马铃薯A病毒在云南省的发生及其P3蛋白序列分析

Occurrence of Potato Virus A and Its P3 Protein Gene Sequence Analysis in Yunnan Province

【目的】了解马铃薯A病毒在云南省的发生分布情况,明确云南省分离物变异情况。【方法】用DAS-ELSIA的方法,检测云南省19个种植马铃薯县(市)的389个样品;对5个地区的阳性样品用RT-PCR扩增马铃薯A病毒P3蛋白基因,克隆测序,使用DNAMAN及DNAStar等软件分析序列,采用MEGA 6.0邻接法(Neighbor-Joining,NJ)构建系统进化树。【结果】DAS-ELISA检测389个样品,PVA的检出率为20.82%。用P3蛋白基因特异性引物从云南5个不同地区的PVA阳性样品中扩增得到1041bp的目的片段;比较分析了云南省5个地区马铃薯A病毒分离物之间,及与国内外13个分离物的P3蛋白和PIPO蛋白同源性,发现云南省5个地区PVA分离物之间P3蛋白和PIPO蛋白氨基酸序列同源性分别为98.8%~99.7%和97.6%~100.0%;与国内外13个分离物P3蛋白和PIPO蛋白的氨基酸序列同源性分别为90.8%~99.1%和90.5%~100.0%。系统进化树显示,云南省5个分离物与湖南省分离物(KF977085)亲缘关系最近;PVA马铃薯分离物和新西兰树番茄分离物之间亲缘关系较远。【结论】PVA在云南省发生普遍;云南省马铃薯A病毒存在一定程度的变异;PVA存在一定的寄主适应性。

【Objective】The present paper aimed to investigate occurrence and distribution of Potato virus A(PVA),and analyze genetic variation of PVA in Yunnan Province.【Method】Using DAS-ELISA,389 potato samples from 19 potato-growing regions of Yunnan Province were tested.Using RT-PCR,P3 protein genes of PVA from 5 positive potato samples were amplified,cloned and sequenced.P3 protein gene sequences of 5 PVA isolates from different regions of Yunnan Province and other 13 PVA isolates were analyzed by DNAMAN and DNAStar,and phylogenetic tree was constructed by Neighbor-joining(NJ)method of MEGA6.0.【Result】The positive sample detection rates of PVA were 20.82%among 389 samples.The DNA fragments of 1041 bp were amplified from 5 positive samples of PVA from different regions of Yunnan Province by RT-PCR with the specific primers for P3 protein gene.The sequence analysis results showed that there were higher amino acid sequence identities 98.8%-99.7%and 97.6%-100.0%of P3 and PIPO proteins between 5 Yunnan PVA isolates than amino acid sequence identities 90.8%-99.1%and 90.5%-100.0%of P3 and PIPO proteins between Yunnan 5 PVA isolates and other 13 PVA isolates.Phylogenetic tree results showed that genetic relationship was the most close between 5 Yunnan isolates andthe Hunan isolate of PVA(KF977085).Contrastly,genetic relationship was most far between PVAisolates from potato and those from tamarillo.【Conclusion】PVA was prevalent in Yunnan province.There was genetic variation between PVA isolates in Yunnan Province.The phylogenetic tree analysis showed that PVA isolates had host adaptability.