Cloning And Sequence Analysis Of Hox1 And PcyA Gene From Cyanobacteria And Construction Of Expression Vector

Heme oxygenasel and ferredoxin oxidoreductase are key enzymes in the synthesis of phycocyanobilin. Their expressed enzymes catalyze heme into phycocyanobilin. Hoxl gene and pcyA gene control the synthesis of Hol and PcyA. CpcE/F catalyze the ligation of phycocyanobilin and apoprotein. Only a combination of color-based, phycocyanin can be optically active. Only when phycocyanobilin and chromophore combined together, can be phycocyanin optically active. Along with the more and more extensive application of the phycocyanin, the cloning of hoxl gene and pcyA gene and their sequencing analysis become to hot topics.The complete hoxl gene including 717bp was cloned from Synechococcus cedrorum by using touchdown PCR technology. Homology analysis based on cyanobacteria hoxl gene showed that the similarity is 99.9% between Synechococcus cedrorum and Synechococcus PCC7942 and 99.2% between Synechococcus cedrorum and Arthrospira platensis. The similarity of deduced amino acid is 99.6% (between Synechococcus cedrorum and Synechococcus PCC7942) and 97.9% (between Synechococcus cedrorum and Arthrospira platensis) respectively. The phylogenetic relationships among eight cyanobacteria were assessed by the methods of neighbor-joining (NJ) and maximum parsimony (MP).The complete pcyA gene including 747bp was cloned from Arthrospira platensis FACHB314 by using PCR technology. Homology analysis based on cyanobacteria pcyA gene showed that the similarity is 99.9% between Arthrospira platensis FACHB314 and Synechocystis sp. PCC 6803. The similarity of deduced amino acid is 99.6%(between Arthrospira platensis FACHB314 and Synechocystis sp. PCC 6803).The expression plasmids vector pUC18-H-P and pSB130-H-P were constructed by using In-FusionTM kit Advantage PCR Cloning Kit. The results of PCR and restriction enzyme double digestion showed that the recombinant plasmids vector pUC18-H-P and pSB130-H-P had been constructed successfully.