红色亚栖热菌TPS/TPP海藻糖合成途径中相关基因的克隆、表达及功能鉴定

Cloning, expression and functional analysis of the genes in TPS/TPP trehalose synthetic pathway of Meiothermus ruber

通过构建红色亚栖热菌(Meiothermus ruberCBS-01)的基因组DNA文库,克隆得到该嗜热菌海藻糖合成途径中的磷酸海藻糖合成酶(TPS)和磷酸海藻糖磷酸酯酶(TPP)基因。以pET21a为表达载体,将磷酸海藻糖合成酶和磷酸海藻糖磷酸酯酶在大肠杆菌中进行表达并纯化,利用薄层层析的方法验证了这两个酶的活性。同时,本研究检测了红色亚栖热菌在各种环境压力下细胞内含物成分的变化情况,发现在高渗环境压力的诱导下,该菌会在胞内积累大量的6-磷酸海藻糖,而并非海藻糖,这为进一步研究TPS/TPP和TreS途径在细胞体内的作用奠定了基础。

By constructing the genomic DNA library of Meiothermus ruber CBS-01, the genes of trehalose phosphate synthase （TPS） and trehalose phosphate phosphatase （TPP） involved in trehalose synthesis were cloned. The genes were cloned into the plasmid pET21a, and expressed in Escherichia coli Rosetta gami （DE3）. The activities of these two purified enzymes were confirmed by thin layer chromatography （TLC）. Meanwhile, we tested the cellular compatible solutes of M. ruber CBS-01 under different environmental pressure, and found that under hyperosmotic pressure, this strain can accumulate trhalose-6-phosphate, but not trehalose. These results can give more insight to future research in the roles of TPS/TPP and TreS pathway.