【作者】 欧师琪；

【导师】 刘学敏；

【作者基本信息】 吉林农业大学 ， 植物病理学， 2005， 硕士

【副题名】大豆胞囊线虫和马铃薯金线虫的RAPD和SCAR标记及禾谷胞囊线虫的RFLP分析

【摘要】 本研究用RAPD技术为大豆胞囊线虫作分子标记并将不稳定的RAPD标记转化为稳定的SCAR标记。用可扩增出大豆胞囊线虫多态性片断的12个随机引物OPA02、OPA03、OPA06、OPA09、OPA13、OPA18、OPB15、OPC06、OPD13、OPG06、OPG08、OPK16寻找大豆胞囊线虫与几种胞囊形线虫间存在的差异,将大豆胞囊线虫的特异性片断进行回收、克隆、测序,在原来随机引物OPA06的10个碱基基础上,增加14个碱基,设计了一对24个碱基的大豆胞囊线虫的SCAR引物SCNFI和SCNRI,同时还设计了马铃薯金线虫的一对特异性引物GrFI和GrRI,通过覆盖全国大豆主产区的38个大豆胞囊线虫群体以及10个禾谷胞囊线虫群体、2个马铃薯金线虫和2个马铃薯白线虫群体对所设计的SCAR引物进行检测,检测率达100%。这说明这个SCAR标记在不同胞囊线虫材料中稳定性高、特异性好,克服了通常的RAPD标记不稳定、不易重复等缺点。用单个胞囊或单头二龄幼虫的DNA作为模板,同样很好的扩增出SCAR标记的特异性片断,为了提高该试验的灵敏性,将上述模板稀释10倍,扩增结果也非常好。试验结果说明,此标记稳定、可靠、操作简单且灵敏度高。可对大豆胞囊线虫单个胞囊、单头二龄幼虫进行鉴定,在利用现有的大豆胞囊线虫特异性引物的基础上,建立快速准确检测和早期诊断大豆胞囊线虫的分子生物学方法和技术规程。 用RFLP-PCR技术研究了中国河南省4个地区9个禾谷胞囊线虫群体核糖体DNA(rDNA)内转录间隔区(ITS)的遗传变异。用PCR技术扩增禾谷胞囊线虫群体的ITS长度为1060bp。用8种限制性内切酶(RE)酶切禾谷胞囊线虫ITS扩增产物,共产生18个酶切片断。ITS-RFLP的研究能直观地观测到禾谷胞囊线虫种内ITS分子多态性,彭德良等对中国和摩洛哥禾谷胞囊线虫的rDNA-ITS进行扩增,得到1060bp的片断,同时使用了HinfⅠ、AluⅠ、AvaⅠ、HindⅢ、CofⅠ、RsaⅠ、HaeⅢ等几种限制性内切酶,酶切结果与本研究得到的结果一致。AvaⅠ和HindⅢ 2种酶不能酶切禾谷胞囊线虫ITS产物。此结果说明与先前研究的中国禾谷胞囊线虫群体不存在差异。更多还原

【Abstract】 The study describes the development of species-specific pairs of PCR primers for the cyst forming nematodes Heterodera glycines and Globodera rostochiensis that amplify species-specific RAPD fragments. Random primers OPA02、 OPA03、 OPA06、 OPA09、 OPA13、 OPA18、 OPB15、 OPC06、 OPD13、 OPG06、 OPG08、 OPK16, acquiring the species-specific fragment that OPA06 amplified. After sequencing the fragments, longer primers were designed to complement the terminal sequences of the polymorphic DNA fragments. The resulting pairs of primers were used to generate the sequence-charaterized amplified regions (SCARs). Using the developed pairs of SCAR primers. SCAR fragments of H.glycines and G. rostochiensis were easily amplified from DNA extractes from single cyst or juveniles of the particular nematode species investigated. The SCAR-PCR-based assays desribed have potential to be optimized for routine practical diagnostic tests. The usefulness of converting RAPD markers into SCAR markers is discussed.The amplification of the rDNA-ITS region of each population of H. avenae (CCN)from Zhengzhou, Henan province,China respectively, yielded one fragment of approximately 1060bp. No PCR products were obtained in the control lacking the DNA template. A total 18 scored fragments were obtained with 8 enzymes used for analysis . AluI did digest CCN ITS products of the nine Henan ,China populations and yieled two fragments, Rsal digested the PCR products of all populations and yielded two fragments. CofI\ MvaI、 HaeIII、 HinfI、 AluI and RsaI produced restriction profiles identical for the all CCN populations. HindIII and AvaI were the two enzymes that did not restrict any the ITS products of cereal cyst nematode.更多还原