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为明确群体感应系统在枯草芽胞杆菌NCD-2菌株抑菌活性和生物膜形成中的作用,通过同源重组技术对群体感应系统中的comA基因进行定位缺失突变,分别比较了NCD-2菌株和comA基因突变子在胞外酶合成、抑菌活性、脂肽抗生素(丰产素)产生以及生物膜形成上的差异,并进一步利用实时荧光定量RT-PCR技术比较了二者生物膜基质编码基因epsA和tasA的表达情况。结果表明,通过同源重组技术获得了comA基因突变子MA-4,同菌株NCD-2相比,其在胞外蛋白酶和纤维素酶的合成能力、对番茄灰霉菌Botrytis cinerea的抑菌活性、丰产素的合成量和生物膜的形成能力上均明显下降;生物膜基质编码基因tasA的表达量下降了70%,而epsA的表达量变化不明显。表明ComA是NCD-2菌株脂肽抗生素和生物膜形成中的重要调控因子。
In order to clarify the role of population-sensing system in the bacteriostatic activity and biofilm formation of Bacillus subtilis NCD-2, the comA gene in population-sensing system was subjected to site-directed mutagenesis by homologous recombination technology. NCD-2 strain and The results showed that the comA gene mutations in the extracellular enzyme synthesis, antibacterial activity, lipopeptide antibiotics (production) and biofilm formation differences, and further use of real-time fluorescent quantitative RT-PCR technology compared the biofilm matrix coding genes epsA And tasA expression. The results showed that the comA gene mutant MA-4 was obtained by homologous recombination technology, its ability of synthesizing extracellular protease and cellulase, its antibacterial activity against Botrytis cinerea, The amount of biosynthesis of biotin and the formation of biofilm decreased significantly. The expression level of tasA, a gene encoding biofilm, decreased by 70% while the expression of epsA did not change significantly. This indicates that ComA is an important regulator of lipopeptide antibiotic and biofilm formation in NCD-2 strain.