马铃薯X病毒湖南分离物的鉴定与分组研究

从湖南石门采集表现重花叶症状的马铃薯叶片中分离纯化到一株线状病毒HN021。经双链RNA(ds—RNA)抽提、寄主反应测定、病毒粒子和内含体的形状观察，初步确定该病毒为马铃薯X病毒(Potato virus X)。以ds—RNA作为模板，用相应引物对HN021分离物的ORF4-UTR-ORF5片段进行RT—PCRP得到1kb左右的双链cDNA片段。对该片段进行克隆和测序，并将测序所得的核苷酸序列与Genbank(登录的11株不同分离物的相应片段的核苷酸序列进行同源性比较和分析。结果表明，HN021与分离自南美洲的三株分离物(COAT，KPA和HB)的同源性为78．4％—79．4％，与其它8株(分离自亚洲、欧洲、大洋洲和北美洲)分离物的同源性为96．4％—97．8％。从氨基酸水平比较，HN021与COAT，KPA和HB三者CP和8kDa蛋白氨基酸序列同源性分别为86．5％—89．0％和74．3％—75．7％，相应地与其它8株分离物的同源性分别为97．1％—98．7％和97．1％—100％。序列分析的结果证实了HN021分离物为马铃薯X病毒，同时表明PVX明显存在两个组(组Ⅰ和组Ⅱ)，HN021和其它来自亚洲、欧洲、大洋洲、北美洲分离物的组Ⅱ，3个南美洲分离物属于组Ⅰ。

Identification and Grouping Study of Potato virus X Hunan Isolate

A filamentous virus HN021 was isolated and purified from mosaic leaves of Solanum tuberosum collected from Shimen, Hunan province. It was primarily identified as potato virus X by double-stranded RNA analysis, host reaction tests and morphological observation of virus particles and inclusions. A fragment of about 1kb size was obtained by RT-PCR, which was performed with ds-RNA as template of cDNA synthesis and respective primers designed for 3' end sequence of PVX. Homological comparison was carried out between the nucleotide sequences of ORF4-UTR-ORF5 regions of HN021 and those of 11 PVX isolates submitted to Genbank. Our result showed that HN021 was 78.4%-79.4% identical to three isolates from South America (COAT, KPA and HB), whereas up to 96.4%-97.8% identical to other eight isolates. From the comparison of sequences of amino acids, the similarities of CP and 8kDa protein were 86.5%-89.0% and 74.3%-75.5% respectively between HN021 and the three isolates from South America, and were 97.1%-98.7% and 97.1%-100.0% respectively between HN021 and other eight isolates. The result of sequence analysis confirmed that isolate HN021 was Potato virus X. Thus, the 12 PVX isolates were divided into two groups according to sequence comparison and analysis. HN021 was assigned to the group II including those of Asian, European, Australian, and North American origin. Whereas, the South-American isolates were classified to group I of PVX.