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

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。

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

The pSAD transit peptide gene, Cytochrome b5 gene and eGFP gene are cloned by PCR technique.Nuclear expression vectors were constructed based on the vectors pMD18-T,pET-28a and pDBle,cyt b5 gene and the fusion genes bG and TbG were inserted into pDBle.The recombinant plasmid pDBle-b5,pDBle-bG and pDBle-TbG were checked by restriction enzyme analysis,PCR and nucleic acid sequencing.The vectors were transferred into Chlamydomonas Reinhardtii(CW15) by glass beads and the transformants were identified with PCR.The transgenic Chlamydomonas were obtained after selecting with zeocin and they were confirmed positive by PCR amplification.PCR analysis of the genomic DNA from transgenic Chlamydomonas showed that the recombinant plasmids have integrated into Chlamydomonas Reinhardtii(CW15) genome.