CN102174481B - Cotton leafworm genetically engineered virus I and construction method thereof - Google Patents

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

No. 1, prodenia litura genetically engineered virus and construction process thereof

Technical field

The present invention relates to microbial technology field, particularly a kind of prodenia litura genetically engineered virus that can prevent and treat the agriculture and forestry injurious insects such as prodenia litura, i.e. No. 1 (SpltMNPV-△ of prodenia litura genetically engineered virus egt- Egfp), specific name is " prodenia litura nuclear polyhedrosis virus genetically engineered strain No.1 " (Genetically modified Spodoptera liturA nucleopolyhedrovirus No.1).This virus is by Chinese Typical Representative culture collection center (CCTCC) preservation, and preservation date is on December 15th, 2010, and deposit number is CCTCC NO:V201027.

Background technology

Prodenia litura is one of primary pest of farm crop, and all there is distribution domestic each provinces and regions.These worm feeding habits are extremely wide, and the known host plant that endangers reaches 99 sections kind more than 290.Along with the change of agricultural tillage system and the fast development of urbanization process, the cultivated area of the dry crops such as vegetables, flowers, greening lawn is more and more in recent years, and causing harm of prodenia litura also is becoming increasingly rampant.The chemical prevention of adopting in current production often falls flat, and easily cause environmental pollution, pesticide residue and person poultry poisoning etc.

Along with the development of national economy and the raising day by day of living standards of the people, people are more and more higher for the specification of quality of various agricultural-food, and particularly the safety issue of product is paid close attention to widely and payes attention to.At present, in the preventing and controlling of various diseases and pests of agronomic crop, the situation of depending on chemical pesticide unduly is still very serious, has brought thus pest resistance, the problem such as wildness and environmental pollution again, directly has influence on the mankind's food safety and living environment.Therefore, greatly develop efficient, wide spectrum, with the good harmless boilogical agricultural chemicals of Environmental compatibility be the needs of agricultural sustainable development.

Many embedding nuclear polyhedrosis virus of prodenia litura ( Spodoptera liturA multicapsid nucleopolyhedrovirus, SpltMNPV) be used for the control of prodenia litura.But because there is the slow-footed defective of desinsection in it, affected it and applied.In order to change the slow-footed defective of wild-type insect baculovirus desinsection, since the nineties in last century, existing multiple recombinant baculovirus comes out both at home and abroad, as disclosed U.S. restructuring Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus (rAcMNPV Stewart L M D et al 1991.Nature in document, 352:85-88), publication number be CN1109105C Chinese invention patent " bollworm genetically engineered virus " (rHaSNPV).Yet, up to now, there is not yet the report that relevant prodenia litura caryogram polyhedrosis gene engineering virus successfully constructs both at home and abroad.

Summary of the invention

The purpose of this invention is to provide a kind of prodenia litura caryogram polyhedrosis gene engineering virus and construction process thereof that can effectively improve wild virus desinsection speed and effect.

The objective of the invention is to realize by following scheme: provide a kind of to wild-type virus SpltMNPV

The prodenia litura genetically engineered virus that lacks and recombinate, its preservation information is: depositary institution: Chinese Typical Representative culture collection center; Deposit number: CCTCC NO:V201027; Classification And Nomenclature: No. 1 (SpltNPV-△ of prodenia litura genetically engineered virus egt- Egfp), lacked in genome egtGene is moulting hormone uridine diphosphoglucose based transferase gene, in disappearance egtThe site of gene is inserted by SpltMNPV

Polyhedron gene phThe marker gene that promotor is controlled is enhanced green fluorescence protein EgfpGene.

No. 1 (SpltNPV-△ of the above-mentioned prodenia litura genetically engineered virus of a kind of structure egt- Egfp) method, comprise the following steps:

(1) with SpltMNPV

DNA is template, amplifies respectively the homologous recombination arm egtUpstream non-coding end be Egt up and egtPartial Fragment and the downstream non-coding end thereof of 3 ' end are Egt down, and amplified production reclaims after purifying warp respectively KpnI / XhoI， XbaI/ SacBe cloned into successively on carrier pBluescript II SK (+) interstitial granules pSK-Egtup-Egtdown in acquisition after the I double digestion;

(2) between these two fragments, adopt molecule clone technology to insert SpltMNPV

Polyhedrin gene promoter P phStart the enhanced green fluorescent protein gene of controlling Egfp, obtain recombinant plasmid pSK-△ egt-P ph- Egfp

(3) with recombinant plasmid pSK-△ egt-P ph- EgfpAnd SpltMNPV Genomic dna cotransfection prodenia litura Spli cell through the allele exchange, obtains disappearance egtThe recombinant virus SpltMNPV-△ of the expressing green fluorescent protein of gene egt- Egfp, then through many wheel fluorescent spot method purification process, obtaining pure recombinant virus is No. 1, prodenia litura genetically engineered virus.

The present invention wild virus ( Spodoptera liturAnucleopolyhedrovirus , SpltMNPV

, GenBank accession number: on basis NC-011616), by gene engineering method, built the restructuring prodenia litura nuclear polyhedrosis virus, i.e. No. 1 (SpltMNPV-△ of prodenia litura genetically engineered virus egt- Egfp) CCTCC NO:V201027, (Ecdysteroid UDP-glucosyltransferase is called for short to have lacked moulting hormone uridine diphosphoglucose based transferase in its genome egt) gene, in disappearance egtThe site of gene, insert by the wild-type virus polyhedron gene ( ph) marker gene controlled of promotor---enhanced green fluorescence protein ( Egfp) gene, improved the desinsection speed of wild virus.

The present invention has following obvious advantage:

1, prodenia litura genetically engineered virus provided by the present invention, lack in its genome egtGene is moulting hormone uridine diphosphoglucose based transferase gene, in disappearance egtThe site of gene, insert by the wild-type virus polyhedron gene ( ph) marker gene controlled of promotor---enhanced green fluorescence protein ( Egfp) gene.Compare with existing wild virus, engineering virus provided by the invention has the advantage such as desinsection speed and better field effect faster.

2, genetically engineered virus provided by the invention can be bred in enormous quantities by the polypide cultural method, be configured to the formulation viral pesticides such as wettable powder or suspended emulsion agent, be used for the control of prodenia litura on the plants such as farm crop, vegetables, flowers and lawn, kept wild prodenia litura viral pesticide and do not injured natural enemy, the environmentally safe and insect advantage such as be difficult for developing immunity to drugs.

Description of drawings

Fig. 1 is the structural representation of No. 1, prodenia litura genetically engineered virus provided by the invention;

Fig. 2 is the building process schematic diagram of No. 1, prodenia litura genetically engineered virus provided by the invention.

Embodiment

Below in conjunction with embodiment and accompanying drawing, the present invention is described further.

Embodiment one:

The present invention is by gene engineering method, have the wild virus of strong virus force ( Spodoptera liturAnucleopolyhedrovirus

, SpltMNPV

, GenBank accession number: on basis NC-011616), lack and recombinate, a kind of prodenia litura genetically engineered virus is provided, having built the restructuring prodenia litura nuclear polyhedrosis virus, be i.e. No. 1 (SpltMNPV-△ of prodenia litura genetically engineered virus egt- Egfp) CCTCC NO:V201027.Referring to accompanying drawing 1, it is a kind of prodenia litura genetically engineered virus that the present embodiment provides, i.e. the structural representation of No. 1, prodenia litura genetically engineered virus; In figure, egtBe moulting hormone uridine diphosphoglucose based transferase gene; P phBe polyhedrin gene promoter; EgfpBe enhanced green fluorescent protein gene.As seen from Figure 1, lack in its genome egtGene is moulting hormone uridine diphosphoglucose based transferase gene, in disappearance egtThe site of gene, insert by the wild-type virus polyhedron gene ( ph) enhanced green fluorescence protein controlled of promotor ( Egfp) gene.The marker gene of this recombinant virus is enhanced green fluorescence protein EGFP, all can monitor and identify by the green fluorescence that green fluorescent protein produces in the purifying of this prodenia litura genetically engineered virus and breeding.

Referring to accompanying drawing 2, it is the building process schematic diagram of No. 1, the prodenia litura genetically engineered virus that provides of the present embodiment; Wherein, egtBe moulting hormone uridine diphosphoglucose based transferase gene; P phBe polyhedrin gene promoter; EgfpBe enhanced green fluorescent protein gene; . egt5 ' for containing egtThe upstream sequence of gene 5 ' end; egt3 ' for containing egtPartial sequence and the upstream sequence thereof of gene 3 ' end; PBluescript

SK (+) is universal support; SpltMNPV

Be wild-type virus; PSK-△ egt-P ph- EgfpBe disappearance egtGene also inserts EgfpThe recombinant transfer vector of gene; SpltMNPV-△ egt- EgfpBe disappearance egtGene also inserts EgfpThe prodenia litura caryogram polyhedrosis gene engineering virus strain of gene, i.e. No. 1 (SpltMNPV-△ of prodenia litura genetically engineered virus egt- Egfp) CCTCC NO:V201027.The construction process of this prodenia litura genetically engineered virus comprises the following steps:

(1) with SpltMNPV

DNA is template, amplifies respectively the homologous recombination arm egtUpstream non-coding end (Egt up) and egtPartial Fragment and the downstream non-coding end (Egt down) thereof of 3 ' end.Amplified production reclaims after purifying warp respectively KpnI / XhoI， XbaI/ SacBe cloned into successively on carrier pBluescript II SK (+) after the I double digestion.Interstitial granules pSK-Egtup-Egtdown in acquisition.

(2) between these two fragments, utilize molecule clone technology to insert SpltMNPV Polyhedrin gene promoter (P ph) start to control enhanced green fluorescent protein gene ( Egfp), obtain recombinant plasmid pSK-△ egt-P ph- Egfp

(3) with pSK-△ egt-P ph- EgfpAnd SpltMNPV

Genomic dna cotransfection prodenia litura (Spli) cell exchanges by allele, obtains disappearance egtThe recombinant virus SpltMNPV-△ of gene and expressing green fluorescent protein egt- EgfpBy many wheels fluorescent spot method purifying, obtaining pure recombinant virus is No. 1 (SpltMNPV-△ of prodenia litura genetically engineered virus egt- Egfp) CCTCC NO:V201027.

Table 1 is No. 1, the prodenia litura genetically engineered virus that provides of the present embodiment (being called for short engineering virus No. 1) and (using dosage is 10 in the bioassay results contrast of wild-type virus ⁷Individual polyhedron/bar larva).

Table 1

Virus strain	LT to 3 instar larvaes 50(day)	LT to 3 instar larvaes 90(day)
			Wild-type virus	3.33	4.95
No. 1, engineering virus	2.48	3.93

Table 2 is No. 1, the prodenia litura genetically engineered virus that provides of the present embodiment (being called for short engineering virus No. 1) and (working concentration is 10 in the land for growing field crops bioassay results contrast of wild-type virus ⁷Individual polyhedron/mL).

Table 2

Result by table 1 shows, is 10 at using dosage ⁷In the situation of individual polyhedron/bar larva, No. 1, engineering virus to 3 age prodenia litura median lethal time (LT ₅₀) shorten 25.3 % than wild virus.Result by table 2 shows, No. 1 insecticidal effect than wild virus of engineering virus obviously improves.Therefore, compare with existing wild virus, engineering virus provided by the invention has the advantage such as desinsection speed and better field effect faster for No. 1.

Viral cultures provided by the present invention is the insect viruses polyhedron, can only adopt live body propagation.Enrichment procedure: with 1 * 10 ⁶Then the concentration infection Spodoptera litura larvae in 3 age of polyhedron/ml is put 27～28 ℃ and was cultivated 5～7 days, collects the worm that dies of illness.Detect survival and also require biological assay, namely detect the activity of virus infection prodenia litura.

The authentication method of the prodenia litura genetically engineered virus that the present embodiment provides is as follows:

(1) utilize the exactness that the restriction endonuclease preliminary evaluation should virus;

(2) design suitable primer, utilize the method for polymerase chain reaction (PCR) further to determine in this virus egtMost of encoding sequence of gene is deleted, foreign gene ( Egfp) correctly insert;

(3) analyze the activity of this viral moulting hormone uridine diphosphoglucose based transferase, determine egtGene is inactivation;

(4) indoor biological activity determination and field control effectiveness test are determined the effect of virus.

Prodenia litura genetically engineered virus provided by the invention can be bred in enormous quantities by the polypide cultural method, be configured to the formulation viral pesticides such as wettable powder or suspended emulsion agent, be used for the control of prodenia litura on the plants such as farm crop, vegetables, flowers and lawn, sterilant has kept wild prodenia litura viral pesticide and has not injured natural enemy, the environmentally safe and insect advantage such as be difficult for developing immunity to drugs.

Claims (1)

1. one kind to wild-type virus SpltMNPV

The prodenia litura genetically engineered virus that lacks and recombinate is characterized in that: its preservation information is: depositary institution: Chinese Typical Representative culture collection center; Deposit number: CCTCC NO:V201027; Classification And Nomenclature: No. 1, prodenia litura genetically engineered virus has lacked in genome egtGene is moulting hormone uridine diphosphoglucose based transferase gene, in disappearance egtThe site of gene is inserted by SpltMNPV

Polyhedron gene phThe marker gene that promotor is controlled is enhanced green fluorescence protein EgfpGene.

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