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Genetic modification method capable of enhancing disinsection efficiency of baculovirus

A baculovirus and genetic modification technology, applied in the field of genetic engineering, can solve problems such as unclear functions, achieve broad application prospects, obvious economic and ecological benefits, and shorten the half-lethal time effect.

Inactive Publication Date: 2015-09-16
JIANGSU UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the functions of Clbi138 and its homologues are still unclear

Method used

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  • Genetic modification method capable of enhancing disinsection efficiency of baculovirus
  • Genetic modification method capable of enhancing disinsection efficiency of baculovirus
  • Genetic modification method capable of enhancing disinsection efficiency of baculovirus

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0025] Clbi138 Gene expression pattern analysis

[0026] 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).

[0027]Trizol reagent was used to extract the RNA from the midgut of the ClbiNPV-infected hornworm (the eggs were collected in the field and then reared artificially), and RT-PCR analysis was performed. The PCR reaction conditions used were: 94°C for 4min; 94°C for 20s, 56°C for 30s , 72 ℃ 1min, 30 cycles; 72 ℃ 5min. The Clbi138 gene has a small amount of transcription 12 hours after the virus infects the host, and the transcription time is 12 hours earlier than that of the polyhedron (Clbipoh) gene in the ClbiNPV genome. Clbi138 The transcription of the gene is gradually enhanced (see figure 1 ).

[0028] 2. Analysis of subcellular localization of Clbi138 protein

[0029] Using pEGFP-N1 vector (purchased...

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Abstract

The invention relates to a genetic modification method capable of enhancing disinsection efficiency of baculovirus, belonging to the field of gene engineering. The method comprises the following steps: amplifying EGFP gene by using a pEGFP-N1 vector as a template, carrying out double enzyme digestion, and connecting into a plasmid pFastBacDUAL to obtain a recombinant plasmid pDUAL-EGFP; designing and synthesizing primers according to the Clbi138 gene sequence and open reading frame thereof; carrying out PCR (polymerase chain reaction) amplification by using greenish brown hawk moth karyotype polyhedrosis virus genome DNA (deoxyribonucleic acid) as a template, recovering the target segment, carrying out enzyme digestion, and connecting into the recombinant plasmid pDUAL-EGFP subjected to double enzyme digestion to obtain a recombinant plasmid pDUAL-EGFP-Clbi138; and transforming a Escherichia coli strain containing silkworm karyotype polyhedrosis virus, culturing, purifying, inoculating into an LB (Langmuir-Blodgett) liquid culture medium, carrying out shake culture, and extracting DNA of the recombinant BmNPV. The experiment proves for the first time that the disinsection efficiency of the recombinant BmNPV is obviously enhanced, the median lethal concentration is reduced by 11 times as compared with the control group, the median lethal time is shortened by 42.9% as compared with the control group, and the liquefaction in the sick polypide is more severe, thereby achieving the effect of enhancing control effects on pests. The recombinant BmNPV has obvious economic and ecological benefits, and has wide application prospects.

Description

technical field [0001] 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 [0002] 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. [0003] 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...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N7/01A01N63/00A01P7/04C12R1/93
Inventor 朱姗颖何华纲时伟汪峰王文兵
Owner JIANGSU UNIV
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