论文部分内容阅读
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 a key enzyme in the regulation of cellular protein and fatty acid content in plant carbon metabolism. By inhibiting the expression of pepc gene, Can improve the cell oil content. The fragments of HR and Crpepc2 were inserted into pSP124s by cloning the partial sequence of the pepc2 gene of Chlamydia reinhardtii and the Chlamydomonas reinhardtii fusion promoter Hsp70A-RBCS2 (referred to as HR), and the reverse expression of Crpepc2 Chlamydomonas reinhardtii expression vector pSP124s-HR-reve-Crpepc2. Plasmid pSP24s and pSP124s-HR-reve-Crpepc2 were transferred into Chlamydomonas reinhardtii cc-503 algae respectively by using a gene gun to obtain empty plasmid and reverse mutant algal strains. QPCR was used to detect the relative expression of pepc2 gene in wild-type, empty-type and reverse-type algae strains of Chlamydomonas reinhardtii. The results showed that the introduction of empty plasmid had little effect on the relative expression of pepc2 gene , Which was 92.95% of the wild type. However, the introduction of the reverse pepc2 gene significantly reduced the relative expression of pepc2 gene in Chlamydomonas reinhardtii, which was only 2.94% of the wild type. On the one hand, this result shows that we established a method for rapid detection of relative expression amount of pepc2 gene in Chlamydomonas reinhardtii by qPCR, and on the other hand, we proved that the method using reverse expression of Crpepc2 (ie “reverse vector technique”) can effectively inhibit The expression of pepc2 gene in Chlamydomonas reinhardtii lay a good foundation for further screening of stable algal strains with high oil content.