甘薯羽状斑驳病毒O株系和RC株系中国分离物全基因组序列分析及其遗传特征

Complete Nucleotide Sequence Analysis and Genetic Characterization of the Sweet potato feathery mottle virus O and RC Strains Isolated from China

【目的】对甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)O株系中国分离物(SPFMV-O-Ch1)和RC株系中国分离物(SPFMV-RC-Ch1)的基因组全序列进行克隆,明确SPFMV-O-Ch1和SPFMV-RC-Ch1的基因组结构特征及其遗传变异情况,为研究甘薯羽状斑驳病毒的致病机制打下基础。【方法】根据GenBank中登录的SPFMV基因组全序列设计2对简并引物和3对特异性引物,利用RT-PCR方法,从感染SPFMV的甘薯叶片中扩增SPFMV O株系和RC株系中国分离物的基因组全长序列,将目的片段分别克隆到pMD19-T载体上,经序列测定、分析和拼接,获得SPFMV-O-Ch1和SPFMV-RC-Ch1的全序列,利用DNAMAN和MEGA7对SPFMV基因组全序列及不同编码区序列进行遗传变异和系统进化树分析,利用RDP软件分析SPFMV基因组重组情况。【结果】经序列测定和拼接,结果表明SPFMV-O-Ch1和SPFMV-RC-Ch1基因组分别包含10922和10851 nt,均包含一个开放阅读框,分别由10557和10482 nt组成,编码一个多聚蛋白,分别由3518和3493个氨基酸残基组成。两个分离物均在P1蛋白内编码一个P1N-PISPO蛋白,在P3蛋白内编码一个P3N-PIPO蛋白。基因组全序列核苷酸一致性分析表明,SPFMV-O-Ch1与SPFMV-RC-Ch1的一致性为87.3%,与GenBank登录的其他分离物基因组全序列一致性为86.0%—95.8%,与Ruk73分离物的一致性最高,为95.8%,与11-1分离物的一致性最低,为86.0%。SPFMV-RC-Ch1与GenBank登录的其他分离物基因组全序列一致性为85.9%—98.7%,与IS90分离物的一致性最高,为98.7%,与Aus1-2B分离物的一致性最低,为85.9%。基于多聚蛋白基因核苷酸序列的遗传进化树分析表明,SPFMV-O-Ch1与Ordinary、10-O和17-O等O株系的分离物形成一个分支,SPFMV-RC-Ch1与S、IS90和CW137等RC株系的分离物形成一个分支。重组分析结果表明,O-Ch1分离物中发现3个重组事件,分别发生在7731—9710、135—10012和4825—6948 nt,RC-Ch1没有发现重组事件。【结论】我国的SPFMV-O-Ch1和SPFMV-RC-Ch1分离物的基因组结构与其他分离物相同,O-Ch1与O株系分离物一致性较高,RC-Ch1与RC株系分离物一致性较高,O-Ch1分离物检测到3个重组事件,RC-Ch1未发现重组事件。

【Objective】The objective of this study is to clone the complete nucleotide sequence of Chinese isolate of Sweet potato feathery mottle virus(SPFMV)O and RC strains,elucidate the genomic structural characterization and variation of SPFMV-O-Ch1 and SPFMV-RC-Ch1,and to lay a foundation for the study of pathogenic mechanism of SPFMV.【Method】According to the SPFMV genome sequences available in GenBank database,2 pairs of degenerate primers and 3 pairs of specific primers were designed,the whole genome of SPFMV O and RC strains isolated from China was amplified by RT-PCR from sweet potato leaves infection with SPFMV and subsequently cloned into vector pMD19-T and sequenced.The complete genome sequences of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates were assembled by using DNAMAN.Genetic variation analyses of complete genomic sequences,polyproteins,and individual protein sequences were performed using DNAMAN.Neighbor-joining phylogenetic tree of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates with other isolates was constructed using MEGA7.0 software.Recombination analyses were carried out using RDP software.【Result】The amplification and sequencing revealed that the complete nucleotide sequence of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates was 10992 nucleotides(nt)and 10851 nt in length,respectively.The viral genome of SPFMV-O-Ch1 isolate contained a single open reading frame(ORF)of 10557 nt encoding a polyprotein of 3518 aa.SPFMV-RC-Ch1 isolate polyprotein consisted of 10482 nt and encoded 3493 aa.Two small ORFs,P1N-PISPO and P3N-PIPO were identified in the P1 and P3 proteins of these two isolates.Pairwise comparisons of the complete genome nucleotide sequence showed that O-Ch1 had 87.3%identity with RC-Ch1 isolate and shared 86.0%-95.8%sequence identity with other SPFMV isolates.It was most closely related to the isolate Ruk73 with 95.8%nt identity and lowest nt identity with 11-1 isolate(86.0%).RC-Ch1 and other SPFMV isolates shared 85.9%-98.7%sequence identity at the complete genome nucleotide sequence level.It had the highest nt identity with IS90 isolate(98.7%),and had lowest nt identity with Aus1-2B isolate(85.9%).Phylogenetic tree analysis based on polyprotein gene indicated that SPFMV-O-Ch1 formed a branch with the isolates of O strain containing Ordinary,10-O and 17-O,and SPFMV-RC-Ch1 formed a branch with the isolates of RC strain containing S,IS90 and CW137.Recombination analysis showed that there were three potential significant recombination events occurred in 7731-9710,135-10012 and 4825-6948 nt of O-Ch1 isolate genome,respectively.No recombination event was detected in the complete genome of RC-Ch1 isolate.【Conclusion】The genomic organizations of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates were same to other isolates.O-Ch1 isolate was closely related to the isolates of O strain and RC-Ch1 isolate was closely related to those isolates of RC strain.Three recombination events were detected in O-Ch1 isolate,but no recombination event was detected in RC-Ch1 isolate.