【作者】 汪屹；

【导师】 张惠展；

【作者基本信息】 华东理工大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 yigP基因存在于许多微生物中,其序列都相对保守。在大肠杆菌中,yigP (Gene ID:948915)基因位于辅酶Q合成相关基因ubiE和ubiB之间。GeneBank注释yigP基因是一个可能的蛋白质编码基因,然而却没有任何的文献进一步支持该结论。目前,并未发现有针对该基因的研究报道。我们前期的研究发现,染色体yigP基因缺陷的菌株必需通过外源载体提供一个完整的yigP基因才能够生存,因此认为yigP是大肠杆菌生长必需基因。此外,还发现yigP上游区域具有启动子活性。使用RACE技术还确认了yigP转录一个252nt的RNA。本论文中,我们发现yigP基因下游297bp的片段足以行使相应功能。利用lacZ作为报告基因,我们精确地鉴定了yigP启动子的边界。同时,结合点突变技术,对启动子的-10区和-35区进行了鉴定,以及发现了启动子上游的一个负转录调控序列。最重要的是,yigP基因对移码突变有免疫能力,这说明yigP并非编码蛋白质,而是一个非编码RNA基因。此外,RT-PCR显示yigP能够与其上下游基因ubiE和ubiB共转录。总之,本研究显示了yigP是一个生长必需的非编码RNA基因更多还原

【Abstract】 YigP gene exists in many species, and the sequence is conserved among bacteria. In Escherichia coli, yigP gene(GeneID:948915) locates between ubiquinone biosynthetic genes ubiE and ubiB. GeneBank annotates yigP as a putative protein-coding gene, however, no research focusing on the yigP gene has been reported yet.Our previous work have confirmed that the strain with yigP inactivation in chromosome must rely on a complementary plasmid containing a wild-type yigP gene, to survive under laboratory growth conditions. Promoter activity was detected in the region of upstream yigP-Using RACE techniques, we confirmed that yigP gene was transcribed to a 252 nt RNA molecule.In this study, we have shown that a 297bp fragment downstream of yigP was enough to complement the chromosomal defective yigP gene. Using lacZ as a report gene, we have identified the precise region of yigP promoter. Meanwhile, the -10 and -35 region of yigP promoter were identified by site mutations and a negative transcriptional regulatory region was also discovered associating with promoter activity. Notably, the yigP gene is immune to frameshift mutations. This indicates yigP is a non-coding RNA gene, rather than a protein coding gene as predicted in the GeneBank. What’s more, our reverse transiption PCR have showed that yigP could cotranscribed with its neighbouring genes ubiE and ubiB.In conclusion, our studies revealed that yigP is an essential non-coding RNA gene.更多还原