基因局部改组技术在ubiA基因突变中的应用

Application of a model for gene local shuffling in ubiA mutation

摘要:

摘要：辅酶Q（coenzyme Q，CoQ）作为线粒体呼吸链中的递氢体具有较高的学术及应用价值。由ubiA基因编码的4-羟苯甲酸聚异戊二烯转移酶（UbiA）是大肠杆菌CoQ生物合成途径的限速步骤，但通过系统突变对其结构进行研究鲜有报道。【目的】应用化学合成的随机序列寡核苷酸，对ubiA基因编码活性区域的DNA序列进行随机突变。【方法】以大肠杆菌MC4100的ubiA基因插入灭活突变株为受体，将化学合成的随机序列替换目标区域序列，进行ubiA基因的局部改组；最终对所得突变株的ubiA改组基因的突变序列及其功

Abstract:

Abstract: As a transhydrogen in the mitochondrial respiratory chain, Coenzyme Q (CoQ) has a critical role in the metabolism. 4-hydroxybenzoate polyprenyltransferase (UbiA), which coded by ubiA gene, is the rate-limiting enzyme in the CoQ biosynthesis progress. However, the relationship between structure and function remains unclear. [Objective] To set up a synthetic oligonucleotide-based shuffling model and apply in the random mutation of DNA sequence of ubiA gene which codes putative active site. [Methods] By using ubiA knockout mutant of E. coli. MC4100 as receptor, we set up a local Shuffling model by replacing target sequence with a random sequence fragment. Sequences of mutants and their function were analyzed. [Results] After local Shuffling and two cycle of screening, we gained seven mutants. Compared to the wild type, most of the mutant amino sequences showed obvious change, and had different change trend of CoQ production. Sequencing result showed that three aspartic acid sites may be highly related to UbiA catalyze activity. [Conclusion] The local Shuffling model we set up is feasible. By applying this model, we preliminary verified several location of key amino acid sites of UbiA.