菠菜叶绿体基因组定点整合表达载体构建及原核表达

The construction and prokaryotic expression of a site-specific integration vector for chloroplast transformation of spinach(Spinacia oleracea L.)

分析菠菜叶绿体基因组全序列,选用了rbcL基因和accD基因的间隔区作为外源基因的定点整合位点,并从菠菜叶绿体基因组中克隆了rbcL基因全长和accD基因的5′端部分,长度分别为1956 bp和1320 bp。以这2个DNA片段作为同源重组片段,以烟草叶绿体基因的启动子Prrn和终止子psbA3′控制外源基因的转录,构建了包含筛选标记基因aadA基因(编码氨基糖苷-3′-腺苷酸转移酶,具有壮观霉素和链霉素抗性)和报告基因GFP(编码绿色荧光蛋白)的菠菜叶绿体基因组定点整合表达载体pRAGA。酶切结果显示构建正确。将该载体转化大肠杆菌,在激光扫描共聚焦显微镜下用488 nm蓝光激发,发现大肠杆菌发出强烈的绿色荧光,而对照菌体没有荧光,表明GFP基因在原核大肠杆菌中已经成功表达。实验结果说明构建的菠菜叶绿体定点整合表达载体pRAGA可以用于菠菜叶绿体转化。

The construction of a site-specific integration and expression vector and its potential utility was reported in the spinach （Spinacia oleracea L. ）chloroplast genetic transfonnation. According to the published chloroplast DNA sequence of spinach, the full length of rbcL gene and the 5＇ end part of accD gene, 1 956 bp and 1 320 bp respectively, were cloned through PCR technique and used as the homologous recombinant fragments in vector construction. The selectable marker aadA gene （encoding aminogly-coside Y-adenylytransferase and confening resistance to spectinomycin and streptomycin） and the reporter gene GFP （encoding green fluorescent protein） were controlled by the promoter Prrn and the terminator psbAY from tobacco, aadA expression cassette and GFP expression cassette were cloned and placed between two homologous recombination fragments to obtain the site-specific integration and expression vector pRAGA for spinach chloroplast transformation. The results of restriction enzyme analysis of obtained vector were in accordance with what was desired. After E, coli was transformed by the vector pRAGA and excited by 488 nm blue light, it was found that E. coli emitted bright green fluorescence under a con-focal laser scanning microscope, while the control E. coli showed no fluorescence. This result indicated that the chloroplast site-specific integration and expression vector pRAGA had been successfully constructed and could be used in prokaryotic chloroplast transformation of spinach.