稻米根中磷烯醇丙酮酸□化酶cDNA的分離及與其它的物種DNA的比較

• Main Author: 陳至達
• Other Authors: 張可中
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/77956987500472780989

碩士 === 東吳大學 === 化學學系 === 88 === In this study, cDNA was successfully synthesized by reverse transcription from mRNA which was isolated from rice roots. Thereafter, a cDNA library with2×106 pfu/ml from rice roots was constructed. A non-degenerate primer pair was used to amplify partial Phosphoenolpyruvate carboxylase(PEFC, EC 4.1.1.31) cDNA fragment from constructed cDNA library by PCR and resulted a 1.1 kb DNA fragment detected in 2% agarose gel. Identified cDNA sequences showed 77%, 84%, and 79% similarities in maize(C3), sorghum(C4), and ice plant(CAM), respectively. A radioactive cDNA probe was made by nick translation of the 1.1 kb cDNA fragment so that full length of PEPC cDNA sequences could be cloned with this radioactive probe. Meanwhile, 3''-RACE was used to identify 3''-end cDNA sequences. The resulting sequences contained the stop codon and 84 bp non-coding region. Likewise, 5''-RACE was applied to examine 5''-end cDNA sequences of PEPC. Update, total 2.5 kb of cDNA sequences including 3''-end nonconding region was determined. This 2.5 kb sequences showed 75%, 74%, and 76% similarities in maize(C3), sorghum (C4), and ice plant(CAM), respectively. Thses results indicated that this root form PEPC might be a house keeping enzyme.