phbB、phbC基因在大肠杆菌中的表达及对马铃薯植株的转化和检测

phbB,phbC GENE EXPRESSION IN E.COLI AND THEIR TRANSFORMATION AND IDENTIFICATION IN POTATO

利用从真养产碱杆菌（Ａｌｃａｌｉｇｅｎｅｓｅｕｔｒｏｐｈｕｓ）Ｈ１６染色体ＤＮＡ中克隆到的催化聚β羟基丁酸酯（ｐｏｌｙβｈｙｄｒｏｘｙｂｕｔｙｒａｔｅ，ＰＨＢ）生物合成的两个关键酶基因：依赖ＮＡＤＰＨ的乙酰乙酰ＣｏＡ还原酶基因（ｐｈｂＢ）以及ＰＨＢ合酶基因（ｐｈｂＣ），同时利用大肠杆菌高效表达载体ｐＫＫ２２３３，构建了嵌合有ｐｈｂＢ和ｐｈｂＣ基因的表达载体ｐＫＣＢ，转入大肠杆菌ＪＭ１０９，通过显微镜观察及气相色谱分析检测ＰＨＢ的合成。在确证克隆基因正确的基础上构建了马铃薯块茎特异性表达载体ｐＰＳＡＧＢ（含ｐｈｂＢ基因）、ｐＢＩＢＧＣ（含ｐｈｂＣ基因）和ｐＰＳＡＧＣＢ（含ｐｈｂＢ和ｐｈｂＣ双基因），转化５个马铃薯品种，经检测获得２０个转基因阳性株系。

Using NADPHdependent acetoacetylCoA reductase gene (phbB) and polyβhydroxybutyrate (PHB) synthase gene (phbC) cloned from Alcaligenes eutrophus H16 and expression vector pKK2233, the authors constructed an E.coli expression vector pKCB containing independent phbB and phbC operators, respectively, and transfered it into E.coli JM109. The microscopy and GC analysis indicated that E.coli JM109 (containing pKCB) induced by IPTG could synthesize polyβhydroxybutyrate (PHB). By DNA processing, three tuberspecific plant expression vectors, pPSAGB (containing phbB), pBIBGC (containing phbC) and pPSAGCB (containing both phbB and phbC), were successfully constructed. In 5 transformed potato cultivars, the authors screened 20 positive lines.