甜菜夜蛾核多角体病毒基因组13.7～21.6m.u.区域的分析

Analysis of Spodoptera exigua Multicapsid Nucleopolyhedrovirus 13.7～21. 6 m.u. Region

甜菜夜蛾核多角体病毒(Spodopteraexiguamulticapsidnucleopolyhedrovirus,SeMNPV)基因组13 7～21 6m u 区域是SeMNPV的重组热点区。以SeMNPV美国分离株SeUS1为模板,长片段PCR对SeUS1的13 7～21 6m u 区域扩增,得到11 3kb,2 0kb和0 7kb3个片段。对照已发表的SeMNPV基因组序列,11 3kb片段是具完整SeMNPV序列基因型的产物,2 0kb和0 7kb片段表明SeUS1中还存在两种缺失突变株。对2 0kb和0 7kb片段的序列分析揭示了这两株突变株在缺失部位的DNA一级结构。对三株重组SeMNPV病毒(SeXD1、SeXD2和SeXD3)的分析发现,它们和野生型病毒SeUS1中的一个基因型一样,均缺失了SeMNPVnt18513～29105之间长10593bp的片段,暗示SeUS1中一株自发形成的缺失突变体在离体细胞中具有复制优势。将该片段基因排列与其它10种基因组序列已测定的杆状病毒比较,发现两个在GroupⅡNPVs中保守的基因簇。最大简约性法对部分基因的系统发育分析表明,缺失片段中某些基因可能存在于杆状病毒分化之前的病毒基因组中。

The PstⅠ D restriction fragment, located between 137 and 216 map units, is a hypervariable region in the genome of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) US1 isolate (SeUS1). Using SeUS1 as the template and synthetic oligonucleotide primers that can amplify the entire PstⅠ D region, three DNA products were obtained by PCR with sizes of 113 kb, 20 kb and 07 kb, respectively. These products represent three major genotypic variants. Sequence analysis and in situ comparison to the complete sequence of SeUS1 identified two genotypes with a deletion/insertion, whereas the third genotype represented intact viral DNA. Meanwhile, three constructed SeMNPV recombinants were found to lack the same SeMNPV 137～216 mu genomic segment. More interestingly, the junction site in all construction mutants is accurately identical to that of the same spontaneous mutant in parental isolate SeUS1.It seems that in vitro either the genomic segment is prone to deletion or that a natural genotype with such a deletion has some replication advantages.SeMNPV 137～216 mu (nt 18 513～29 105) segment contains some auxiliary genes, such as cathepsin(v_cath), chitinase(chiA), gp37, protein tyrosine phosphatase(ptp2),ecdysteroid UDP glucosyl transferase(egt), and other unidentified ORFs. Comparison of the gene organization in the deletion segment of SeMNPV to that of 10 other baculoviruses, which the complete genome sequences have been analyzed to date, show some conservation gene order in this segment in Nucleopolyhedrovirus Group Ⅱ taxa. Phylogenetic analysis of v_cath, chiA, gp37, egt indicates these gene sequences show a deep division between the GVs and NPVs, NPVs can be further divided into two groups. The genomic segment seems to be present in the genome of the most recent common ancestor baculoviruses even if it is hypervariable.