摘要
根据GenBank已公布的种子特异性的Oleosin蛋白基因启动子序列设计合成引物,利用PCR技术从油菜总基因组DNA中扩增出Oleosin基因启动子序列(SOP),将该序列克隆到pGM-T载体中,经鉴定获得pGM-T-SOP重组载体。测序和序列分析表明,该启动子序列由899 bp核苷酸组成,其核苷酸序列与GenBank中的Oleosin基因启动子序列同源性高达95.6%。分别用限制性内切酶HindⅢ和BamH I双酶切重组质粒pGMT-SOP和双元植物表达载体pBI121,分别回收pGMT-SOP重组质粒中的SOP小片段和pBI121植物表达载体中去掉CaMV35S组成型启动子的大片段,经连接、转化和鉴定,获得由SOP驱动报告基因GUS的新型植物表达载体pBI121-SOP,为外源基因在油菜种子中的定位表达研究奠定基础。
A pair of primers was designed according to the sequence of oleosin protein promoter, which showed seeds oil-body specificity, and a fragment of oleosin gene in the promoter region was amplified by polymerase chain reaction （PCR） using the genomic DNA of Brassica napus as template. The fragment was cloned into pGM-T vector, and a new recombined vector named pGM-T-SOP was obtained after analyzed with PCR and restriction digestion. The results of sequence analysis indicated that this fragment had 95.6 % homology compared with the reported promoter in Genbank. A new plant expression vector named pBI121-SOP in which the reporter gene GUS is drived by SOP was constructed after cutting two vectors pGMT-SOP and pBI121 with two restriction enzymes Hind 111 and BamH I, subsequently recovering the small SOP fragment from pGMT-SOP recombined vector and the long fragment excised CaMV35S promoter from pBI121 plant expression vector, and then ligation, transformation and identification. And then foundation has been set up for further research work in expression and function of this promoter.
出处
《华北农学报》
CSCD
北大核心
2007年第2期6-10,共5页
Acta Agriculturae Boreali-Sinica
基金
湖北省教育厅项目(Q200513002)
国家"863"计划项目(2002AA241031)
关键词
种子特异表达启动子
克隆
油菜
Seeds oil-body-specific promoter
Cloning
Brassica napus.
