棉铃虫核型多角体病毒朝鲜株的部分基因组克隆载体的构建与序列分析
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摘要
棉铃虫核型多角体病毒(Helicovera armigera nucleocapsid nucleopolyhedrovirus，简称HaNPV)属于杆状病毒科，核型多角体病毒属。其中又分为两个亚属，即单核衣壳NPV亚属(SNPV)和多核衣壳NPV亚属(MNPV)。
经过对棉铃虫核型多角体病毒的多角体生物学特性、繁殖、形态结构和基因组几种限制性内切酶酶切图谱初步分析，每个分离株(isolate)或变异株(variant)的杀虫毒力并不完全相同。
通过生物活性实验，已确定了棉铃虫单粒包埋核型多角体(HaSNPV)的杀虫毒力比棉铃虫多粒包埋型核型多角体(HaMNPV)的杀虫毒力高得多。
到目前为止，棉铃虫单粒包埋核型多角体病毒(HaSNPV)中发现一些分离株，它们包括G4分离株，上海株，博茨瓦那株(HaS-BW)，印度株(HaS-IN)和南非株(HaS-SA)。
为了充分利用棉铃虫单粒包埋核型多角体病毒资源，为了开展害虫生物防治提供依据，对首次在朝鲜分离得到的棉铃虫单粒核型多角体病毒进行了研究。
本文从形态结构，结构多肽，限制性内切酶酶切图谱等方面进行了研究。多角体直径0.7～2μm。病毒粒子大小为326nm×69nm。经SDS-PAGE分析，棉铃虫多角体蛋白分子量为28.7kD。病毒粒子结构多肽与曾报道的G4株(湖北株)和HzSNPV(美洲株)比较类似。棉铃虫核型多角体病毒朝鲜株(HaSNPV-KO)的基因组BamHI，EcoRI，HindⅢ和PstI消化后，得到的内切酶图谱表现，与已报道的几个分离株类似，分子大小均为130.18kb。
棉铃虫单粒包埋核型多角体病毒G4分离株(Gene Bank 271059)，上海株(Gene Bank 303045)和美洲株(HzSNPV)(Gene Bank，334030)的全序列已测完，而且对G4分离株和美洲株基因组结构的分析进行得比较深。G4分离株基因组大小为131403bp，上海株为130760bp，美洲株为130869bp。
G4株与美洲株基因组分析结果表明，G4株有135个ORF，而美洲株有
139个O灯。据上述结果，可见，即使属于同一种病毒，每个分离株基因组
之间具有一定的差异。
本文进行了棉铃虫核型多角体病毒朝鲜分离株基因组BamHI一I片段全序
列，BamHI一H片段全序列和B田刀Hl一G片段部分序列的测序，而且与已报道的
G4株，上海株和美洲株比较分析。
棉铃虫核型多角体病毒朝鲜分离株基因组BamHI一I片段包括p26基因、
Plo基因和p74基因的3’末端，BamHI一H片段包括gP37基因、hrZ同源序
列和br。一b基因的5’末端。B田nHI一G片段部分序列包括bro一b基因的3’
末端、hr3同源序列、he65基因、iaPZ基因和HaORF63基因的3’末端。
上述的p26基因、plo基因和p74基因的3’末端、即37基因、bro一b
基因、he65基因、iapZ基因和 HaORF63基因的3’末端与曾测完的三个棉铃
虫单粒包埋核型多角体病毒分离株(G4株、美洲株和上海株)的同源性比较
结果表明，它们之间有极高的同源性，甚至有的基因的核昔酸序列完全相同。
hrZ同源序列由1150个核昔酸组成，朝鲜株的hrZ序列比上海株多243
个bp，朝鲜株的hr2序列与病毒美洲株的hr2序列有90.5%的同源性，美洲
株的hrZ序列比朝鲜株多390个bP。
棉铃虫核型多角体朝鲜株hr3序列核普酸序列的大小共为 759bp，与G4
株一样。朝鲜株与G4株(759bP)的hr3核营酸序列完全相同，与美洲株(492bP)
有94.4%的同源性。
用nNAsls Max和scanprosite软件，进行了对p26基因、plo基因、gp37
基因、bro一b基因、he65基因和iaPZ基因编码的氨基酸蛋白的二级结构和疏
水性的分析。
Helicovera armigera nucleopolyhedrosis(HaNPV) is a member of the family Baculovirus, is designated as single (S) or multiple (M) depending on the potential number of nucleocapsid packaged in a virion.
H. armigera single nucleocapsid nucleopolyhedrosis(HaSNPV) were isolated in differente countries, they are G4, HaS-BW, HaS-ZN, SHANGHAI isolate and HaS-SA.
Due to its high virulence, HaSNPV has been adopted for mass production as a viral pesticide and has been widely used to control the insect pests in China and in other countries.
Biochemical, serological and molecular studies revealed that the various SNPVs have a high degree of similarity. But Helicovera SNPVs containg different genotypes share, show differences in virulence and rstiction enzyme profiles.
HaSNPV-KO was studied on the shape and structure, structural polypeptide, restriction pattern.
Diameter of polyhedron is 0.7-2.0μm, size of virion is 326nm.69nm. Assayed by SDS-PAGE, polyhedron of HaSNPV-KO contained a single polypeptide whose molecular is 28.7kDa, genome is 130.18kb. Genome digested by BamHI, EcoRI, HindIII and PstI, map attained is very similar to the other isolates reported.
Insight into the molecular characteristics that specify the biological properties of HaSNPV is important not only to understand the basis of host range and virulence, but also for the successful and bio-safe genetic modification of this virus. Despite the extensive usage of HaSNPV in pest control over many decades, the molecular knowledge of the HaSNPV genome is limited to viral DNA restriction
enzymes profile analysis.
The nucleotide sequence of G4 isolate(Gene Bank 271059), HzSNPV(Gene Bank 334030) and SHANGHAI isolate(Gene Bank 303045) have been determined.
The genome of G4 was assembled into a conyiguous sequence of 131403bp.
The size are very similar to the HzSNPV(130869bp) and SHANGHAI isolate(130760bp).
In order to investigate the genomic organization of the HaSNPV-KO, determind BamHI-I fragment, BamHI-H fragment and BamHI-G fragment, compared with the other isolates.
The BamHI-I fragment contained p26 gene, pl0 gene and 3'termini of p74 gene; the BamHI-H fragment contained gp37 gene,hr2 homologous region and 5'termini of bro-b gene; the 3'termini of bro-b gene, he65 gene,hr3 homopogous region, iap2 gene and 3'termini of HaORF63 gene were localized BamHI-G fragment of genome.
Nucleotide sequence aligment indicated that these gene has an extremely high degree of indentity with that of other isolate.
hr2 homologous region have 1150bp,which is equal with the G4 isolate. And hr3 homologous region have 75 9bp,which is equal with the G4 isolate.
The secondary protein structure prediction and hydrophobic amino acid of the p26 gene, pl0 gene, gp37 gene, bro-b gene, he65 gene and iap2 gene were performed with computer programs.
经过对棉铃虫核型多角体病毒的多角体生物学特性、繁殖、形态结构和基因组几种限制性内切酶酶切图谱初步分析，每个分离株(isolate)或变异株(variant)的杀虫毒力并不完全相同。
通过生物活性实验，已确定了棉铃虫单粒包埋核型多角体(HaSNPV)的杀虫毒力比棉铃虫多粒包埋型核型多角体(HaMNPV)的杀虫毒力高得多。
到目前为止，棉铃虫单粒包埋核型多角体病毒(HaSNPV)中发现一些分离株，它们包括G4分离株，上海株，博茨瓦那株(HaS-BW)，印度株(HaS-IN)和南非株(HaS-SA)。
为了充分利用棉铃虫单粒包埋核型多角体病毒资源，为了开展害虫生物防治提供依据，对首次在朝鲜分离得到的棉铃虫单粒核型多角体病毒进行了研究。
本文从形态结构，结构多肽，限制性内切酶酶切图谱等方面进行了研究。多角体直径0.7～2μm。病毒粒子大小为326nm×69nm。经SDS-PAGE分析，棉铃虫多角体蛋白分子量为28.7kD。病毒粒子结构多肽与曾报道的G4株(湖北株)和HzSNPV(美洲株)比较类似。棉铃虫核型多角体病毒朝鲜株(HaSNPV-KO)的基因组BamHI，EcoRI，HindⅢ和PstI消化后，得到的内切酶图谱表现，与已报道的几个分离株类似，分子大小均为130.18kb。
棉铃虫单粒包埋核型多角体病毒G4分离株(Gene Bank 271059)，上海株(Gene Bank 303045)和美洲株(HzSNPV)(Gene Bank，334030)的全序列已测完，而且对G4分离株和美洲株基因组结构的分析进行得比较深。G4分离株基因组大小为131403bp，上海株为130760bp，美洲株为130869bp。
G4株与美洲株基因组分析结果表明，G4株有135个ORF，而美洲株有
139个O灯。据上述结果，可见，即使属于同一种病毒，每个分离株基因组
之间具有一定的差异。
本文进行了棉铃虫核型多角体病毒朝鲜分离株基因组BamHI一I片段全序
列，BamHI一H片段全序列和B田刀Hl一G片段部分序列的测序，而且与已报道的
G4株，上海株和美洲株比较分析。
棉铃虫核型多角体病毒朝鲜分离株基因组BamHI一I片段包括p26基因、
Plo基因和p74基因的3’末端，BamHI一H片段包括gP37基因、hrZ同源序
列和br。一b基因的5’末端。B田nHI一G片段部分序列包括bro一b基因的3’
末端、hr3同源序列、he65基因、iaPZ基因和HaORF63基因的3’末端。
上述的p26基因、plo基因和p74基因的3’末端、即37基因、bro一b
基因、he65基因、iapZ基因和 HaORF63基因的3’末端与曾测完的三个棉铃
虫单粒包埋核型多角体病毒分离株(G4株、美洲株和上海株)的同源性比较
结果表明，它们之间有极高的同源性，甚至有的基因的核昔酸序列完全相同。
hrZ同源序列由1150个核昔酸组成，朝鲜株的hrZ序列比上海株多243
个bp，朝鲜株的hr2序列与病毒美洲株的hr2序列有90.5%的同源性，美洲
株的hrZ序列比朝鲜株多390个bP。
棉铃虫核型多角体朝鲜株hr3序列核普酸序列的大小共为 759bp，与G4
株一样。朝鲜株与G4株(759bP)的hr3核营酸序列完全相同，与美洲株(492bP)
有94.4%的同源性。
用nNAsls Max和scanprosite软件，进行了对p26基因、plo基因、gp37
基因、bro一b基因、he65基因和iaPZ基因编码的氨基酸蛋白的二级结构和疏
水性的分析。
Helicovera armigera nucleopolyhedrosis(HaNPV) is a member of the family Baculovirus, is designated as single (S) or multiple (M) depending on the potential number of nucleocapsid packaged in a virion.
H. armigera single nucleocapsid nucleopolyhedrosis(HaSNPV) were isolated in differente countries, they are G4, HaS-BW, HaS-ZN, SHANGHAI isolate and HaS-SA.
Due to its high virulence, HaSNPV has been adopted for mass production as a viral pesticide and has been widely used to control the insect pests in China and in other countries.
Biochemical, serological and molecular studies revealed that the various SNPVs have a high degree of similarity. But Helicovera SNPVs containg different genotypes share, show differences in virulence and rstiction enzyme profiles.
HaSNPV-KO was studied on the shape and structure, structural polypeptide, restriction pattern.
Diameter of polyhedron is 0.7-2.0μm, size of virion is 326nm.69nm. Assayed by SDS-PAGE, polyhedron of HaSNPV-KO contained a single polypeptide whose molecular is 28.7kDa, genome is 130.18kb. Genome digested by BamHI, EcoRI, HindIII and PstI, map attained is very similar to the other isolates reported.
Insight into the molecular characteristics that specify the biological properties of HaSNPV is important not only to understand the basis of host range and virulence, but also for the successful and bio-safe genetic modification of this virus. Despite the extensive usage of HaSNPV in pest control over many decades, the molecular knowledge of the HaSNPV genome is limited to viral DNA restriction
enzymes profile analysis.
The nucleotide sequence of G4 isolate(Gene Bank 271059), HzSNPV(Gene Bank 334030) and SHANGHAI isolate(Gene Bank 303045) have been determined.
The genome of G4 was assembled into a conyiguous sequence of 131403bp.
The size are very similar to the HzSNPV(130869bp) and SHANGHAI isolate(130760bp).
In order to investigate the genomic organization of the HaSNPV-KO, determind BamHI-I fragment, BamHI-H fragment and BamHI-G fragment, compared with the other isolates.
The BamHI-I fragment contained p26 gene, pl0 gene and 3'termini of p74 gene; the BamHI-H fragment contained gp37 gene,hr2 homologous region and 5'termini of bro-b gene; the 3'termini of bro-b gene, he65 gene,hr3 homopogous region, iap2 gene and 3'termini of HaORF63 gene were localized BamHI-G fragment of genome.
Nucleotide sequence aligment indicated that these gene has an extremely high degree of indentity with that of other isolate.
hr2 homologous region have 1150bp,which is equal with the G4 isolate. And hr3 homologous region have 75 9bp,which is equal with the G4 isolate.
The secondary protein structure prediction and hydrophobic amino acid of the p26 gene, pl0 gene, gp37 gene, bro-b gene, he65 gene and iap2 gene were performed with computer programs.
引文
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