摘要
为构建一种新型基因敲除质粒,以pSET4s和pK18mobsacB质粒为基础,通过PCR和酶切连接的方法,构建了新质粒pSETsacB,然后以pK18mobsacB质粒为对照,评价新建质粒对达卡气单胞菌kdpE基因的有痕敲除(带有四环素筛选标记)和无痕敲除效率.结果表明,两种质粒在有痕敲除效率上无差别,但在无痕敲除上,pSETsacB的敲除效率显著高于对照质粒pK18mobsacB.由于pSETsacB为穿梭性温敏质粒,因此,该质粒可望在革兰氏阳性和阴性菌的遗传机制研究中皆可发挥作用,本研究同时也为检视kdpE基因在达卡气单胞菌中的功能提供了前期的突变体材料.
Abstract
In order to construct a novel gene-knockout plasmid,a novel plasmid pSETsacB was constructed based on the plasmids pSET4s and pK18mobsacB by using PCR and cloning techniques.Then pK18mobsacB was used as control,the knockout efficiency of kdpE gene in Aeromonas dhakensis was evaluated via the newly constructed plasmid with or without tetr marker.The results showed that there was no difference in the knockout efficiency between the two plasmids with tetr marker,but the efficiency of scarless deletion by pSETsacB was significantly higher than that of the control plasmid pK18mobsacB.pSETsacB is a thermosensitive shuttle plasmid,and is hence expected to play a role in the genetic mechanism of Gram-positive and Gram-negative bacteria.This study also provides important mutant material for investigating the kdpE gene of A.dhakensis.
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