盐单胞菌假定蛋白基因的功能预测与克隆表达及耐盐相关性  

Functional prediction and clonal expression of hypothetical protein gene and validation of salt tolerance correlation in the genus Halomonas

作　　者：舒志万 王智博 陶宇杰 王嵘 沈国平 邢江娃 朱德锐 SHU Zhiwan;WANG Zhibo;TAO Yujie;WANG Rong;SHEN Guoping;XING Jiangwa;ZHU Derui(Center for Basic Medical Research,College of Medicine,Qinghai University,Xining 810016,China)

机构地区：[1]青海大学医学院基础医学研究中心,西宁810016

出　　处：《生物学杂志》2023年第6期12-18,共7页Journal of Biology

基　　金：国家自然科学基金项目(32260019);青海省基础应用研究计划项目(2020ZJ767)。

摘　　要：为挖掘盐单胞菌盐胁迫过程中存在的相关新型未知基因,并明确其功能,以野生菌株Halomonas campaniensis strain XH26为研究对象,通过盐梯度转录组学数据筛选出上调表达的新型基因orf03282(300 bp),利用生物信息学工具进行基因序列、蛋白理化性质、跨膜结构域和二级结构等分析。通过克隆目的基因和构建异源表达载体,探究相关功能。结果表明:随盐度增加,基因orf03282的表达量呈上调表达趋势。在盐单胞菌属中,orf03282的基因序列进化保守,与菌株H.campaniensis LS21和H.alkaliphila X3的相似性最高,分别为99.67%和99.33%。预测蛋白Orf03282为不稳定的疏水性单次跨膜蛋白,定位于细胞外膜。蛋白Orf03282能在菌株E.coil BL21体内实现异源表达,可提高E.coil BL21胞内的Ca^(2+)含量和增强菌株的盐耐受性。推测蛋白Orf03282的生理功能可能与Ca^(2+)离子转运相关,通过增加胞内Ca^(2+)浓度提高细胞的耐盐能力。To uncover novel unknown genes relevance during salt stress in Halomonas and to clarify their functions,in this study,the wild strain Halomonas campaniensis strain XH26 was used as the target.The novel gene orf03282(300 bp)up-regulated in expression was screened by salt gradient transcriptomics data and was analyzed for gene sequence,protein physicochemical properties,transmembrane structural domains and secondary structure using bioinformatics tools.Initial exploration of relevant functions through cloning of target genes and construction of heterologous expression vectors.The results showed that the expression of gene orf03282 tended to be up-regulated with increasing salinity.Among the genus Halomonas,the gene sequence of orf03282 was evolutionarily conserved and showed the highest similarity with strains H.campaniensis LS21 and H.alkaliphila X3,with 99.67%and 99.33%,respectively.The predicted protein Orf03282 was an unstable hydrophobic single transmembrane protein localized to the outer cell membrane.The protein Orf03282 was heterologously expressed in E.coil BL21 and could increase the intracellular Ca^(2+)content of E.coil BL21 and enhance the salt tolerance of the strain.Therefore,the physiological function of protein Orf03282 might be related to Ca^(2+)ion transport,increasing the salt tolerance of cells by increasing the intracellular Ca^(2+)concentration.

关 键 词：Halomonas campaniensis 假定蛋白 生物信息学分析 功能注释 耐盐性验证 

分 类 号：Q51[生物学—生物化学]