含铁蛋白cyb5融合基因莱茵衣藻核转化表达载体的构建及转化

Construction of Cytochrome b5 Nuclear Expression Vectors and Transformation in Chlamydomonas Reinhardtii

旨在构建并转化细胞色素cytb5基因的衣藻核转化表达载体,为其在衣藻叶绿体中对铁的利用情况研究奠定基础。将克隆的PsaD信号肽、细胞色素b5和增强型绿色荧光蛋白基因的基因融合,插入到pDBle载体中;采用玻璃珠法,将重组子导入莱茵衣藻(CW15)中;经博来霉素筛选获得转基因植株并鉴定。本项研究通过分子克隆技术获得了衣藻自身来源的PsaD信号肽、裂殖酵母来源的细胞色素b5和常用增强型绿色荧光蛋白基因片段;重组质粒pDBle-b5、pDBle-bG和pDBle-TbG测序完全正确;经博来霉素抗性筛选,获得转化衣藻单克隆;通过PCR检测转化衣藻基因组DNA,扩增片段与预期相符。实验结果表明,成功构建了pDBle-b5、pDBle-bG和pDBle-TbG衣藻核转化表达载体,重组质粒已整合到莱茵衣藻基因组中。

Nuclear expression vectors containing cytochrome gene, cytb5, were constructed and transformed into Ch]amydomonas Reinhardtii CW15, in order to establish a solid basis for the study on utilizing iron in the chloroplast of Cblamydomonas Reinbardtii. The PsaD transit peptide （TP）, cytb5 （bS）, and enhanced green fluorescent protein gene （egfp） were fused together, being inserted into the pDBle vector. By utilizing glass beads, the recombinant plasmids were transformed into ChIamydomonas Reinhardtii CW15. Subsequently, transgenic ChIamydomonas were validated upon the Zeocin selection and following PCR. In this study, using molecular cloning technology, PsaD transit peptide gene was obtained from Chlamydomonas, cytb5 fiom Schizosaccharomyces pombe, and egfp which was commonly employed. Recombinant plasmids pDBle-b5, pDBle-bG and pDBle-TbG were all completely checked by sequencing. Upon the Zeocin screen, single clones were obtained. After PCR validation of exogenous genes on Cblamydomonas genome, amplified fragments were detected in accordance with expected lengths. All results suggest expression vectors, pDBle-b5, pDBle-bG and pDBle-TbG, for the nuclear transformation in Cblamydomonas Reinhardtii, were constructed successfully, and they were already integrated into Chlamydomonas Reinhardtii CW15 genome.