生物膜形成相关基因gltB、serA和fliZ对枯草芽孢杆菌Bs916多细胞行为和防效的调控
发布时间：2018-08-05 18:33
【摘要】：枯草芽孢杆菌作为一种重要的生防细菌资源,能够分泌多种抗真菌的脂肽类抗生素,被广泛用于多种植物病害的生物防治。近年来,细菌生物膜在防治植物病害中的重要作用被逐步认识,其精细调控网络逐步清晰,其多细胞行为也逐步被研究。为进一步阐述枯草芽孢杆菌Bs916生物膜在防病过程的重要作用与调控机制,在Bs916全基因组测序精细图的前提下进行如下研究:1、构建了 Bs916转座子随机插入突变株库,筛选对水稻细菌性条斑病菌抑菌能力发生显著改变的突变株并克隆插入位点的基因,获得Bs916与抗细菌活性相关基因。以Bs916生物膜形成表型作为对照,从抑制水稻细菌性条斑病菌能力发生显著变化突变株中筛选生物膜形成缺陷的突变株。PCR和Southern Blot检测表明Bs916随机插入突变株库被成功构建,85%突变株以转座子单拷贝形式插入。筛选到30株对水稻细菌性条斑病菌抑制能力发生显著改变的突变株,克隆到21个插入位点基因序列并测序分析,生物信息学分析结果显示这些基因与感受态形成、鞭毛运动和抗菌次生代谢产物的合成相关。从抑菌能力变化的30株突变株中筛选出10株生物膜形成具有缺陷的突变株,进一步筛选得到3株生物膜形成具有严重缺陷突变株△gltB、△fliZ和△serA,为深入研究Bs916生物膜形成及调控机制提供试验材料。2、分别检测了 Bs916及3种脂肽类抗生素单敲除突变株(△bac、△srf和△fen)分泌杆菌霉素L、表面活性素和泛革素产量。以水稻纹枯病菌Rhizoctonia solani为靶标菌株,Bs916为对照菌株,确认3种脂肽类抗生素对R.solani的抑制作用。试验结果确认Bs916能够分泌3种脂肽类抗生素杆菌霉素L、表面活性素和泛革素;Bs916丧失杆菌霉素L的突变株△bac抑菌能力显著下降,同时丧失杆菌霉素L和表面活性素的突变菌株△srf基本丧失了对R.solani的抑制作用,丧失泛革素的突变株△fen对R.solani仍具有一定的抑制作用。研究结果确认了 3种脂肽抗生素对水稻纹枯病菌R.solani的抑制作用,其中杆菌霉素L起主要作用;同时发现Bs916缺失表达srfAA后,丧失了表面活性素和杆菌霉素L的合成能力。3、构建了 3个生物膜调控相关基因gltB、fliZ和serA的定向敲除突变株△gltB、△fliZ和△serA。检测了 3个突变株和Bs916的生物膜形成能力和对水稻纹枯病的防治效果。试验结果显示,3个突变株的生物膜形成有严重缺陷,仅具有平面结构,而对照菌株Bs916能够形成完整的三维结构的生物膜;3个突变株△gltB、△fliZ和△serA对水稻纹枯病的防治效果和Bs916相比均显著下降。此外,Bs916在被水稻纹枯病菌侵染的伤口部位呈现出明显的群集效应,绿色荧光明显。3个突变株均无明显的群集效应,菌体数量不足,菌落分布无规律。试验结果表明:3个生物膜调控相关基因对Bs916的多细胞行为具有显著的调控作用。4、检测了 3个突变株△gltB、△fliZ和△serA和Bs916中3种脂肽类抗生素杆菌霉素L、表面活性素和泛革素的产量。与Bs916相比,△gltB不分泌杆菌霉素L和泛革素,表面活性素产量提高了 5倍,△fliZ和AserA中3种脂肽类抗生素产量变化不明显。初步认为可能是突变株△gltB由于杆菌霉素L和泛革素的缺失、生物膜形成缺陷导致定殖能力减弱,共同造成对水稻纹枯病防效下降的结果;突变株△fliZ和AserA由于生物膜形成缺陷导致定殖能力减弱,造成对水稻纹枯病防效下降。5、研究了gltB调控Bs916生物膜形成的作用机理。根据RT-PCR分析发现突变株△gltB中聚谷氨酸合成的必须基因capB (ysC)转录水平下调5倍,检测了 Bs916和△gltB生物膜形成过程中谷氨酸利用能力和聚谷氨酸合成能力。AgltB和Bs916相比利用谷氨酸形成聚谷氨酸的能力显著降低。通过外源添加聚谷氨酸恢复△gltB生物膜形成结果显示添加终浓度为10g/L的聚谷氨酸可以使△gltB的生物膜形成部分恢复;添加终浓度为20g/L的聚谷氨酸时,生物膜形成基本恢复。Bs916分别添加终浓度是10g/L和20g/L的聚谷氨酸,其生物膜形成得到增强。在Bs916和AgltB中分别添加终浓度为30g/L的聚谷氨酸时,由于浓度过高抑制其生物膜的形成。通过菌体相邻培养证明Bs916分泌的聚谷氨酸能够恢复AgltB的生物膜形成。gltB通过调控谷氨酸形成聚谷氨酸的途径参与调控Bs916的生物膜形成过程。6、对突变株△gltB、△fliZ和△serA和Bs916进行转录组测序,对差异表达基因和代谢途径进行分析。差异表达基因结果显示:△gltB、△fliZ和AserA和Bs916比较,差异基因数量相差显著,其中△fliZ上下调基因数目差异最显著。差异基因GO分析结果显示:3个突变株和Bs916相比,发生变化的二级子目录分类蛋白主要是细胞过程、细胞和细胞组分。差异基因KEGG富集分析结果显示:3个突变株和Bs916相比,发生变化的代谢过程主要是氨基酸代谢、碳水化合物代谢和膜运输过程,均与Bs916生物膜形成及能量代谢和物质转运相关。进一步富集在前3个共有过程部分主要基因结果显示:yfmT、yhfS和alsS基因均同时参加两个重要的代谢过程,且在同一个过程由不同基因引起的导致生物膜减弱时的表达趋势明显不同,预测上述3个基因在Bs916的生物膜调控网络中具有重要的调控作用。
[Abstract]:Bacillus subtilis, as an important Biocontrol Bacterial resource, can secrete a variety of anti fungal lipopeptide antibiotics and is widely used in the biological control of various plant diseases. In recent years, the important role of bacterial biofilm in plant disease prevention is gradually recognized. Its fine regulation network is gradually clear, and its multicellular behavior is gradually developed. In order to further elaborate the important role and regulation mechanism of Bacillus subtilis Bs916 biofilm in the disease control process, the following study was carried out on the premise of the fine mapping of Bs916 whole genome sequencing: 1, a random insert mutant library of Bs916 transposon was constructed to screen the mutation of bacteriostasis ability of bacterial leaf spot pathogen of rice. Bs916 and anti bacterial activity related genes were obtained by cloning the gene of the insertion site. The phenotype of Bs916 biofilm formation was used as the control. The mutant strain.PCR and Southern Blot screening of the biofilm formation defects were screened from the significant changes in the mutant strain of the bacterial leaf spot pathogen of rice. The results showed that the random insertion mutant library of Bs916 was successful. The 85% mutant was inserted in the form of a single copy of the transposon. 30 mutant strains that significantly changed the inhibition ability of the bacterial spot pathogen of rice were screened and cloned to 21 insertion sites and sequenced. The bioinformatics analysis showed that these genes were formed with the sensory state, flagellum movement and secondary metabolites. From 30 mutant strains of bacteriostasis, 10 biofilms were screened to form defective mutants, and 3 biofilms were screened to form serious defective mutants, Delta gltB, Delta fliZ and delta serA, which provided an experimental material.2 for in-depth study of the formation and regulation mechanism of Bs916 biofilm, and respectively detected Bs916 and 3 lipopeptide antibiotics single knockout mutant strains (delta BAC, delta SRF and delta Fen) secreted mycophencin L, surfactants and pan gram yield. Rhizoctonia solani of Rhizoctonia Solanum was used as the target strain and Bs916 as the control strain. The inhibitory effect of 3 lipopeptides on R.solani was confirmed. The results confirmed that Bs916 could secrete 3 kinds of lipopeptides. Antibiotic mycophencin L, surfactants and pan gram; Bs916 loss of baccomycin L mutant strain delta BAC significantly decreased, while the loss of Mycobacterium L and surfactants mutant strain delta SRF basically lost the inhibition of R.solani, the loss of Pan gram mutants delta Fen still has a certain inhibitory effect on R.solani. The results confirmed the inhibitory effect of 3 lipoprotein antibiotics on Rice Sheath Blight strain R.solani, in which baccomycin L played a major role. At the same time, it was found that after Bs916 deletion expressed srfAA, the synthesis ability of the surfactant and baccomycin L was lost.3, and a directional knockout mutant of the 3 biofilm regulated genes gltB, fliZ and serA was constructed. Delta gltB, Delta fliZ and delta serA. were used to detect the biofilm formation ability of 3 mutant strains and Bs916 and the control effect on rice sheath blight. The results showed that the biofilms of the 3 mutant strains had serious defects, only a planar structure, while the control strain Bs916 could form a complete three-dimensional structure of the biofilm; 3 mutant strains Delta gltB, delta fli The effect of Z and delta serA on the control of rice sheath blight was significantly lower than that of Bs916. In addition, Bs916 had a distinct cluster effect on the wound sites infected by the rice Rhizoctonia Rhizoctonia, and the green fluorescent obviously.3 mutant had no obvious cluster effect, the number of the bacteria was insufficient and the distribution of the colony was irregular. The results of the experiment showed that 3 biological membranes were adjusted. Control related genes had a significant regulatory effect on the multicellular behavior of Bs916,.4, 3 mutant strains of delta gltB, Delta fliZ and delta serA and Bs916 were detected for the production of 3 kinds of lipopeptide antibiotic mycophenycin L, surfactants and pan gram. Compared with Bs916, Delta gltB non secretocin L and pan gram, the production of surfactants increased by 5 times, Delta The yield changes of 3 kinds of lipopeptide antibiotics in fliZ and AserA were not obvious. It was preliminarily believed that the mutant strain Delta gltB was due to the absence of baccomycin L and pan gram, the biofilm formation defect resulted in the weakening of the colonization ability and the result of the decrease in the prevention of rice sheath blight. The mutant strain Delta fliZ and AserA caused the colonization due to the formation defects of the biofilm. The ability to reduce the resistance to rice sheath blight was reduced by.5, and the mechanism of gltB regulation of Bs916 biofilm formation was studied. According to RT-PCR analysis, the transcriptional level of the essential gene capB (ysC) of the polyglutamic acid synthesis in the mutant Delta gltB was down to 5 times, and the ability of glutamic acid utilization and polyglutamine during the formation of Bs916 and delta gltB raw membrane was detected. The ability of acid synthesis.AgltB and Bs916 to form polyglutamic acid by glutamic acid is significantly lower than that of glutamic acid. The result of the formation of delta gltB biofilm by adding polyglutamic acid shows that polyglutamic acid with a final concentration of 10g/L can make a partial recovery of the biofilm of delta gltB, and the biofilm form of a polyglutamic acid with a final concentration of 20g/L is a biofilm The formation of polyglutamic acid of 10g/L and 20g/L was added to.Bs916, and the formation of biofilm was enhanced. When polyglutamic acid with final concentration of 30g/L was added to Bs916 and AgltB, the formation of the biofilm was inhibited by high concentration. The polyglutamic acid secreted by Bs916 could restore the birth of AgltB by the adjacent culture of the mycelium. The biofilm formation.GltB participates in the regulation of the formation of polyglutamic acid by regulating glutamic acid and participates in the regulation of the biofilm formation of Bs916,.6. The mutant strain Delta gltB, Delta fliZ, Delta serA and Bs916 are sequenced to analyze the differentially expressed genes and metabolic pathways. The differential expression gene results show that Delta gltB, Delta fliZ and AserA and Bs916 are compared. The difference of the number of different genes was significant, and the difference of the number of down regulated genes on the delta fliZ was the most significant. The difference gene GO analysis showed that the 3 mutants and Bs916 were mainly cell processes, cell and cell components. The results of differential gene KEGG enrichment analysis showed that 3 mutant strains were compared with Bs916. The metabolic processes of the biological changes are mainly amino acid metabolism, carbohydrate metabolism and membrane transport process, which are related to the formation of Bs916 biofilm, energy metabolism and material transport. Further enrichment in the first 3 common processes showed that the yfmT, yhfS and alsS genes both participated in the two important metabolic processes at the same time, and in the same one. The expression trend of the biofilm induced by different genes is obviously different. It is important to predict the 3 genes in the biofilm regulation network of Bs916.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S476.1
[Abstract]:Bacillus subtilis, as an important Biocontrol Bacterial resource, can secrete a variety of anti fungal lipopeptide antibiotics and is widely used in the biological control of various plant diseases. In recent years, the important role of bacterial biofilm in plant disease prevention is gradually recognized. Its fine regulation network is gradually clear, and its multicellular behavior is gradually developed. In order to further elaborate the important role and regulation mechanism of Bacillus subtilis Bs916 biofilm in the disease control process, the following study was carried out on the premise of the fine mapping of Bs916 whole genome sequencing: 1, a random insert mutant library of Bs916 transposon was constructed to screen the mutation of bacteriostasis ability of bacterial leaf spot pathogen of rice. Bs916 and anti bacterial activity related genes were obtained by cloning the gene of the insertion site. The phenotype of Bs916 biofilm formation was used as the control. The mutant strain.PCR and Southern Blot screening of the biofilm formation defects were screened from the significant changes in the mutant strain of the bacterial leaf spot pathogen of rice. The results showed that the random insertion mutant library of Bs916 was successful. The 85% mutant was inserted in the form of a single copy of the transposon. 30 mutant strains that significantly changed the inhibition ability of the bacterial spot pathogen of rice were screened and cloned to 21 insertion sites and sequenced. The bioinformatics analysis showed that these genes were formed with the sensory state, flagellum movement and secondary metabolites. From 30 mutant strains of bacteriostasis, 10 biofilms were screened to form defective mutants, and 3 biofilms were screened to form serious defective mutants, Delta gltB, Delta fliZ and delta serA, which provided an experimental material.2 for in-depth study of the formation and regulation mechanism of Bs916 biofilm, and respectively detected Bs916 and 3 lipopeptide antibiotics single knockout mutant strains (delta BAC, delta SRF and delta Fen) secreted mycophencin L, surfactants and pan gram yield. Rhizoctonia solani of Rhizoctonia Solanum was used as the target strain and Bs916 as the control strain. The inhibitory effect of 3 lipopeptides on R.solani was confirmed. The results confirmed that Bs916 could secrete 3 kinds of lipopeptides. Antibiotic mycophencin L, surfactants and pan gram; Bs916 loss of baccomycin L mutant strain delta BAC significantly decreased, while the loss of Mycobacterium L and surfactants mutant strain delta SRF basically lost the inhibition of R.solani, the loss of Pan gram mutants delta Fen still has a certain inhibitory effect on R.solani. The results confirmed the inhibitory effect of 3 lipoprotein antibiotics on Rice Sheath Blight strain R.solani, in which baccomycin L played a major role. At the same time, it was found that after Bs916 deletion expressed srfAA, the synthesis ability of the surfactant and baccomycin L was lost.3, and a directional knockout mutant of the 3 biofilm regulated genes gltB, fliZ and serA was constructed. Delta gltB, Delta fliZ and delta serA. were used to detect the biofilm formation ability of 3 mutant strains and Bs916 and the control effect on rice sheath blight. The results showed that the biofilms of the 3 mutant strains had serious defects, only a planar structure, while the control strain Bs916 could form a complete three-dimensional structure of the biofilm; 3 mutant strains Delta gltB, delta fli The effect of Z and delta serA on the control of rice sheath blight was significantly lower than that of Bs916. In addition, Bs916 had a distinct cluster effect on the wound sites infected by the rice Rhizoctonia Rhizoctonia, and the green fluorescent obviously.3 mutant had no obvious cluster effect, the number of the bacteria was insufficient and the distribution of the colony was irregular. The results of the experiment showed that 3 biological membranes were adjusted. Control related genes had a significant regulatory effect on the multicellular behavior of Bs916,.4, 3 mutant strains of delta gltB, Delta fliZ and delta serA and Bs916 were detected for the production of 3 kinds of lipopeptide antibiotic mycophenycin L, surfactants and pan gram. Compared with Bs916, Delta gltB non secretocin L and pan gram, the production of surfactants increased by 5 times, Delta The yield changes of 3 kinds of lipopeptide antibiotics in fliZ and AserA were not obvious. It was preliminarily believed that the mutant strain Delta gltB was due to the absence of baccomycin L and pan gram, the biofilm formation defect resulted in the weakening of the colonization ability and the result of the decrease in the prevention of rice sheath blight. The mutant strain Delta fliZ and AserA caused the colonization due to the formation defects of the biofilm. The ability to reduce the resistance to rice sheath blight was reduced by.5, and the mechanism of gltB regulation of Bs916 biofilm formation was studied. According to RT-PCR analysis, the transcriptional level of the essential gene capB (ysC) of the polyglutamic acid synthesis in the mutant Delta gltB was down to 5 times, and the ability of glutamic acid utilization and polyglutamine during the formation of Bs916 and delta gltB raw membrane was detected. The ability of acid synthesis.AgltB and Bs916 to form polyglutamic acid by glutamic acid is significantly lower than that of glutamic acid. The result of the formation of delta gltB biofilm by adding polyglutamic acid shows that polyglutamic acid with a final concentration of 10g/L can make a partial recovery of the biofilm of delta gltB, and the biofilm form of a polyglutamic acid with a final concentration of 20g/L is a biofilm The formation of polyglutamic acid of 10g/L and 20g/L was added to.Bs916, and the formation of biofilm was enhanced. When polyglutamic acid with final concentration of 30g/L was added to Bs916 and AgltB, the formation of the biofilm was inhibited by high concentration. The polyglutamic acid secreted by Bs916 could restore the birth of AgltB by the adjacent culture of the mycelium. The biofilm formation.GltB participates in the regulation of the formation of polyglutamic acid by regulating glutamic acid and participates in the regulation of the biofilm formation of Bs916,.6. The mutant strain Delta gltB, Delta fliZ, Delta serA and Bs916 are sequenced to analyze the differentially expressed genes and metabolic pathways. The differential expression gene results show that Delta gltB, Delta fliZ and AserA and Bs916 are compared. The difference of the number of different genes was significant, and the difference of the number of down regulated genes on the delta fliZ was the most significant. The difference gene GO analysis showed that the 3 mutants and Bs916 were mainly cell processes, cell and cell components. The results of differential gene KEGG enrichment analysis showed that 3 mutant strains were compared with Bs916. The metabolic processes of the biological changes are mainly amino acid metabolism, carbohydrate metabolism and membrane transport process, which are related to the formation of Bs916 biofilm, energy metabolism and material transport. Further enrichment in the first 3 common processes showed that the yfmT, yhfS and alsS genes both participated in the two important metabolic processes at the same time, and in the same one. The expression trend of the biofilm induced by different genes is obviously different. It is important to predict the 3 genes in the biofilm regulation network of Bs916.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S476.1
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