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作 者:李海娟 徐玲玲[1] Li Haijuan;Xu Lingling(Xi'an University,Xi'an,710065)
机构地区:[1]西安文理学院,西安710065
出 处:《基因组学与应用生物学》2020年第3期1143-1149,共7页Genomics and Applied Biology
基 金:国家自然科学基金(No.31700059);陕西省自然科学基金(No.2018JQ3037);陕西省高校科协青年人才托举计划项目(No.20180210)共同资助。
摘 要:在低拷贝质粒中,质粒分离基因位点parABS对质粒DNA的子细胞精确分离有重要意义。噬热栖热菌(Thermus thermophilus)为多倍体,其染色体上表达有一个parABS同系物,命名为parABSTth,包含parATth和parBTth两个基因,以及一个候选parSTth的DNA序列(5’-TGTTTCCCGTGAAACA-3’)。parABSTth基因位点的功能与机制尚未清楚。本研究首先在大肠杆菌Escherichia coli中克隆并表达了T. thermophilus parABSTth位点中的parBTth基因。利用Ni-IDA亲和层析方法纯化得到了重组蛋白ParBTth;经SDS-PAGE测定,该重组蛋白纯度达到了近99%,通过凝胶迁移实验(EMSA)验证了重组ParBTth蛋白的功能。结果显示,重组ParBTth能与候选parSTthDNA序列特异性相结合。因此,异源表达的重组ParBTth蛋白是具有生物学活性的。该结果也验证了候选parSTthDNA序列确实为T. thermophilus的parABSTth基因位点中特异性的parS序列。本研究为揭示多倍体细菌染色体的子细胞分离过程原理提供了一定的基础理论依据。In low-copy-number plasmids, the partitioning gene locus parABS is important for accurate plasmid segregation into daughter cells. Thermus thermophilus is polyploid, the chromosome of T. thermophilus encodes a parABS homolog, termed here parABSTth, which contains two genes parATthand parBTth, and a candidate DNA sequence parSTth(5’-TGTTTCCCGTGAAACA-3’). The function and mechanism of the parABSTthgene locus are not clear.In this manuscript, we initially cloned and expressed the parBTthgene in the parABSTthlocus of T. thermophilus in Escherichia coli. Using Ni-IDA affinity chromatography method, the recombinant protein ParBTthwas purified and obtained;SDS-PAGE was used to analyze the purity of the recombinant protein, which showed near 99% purity.The electrophoretic mobility shift assay(EMSA) was performed to analyze the function of the recombinant ParBTth protein. The result showed the recombinant ParBTthcould bind the candidate parSTthsequence specifically.Therefore, the heterologously expressed recombinant ParBTthprotein was biologically active. The results also confirmed that the candidate parSTthsequence was indeed the specific parS sequence of the parABSTthgene locus of T. thermophilus. This study provides some theoretical basis for revelation of the process and mechanism of chromosome segregation into daughter cells in polyploid bacteria.
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