3种大蒜中洋葱黄矮病毒的RT-PCR分子鉴定

Molecular Identification of Onion Yellow Dwarf Virus from 3 Kinds of Garlic by Using RT-PCR

洋葱黄矮病毒(Onion yellow dwarf virus,OYDV)是危害大蒜产量和品质的主要病毒之一。快速有效的病毒检测方法可为大蒜病毒病的研究和有效防御提供理论与技术资料。本研究利用反转录-聚合酶链式反应(RT-PCR)技术对甘肃"成县迟蒜"和山东"鲁蒜王2号"中OYDV外壳蛋白基因进行特异性扩增,并克隆到载体PMD18-TEasy Vector上进行核苷酸序列测定及分析。成功分离得到OYDV的两个DNA片段——GSCCS和SDLSW2,与GenBank中已报道的AB000837.1、AB000838.1和AJ409311.1等22个OYDV DNA片段的核苷酸相似性分别为81%～98%和81%～84%,氨基酸相似性分别为85%～99%和89%～94%,二者之间核苷酸相似性为84%,氨基酸相似性为89%。系统进化树显示不同DNA片段可聚为3个组群,GSCCS与中国山东金乡OYDV DNA片段同属一组,SDLSW2与其它16个OYDV DNA片段同属一组,表明OYDV在甘肃"成县迟蒜"和山东"鲁蒜王2号"中均存在,但具有一定差异。本实验确定了OYDV的基因变异程度,为进一步研究病毒进化和变异提供了有利条件。

Onion yellow dwarf virus（OYDV） is one of the main virus which is harmful to the yield and quality of garlic.The rapid and effective method of virus detection could provide theory and technology for the virus fundamental study and disease protection.The coat protein gene of OYDV was amplified from garlic named＂Gansuchengxianchisuan＂ and ＂Shandonglusuanwang No.2＂ by RT-PCR,and the purified PCR products were cloned into T-vector for sequencing.The result showed that two OYDV DNA fragments（GSCCS and SDLSW2） were obtained.Comparison with the other twenty-two DNA fragments of AB000837.1,AB000838.1 and AJ409311.1 etc reported in GenBank respectively showed that the nucleotide sequence identities were 81%～98% and 81%～84%,and the amino acids sequence identities were 85%～99% and 89%～94%.The nucleotide sequence identity between GSCCS and SDLSW2 was 84%,and the amino acids identity between the two fragments was 89%.Phylogenetic analysis showed that all the OYDV DNA fragments were classified into 3 groups.GSCCS was placed in the same clade with OYDV DNA fragment from Jinxiang of Shandong;and SDLSW2 was placed in the same clade with other 16 OYDV DNA fragments,indicated that both of the two cultivars included OYDV and there were some differences between GSCCS and SDLSW2.Our work determined the extent of OYDV genetic variation and paved the way for the further study on its evolution and mutation.