烟草坏死病毒A大豆分离物的分子鉴定

Characterization of Tobacco necrosis virus A,an isolate infecting soybean(Glycine max)

对曹琦和濮祖芹早期分离到的烟草坏死病毒大豆分离物的生物学、血清学和外壳蛋白的序列进行了进一步研究。该分离物能侵染8科29种植物,除系统侵染大豆和本生烟外,其余寄主均为局部侵染。电镜下病毒粒子呈球状,直径约28nm。基因组为单组分RNA,大小约为3.7 kb,具有2条亚基因组,分别约为1.6 kb和1.3 kb。外壳蛋白亚基的分子量约为30 kDa。血清学试验表明,该分离物与TNV柳树分离物的抗血清呈特异反应,与同属坏死病毒属(Necrovirus)的烟草坏死病毒D(TNV-D)和甜菜黑色焦枯病毒(BBSV)无血清学关系。利用简并引物通过RT-PCR克隆了该分离物的外壳蛋白基因。序列分析表明,该分离物与烟草坏死病毒A(TNV-A)、TNV-D和TNV-DH的外壳蛋白分别具有88.77%、45.13%和45.49%的氨基酸序列一致性。因此,该大豆分离物属于TNV-A的一个新株系,命名为TNV-AC。

A virus isolated from soybean （Glycine max （L.） Merr. ） collected in Nanjing exhibited serious necrosis symptom. It could be mechanically transmitted to a range of 29 plant species of 8 families in greenhouse, but caused systemic infection only on soybean and Nicotiana benthamiana. The virus has an; icosahedral particle in a diameter of 28 nm and contains one RNA component of 3.7 kb capsidated by a single type coat protein of 30 kDa. Besides of the genomic RNA, two subgenomic RNA components of approximately 1.6 kb and 1.3 kb respectively, were found in infected leaves. The virions reacted specifically with antiserum against a willow isolate of Tobacco necrosis virus （TNV）, but not with Tobacco necrosis virus D （TNV-D） and Beet black scorch virus （ BBSV）, another member of genus Necrovirus. The cDNA of its coat protein （CP） gene was synthesized and sequenced using degenerate primers. The results showed that the fragment was 831 nt long, encoding a predicted protein of 276 amino acids and shared 88.77%, 45.13% and 45.49% identity to that of TNV-A, TNV-D and TNV-Dn respectively. Based on biological, morphological and serological properties, the alignment comparison of amino acid sequence of CP provided the conclusion that the soybean isolate is a new strain of Tobacco necrosis virus A （TNV-A） in genus Necrovirus of family Tombusviridae, designated as TNV-A^C.