莱茵衣藻PEPC基因转化与表达分析

发布时间：2019-04-02 06:31

【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。
[Abstract]:In order to obtain stable phenotypic engineering algae and realize the strategy of genetic engineering to accelerate the development of microalgae biofuel industry, In this study, the endogenous phosphoenolpyruvate carboxylase gene was transferred to Chlamydomonas Rhine cells by homologous recombination. Based on the pHyg3 plasmid, the recombinant vectors of ppc1 and ppc2 were constructed and transformed into Chlamydomonas rhamnoides CC400 and CC406 by glass bead method. The strain was screened with hygromycin-resistant solid medium to obtain the target fragment. The results showed that the target fragment could exist stably on the genome in transformed algal cells. When the homologous fragment length is 0.5 kb, the relatively long fragment is not conducive to integration into the genome. The transformed fragment could be amplified from the genome of the transformed algae plant, indicating that the target fragment was integrated into the genome in the form of non-homologous recombination. After the transformed algal cells were subcultured for many times, the transformed foreign fragments could still be detected. This study provides an effective method for the study of transgenic engineering and homologous recombination strategy of microalgae cells.
【作者单位】：上海海洋大学水产与生命学院;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;
【基金】：国家自然科学基金项目(31201975,31172389)资助
【分类号】：Q943.2

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