Differential Gene Analysis Of Halophilic Salt-excited Transcription And Functional Verification Of Novel Up-regulated Genes

Objective Exploring the co-occurrence patterns of microbial community in Qinghai Lake,and constructing engineering strains containing novel up-regulated genes and verifying the ability of salt tolerance.Methods We applied high-throughput sequencing of the 16 S rRNA genes to analyze the community structure of bacteria(V3-V4 regions)and archaea(V3-V5 regions)in Qinghai Lake;The Spearman and Pearson correlation coefficients between environmental factors and species were calculated;Based on network analysis,the co-occurrence pattern between bacteria in samples and archaea and correlations between species were analyzed.To analyze the transcriptome sequencing results obtained by the Qinghai Lake model strain Halomonas sp.QHL1 under salt challenge conditions(high salt,medium salt and low salt)and obtain up-regulated genes.The recombinant strains containing the novel genes nug89,nug90,nug91,nugF4 and nugF5 were constructed by molecular cloning technology and induced by IPTG.Analysis the salt tolerance of recombinant strains.Results 1)3 phyla,66 classes and 395 genera of bacteria were detected.The dominant genera were Loktanella(11.84%),Nitrincola(6.78%)and Pseudarthrobacter(3.72%).For archaea,12 phyla,19 classes and 68 genera were detected.The dominant genera were Woesearchaeota DHVEG-6 norank(34.44%),Methanosarcina(24.05%)and Soil Crenarchaeotic Group norank(19.04%).Proteobacteria,Bacteroidetes and Actinobacteria,the core phyla of bacteria,exist in an ecological relationship of mutualism.In the core group Euryarchaeota of archaea,Methanomicrobia and Methanobacteria associated with methane metabolism are competitive with Halobacteria,while members of the genus Halobacteria and the genus of methane metabolism are internally symbiotic.2)The recombinant strain E.coli BL21 was successfully constructed(containing recombinant plasmids pET28a-nug89,pET28a-nug90,pET28a-nug91,pET28a-nugF4 and pET28a-nugF5,respectively).Heterologous expression of the nug89,nug90,nug91,nugF4 and nugF5 genes was achieved.The recombinant strain E.coli BL21/pET28a-nug90 had the highest growth rate when NaCl?0.2 M.At 0.2 M<NaCl?0.6 M,the recombinant strain E.coli BL21/pET28a-nugF5 had the highest growth.When the NaCl was 0.8 M,only the recombinant strains E.coli BL21/pET28a-nug89,E.coli BL21/pET28a-nugF5 and E.coli BL21/pET28a-nugF4 were grown,and the latter had the highest growth.Conclusion The microbial community structure in Qinghai Lake is complex,and the species are affected by many environmental factors.Ecological relationship of most bacterial populations is types of mutualism.The genus associated with methane metabolism competes with the population of the genus Halobacter.The recombinant strains E.coli BL21/pET28a-nugF4 and E.coli BL21/pET28a-nugF5 were more resistant to high salt than recombinant strains E.coli BL21/pET28a-nug89,E.coli BL21/pET28a-nug90 and E.coli BL21/pET28a-nug91.