施氏假单胞菌75 mRNA差异显示PCR方法的建立

Establishment of a Procaryote DDRT-PCR Method by Strain Pseudomonas stutzeri 75

为丰富原核生物基因表达差异的研究方法,将应用于真核生物中的差异显示PCR方法应用于原核生物基因差异表达的研究,以1株有机磷农药乐果的降解菌施氏假单胞菌(Pseudomonas stutzer)75为研究材料,以5′末端依赖性的RNA外切酶(Terminator Exonuclease)降解其中的16S rRNA和tRNA,并用随机引物替代锚定引物,改良真核生物差异显示PCR,建立原核生物mRNA差异显示PCR的试验体系。以LB培养基培养的菌株为对照组,以无机盐培养基(乐果为唯一碳源)为试验组。结果表明:提取总RNA,通过5′-磷酸基团依赖的核酸外切酶降解法获得mRNA,经逆转录,用双随机引物扩增获得253个差异基因,16S rRNA、tRNA的污染较少,其中之一为硫酸盐运输蛋白基因。

To set up a technique for the differential display of procaryote gene, a strain of P. stutzeri 75 isolated from an environment polluted by organophosphorus pesticide was taken as sample. Terminator 5＇-phosphate-dependent exonuclease enzyme was used to degrade 16S rRNA and tRNA of bacteria. The enriched mRNA of P. stutzeri 75 was reversely transcripted by a random primer. A pair of random primers were taken for the next PCR process and the method of DDRT-PCR for procaryote gene was established. The strain,P, stutzeri75, was cultivated in Luria-Bertani （LB） medium or an inorganic salt medium only with dimethoate as carbon source. Both samples were collected for total RNA isolation. mRNA was further purified and transcriped. A total of 253 differential genes were obtained with a low background of rRNA and tRNA, one of them was transport protein gene of sulfate.