pSETsac B质粒构建及其对达卡气单胞菌kdp E基因的有痕与无痕敲除效率评价  

Construction of pSETsacB plasmid and evaluation of kdpE gene knockout efficiency with and without markers in Aeromonas dhakensis

作　　者：凌博 陈丽青 王宇 杨诺 范丽霞[3] 郭桂英[4] 李雪松 曾纪锋[1] 李迁[5] 郑继平[3] LING Bo;CHEN Liqing;WANG Yu;YANG Nuo;FAN Lixia;GUO Guiying;LI Xuesong;ZENG Jifeng;LI Qian;ZHENG Jiping(School of Animal Science and Technology,Hainan University,Haikou,Hainan 570228;Biotechnology Research Institute,Shanghai Academy of Agricultural Sciences,Shanghai 201106;School of Life Sciences,Hainan University,Haikou,Hainan 570228;School of Science,Hainan University,Haikou,Hainan 570228;Network and Technology Center,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]海南大学动物科技学院,海口570228 [2]上海市农业科学院生物技术研究所,上海201106 [3]海南大学生命科学学院,海口570228 [4]海南大学理学院,海口570228 [5]海南大学网络与技术中心,海口570228

出　　处：《热带生物学报》2024年第4期452-459,共8页Journal of Tropical Biology

基　　金：国家自然科学基金(32060788,32060131);海南省自然科学基金(321MS007,821MS029,821RC1052,2019RC084)。

摘　　要：为构建一种新型基因敲除质粒,以pSET4s和pK18mobsac B质粒为基础,通过PCR和酶切连接的方法,构建了新质粒pSETsac B,然后以pK18mobsac B质粒为对照,评价新建质粒对达卡气单胞菌kdp E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,pSETsac B的敲除效率显著高于对照质粒pK18mobsac B。由于p SETsac B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kdp E基因在达卡气单胞菌中的功能提供了前期的突变体材料。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.

关 键 词：pSETsac B kdp E基因 基因敲除 

分 类 号：Q784[生物学—分子生物学]