“细菌型”pepc2基因反向表达载体构建及在莱茵衣藻中的表达  

The expression of recombinant vector of reverse “bacterial-type”pepc2 gene in Chlamydomonas reinhardtii

作　　者：田琪琳[1] 施定基[1,2] 贾晓会[1] 米华玲[3] 黄希文[1] 何培民[1] 

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]中国科学院植物研究所,北京100093 [3]中国科学院上海生命科学研究院植物生理生态研究所,上海200032

出　　处：《上海海洋大学学报》2013年第5期665-671,共7页Journal of Shanghai Ocean University

基　　金：国家高技术研究发展计划(2009AA064401);上海市科学技术委员会项目(12391901700)

摘　　要：磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase,PEPC,EC 4.1.1.31)在植物碳代谢中处于代谢纽的关键位置,是调节细胞蛋白质和脂肪酸含量的关键酶,通过抑制pepc基因的表达,可以提高细胞的含油率。通过克隆莱茵衣藻"细菌型"pepc2基因的部分序列(简称Crpepc2)和莱茵衣藻融合启动子Hsp70A-RBCS2(简称HR),将HR和Crpepc2片段插入pSP124s中,获得Crpepc2反向表达的莱茵衣藻高效表达载体pSP124s-HR-reve-Crpepc2。利用基因枪将pSP24s和pSP124s-HR-reve-Crpepc2分别转入莱茵衣藻cc-503藻株,得到空质粒型和反向型突变藻株。利用qPCR检测莱茵衣藻野生型、空质粒型和反向型藻株"细菌型"pepc2基因的相对表达量,结果表明空质粒的导入对莱茵衣藻pepc2基因的相对表达量影响很小,为野生型的92.95%;而反向型pepc2基因的导入明显降低了莱茵衣藻pepc2基因的相对表达量,仅为野生型的2.94%。该结果一方面说明我们建立了利用qPCR快速检测莱茵衣藻pepc2基因相对表达量的方法,另一方面也证明利用Crpepc2反向表达的方法(即"反向载体技术")可以有效的抑制莱茵衣藻pepc2基因的表达,为进一步筛选稳定的含油量高的藻株奠定了良好的基础。Phosphoenolpyruvate carboxylase （ PEPC, EC 4.1.1.31 ） is located at the key site of plant carbon metabolism pathways which can regulate the protein content and the lipid content in cell, and the down- regulation of PEPC expression caused increased lipid accumulation. In this study, we cloned the fragment of the ＂bacterial-type＂ pepc2 gene from Chlamydoraonas reinhardtii （named Crpepc2 ）and the Hsp70A-RBCS2 promoter （named HR）, inserted the HR and Crpepc2 into pSP124s vector, then the reverse recombinant vector pSP124s-HR-reve-Crpepc2 was obtained. The pSP124s and pSP124s-HR-reve-Crpepc2 vector were transformed into C. reinhardtii cc-503 strain through biolistic, respectively, the blank strain and reverse strain were obtained. The relative expression of pepc2 gene was measured in wild strain, blank strain and reverse strain by using qPCR. The date showed that the pSP24s in the blank strain didn＇ t obviously influence the relative expression of pepc2 gene, which was 92.95% of that of the wild strain, and the reve-Crpepc2 in the reverse strain significantly inhibited the relative expression of pepc2 gene, which was only 2.94% of that of the wild strain. This result indicated that we have established the method to detect the relative expression of pepc2 gene in C. reinhardtii by using qPCR, and it also proved that ＂reverse vector method （RVM）＂ could effectively inhibit the expression of pepc2 gene in C. reinhardtii. These findings may lay a good foundation for screening high-lipid content reverse strain.

关 键 词：莱茵衣藻 磷酸烯醇式丙酮酸羧化酶 qPCR 反向载体技术 生物柴油 

分 类 号：Q785[生物学—分子生物学]