优化噬热栖热菌的遗传转化体系  

Optimization of the genetic transformation system in Thermus thermophilus

作　　者：李海娟 徐玲玲[1] LI Hai-juan;XU Ling-ling(College of Biological and Environmental Engineering,Xi'an University,Xi’an 710065,China)

机构地区：[1]西安文理学院生物与环境工程学院,陕西西安710065

出　　处：《生物技术》2019年第1期63-68,共6页Biotechnology

基　　金：国家自然科学基金项目(31700059);陕西省自然科学基金项目(2018JQ3037);陕西省高校科协青年人才托举计划项目(20180210)

摘　　要：[目的]优化噬热栖热菌Thermus thermophilus的转化体系。[方法]将质粒DNA的形态、浓度及转化时间作为变量设计噬热栖热菌T.thermophilus的转化体系,以通过直接双交换同源重组法获取Δpyr E突变体的概率为依据判定转化效率。[结果]在转化时间为2 h,使用3.0μg/m L线性质粒DNA,获取表观Δpyr E的概率为3.44×10^(-5);而使用同样浓度的超螺旋质粒DNA,该概率可达1.03×10^(-3);说明超螺旋质粒的转化效率高于线状质粒。质粒DNA的使用浓度及转化反应时间对转化效率亦有影响,但并非完全成正相关。使用浓度为15μg/m L超螺旋质粒DNA,在转化时间为3 h时,获取表观Δpyr E的概率达到最大值(1.36×10^(-2));超过该阈值,转化效率降低。[结论]在T.thermophilus中,通过优化转化体系将基因无痕敲除突变体获取概率提高到10^(-2)。[Objective]Optimization of the genetic transformation system in Thermus thermophilus.[Method]The plasmid DNA form,the plasmid DNA concentration and the transformation reaction time were set as variables to design the genetic transformation system of T.thermophilus,and the frequency of obtainingΔpyr E mutant via direct double-crossover homologous recombination method was used as a criterion to determine the transformation efficiency.[Result]When the transformation time was set at 2 h,the frequency of obtaining apparentΔpyr E was 3.44×10^-5when using 3.0μg/m L linearized plasmid DNA for transformation;while the value could reach 1.03×10^-3 when a same amount of supercoiled plasmid DNA was used.This indicated that the transformation efficiency of the supercoiled plasmid was higher than that of the linearized plasmid.The concentration of the plasmid DNA and the transformation reaction time also influenced the transformation efficiency,however,they were not always positively correlated.When the supercoiled plasmid DNA was used at a concentration of 15μg/m L and the transformation reaction time was 3 h,the frequency of obtaining the apparentΔpyr E mutants reached the highest level(1.36×10^-2).And the transformation frequencies were decreased when the plasmid concentration or the transformation time was beyond the threshold.[Conclusion]Through optimization of the genetic transformation system,the frequency of obtaining gene clean deletion mutant was increased to 10^-2 in T.thermophilus.

关 键 词：噬热栖热菌 遗传转化系统 双交换性同源重组法 基因无痕敲除突变体 

分 类 号：Q93-31[生物学—微生物学]