斜纹夜蛾核型多角体病毒日本B株pif基因及C1株分子鉴定研究
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摘要
斜纹夜蛾核型多角体病毒(Spodoptera litura multicapsid nucleopolyhedrovirus,SpltMNPV)属于杆状病毒科(Baculoviridae)包涵体杆状病毒亚科(Eubaculovirinae)核型多角体病毒属(Nucleopolyhedrovirus, NPV)A亚群。国内于1972年在广州地区首次从野外死亡的斜纹夜蛾虫体分离到这种病毒。其宿主斜纹夜蛾(Spodoptera litura, Spli)是鳞翅目夜蛾科的杂食性害虫,分布范围广,是我国粮棉、油等经济作物和蔬菜的重要害虫之一。应用SpltMNPV防治斜纹夜蛾是一种有效的生物防治方法,而通过基因工程手段构建新的高效杀虫病毒是改良生物病毒杀虫剂的重要途径。从分子水平深入地阐明SpltMNPV在体内的侵染、增殖规律、建立SpltMNPV载体表达系统是达到上述目的的理论基础和技术准备。因此,近年来对SpltMNPV的研究受到广泛关注,有关该病毒的序列测定、基因结构、功能和表达调控等分子生物学研究工作进展迅速,特别是对一些重要基因的结构分析,有助于筛选毒力较强的杀虫毒株,并为这一病毒杀虫剂的改良和发展以及组建昆虫杆状病毒表达载体奠定基础。本研究在Spli-SpltMNPV的模型中对三个经口感染相关基因进行了初步的研究,并尝试构建了带有绿色荧光蛋白标记的转移载体,主要工作及成果如下:
1、克隆了SpltMNPV日本B株三个经口感染因子(per os infectivity Factor, pif、pif-2、pif-3)基因,大小分别为1581 bp、1278 bp和603 bp,其推导的氨基酸序列分别为527 aa( GenBank登录号为FJ384665),426 aa(GenBank登录号为FJ384666)和201 aa (GenBank登录号为FJ384667)。根据推导的氨基酸序列进行了蛋白质的二级结构预测和分子系统发育分析。
2、在大肠杆菌内表达了pif-2基因,并对其进行了转录时相分析。将pif-2克隆至原核表达载体pET32a(+),经IPTG诱导,SDS-PAGE显示在69kD处有特异性表达条带,Western blot分析结果为目的蛋白。SpltMNPV感染Spli细胞,收集不同时间点的细胞,运用RT-PCR法检验pif-2在细胞内的转录时相。结果显示pif-2在细胞内从3 h持续到72 h都有转录本的存在。
3、构建了以增强型绿色荧光蛋白基因(egfp)为标记的、pif-2失活的重组斜纹夜蛾核型多角体病毒。分别扩增出pif-2读码框上下游的不同片段及ph启动子启动的egfp ,将三个片段按顺序克隆进pUC19载体,构建了转移载体pSplt-△pif-2-egfp。将pSplt-△pif-2-egfp与野生型SpltMNPV DNA共转染Spli细胞,通过同源重组和有限稀释法筛选,获得了以ph-egfp基因替代pif-2的重组病毒SpltMNPV-△pif-2-egfp,为SpltMNPV pif-2的功能研究打下了基础。
4、首次从分子层次研究了SpltMNPV-JP-C1的DNA构成。将纯化的C1株基因组酶切,利用半补齐法获得了C1株的基因组文库。从文库中随机抽取8个克隆测序,将测序结果与中国G2株和日本C3株进行序列比对,结果证明,所有的测序结果与中国G2株都有较高的相似性,而与SpltNPV日本株C3同源性极低。
Spodoptera litura (Lepidoptera:Noctuidae) multicapsid nucleopolyhedrovirus, SpltMNPV,which,in china, was firstly isolated from cadavers of Spodoptera litura in Guangzhou, belongs to the subgroup A of Nucleopolyhedrovirus.Spodoptera litura, the host of the virus is a kind of polyphagous insect which is harmful to economic crop and vegetables such as alimentary crop,cotton,oil plants and so on. And it is also one of the primary insects pests around the world. Present, SpltMNPV is considered as the most effective insecticide against the insect, therefore, the researches on it are becoming more and more popular. Those years, some of the studies about the virus have been greatly improved, such as the sequence determination, the analysis of its gene structure, its function and regulation of expression. Those progresses, especially the analysis of some important gene structures of it, have contributed to the selecting of the strain with stronger virulence, the improving and developing of this viral insecticide, and the constructing of the baculovirus expression vector.In order to research on Spli-SpltMNPV system, there of per os infectivity gene was cloned and the novel baculovirus transfer vector containing egfp was constructed.The main conclusions are as followed:
(1)The per os infectivity factors of B strains of SpltMNPV from Japan are composed of 1581、1278 and 603 nucleic acids, which codes a peptide of 527、426 and 201 amino acids.These genes sequences have been submitted to the NCBI GenBank and the accession numbers are FJ384665(pif) FJ384666(pif-2) FJ384667(pif-3).Proteins, second structure and the molecule phylogeny are analyzed based on these sequences of amino acids.
(2)The pif-2 gene was highly expressed in the E.coli and analyzed its phase of transcription.The pif-2 gene was cloned into pET32a(+) and induced in E.coli by IPTG. A specific band was detected at 69 kD,identified by Western Blotting.Collect the cells at the different time,which were infected by the SpltMNPV BV. The phase of transcription was analyzed by the method of RT-PCR.The exist of transcript of pif-2 was found from 3h to 72h.
(3)The recombinant virus marked by egfp was constructed, pif-2 was inactive. The fragment of ph-egfp was inserted into this vector between the 5’end and 3’end-flanking fragments of SpltMNPV pif-2 gene tandem linked into pUC19. The spli cells were cotransfected with pSplt-△pif-2-egfp and the wild SpltMNPV genome DNA. The recombinant virus containing egfp was selected with the limited dilution method.
(4) SpltMNPV-JP-C1 was invested at the level of molecule.The pure genome of C1 was cut by enzyme and constructed the genomic library.8 random clones was sequenced.The sequences were compared with the G2 and C3.From the result we can found that compared with the C3,ll the clones were hightly homologous with the G2.
1、克隆了SpltMNPV日本B株三个经口感染因子(per os infectivity Factor, pif、pif-2、pif-3)基因,大小分别为1581 bp、1278 bp和603 bp,其推导的氨基酸序列分别为527 aa( GenBank登录号为FJ384665),426 aa(GenBank登录号为FJ384666)和201 aa (GenBank登录号为FJ384667)。根据推导的氨基酸序列进行了蛋白质的二级结构预测和分子系统发育分析。
2、在大肠杆菌内表达了pif-2基因,并对其进行了转录时相分析。将pif-2克隆至原核表达载体pET32a(+),经IPTG诱导,SDS-PAGE显示在69kD处有特异性表达条带,Western blot分析结果为目的蛋白。SpltMNPV感染Spli细胞,收集不同时间点的细胞,运用RT-PCR法检验pif-2在细胞内的转录时相。结果显示pif-2在细胞内从3 h持续到72 h都有转录本的存在。
3、构建了以增强型绿色荧光蛋白基因(egfp)为标记的、pif-2失活的重组斜纹夜蛾核型多角体病毒。分别扩增出pif-2读码框上下游的不同片段及ph启动子启动的egfp ,将三个片段按顺序克隆进pUC19载体,构建了转移载体pSplt-△pif-2-egfp。将pSplt-△pif-2-egfp与野生型SpltMNPV DNA共转染Spli细胞,通过同源重组和有限稀释法筛选,获得了以ph-egfp基因替代pif-2的重组病毒SpltMNPV-△pif-2-egfp,为SpltMNPV pif-2的功能研究打下了基础。
4、首次从分子层次研究了SpltMNPV-JP-C1的DNA构成。将纯化的C1株基因组酶切,利用半补齐法获得了C1株的基因组文库。从文库中随机抽取8个克隆测序,将测序结果与中国G2株和日本C3株进行序列比对,结果证明,所有的测序结果与中国G2株都有较高的相似性,而与SpltNPV日本株C3同源性极低。
Spodoptera litura (Lepidoptera:Noctuidae) multicapsid nucleopolyhedrovirus, SpltMNPV,which,in china, was firstly isolated from cadavers of Spodoptera litura in Guangzhou, belongs to the subgroup A of Nucleopolyhedrovirus.Spodoptera litura, the host of the virus is a kind of polyphagous insect which is harmful to economic crop and vegetables such as alimentary crop,cotton,oil plants and so on. And it is also one of the primary insects pests around the world. Present, SpltMNPV is considered as the most effective insecticide against the insect, therefore, the researches on it are becoming more and more popular. Those years, some of the studies about the virus have been greatly improved, such as the sequence determination, the analysis of its gene structure, its function and regulation of expression. Those progresses, especially the analysis of some important gene structures of it, have contributed to the selecting of the strain with stronger virulence, the improving and developing of this viral insecticide, and the constructing of the baculovirus expression vector.In order to research on Spli-SpltMNPV system, there of per os infectivity gene was cloned and the novel baculovirus transfer vector containing egfp was constructed.The main conclusions are as followed:
(1)The per os infectivity factors of B strains of SpltMNPV from Japan are composed of 1581、1278 and 603 nucleic acids, which codes a peptide of 527、426 and 201 amino acids.These genes sequences have been submitted to the NCBI GenBank and the accession numbers are FJ384665(pif) FJ384666(pif-2) FJ384667(pif-3).Proteins, second structure and the molecule phylogeny are analyzed based on these sequences of amino acids.
(2)The pif-2 gene was highly expressed in the E.coli and analyzed its phase of transcription.The pif-2 gene was cloned into pET32a(+) and induced in E.coli by IPTG. A specific band was detected at 69 kD,identified by Western Blotting.Collect the cells at the different time,which were infected by the SpltMNPV BV. The phase of transcription was analyzed by the method of RT-PCR.The exist of transcript of pif-2 was found from 3h to 72h.
(3)The recombinant virus marked by egfp was constructed, pif-2 was inactive. The fragment of ph-egfp was inserted into this vector between the 5’end and 3’end-flanking fragments of SpltMNPV pif-2 gene tandem linked into pUC19. The spli cells were cotransfected with pSplt-△pif-2-egfp and the wild SpltMNPV genome DNA. The recombinant virus containing egfp was selected with the limited dilution method.
(4) SpltMNPV-JP-C1 was invested at the level of molecule.The pure genome of C1 was cut by enzyme and constructed the genomic library.8 random clones was sequenced.The sequences were compared with the G2 and C3.From the result we can found that compared with the C3,ll the clones were hightly homologous with the G2.
引文
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[1]姚文辉斜纹夜蛾的生物学特性。华东昆虫学报, 2005, 14(2); 122～127。
[2]吕鸿声昆虫病毒分子生物学。北京:中国农业科技出版社,1998。
[3] 0’Reilly D R, Miller L K. Improvement of a baculovirus pesticide by deletion of the TGE gene. Biotechnology,1991,9:1086-1089.
[4] Kikhno I, Gutiérrez S, Croizier L,et al. 2002, Characterization of pif, a gene required for the per os infectivity of Spodoptera littoralis nucleopolyhedrovirus.J Gen Virol, 83:3013-3022.
[5] Minggang Fang,Yingchao Nie,Qian Wang,et al.2006,Open reading frame 132 of Heliocoverpa armigera nucleopolyhedrovirus encodes a functional per os infectivity factor(PIF-2).Journal of General Virology,87:2563-2569.
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