluorescent quantitative PCR (qRT-PCR).

Second, experimental results

The transcription levels of the plutella xylostella GNBP3 gene in 4 different developmental stages and 6 different tissues were detected by qRT-PCR, and the results are shown in FIG. 2. As can be seen from FIG. 2A, the Plutella xylostella GNBP3 gene is expressed in 4 instars of the Plutella xylostella, the relative expression level of the GNBP3 gene is high in the pupal stage, and the expression level of the gene is low in the egg stage, namely, the larva and the imago; as can be seen from FIG. 2B, the plutella xylostella GNBP3 gene has the highest expression level in the midgut, followed by the adipose body and epidermis, and the plutella xylostella GNBP3 gene has a low expression level in other tissue sites, such as blood cells, head and Ma tube. And the expression of blood cells is higher than that of the head, and the expression level in the Malpighian tube is the lowest. The results show that the expression level of the plutella xylostella GNBP3 is different at different positions, and the involved functions are different.

Example 3 detection of sensitivity of Plutella xylostella GNBP3 Gene to fungi

First, experiment method

Three entomogenous fungi stored in the laboratory, Beauveria bassiana (Beauveria bassiana), Metarhizium anisopliae (Metarhizium anisopliae) and Isaria fumosorosea (Isaria fumosorosea), were transferred to PDA plates and then cultured in a 25 ℃ incubator (12L:12D) for 7 days. After sporulation, 20mL of 0.05% Tween-80 solution was added to the plate, the spores on the plate were gently scraped with an inoculating loop, the prepared solution was poured into a beaker, and stirred with a magnetic stirrer for 30 min. After the spores are completely dispersed, filtering the bacterial liquid by using double-layer medical gauze to obtain a spore suspension. Counting the mother liquor spore concentration by a hemocytometer, preparing 1.0 × 10 by 0.05% Tween-80 solution⁸CFU/mL spore suspension, ready for use.

Selecting diamondback moth larvae with consistent development age of 4 on the first day, and treating the diamondback moths by a body wall soaking method. The specific method comprises the following steps: soaking 4-year-old diamondback moth in spore suspension (1.0 × 10)⁸CFU/mL) for 10s, sucking off excessive liquid by using filter paper, normally feeding, sampling every 12h until 72h, obtaining plutella xylostella fat bodies by means of dissection on ice, and storing in Trizol. 0.05% Tween 80 treated Plutella xylostella was used as a negative control, and samples were taken from 12-72h, dissected on ice to obtain Plutella xylostella fat bodies, and stored in Trizol. All samples were total RNA extracted and reverse transcribed to first strand cDNA. RT-qPCR is utilized to detect the time expression pattern of the plutella xylostella GNBP3 gene under the induction of three different fungi.

Second, experimental results

The result of RT-qPCR detection of the Plutella xylostella GNBP3 gene is shown in FIG. 3. As can be seen from FIG. 3, the gene transcription level of the three entomogenous fungi strongly inducing the diamondback moth GNBP3 reaches the highest level within 48h, wherein the gene transcription level of the beauveria bassiana inducing the diamondback moth GNBP3 is the best. Within 12-72h, the beauveria bassiana strongly induces the gene transcription level of the diamondback moth GNBP3, the highest peak is brought within 48h, and compared with a control, the gene transcription level is improved by 25 times. At 12-48h, the gene transcription level induction of the isaria fumosorosea to the plutella xylostella GNBP3 is enhanced all the time, and the gene transcription level begins to decrease at 60-72 h. After the metarhizium anisopliae infects the plutella xylostella, the GNBP3 gene expression level is obviously enhanced, and compared with a control, the expression level is improved by 20 times. RT-qPCR detection results show that the three entomogenous fungi can strongly induce the expression of the plutella xylostella GNBP3 gene.

Example 4 construction of RNA interference vector for Plutella xylostella Pattern recognition receptor GNBP3

First, experiment method

Acquisition of Plutella xylostella GNBP3 and GFP fragments

A500 bp sequence is selected according to the conserved region of the GNBP3 gene, two pairs of primers dsGNBP3-F1, dsGNBP3-F2, dsGNBP3-R1 and dsGNBP3-R2 are designed, wherein F1 has an enzyme cutting site XhoI (CTCGAG), R1 has an enzyme cutting site HindIII (AAGCTT), F2 has an enzyme cutting site Bgl II (AGATCT), R2 has an enzyme cutting site Kpn I (GGTACC), and the primer sequences are shown in a sequence table 1. In order to construct pSilent-GFP plasmid, 4 primers, GFP-F1, GFP-F2, GFP-R1 and GFP-R2 were designed, wherein F1 has cleavage site Xho I (CTCGAG), R1 has cleavage site HindIII (AAGCTT), F2 has cleavage site Bgl II (AGATCT), and R2 has cleavage site Kpn I (GGTACC). The specific primer sequences are shown in Table 1, and the target genes GNBP3 and GFP are amplified by RT-PCR according to F1/R1 and F2/R2 pairs. And (3) recovering a PCR product, cloning a target fragment T, transforming a plasmid into an escherichia coli DH5 alpha strain, carrying out PCR verification and double enzyme digestion identification, and finally sending the obtained product to a company for sequencing identification to obtain a positive clone so as to ensure that correct enzyme digestion sites are introduced into two ends of a target fragment.

Second, experimental results

The RT-PCR amplified the product of the Plutella xylostella GNBP3 fragment, and the result is shown in FIG. 4, and the fragment size coincided with the expected size. The GNBP3 fragment was further cloned into a T vector, and the results of double-restriction enzyme identification are shown in FIG. 4. The nucleotide sequence of the target fragment is shown as SEQ ID NO: 3, further sequencing verification ensured that the correct XhoI/Hind III and BglII/KpnI cleavage sites were introduced at both ends of the GNBP3 sequence. For the GFP gene, the protocol was identical to that of GNBP3, again ensuring that the GFP ends of the sequence bear respective XhoI/HindIII and BglII/KpnI cleavage sites. The plasmid with the target genes GNBP3 and GFP was stored at-20 ℃ and the corresponding glycerol was stored at-80 ℃ for further use.

Secondly, construction of recombinant plasmids pSilent-GNBP3 and pSilent-GFP

The correct exogenous fragment GNBP3 (whose nucleotide sequence is set forth in SEQ ID NO: 3) and pSilent-1 plasmid are simultaneously subjected to XhoI/HindIII double enzyme digestion, the gel is cut to recover a target fragment GNBP3 (the plasmid is connected with the pSilent-1 plasmid after enzyme digestion, transformed, verified by PCR, extracted recombinant plasmid is subjected to double enzyme digestion and verification, the result is shown in figure 4. the plasmid with correct enzyme digestion and verification is further sent to a company for sequencing and verification, the recombinant plasmid with correct sequencing and verification is stored in a plasmid and glycerol strain, the recombinant plasmid and the GNBP3 gene are subjected to double enzyme digestion by BglII/KpnI, recovered, connected, transformed, verified by PCR, extracted recombinant plasmid is subjected to BglII/KpnI double enzyme digestion and verification, the company sequencing further confirms that the GNBP3 sequence is correctly introduced into an expression vector pSilent-1, the successful construction of a recombinant interference vector pSilent-GNBP3 is illustrated, and the method of the recombinant vector pSilent-GFP refers to the construction process of pSilent-GNBP 3.

An enzyme digestion reaction system:

the double-restriction reaction system with the restriction enzyme site GNBP3 fragment is 100 μ L: (1) dd H₂O52. mu.L, (2) 30. mu.L of the recovered product, (3) 10. mu.L of 10 XBuffer, (4) 4. mu.L of XhoI (BglII), and (5) 4. mu.L of Hind III (KpnI);

pSilent-1 double enzyme digestion reaction system 100. mu.L: (1) dd H₂O67. mu.L, (2) Psilent plasmid 15. mu.L, (3)10 XBuffer 10. mu.L, (4) XhoI (BglII) 4. mu.L, and (5) Hind III (KpnI) 4. mu.L.

And (3) after enzyme digestion is carried out for 5 hours at 37 ℃, 1.0% agarose gel electrophoresis detection is carried out on the enzyme digestion product, and the plasmid and the PCR fragment are respectively reclaimed after tapping. And (3) connecting the pSilent-1 plasmid digestion product with the PCR product digestion product, wherein the connection system is as follows: (1) PCR product digestion product 14. mu.L, (2) pSilent plasmid digestion product 3. mu.L, (3) T4 DNA Ligase 1. mu.L, and (4) T4 DNA Ligase Buffer 2. mu.L.

The reaction ligation mixture was left at 16 ℃ overnight for ligation. Transforming the ligation product into escherichia coli DH5 alpha competent cells, selecting single clone, shaking bacteria, extracting plasmids, carrying out double enzyme digestion (Xho I/Hind III) to identify the correct recombinant plasmids, and sending the recombinant plasmids to Aiji biotechnology limited for sequencing.

The plasmid with correct sequencing is cut with PCR product with cutting site BglII/Kpn I separately. And (3) carrying out enzyme digestion at 37 ℃ for 5h, and carrying out 1.0% agarose gel electrophoresis recovery on the enzyme digestion product after the reaction is finished. After the digestion product of the plasmid with correct sequencing is connected with the digestion product of the PCR product, the obtained product is transformed into a competent cell of Escherichia coli DH5 alpha, and the competent cell is selected to be monoclonal, shaken, extracted with plasmid, and subjected to double enzyme digestion (Bgl II/Kpn I) to identify the correct recombinant plasmid, and then the recombinant plasmid is sent to Aiji biotechnology Limited for sequencing. And preserving the glycerol bacteria and the recombinant plasmid for later use.

Example 5 verification of the Plutella xylostella RNA interference vector pSilent-GNBP3

First, experiment method

1. Preparation of beauveria bassiana protoplast

Culturing Beauveria bassiana in PDA culture medium at 25 deg.C and relative humidity of 80% in dark for about 3 days; after sporulation, transferring the spores into a 200ml triangular flask containing 100ml PD medium, and carrying out shaking culture at 25 ℃ and 120rpm for 15 h; filtering the cultured bacteria liquid into a 50mL centrifuge tube under the aseptic condition; centrifuging at 44 deg.C and 5000rpm for 10min, and removing supernatant; resuspending the precipitate with 0.7mol/L Na Cl solution, centrifuging at 4 deg.C and 5,000rpm for 8min, and discarding the supernatant; adding the enzymolysis liquid into a 50ml centrifuge tube, and carrying out constant temperature oscillation at 30 ℃ and 100rpm for enzymolysis for 8 h; after the hypha is subjected to enzymolysis, filtering the mixed solution; transferring the filtrate into a new centrifuge tube, and centrifuging at 4 ℃ and 4000rpm for 0 min; discarding the supernatant; resuspending and precipitating 0.7mol/L NaCl solution at 4 ℃, and centrifuging at 4000rpm for 8 min; discarding the supernatant; resuspending the precipitate with 5mL of STC solution at 4 deg.C, centrifuging at 4000rpm for 5min, and discarding the supernatant; adding 1mL of STC solution into a centrifuge tube to obtain the protoplast.

2. Beauveria bassiana protoplast transformed by recombinant plasmid

Adding 10 μ g of recombinant plasmids pSilent-GNBP3 and pSilent-GFP into a mixture containing 1ml of protoplast; adding 1.74mL of PTC solution along the tube wall, and standing on ice for 20 min; adding 2mL of regeneration liquid culture medium into the centrifuge tube, and placing for 2min on ice; adding 4mL of regeneration liquid culture medium into the centrifuge tube, and placing for 2min on ice; the centrifuge tube was charged with 4mL of regeneration broth, 10. mu.L of Amp + (50. mu.g/mL) was added, and regeneration was performed at 25 ℃ and 120rpm at 45 ℃ for about 14 h. The regenerated culture was centrifuged at 4000rpm for 15min at 4 ℃ and the supernatant was discarded to leave about 5mL of liquid in the tube, the regeneration was examined under a microscope, diluted with the regenerated liquid medium as appropriate, 200. mu.L of the solution was applied to a regenerated solid medium (600. mu.g/mL hygromycin B, 50. mu.g/mL Amp +) and cultured in the dark at 25 ℃ for about 3 days.

3. Screening of recombinant Beauveria bassiana

Picking single colony from the cultured plate, culturing on a regeneration solid culture medium in the dark at 25 ℃ for about one week; after hyphae are produced, taking the hyphae as a template, and carrying out PCR amplification by using a primer used when an RNA expression vector is constructed. And (3) carrying out 1.0% agarose gel electrophoresis on the 5 mu L PCR product, and observing whether an expected large and small target band appears under an ultraviolet lamp. The strain with the PCR amplification result is further subjected to sequencing identification by Guangzhou Egyptian biotechnology Limited. The glycerol bacteria are preserved in the fungus beads with the correct sequence and preserved at-80 ℃ for later use.

4. Culture of recombinant Beauveria bassiana

Inoculating original Beauveria bassiana and recombinant Beauveria bassiana (pSilent-GNBP3, pSilent-GFP) on a PDA plate, culturing in an incubator at 25 ℃, after the plate is full of recombinant Beauveria bassiana spores, washing off the spores by 0.05% Tween 80, filtering by double-layer gauze to remove impurities, pouring the prepared solution into a beaker, and stirring for 30min by a magnetic stirrer. After the spores are completely dispersed, filtering the bacterial liquid by using double-layer medical gauze to obtain a spore suspension. Counting the mother liquor spore concentration by a hemocytometer, preparing 1.0 × 10 by 0.05% Tween-80 solution⁶CFU/mL spore suspension, namely the spore suspension of the recombinant beauveria bassiana pSilent-GNBP3 and the spore suspension of the recombinant beauveria bassiana pSilent-GFP, is obtained for later use.

5. Treatment of recombinant Beauveria bassiana and collection of samples

Collecting the same batch of well-developed fourth-instar first-day larvae of diamondback moth, and respectively carrying pSilent-GNBP3 recombinant Beauveria bassiana spore suspension (1 × 10)⁶CFU/ml) and pSilent-GFP recombinant Beauveria bassiana spore suspension (1X 10)⁶CFU/ml) treatment of plutella xylostella. Completely soaking in spore suspension of recombinant strain for 10s, sucking excessive water with filter paper, feeding under normal condition, dissecting coat, midgut and fat of diamondback moth after 12hThree tissues were sampled every 12h until after 60 h. Diluting original Beauveria bassiana spore solution with 0.05% Tween 80 to 1 × 10⁶CFU/ml was used as a negative control containing pSilent-GFP (1X 10)⁶CFU/ml) of recombinant beauveria spores as non-interfering RNA control.

6. Transcriptional expression Pattern of the GNBP3 Gene

Three tissue samples of plutella xylostella are collected, and the total RNA and the first strand of the synthesized cDNA are extracted. Two pairs of fluorescent quantitative PCR primers are respectively designed according to the full-length cDNA sequence of the diamondback moth GNBP3 and the cDNA sequence of the diamondback moth RPS13 gene. The GNBP3 quantitative primers are GNBP3 primers GNBP3-qF and GNBP3-qR, the Rps13 primers are Rps13-qF and Rps13-qR, the primer sequences are shown in Table 1, the RT-qPCR reaction system is 25 mu L: ddH2O 8.5.5 μ L, SYBR Premix ExTaq II 12.5 μ L, upstream primer 1 μ L, downstream primer 1 μ L, template 1 μ L. After the reaction system is prepared, the mixture is mixed evenly and is quickly centrifuged in a short time. The reaction was carried out on a Bio-CFX 96TM apparatus under the following conditions: 95 ℃ for 30s,40 cycles 95 ℃ for 5s,60 ℃ for 10 s. RT-qPCR was used to detect the expression of the GNBP3 gene in three tissues.

7. RT-qPCR detection of expression of antibacterial peptide gene

To further verify whether the GNBP3 gene modulates a terminal effector in the Toll signaling pathway. The respective primer sequences of 6 antibacterial peptide genes of the plutella xylostella, namely Cecropin1, Cecropin2, Cecropin3, Moricin, Lysozyme1 and Lysozyme2, are selected and shown in the table 1. The transcription levels of 6 downstream effector genes in three tissues of the plutella xylostella body are detected by using RT-qPCR.

Second, experimental results

1. Recombinant beauveria bassiana

The correctly identified recombinant Beauveria bassiana pSilent-GNBP3 was cultured in PDA, and the results are shown in FIG. 5, which shows that the growth state of the recombinant Beauveria bassiana pSilent-GNBP3 was good. The result of the spore suspension of the recombinant Beauveria bassiana pSilent-GNBP3 is shown in FIG. 5. The graph shows that the fungal spores are plump, and the experimental plutella xylostella can be treated.

2. Expression pattern detection of plutella xylostella GNBP3

After 4-year-old plutella xylostella is treated by the recombinant beauveria bassiana, the mRNA transcription level of the GNBP3 gene is detected by RT-qPCR, and the result is shown in FIG. 6. As can be seen from fig. 6, the plutella xylostella GNBP3 gene was significantly reduced in the midgut, epidermis and adipose body, with the expression level of the epidermis being most reduced to a very significant level of difference (p < 0.01). In the middle intestine, the GNBP3 gene was expressed at low levels for 12-60h and was inhibited from expression compared to the control. In the epidermis, after the recombinant beauveria bassiana treats the plutella xylostella, the GNBP3 gene is obviously inhibited and expressed within 12-60h, and the difference is extremely obvious (p is less than 0.01); in fat bodies, after the recombinant beauveria bassiana is used for treating the plutella xylostella, the GNBP3 gene is obviously inhibited and expressed within 12-48h, the difference obvious level (p is less than 0.05) is achieved, and the GNBP3 gene is not obviously reduced in 60 h. The result shows that the recombinant beauveria bassiana can interfere with GNBP3, so that the expression of the GNBP3 gene is reduced.

2. Expression pattern detection of diamondback moth antibacterial peptide gene

After 4-year-old plutella xylostella is treated by the recombinant beauveria bassiana, the mRNA transcription level of the antibacterial peptide genes (Cecropin1, Cecropin2, Cecropin3, Moricin, Lysozyme1 and Lysozyme2) is detected by RT-qPCR, and the result is shown in FIGS. 7-9. As can be seen from FIGS. 7 to 9, the transcription levels of the plutella xylostella antimicrobial peptide genes are all reduced, but the expression patterns of the plutella xylostella antimicrobial peptide genes are different. As can be seen from FIG. 7, the expression of the Cecropin1 was strongly down-regulated in both the adipose body and the epidermis of plutella xylostella. Downregulation was greatest in the adipose body, with Cecropin1 downregulating levels up to 95-fold in adipose body and 11-fold in epidermis. The expression of the antibacterial peptide Cecropin2 was strongly down-regulated in all three tissues of plutella xylostella. Cecropin2 down-regulated in fat up to 32-fold, epidermis down-regulated 9-fold and midgut down-regulated 3-fold.

As can be seen from FIG. 8, the expression of the antimicrobial peptides Cecropin3 and Moricin was strongly down-regulated in the fat body, epidermis and midgut of plutella xylostella. Downregulation was greatest in the adipose body, with Cecropin3 downregulating levels up to 50-fold in adipose body and 11-fold in epidermis. The expression of the antibacterial peptide Cecropin2 was strongly down-regulated in all three tissues of plutella xylostella. Cecropin2 down-regulated in fat up to 32-fold, epidermis down-regulated 9-fold and midgut down-regulated 3-fold.

As can be seen from FIG. 9, the expression of the antimicrobial peptides Lysozyme1 and Lysozyme1 was strongly down-regulated in the fat body, epidermis and middle intestine of plutella xylostella. The expression level of the antibacterial peptide genes is strongly inhibited and expressed, particularly in epidermis and fat body, all the antibacterial peptide genes are strongly inhibited and expressed, particularly, the antibacterial peptide genes Cecropin2, Lysozyme1 and Lysozyme2 are strongly inhibited and expressed in fat body, and compared with a control, the expression level of the antibacterial peptide genes reaches more than 30 times, 90 times and 100 times. The PxGNBP3 gene can be inhibited to express, and further inhibit the expression of downstream antibacterial peptide genes.

Example 6 bioassay of the Plutella xylostella RNA interference vector pSilent-GNBP3

First, experiment method

1. Insecticidal effect of recombinant beauveria bassiana with different concentrations

The constructed recombinant beauveria bassiana pSilent-GNBP3 is prepared into a concentration of 10⁴、10⁵、10⁶、10⁷Spore/ml spore suspension.

Selecting the larvae of the 3 rd day of the healthy plutella xylostella which are consistent in development. The prepared spore suspension 10⁴ CFU/ml、10⁵CFU/ml、10⁶CFU/ml、10⁷CFU/ml treated 100 plutella xylostella heads separately. The body wall soaking method is adopted, namely the diamondback moths are completely soaked in respective spore suspension for 10s, the diamondback moths are normally raised after excessive liquid is absorbed by filter paper, the culture is recorded once every 24h, and the death number and survival number of the diamondback moths are recorded every day until 168 h. The same treatment was done with 0.05% tween 80 and beauveria as a control. And (4) counting the survival rate of the diamondback moth under the recombinant beauveria bassiana with different concentrations, independently repeating the experiment for three times, and performing biological statistics.

2. Synergistic effect of recombinant beauveria bassiana on bacillus thuringiensis Bt and serratia marcescens Sm

Inoculating glycerol strain of Serratia marcescens (S.marcescens) and Bacillus thuringiensis (B.thuringiensis) in laboratory to fresh LB culture medium without any resistance, culturing seed strain at 37 deg.C and 240rpm overnight, inoculating seed strain 1:100 to fresh LB culture medium without any resistance, centrifuging at 37 deg.C and 240rpm, OD600 ═ 0.8, 12000g for 10min, collecting thallus, washing thallus with PBS twice, counting mother liquor thallus concentration with hemocytometer, diluting with PBS to 1.0 × 10⁷CFU/mL is ready for use. Will constructThe constructed recombinant beauveria bassiana pSilent-GNBP3 are respectively prepared into the concentration of 10⁴CFU/ml spore suspension. Selecting the larvae of the 3 rd day of the healthy plutella xylostella which are consistent in development. The prepared spore suspension 10⁴CFU/ml processed 200 plutella xylostella. Soaking diamondback moth in the spore suspension for 10s, sucking off excessive liquid with filter paper, and culturing for 24 hr, and treating with 0.05% Tween 80 and Beauveria bassiana. The insect body is further fed with Bacillus thuringiensis (1.0 × 10)⁷CFU/ml) and Serratia marcescens (1.0X 10)⁷CFU/ml). The feeding method comprises the following steps: the bacteria were separately cultured in the ratio of 1: 4 (mL: g), namely 4g of feed, is added with 1mL of bacterial liquid to breed the plutella xylostella, and new feed mixed with the bacterial liquid is added every day. The number of dead and alive plutella xylostella was recorded every 12h, up to 84 h. For the control group of diamondback moth, after the diamondback moth is treated with 0.05% Tween 80 and beauveria bassiana for 24h, the same feed ratio is used to contain Bacillus thuringiensis (1.0 × 10) in reference to the method for recombining beauveria bassiana⁷CFU/ml) and Serratia marcescens (1.0X 10)⁷CFU/ml) were fed to Plutella xylostella, and statistics of Bacillus thuringiensis (1.0 × 10) were performed⁷CFU/ml) and Serratia marcescens (1.0X 10)⁷CFU/ml), the experiment was repeated three times independently, and biological statistics were performed.

Second, experimental results

1. Bioassay of different concentrations of recombinant Beauveria bassiana treated plutella xylostella

The prepared spore suspension 10⁴CFU/ml、10⁵CFU/ml、10⁶CFU/ml、10⁷CFU/ml respectively treats 100 plutella xylostella, counts the death number of the plutella xylostella in 7 days, and performs biological statistics, and the result is shown in figure 10. From FIG. 10, it can be seen that the survival rate of the diamondback moth can be significantly reduced by the recombinant beauveria bassiana 10 under the treatment of the diamondback moth at four concentrations⁷After the diamondback moth is treated by CFU/ml, the death rate is highest, and the time is shortest. The concentration of the recombinant beauveria bassiana strain is 10⁴The survival rate of spore suspension of spore/ml is 0, day 7, and the concentration of recombinant Beauveria bassiana strain is 10⁵Spores/mlThe time period after the suspension treatment at which the survival rate was 0 was 6 days, the concentration of the recombinant Beauveria bassiana strain was 10⁶The time at which the survival rate after treatment with the spore suspension of spores/ml was 0 was day 5; the concentration of the recombinant beauveria bassiana strain is 10⁷Spore/ml spore suspension treatment survival rate 0 time 4 days, and original beauveria bassiana concentration 10⁴、10⁵、10⁶、10⁷The survival rates of sporulated diamondback moth on 7 days are 58.33%, 48.7%, 41.18% and 30.17%, respectively, and the concentration of the original beauveria bassiana spore suspension is 10 to make the survival rate of diamondback moth on 5 days be 0⁹Spores/ml. The results show that compared with the original beauveria bassiana, the recombinant strain GNBP3 has a greater influence on the survival rate of the plutella xylostella and can significantly shorten the death time of the plutella xylostella.

2. Synergistic effect of recombinant beauveria bassiana on other microorganisms

Preparing spore suspension 10 from recombinant Beauveria bassiana pSilent-GNBP3 and original Beauveria bassiana⁴CFU/ml, respectively treating 200 larvae of 3 rd larva, namely diamondback moth on the first day. Meanwhile, 200 heads of plutella xylostella of healthy 3-instar larvae on the first day are selected, treated by 0.05% of Tween 80, and after 24 hours, the death number and survival number are counted. And (4) selecting 100 diamondback moths from the three treatments, feeding the diamondback moths with the bacteria-carrying feed every 12 hours until 84 hours, and recording the survival number and the death number of the diamondback moths. The cumulative death number of the plutella xylostella after 84h is counted, and biological statistics are carried out, and the result is shown in figure 11. As can be seen from FIG. 11, the insecticidal effects of the two microorganisms can be significantly improved by the recombinant Beauveria bassiana pSilent-GNBP3, and the synergistic effect of the recombinant Beauveria bassiana pSilent-GNBP3 on the Bacillus thuringiensis Bt is better than that of the Serratia marcescens Sm. Spore suspension 10 in recombinant Beauveria bassiana pSilent-GNBP3⁴The survival rate of the diamondback moth treatment is 0 at the 7 th day when the diamondback moth is treated by CFU/ml. When the spore suspension 10 of the recombinant beauveria bassiana pSilent-GNBP3 is cultured⁴When the diamondback moth is treated by CFU/ml and Bt, the treatment survival rate of the diamondback moth is 0 at the 4 th day, and the concentration of the recombinant beauveria bassiana strain is 10⁷The time to treatment of spore suspension at 0 viability for spores/ml was consistent at day 4. At this time, the process of the present invention,the survival rate of the plutella xylostella treated by the Bt alone is 30%, which shows that the combination of the recombinant beauveria bassiana pSilent-GNBP3 spore suspension and the Bt can greatly improve the insecticidal effect and play a good synergistic effect. When the recombinant beauveria bassiana pSilent-GNBP3 spore suspension 10⁴When the diamondback moth is treated by the CFU/ml and the serratia marcescens Sm together, the treatment survival rate of the diamondback moth is 0 for 4.5 days, and the concentration of the recombinant Beauveria bassiana strain is 10⁶The time to kill the pests was half a day shorter than the 5 th day when the spore suspension at spore/ml was treated for a survival rate of 0. The result shows that when the recombinant beauveria bassiana pSilent-GNBP3 spore suspension is combined with the serratia marcescens Sm, the insecticidal effect is greatly improved, and the composition is a good synergist. Therefore, the recombinant beauveria bassiana not only can independently improve the insecticidal effect, but also can be used as a synergist to improve the microbial insecticide, thereby enlarging the insecticidal range and being environment-friendly.

Sequence listing

<110> southern China university of agriculture

<120> recombinant fungus expressed by targeted silent pest pattern recognition protein GNBP3 gene and application thereof in pest control

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1485

<212> DNA

<213> Plutella xylostella

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agagcatctg tacttgtttt gtgcgttttt gctattcatt tcgtgactgg tcaatctgga 60

caatctggac acgcgtatga aatacctgat gtgcgagtac aagtgtttca acctaaagga 120

ttgaggatgt caataccaga cacaccaggc ataggcctga tggcgatcca gctgaacctg 180

aacagaaaaa tagcttcccc aatcgtcggc gagtacagcg gggaggtcct atctccagtc 240

aacggcagat ggacttacga ccttcccgac ttagaactga agatggatga cgtcatcaac 300

tattacatct tcgtctcctt caacgggaaa ggatacctga aagacgactt gacgtttaaa 360

gtcagtaagt tcgacaatga aatgcaagaa accacgttgc cgtcagtgca agagtgtcgt 420

ccatcagtga ccacggtcct gggcaagccg gctcgctgcg cgggagacac cgtcttctat 480

gacgacttcg agtcactgga cacagcgtac gtttggcaga cggaacacaa gattgctttg 540

gatcatcctg aatacccgtt tatttcgtac cagcgttcag cggtggaagt ccaaaaaggc 600

aatttaatca taaagccgag gttacagcaa actgaacctt ctttcaatgc tgacagcatg 660

tacagtgggg cactgaattt aaacgactgg tgcactgggc cgccgatgac gtgtgagatg 720

tccgcgtcgg gggtcaacgt cctgccgccc gtgctctccg gcagggtcac gaataaaaac 780

ttcaccttca gatacggaac cgtcgagatc cgggccaagt tgccgcaggg ggactggctg 840

tatccagaat tacttctggt tccgtttact cataagtacg gcgtcgctcc atatgcttcc 900

ggattaatca aaattgctac agccagaggc aacttgaatc tttccagcag ttctcaagat 960

tacggcaata aggtgctgta cggaggcccg atcctcgagc gccggtgccg cgacgcgctc 1020

cagcgcactc acgcggtgac tacagaagag tgggggaaag atttccacac gtattctttg 1080

aaatggacac cagacctcat agtactgaaa gtagatggca tcgaatggtc ccggtacagt 1140

cctggtgaca agggcatggt gggacttcta ccggagagct gtcagacgtc agctgtcaga 1200

taccaggccg gaaccaagat ggcgcctttt gaccaagaat ttaccctatc cctggggcta 1260

gcggcgggtg gtgtgaccga gttccctgac ctgcctcaga agccctggag agactcagcc 1320

agcaaagcca agctccactt ctggcaagct ctggactcct ggtaccagac ctggggacaa 1380

aacgctaggc ttgtcgtgaa ttatgtcaaa gttactgctg tttaggatta aaatacctat 1440

tgtttttcaa atgtgaaaga aaaaaaaaaa aaaaaaaaaa aaaaa 1485

<210> 2

<211> 432

<212> PRT

<213> Plutella xylostella

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Met Ser Ile Pro Asp Thr Pro Gly Ile Gly Leu Met Ala Ile Gln Leu

1 5 10 15

Asn Leu Asn Arg Lys Ile Ala Ser Pro Ile Val Gly Glu Tyr Ser Gly

20 25 30

Glu Val Leu Ser Pro Val Asn Gly Arg Trp Thr Tyr Asp Leu Pro Asp

35 40 45

Leu Glu Leu Lys Met Asp Asp Val Ile Asn Tyr Tyr Ile Phe Val Ser

50 55 60

Phe Asn Gly Lys Gly Tyr Leu Lys Asp Asp Leu Thr Phe Lys Val Ser

65 70 75 80

Lys Phe Asp Asn Glu Met Gln Glu Thr Thr Leu Pro Ser Val Gln Glu

85 90 95

Cys Arg Pro Ser Val Thr Thr Val Leu Gly Lys Pro Ala Arg Cys Ala

100 105 110

Gly Asp Thr Val Phe Tyr Asp Asp Phe Glu Ser Leu Asp Thr Ala Tyr

115 120 125

Val Trp Gln Thr Glu His Lys Ile Ala Leu Asp His Pro Glu Tyr Pro

130 135 140

Phe Ile Ser Tyr Gln Arg Ser Ala Val Glu Val Gln Lys Gly Asn Leu

145 150 155 160

Ile Ile Lys Pro Arg Leu Gln Gln Thr Glu Pro Ser Phe Asn Ala Asp

165 170 175

Ser Met Tyr Ser Gly Ala Leu Asn Leu Asn Asp Trp Cys Thr Gly Pro

180 185 190

Pro Met Thr Cys Glu Met Ser Ala Ser Gly Val Asn Val Leu Pro Pro

195 200 205

Val Leu Ser Gly Arg Val Thr Asn Lys Asn Phe Thr Phe Arg Tyr Gly

210 215 220

Thr Val Glu Ile Arg Ala Lys Leu Pro Gln Gly Asp Trp Leu Tyr Pro

225 230 235 240

Glu Leu Leu Leu Val Pro Phe Thr His Lys Tyr Gly Val Ala Pro Tyr

245 250 255

Ala Ser Gly Leu Ile Lys Ile Ala Thr Ala Arg Gly Asn Leu Asn Leu

260 265 270

Ser Ser Ser Ser Gln Asp Tyr Gly Asn Lys Val Leu Tyr Gly Gly Pro

275 280 285

Ile Leu Glu Arg Arg Cys Arg Asp Ala Leu Gln Arg Thr His Ala Val

290 295 300

Thr Thr Glu Glu Trp Gly Lys Asp Phe His Thr Tyr Ser Leu Lys Trp

305 310 315 320

Thr Pro Asp Leu Ile Val Leu Lys Val Asp Gly Ile Glu Trp Ser Arg

325 330 335

Tyr Ser Pro Gly Asp Lys Gly Met Val Gly Leu Leu Pro Glu Ser Cys

340 345 350

Gln Thr Ser Ala Val Arg Tyr Gln Ala Gly Thr Lys Met Ala Pro Phe

355 360 365

Asp Gln Glu Phe Thr Leu Ser Leu Gly Leu Ala Ala Gly Gly Val Thr

370 375 380

Glu Phe Pro Asp Leu Pro Gln Lys Pro Trp Arg Asp Ser Ala Ser Lys

385 390 395 400

Ala Lys Leu His Phe Trp Gln Ala Leu Asp Ser Trp Tyr Gln Thr Trp

405 410 415

Gly Gln Asn Ala Arg Leu Val Val Asn Tyr Val Lys Val Thr Ala Val

420 425 430

<210> 3

<211> 498

<212> DNA

<213> Plutella xylostella

<400> 3

acgaataaaa acttcacctt cagatacgga accgtcgaga tccgggccaa gttgccgcag 60

ggggactggc tgtatccaga attacttctg gttccgttta ctcataagta cggcgtcgct 120

ccatatgctt ccggattaat caaaattgct acagccagag gcaacttgaa tctttccagc 180

agttctcaag attacggcaa taaggtgctg tacggaggcc cgatcctcga gcgccggtgc 240

cgcgacgcgc tccagcgcac tcacgcggtg actacagaag agtgggggaa agatttccac 300

acgtattctt tgaaatggac accagacctc atagtactga aagtagatgg catcgaatgg 360

tcccggtaca gtcctggtga caagggcatg gtgggacttc taccggagag ctgtcagacg 420

tcagctgtca gataccaggc cggaaccaag atggcgcctt ttgaccaaga atttacccta 480

tccctggggc tagcggcg 498

<210> 4

<211> 166

<212> PRT

<213> Plutella xylostella

<400> 4

Thr Asn Lys Asn Phe Thr Phe Arg Tyr Gly Thr Val Glu Ile Arg Ala

1 5 10 15

Lys Leu Pro Gln Gly Asp Trp Leu Tyr Pro Glu Leu Leu Leu Val Pro

20 25 30

Phe Thr His Lys Tyr Gly Val Ala Pro Tyr Ala Ser Gly Leu Ile Lys

35 40 45

Ile Ala Thr Ala Arg Gly Asn Leu Asn Leu Ser Ser Ser Ser Gln Asp

50 55 60

Tyr Gly Asn Lys Val Leu Tyr Gly Gly Pro Ile Leu Glu Arg Arg Cys

65 70 75 80

Arg Asp Ala Leu Gln Arg Thr His Ala Val Thr Thr Glu Glu Trp Gly

85 90 95

Lys Asp Phe His Thr Tyr Ser Leu Lys Trp Thr Pro Asp Leu Ile Val

100 105 110

Leu Lys Val Asp Gly Ile Glu Trp Ser Arg Tyr Ser Pro Gly Asp Lys

115 120 125

Gly Met Val Gly Leu Leu Pro Glu Ser Cys Gln Thr Ser Ala Val Arg

130 135 140

Tyr Gln Ala Gly Thr Lys Met Ala Pro Phe Asp Gln Glu Phe Thr Leu

145 150 155 160

Ser Leu Gly Leu Ala Ala

165

Claims

1. Any one of a diamondback moth pattern recognition receptor protein gene GNBP3, a fragment of the diamondback moth pattern recognition receptor protein gene GNBP3 and a diamondback moth pattern recognition receptor protein GNBP3 is used as a pest control target;

the nucleotide sequence of the diamondback moth pattern recognition receptor protein gene GNBP3 is shown as SEQ ID NO: 1, and the amino acid sequence of the diamondback moth pattern recognition receptor protein GNBP3 is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the fragment of the diamondback moth pattern recognition receptor protein gene GNBP3 is shown as SEQ ID NO: 3, respectively.

2. A recombinant expression vector capable of suppressing transcription of a fragment of the diamondback moth pattern recognition receptor protein gene GNBP3or the diamondback moth pattern recognition receptor protein gene GNBP3 according to claim 1 or silencing the diamondback moth pattern recognition receptor protein gene GNBP3 according to claim 1.

3. A recombinant bacterium carrying the recombinant expression vector of claim 2.

4. Use of the recombinant expression vector of claim 2 or the recombinant bacterium of claim 3 for controlling pests.

5. A method for controlling pests, characterized by inhibiting transcription of a fragment of the diamondback moth pattern recognition receptor protein gene GNBP3or the diamondback moth pattern recognition receptor protein gene GNBP3 according to claim 1.

6. The use of the recombinant bacterium of claim 3 as a synergist in the preparation of pesticides or in the control of pests.