叶绿体转化三角褐指藻表达外源蛋白

Foreign Protein Expression in Chloroplast Transformation of Phaeodactylum tricornutum

摘要:

为建立三角褐指藻(Phaeodactylum tricornutum)叶绿体表达体系，从其叶绿体基因组中克隆了rns-trnI、trnA-rnl序列作为遗传转化同源重组序列，并以氯霉素抗性基因(CAT)表达盒作为筛选标记，以及绿色荧光蛋白基因(GFP)表达盒作为报告基因。以TA克隆载体pMD19-T为基础，将CAT表达盒以及GFP表达盒连接到rns-trnI /trnA-rnl两个同源重组片段之间，构建得到含有报告基因GFP表达盒的三角褐指藻叶绿体表达载体，利用电穿孔法将载体导入三角褐指藻叶绿体。对转化藻进行抗性筛选以及报告基因的检测，结果表明，外源基因CAT在转化藻中得到表达并提供抗性, 报告基因GFP成功地在叶绿体中表达。

Abstract:

To achieve foreign protein expression in the chloroplast of Phaeodactylum tricornutum, a chloroplast transformation vector was constructed. The sequences rns-trnI and trnA-rnl from P. tricornutum chloroplast genome were cloned and used as homologous recombination elements. Chloramphenicol acetyltransferase gene (CAT) expression cassette conferring chloramphenicol resistance was employed as selection marker, and green fluorescent protein gene (GFP) as reporter gene. Based on the TA cloning vector pMD19-T, CAT and GFP expression cassette were cloned in between the two homologous recombination elements. The resultant chloroplast transformation vector was transferred into the chloroplast of P. tricornutum by electroporation method. Transplastomic P. tricornutum cells were obtained upon chloramphenicol selection and reporter GFP could be detected in the chloroplasts, indicating that chloroplast expression system was successfully achieved.