Cloning And Prokaryotic Expression Of Polysaccharide Metabolism-related Differently Expressed Genes From Nostoc Flagelliforme Under Salt Stress

Nostoc flagelliforme is a photosynthetic blue-green algae.It has high edible and officinal value.The polysaccharide secreted by Nostoc flagelliforme protects cells from the hostile environment during the growth and metabolism.It was found that the polysaccharide has the antioxidant,antiviral,antitumor and immunity enhancement activities,which shows that it has high medicinal value.The production of polysaccharide increased significantly under salt stress,compared with normal cultivation.The transcriptome sequencing results by RNA-Seq illustrated that numbers of genes involved in different physical processes were differentially expressed in Nostoc flagelliforme under salt stress contrast to normal cultivation.In this study,five differentially expressed genes proposed to participated in polysaccharide metabolism in Nostoc flagelliforme were cloned with the DNA of Nostoc flagelliforme as templates.Then the genes were analyzed by bioinformatics.Moreover,these genes were transferred to the plasmid,p ET28 a,for highly-efficient expression and the cultivation conditions were optimized.This study will enrich more genes data of Nostoc flagelliforme and provide the theoretical basis for studying the metabolic regulation of the polysaccharide in Nostoc flagelliforme.The five genes GT1(encoded for glycosyl transferase1),GT2(encoded for glycosyl transferase2),GT3(encoded for glycosyl transferase3),GMD(encoded for GDP-mannose 4,6-dehydratase),and PEP(encoded for polysaccharide export protein)were cloned and sequenced.Then the genes were analyzed by bioinformatics methods and the results were shown as follows:The lengths of GT1,GT2,GT3,GMD and PEP were 1290 bp,1173 bp,948 bp,1080 bp and 1467 bp,respectively.Each of the nucleotide sequences and amino acid sequences was highly conserved.The molecular weight and isoelectric point of GT1,GT2,GT3,GMD and PEP were 47.54 k Da,43.16 k Da,35.99 k Da,41.08 k Da,51.28 k Da and9.33,7.64,6.34,5.73,5.50,respectively.GT1,GT2,GT3,GMD and PEP were hydrophilic protein with no transmembrane domain.The secondary structure of GT1,GT2,GT3 were mainly randomly coiled and alpha helix.The secondary structure of GMD and PEP was randomly coiled,alpha helix and strands.GT1,GT2,GT3,GMD were PCR amplified and connected with p ET28 a.Then the genes were expressed in E.coli BL21 with IPTG as inducer,respectively.The concentration of IPTG,the OD when IPTG added and the culture temperature were chosen as the variate to do the single factor experiment.SDS-PAGE was used to screen the best condition of target protein expression.The results showed that the best condition of protein expression was that the OD was 0.8,the concentration of IPTG was 1 m M and the culture temperature after IPTG added was 16?.The size of four expressed target proteins was as expected under the best condition.The results laid a foundation for further reasearch on structure and function of GT1,GT2,GT3,GMD and the role in metabolic regulation of the polysaccharide from Nostoc flagelliforme.The research provided a theoretical and experimental basis for studying the metabolic regulation of the polysaccharide in Nostoc flagelliforme.