| The Antarctic has an extreme environment where the pole-end temperature,radiation,and desiccation limit most of life activities.Through long term adaptation and evolution,the microbial populations have formed special biodiversity and genetic backgrounds,developed various metabolic pathways producing enzymes and active substances with special functions.In order to investigate the biodiversity and community structure of the microorganisms in the Antarctic soil,we collected 8 soil samples from Antarctic ASPA113 and ASPA125 region as study objectives during the 31 nd Antarctic Scientific Expedition.With metagenomic high-throughput sequencing our study enables to analyze uncultivated microbial populations that may play important roles in the environment.The most diverse soil sample was carried forward to the microorganism isolation under different cultural conditionsand the morphology of the isolated clones was observed to screen 70 single bacteria.We constructed DNA library based on ddRAD technology and identified the species of the 70 single bacteria.We selected 11 isolated bacteria strains cultivated in Gibbson medium to cultivate under salt stress and 3 strains were characterized with salt tolerance.We assembled and drew the whole genome circle diagram of the NO.242 bacteria(Pseudomonas sp.TKP)based on the sequencing data of NGS on Illumina Hiseq 2500 platform and long single molecular sequencing on Nanopore platform.In addition,transcriptome sequencing was performed to further explore the functional genes related to the regulation mechanism of salt tolerance.Initially we obtained the diversity information through metagenomic high-throughput sequencing.The microbes identified from the 8 soil samples belong to 9 phyla,14 classes,27 orders,41 families,52 genera,74 species.Among them,bacteria accounted for the largest proportion,followed by fungi and DNA viruses.Antarctic soil microorganisms include Acidobacteria,Actinobacteria,Bacteroidetes,Cyanobacteria,DeinococcusThermus,Firmicutes,Proteobacteria,Ascomycota,Viruses-noname representative groups of nine phyla;dominant flora including Granulicella,Candida,Pseudomonas.The study of environmental microbial communities through metagenomic high-throughput sequencing provides support for further investigations of metabolic potentials of microbiomes,in addition to their taxonomic composition.This greatly improves the ability to interpret and predict functional interactions and population dynamics of microbiomes.Using traditional environmental microorganism separation method,we selected 4 media and 3 temperatures to isolate the soil diluted with different concentrations.We isolated 70 bacteria strains,observed their morphology,and performed high-throughput sequencing based on ddRAD technology.The results showed that there were 8 genera including: Pseudomonas,Sphingomonas,Arthrobacter,Burkholderia,Bacillus,Rhodococcus,Collimonas,Variovorax,among which Pseudomonas is an absolute dominant flora,followed by Sphingomonas,Arthrobacter,and Burkholderia.From salt stress cultivation results of the 11 bacteria strains isolated on Gibbson medium,3 salt-tolerant bacteria were screened,of which the transcriptome sequencing was performed and differentially expressed genes were analyzed.The results showed that,a large number of gene expression changed significantly in salt-tolerant bacteria under salinity stress conditions,compared with standard Gibbson culture conditions.461(7.91%)genes of 242 bacteria were significantly up-regulated,417(7.15%)genes were significantly downregulated;44(8.44%)genes of 248 bacteria were significantly up-regulated,26(4.99%)genes were significantly down-regulated;280 bacteria 499(8.35%)genes were significantly up-regulated and 417(9.03%)genes were significantly down-regulated.Genes related to energy production and conversion of bacteria No.242 under salt stress were significantly down-regulated,and genes related to inorganic ion transport and metabolism were significantly up-regulated,which reflected the molecular mechanism of Antarctic bacteria to adapt to salt absorption environment.The proA,proB,and proC three proline synthesis genes contained in this bacterium were up-regulated under salt stress conditions,encoding the production and metabolism of proline,and obtaining a compatible substance of proline to resist the high salt environment.The whole genome information of bacteria No.242(Pseudomonas sp.TKP)was also obtained based on the second and third generation genome data.It was found that the bacteria contains uvr A,uvrB,uvrC radiation resistance related genes to adapt to the high radiation environment of Antarctica.Transcriptome sequencing and stress-related gene analysis in our study can help people to understand the stress resistance mechanisms of Antarctic soil microorganisms and may accelerate the actual development of genetic resources. |
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