CN102174481B - Cotton leafworm genetically engineered virus I and construction method thereof - Google Patents
Cotton leafworm genetically engineered virus I and construction method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microbes, in particular relating to a genetically engineered virus I (SpltMNPV-Delta egt-egfp) capable of preventing and controlling farming and forestry pests such as cotton leafworm. The virus is preserved in the China Center for Type Culture Collection (CCTCC) with the collection number of CCTCC NO: V201027. In the invention, a genetic engineering method is utilized to delete and recombine the wild virus so as to obtain the cotton leafworm genetically engineered virus. The genome of the virus loses an egt gene, namely an ecdysteroid uridine diphosphate glucosyltransferase gene; and a marker gene, namely an enhanced green fluorescent protein (egfp) gene is inserted to the locus losing the egt gene, wherein the marker gene is controlled by the wild virus polyhedrin (ph) promoter. Compared with the wild virus, the genetically engineered virus has the advantages of higher pesticidal speed, better field application effect and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a prodenia litura genetic engineering virus capable of preventing and controlling agricultural and forestry pests such as prodenia litura, namely a prodenia litura genetic engineering virus No.1 (SpltMNPV-Delta)egt-egfp) The classification name is "Prodenia litura nuclear polyhedrosis virus engineered strain No. 1" (genetic modified) Spodoptera litura nucleosolvalhydrovirus No. 1). The virus is preserved by China Center for Type Culture Collection (CCTCC), the preservation date is 12 months and 15 days in 2010, and the preservation number is CCTCC NO: V201027.
Background
The prodenia litura is one of main pests of crops, and is distributed in various domestic provinces. The insect feeding ability is very wide, and the known harmful host plants can reach more than 290 families of 99 families. With the change of agricultural cultivation system and the rapid development of urbanization process in recent years, the planting area of dry land crops such as vegetables, flowers, green lawns and the like is more and more increased, and the pest of prodenia litura is more and more rampant day by day. The chemical prevention and control adopted in the current production often cannot achieve the expected effect, and environmental pollution, pesticide residue, human and animal poisoning and the like are easily caused.
With the continuous development of national economy and the increasing improvement of the living standard of people, the quality requirements of people on various agricultural products are higher and higher, and particularly, the safety problem of the products is widely concerned and valued. At present, in the prevention and treatment work of various crop diseases and insect pests, the situation of excessively depending on chemical pesticides is still serious, so that the problems of pest resistance, rampant, environmental pollution and the like are caused, and the food safety and living environment of human beings are directly influenced. Therefore, the vigorous development of pollution-free biopesticides with high efficiency, broad spectrum and good environmental compatibility is a demand for sustainable agricultural development.
Spodoptera litura multigrain embedded nuclear polyhedrosis virus (BmCPV) (II)Spodoptera litura multicapsid nucleolyticus, SpltMNPV) has been used for the control of spodoptera litura. But the popularization and the application of the pesticide are influenced due to the defect of low insecticidal speed. In order to overcome the defect that wild type insect baculovirus has low insecticidal speed, various recombinant insect baculovirus have been published at home and abroad since the last 90 th century, such as American recombinant Autographa californica nuclear polyhedrosis virus (rAcMNPV Stewart L M D et al 1991.Nature,352:85-88) disclosed in the literature and Chinese patent invention 'Chinese cotton bollworm genetic engineering virus' (rHaSNPV) with the publication number of CN 1109105C. However, so far, no report about the successful construction of the prodenia litura nuclear polyhedrosis gene engineering virus is found at home and abroad.
Disclosure of Invention
The invention aims to provide a prodenia litura nuclear polyhedrosis gene engineering virus capable of effectively improving the insecticidal speed and effect of wild viruses and a construction method thereof.
The purpose of the invention is realized according to the following scheme: provides a method for culturing wild virus SpltMNPV
The preservation information of the deleted and recombined prodenia litura genetic engineering virus is as follows: the preservation unit: inNational type culture collection; the preservation number is: CCTCC NO: V201027; and (3) classification and naming: SpltNPV-delta (SpltNPV-delta) Spodoptera litura gene engineering virus No.1egt -egfp) In the genome is deletedegtDeletion of the gene, ecdysone uridine diphosphate glucosyltransferase geneegtAt the site of the gene, SpltMNPV was inserted
Polyhedrin gene of (2)phPromoter-controlled marker gene, i.e. enhanced green fluorescent proteinegfpA gene.
Construction of the prodenia litura gene engineering virus No.1 (SpltNPV-delta)egt -egfp) The method comprises the following steps:
(1) using SpltMNPV
DNA as template, respectively amplifying homologous recombination armsegtUpstream non-encoding end, Egt up andegt part of the fragment at the 3' end and the downstream non-coding end, namely Egt down, are respectively subjected to recovery and purification of amplification productsKpn I /Xho I， Xba I/ SacAfter double enzyme digestion, the I is sequentially cloned to a vector pBluescript II SK (+), and an intermediate plasmid pSK-Egtup-Egtdown is obtained;
(2) between the two fragments, SpltMNPV is inserted by molecular cloning
Polyhedrin gene promoter P ofphEnhanced green fluorescent protein gene for starting controlegfpObtaining the recombinant plasmid pSK-deltaegt-Pph- egfp；
(3) The recombinant plasmid pSK-Deltaegt-Pph- egfpAnd SpltMNPVProdenia litura Spl cotransfected by genomic DNAi cells, by allelic exchange of genes, obtaining deletionsegtGene expression green fluorescent protein recombinant virus SpltMNPV-deltaegt-egfpAnd then purified by a multi-round fluorescent spot method to obtain a pure recombinant virus, namely the prodenia litura gene engineering virus No. 1.
The present invention relates to wild virus: (A)Spodoptera lituranucleopolyhedrovirus ,SpltMNPV 
GenBank accession No.: NC-011616), and constructing recombinant Splitura nucleopolyhedrosis virus, i.e. SpltMNPV-Delta, Gene engineering Virus No.1egt-egfp) CCTCC NO: V201027, wherein ecdysone uridine diphosphate glucosyltransferase (Ecdyssteroid UDP-glucopyranosyl transferase, abbreviation) is deleted in genomeegt) Gene in deletionegtAt the site of the gene, the polyhedrin gene of wild-type virus is inserted (ph) Promoter-controlled marker gene-enhanced green fluorescent protein: (egfp) The gene improves the insecticidal speed of the wild virus.
The invention has the following obvious advantages:
1. the genome of the prodenia litura gene engineering virus provided by the invention is deletedegtDeletion of the gene, ecdysone uridine diphosphate glucosyltransferase geneegtAt the site of the gene, the polyhedrin gene of wild-type virus is inserted (ph) Promoter-controlled marker gene-enhanced green fluorescent protein(s) ((R))egfp) A gene. Compared with the existing wild virus, the engineering virus provided by the invention has the advantages of higher insecticidal speed, better field application effect and the like.
2. The genetic engineering virus provided by the invention can be propagated in large batch by a worm body culture method, can be prepared into wettable powder or suspoemulsion and other dosage form virus insecticides, is used for preventing and controlling prodenia litura on crops, vegetables, flowers, lawns and other plants, and keeps the advantages of no harm to natural enemies, environmental safety, difficult generation of drug resistance to pests and the like of wild prodenia litura virus insecticides.
Drawings
FIG. 1 is a schematic structural diagram of a Prodenia litura genetic engineering virus No.1 provided by the present invention;
FIG. 2 is a schematic diagram of the construction process of the Prodenia litura genetic engineering virus No.1 provided by the invention.
Detailed Description
The invention is further described below with reference to examples and figures.
The first embodiment is as follows:
the present invention relates to a gene engineering method for producing wild virus (B) with strong virulenceSpodoptera lituranucleopolyhedrovirus 
，SpltMNPV 
GenBank accession No.: NC-011616), provides a prodenia litura genetic engineering virus, constructs a recombinant prodenia litura nuclear polyhedrosis virus, namely the prodenia litura genetic engineering virus No.1 (SpltMNPV-delta)egt-egfp) CCTCC NO: V201027. Referring to the attached drawing 1, it is a schematic structural diagram of a prodenia litura genetically engineered virus, i.e., a prodenia litura genetically engineered virus No.1, provided in this embodiment; in the figure, the position of the upper end of the main shaft,egtis ecdysone uridine diphosphate glucosyltransferase gene; pphIs polyhedrin gene promoter;egfpis enhanced green fluorescent protein gene. As can be seen from FIG. 1, the base thereofDeletion in genomeegtDeletion of the gene, ecdysone uridine diphosphate glucosyltransferase geneegtAt the site of the gene, the polyhedrin gene of wild-type virus is inserted (ph) Promoter-controlled enhanced green fluorescent protein: (egfp) A gene. The marker gene of the recombinant virus is enhanced green fluorescent protein EGFP, and can be monitored and identified by green fluorescence generated by the green fluorescent protein in the purification and proliferation processes of the prodenia litura genetic engineering virus.
Referring to fig. 2, it is a schematic diagram of the construction process of the spodoptera litura genetically engineered virus No.1 provided in this example; wherein,egtis ecdysone uridine diphosphate glucosyltransferase gene; pphIs polyhedrin gene promoter;egfpis an enhanced green fluorescent protein gene; .egt 5' is a radical containingegtUpstream sequence of 5' end of gene;egt 3' is containingegtPartial sequence of gene 3' end and its upstream sequence; pBluescript
SK (+) is a general carrier; SpltMNPV
Is a wild type virus; pSK-Deltaegt-Pph- egfpIs absent ofegtGene insertionegfpRecombinant transfer vectors for genes; SpltMNPV-deltaegt- egfpIs absent ofegtGene insertionegfpGene prodenia litura nuclear polyhedrosis gene engineering virus strain, namely prodenia litura gene engineering virus No.1 (SpltMNPV-delta)egt-egfp) CCTCC NO: V201027. The construction method of the prodenia litura genetic engineering virus comprises the following steps:
(1) using SpltMNPV
DNA as template, respectively amplifying homologous recombination armsegtUpstream non-encoding end (Egt up) andegt 3' terminal partial fragment and method for producing the sameDownstream non-coding end (Egt down). The amplification products are respectively subjected to recovery and purificationKpn I /Xho I， Xba I/ SacThe DNA fragment I is cloned to a vector pBluescript II SK (+) after double enzyme digestion. Obtaining an intermediate plasmid pSK-Egtup-Egtdown.
(2) Between these two fragments, SpltMNPV was inserted using molecular cloning techniquesPolyhedrin gene promoter (P)ph) Enhanced green fluorescent protein gene for promoting control: (egfp) Obtaining the recombinant plasmid pSK-deltaegt-Pph- egfp。
(3) pSK-Deltaegt-Pph- egfpAnd SpltMNPV
Transfecting spodoptera litura (Spli) cells with genome DNA, and obtaining deletion through gene allelic exchangeegtRecombinant virus SpltMNPV-delta of gene and expression green fluorescent proteinegt- egfp. Purifying by multiple rounds of fluorescent spot method to obtain pure recombinant virus, i.e. SpltMNPV-Deltaegt-egfp）CCTCC NO:V201027。
Table 1 shows the comparison of the bioassay results of the Prodenia litura genetically engineered virus No.1 (engineering virus No.1 for short) and the wild type virus provided in this example (the dosages are all 10)7Individual polyhedra/larvae).
TABLE 1
| Viral strains | LT against 3-instar larvae50(day) | LT against 3-instar larvae90(day) |
| Wild type virus | 3.33 | 4.95 |
| Engineered Virus No.1 | 2.48 | 3.93 |
Table 2 shows the comparison of field bioassay results of the Prodenia litura genetically engineered virus No.1 (engineering virus No.1 for short) and the wild type virus provided in this example (the concentrations used are all 10%7polygon/mL).
TABLE 2
As shown by the results in Table 1, the dosage at 10 was used7Half-lethal time of engineered virus No.1 against 3-instar Prodenia Litura (LT) in case of single polyhedron/single larva50) The reduction is 25.3% compared with the wild virus. The results in Table 2 show that the insecticidal effect of the engineering virus No.1 is obviously improved compared with that of the wild virus. Therefore, compared with the existing wild virus, the engineering virus No.1 provided by the invention has the advantages of higher insecticidal speed, better field application effect and the like.
The virus culture provided by the invention is insect virus polyhedron, and only living body proliferation can be adopted. The proliferation method comprises the following steps: at 1 × 106Infecting 3-year spodoptera litura larvae at the concentration of polyhedra/ml, culturing at 27-28 ℃ for 5-7 days, and collecting dead and diseased insects. Detection of survival also requires a bioassay, i.e. detection of diseaseActivity of virulent infection of prodenia litura.
The identification method of the prodenia litura genetic engineering virus provided by the embodiment is as follows:
(1) primarily identifying the correctness of the virus by using restriction endonuclease;
(2) designing suitable primers, and further determining virus content by Polymerase Chain Reaction (PCR)egtMost of the coding sequence of the gene has been deleted, and a foreign gene (egfp) Has been inserted correctly;
(3) analyzing the activity of ecdysone uridine diphosphate glucosyltransferase of the virus to determineegtThe gene has been inactivated;
(4) and determining the effect of the virus by indoor biological activity measurement and field efficacy test.
The prodenia litura genetic engineering virus provided by the invention can be propagated in large batch by a worm body culture method, can be prepared into virus insecticides in dosage forms of wettable powder or suspoemulsion and the like, is used for preventing and controlling prodenia litura on plants such as crops, vegetables, flowers, lawns and the like, and the insecticides retain the advantages that wild prodenia litura virus insecticides do not harm natural enemies, are safe to environment, are not easy to generate drug resistance to pests and the like.
Claims (1)
1. SpltMNPV for wild type virus
The prodenia litura genetic engineering virus subjected to deletion and recombination is characterized in that: the preservation information is as follows: the preservation unit: china center for type culture Collection; the preservation number is: CCTCC NO: V201027; and (3) classification and naming: spodoptera litura gene engineering virus No.1, which is deleted in genomeegtDeletion of the gene, ecdysone uridine diphosphate glucosyltransferase geneegtAt the site of the gene, SpltMNPV was inserted
Polyhedrin gene of (2)phPromoter-controlled marker gene, i.e. enhanced green fluorescent proteinegfpA gene.
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Non-Patent Citations (6)
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