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標題: 立枯絲核菌第四菌絲融合群(Rhizoctonia solani AG4)致病株間cbh1基因片段選殖及研究
Cloning and Characterization on the Fragment of the Exo-cellulase Gene cbh1 in Diffefent Pathogenicity Isolates of Rhizoctonia solani AG-4
作者: 董世瑋
Tung, Shih-Wei
關鍵字: Rhizoctonia solani;立枯絲核菌;cbh1;纖維素外切酵素
出版社: 植物學系
立枯絲核菌(Rhizoctonia solani Kühn)為世界性分布之土媒植物病原真菌,會造成寄主植物幼苗倒伏、莖腐及葉斑。由於各地環境不同,其生長條件、形態、寄主植物與致病性均有明顯差異。目前對於其致病性基因與影響其致病性強弱的機制仍不清楚。
在目前對於影響立枯絲核菌,強弱致病株的研究,除了以RAPD方式選殖致病性基因、探討強弱致病株內ITS的不同、菌體內線型質體對致病性強弱的影響……等,為了從另一方向探討此菌致病性差異。本實驗選取9株台灣地區之立枯絲核菌第四融合群(AG4)菌株與1株HG1標準菌株共十株進行纖維素分解酵素活性測試後,利用設計自 Phanerochaete chrysosporium cbh-1 , Neurospora crassa cbh-1, Pencilium janthinellum cbh-1基因之一對引子,先對立枯絲核菌cbh-1基因利用PCR方式進行增幅反應,將產物選殖入pGEM-T Easy載體定序後,根據定序結果再設計一對專一性較高引子增幅其他無法以第一組引子作用菌株cbh1基因,並對結果進行定序。比較強弱致病株間cbh-1基因序列序列差異,了解強弱致病株在cbh-1基因上是否具有明顯差異。

Rhizoctonia solani Kühn 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. Until now, the pathogenicity gene is unknown.
At present, lots methods are used to research about Rhizoctonia solani pathogenicity gene(s), like random amplified polymorphic DNA (RAPD), ITS sequences, linear form plasmid….etc. In this study, we have chosen ten local, different virulent isolates belonging to anastomosis group 4 (AG4) and a standard isolates from Japan belonging to AG4 and tested its cellulase activity. Then by the pair of primers designing from Phanerochaete chrysosporium cbh-1 , Neurospora crassa cbh-1, Pencilium janthinellum cbh-1 and polymerase chain reaction, we cloned the exo-cellulase gene of Rhizoctonia solani into plasmid pGEM®-T Easy Vector and sequence the gene. According to the sequencing results, re-design a more specific to R. solani cbh1 primer to amplify the isolates with the cbh1 can not be amplified by the first pair primer. Then sequence the result again. Bottom on the data we try to find the relationship between virulent, pathogenicity and variation of exo-cellulase gene.
Appears in Collections:生命科學系所

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