Cloning And Expression Of The Main Subunit Genes Of Phycoerythrin And Phycocyanin From Porphyra Haitanensis

Phycoerythrin and phycocyanin,two classes of phycobiliprotein, act as substances of light-harvesting and transferring energy to photosystem reaction centers in algal photosynthesis;also as some kind of valuable nutritious components as store proteins ; moreover, researches revealed that all phycobiliproteins had extensive applied perspective in quite a lot of fields, such as immunofluorescence technique, anti-tumour medicine, health care and treatment medicine, food, fine chemical industry and cosmetic industry.Based on the sequences of phycoerythrin and phycocyanin genes in GenBank,the pairs of degenerate primers were designed and synthesized for cloning these genes of Porphyra haitannesis.For phycoerythrin, a specific fragment about 1103 bp in size was obtained after PCR amplification using the cpDNA of P. haitannesis as template.The structure of this fragment was arranged as cpeB-intergenic region-cpeA with 534 bp,74 bp,and 495 bp respectively.By comparing the phycoerythrin gene sequences of P. haitanensis with the ones of other species in GenBank,the homologies were from 95.4 % to 74.1 % with these eleven species of RhodoPhyta, and 62.2 % with Fremyella diplosiphon.For phycocyanin, a specific fragment about 1071 bp in size was obtained after PCR amplification using the cpDNA of P. haitannesis as template.The structure of this fragment is arranged as cpcB-intergenic region-cpcA with 519 bp,63 bp,and 489 bp respectively.By comparing the phycoerythrin gene sequences of P. haitanensis with the ones of other species in GenBank,the homologies were from 91.1 % to 73.2 % with these seven species of RhodoPhyta, and 68.5 % to 58.1 % with the other four species of Cyanophyta.The homologous was even higher in the coding region than in the intergenic region for both of the two genes.We used the homology among cpcAB/cpeAB in different algaes to estimate the genetic distance, more, we protracted the phylogenetic tree to compare their kindred. As the basic work for further research, we moded the 3D structure of subunits of phycoerythrin and phycocyanin with software D.S. Modeling. And we also evaluated the nutrition of phycoerythrin and phycocyanin.We constructed the recombinant prokaryotic expression plasmids: pTO-T7-cpcAB and pTO-T7-cpeAB, which contained the target genes cpcAB and cpeAB cloned from the P. haitanensis chloroplast genome respectively. Then we transformed them into E.coli BL21(DE3),induced them to express with IPTG,and examined and analyzed the expression products by SDS-PAGE. The results revealed that the subunits ofαandβwere all expressed. The molecular weight of subunitαwas about 19 kDa and 17.5 kDa for subunitβ.The expression products accounted for more than 50% of the total E. coli. proteins after 3 hours inducing at 37℃.In order, the products above were taken DOC-washing,0.6% SKL-solving,Sephadex G-100 gel filtration,and at last SDS-PAGE analysis. The result revealed that the proteins we got had been electrophoretically prue.