摘要
本实验借助宽宿主载体pBBR1MCS-5,构建在弱氧化葡糖杆菌(Gluconobacter suboxydans)中表达FLP重组酶的表达载体,转化弱氧化葡糖杆菌,获得阳性转化子;采用两步法RT-PCR(reverse transcription-polymerase chain reaction)验证,结果表明:flp基因在宿主细胞中转录表达;将pBBR1MCS-psldh-flp重组质粒转化至带有四环抗性的突变菌株JGDH-,PCR验证表明带有FTR位点的抗性标记得到有效删除。重组酶FLP在Gluconobacter suboxydans的表达提供了一种循环使用选择标记基因进行多个位点基因敲除或置换的方法。
In this paper, the broad-host vector pBB1MCS-5 was used to construct an expression vector for flippase recombination enzyme(FLP). The expression vector was transformed into Gluconobacter suboxydans, and the positive transformants were screened. The results of validation using a two-step RT-PCR method showed that flp was transcribed and expressed in the host cell. The pBBR1MCS-psldh-flp recombinant plasmid was transformed into tetracyclic-resistant JGDH-mutant strains, and PCR verification showed that the sites with FTR resistance marker had been effectively removed. Recombination FLP expression in Gluconobacter suboxydans provides a method that circularly uses the selectable marker gene to knock out or replace multiple locus genes.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2014年第11期204-208,共5页
Food Science
作者简介
刘静文(1988-),女,硕士研究生,研究方向为代谢工程。E-mail:liujingwen_1988@126.com。
