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

Abstract:

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 Chlamydomonas 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.