Expression Characteristics Of The Temperature Adaptation Related Hypothetical Genes In The Antarctic Psychrotrophic Bacterium Psychrobacter Sp.G

Antarctic ecosystem is characterized by extreme cold, dry, poor nutrient availability and strong radiation, and there are abundant of microorganisms which play vital roles in the material circulation of the Antarctic ecosystem. The previous transcriptome analysis of the Antarctic psychrotrophic bacterium Psychrobacter sp. G showed that, after the cold shock（0 ℃） / heat shock（30 ℃） stress, the expression levels of 11 and 46、12 and 48 genes were significantly up-regulated and down-regulated respectively, including many genes affiliated with unknown function. This article conducted gene cloning and sequence analysis of three unknown function genes with significant regulations; furthermore, qRT-PCR and western blotting were used to analyze their expression patterns under different temperature/salinity stresses at both transcriptional and translational level. The study is helpful to clarify the adaptation mechanism of Psychrobacter sp. G in the severe environment of Antarctic.Transcriptomic analysis showed that, after exposed to high temperature（30 ℃）, the expression of hypothetical gene PSYCG₀3870（AGP48309.1） was significantly up-regulated（log2FC=2.5, FDR≤0.05）. The ORF of this hypothetical protein gene was 177 bp, the upstream and downstream regulation sequences included-10、-35、TIS、RBS and a polyadenylation signal sequence AATAAA located in the downstream of termination codon（TAA）. Hydrophobic analysis of amino acid sequences showed that the C end of the deduced protein had a strong hydrophobic region（score﹥0）, and no transmembrane area was identified. BLASTP analysis indicated that the function protein with the highest similarity was unnamed protein product（CCW68256.1, 37%）. In the mRNA pattern, the expression of PSYCG₀3870 gene was significantly inhibited by low temperature and induced by high temperature; under the salinity stress, the expression of the gene could be inhibited by both low salinity and high salinity; and under the combined stress, the expression levels of gene could be increased by low temperature, low and high salinity; however, the expression of the gene was inhibited with the high temperature, low and high salinity. In the protein pattern, the expression of the protein could be enhanced by both low and high temperature; under the salinity stress, the expression of the protein could be inhibited by low salinity and induced by high salinity; and under the combined stress, the expression of hypothetical protein PSYCG₀3870 changed little at 6 h under the condition of high temperature, low salinity（30 ℃, 15） and was all suppressed except high temperature, high salinity（30 ℃, 90） in 2 h and 6 h.Transcriptomic analysis showed that, after exposed to low temperature（0 ℃）, the expression of hypothetical gene PSYCG₁0180（AGP49518.1） was significantly up-regulated（log2FC=4.1, FDR≤0.05）. The ORF of this hypothetical protein gene was 624 bp, the upstream and downstream regulation sequences included-10、-35、TIS、RBS and a downstream box of 14 bp located in the downstream of initiation codon（ATG）. Hydrophobic analysis of amino acid sequences showed that the C end of the deduced protein had a strong hydrophobic region（score﹥0） and typical transmembrane area existed in 181-203 AA. BLASTP analysis indicated that the known function protein with the highest similarity was blue light sensor protein（WP₀10201745.1, 37%）. In the mRNA pattern, the expression of PSYCG₁0180 gene was significantly induced by low temperature and inhibited by high temperature; under the salinity stress, the expression of the gene could be significantly inhibited by all salinity; and under the combined stress, the expression levels of gene could be significantly induced by low temperature, low salinity（0 ℃, 15） and inhibited by high temperature, high salinity（30 ℃, 90）. In the protein pattern, under the temperature stress, the expression of the protein was induced in short time（6 h）, and was inhibited by long time（12 h）; under the salinity stress, the expression of the protein could be inhibited by both low salinity and high salinity; and under the combined stress, the expression of hypothetical protein PSYCG₁0180 was all suppressed in except high temperature, low salinity（30 ℃, 15） in 2 h.Transcriptomic analysis showed that, after exposed to low temperature（0 ℃）, the expression of hypothetical gene PSYCG₀6740（AGP48869.1） was significantly down-regulated（log2FC=-6.4, FDR≤0.05）. The ORF of this hypothetical protein gene was 1236 bp, the upstream and downstream regulation sequences included-10、-35、TIS、RBS and AT-rich UP element located in the upstream of-35 region, and a downstream box of 14 bp located in the downstream of initiation codon（ATG）. Hydrophobic analysis of amino acid sequcences showed that the deduced protein had no obvious hydrophobic region and typical transmembrane area. BLASTP analysis indicated that the known function protein with the highest similarity was ATP-grasp domain-containing protein（WP₀11513612.1, 97%）. In the mRNA pattern, the expression of PSYCG₀6740 gene was inhibited by low temperature, and was induced by high temperature; under the salinity stress, the expression of the gene was inhibited by both low salinity and high salinity; and under the combined stress, the expression levels of gene was extremely significantly induced by low temperature, low salinity（0 ℃, 15） and inhibited by high temperature, high salinity（30 ℃, 90）. In the protein pattern, the expression of the protein was inhibited by low temperature, and was enhanced by high temperature; under the salinity stress, it could be inhibited or induced by both low salinity and high salinity; and under the combined stress, the expression of hypothetical protein PSYCG₀6740 was induced in short time（2 h）, and was inhibited by long time（12 h）.