枯草杆菌生物素操纵子基因的克隆与功能表达研究
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摘要
本研究以大肠杆菌（E. coli）克隆载体pUC19为模板，分别用两对引物扩增了含有复制起始序列（ori）和复制蛋白基因（rep）的700bp 片段，以枯草芽孢杆菌（B.subtilis）质粒载体pGDV1为基本骨架，构建了三个带有氯霉素抗性基因的大肠杆菌－枯草芽孢杆菌小型穿梭载体pGDVM、pGDVM1和pGDVM2。经大肠杆菌－枯草杆菌反复穿梭实验和细菌连续培养分析检测，结果证明：含大肠杆菌pUC19复制起始区（ori）和复制起始蛋白基因（rep）的700bp克隆片段在大肠杆菌中具有完全的复制功能；构建的三个质粒载体在大肠杆菌和枯草杆菌中能稳定地遗传。这三个穿梭质粒载体分子较小，仅3kb左右；具有较高的拷贝数（在枯草杆菌中约150-200拷贝数），带有多个不同的克隆位点，方便插入目标基因，并且带有氯霉素抗性基因，可以为受体菌提供理想的抗性筛选标记。该系列载体可以作为枯草杆菌理想的基因工程菌构建骨架。
本研究成功地克隆了枯草杆菌生物素操纵子基因bioB、bioW 和基因簇bioWAFDB片段；以大肠杆菌表达质粒载体pEXT20为骨架，分别构建了bioW、bioB和bioWAFDB诱导型表达载体pEXT20bioW、pEXT20bioB和pEXT20bioWAFDB，可以通过IPTG诱导，在大肠杆菌中进行表达。通过它们与大肠杆菌生物素生物合成基因系列bioF、bioB、bioC、bioD和bioA缺陷株R874、R875、R876、R877和R879的遗传互补实验，证明：枯草杆菌生物素基因bioA、bioB、bioD和 bioF在大肠杆菌中功能正常； bioW基因（编码庚二酰CoA合成酶）的表达载体对大肠杆菌缺陷株没有遗传互补作用， 但是bioW基因编码的庚二酰CoA合成酶在大肠杆菌中有催化活性，可以催化外源的庚二酸合成庚二酰CoA，为生物素合成途径提供底物。经过IPTG不同水平的诱导表达实验，发现：枯草杆菌生物素基因bioWAFDB的过量表达对受体菌的生长有严重抑制作用，该抑制作用主要是由bioW引起，并且不能通过添加二氨基庚二酸而解除。枯草杆菌bioB基因诱导表达产物未对大肠杆菌的生长造成抑制，实验结果为枯草杆菌生物素基因工程菌的构建及生物素发酵生产提供了有价值的理论依据。
Two DNA fragments, which are about 700bps and contain ori and rep of E.coli, were cloned from pUC19 using two pairs of primers respectively. Three E.coli-B.subtilis shuttle vectors pGDVM，pGDVM1 and pGDVM2whith chloramphenicol resistance, were then constructed with the framework pGDV1 , a plasmid come from B.subtilis strain 1E60. It indicates that the cloned 700 bp DNA fragments contains ori and rep of E.coli have a complete ability of replication, and the three shuttle vectors can inherit and clone steadily via continuous culture and times of shuttle experiments between E. coli and B. subtilis, This series of shuttle vectors based on plasmid pGDV1 , are numerous to 150-200 copies in B. subtilis . The length of vectors are only about 3kb ,they contain multiple clone site and chloramphenicol resistance gene,can act as ideal framework for B.subtis genetic engineering.
Biotin operon genes bioB,bioW and gene cluster bioWAFDB in B. subtilis have been cloned and inserted into the downstream of Ptac promoter of E.coli expression vector pEXT20, and yield induced expression vectors —pEXT20bioW, pEXT20bioB and pEXT20bioWAFDB . Five E. coli mutant strains, R874,R875,R876,R877 and R879 with defect of bioF, bioB, bioC,bioD and bioA respectively, were used for genetic complement analysis of biotin genes. The results show that four biotin biosynthetic genes bioF, bioB, bioD and bioA in B. subtilis can express in E. coli, but the expression vector of bioW can not competent the bioC gene defect Without supplement of PADK. Inducing of IPTG under high level, the growth of host strains of pEXT20bioWAFD is inhibited. The growth inhibition results from the bioW gene but not from the overexpression of bioB.
本研究成功地克隆了枯草杆菌生物素操纵子基因bioB、bioW 和基因簇bioWAFDB片段；以大肠杆菌表达质粒载体pEXT20为骨架，分别构建了bioW、bioB和bioWAFDB诱导型表达载体pEXT20bioW、pEXT20bioB和pEXT20bioWAFDB，可以通过IPTG诱导，在大肠杆菌中进行表达。通过它们与大肠杆菌生物素生物合成基因系列bioF、bioB、bioC、bioD和bioA缺陷株R874、R875、R876、R877和R879的遗传互补实验，证明：枯草杆菌生物素基因bioA、bioB、bioD和 bioF在大肠杆菌中功能正常； bioW基因（编码庚二酰CoA合成酶）的表达载体对大肠杆菌缺陷株没有遗传互补作用， 但是bioW基因编码的庚二酰CoA合成酶在大肠杆菌中有催化活性，可以催化外源的庚二酸合成庚二酰CoA，为生物素合成途径提供底物。经过IPTG不同水平的诱导表达实验，发现：枯草杆菌生物素基因bioWAFDB的过量表达对受体菌的生长有严重抑制作用，该抑制作用主要是由bioW引起，并且不能通过添加二氨基庚二酸而解除。枯草杆菌bioB基因诱导表达产物未对大肠杆菌的生长造成抑制，实验结果为枯草杆菌生物素基因工程菌的构建及生物素发酵生产提供了有价值的理论依据。
Two DNA fragments, which are about 700bps and contain ori and rep of E.coli, were cloned from pUC19 using two pairs of primers respectively. Three E.coli-B.subtilis shuttle vectors pGDVM，pGDVM1 and pGDVM2whith chloramphenicol resistance, were then constructed with the framework pGDV1 , a plasmid come from B.subtilis strain 1E60. It indicates that the cloned 700 bp DNA fragments contains ori and rep of E.coli have a complete ability of replication, and the three shuttle vectors can inherit and clone steadily via continuous culture and times of shuttle experiments between E. coli and B. subtilis, This series of shuttle vectors based on plasmid pGDV1 , are numerous to 150-200 copies in B. subtilis . The length of vectors are only about 3kb ,they contain multiple clone site and chloramphenicol resistance gene,can act as ideal framework for B.subtis genetic engineering.
Biotin operon genes bioB,bioW and gene cluster bioWAFDB in B. subtilis have been cloned and inserted into the downstream of Ptac promoter of E.coli expression vector pEXT20, and yield induced expression vectors —pEXT20bioW, pEXT20bioB and pEXT20bioWAFDB . Five E. coli mutant strains, R874,R875,R876,R877 and R879 with defect of bioF, bioB, bioC,bioD and bioA respectively, were used for genetic complement analysis of biotin genes. The results show that four biotin biosynthetic genes bioF, bioB, bioD and bioA in B. subtilis can express in E. coli, but the expression vector of bioW can not competent the bioC gene defect Without supplement of PADK. Inducing of IPTG under high level, the growth of host strains of pEXT20bioWAFD is inhibited. The growth inhibition results from the bioW gene but not from the overexpression of bioB.
引文
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