CAJ | 학술논문

为了验证枯草芽孢杆菌（ Bacillus subtilis）中ClpQY基因的功能，以枯草芽孢杆菌基因组为模板，PCR扩增出ClpQY基因上下游同源序列840 bp和983 bp的片段，并与自杀质粒pMAD载体连接，构建重组质粒pMAD－up－down，运用化学转化法通过枯草芽孢杆菌菌株PY79，采用LOOP IN和LOOP OUT的方法，获得不带抗性，且ClpQY完全敲除的枯草芽孢杆菌3610菌株。产孢试验表明，该基因在芽孢生成中为非必需基因，而在生物膜的形成过程中具有调节作用。高温生长试验显示，该基因对细胞逆境条件下的生长存在一定的调控功能。
위료험증고초아포간균（ Bacillus subtilis）중ClpQY기인적공능，이고초아포간균기인조위모판，PCR확증출ClpQY기인상하유동원서렬840 bp화983 bp적편단，병여자살질립pMAD재체련접，구건중조질립pMAD－up－down，운용화학전화법통과고초아포간균균주PY79，채용LOOP IN화LOOP OUT적방법，획득불대항성，차ClpQY완전고제적고초아포간균3610균주。산포시험표명，해기인재아포생성중위비필수기인，이재생물막적형성과정중구유조절작용。고온생장시험현시，해기인대세포역경조건하적생장존재일정적조공공능。
In order to verify the ClpQY gene in Bacillus subtilis, with Bacillus subtilis genome as template, ClpQY gene upstream and downstream homologous sequences 840 bp and 983 bp fragments were amplified by PCR and connected with the plas?mid pMAD. The recombinant plasmid pMAD?up?down was transformed into Bacillus subtilis strain PY79 competent cells containing the up and down sequences of ClpQY gene without ClpQY and the sequences of pMAD by double exchange of ho?mologous recombination. Then, the genome of PY79 Looped in pMAD?up?down was transformed into Bacillus subtilis strain 3610. The ClpQY gene was deleted completely by efficient homologous recombination because of large fragments of PY79 genome. This deletion strain was scarless knock out and there was no effect of resistance gene and no polarity effect caused by the existence of part sequences of ClpQY gene to the downstream genes, which could objectively reflect with the function of the gene. There was little effect on sporulation. The heat stress experiment showed that deletion strain had higher growth than wild type and ClpQY gene might regulate transcript factors in maintaining cellular stability.