Cloning Expression And Functional Identification Of Salt Stress-related Genes From Nostoc Flagelliforme

Nostoc flagelliforme is a terrestrial cyanobacteria with stress-tolerance,which lives in poor condition.Its growth is suppressed and the production of polysaccharide increased under salt stress.I t has high officinal and edible value.The differential expressed g enes related to salt stress were screened by RNA-Seq technology,and they were carried out to cloning expression and functional i dentification.The result may laid the foundation for further study on the gene function and molecular mechanism of identification.The TrkH family potassium absorption protein gene,TrkA-N p otassium channel protein gene,TrkA-N1 potassium channel protein gene,TrkA-N domain protein gene and DnaK protective protein g ene sequencing and bioinformatics analysis showed that:All genes sequence size were respectively 1335 bp,696 bp,1692 bp,678 bp and 1905 bp.All sequences were highly conserve d.The molecular weight of protein was respectively 48.30 kDa,31.41 kDa,61.48 kDa,28.52 kDa and 69.85 kDa.The theoretical isoel ectric points were respectively 9.07,5.82,6.98,5.90 and 4.86.The T rkH family potassium absorptive protein contained 10 transmembr ane regions and the TrkA-N1 potassium channel protein containe d two transmembrane regions.TrkA-N potassium channel protein,T rkA-N domain protein and DnaK protective molecular protein co ntained no transmembrane regions.All of the 5 proteins were hyd rophilic proteins.TrkH family potassium absorptive protein had 17 serine phosphorylation sites,15 threonine phosphorylation sites a nd 2 tyrosine phosphorylation sites.TrkA-N potassium channel pr otein had 10 serine phosphorylation sites,6 threonine phosphoryla tion sites and 2 tyrosine phosphorylation sites.TrkA-N1 potassium channel protein had 19 serine phosphorylation sites,12 threonine phosphorylation sites and 6 tyrosine phosphorylation sites.TrkA-N domain protein had 11 serine phosphorylation sites,6 threonine phosphorylation sites and 2 tyrosine phosphorylation sites.DnaK protective molecular protein had 29 serine phosphorylation sites,22 threonine phosphorylation sites and 3 tyrosine phosphorylation sites.The second structure of DnaK protective molecular protein,TrkA-N potassium channel protein,TrkA-N1 potassium channel pr otein and TrkA-N domain protein were mainly alpha helix and f old.The second structure of DnaK protective molecular protein m ainly were alpha helix,random curling and folding.TrkA-N potassium channel protein,TrkA-N domain protein an d DnaK protective molecular protein were all successfully expres sed,and the bands were in accordance with the expected size.The conditions of expression were as follows:Adding IPTG of conce ntration was ImM to the bacterial fluid as OD value was 0.8,co ntinued to culture for 20 hours at 37 degrees and 120rpm.TrkH family potassium absorptive protein and TrkA-N1 potassium chan nel protein were not expressed.The results provided an idea for t he construction of recombinant high expression vector.