莱茵衣藻PEPC基因转化与表达分析  

Analysis of Transformation and Expression of PEPC Gene in Chlamydomonas reinhardtii

作　　者：王秀媛[1] 于水燕[2] 周志刚[1] 

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]上海辰山植物园,中国科学院上海辰山植物科学研究中心,上海市资源植物功能基因组学重点实验室,上海201602

出　　处：《基因组学与应用生物学》2017年第3期911-920,共10页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31201975,31172389)资助

摘　　要：为了获得表型稳定的工程藻株,实现基因工程改造策略加快微藻生物燃料产业发展的步伐,本研究应用同源重组的方法将内源性的磷酸烯醇式丙酮酸羧化酶的编码基因转到莱茵衣藻细胞中,期望获得表型稳定的工程藻株。基于pHyg3质粒我们分别构建了ppc1和ppc2的重组载体,采用玻璃珠法转化莱茵衣藻CC400和CC406藻株,应用潮霉素抗性的固体培养基筛选得到转入目的片段的藻株。研究表明,转化的藻细胞中,目的片段可在基因组上稳定存在。同源片段长度在0.5~1 kb时,相对较长的片段不利于整合到基因组中。从转化藻株的基因组中能够扩增得到转入的片段,说明转入的目的片段以非同源重组的形式整合到基因组上。转化的藻细胞多次传代培养后,仍能检测到转入的外源片段。本研究进一步为微藻细胞的转基因工程和同源重组策略的研究提供了行之有效的方法。In order to obtain engineering algal strains with stable phenotype and implement the strategy of genetic engineering to accelerate the pace of microalgae biofuels industry, this research used homologous recombination method to transform the encoding genes of endogenous phosphoenolpyruvate carboxylase into Chlamydomonas reinhardtii cells, hoping to obtain engineering algal strains with stable phenotype. The recombinant plasmids of ppcl and ppc2 were constructed based on the pHyg3 plasmid, CC400 and CC406 strains in C. reinhardtii were transformed by glass bead method, and the strains transferred to the target fragment were screened out by solid medium with hygromycin resistance. The results showed that in transformed algal cells, the target fragment was stably present on the genome. When the length of the homologous fragment was between 0.5 kb and 1 kb, the rela- tively long fragment was not conducive to be integrated into the genome. The genome of the transformed algal strains could be amplified to obtain transferred target fragments, which suggested that the exogenous fragments in- tegrated into the mutant genome in the form of non-homologous recombination. The exogenous fragments still could be detected in the transformed algae cells after several passages. This research further provided an effective method for the transgenic engineering and homologous recombination strategy ofmicroalgae cells.

关 键 词：微藻 基因转化 磷酸烯醇式丙酮酸羧化酶 

分 类 号：Q943.2[生物学—植物学]