藏红花病毒病原的分子鉴定  

Molecular identification of viral pathogens in Crocus sativus

作　　者：谢礼[1,2] 吕明芳 董峰丽[1] 饶君凤[3] 毛碧增[1] 洪健[1] 

机构地区：[1]浙江大学生物技术研究所,浙江杭州310058 [2]浙江省农业科学研究院病毒学与生物技术研究所,浙江杭州310021 [3]杭州职业技术学院,浙江杭州310018

出　　处：《中草药》2013年第8期1033-1036,共4页Chinese Traditional and Herbal Drugs

基　　金：浙江省杭州市农业科研攻关项目(20110232B16);浙江省中药材农业新品种选育重大科技专项(2012C12912-7)

摘　　要：目的研究栽培藏红花的病毒病原。方法综合运用病毒粒子形态和细胞病理学电镜观察、DAS-ELISA检测、RT-PCR检测及序列测定等技术进行病原鉴定。结果透射电镜负染色观察到病株汁液含有600～900 nm的线状病毒粒子;超薄切片观察到病株细胞质内有大量线状病毒粒子、II型风轮状内含体和电子致密无定型体,符合菜豆黄花叶病毒Beamyellow mosaic virus(BYMV)的病理学特征。应用BYMV抗体进行病株DAS-ELISA检测结果为阳性,应用马铃薯Y病毒属特异性引物Sprimer和M4对病株进行RT-PCR检测结果为阳性,对阳性结果进行分子克隆及序列测定发现目标序列与BYMV有99%的同源性。结论综合检测结果判明侵染浙江藏红花的病毒病原为BYMV。Objective To investigate the viral pathogens in cultivated Crocus sativus. Methods Viral pathogen identification was carded out by the observation of virus particle morphology and cytopathology as well as the detection of DAS-ELISA, RT-PCR, and sequencing. Results Linear virus particles of 600--900 nm in length were observed in C. sativus by negative staining under transmission electron microscope （TEM）. Bundles of linear virus particles, cylindrical inclusion bodies of subdivision II, and amorphous inclusion bodies were observed in the cells of C. sativus under TEM after ultrathin-section. These observations resembled the cytopathology of infectious Bean yellow mosaic virus （BYMV）. Positive result of DAS-ELISA was obtained from the leaves of C. sativus by using monoclonal antiserum against BYMV capsid protein. Positive result of RT-PCR induced by the Potyvirus specific primers （Sprimer and M4） was also obtained. Sequencing after RT-PCR revealed that the viral sequence in this diseasedC, sativus had a homology of 99% with the BYMV sequence. Conclusion The pathogenic virus of this C. sativus disease is identified as BYMV.

关 键 词：藏红花 病原鉴定 菜豆黄花叶病毒 分子鉴定 分子克隆 

分 类 号：R282.12[医药卫生—中药学]