广西家蚕核型多角体病毒gp64基因遗传多态性及进化分析

Genetic polymorphism and evolution of gp64 gene from Bombyx mori nucleopolyhedrovirus in Guangxi

【目的】了解广西家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)gp64基因的遗传多态性及进化特点,掌握BmNPV在广西蚕区的流行和传播情况,揭示BmNPV种群维持遗传多样性的模式与机制。【方法】对20株广西BmNPV毒株的gp64基因进行测序分析,根据gp64基因构建遗传进化树及绘制毒株流行分布图,并比对不同毒株的致病力。【结果】广西BmNPV毒株gp64基因开放阅读框(ORF)长度存在3种情况(1590、1593和1599 bp),分别编码含529、530和532个氨基酸残基的GP64蛋白。20株广西BmNPV毒株与标准参考T3株的gp64基因核苷酸序列同源性在97.6%~99.2%,其推导氨基酸序列同源性在96.4%~99.6%。在广西BmNPV毒株gp64基因近N端分别出现GCG缺失和GCGCCG/GTGCCG插入突变,发生核苷酸替换突变的位点数目在13~28个,但大部分为同义替换,对编码蛋白的三聚体空间构象无明显影响。广西BmNPV毒株gp64基因编码蛋白的N-糖基化位点为3~4个;除GXZS株外,所有毒株的O-糖基化位点均为2个,且预测位点一致。基于gp64基因构建的遗传进化树显示,几乎所有的广西BmNPV毒株聚类于Clade Ⅰ分群,其又被分为2个主要亚群(Sub-clade Ⅰ和Sub-clade Ⅱ);而几乎所有的国外参考毒株聚类于Clade Ⅱ分群。广西蚕区的BmNPV流行分布呈集中性与分散性并存;GXUA株对四龄和五龄起蚕的半数致死量(LD_(50))分别为3.3和3.1,而GXZZ株对四龄和五龄起蚕的LD_(50)分别为5.5和5.3,说明GP64蛋白糖基化位点较少的BmNPV毒株表现出较弱的致病力。【结论】广西BmNPV毒株gp64基因在进化过程中其信号肽区出现明显变异,发生同义突变的频率较高,形成较独立的进化分群,毒株间的致病力差异可能与GP64蛋白糖基化位点不同有关,说明广西BmNPV毒株具有不同的基因型和表型,在一定程度上维持了BmNPV野生群体的遗传多样性。

【Objective】This research was aimed to understand the genetic polymorphism and evolving characteristic of the gp64 genes derived from Bombyx mori nucleopolyhedrovirus(BmNPV)in Guangxi,study the condition of Bm-NPV epidemic and spread in Guangxi sericultural regions,andreveal the patterns and mechanisms of maintaining genetic diversity in BmNPV population.【Method】The gp64 gene of 20 Guangxi BmNPV strains were sequenced and analyzed.The phylogenetic tree was constructed according to gp64 gene and the epidemic distribution map was drawn.The pathogenicity of different strains was also compared.【Result】The open reading frame(ORF)length of gp64 genes of these Guangxi BmNPV strains showed three conditions(1590,1593 and 1599 bp),which encoded GP64 proteins of 529,530 and 532 amino acid residues,respectively.The homologies of nucleotide sequence in gp64 genes of 20 Guangxi BmNPV strains was 97.6%-99.2%and the homologies of deduced amino acid sequence was 96.4%-99.6%compared with standard reference T3 strain.The mutations of GCG deletion and GC/TGCCG insertion were found near the N terminal of gp64 gene of Guangxi BmNPV strains.The site mutation numbers of nucleotide substitution were between 13 to 28,but most of them were synonymous substitutions which had little effect on the trimeric spatial conformation of the coded protein.There were three to four N-glycosylation sites in decoded amino acids of gp64 genes of Guangxi BmNPV strains.There were two O-glycosylation sites in all strains except for GXZSand the predicted sites were all the same.Almost all the Guangxi BmNPV strains were clustered as CladeⅠwhich was further separated into two major groups(Sub-clade Ⅰ and Sub-clade Ⅱ)in the phylogenetic tree constructed based on gp64 gene,while almost all the reference strains from abroad were clustered as CladeⅡ.The BmNPV epidemic distribution showed coexistence of concentration and scatteration.The median lethal dose(LD_(50))of GXUA strains for 4^(th)and 5^(th) instar silkworm were 3.3 and 3.1,while LD_(50) of GXUA strains for 4^(th)and 5^(th) instar silkworm were 5.5 and 5.3.It indicated that BmNPV strain with less glycosylation site in GP64 protein glycosylation site showed weak pathogenicity.【Conclusion】A major mutation in signal peptide of gp64 genes of Guangxi BmNPV strains occurs,frequency of synonymous mutation is high and a relatively independent cluster is formed during the evolution.The diverse pathogenicity of BmNPV strains may be related to the differentiation of GP64 protein glycosylation sites.It shows that there are diverse genotypes and phenotypes among the Guangxi BmNPV strains.These diversities sustain the genetic diversity in BmNPV wild population to a certain extent.