Evidence For Nostoc Sphaeroides Kützing Genome And Its Phycocyanin Gene Cloning And Expression

Nostoc sphaeroides Kützing,is specifically well-known dual-use algae for food and medicine.With the development of economy and society,the value of Nostoc sphaeroides Kützing as a natural antioxidant resource becomes more and more prominent.In this study,the whole genome was analyzed and cloned the phycocyanin(PC)of the Nostoc sphaeroides Kützing from Enshi region,using the third generation genome sequencing and modern molecular biology techniques and to expresse them in E.coli.The main results of our study are as follows:(1)Using the purified fresh Nostoc sphaeroides kützing,the total DNA was extracted for genome sequencing,and the whole genome sequence of Nostoc sphaeroides kützing of Enshi was 66.8 M bp,contained one chromosome and four large plasmids;the G+C content of its genome is 41.6%.The predicted number of genes encoding proteins in the genome was 7 125,accounting for 98.9% of the total number of genes.In addition,254 and 196 genes involved in essiential amino acid and vitamin metabolism,e.g.biotin,were found in the genome,which are important evidence of their antioxidant value.(2)The phycocyanin subunit gene was cloned for the first time by designing gene specific primers.It was found that the length of Ns-PC-? and Ns-PC-? gene is 492 bp and 522 bp,respectively,encoding protein 163 and 173 amino acids respectively.Ns-PC-? and Ns-PC-? exist in the form of double cistrons,and Ns-PC-? is located downstream of Ns-PC-?.Simulating its advanced structure,it is found that Ns-PC-? and Ns-PC-? single subunits contain seven alpha-helixes.In addition to the N-terminal alpha-helix,several other alpha-helixes further fold to form a hydrophobic core region.Moreover,Ns-PC-? and Ns-PC-? single subunits are well aligned to form a stable and closed monomer with an arc of nearly 2?/3.(3)Prokaryotic expression showed that the molecular weights of Ns-PC-? and Ns-PC-? were nearly 20 kDa.To isolate and purify the prokaryotic proteins of Ns-PC-? and Ns-PC-?,and to identify their antioxidant activities.The results showed that both subunits had strong antioxidant activity.And to the ABTS+.,the scavenging capacity of free radicals is proportional to the concentration of added protein Ns-PC-? and Ns-PC-?.