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

Abstract

The present invention relates to a kind of genetic modification methods improving baculoviral insecticidal efficiency, belong to genetic engineering field；The present invention is connected into plasmid pFastBacDUAL using pEGFP N1 carriers as template amplification EGFP gene after double digestion, obtains recombinant plasmid pDUAL EGFP；According toClbi138Gene order and its open reading frame design synthetic primer；Using greenish brown hawk moth nuclear polyhedrosis virus genomic DNA as template, PCR amplification is carried out, target fragment is recycled, is connected into the recombinant plasmid pDUAL EGFP through same double digestion after double digestion, obtains recombinant plasmid pDUAL EGFP Clbi138；The coli strain containing bombyx mori nuclear polyhydrosis virus is converted, LB liquid medium, shaken cultivation, extraction recombination BmNPV DNA are inoculated in after culture purified；And confirm that the insecticide efficiency of recombination BmNPV significantly increases for the first time by testing, half lethal concentration reduces 11 times than control group, LD50 time shortens 42.9% than control group, and liquefaction is more serious in disease pest body, to reach the control effect for improving insect pest, with apparent economy and ecological benefits, have broad application prospects.

Description

A kind of genetic modification method improving baculoviral insecticidal efficiency

Technical field

The present invention relates to a kind of genetic modification methods improving baculoviral insecticidal efficiency, belong to genetic engineering field.

Background technology

Using prevention and cure of viruses pest, not only control effect is good, and to safety of human and livestock, to natural enemies security, do not pollute ring Border does not make pest develop immunity to drugs.In addition, virus can accumulate in the environment, forms epidemic disease in pest population and control for a long time Pest insect density processed has apparent economy and ecological benefits, is current more satisfactory and promising Biocidal Agent.

Baculovirus insecticides are free from environmental pollution with its, do not destroy the ecological balance, are not easy to natural enemy and safety of human and livestock, pest It generates resistance, can become the hot spot in biological control research the advantages that field controls pest population for a long time, have in production Wide application prospect.But the shortcomings of desinsection speed is slow restricts its further genralrlization and application.Technique for gene engineering is enhancing Baculoviral insecticidal effect provides potential possibility.Currently, the research for improving recombinant baculovirus desinsection toxicity mainly collects In in diuretic hormone（DH）Gene, juvenile hormone ester（JHE）Gene, moulting hormone（MH）Gene, chitinase（Chitinase） Gene, synergistic protein（enhancin）Gene, Bt genes, neurotoxin gene, plant proteinase inhibitor gene etc.（Lv Hong Sound, insect viruses molecular biology, Chinese agriculture Science Press, 1998, Beijing）, but most genes improve baculoviral Insecticidal effect is still undesirable.

Nuclear polyhedrosis virus (NPV) to lepidopterous larvae have very high pathogenicity, be in insect viruses type most Potentiality person, and the most widely used one kind of research.Currently, the insect viruses preparation of domestic and international merchandized handling, the overwhelming majority It is NPV（The Journal of Shanxi Agricultural University such as man of virtue and ability 1998 is fastened on the left sides exploitation of nucleopolyhedrovirus insecticide, 1（4）：302- 305）.Bombyx mori nuclear polyhydrosis virus（BmNPV）Belong to A type baculoviral category, is one kind and host domain of baculoviral One of wider baculoviral can infect a variety of agriculture and forestry injurious insects.To make baculoviral become ideal biological insecticides, life is utilized Object technology is transformed wild-type virus the approach for the most effective optimal foreground for being current raising baculoviral insecticidal effect.

Greenish brown hawk moth nuclear polyhedrosis virus（Clanis bilineata nucleopolyhedrovirus；Shan-Ying Zhu, Jian-Ping Yi, Wei-De Shen, Li-Qun Wang, Hua-Gang He, Yong Wang, Bing Li and Wen-Bing Wang. Genomic sequence, organization and characteristics of a New nucleopolyhedrovirus isolated from Clanis bilineata larva, BMC Genomics, 2009,10：91）'sClbi138Gene（Accession number in GenBank is DQ504428）3111 bases of overall length derive 1036 amino acid of protein sequence overall length, be respectively provided with a signal peptide in N-terminal and C-terminal（SP）And transmembrane domain （TM）, and in inside, there are a metalloproteinases characteristic sequences, i.e., relevant HEXXH blocks are combined with zinc ion.To complete It is retrieved at the baculoviral of genome sequencing, it is found that Clbi138 homologues are guarded in all II classes α-baculovirals In the presence of being also described in amsacta moorei entomopoxvirus in addition（AmEPV）Deng.But the function of Clbi138 and its homologue is still unknown Really.

Present invention offer is a kind of to utilize greenish brown hawk moth nuclear polyhedrosis virusClbi138Gene improves baculoviral silkworm caryogram Polyhedrosis virus（BmNPV）The method of insecticide efficiency, bombyx mori nuclear polyhydrosis virus（BmNPV）There is no Clbi138 in genome Homologue, therefore by Clbi138 channel genes BmNPV, to study the function of Clbi138, and confirmed for the first time by experimentClbi138Gene improves baculoviral insecticidal efficiency, and being studied by rite-directed mutagenesis confirms HEXXH blocks to Clbi138 albumen The importance of biological function, to confirmClbi138A kind of metalloproteinases of gene code.

Invention content

It is an object of the invention to disclose a kind of utilization greenish brown hawk moth nuclear polyhedrosis virusClbi138Gene improves rod-shaped disease The genetic modification method of malicious insecticide efficiency.

It is of the present invention to come from greenish brown hawk moth nuclear polyhedrosis virus（ClbiNPV）'sClbi138Gene（GenBank is stepped on Record number is DQ504428, and shown in SEQ ID NO.1, amino acid sequence is as shown in SEQ ID NO.2）, come from greenish brown hawk moth caryogram Polyhedrosis virus, the open reading frame of the gene（ORF）Overall length is 3111 bases, encodes 1036 amino acid, mould containing HEXXH Sequence studies confirmation for the site-directed point mutation of HEXXH blocks,Clbi138Gene has metal proteinase activity.

Clbi138 and its homologue are widely present in II class α baculovirals, but its function is still not clear, due to Clbi138 and its homologue all have with the relevant conservative HEXXH blocks of metalloproteinases, easily speculate them also and have and is similar Function；Other sequences are also easily substituted in Clbi138 and its homologue, to generate the still mutation with biological activity Body.

The present invention uses following technical scheme to reach above-mentioned purpose：

Present invention offer is a kind of to utilize greenish brown hawk moth nuclear polyhedrosis virusClbi138Gene improves baculoviral insecticidal efficiency Method, this method mainly includes the following steps that：

（1）Using pEGFP-N1 carriers as template, EGFP gene is expanded using round pcr, after KpnI and XhoI double digestions It is connected into donor plasmid pFastBacDUAL, forms recombinant plasmid pDUAL-EGFP；

（2）According toClbi138The open reading frame of gene designs and synthesizes pair of primers P3（SEQ ID NO.5）、P4 （SEQ ID NO.6）, underscore part is BamHI and KpnI restriction enzyme sites；1 primer P5 is redesigned according to Clbi138 genes （SEQ ID NO.7）, underscore part is the restriction enzyme site of HindIII.

（3）With greenish brown hawk moth nuclear polyhedrosis virus（ClbiNPV）Genomic DNA is carried out as template by primer of P3/P5 After PCR amplification, agarose gel electrophoresis gel extraction target fragment is connected into after BamHI and HindIII double digestions through same Double digestion recombinant plasmid pDUAL-EGFP forms recombination donor plasmid pDUAL-EGFP-Clbi138.

（4）PDUAL-EGFP, pDUAL-EGFP-Clbi138 is taken to convert the Escherichia coli DH10B bacterium containing BmNPV respectively Strain is coated on LB plating mediums, and static gas wave refrigerator keeps locus coeruleus colour developing abundant, picking white colony, further in LB tablet cultures It crosses and purifies on base；The white colony of purifying is inoculated in the LB liquid medium containing kanamycins, tetracycline, gentamicin, 37 DEG C of 18 ~ 20h of shaken cultivation, extraction recombination BmNPV DNA（vBm^EGFP、vBm^EGFP/Clbi138）.

Beneficial effects of the present invention：

This method will come from greenish brown hawk moth nuclear polyhedrosis virus（ClbiNPV）'sClbi138Gene（SEQ ID NO.1）It leads Enter bombyx mori nuclear polyhydrosis virus（BmNPV）Afterwards, the insecticide efficiency for recombinating BmNPV significantly increases, half lethal concentration（LC₅₀）For 12.51 pfu reduce 11 times, LD50 time than control group（LT₅₀）For 84.12 h, shorten 42.9%, and disease than control group Liquefaction is more serious in polypide.Therefore, the protein of Clbi138 genes and its coding has important application in the prevention and control field of pest Value.

Heretofore described Clbi138 is a kind of metalloproteinases, can participate in the degradation of disease pest in-vivo tissue.It will Clbi138 channel genes bombyx mori nuclear polyhydrosis virus（BmNPV）The half lethal concentration of recombinant virus is can significantly reduce, evil is shortened The median lethal time of worm, and disease pest in-vivo tissue is accelerated to degrade, to reach the control effect for improving insect pest, there is apparent warp Ji and ecological benefits, have broad application prospects.

Description of the drawings

Fig. 1：Clbi138The RT-PCR results of gene.M：DL2000 DNA marker；1 ~ 8 is the RT- of Clbi138 genes The time of PCR product, ClbiNPV infection larvas is followed successively by 0h, 4h, 8h, 12h, for 24 hours, 48h, 72h, 96h；9 ~ 16 be Clbipoh The RT-PCR products of gene, infection time are followed successively by 0h, 4h, 8h, 12h, for 24 hours, 48h, 72h, 96h.

Fig. 2：The rite-directed mutagenesis of Clbi138 HEXXH blocks.Figure is the mutation position by site-directed point mutation technology creation The sequencing identification of point.

Fig. 3：The dead silkworm of baculoviral virus infection.A：vBm^EGFP/Clbi138Infected silkworm（After death for 24 hours）；

B：vBm^EGFPInfected silkworm（After death for 24 hours, control group）.

Fig. 4：The virology effect of Clbi138 HEXXH blocks mutation.1-4：It is followed successively by vBm^EGFP、vBm ^{EGFP/Clbi138M1}、 vBm ^{EGFP/Clbi138M2}And vBm ^{EGFP/Clbi138M3}The silkworm of infection 3 days（Without dead individuals）, 5：vBm ^EGFP/Clbi138Infect 3 days just Dead silkworm.

Fig. 5：Clbi138 and its homologue Multiple Sequence Alignment（Part）.Shade show conserved sequence, shown in double underline It is the activated centre of metalloproteinases for highly conserved HEXXH blocks.

Specific implementation mode

Clbi138The expression pattern analysis of gene

According to ClbiNPV genome C lbi138 genes（Accession number in GenBank is DQ504428）Sequence design is simultaneously Synthesize pair of primers P1（SEQ ID NO.3）、P2（SEQ ID NO.4）.

Using the sparrow line hawkmoth of Trizol reagents extraction ClbiNPV infection（Collect artificial feeding after worm's ovum in field）Middle intestines RNA, carries out RT-PCR analyses, and used PCR reaction conditions are： 94℃ 4min；94 DEG C of 20s, 56 DEG C of 30s, 72 DEG C 1min, 30 cycles；72℃ 5min.Clbi138 genes have a small amount of transcription when virus infects host 12h, transcribe the time Than polyhedral body in ClbiNPV genome（Clbipoh）Gene morning 12h, with the extension of infection time,Clbi138Gene turns Record gradually increases（See Fig. 1）.

The Subcellular Localization of 2.Clbi138 albumen

With pEGFP-N1 carriers（Purchased from TAKARA companies）For template, EGFP is expanded using round pcr（Enhanced green is glimmering Photoprotein）Gene is connected into donor plasmid pFastBac1 after KpnI and HindIII double digestions（Purchased from Invitrogen companies）, Form recombinant plasmid pFB-EGFP.

According toClbi138The open reading frame of gene（ORF）Design and synthesize pair of primers P3（SEQ ID NO.5）、P4 （SEQ ID NO.6）.Underscore part is BamHI and KpnI restriction enzyme sites.

PCR amplification is carried out using ClbiNPV genomic DNAs template, PCR reaction conditions are： 94℃ 4min； 94℃ 20s, 50 DEG C of 30s, 72 DEG C of 3min, 32 cycles；72℃ 5min.After 1.0% agarose gel electrophoresis, gel extraction mesh Segment be connected into the above-mentioned recombinant plasmid pFB-EGFP through same double digestion after BamHI and KpnI double digestions, form recombination After donor plasmid pFB-Clbi138-EGFP, transfection BmN cells 48h, observed under laser confocal microscope, green florescent signal Positioned at nucleus periphery, it can determine whether that Clbi138 albumen can be positioned at the nuclear membrane of BmN cells according to the core areas dyed DAPI.

The structure of the recombinant baculovirus of the gene containing Clbi138

With pEGFP-N1 carriers（Purchased from TAKARA companies）For template, EGFP gene is expanded using round pcr, through KpnI and Donor plasmid pFastBacDUAL is connected into after XhoI double digestions（Purchased from Invitrogen companies）, form recombinant plasmid pDUAL- EGFP。

Bombyx mori nuclear polyhydrosis virus（BmNPV）There is no Clbi138 homologues in genome, therefore can be by Clbi138 bases Because importing BmNPV, to study the function of Clbi138.

1 primer P5 is redesigned according to Clbi138 genes（SEQ ID NO.7）, underscore part is the digestion of HindIII Site.

Using ClbiNPV genomic DNAs template, PCR amplification is carried out by primer of P3/P5, PCR reaction conditions are： 94 ℃ 4min；94 DEG C of 20s, 50 DEG C of 30s, 72 DEG C of 3min, 32 cycles； 72℃ 5min.1.0% Ago-Gel electricity After swimming, gel extraction target fragment is connected into the above-mentioned recombinant plasmid through same double digestion after BamHI and HindIII double digestions PDUAL-EGFP forms recombination donor plasmid pDUAL-EGFP-Clbi138, and wherein EGFP gene is located at p10 promoters downstream, Clbi138 genes are located at polyhedrosis gene（PH）Promoter downstream.

5 μ L pDUAL-EGFP, pDUAL-EGFP-Clbi138 is taken to convert the Escherichia coli DH10B containing BmNPV respectively Bacterial strain is coated on LBac tablets, and 37 DEG C of 24~48 h of static gas wave refrigerator keep locus coeruleus colour developing abundant, and picking white colony further exists The flat lining out purifying of LBac.The white colony of purifying is inoculated in LB liquid medium（Containing 50 μ g/mL of kanamycins, Fourth Ring 50 μ g/mL of element, 70 μ g/mL of gentamicin）, 37 DEG C of 18 ~ 20h of shaken cultivation, extraction recombination BmNPV DNA（vBm^EGFP、vBm^{EGFP /Clbi138}）.Using lipofection, BmNPV will be recombinated（vBm^EGFP、vBm^EGFP/Clbi138）Transfect BmN cells, continue culture 3 ~ 5 days, low-speed centrifugal collected cell supernatant, after repeatedly multiple sense amplification, collected the BV particles in supernatant.By green fluorescence Signal, using chain termination method measure two kinds of recombinant viruses titre, respectively 1.2 × 10⁷PFU/mL, 1.6 × 10⁷PFU/mL 。

Clbi138The rite-directed mutagenesis of gene HEXXH blocks

According toClbi1383 groups of primer P6 of gene order design synthesis（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）.Using weight Folded PCR methods carry out site-directed point mutation.

With wild typeClbi138Gene is template, is expanded with primer P6 and P5, purpose is recycled from Ago-Gel Segment 138M1-1；It is expanded with primer P3 and P7, target fragment 138M1-2 is recycled from Ago-Gel；Equal proportion mixes Glue recovery product 138M1-1 and 138M1-2, are expanded with P6 and P7, obtain mutationClbi138M1Gene.

With same method, mutation is created using primer P8 and P9Clbi138M2Gene is created using primer P10 and P11 MutationClbi138M3Gene.

By three mutatorsClbi138M1、Clbi138M2、Clbi138M3Sequencing identification is carried out, expected point is contained Mutational site（See Fig. 2）.In the coded product of three mutators, HEXXH blocks sport respectively AEXXH, HAXXH, HEXXA。

By the method in step 3,3 are mutated respectivelyClbi138BmNPV of the channel genes containing EGFP gene is formed Recombinant virus vBm^{EGFP/Clbi138M1}、vBm^{EGFP/Clbi138M2}、vBm^{EGFP/Clbi138M3}。

Clbi138Gene can enhance the verification of the insecticide efficiency of recombinant baculovirus

Respectively with the recombinant virus BV of identical titre（vBm^EGFP、vBm^EGFP/Clbi138、vBm^{EGFP/Clbi138M1}、vBm^{EGFP /Clbi138M2}、vBm^{EGFP/Clbi138M3}）Five age silkworms are injected, per boss silkworm injection 1 × 10⁴PFU, every group of injection 50, is adding mulberry leaf just Often raising, observes and counts The dead quantity.

Result of study shows vBm^EGFP/Clbi138The half death time of the silkworm of infection（LT₅₀）It is all dead for 84.12h It is 88h to die the time, and vBm^EGFP, infection control group LT₅₀For 147.16h, whole death times are 154h, Clbi138 bases The expression of cause can make LT₅₀Shorten 63h, i.e. the silkworm death time shortens 42.9%（It is shown in Table 1）.

Observation is carried out for dead silkworm for 24 hours also found, vBm^EGFP/Clbi138The tissue liquefaction of infected silkworm is even more serious （See Fig. 3）.

Therefore, the mechanism that Clbi138 albumen shortens the death time may be Clbi138 protein exhibits metalloprotein enzyme activity Property, host tissue is seriously destroyed, so as to cause host's quick death.

In addition, with different virus population（10,20,50,100,200,500 pfu）Inject five age silkworms, vBm^{EGFP /Clbi138}Half lethal concentration（LC₅₀）For 12.51 pfu, and vBm^EGFPThe LC of control group₅₀For 141.21 pfu（It is shown in Table 1）.

It can be seen that Clbi138 can greatly enhance the toxicity of baculoviral, there is potential application in control of insect Value.

To sum up the study found that containing mutationClbi138Three kinds of recombinant virus vBm of gene^{EGFP/Clbi138M1}、vBm^{EGFP /Clbi138M2}、vBm^{EGFP/Clbi138M3}Insecticide efficiency, with vBm^EGFPControl group is without significant difference（See Fig. 4）, show HEXXH blocks In the variations of three conserved positions can causeClbi138The forfeiture of gene function.HEXXH blocks are the activity of metalloproteinases Center, the expression of wild type Clbi138 can accelerate the degradation of disease pest in-vivo tissue, and the variation of HEXXH blocks makes the function lose It loses, therefore,Clbi138The coded product of gene is a kind of metalloproteinases dependent on conservative HEXXH blocks.

The insecticide efficiency of 1 recombinant baculovirus of table

The bioinformatic analysis of 6.Clbi138

Clbi138 and homologue are the characteristic sequences of II class α baculovirals, and are present in all II classes α baculovirals. There are one signal peptides in N-terminal tool for most of Clbi138 homologues（SP）, in C-terminal tool, there are one transmembrane domains（TM）.Multisequencing It compares and finds, Clbi138 homologues have the amino acid sequence that many places are guarded, and especially all contain HEXXH blocks（See Fig. 5）, The block participates in the combination of zinc ion, is the characteristic sequence of metalloproteinases.It is even more serious that Clbi138 makes disease pest tissue liquefy Reason, it may be possible to which Clbi138 has played metal proteinase activity.In view of Clbi138 homologues and Clbi138 protein sequences Similitude and possessed conservative HEXXH blocks, easily speculating Clbi138 homologues also, there is raising baculoviral to kill The function of worm efficiency.

SEQUENCE LISTING

<110>Jiangsu University

<120>A kind of genetic modification method improving baculoviral insecticidal efficiency

<130>A kind of genetic modification method improving baculoviral insecticidal efficiency

<160> 13

<170> PatentIn version 3.3

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<212> DNA

<213>Greenish brown hawk moth nuclear polyhedrosis virus（Clanis bilineata nucleopolyhedrovirus）

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<213>Greenish brown hawk moth nuclear polyhedrosis virus（Clanis bilineata nucleopolyhedrovirus）

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

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Asn Val Phe Ala Glu Cys Thr Asn Tyr Ile Gln Ile Asn Phe Glu Asp

530 535 540

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 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 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

gcgaagcttt tattcaaaca tatgtaattt tg 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.