一步法构建莱茵衣藻叶绿体表达载体

One-step Construction of Chloroplast Expression Vectorinchloroplast of Chlamydomonas Reinhardtii

为简化莱茵衣藻叶绿体表达载体构建步骤,将莱茵衣藻叶绿体中表达所需的DNA元件组合,构建入可一步TA克隆插入靶基因的载体pTBNK,可直接用于在莱茵衣藻叶绿体表达靶基因。将EGFP克隆至表达载体pTBNK,经蓝白班筛选获得阳性转化子pTBNK-EGFP,通过基因枪转化衣藻叶绿体并获得阳性克隆。通过PCR鉴定,EGFP成功整合至衣藻叶绿体基因组中。

To simplify procedures of constructing expression vector in chloroplast of Chlamydomonas reinhardtii,DNA ele⁃ments for gene clone and gene expression were integrated into the plasmid pTBNK,which can be used for expressing heterolo⁃gous genes in chloroplast of Chlamydomonas reinhardtii after one step TA clone.Green fluoresce protein encoding gene was in⁃serted into pTBNK.After blue-white selection,recombinant plasmid pTBNK-EGFP was obtained.pTBNK-EGFP was success⁃fully introduced into chloroplast of Chlamydomonas reinhardtii by gene gun and PCR analysis confirmed egfp was integrated in⁃to chloroplast of Chlamydomonas reinhardtii.