摘要
为分析编码多亚基钠/氢逆向转运蛋白Group 2型mrp(mrp2)操纵子耐盐碱能力,首先构建mrp2自杀质粒p K18mobsac B-mrp2-Nco I,电转化野生型菌株-松嫩盐单胞菌(Halomonas songnenensis)NEAU-ST10-39T,基于以上基因敲除原理获得mrp2敲除菌株。PCR验证及测序结果表明,成功获得mrp2敲除菌株,并将其命名为NEAU-ST10-39T-△mrp2。为进一步验证突变株正确性,构建重组穿梭载体p BBR1-MCS5-mrp2,转化敲除菌株NEAU-ST10-39T-△mrp2,获得转化子命名为NEAU-ST10-39T-△mrp2/p BBR1-MCS5-mrp2。耐盐碱测试显示,NEAU-ST10-39T-△mrp2/p BBR1-MCS5-mrp2表现出与野生型菌株类似耐盐碱能力,进一步证实敲除菌株NEAUST10-39T-△mrp2构建正确;随Na Cl浓度和p H升高,敲除菌株NEAU-ST10-39T-△mrp2生长受到抑制,表明mrp2对宿主菌NEAU-ST10-39T在高盐碱条件下生长具有重要作用。电转化方法可避免p K18mobsac B在三亲本接合过程中受体菌抗性筛选、突变株纯化弊端,简化质粒导入宿主程序;NEAU-ST10-39T-△mrp2成功构建为敲除盐单胞菌中其他耐盐碱基因和揭示该基因耐盐碱机制奠定基础。
In order to analyze of the halo- alkaline- tolerant capacity of mrp2 operon encoding aGroup 2 multi- subunit Na ＋/H ＋ antiporter in the host Halomonas songnenensis NEAU- ST10- 39T, thesuicide plasmid of mrp2, pK18mobsacB- mrp2- NcoI, was constructed and then electroporated into thewild type NEAU-ST10-39T. Finally, the mrp2-knockout mutant designated NEAU-ST10-39T-△mrp2 was obtained by using the homologous recombination and counter-selection on the plate containing 10% sucrose. PCRverification and sequencing analysis showed that mrp2 succeeded in being knockout in the mutant NEAU-ST10-39T- △mrp2. For the confirmation of this result, mrp2 was constructed into a broad- host- range shuttle vector,pBBR1- MCS5, and the resultant construct designated pBBR1- MCS5- mrp2 was electroporated into the mutantNEAU- ST10- 39T- △mrp2. The resultant transformant was designated NEAU- ST10- 39T- △mrp2/pBBR1- MCS5-mrp2. Growth test for the halo-alkaline tolerance showed that the transformant NEAU-ST10-39T-△mrp2/pBBR1-MCS5- mrp2 exhibited the similar growth to the wild type strain NEAU- ST10- 39T. In contrast, the growth of themutant NEAU-ST10-39T-△mrp2 was inhibited under the highly-saline or highly alkaline conditions, revealing thatmrp2 played a critical role in the halo- alkaline tolerance of the host NEAU- ST10- 39T. Successful application ofgene knockout through the electroporation and construction of the mutant NEAU-ST10-39T-△mrp2 will be veryhelpful for carrying out the knockout of other halo- alkaline tolerant genes from Halomonas genus and furtheranalysis of the halo-alkaline tolerance of mrp2 in the host NEAU-ST10-39T.
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2017年第12期11-20,共10页
Journal of Northeast Agricultural University
基金
国家自然科学基金项目(31570045)
黑龙江省自然科学基金项目(C201417)
博士后科研启动资金(LBH-Q14022)
