斜纹夜蛾核型多角体病毒Ⅱ型（SpltMNPV Ⅱ）BV、ODV组成型蛋白质谱分析和4种ORF的基因分析
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摘要
斜纹夜蛾(Spodoptera litura, Spli)属鳞翅目夜蛾科,是一种杂食性农业和林业害虫,主要危害甘蓝、白菜、藕、芋、蕹菜、苋菜、马铃薯、茄子、辣椒、番茄、豆类、瓜类以及葱韭等。由于斜纹夜蛾的食性杂、世代重叠、抗性强,因此防治难度高。在各种化学合成杀虫剂严重污染环境,害虫对化学合成杀虫剂抗性日益增强的情况下,生物防治越显重要和迫切。从自然界中寻找和筛选新的高毒力的病毒株系是开发NPV生物杀虫剂的重要途径。SpltMNPVⅡ是一种繁殖率和毒力极强的新的病毒变异株。该病毒株的基因组全序列已经测定完成。但病毒BV和ODV的组成蛋白并不清楚,论文对SpltMNPVⅡBV和ODV进行了质谱分析,确定了BV和ODV蛋白组成,并对组成蛋白基因的中的ORF6、ORF13、ORF57和ORF146进行了启动子活性分析,转录时相和表达时相分析,对ORF57基因的部分功能进行了鉴定。
主要研究结果与发现:
1.用二级质谱分析方法对SpltMNPVⅡBV和ODV的组成蛋白进行了研究,确定了SpltMNPVⅡBV由42种蛋白组成,通过与已知杆状病毒同源基因的比较,确定了其中26种蛋白基因的部分功能;SpltMNPVⅡODV由46种蛋白组成,与已知杆状病毒同源基因的比较,确定了其中24种蛋白基因的部分功能。
2.对SpltMNPVⅡORF6、ORF13、ORF57和ORF146基因结构进行了分析。ORF6基因全长1509 bp,编码502 aa,预测蛋白分子量为55.0 kDa。该基因既有早期启动子基序CAGT,又有晚期启动子基序ATAAG,推测该基因可能是一个在病毒生命周期中早晚期都表达的基因;ORF13基因读码框全长333 bp,编码110 aa,理论预测蛋白分子量为12.2 kDa。起始密码子ATG上游发现有一个TATA box和杆状病毒晚期启动子基序ATAAG,没有发现明显的早期启动子基序;ORF57基因全长1098 bp,编码365 aa,预测蛋白分子量为42.6 kDa。起始密码子ATG上游发现有一个TATA box,在起始密码子上游没有发现明显的早期和晚期启动子基序;ORF146基因涵盖了1383 bp的读码框,编码460 aa,理论预测蛋白分子量为50.2 kDa。该基因起始密码子ATG上游发现有一个TATA box,一个晚期启动子基序ATAAG。
3.对SpltMNPVⅡORF6、ORF13、ORF57和ORF146基因的启动子活性和转录时相进行了分析。发现这4个基因是在病毒生命周期的早期和晚期都有转录的基因。其中ORF13和ORF146基因能被SpltMNPVⅡ病毒编码的反式作用因子所激活,在感染后期能提高转录水平。而ORF6基因也能被SpltMNPVⅡ病毒编码的反式作用因子所激活,但是为负调控,在感染后期转录水平有明显下降。
4.对SpltMNPVⅡORF6、ORF13和ORF146基因的部分序列进行了原核表达,表达产物经纯化和质谱鉴定后,免疫豚鼠制作了多克隆抗体。抗体效价用ELISA进行测定,3个基因的表达产物制作的抗体效价都在1:2500以上。
5.以制备的多克隆抗体为一抗,对SpltMNPVⅡORF6、ORF13和ORF146基因在宿主细胞的表达产物进行了表达时相分析。0RF6基因产物最早在病毒感染细胞后8 h出现,0RF146基因产物最早在病毒感染细胞后4 h出现,并且后期都有表达,说明这两个基因都是早期和晚期均表达的基因,与它们的启动子分析和转录时相分析结果一致。而ORF13最早在出现病毒感染细胞后12 h,并一直持续到后期,说明ORF13为晚期表达基因。但是ORF13的启动子活性分析和转录时相分析都表明该基因是个早晚期都表达的基因。可能是由于该基因在表达的初始阶段,蛋白合成量和积累水平比较低,还不足以检测出来的缘故。
6.克隆和表达了SpltMNPVⅡORF57基因的完整ORF,纯化的表达产物用质谱进行了鉴定。采用同位素法和痕量磷特异检测方法(孔雀石绿检测试剂法)确定了ORF57基因是一个双功能酶基因,该基因编码的蛋白既具有多聚核苷酸激酶功能,又具有3’磷酸酶功能。
Spodoptera litura(Spli) is an omnivorous agricultural and forestry pests of Lepidoptera Noctuidae, mainly against cruciferous vegetables, beans, melons, onions and other crops. As the Spodoptera litura feeding miscellaneous, generations overlap, and strong resistance, it is difficult to control. Chemical synthesis of various pesticides are serious pollution of the environment and pest resistance to chemical synthetic pesticides growing circumstances, biological control is more and more important and urgent. From natural search and selection of new highly virulent virus strain is another important way to develop biological pesticides NPV. SpltMNPVⅡis a kind of reproduction rate and the highly virulent new virus variants. The whole genome sequence has been determined complete. However, the composition of the BV and ODV were unknown, to determine the composition of the BV and ODV, we analyse BV and ODV by mass spectrometry, the BV and ODV structutal proteins were determined, and the promoter activity analysis, transcription phase analysis and expression phase analysis have been done on the ORF6, ORF13, ORF57 and ORF146 genes,as well,the function of the ORF57 gene was identified. The major findings are as follows:
1, The constitutive protein of SpltMNPVⅡBV and ODV were determined by mass spectrometry .The SpltMNPVⅡBV was composed of 42 different protein, compared with known homology baculovirus gene, 26 species of which gene function were identified; SpltMNPVⅡODV was composed of 46 kinds of proteins, compared with known homology baculovirus gene, 24 species of which gene function were identified.
2, SpltMNPVⅡORF6, ORF13, ORF57 and ORF146 gene structure analysis have been done, ORF6 gene was 1509bp, encoding 502aa, predicted protein molecular weight of 55.0kDa, both early promoter motif CAGT and late promoter motif ATAAG were found, suggesting that the gene may be a gene that both express in early and late phase in the viral life cycle. ORF13 gene opening reading frame has a length of 333 bp, encoding 110 amino acids, predicted protein molecular weight of 12.2kD. A TATA box was found upstream of Start codon ATG, late promoter motif ATAAG was found upstream of Start codon ATG, there is no obvious early promoter motif. ORF57 gene is 1098bp, encoding 365aa, predicted protein molecular weight of 42.6kDa. A TATA box was found upstream of Start codon ATG, we did not find significant early and late promoter motifs upstream of the start codon. ORF146 gene covers 1383 bp open reading frame, encoding 460 amino acids, predicted protein molecular weight of 50.2kD. A TATA box and a late promoter motif ATAAG were found upstream of the gene start codon ATG.
3, SpltMNPVⅡORF6, ORF13, ORF57 and ORF146 gene promoter activity and transcription phase were analyzed. Both early and late gene transcription were found in the virus life cycle,. ORF13 and ORF146 genes which could be activated by SpltMNPVⅡtrans-acting factors in the late stage of infection, which can increase the transcription level. The ORF6 gene can also be activated by SpltMNPVⅡtrans-acting factors, but the transcription level in the late stage of infection was decreased significantly.
4, Part of SpltMNPVⅡORF6, ORF13 and ORF146 gene sequences were expressed by prokaryotic expression system,which products were purified and idenified by mass spectrometry,.The immunization of guinea pigs produced a polyclonal antibody., antibody titers were determined by ELISA, antibody titers of polyclonal antibodies which produced from the three gene expression product were high, more than in 1:2500.
5, Using polyclonal antibodies as first antibody, products from SpltMNPVⅡORF6, ORF13 and ORF146 gene expression in the host cell were analyzed. 0RF6 gene expressed in infected cells as early as 8 h after infection, 0RF146 gene expressed as early as 4 h after infection, these two genes were expressed both early and late phase, and their Promoter analysis and special libraries is consistent with the results. The ORF13 first infected cells in the event of 12h, and continued until the late phase,which shown ORF13 was the late gene. But the ORF13 promoter activity analysis and transcription phase analysis demonstrated that the gene was expressed both early and late phase. This estimate is due to the gene product, in the initial stage of protein synthesis was low which not enough to detect.
6, We cloned and expressed the SpltMNPVⅡORF57 gene complete ORF, the expression product was purified and identified using mass spectrometry. With the isotope method and specific detection of trace phosphorus (malachite green reagent method) to determine the ORF57 gene encoding a protein has both polynucleotide kinase function and 3 'phosphatase function. ORF57 gene is a bifunctional enzyme gene.
主要研究结果与发现:
1.用二级质谱分析方法对SpltMNPVⅡBV和ODV的组成蛋白进行了研究,确定了SpltMNPVⅡBV由42种蛋白组成,通过与已知杆状病毒同源基因的比较,确定了其中26种蛋白基因的部分功能;SpltMNPVⅡODV由46种蛋白组成,与已知杆状病毒同源基因的比较,确定了其中24种蛋白基因的部分功能。
2.对SpltMNPVⅡORF6、ORF13、ORF57和ORF146基因结构进行了分析。ORF6基因全长1509 bp,编码502 aa,预测蛋白分子量为55.0 kDa。该基因既有早期启动子基序CAGT,又有晚期启动子基序ATAAG,推测该基因可能是一个在病毒生命周期中早晚期都表达的基因;ORF13基因读码框全长333 bp,编码110 aa,理论预测蛋白分子量为12.2 kDa。起始密码子ATG上游发现有一个TATA box和杆状病毒晚期启动子基序ATAAG,没有发现明显的早期启动子基序;ORF57基因全长1098 bp,编码365 aa,预测蛋白分子量为42.6 kDa。起始密码子ATG上游发现有一个TATA box,在起始密码子上游没有发现明显的早期和晚期启动子基序;ORF146基因涵盖了1383 bp的读码框,编码460 aa,理论预测蛋白分子量为50.2 kDa。该基因起始密码子ATG上游发现有一个TATA box,一个晚期启动子基序ATAAG。
3.对SpltMNPVⅡORF6、ORF13、ORF57和ORF146基因的启动子活性和转录时相进行了分析。发现这4个基因是在病毒生命周期的早期和晚期都有转录的基因。其中ORF13和ORF146基因能被SpltMNPVⅡ病毒编码的反式作用因子所激活,在感染后期能提高转录水平。而ORF6基因也能被SpltMNPVⅡ病毒编码的反式作用因子所激活,但是为负调控,在感染后期转录水平有明显下降。
4.对SpltMNPVⅡORF6、ORF13和ORF146基因的部分序列进行了原核表达,表达产物经纯化和质谱鉴定后,免疫豚鼠制作了多克隆抗体。抗体效价用ELISA进行测定,3个基因的表达产物制作的抗体效价都在1:2500以上。
5.以制备的多克隆抗体为一抗,对SpltMNPVⅡORF6、ORF13和ORF146基因在宿主细胞的表达产物进行了表达时相分析。0RF6基因产物最早在病毒感染细胞后8 h出现,0RF146基因产物最早在病毒感染细胞后4 h出现,并且后期都有表达,说明这两个基因都是早期和晚期均表达的基因,与它们的启动子分析和转录时相分析结果一致。而ORF13最早在出现病毒感染细胞后12 h,并一直持续到后期,说明ORF13为晚期表达基因。但是ORF13的启动子活性分析和转录时相分析都表明该基因是个早晚期都表达的基因。可能是由于该基因在表达的初始阶段,蛋白合成量和积累水平比较低,还不足以检测出来的缘故。
6.克隆和表达了SpltMNPVⅡORF57基因的完整ORF,纯化的表达产物用质谱进行了鉴定。采用同位素法和痕量磷特异检测方法(孔雀石绿检测试剂法)确定了ORF57基因是一个双功能酶基因,该基因编码的蛋白既具有多聚核苷酸激酶功能,又具有3’磷酸酶功能。
Spodoptera litura(Spli) is an omnivorous agricultural and forestry pests of Lepidoptera Noctuidae, mainly against cruciferous vegetables, beans, melons, onions and other crops. As the Spodoptera litura feeding miscellaneous, generations overlap, and strong resistance, it is difficult to control. Chemical synthesis of various pesticides are serious pollution of the environment and pest resistance to chemical synthetic pesticides growing circumstances, biological control is more and more important and urgent. From natural search and selection of new highly virulent virus strain is another important way to develop biological pesticides NPV. SpltMNPVⅡis a kind of reproduction rate and the highly virulent new virus variants. The whole genome sequence has been determined complete. However, the composition of the BV and ODV were unknown, to determine the composition of the BV and ODV, we analyse BV and ODV by mass spectrometry, the BV and ODV structutal proteins were determined, and the promoter activity analysis, transcription phase analysis and expression phase analysis have been done on the ORF6, ORF13, ORF57 and ORF146 genes,as well,the function of the ORF57 gene was identified. The major findings are as follows:
1, The constitutive protein of SpltMNPVⅡBV and ODV were determined by mass spectrometry .The SpltMNPVⅡBV was composed of 42 different protein, compared with known homology baculovirus gene, 26 species of which gene function were identified; SpltMNPVⅡODV was composed of 46 kinds of proteins, compared with known homology baculovirus gene, 24 species of which gene function were identified.
2, SpltMNPVⅡORF6, ORF13, ORF57 and ORF146 gene structure analysis have been done, ORF6 gene was 1509bp, encoding 502aa, predicted protein molecular weight of 55.0kDa, both early promoter motif CAGT and late promoter motif ATAAG were found, suggesting that the gene may be a gene that both express in early and late phase in the viral life cycle. ORF13 gene opening reading frame has a length of 333 bp, encoding 110 amino acids, predicted protein molecular weight of 12.2kD. A TATA box was found upstream of Start codon ATG, late promoter motif ATAAG was found upstream of Start codon ATG, there is no obvious early promoter motif. ORF57 gene is 1098bp, encoding 365aa, predicted protein molecular weight of 42.6kDa. A TATA box was found upstream of Start codon ATG, we did not find significant early and late promoter motifs upstream of the start codon. ORF146 gene covers 1383 bp open reading frame, encoding 460 amino acids, predicted protein molecular weight of 50.2kD. A TATA box and a late promoter motif ATAAG were found upstream of the gene start codon ATG.
3, SpltMNPVⅡORF6, ORF13, ORF57 and ORF146 gene promoter activity and transcription phase were analyzed. Both early and late gene transcription were found in the virus life cycle,. ORF13 and ORF146 genes which could be activated by SpltMNPVⅡtrans-acting factors in the late stage of infection, which can increase the transcription level. The ORF6 gene can also be activated by SpltMNPVⅡtrans-acting factors, but the transcription level in the late stage of infection was decreased significantly.
4, Part of SpltMNPVⅡORF6, ORF13 and ORF146 gene sequences were expressed by prokaryotic expression system,which products were purified and idenified by mass spectrometry,.The immunization of guinea pigs produced a polyclonal antibody., antibody titers were determined by ELISA, antibody titers of polyclonal antibodies which produced from the three gene expression product were high, more than in 1:2500.
5, Using polyclonal antibodies as first antibody, products from SpltMNPVⅡORF6, ORF13 and ORF146 gene expression in the host cell were analyzed. 0RF6 gene expressed in infected cells as early as 8 h after infection, 0RF146 gene expressed as early as 4 h after infection, these two genes were expressed both early and late phase, and their Promoter analysis and special libraries is consistent with the results. The ORF13 first infected cells in the event of 12h, and continued until the late phase,which shown ORF13 was the late gene. But the ORF13 promoter activity analysis and transcription phase analysis demonstrated that the gene was expressed both early and late phase. This estimate is due to the gene product, in the initial stage of protein synthesis was low which not enough to detect.
6, We cloned and expressed the SpltMNPVⅡORF57 gene complete ORF, the expression product was purified and identified using mass spectrometry. With the isotope method and specific detection of trace phosphorus (malachite green reagent method) to determine the ORF57 gene encoding a protein has both polynucleotide kinase function and 3 'phosphatase function. ORF57 gene is a bifunctional enzyme gene.
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