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安徽省水稻黑条矮缩病毒和南方水稻黑条矮缩病毒的 分子检测和序列分析
李祥宇,闫德龙,郑兆阳,陈莉,刘家成,丁克坚,江彤
0
(安徽农业大学植物保护学院,合肥 230036;安徽省植保总站,合肥 230001)
摘要:
近年来,水稻黑条矮缩病毒(RBSDV)和南方水稻黑条矮缩病毒(SRBSDV)在安徽各稻区危害日益严重,生产上仅凭症状难以区分2种病毒。本研究建立的RT-PCR方法可以实现一次性检测和鉴定RBSDV和SRBSDV 2种病毒,根据RBSDV和SRBSDV S9保守序列设计3个引物,不仅可以从单独感染RBSDV或SRBSDV的水稻样本中分别扩增出1条大小不同的特异性条带,而且还可以从感染RBSDV和SRBSDV的混合水稻样本中一次性扩增出2条大小不同的特异性条带。从安徽省庐江县、郎溪县、怀宁县和宣州市4个地区水稻田采集表现明显矮缩病症状的水稻样本,RT-PCR检测结果表明,庐江和郎溪水稻样本受到RBSDV侵染,怀宁和宣州水稻样本受到SRBSDV侵染。选取庐江县、郎溪县、怀宁县和宣州市各1个水稻样本,利用RT-PCR扩增出特异性条带,再分别克隆和测序。序列分析表明,庐江、郎溪2个样本的核苷酸序列与RBSDV-Shandong和RBSDV-Zhjr S9部分片段序列相似性高达99.3%~99.8%,且与RBSDV的亲缘关系最近,说明庐江和郎溪样本感染的病毒是RBSDV的2个分离物;怀宁、宣州2个样本的核苷酸序列与SRBSDV-Shangdong和RBSDV-2 S9部分片段序列相似性高达99.5%,且与SRBSDV亲缘关系最近,说明怀宁和宣州样本感染的病毒是SRBSDV的2个分离物。
关键词:  水稻黑条矮缩病毒  南方水稻黑条矮缩病毒  RT-PCR检测  克隆  序列分析
DOI:
基金项目:安徽省教育厅自然科学基金重点项目(KJ2012A113, KJ2011A120), 国家科技支撑项目(2012BAD04B09, 2012BAD07B02)和安徽省自然科学基金(11040606M68)共同资助。
Molecular detection and sequence analysis of Rice Black-Streaked Dwarf Virus and Southern Rice Black-Streaked Dwarf Virus in Anhui Province
LI Xiang-yu,YAN De-long,ZHENG Zhao-yang,CHEN Li,LIU Jia-cheng,DING Ke-jian,JIANG Tong
(School of Plant Protection, Anhui Agricultural University, Hefei 230036;Anhui General Station of Plant Protection, Hefei 230001)
Abstract:
In recent years, Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) increasingly caused serious damage in the rice areas of Anhui Province. However, it was very difficult to distinguish the two viruses in the field samples only by the symptoms. The RT-PCR method established by our research can realize one-time detection and identification of two virus species of RBSDV and SRBSDV. Three primers were designed based on the conserved sequence of S9 segment of RBSDV and SRBSDV. Not only one different size specific band can be amplified respectively from the rice samples infected with single RBSDV or SRBSDV, but also two different size specific bands can be amplified from the mixed rice samples infected with RBSDV and SRBSDV. The rice samples showed obvious dwarf symptoms were collected from the paddy fields of 4 areas, Lujiang, Langxi, Huaining and Xuanzhou of Anhui province. The detection results of RT-PCR showed that the rice samples from Lujiang and Langxi were infected with RBSDV, and the rice samples from Huaining and Xuanzhou were infected with SRBSDV. Each one rice sample from Lujiang, Langxi, Huaining and Xuanzhou were selected. Specific bands were amplified by RT-PCR, and then each one was cloned and sequenced respectively. Sequence analysis indicated that the nucleotide sequences of two samples from Lujiang and Langxi shared the highest sequence similarity (99.3%- 99.8%) with S9 partial segment of RBSDV-Shangdong and RBSDV-Zhijr, and the two nucleotide sequences were most closely related to RBSDV. It illustrated that viruses infected samples from Lujaing and Langxi were two isolates of RBSDV. The nucleotide sequence of two samples from Huaining and Xuanzhou shared the highest sequence similarity (99.5%) with S9 partial segment of SRBSDV-Shangdong and RBSDV-2, and the two nucleotide sequences were most closely related to SRBSDV. It illustrated that viruses infected samples from Huaining and Xuanzhou were two isolates of SRBSDV.
Key words:  Rice black-streaked dwarf virus  Southern rice black-streaked dwarf virus  RT-PCR detection  clone  sequence analysis

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