解淀粉芽孢杆菌PEBA20群体感应基因簇的克隆及相关特性研究
发布时间：2018-08-04 10:04
【摘要】：本研究对解淀粉芽孢杆菌PEBA20菌株的群体感应相关基因进行基因克隆并作出生物信息学分析,以期了解其基因结构特征;利用同源重组法构建了comA基因缺失突变株进而验证该基因及群体感应系统对生防活性、生物膜形成的影响。主要研究结果如下:1.从Bacillus amyloliquefaciens PEBA20中克隆了群体感应系统基因comQXPA,通过序列比对分析发现,comA基因的保守性是最高的,其次是comP,comX保守性最低。对蛋白功能进行预测发现预期功能相适应的结构。2.构建了comA基因缺失突变株,comA基因同源前臂和同源后臂的连接质粒comAF-comAB与自杀质粒pk18mobsacB经酶切、T4连接酶连接得到重组质粒comAFcomAB-pk18mobsacB。将构建得到的重组质粒经电转化方法转化入B.amyloliquefaciens PEBA20感受态细胞中,采用卡那筛选法及继代培养大量筛选获得comA基因缺失的PEBA20突变株。以缺失株DNA为模板进行PCR筛选阳性克隆,并通过测序进一步确定获得comA基因缺失突变株。3.对comA基因缺失突变株的生物膜表型进行检测,发现固气界面comA基因缺失突变株在NB平板上初期为白色平滑圆形的小菌落,表面无褶皱。之后菌落外围呈放射状,内部呈现一个圆圈,圆圈边缘界限明显呈白色;随着时间的推移菌落边缘扩大速度明显高于内部小圆圈的速度;后期菌落不再扩大,菌落边缘分化程度明显,白色边缘与透明边缘交替出现;气液界面12h后已出现浑浊,至24h时已形成一层平整的薄薄的生物膜,36h生物膜已出现破裂降解,生物膜不完整,底部出现菌体沉淀;随着时间的推移生物膜逐渐降解完全菌体在底部沉淀。表明comA基因影响了生物膜形成及其复杂程度。4.检测B.amyloliquefaciens PEBA20野生株和comA基因缺失突变株对四种真菌的抑菌活性。结果显示,与野生株相比,comA基因缺失突变株对四种指示真菌抑菌效果均下降。这表明comA基因的缺失突变影响了生防细菌对指示真菌的抑菌活性作用。
[Abstract]:In this study, the genes related to population induction of Bacillus amylolyticus PEBA20 strain were cloned and bioinformatics analysis was made in order to understand the structural characteristics of the genes. The comA gene deletion mutant was constructed by homologous recombination method to verify the effect of the gene and the population sensing system on biocontrol activity and biofilm formation. The main results are as follows: 1. The population sensing system gene comQXPA was cloned from Bacillus amyloliquefaciens PEBA20, and the sequence alignment analysis showed that the conserved character of comcomA gene was the highest, followed by the lowest conservation of comcomX gene. The function of protein was predicted and the structure of expected function was found. 2. 2. The ligation plasmid comAF-comAB of the homologous forearm and hind arm of the comA gene deletion mutant was constructed and ligated with the suicide plasmid pk18mobsacB to obtain the recombinant plasmid comAFcomAB-pk18mobsacB. The constructed recombinant plasmid was transformed into B.amyloliquefaciens PEBA20 receptive cells by electrotransformation. A large number of PEBA20 mutants with comA gene deletion were obtained by Karna screening and subculture. The deletion strain DNA was used as template to screen the positive clones of PCR, and the comA gene deletion mutant. 3 was further confirmed by sequencing. The biofilm phenotype of the comA gene deletion mutant was detected. It was found that the comA gene deletion mutant on the solid gas interface was a white, smooth, circular colony at the initial stage of the NB plate and had no fold on the surface. The outer circle of the colony was radial, the inner circle appeared a circle, the boundary of the circle was obviously white; over time, the expanding speed of the colony edge was obviously higher than that of the inner small circle; the later colony no longer expanded. The differentiation degree of colony edge was obvious, the white edge and transparent edge appeared alternately, the gas-liquid interface appeared turbidity after 12 hours, and by 24 hours, a thin and flat biofilm had been formed, and the biofilm had broken down and degraded, and the biofilm was incomplete. At the bottom, the bacteria were precipitated, and the biofilm gradually degraded at the bottom. The results showed that comA gene affected biofilm formation and its complexity. The antimicrobial activity of B.amyloliquefaciens PEBA20 wild strain and comA gene deletion mutant against four fungi was detected. The results showed that compared with wild strains, the inhibitory effect of the mutant on four indicator fungi was decreased. This indicated that the deletion of comA gene affected the bacteriostatic activity of biocontrol bacteria against indicator fungi.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q78;S476.1
本文编号：2163531
[Abstract]:In this study, the genes related to population induction of Bacillus amylolyticus PEBA20 strain were cloned and bioinformatics analysis was made in order to understand the structural characteristics of the genes. The comA gene deletion mutant was constructed by homologous recombination method to verify the effect of the gene and the population sensing system on biocontrol activity and biofilm formation. The main results are as follows: 1. The population sensing system gene comQXPA was cloned from Bacillus amyloliquefaciens PEBA20, and the sequence alignment analysis showed that the conserved character of comcomA gene was the highest, followed by the lowest conservation of comcomX gene. The function of protein was predicted and the structure of expected function was found. 2. 2. The ligation plasmid comAF-comAB of the homologous forearm and hind arm of the comA gene deletion mutant was constructed and ligated with the suicide plasmid pk18mobsacB to obtain the recombinant plasmid comAFcomAB-pk18mobsacB. The constructed recombinant plasmid was transformed into B.amyloliquefaciens PEBA20 receptive cells by electrotransformation. A large number of PEBA20 mutants with comA gene deletion were obtained by Karna screening and subculture. The deletion strain DNA was used as template to screen the positive clones of PCR, and the comA gene deletion mutant. 3 was further confirmed by sequencing. The biofilm phenotype of the comA gene deletion mutant was detected. It was found that the comA gene deletion mutant on the solid gas interface was a white, smooth, circular colony at the initial stage of the NB plate and had no fold on the surface. The outer circle of the colony was radial, the inner circle appeared a circle, the boundary of the circle was obviously white; over time, the expanding speed of the colony edge was obviously higher than that of the inner small circle; the later colony no longer expanded. The differentiation degree of colony edge was obvious, the white edge and transparent edge appeared alternately, the gas-liquid interface appeared turbidity after 12 hours, and by 24 hours, a thin and flat biofilm had been formed, and the biofilm had broken down and degraded, and the biofilm was incomplete. At the bottom, the bacteria were precipitated, and the biofilm gradually degraded at the bottom. The results showed that comA gene affected biofilm formation and its complexity. The antimicrobial activity of B.amyloliquefaciens PEBA20 wild strain and comA gene deletion mutant against four fungi was detected. The results showed that compared with wild strains, the inhibitory effect of the mutant on four indicator fungi was decreased. This indicated that the deletion of comA gene affected the bacteriostatic activity of biocontrol bacteria against indicator fungi.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q78;S476.1
【共引文献】
相关期刊论文 前1条
1 贾翠丽;张华伟;王斌斌;朱宏吉;乔建军;;革兰氏阳性菌自然感受态生理机制的研究进展[J];中国生物工程杂志;2015年06期
，本文编号：2163531
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