枯草芽孢杆菌bioW基因在大肠杆菌中的表达及其对宿主生长的抑制作用  

Expression of the B.subtills bioW gene in E.coli and its growth inhibition of host

作　　者：杨明明[1,2] 杨朝霞[2] 张西锋[2] 王晶[2] 刘锦妮[2] 岑沛霖[1] 

机构地区：[1]浙江大学材料与化学工程学院,浙江杭州310027 [2]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2009年第1期56-60,共5页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家重点新产品计划项目(2003ED760039)

摘　　要：【目的】克隆枯草芽孢杆菌(B.subtilis)生物素基因(bioW),在bioW基因缺陷型大肠杆菌中进行表达,探讨其对bioW基因缺陷型大肠杆菌的遗传互补作用和对宿主的生长抑制作用,为生物素基因工程菌的构建奠定基础。【方法】用大肠杆菌表达载体pEXT20和枯草芽孢杆菌bioW基因构建表达载体pEXT20-bioW,并将其转化到大肠杆菌DH5α和生物素基因缺陷型大肠杆菌株R876中,构建重组菌DH5α(pEXT20-bioW)和R876(pEXT20-bioW)。分别将R876、DH5α(pEXT20-bioW)、R876(pEXT20-bioW)在不同培养基和不同IPTG浓度下进行培养,检测bioW基因对bioW基因缺陷型大肠杆菌株的遗传互补和生长抑制作用。【结果】在生物素限制性培养基中,只有同时加入庚二酸和IPTG时,R876(pEXT20-bioW)才能生长。在不含IPTG的LB培养基中,R876(pEXT20-bioW)生长正常;在添加不同浓度IPTG的LB培养基中,DH5α(pEXT20-bioW)和R876(pEXT20-bioW)生长均受到不同程度的抑制。【结论】bioW基因对bioW基因缺陷型大肠杆菌有遗传互补作用。在大肠杆菌中,bioW基因的表达对宿主有生长抑制作用。[Objective] In order to construct biotin genetic engineering bacteria, the Bacillus subtilis bloW gene involved in biotin biosynthesis pathway was investigated in this study. [Method] The B. subtilis blow gene was PCR amplified from B. subtilis chromosomal DNA and,the expression vector pEXT20-bioW was constructed by cloning the bioW into the E. coli expression pEXT20. Then, the pEXT20-bioW was transformed into the E. coli R876 and E. coli DH5α to examine genetic complementation respectively. [Result] When both pimelic acid and IPTG were supplemented in the biotin-free medium,R876（pEXT20-bloW）could grow,conforming that the blow complemented the defect of bioC. In the LB medium （without IPTG）, E. coli R876 （pEXT20-bioW） grew normally, the growth of both DH5α（pEXT20-bioW）and R876（ had genetic complementation to the bacteria lacking however,when high concentration IPTG was added, pEXT20-bioW）was inhibited. [Conclusion] The bioW of biotin gene and the growth of E. coli harbouring pEXT20-bioW was inhibited.

关 键 词：枯草芽孢杆菌 大肠杆菌 bioW基因 生长抑制 遗传互补 

分 类 号：Q563.7[生物学—生物化学]