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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (25): 219-223.

• 林学 园艺 园林 • 上一篇    下一篇

复合侵染甘蔗的病原病毒RT-PCR检测

秦碧霞 蔡健和 魏源文 莫磊兴 陆秀红 刘志明   

  • 收稿日期:2011-03-25 修回日期:2011-05-23 出版日期:2011-10-05 发布日期:2011-10-05
  • 基金资助:

    广西自然科学基金项目;国家科技支撑计划项目;广西科学研究与技术开发计划项目

Detection of Viruses Co-infected Sugarcane by RT-PCR

  • Received:2011-03-25 Revised:2011-05-23 Online:2011-10-05 Published:2011-10-05

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摘要：

根据甘蔗花叶病毒（sugarcane mosaic virus，SCMV）、高粱花叶病毒（sorghum mosaic virus，SrMV）和甘蔗黄叶病毒（sugarcane yellow leaf virus，SCYLV）外壳蛋白（CP）基因序列分别设计合成3对特异引物SCMV–F/R、SrMV-F/R和SCYLV-F/R，以表现花叶和黄叶复合症状的甘蔗叶片总RNA为模板，分别进行3种病毒单一RT-PCR检测，在SrMV和SCYLV特异引物RT-PCR反应体系中分别扩增到约850 bp和630 bp特异性片段。序列测定及同源性比对结果显示，850 bp片段测序所得序列与浙江象山、临平和余杭SrMV分离物（GenBank登录号分别为AJ310194、AJ310195和AJ310198）对应序列同源性95%，630 bp片段测序所得序列与广东、广西、福建SCYLV分离物（GenBank登录号分别为GU190165、GU190162、GU190159）对应序列同源性99%，表明该样品同时感染SrMV和SCYLV。在此基础上建立同时检测SrMV和SCYLV的一步双重RT-PCR体系，可检测10-6 g病叶组织中的病毒，田间样品检测效果良好。

关键词: 多胺, 多胺, D-精氨酸, 渗透胁迫, 抗旱性, 水稻

Abstract:

Three sets of specific primers (SCMV-F/R, SrMV-F/R, SCYLV-F/R) for each virus based on the CP gene sequence of sugarcane mosaic virus (SCMV), sorghum mosaic virus (SrMV) and sugarcane yellow leaf virus (SCYLV). Total RNA was extracted from sugarcane leaf with mosaic and yellow leaf symptom. The expected 850 bp and 630 bp fragment was amplified by RT-PCR with SrMV-F/R and SCYLV-F/R, respectively. Sequence analysis indicated 850 bp segment shared identities of 95% homologus with SrMV isolates from Xiangshan, Linping and Yuhang in Zhejiang Province (GenBank accession numbers AJ310194, AJ310195 and AJ310198). 630 bp segment shared identities of 99% homologus with SCYLV isolates from Guangdong, Guangxi and Fujian (GenBank accession numbers GU190165, GU190162, GU190159). Sequence analysis confirmed the sugarcane was co-infected by SrMV and SCYLV. A double RT-PCR was applied to detecting SrMV and SCYLV base on single RT-PCR. Sensibility test showed it could simultaneously detect out SrMV and SCYLV from 10-6 g sugarcane leafs infected by virus. It was proved simple, rapid and sensitive for detection of SrMV and SCYLV.