SpltMNPV重组病毒的构建和SpltMNPV-JP-G_（1-2）iap基因的克隆
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摘要
斜纹夜蛾(Spodoptera litura)是各种农作物和绿化植物的重要害虫,能够危害109科389种寄主植物。目前,生产上对于斜纹夜蛾的防治主要侧重于化学防治,但效果并不理想。从保持生态平衡和降低化学农药对环境污染的战略考虑,利用斜纹夜蛾核型多角体病毒(spodoptera litura multicapsid nucleopolyhedrovirus,SpltMNPV)防治农作物害虫已成为国内外生物防治的重要内容之一。但是,它也有一定的局限性,在野外条件下,杀虫速度较慢,对高龄害虫需用量大以及宿主域窄等缺点限制了其广泛应用。为了克服上述不足,最重要的方法之一就是应用基因工程和分子生物学等手段,通过缺失某一基因并表达昆虫选择性毒素基因。
本研究对野生型斜纹夜蛾核型多角体病毒(SpltMNPV)日本株G_(1-2)的凋亡抑制基因-iap基因进行克隆与序列分析;对野生型斜纹夜蛾核型多角体病毒(SpltMNPV)日本株G_(1-2)和G_(10-3)进行改造,通过缺失polh或egt基因并插入由多角体启动子pph启动外源毒素.蝎毒素基因以及增强型绿色荧光蛋白标记基因(enchanced greenfluorescent protein gene,egfp)基因,构建了新的基因工程重组病毒。
主要工作成果如下:
1、克隆了SpltMNPV-JP-G_(1-2)的凋亡抑制基因iap,该基因大小为411bp,其编码区共编码136个氨基酸,预计蛋白分子量15.67kD(GenBank的登录号为FJ384668)。采用生物信息学方法对iap基因进行了分子系统发育分析。
2、利用昆虫病毒分子生物学和基因工程理论、方法和技术对具有强烈杀虫活性的斜纹夜蛾核型多角体病毒日本株进行基因工程改造。成功构建了8个SpltMNPV重组转移载体,为构建和筛选重组斜纹夜蛾杆状病毒杀虫剂奠定了基础。
3、进行了基因工程重组斜纹夜蛾核型多角体病毒的尝试。使用10个重组转移载体与野生型SpltMNPV DNA共转染Spli细胞,通过同源重组和有限稀释法筛选,初步获得了三个重组病毒SpltMNPV-△egtG_(1-2)-p10-egfp-pph-Bt、SpltMNPV-△egtG_(1-2)-p10-egfp-pph-BmK ITal和SpltMNPV-△egtG_(10-3)-pph-egfp,为进一步开展斜纹夜蛾的生物防治研究打下了良好的基础。
Spodoptera litura((Lepidoptera:Noctuidae)) is one of the most serious pest around the world.They are highly polyphagous,attacking 389 specieses,109 families of agricultural crops and greening plants.Recently,chemical pesticides are frequently used to control pest but has little effect.Considing to protect the environmental and to balance ecological environment,Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) has been developed as a commercial biopesticide to control the crop inscets in the world.However,SpltMNPV also has definite restrictions in outdoors condition,a slowish speed on killing pest and more quantity and narrow host specificity limit to use in large scale.In order to solve the above problems,one of the most approaches to improve the insecticidal properties of SpltMNPV is by means of genetic engineering and molecular biology to lack a certain gene and express the gene of the insect-selective toxins.
The sequence of SpltMNPV-JP-G_(1-2) iap gene was cloned and analysed in this research.SpltMNPV-JP-G_(1-2) and G_(10-3) were recombined and a new recombinant virus was constrcucted by lacking of polh or egt gene and inserting insect-selective toxin
Bmk ITa1 and egfp under the pph promoter.
The main conclusions are as followed:
1.The iap gene was cloned from SpltMNPV-JP-G_(1-2) strain genome.This gene that has been submitted to the NCBI GenBank and the accession numbers is FJ384668.It's 411bp long and potential to encode 136 amino acids,a polypeptide with predicted molecular weight of 15.67 kD.Analyzing nucleotide and amino acid sequence of gene iap in SpltMNPV Japanese strain(G_(1-2))was done at the molecular biological level.
2.Genetic engineering and molecular biology approaches have been developed to enhance the insecticidal properties of SpltMNPV-JP.Eight SpltMNPV recombinant transfer vectors have been constructed successfully and to lay the foundation for constructing and filtrating the insecticide of baculovirus.
3.Genetic recombinant SpltMNPV has been carried out.The spli cells were cotransfected with ten transfer vectors and the wild SpltMNPV genome DNA.With homologous recombination and limiting dilution assay,three recombinant virus, SpltMNPV-ΔegtG_(1-2)-p10-egfp-pph-Bt,SpltMNPV-Δegt G_(1-2)-p10-egfp-pph-BmK ITa1 and SpltMNPV-Δegt G_(10-3)-pph-egfp,were gained.The results lay the foundation for the improvement and development of this virus insecticide and further study of biological control.
本研究对野生型斜纹夜蛾核型多角体病毒(SpltMNPV)日本株G_(1-2)的凋亡抑制基因-iap基因进行克隆与序列分析;对野生型斜纹夜蛾核型多角体病毒(SpltMNPV)日本株G_(1-2)和G_(10-3)进行改造,通过缺失polh或egt基因并插入由多角体启动子pph启动外源毒素.蝎毒素基因以及增强型绿色荧光蛋白标记基因(enchanced greenfluorescent protein gene,egfp)基因,构建了新的基因工程重组病毒。
主要工作成果如下:
1、克隆了SpltMNPV-JP-G_(1-2)的凋亡抑制基因iap,该基因大小为411bp,其编码区共编码136个氨基酸,预计蛋白分子量15.67kD(GenBank的登录号为FJ384668)。采用生物信息学方法对iap基因进行了分子系统发育分析。
2、利用昆虫病毒分子生物学和基因工程理论、方法和技术对具有强烈杀虫活性的斜纹夜蛾核型多角体病毒日本株进行基因工程改造。成功构建了8个SpltMNPV重组转移载体,为构建和筛选重组斜纹夜蛾杆状病毒杀虫剂奠定了基础。
3、进行了基因工程重组斜纹夜蛾核型多角体病毒的尝试。使用10个重组转移载体与野生型SpltMNPV DNA共转染Spli细胞,通过同源重组和有限稀释法筛选,初步获得了三个重组病毒SpltMNPV-△egtG_(1-2)-p10-egfp-pph-Bt、SpltMNPV-△egtG_(1-2)-p10-egfp-pph-BmK ITal和SpltMNPV-△egtG_(10-3)-pph-egfp,为进一步开展斜纹夜蛾的生物防治研究打下了良好的基础。
Spodoptera litura((Lepidoptera:Noctuidae)) is one of the most serious pest around the world.They are highly polyphagous,attacking 389 specieses,109 families of agricultural crops and greening plants.Recently,chemical pesticides are frequently used to control pest but has little effect.Considing to protect the environmental and to balance ecological environment,Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) has been developed as a commercial biopesticide to control the crop inscets in the world.However,SpltMNPV also has definite restrictions in outdoors condition,a slowish speed on killing pest and more quantity and narrow host specificity limit to use in large scale.In order to solve the above problems,one of the most approaches to improve the insecticidal properties of SpltMNPV is by means of genetic engineering and molecular biology to lack a certain gene and express the gene of the insect-selective toxins.
The sequence of SpltMNPV-JP-G_(1-2) iap gene was cloned and analysed in this research.SpltMNPV-JP-G_(1-2) and G_(10-3) were recombined and a new recombinant virus was constrcucted by lacking of polh or egt gene and inserting insect-selective toxin
Bmk ITa1 and egfp under the pph promoter.
The main conclusions are as followed:
1.The iap gene was cloned from SpltMNPV-JP-G_(1-2) strain genome.This gene that has been submitted to the NCBI GenBank and the accession numbers is FJ384668.It's 411bp long and potential to encode 136 amino acids,a polypeptide with predicted molecular weight of 15.67 kD.Analyzing nucleotide and amino acid sequence of gene iap in SpltMNPV Japanese strain(G_(1-2))was done at the molecular biological level.
2.Genetic engineering and molecular biology approaches have been developed to enhance the insecticidal properties of SpltMNPV-JP.Eight SpltMNPV recombinant transfer vectors have been constructed successfully and to lay the foundation for constructing and filtrating the insecticide of baculovirus.
3.Genetic recombinant SpltMNPV has been carried out.The spli cells were cotransfected with ten transfer vectors and the wild SpltMNPV genome DNA.With homologous recombination and limiting dilution assay,three recombinant virus, SpltMNPV-ΔegtG_(1-2)-p10-egfp-pph-Bt,SpltMNPV-Δegt G_(1-2)-p10-egfp-pph-BmK ITa1 and SpltMNPV-Δegt G_(10-3)-pph-egfp,were gained.The results lay the foundation for the improvement and development of this virus insecticide and further study of biological control.
引文
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