小菜粉蝶颗粒体病毒lef-3基因的克隆与分析

 【目的】研究杆状病毒lef-3基因的起源与进化,从分子水平明确病毒之间的亲缘关系。【方法】通过常规PCR方法获得小菜粉蝶颗粒体病毒晚期基因表达调控因子lef-3的基因片段,克隆后测序,然后利用软件对lef-3及编码序列进行生物信息学分析。【结果】克隆得到的PiraGV lef-3基因ORF序列中存在4个突变位点,但氨基酸性质未发生改变,推导PiraGV LEF-3蛋白含399个氨基酸残基,分子量为3.99 kD;通过高级结构预测及其编码序列与其它杆状病毒的LEF-3同源性比对表明,该基因可能编码单链DNA结合蛋白;BLAST比对发现lef-3基因只存在于鳞翅目昆虫为宿主的杆状病毒基因组;进化分析表明LEF-3可分为3组,其中GV属的LEF-3聚类为1个组,NPV属的LEF-3聚类为2个组;GV的lef-3基因编码区的选择压力分析表明,大多数lef-3基因间相比较表现为负向选择,但不同lef-3基因间的Ka/Ks不同,也有正向选择;起源分析表明,杆状病毒lef-3基因可能来源于细菌。【结论】获得了小菜粉蝶颗粒体病毒（PiraGV）lef-3基因,通过生物信息学分析推测出了lef-3基因的起源进化规律。

Cloning and Sequence Analysis of lef-3 Gene from Pieris rapae Granulovirus

[Objective] To better understand the origination and evolution of lef-3 gene from bacnlovirus genome and determine the relationships between various viruses at molecular level.[Method] Late expression factor 3 gene (lef-3) fragments of Pieris rapae granulovirus was obtained through conventional PCR method,sequenced after being cloned into T-vector.Then,bioinformatics analysis on lef-3 gene and its encoding sequences were conducted by using bio-softs.[Result] The DNA fragment was cloned and 4 mutations were appeared ,which did not alter the sequences of amino acids.It was inferred that PiraGV LEF-3 protein contained 399 amino acids ,the total molecular of which was 3.99 kD.Prediction of the lef-3 advanced structure and homology comparison between its encoding sequences and other LEF-3 genes from various baculoviruses showed that the lef-3 gene might encode the single strand DNA-binding protein .The result of blast revealed that the lef-3 gene only existed in Lepidoptera host for the baculovirus genome,and the evolution analysis illustrated that lef-3 gene could be divided into three groups including one GV genus and two NPV genus.The selection pressure analysis of GV's lef-3 gene coding region showed that the majority of lef-3 genes performed negative selection,while the Ka/Ks differed from different lef-3 genes,to some extent ,which also performed positive selection.The origination analysis revealed that lef-3 gene of baculovirus might derive from bacteria.[Conclusion] The lef-3 gene of PiraGV was cloned successfully and speculations upon the possible pattern of origination and evolution were made through bioinformatics analysis.