CN104911201B - A kind of genetic modification method improving baculoviral insecticidal efficiency - Google Patents

Abstract

The invention relates to a genetic transformation method for improving the insecticidal efficiency of baculovirus, belonging to the field of genetic engineering; the invention uses pEGFP-N1 carrier as a template to amplify the EGFP gene, and after double enzyme digestion, it is connected into the plasmid pFastBacDUAL to obtain the recombinant plasmid pDUAL-N1 EGFP; according to the sequence of Clbi138 gene and its open reading frame, design and synthesize primers; use the Genomic DNA of Hornmotha nucleopolyhedrosis virus as a template, carry out PCR amplification, recover the target fragment, and connect it into the same double-digested DNA after double-enzyme digestion. The recombinant plasmid pDUAL‑EGFP was obtained as the recombinant plasmid pDUAL‑EGFP‑Clbi138; transformed into an Escherichia coli strain containing the silkworm nuclear polyhedrosis virus, cultured and purified, inoculated in LB liquid medium, shaken and cultured, and the recombinant BmNPV DNA was extracted; and passed the experiment for the first time It is confirmed that the insecticidal efficiency of recombinant BmNPV is significantly enhanced, the half-lethal concentration is 11 times lower than that of the control group, the half-lethal time is 42.9% shorter than that of the control group, and the liquefaction of the pests is more serious, so as to improve the control effect of insect pests and have obvious economic benefits. And ecological benefits, has broad application prospects.

Description

A genetic modification method for improving the insecticidal efficiency of baculovirus

technical field

The invention relates to a genetic modification method for improving the insecticidal efficiency of baculovirus, which belongs to the field of genetic engineering.

Background technique

The use of viruses to control pests not only has a good control effect, but also is safe for humans and animals, safe for natural enemies, does not pollute the environment, and does not make pests resistant to pesticides. In addition, the virus can accumulate in the environment, form an epidemic in the pest population and control the pest population density for a long time, which has obvious economic and ecological benefits, and is currently an ideal and promising biopesticide.

Baculovirus insecticides have become a hotspot in biological control research due to their advantages of not polluting the environment, not destroying ecological balance, being safe to natural enemies and humans and animals, not easy to produce resistance to pests, and being able to control pest populations in the field for a long time. It has broad application prospects. However, the disadvantages such as slow insecticidal speed restrict its further popularization and application. Genetic engineering technology provides a potential possibility to enhance the insecticidal effect of baculovirus. At present, research on improving the insecticidal toxicity of recombinant baculoviruses mainly focuses on the diuretic hormone (DH) gene, juvenile hormone ester (JHE) gene, ecdysone (MH) gene, chitinase (Chitinase) gene, synergistic protein (enhancin) gene, Bt gene, neurotoxin gene, plant protease inhibitor gene etc. not ideal.

Nuclear polyhedrosis virus (NPV) has high pathogenicity to Lepidoptera larvae, is the most potential among insect virus types, and is also the most widely used type of research. At present, most of the commercially produced insect virus preparations at home and abroad are NPV (Nuclear polyhedrosis virus insecticide development. Zuo Shuanyan et al. Journal of Shanxi Agricultural University. 1998, 1(4): 302-305 ). Bombyx mori nuclear polyhedrosis virus (BmNPV) belongs to the genus Baculovirus A, is a type of baculovirus, and is also one of the baculoviruses with a wide host domain. It can infect a variety of agricultural and forestry pests. In order to make baculovirus an ideal biological insecticide, the most effective and promising way to improve the insecticidal effect of baculovirus is to modify the wild-type virus by using biotechnology.

Clanis bilineata nucleopolyhedrovirus; Shan-YingZhu, Jian-Ping Yi, Wei-De Shen, Li-Qun Wang, Hua-Gang He, Yong Wang, Bing Liand Wen-Bing Wang. Genomic sequence, organization and characteristics of a new nucleopolyhedrovirus isolated from Clanis bilineata larva, BMC Genomics, 2009, 10:91) the Clbi138 gene (the accession number in GenBank is DQ504428) has a full length of 3111 bases, and its deduced protein sequence has a full length of 1036 Amino acids have a signal peptide (SP) and a transmembrane domain (TM) at the N-terminus and C-terminus respectively, and there is a metalloprotease characteristic sequence inside, that is, the HEXXH module related to zinc ion binding. A search of baculoviruses whose whole genomes have been sequenced revealed that Clbi138 homologues are conserved in all class II α-baculoviruses, and also found in Ameppox virus (AmEPV) and others. However, the functions of Clbi138 and its homologues are still unclear.

The invention provides a method for improving insecticidal efficiency of the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) by using the Clbi138 gene of the bean moth nucleopolyhedrosis virus (BmNPV). There is no Clbi138 homology in the silkworm nuclear polyhedrosis virus (BmNPV) genome Therefore, the Clbi138 gene was introduced into BmNPV to study the function of Clbi138, and it was first confirmed by experiments that the Clbi138 gene improved the insecticidal efficiency of baculovirus, and the importance of the HEXXH motif to the biological function of the Clbi138 protein was confirmed by site-directed mutagenesis studies, thus It was confirmed that the Clbi138 gene encodes a metalloprotease.

Contents of the invention

The object of the invention is to disclose a genetic modification method for improving the insecticidal efficiency of baculovirus by utilizing the Clbi138 gene of the bean hornworm nuclear polyhedrosis virus.

The Clbi138 gene (GenBank accession number is DQ504428, shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2) from bean hornworm nuclear polyhedrosis virus (ClbiNPV) according to the present invention comes from soybean The hawkmoth nuclear polyhedrosis virus, the open reading frame (ORF) of the gene is 3111 bases long, encodes 1036 amino acids, and contains the HEXXH motif. The site-directed mutation study of the HEXXH motif has confirmed that the Clbi138 gene has metal protease activity.

Clbi138 and its homologues are widely found in class II α-baculoviruses, but their functions are still unclear. Since Clbi138 and its homologues all have the conserved HEXXH motif associated with metalloproteases, it is easy to speculate that they also have similar function; other sequences in Clbi138 and its homologues are also easily substituted, resulting in mutants that still have biological activity.

The present invention adopts the following technical solutions to achieve the above object:

The present invention provides a method for improving the insecticidal efficiency of baculovirus by using the gene of the bean hornworm nucleopolyhedrosis virus Clbi138 . The method mainly includes the following steps:

(1) Using the pEGFP-N1 vector as a template, the EGFP gene was amplified by PCR technology, digested with KpnI and XhoI, and then ligated into the donor plasmid pFastBacDUAL to form the recombinant plasmid pDUAL-EGFP;

(2) Design and synthesize a pair of primers P3 (SEQ ID NO.5) and P4 (SEQ ID NO.6) according to the open reading frame of the Clbi138 gene, the underlined parts are BamHI and KpnI restriction sites; redesign according to the Clbi138 gene One primer P5 (SEQ ID NO.7), the underlined part is the restriction site of HindIII.

(3) Using the genomic DNA of the bean hornworm nuclear polyhedrosis virus (ClbiNPV) as a template and using P3/P5 as primers for PCR amplification, the target fragment was recovered by agarose gel electrophoresis and double-digested by BamHI and HindIII Afterwards, the recombinant plasmid pDUAL-EGFP was connected with the same double digestion to form the recombinant donor plasmid pDUAL-EGFP-Clbi138.

(4) Take pDUAL-EGFP and pDUAL-EGFP-Clbi138 to transform Escherichia coli DH10B strain containing BmNPV respectively, spread on LB plate medium, culture statically to make the blue spots fully develop color, pick white colonies, and further culture on LB plates Purify by streaking on the substrate; inoculate the purified white colonies in LB liquid medium containing kanamycin, tetracycline, and gentamicin, culture with shaking at 37°C for 18-20 hours, and extract recombinant BmNPV DNA (vBm ^EGFP , vBm ^{EGFP/ Clbi138} ).

Beneficial effects of the present invention:

In this method, after the Clbi138 gene (SEQ ID NO.1) from the bean hornworm nucleopolyhedrosis virus (ClbiNPV) was introduced into the silkworm nucleopolyhedrosis virus (BmNPV), the insecticidal efficiency of the recombinant BmNPV was significantly enhanced, and the half-lethal concentration ( LC ₅₀ ) was 12.51 pfu, which was 11 times lower than that of the control group, and the median lethal time (LT ₅₀ ) was 84.12 h, which was 42.9% shorter than that of the control group, and the liquefaction in the pest body was more serious. Therefore, the Clbi138 gene and its encoded protein have important application value in the field of pest control.

The Clbi138 described in the present invention is a metalloprotease that can participate in the degradation of tissues in the body of diseased insects. Introducing the Clbi138 gene into the silkworm nuclear polyhedrosis virus (BmNPV) can significantly reduce the half-lethal concentration of the recombinant virus, shorten the half-lethal time of the pest, and accelerate the degradation of the internal tissue of the pest, so as to improve the control effect of the pest, and has obvious economic benefits. And ecological benefits, has broad application prospects.

Description of drawings

Figure 1: RT-PCR results of Clbi138 gene. M: DL2000 DNA marker; 1~8 are RT-PCR products of Clbi138 gene, and the time of ClbiNPV infection larvae is 0h, 4h, 8h, 12h, 24h, 48h, 72h, 96h; 9~16 are RT-PCR products of Clbipoh gene PCR products, the infection time is 0h, 4h, 8h, 12h, 24h, 48h, 72h, 96h.

Figure 2: Site-directed mutagenesis of the Clbi138 HEXXH motif. The picture shows the sequencing identification of mutation sites created by gene site-directed mutagenesis technology.

Figure 3: Bombyx mori killed by baculovirus infection. A: vBm ^EGFP/Clbi138 infected silkworm (24h after death);

B: Bombyx mori infected with vBm ^EGFP (24 hours after death, control group).

Figure 4: Virological effects of Clbi138 HEXXH motif mutations. 1-4: Bombyx mori infected with vBm ^EGFP , vBm ^{EGFP/Clbi138M1} , vBm ^{EGFP/Clbi138M2} and vBm ^{EGFP/Clbi138M3} for 3 days (no dead individuals), 5: Bombyx mori that had just died after 3 days of infection with vBm ^EGFP/Clbi138 .

Figure 5: Multiple sequence alignment of Clbi138 and its homologues (partial). The shaded shows the conserved sequence, and the double underline shows the highly conserved HEXXH motif, which is the active center of the metalloprotease.

Detailed ways

Analysis of the expression pattern of Clbi138 gene

A pair of primers P1 (SEQ ID NO.3) and P2 (SEQ ID NO.4) were designed and synthesized according to the sequence of the Clbi138 gene of the ClbiNPV genome (the accession number in GenBank is DQ504428).

Trizol reagent was used to extract the RNA of the midgut of Mangnathus moth infected by ClbiNPV (the eggs were collected in the field and reared artificially), and then analyzed by RT-PCR. The PCR reaction conditions used were: 94°C for 4min; 94°C for 20s, 56°C for 30s , 72°C for 1min, 30 cycles; 72°C for 5min. The Clbi138 gene was slightly transcribed 12 hours after the virus infected the host, and the transcription time was 12 hours earlier than the polyhedron (Clbipoh) gene in the ClbiNPV genome. As the infection time prolongs, the transcription of the Clbi138 gene gradually increased (see Figure 1).

2. Subcellular localization analysis of Clbi138 protein

Using the pEGFP-N1 vector (purchased from TAKARA Company) as a template, the EGFP (Enhanced Green Fluorescent Protein) gene was amplified by PCR technology, digested with KpnI and HindIII, and then ligated into the donor plasmid pFastBac1 (purchased from Invitrogen Company), The recombinant plasmid pFB-EGFP was formed.

According to the open reading frame (ORF) of Clbi138 gene, a pair of primers P3 (SEQ ID NO.5) and P4 (SEQ ID NO.6) were designed and synthesized. The underlined part is the restriction site of BamHI and KpnI.

PCR amplification was performed using ClbiNPV genomic DNA as a template. The PCR reaction conditions were: 94°C for 4min; 94°C for 20s, 50°C for 30s, 72°C for 3min, 32 cycles; 72°C for 5min. After 1.0% agarose gel electrophoresis, the target fragment was recovered by cutting the gel, and after double digestion with BamHI and KpnI, it was connected into the above-mentioned recombinant plasmid pFB-EGFP that had been cut with the same double digestion to form the recombinant donor plasmid pFB-Clbi138-EGFP, 48 hours after the transfection of BmN cells, the green fluorescent signal was located around the nucleus when observed under a laser confocal microscope. According to the nuclear area stained with DAPI, the Clbi138 protein could be localized in the nuclear membrane of BmN cells.

Construction of Recombinant Baculovirus Containing Clbi138 Gene

Using the pEGFP-N1 vector (purchased from TAKARA) as a template, the EGFP gene was amplified by PCR technology, digested with KpnI and XhoI, and then ligated into the donor plasmid pFastBacDUAL (purchased from Invitrogen) to form the recombinant plasmid pDUAL-EGFP.

There is no Clbi138 homologue in the silkworm nuclear polyhedrosis virus (BmNPV) genome, so the Clbi138 gene can be introduced into BmNPV to study the function of Clbi138.

A primer P5 (SEQ ID NO.7) was designed based on the Clbi138 gene, and the underlined part is the restriction site of HindIII.

Using ClbiNPV genomic DNA as a template and P3/P5 as primers for PCR amplification, the PCR reaction conditions were: 94°C for 4min; 94°C for 20s, 50°C for 30s, 72°C for 3min, 32 cycles; 72°C for 5min. After 1.0% agarose gel electrophoresis, the target fragment was recovered by cutting the gel, and after double digestion with BamHI and HindIII, it was connected into the above-mentioned recombinant plasmid pDUAL-EGFP that had been cut with the same double enzymes to form the recombinant donor plasmid pDUAL-EGFP-Clbi138, The EGFP gene is located downstream of the p10 promoter, and the Clbi138 gene is located downstream of the polyhedron gene (PH) promoter.

Take 5 μL of pDUAL-EGFP and pDUAL-EGFP-Clbi138 to transform the Escherichia coli DH10B strain containing BmNPV, spread it on the LBac plate, and culture it statically at 37°C for 24-48 h, so that the color of the blue spot is fully developed, and the white colony is picked, and further in the Streak purification on LBac plates. The purified white colony was inoculated in LB liquid medium (containing kanamycin 50 µg/mL, tetracycline 50 µg/mL, gentamicin 70 µg/mL), cultured with shaking at 37°C for 18-20 h, and extracted recombinant BmNPV DNA (vBm ^EGFP , ^{vBmEGFP /Clbi138} ). Using liposome transfection method, transfect BmN cells with recombinant BmNPV ( ^vBmEGFP , ^{vBmEGFP/Clbi138} ), continue to culture for 3~5 days, collect cell supernatant by low-speed centrifugation, and collect BV particles in the supernatant. With the aid of the green fluorescent signal, the titers of the two recombinant viruses were determined by the terminal termination method, and they were 1.2×10 ⁷ PFU/mL and 1.6×10 ⁷ PFU/mL, respectively.

Site-directed Mutation of HEXXH Motif in Clbi138 Gene

According to the Clbi138 gene sequence, three sets of primers P6 (SEQ ID NO.8) and P7 (SEQ ID NO.9), P8 (SEQ ID NO.10) and P9 (SEQ ID NO.11), P10 (SEQ ID NO. .12) and P11 (SEQ ID NO.13). Gene site-directed mutagenesis was performed by overlapping PCR.

Use the wild-type Clbi138 gene as a template, amplify with primers P6 and P5, and recover the target fragment 138M1-1 from the agarose gel; amplify with primers P3 and P7, and recover the target fragment 138M1-1 from the agarose gel 2. The products 138M1-1 and 138M1-2 were recovered by mixing gels in equal proportions, and amplified with P6 and P7 to obtain the mutated Clbi138M1 gene.

In the same way, primers P8 and P9 were used to create a mutated Clbi138M2 gene, and primers P10 and P11 were used to create a mutated Clbi138M3 gene.

The three mutant genes Clbi138M1 , Clbi138M2, and Clbi138M3 were sequenced and identified, all of which contained expected point mutation sites (see Figure 2). In the coding products of the three mutant genes, the HEXXH motifs were mutated into AEXXH, HAXXH, and HEXXA, respectively.

According to the method in step 3, respectively introduce the three mutated Clbi138 genes into the BmNPV containing the EGFP gene to form recombinant viruses vBm ^{EGFP/Clbi138M1} , vBm ^{EGFP/Clbi138M2} , and vBm ^{EGFP/Clbi138M3} .

Verification that Clbi138 gene can enhance the insecticidal efficiency of recombinant baculovirus

Five instar silkworms were injected with the same titer of recombinant virus BV (vBm ^EGFP , vBm ^EGFP/Clbi138 , vBm ^{EGFP/Clbi138M1} , vBm ^{EGFP /Clbi138M2} , vBm ^{EGFP/Clbi138M3} ), and each silkworm was injected with 1×10 ⁴ PFU, each group Inject 50 heads, add mulberry leaves to raise normally, observe and count the number of deaths.

^The results showed that the half time _of death (LT ₅₀ ) of the silkworm infected with vBm ^EGFP/Clbi138 was 84.12 hours, and the total death time was 88 hours. The expression of Clbi138 gene can shorten LT ₅₀ by 63h, that is, the death time of silkworm can be shortened by 42.9% (see Table 1).

Observation of silkworms that died for 24 hours also found that the tissue liquefaction of silkworms infected with vBm ^EGFP/Clbi138 was more serious (see Figure 3).

Therefore, the mechanism by which Clbi138 protein shortens the death time may be that Clbi138 protein exerts metalloprotease activity and severely damages the host tissue, thereby leading to the rapid death of the host.

In addition, when five-instar silkworms were injected with different numbers of virus particles (10, 20, 50, 100, 200, 500 pfu), the half-lethal concentration (LC ₅₀ ) of vBm ^{EGFP /Clbi138} was 12.51 pfu, while the LC ₅₀ of the vBm ^EGFP control group was 12.51 pfu. It is 141.21 pfu (see Table 1).

It can be seen that Clbi138 can greatly enhance the toxicity of baculovirus, and has potential application value in pest control.

In summary, it was found that the insecticidal efficiency of the three recombinant viruses vBm ^{EGFP/Clbi138M1} , vBm ^{EGFP /Clbi138M2} , and vBm ^{EGFP/Clbi138M3} containing the mutated Clbi138 gene was not significantly different from that of the vBm ^EGFP control group (see Figure 4), indicating that the HEXXH model The mutations of the three conserved sites in the sequence will lead to the loss of the function of the Clbi138 gene. The HEXXH motif is the active center of metalloproteases. The expression of wild-type Clbi138 can accelerate the degradation of tissues in the pest, while the variation of the HEXXH motif can cause the loss of this function. Therefore, the coding product of the Clbi138 gene is a kind of protein that depends on the conserved HEXXH Modular metalloproteases.

Table 1 Insecticidal efficiency of recombinant baculovirus

6. Bioinformatics analysis of Clbi138

Clbi138 and homologues are sequences unique to class II alpha baculoviruses and are present in all class II alpha baculoviruses. Most Clbi138 homologues have a signal peptide (SP) at the N-terminus and a transmembrane domain (TM) at the C-terminus. Multiple sequence alignments revealed that Clbi138 homologues have multiple conserved amino acid sequences, especially all containing the HEXXH motif (see Figure 5), which is involved in the binding of zinc ions and is a characteristic sequence of metalloproteases. The reason why Clbi138 made the tissue liquefaction more serious may be that Clbi138 exerted metalloprotease activity. In view of the similarity between the Clbi138 homolog and the Clbi138 protein sequence, as well as the conserved HEXXH motif, it is easy to speculate that the Clbi138 homologue also has the function of improving the insecticidal efficiency of baculovirus.

SEQUENCE LISTING

<110> Jiangsu University

<120> A Genetic Modification Method for Improving the Insecticidal Efficiency of Baculovirus

<130> A Genetic Modification Method for Improving the Insecticidal Efficiency of Baculovirus

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Val Thr Phe Ile Met Thr Pro His Lys Ile Ile Met Ala Asn Val Tyr

545 550 555 560

Thr Asn Asp Ser Arg Ser Val Ser Phe Ala Gln His Glu Ile Arg Phe

565 570 575

Asn Arg His Lys Leu Ser Arg Phe Asp Tyr Asp Trp Phe Leu Asn Gly

580 585 590

Leu Ile Lys Gln Thr Leu Ile Tyr Leu Gly Asp Val Tyr Asn Tyr Ile

595 600 605

Gly Ile Asp Asn Thr Ala Tyr Asn Tyr Arg Pro Glu Thr Ile Phe Cys

610 615 620

Gln Lys Gln Thr Gln Asn Pro Glu Leu Gly Ile Ile Glu Phe Val Ser

625 630 635 640

Lys Thr Asn Val Trp Ser Asn Phe Phe Asn Asn Met Thr Val Ala Glu

645 650 655

Ala Arg Gln His Ile Arg Asn Phe Val Lys Ser Lys Glu Asp Cys Ala

660 665 670

Thr Phe Leu Asn Pro Val Val Val Asn Asp Asp Ile Ala Ala Asn Val

675 680 685

Pro Gln Tyr Leu Lys Asn Tyr Ala Tyr Arg Ile Asn Asn Val Val Thr

690 695 700

Ile Asn Ile Leu Asn Lys Arg Asp Val Tyr Ile Lys Leu Asp Phe Arg

705 710 715 720

Asn Asn Thr Ile Leu His Leu Ala Ala Leu His Asn Pro Asn Thr Tyr

725 730 735

Val Gln Leu Ser Asn Gln Phe Ser Lys Glu Cys Asn Ser Leu Leu Asn

740 745 750

Tyr Asp Asn Tyr Thr Ser Asn Gln Leu Tyr Gln Phe Tyr Asn Asn Tyr

755 760 765

Lys Leu Ser Thr Gly Val Val Lys Val Lys Tyr Cys Phe Lys Tyr Ile

770 775 780

Lys Thr Gln Pro Lys Gln Asn Ser Leu Val Pro Asn Ile Ile Thr Thr

785 790 795 800

Leu Leu Pro Ala Lys Thr Thr Phe Asn Leu Thr Thr Ile Ile Pro Lys

805 810 815

Tyr Ile Asn Asp Asp Asn Lys Asn Val Thr Ser Ser Thr Asp Asn Asp

820 825 830

Asn Glu Asn Val Thr Ser Ser Ser Thr Asp Gln Pro Val Thr Ile Arg

835 840 845

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

850 855 860

Thr Leu Ala Ile Leu Thr Thr Tyr Asn Tyr Val Ser Glu Asn Glu Asn

865 870 875 880

Asn Lys Ser Asn Asn Asn Asn Asn Asn Gln Asn Tyr Lys Thr Arg Phe Val

885 890 895

Pro Val Lys Leu Glu Ser Asp Arg Leu Val Pro Val Lys Leu Glu Ser

900 905 910

Asp Gln Lys Val Phe Gly Asn Asp Glu Asn Glu Glu Glu Thr Leu Tyr

915 920 925

Leu Asn Asp Asn Thr Thr Ala Pro Trp Thr Lys Glu Ala Ile Ala Ala

930 935 940

Ala Ala Ala Ala Ala Val Ala Pro Ser Asn Ile Ser Tyr Ile Asn Ile

945 950 955 960

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

965 970 975

Ile Leu Val Asn Ile Thr Tyr Ile Thr Ile Thr Ile Val Ile Tyr Ala

980 985 990

Lys Cys Cys Ala Ile Lys Lys Thr Thr His Ile Asn Lys Asn Thr Lys

995 1000 1005

His Lys Phe Asn Lys Thr Lys Phe Tyr Ser Asn Asn Ser Ile Glu

1010 1015 1020

Glu Lys Ser Asp Thr Glu Ala Lys Leu His Met Phe Glu

1025 1030 1035

<210> 3

<211> 27

<212>DNA

<213> Artificial sequence

<400> 3

cagatcaacc tgtaacaatt cgaccta 27

<210> 4

<211> 27

<212>DNA

<213> Artificial sequence

<400> 4

ttattcaaac atatgtaatt ttgcttc 27

<210> 5

<211> 34

<212>DNA

<213> Artificial sequence

<400> 5

gcgggatcca tgttttttgt gaatacaatt atca 34

<210> 6

<211> 31

<212>DNA

<213> Artificial sequence

<400> 6

ccgggtacct tcaaacatat gtaattttgc t 31

<210> 7

<211> 32

<212>DNA

<213> Artificial sequence

<400> 7

gcgaagcttttattcaaaca tatgtaatttttg 32

<210> 8

<211> 29

<212>DNA

<213> Artificial sequence

<400> 8

ctagaaacta tggcgccgaa ttgcaccac 29

<210> 9

<211> 29

<212>DNA

<213> Artificial sequence

<400> 9

gtggtgcaat tcggcgccat agtttctag 29

<210> 10

<211> 28

<212>DNA

<213> Artificial sequence

<400> 10

actatggcca cgccttgcac cacgccat 28

<210> 11

<211> 28

<212>DNA

<213> Artificial sequence

<400> 11

atggcgtggt gcaaggcgtg gccatagt 28

<210> 12

<211> 28

<212>DNA

<213> Artificial sequence

<400> 12

cacgaattgc acgccgccat tctgtact 28

<210> 13

<211> 28

<212>DNA

<213> Artificial sequence

<400> 13

agtacagaat ggcggcgtgc aattcgtg 28

Claims (4)

1. a kind of genetic modification method improving baculoviral insecticidal efficiency, which is characterized in that include the following steps：

（1）Using pEGFP-N1 carriers as template, PCR amplification EGFP gene is connected into donor plasmid after double digestion PFastBacDUAL forms recombinant plasmid pDUAL-EGFP；

（2）According toClbi138Gene order and its open reading frame design and synthesize primer；

（3）With greenish brown hawk moth nuclear polyhedrosis virus（ClbiNPV）Genomic DNA is as template, with step（2）The primer of synthesis into After row PCR amplification, agarose gel electrophoresis gel extraction target fragment is connected into the recombination through same double digestion after double digestion Plasmid pDUAL-EGFP forms recombination donor plasmid pDUAL-EGFP-Clbi138；

（4）PDUAL-EGFP, pDUAL-EGFP-Clbi138 conversion is taken to contain bombyx mori nuclear polyhydrosis virus respectively（BmNPV） Escherichia coli DH10B bacterial strains, be coated on LB plating mediums, static gas wave refrigerator keeps locus coeruleus colour developing abundant, picking white colony, into One step is crossed purifying on LB plating mediums；The white colony of purifying is inoculated in containing kanamycins, tetracycline, gentamicin LB liquid medium, after shaken cultivation, extraction recombination BmNPV DNA are denoted as vBm respectively^EGFP、vBm^EGFP/Clbi138；

Include to come from greenish brown hawk moth nuclear polyhedrosis virus by the recombinant bombyx mori nuclear polyhedrosis virus that the genetic modification method obtains （ClbiNPV）'sClbi138Gene, nucleotide sequence is as shown in SEQ ID NO.1, amino acid sequence such as SEQ ID Shown in NO.2；

Step（3）Described in recombination donor plasmid pDUAL-EGFP-Clbi138 in, EGFP gene is located under p10 promoters Trip, Clbi138 genes are located at polyhedrosis gene PH promoters downstream.

2. a kind of genetic modification method improving baculoviral insecticidal efficiency according to claim 1, which is characterized in that Step（2）Described in primer be P3 and P5, nucleotide sequence is as shown in SEQ ID NO.5 and SEQ ID NO.7.

3. the recombinant bombyx mori nuclear polyhedrosis virus that the method according to claim 11 obtains, wherein the Clbi138 eggs expressed There is metal proteinase activity in vain, the degradation of disease pest in-vivo tissue can be accelerated.

4. the recombinant bombyx mori nuclear polyhedrosis virus that the method according to claim 11 obtains answering in preparing insecticide With.