二氢黄酮还原酶基因的克隆与转基因烟草的获得

以蒺藜苜蓿(Medicagotrunctulacv.5160)幼果总RNA为模板,采用RT-PCR技术克隆到二氢黄酮还原酶(DFR)基因的cDNA序列,所获得的cDNA序列全长1018 bp,具有完整的ORF,编码337个氨基酸。Blast分析表明,该片段与GenBank中注册的DFR基因同源性为99.80%。以植物表达载体pBI121为基础,构建了Ca MV35S启动子驱动的DFR基因植物表达载体pBIDFR;采用直接转化法将pBIDFR导入根癌农杆菌EHA105,用该菌株对普通烟草进行遗传转化获得6株转基因植株。

Cloning and Construction of Plant Expression Vector of Dihydroflavonol Reductase Gene (DFR) and Transgenic Nicotiana tabacum

The dihydroflavonol reductase (DFR) gene was cloned from young fruit of Medicago truncatula cv.5160 by RT-PCR and the coding sequences of this gene were analyzed.The result indicated that the full-length cDNA is 1 018 bp and contain open reading frame (ORF) encoding 337 amino-acids.The sequences were analyzed with software Blast and exhibited homologous 99.80% with DFR from GenBank.Then the promoter CaMV35S driven,plant expression vector pBIDFR with DFR was constructed based on the vector pBI121.By direct DNA transfer,pBIDFR was transferred into Agrobacterium tumefacien EHA105.Then transfer the new engineering bacterium to Nicotiana tabacum and six transgenic tobacco plants were obtained.