Corynebacterium glutamicum组成型启动子及核糖体结合位点的研究

Study of Constitutive Promoter and RBS in Corynebacterium glutamicum

谷氨酸棒杆菌(Corynebacterium glutamicum,C.glutamicum)是一种广泛用于工业生产的生物安全性菌株。本研究旨在丰富组成型启动子表达元件库,从C.glutamicum ATCC 13032中克隆了5种基因启动子,分别为锰超氧化物歧化酶基因启动子(psod)、延伸因子基因启动子(ptuf)、三磷酸甘油醛脱氢酶基因启动子(pgap)、苹果酸合成酶基因启动子(pms)和二氢吡啶二羧酸合酶基因启动子(pa16)。通过构建工具质粒,以绿色荧光蛋白基因(gfp)为报告基因,研究了这5种基因启动子的启动活性。结果表明5种基因启动子的启动活性由高到低依次为pa16、psod、pms、ptuf和pgap,荧光强度分别为465 RFU/OD_(600)、420 RFU/OD_(600)、305 RFU/OD_(600)、200 RFU/OD_(600)和175 RFU/OD_(600)。此外,基于已构建的载体pa16gfp-p XMJ19,以卡那霉素为报告基因,构建了包含两个核糖体结合位点序列(CGAAAGGATTTTTTACCC及CAGGAGGACATACA)的psod和ptuf的验证质粒,为构建不同C.glutamicum工程菌提供了可选择性调控元件。

Corynebacterium glutamicum （C. glutamicum） is a kind ofbio-security strain, which is widely used in industrial production. With the aim of enriching the constitutive promoter expression-element library, we cloned five gene promoters from C. glutamicum ATCC 13032, including manganese superoxide dismutase gene promoter （psod）, elongation factor TU gene promoter （ptu]）, glyceraldehyde 3-phosphate dehydrogenase gene promoter （pgap）, malate synthase gene promoter （pros） and dihydrodipicolinate synthase gene promoter （pa16）. We studied the activity of five gene promoters by constructing tool plasmid and taking green fluorescent protein gene （gfp） as the reporter gene. The results indicated that the activity of five gene promoters in order from high to low was pal6, psod, pros, ptufandpgap. Besides, the fluorescence intensity were 465 RFU/OD600, 420 RFU/OD600, 305 RFU/OD600, 200 RFU/OD600 and 175 RFU/OD600, respectively. In addition, based on the constructed vector pal6gfp-pXMJ19, we constructed verify plasmids of psod and ptuf that contained 2 ribosome binding site sequences （CGAAAGGATTTTTTACCC and CAGGAGGACATACA） with Kanamycin as the reporter gene, which provided optional regulatory elements for constructing different C. Glutamicum engineering strains.