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標題: 立枯絲核菌菌株CHU341之線形質體pCHU341的定序策略
Sequencing strategies of a linear plasmid-like DNA pCHU341 in Rhizoctonia solani strain CHU341
作者: 凌玉龍
Ling, Yu-Long
關鍵字: Rhizoctonia solani
linear plasmid
出版社: 植物學系
摘要: 本研究利用真菌線狀質體 DNA鹼性溶離法,篩選立枯絲核菌質體DNA存在 :共計發現十一株帶有質體DNA存在,三株檢測不到質體DNA。所抽取得到 之質體大小均約2.6Kb,並有二倍體存在。而在抽取真菌質體 DNA之方法 比較,假若只用於限制▇圖譜建立時,可使用真菌總體DNA抽取法,以獲 得大量質體DNA供作研究;但若要作質體 DNA選殖研究,就必需採用真菌 質體 DNA鹼性溶離法獲得質體DNA,減少染色體DNA之干擾。純化之 pCHU341使用限制▇切割後,連接各質體DNA片段,次選殖於定序載體上, 並定序之。目前已將 pCHU341之BamH I片段定序完畢,共564bp。本研究 中為選殖質體DNA末端序列,嘗試利用單一 primer進行質體DNA末端序列 之PCR倍增,以嘗試提供一簡單可行之方法,選殖質體 DNA末端序列,最 後並討論其它兩種選殖質體末端序列方法之可行性與實用性。
The alkaline lysis method for linear plasmid DNA is used to isolate the plasmid DNA from Rhizoctonia solani CHU341. The plasmids in fourteen strains of R. solani were tested.It indicates that 11 strains of the fungi contain plasmid. The size of the extracted plasmid, pCHU341, is about 2.6 kb and some strains have dimer-type plasmids. DNAs of R. solani were extracted by two ways: 1. Extracting total DNA in order to get much amount of plasmid DNA for restriction enzymes mapping study. 2. Using the alkaline lysis method for DNA cloning,it reduces the contamination from chromosomal DNA. The purified DNA of pCHU341 was digested with restriction enzymes. The DNA was subcloned and sequenced with the ing vector. The BamH I fragment of pCHU341 has beenin 564bp. In order to clone the terminal fragment of pCHU341, the methods of polymerase chain reaction and single primer to amplify the DNA of terminal fragment of pCHU341 were used.The probability and practicality of these two methods to clone the terminal fragment DNA of pCHU341 were discussed.
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