具群体感应抑制作用短小芽孢杆菌F3-1菌株的诱变育种  

Mutation breeding of Bacillus pumilus strain F3-1 with quorum sensing inhibitory effects

作　　者：彭孟凡 范斌[1,2] 张庆华[1,3] 宋增福[1,2] PENG Meng-fan;FAN Bin;ZHANG Qing-hua;SONG Zeng-fu(National Demonstration Center for Experimental Fisheries Science Education(Shanghai Ocean University),Shanghai 201306,China;National Pathogen Collection for Aquatic Animals(Shanghai Ocean University),Shanghai 201306,China;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education(Shanghai Ocean University),Shanghai 201306,China)

机构地区：[1]水产科学国家级实验教学示范中心(上海海洋大学),上海201306 [2]国家水生动物病原库(上海海洋大学),上海201306 [3]水产种质资源发掘与利用教育部重点实验室(上海海洋大学),上海201306

出　　处：《南方农业学报》2018年第7期1423-1431,共9页Journal of Southern Agriculture

基　　金：上海市协同创新中心基金项目(ZF1206);南京创业人才321计划项目(D-8005-14-0005)

摘　　要：【目的】获得群体感应(QS)信号分子降解能力更强的突变短小芽孢杆菌(Bacillus pumilus),增强其在水产细菌性病害防治中的效果,为水产养殖业利用细菌QS淬灭机制防治细菌性疾病提供借鉴。【方法】以具有降解QS信号分子特性的短小芽孢杆菌F3-1为出发菌株、紫外线和亚硝酸钠(Na NO2)为诱变剂进行诱变选育;以紫色杆菌(Chromobacteria violaceum ATCC12472)为报告菌株筛选正向突变,经连续传代25代后测定其遗传稳定性;采用单因素试验测定培养时间、p H、盐度和温度对突变菌株降解QS信号分子能力的影响,同时通过扩增QS抑制基因aii A及SDS-PAGE分析验证突变菌株的蛋白表达情况。【结果】两种诱变方法共获得205株突变菌株,以紫色杆菌为报告菌株通过紫外诱变筛选出具有QS抑制作用的正向突变株4株,编号分别为FF1-2、FF3-2、FF3-3和FF4-1。其中,突变菌株FF1-2的蛋白产量达47.48±2.87μg/m L,约是出发菌株F3-1的3.12倍,对紫色杆菌的褪色圈直径是出发菌株F3-1的2.96倍,抑制紫色素的能力较出发菌株F3-1提高64%,说明该突变菌株具有很强的QS抑制能力;连续传代25代后,突变菌株FF1-2的产蛋白能力及对紫色素的抑制作用无显著变化(P>0.05)。突变菌株FF1-2在培养时间为40 h、温度30℃、盐度0.5%、p H 7.0时,紫色素褪色效果最佳。从出发菌株F3-1和突变菌株FF1-2中均能扩增获得753 bp的aii A基因,且通过SDS-PAGE分析验证二者在28 k D处均呈现出特异条带。【结论】采用诱变育种技术筛选得到的突变菌株FF1-2能显著提高QS抑制能力,且该抑制能力能稳定遗传,即通过诱变育种技术提高短小芽孢杆菌的产酶量及酶活力具有可行性。【Objective】Mutant Bacillus pumilus with stronger quorum sensing(QS)signal molecule degradation was obtained,and its effects in prevention and treatment of bacterial diseases in aquatic products were enhanced,which could provide reference for aquaculture using QS quenching mechanism for prevention of bacterial diseases.【Method】B.pumilus F3-1 with characters of QS quenching signal molecular was used as the original strain for mutation breeding by ultraviolet and sodium nitrite(NaNO2)as mutagen.The forward mutation was screened by Chromobacteria violaceum ATCC12472 as a reporter strain,and its genetic stability was determined after serial passage for 25 generations.Single factor test was used to determine the effects of culture time,pH,salinity and temperature of ultraviolet mutagenesis on the ability of mutant strains to degrade QS signaling molecules.At the same time,the protein expression of mutant strains was verified by amplification of QS inhibitor gene aiiA and SDS-PAGE analysis.【Result】A total of 205 mutant strains were ob tained by two mutagenesis methods.Four positive mutant strains with QS inhibition were screened by C.violaceum as reporter strains,numbered FF1-2,FF3-2,FF3-3 and FF4-1.Among them,the protein yield of mutant strain FF1-2 was 47.48±2.87μg/mL,which was as 3.12 times as that of the original strain F3-1,and the diameter of the fading circle of C.violaceum was as 2.96 times as that of the original strain F3-1.The ability to inhibit violacein increased by 64%than that of the original strain F3-1,indicating that the mutant strain had strong QS inhibition ability.After 25 inoculations,the protein yield and the inhibition to violacein of the mutant strain FF1-2 was not significantly different(P>0.05).When mutant strain FF1-2 was cultured under the following conditions:culture time 40 h,temperature 30℃,salinity 0.5%,pH 7.0,the fading effects of violacein were the best.A 753 bp gene aiiA was amplified from both the original strain F3-1 and the mutant strain FF1-2,and it was confirmed b

关 键 词：短小芽孢杆菌 群体感应(QS) 诱变育种 AIIA基因 紫色杆菌 

分 类 号：S942.3[农业科学—水产养殖]