Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22200
標題: 以RAPD方法選殖立枯絲核菌第四融合群(AG-4)之致病相關性基因
Cloning the pathogenic gene of Rhizoctonia solani AG-4 by RAPD
作者: 何俊成
Ho-Chun-Cheng
關鍵字: RAPD
逢機增幅多型性核酸分析
Cloning
pathogenic gene
選殖
致病相關性基因
出版社: 植物學系
摘要: 立枯絲核菌(Rhizoctonia solani)為一世界性分佈之土媒植物病原真菌,會造成寄主植物幼苗倒伏、莖腐、根腐及葉斑,因各地環境不同,其生長條件、形態、寄主植物與致病性都有明顯的差異。目前對於造成其具致病性的基因還不是很清楚,本實驗室之前的研究中,以OPA-OPF六組逢機增幅多型性核酸引子(200餘條)對CHU341及CHU344之total DNA進行逢機增幅多型性核酸分析(RAPD)測試,結果以OPC組所增殖出之條帶最能明顯區分出CHU341及CHU344二菌株之基因型。在以OPC系列12條引子對15株台灣地區之立枯絲核菌第四融合群(R. solani AG-4)菌株,及HG-1標準菌株共16株供試菌株進行RAPD實驗,增幅之產物經洋菜凝膠電泳分析後之結果,進行歸群分析可得致病性菌株14株與無致病性菌株2株在OPC-05為引子的實驗中,有9株致病性菌株CHU341、CHU352、CHU348、HG-I、CHU356、CHU357、CHU359、CHU360、CHU361、CHU362在600bp及900bp的片段上與無致病性菌株有明顯差異。 本實驗即是將強致病性菌株CHU352的600bp及900bp片段,從洋菜凝膠中回收,並以pGEM-3Zf(+)為載體,經限制切割酵素SmaI切割,與600bp及900bp片段進行平齊式連接後,送入大腸桿菌(Escherichia coli) XL1Blue-MRF!中,共獲得600bp之轉形株8株、900bp之轉形株4株;分別進行核酸定序分析,600bp獲得五種不同的核酸序列,而900bp亦獲得四種不同的核酸序列,將所得之核酸序列送入GeneBank中比對,以期找出與其致病性有相關的基因。
Rhizoctonia solani Kuhn is a world-wide soil-born pathogenic fungus which can cause damping-off, stem rot, root rot and leaf spot. It show tremendous variation in characteristics such as geographic location, morphology, host specificity and pathogenicity. The pathogenicity gene is unknown, but we are looking for the gene by random amplified polymorphic DNA methed with six primer kits, 200 primers, to analysis the total DNA of CHU341 and CHU344. The results indicate OPC primer kits could distingulish the genotype between CHU341 and CHU344. By the RAPD method ,PCR amplify with 12 primers of OPC to test the 15 isolates of R. solani of AG-4 from Taiwan and one standard strain HG-1, the PCR products were analysis by electrophoresis and cluster grouping, the results show that fourteen strains were virulent and two strains were non-virulent by the OPC-05 primer. The nine virulent strains , CHU341, CHU352, CHU348, HGI, CHU356, CHU357, CHU359, CHU360, CHU361 and CHU362 have obvious different from the non-virulent strains in fragments of 600 bp and 900 bp. We recover the PCR products of 600 bp and 900 bp in hypovirulent strain, CHU352, by electrophoresis in agarose gel. Using pGEM-3Zf(+) as vector cut with SmaⅠ, the fragments of 600 bp or 900 bp was ligated with vector and electro-transform into Escherichia coli, XL1 Blue-MRF''. We obtained eight transformation strains with 600 bp fragment and four transformation strains with 900 bp fragment. And then, analysis sequences of the 12 clones , we obtain five different sequences in the 600bp fragment and four different sequences in the 900bp fragment. Finally, all the sequences were searching in GeneBank. We hope to obtain a partial sequences related pathogenicity gene in our RAPD clones.
URI: http://hdl.handle.net/11455/22200
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