生物膜形成相關(guān)基因gltB、serA和fliZ對(duì)枯草芽孢桿菌Bs916多細(xì)胞行為和防效的調(diào)控
發(fā)布時(shí)間：2018-08-05 18:33
【摘要】：枯草芽孢桿菌作為一種重要的生防細(xì)菌資源,能夠分泌多種抗真菌的脂肽類(lèi)抗生素,被廣泛用于多種植物病害的生物防治。近年來(lái),細(xì)菌生物膜在防治植物病害中的重要作用被逐步認(rèn)識(shí),其精細(xì)調(diào)控網(wǎng)絡(luò)逐步清晰,其多細(xì)胞行為也逐步被研究。為進(jìn)一步闡述枯草芽孢桿菌Bs916生物膜在防病過(guò)程的重要作用與調(diào)控機(jī)制,在Bs916全基因組測(cè)序精細(xì)圖的前提下進(jìn)行如下研究:1、構(gòu)建了 Bs916轉(zhuǎn)座子隨機(jī)插入突變株庫(kù),篩選對(duì)水稻細(xì)菌性條斑病菌抑菌能力發(fā)生顯著改變的突變株并克隆插入位點(diǎn)的基因,獲得Bs916與抗細(xì)菌活性相關(guān)基因。以Bs916生物膜形成表型作為對(duì)照,從抑制水稻細(xì)菌性條斑病菌能力發(fā)生顯著變化突變株中篩選生物膜形成缺陷的突變株。PCR和Southern Blot檢測(cè)表明Bs916隨機(jī)插入突變株庫(kù)被成功構(gòu)建,85%突變株以轉(zhuǎn)座子單拷貝形式插入。篩選到30株對(duì)水稻細(xì)菌性條斑病菌抑制能力發(fā)生顯著改變的突變株,克隆到21個(gè)插入位點(diǎn)基因序列并測(cè)序分析,生物信息學(xué)分析結(jié)果顯示這些基因與感受態(tài)形成、鞭毛運(yùn)動(dòng)和抗菌次生代謝產(chǎn)物的合成相關(guān)。從抑菌能力變化的30株突變株中篩選出10株生物膜形成具有缺陷的突變株,進(jìn)一步篩選得到3株生物膜形成具有嚴(yán)重缺陷突變株△gltB、△fliZ和△serA,為深入研究Bs916生物膜形成及調(diào)控機(jī)制提供試驗(yàn)材料。2、分別檢測(cè)了 Bs916及3種脂肽類(lèi)抗生素單敲除突變株(△bac、△srf和△fen)分泌桿菌霉素L、表面活性素和泛革素產(chǎn)量。以水稻紋枯病菌Rhizoctonia solani為靶標(biāo)菌株,Bs916為對(duì)照菌株,確認(rèn)3種脂肽類(lèi)抗生素對(duì)R.solani的抑制作用。試驗(yàn)結(jié)果確認(rèn)Bs916能夠分泌3種脂肽類(lèi)抗生素桿菌霉素L、表面活性素和泛革素;Bs916喪失桿菌霉素L的突變株△bac抑菌能力顯著下降,同時(shí)喪失桿菌霉素L和表面活性素的突變菌株△srf基本喪失了對(duì)R.solani的抑制作用,喪失泛革素的突變株△fen對(duì)R.solani仍具有一定的抑制作用。研究結(jié)果確認(rèn)了 3種脂肽抗生素對(duì)水稻紋枯病菌R.solani的抑制作用,其中桿菌霉素L起主要作用;同時(shí)發(fā)現(xiàn)Bs916缺失表達(dá)srfAA后,喪失了表面活性素和桿菌霉素L的合成能力。3、構(gòu)建了 3個(gè)生物膜調(diào)控相關(guān)基因gltB、fliZ和serA的定向敲除突變株△gltB、△fliZ和△serA。檢測(cè)了 3個(gè)突變株和Bs916的生物膜形成能力和對(duì)水稻紋枯病的防治效果。試驗(yàn)結(jié)果顯示,3個(gè)突變株的生物膜形成有嚴(yán)重缺陷,僅具有平面結(jié)構(gòu),而對(duì)照菌株Bs916能夠形成完整的三維結(jié)構(gòu)的生物膜;3個(gè)突變株△gltB、△fliZ和△serA對(duì)水稻紋枯病的防治效果和Bs916相比均顯著下降。此外,Bs916在被水稻紋枯病菌侵染的傷口部位呈現(xiàn)出明顯的群集效應(yīng),綠色熒光明顯。3個(gè)突變株均無(wú)明顯的群集效應(yīng),菌體數(shù)量不足,菌落分布無(wú)規(guī)律。試驗(yàn)結(jié)果表明:3個(gè)生物膜調(diào)控相關(guān)基因?qū)s916的多細(xì)胞行為具有顯著的調(diào)控作用。4、檢測(cè)了 3個(gè)突變株△gltB、△fliZ和△serA和Bs916中3種脂肽類(lèi)抗生素桿菌霉素L、表面活性素和泛革素的產(chǎn)量。與Bs916相比,△gltB不分泌桿菌霉素L和泛革素,表面活性素產(chǎn)量提高了 5倍,△fliZ和AserA中3種脂肽類(lèi)抗生素產(chǎn)量變化不明顯。初步認(rèn)為可能是突變株△gltB由于桿菌霉素L和泛革素的缺失、生物膜形成缺陷導(dǎo)致定殖能力減弱,共同造成對(duì)水稻紋枯病防效下降的結(jié)果;突變株△fliZ和AserA由于生物膜形成缺陷導(dǎo)致定殖能力減弱,造成對(duì)水稻紋枯病防效下降。5、研究了gltB調(diào)控Bs916生物膜形成的作用機(jī)理。根據(jù)RT-PCR分析發(fā)現(xiàn)突變株△gltB中聚谷氨酸合成的必須基因capB (ysC)轉(zhuǎn)錄水平下調(diào)5倍,檢測(cè)了 Bs916和△gltB生物膜形成過(guò)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顯著降低。通過(guò)外源添加聚谷氨酸恢復(fù)△gltB生物膜形成結(jié)果顯示添加終濃度為10g/L的聚谷氨酸可以使△gltB的生物膜形成部分恢復(fù);添加終濃度為20g/L的聚谷氨酸時(shí),生物膜形成基本恢復(fù)。Bs916分別添加終濃度是10g/L和20g/L的聚谷氨酸,其生物膜形成得到增強(qiáng)。在Bs916和AgltB中分別添加終濃度為30g/L的聚谷氨酸時(shí),由于濃度過(guò)高抑制其生物膜的形成。通過(guò)菌體相鄰培養(yǎng)證明Bs916分泌的聚谷氨酸能夠恢復(fù)AgltB的生物膜形成。gltB通過(guò)調(diào)控谷氨酸形成聚谷氨酸的途徑參與調(diào)控Bs916的生物膜形成過(guò)程。6、對(duì)突變株△gltB、△fliZ和△serA和Bs916進(jìn)行轉(zhuǎn)錄組測(cè)序,對(duì)差異表達(dá)基因和代謝途徑進(jìn)行分析。差異表達(dá)基因結(jié)果顯示:△gltB、△fliZ和AserA和Bs916比較,差異基因數(shù)量相差顯著,其中△fliZ上下調(diào)基因數(shù)目差異最顯著。差異基因GO分析結(jié)果顯示:3個(gè)突變株和Bs916相比,發(fā)生變化的二級(jí)子目錄分類(lèi)蛋白主要是細(xì)胞過(guò)程、細(xì)胞和細(xì)胞組分。差異基因KEGG富集分析結(jié)果顯示:3個(gè)突變株和Bs916相比,發(fā)生變化的代謝過(guò)程主要是氨基酸代謝、碳水化合物代謝和膜運(yùn)輸過(guò)程,均與Bs916生物膜形成及能量代謝和物質(zhì)轉(zhuǎn)運(yùn)相關(guān)。進(jìn)一步富集在前3個(gè)共有過(guò)程部分主要基因結(jié)果顯示:yfmT、yhfS和alsS基因均同時(shí)參加兩個(gè)重要的代謝過(guò)程,且在同一個(gè)過(guò)程由不同基因引起的導(dǎo)致生物膜減弱時(shí)的表達(dá)趨勢(shì)明顯不同,預(yù)測(cè)上述3個(gè)基因在Bs916的生物膜調(diào)控網(wǎng)絡(luò)中具有重要的調(diào)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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S476.1
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