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Mapping and sequencing of linear plasmid DNA in Rhizoctonia solani
CHU352為材料, 對其線形、分子量約 2.6 kb 質體 pCHU352進行限制圖
PvuⅡ, BclⅠ, KpnⅠ, EcoRⅠ, SmaⅠ, BglⅡ, BamHⅠ )切割後, 此
2.6 Kb 大小的質體被略分為三種型式。此三種型式的質體DNA除了EcoRⅠ
切位外, 並不具有其他共同切位。 pCHU352在 proteinase K 處理後,
對 exonucleaseⅢ有不敏感性(insensitive), 顯示其端點並非結合蛋
白( binding protein); 經變性處理後分析其pattern大小及位置並無變
化,且質體對S1 nuclease不敏感, 間接證明了兩端的髮夾形結構。
pCHU352經AvaⅠ, HindⅢ, SacⅠ, XhoⅠ切割後分別被選殖在 E.coli
的 pBluescript KS(+) 或 pGEM-3zf(+),其中 1.5Kb大小的 HindⅢ片段
Plasmid pCHU352 is a linear DNA element of Rhizoctonia solani AG4, a strong pathogenic strain, CHU352. The molecular weight of pCHU352 is 2.6 Kb. This study was tried to analyze the restriction maps of plasmid pCHU352 and clone the restriction fragments from pCHU352 for DNA sequencing. Restriction map of the plasmid was constructed by digested with different enzymes ( PvuⅡ, BclⅠ, BamHⅠ, KpnⅠ, EcoRⅠ, BglⅡ, SmaⅠ), and the result suggested that there exist three types of plasmids. Distribution of the restriction sites were different among the three plasmid DNAs except the EcoRⅠ cutting site. The plasmid DNAs were resisted to exonuclease Ⅲ after treated with proteinase K, in contrast, treated with S nuclease will make the plasmid DNAs be sensitive to exonuclease Ⅲ. This result indicated that the linear plasmid has hairpin loops, not terminial binding protein at the both termini. A 1.5 kb HindⅢ fragment of pCHU352 was cloned to Escherichia coli vector and it could be subcloned by exonucleaseⅢ deletion or restriction enzyme digestion. The sequences of this fragment were compared with the plasmid isolated from R. solani pathogenic strain CHU341.
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