【作者】 高强；

【导师】 戴良英； 吴品珊； 朱水芳；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2004， 硕士

【摘要】 小麦矮化腥黑穗病菌(Tilletia controversa kühn,简称TCK)引起的小麦矮化腥黑穗病(Dwarf bunt of wheat)是麦类黑穗病中危害最大，防治最难的一种病害，常对小麦生产造成巨大危害，同时也是我国一类检疫性病害。但是在我国日常的口岸检疫工作中我们发现小麦网腥黑穗病菌T．caries(TCT)和小麦光腥黑穗病菌T．foetida(TFL)与小麦矮腥黑穗病菌T．controversa(TCK)的冬孢子形态非常相似，这给检疫鉴定工作带来了很大的麻烦。几十年来，国内外一些学者主要从腥黑穗病菌的形态学、血清学、生化等方面进行了大量的研究探索，并取得了巨大的成果，但从分子生物学水平上对他们进行区分国内还未见报道。 利用两种分子生物学方法结合实时荧光PCR技术手段对TCK和它的两种近似种TCT和TFL进行了鉴别研究。利用RAPD方法，在目前筛选的引物中能够找出引物OperonA01(CAGGCCCTTC)鉴别出TCK和TFL，但不能鉴别出TCK和TCT；利用核糖体基因分析法，在核糖体基因的外部转录间隔区(externally transcribed spacer，ETS)和非转录间隔区(non- transcribed spacer，NTS)分别找到了TCK不同于TCT和TFL的特异性序列。并根据TCK的ETS上的特异性序列设计了Taqman探针，利用实时荧光PCR技术通过优化实时荧光PCR反应条件实现了从TCK、TCT、TFL中检测出TCK。 利用基于Taqman探针的实时荧光PCR技术可以快速、准确的实现对TCK的检测，可避免常规检测方法造成的误检、漏检，且这种检测方法相对于常规检测方法更为灵敏，整个检测过程只要2个小时，由于采用全封闭反应管及光电传导系统，不用跑凝胶电泳，因而降低了实验室常见的污染问题。更多还原

【Abstract】 Dwarf bunt of wheat is the most serious and difficult controlled disease which often make; great damages in the production of wheat, it is also a kind of quarantined diseasem in China. But the teliospore morphology of T. caries (TCT), T. foetida (TFL) and T. controversa(TCK.) is found very similar. So it bring us great difficult on the quarantined identification. Many research have been done on the morphology, serology and biochemistry of TCK, TCT and TFL for many years and acquired great progresses. But there are no reports that have been found on the molecular biology in China.T. caries(TCT) > T. foetida(TFL)and T. controversa(TCK) have been differentiated by using two methods of molecular biology which combine Real-time Fluorescent PCR in this paper. Primer OperonA01 have been found to differentiated TCK and TFL but can ’ t to differentiated TCK and TCT by using RAPD. Special sequences have been found at the externally transcribed spacer(ETS) and at the non-transcribed spacer (NTS) of ribosome gene by analytical method of ribosome gene sequence . Taqman probe have been designed according to the special sequences of TCK ETS sequence. TCK has been detected by using Real-time Fluorescent PCR.TCK can be deteced rapidly, accurately, relatively sensitive and effectively by Real-time Fluorescent PCR which based on probe Taqman .It reduced pollution due to adopting full-closed reaction tube and photoelectricity conduct system during the experiment.更多还原