外源glnA基因干扰大肠杆菌的代谢

Metabolism of Escherichia coli is interfered by Bacillus subtilis glnA gene

摘要:

采用分子生物学的方法构建了含Bacillus subtilis glnA基因的重组菌株Escherichia coli DH5a(pMD19-glnA), 用毛细管电泳和核磁共振对重组菌株的转化谷氨酸的产物进行定性鉴定, 并进一步通过荧光定量RT-PCR测定谷氨酰胺合成酶基因(glnA)mRNA水平的相对表达量, 最后用SDS-聚丙烯酰胺凝胶电泳对蛋白的相对表达情况进行了分析。结果表明重组菌株并没有增加谷氨酰胺的产量, 而是明显增加了g-氨基丁酸(GABA)的产量。实验表明重组菌株中的glnA基因可以正常转录, 但是谷氨酰胺合成酶的蛋白表达量并没有增加。这种外源基因干扰大肠杆菌代谢的现象值得进一步研究。

关键词:谷氨酰胺合成酶, 毛细管电泳, 荧光定量RT-PCR, SDS-聚丙烯酰胺凝胶电泳

Abstract:

A recombinant strain Escherichia coli DH5a(pMD19-glnA) including Bacillus subtilis glnA gene was constructed. Capillary electrophoresis and nuclear magnetic resonance were used to determine qualitatively the product of transformation by recombinant strain, and the relative level of mRNA expression of glnA was also determined by fluorescence quantitative RT-PCR. Subsequently, SDS-PAGE (polyacrylamide gel electrophoresis) was used to analysis the relative level of protein. Surprisingly, there was no increase of glutamine production in this recombinant strain, but an obvious increase in the GABA (g-aminobutyric acid ) production. It was showed in the experiment that protein expression of the glutamine synthetase did not increase, although glnA gene can be transcribed normally in this recombined strain. The phenomenon of exogenous glnA gene interfering metabolism of Escherichia coli was worthy of further study.

Key words:glutamine synthetase (GS), capillary electrophoresis, fluorescence quantitative RT-PCR, SDS-PAGE