摘要
采用同源重组法高效构建枯草芽孢杆菌转酮酶(tkt)缺失突变株。以大肠杆菌(E.coliDH5α)质粒pBlUSKM为框架,构建出基于枯草杆菌(B.S104)tkt基因位点的整合载体pb-Trs-n,将此载体重组到Bacillus subtilis104中,从新霉素抗性平板上挑取转化子,整合载体pb-Trs-n的测序结果与Kunst.F报道的tkt基因高度同源(98.9%)。同源重组后,B.S104染色体上的tkt基因(2 004bp)部分与载体pb-Trs-n的neo基因(1 197bp)发生了同源交换,确定了该转化子为枯草芽孢杆菌转酮酶缺失突变株(tkt-,neo),该方法构建枯草芽孢杆菌转酮酶(tkt)缺失突变株是可行的,为D-核糖工程菌的研究奠定了基础。
The Bacillus subtilis tkt Mutant was constructed by homologous recombination in bacteria.The delivery vector pb- Trs- n which can be integrated into tkt locus within the B. subtilis chromosome by plastid of the E. coli DH5α.The vector was recombined to B. S 104 and transformants was selected by neomycin resistance medium. And the delivery vector pb- Trs - n was identified high identity（98.9% ）with tkt gene by reported of Kunst. F. And the part of tkt homologous gene was exchanged by neo gene. The recombinant gene was verified by genome PCR identlfication, and validated as a B. subtilis（ tkt - , neo）. It＇ s a offers method to acquire B. subtilis（tkt） mutant for the D - ribose in research and production.
出处
《生物技术》
CAS
CSCD
2006年第6期23-26,共4页
Biotechnology
基金
湖北省工业微生物重点实验室开放基金项目资助(No.20040405)
