【作者】 张琼；

【导师】 喻子牛；

【作者基本信息】 华中农业大学 ， 微生物学， 2001， 博士

【摘要】 本文围绕苏云金芽胞杆菌，从三个方面进行了研究，取得结果如下： 1．苏云金芽胞杆菌自身诱导物抑制蛋白Aii及其对植物病原菌致病性的影响 自身诱导物（autoinducer，以下简称AI）分子是一种广泛存在于革兰氏阴性植物病原菌的细胞间信号分子，与病原菌的致病性有着直接或问接的关系。对AI分子活性具有抑制作用的化学物质或细菌，对病原菌的致病性也将产生影响。 本研究经大量筛选，发现苏云金芽胞杆菌和蜡状芽胞杆菌的培养物可抑制酰基高丝氨酸内酯（acyl-homoserine-lactone，简称AHL）类AI分子的活性，其活性物质是一种蛋白质，称之为Aii蛋白。检测蛋白Aii及其编码基因aff在芽胞杆菌和苏云金芽胞杆菌不同亚种中的分布发现，除苏云金芽胞杆菌和蜡状芽胞杆菌外，枯草芽胞杆菌和球形芽胞杆菌有较弱的Aii活性。用PCR方法也检测到，在上述四种细菌中存在aii基因。而巨大芽胞杆菌的培养物没有Aii活性，Southern杂交结果表明无aii基因的存在。在苏云金芽胞杆菌不同亚种中均可检测到Aii蛋白活性和aii基因。在Southern杂交的基础上，将检测的43个菌株分组，对其中22个菌株的aii基因进行DNA序列测定，结果表明，不同菌株aii基因的DNA序列同源性为85.4％—100％；而Aii蛋白的氨基酸序列同源性为88.1％—100％。 研究中还发现，苏云金芽胞杆菌Aii蛋白是一种非分泌型蛋白。aii基因位于染色体上，在培养初期开始转录，一直延续到芽胞形成前期。该蛋白不仅可作用于多种提纯的AHL类信号分子，对细菌培养液中的AHL类分子的活性也有抑制作用。以胡萝卜软腐欧文氏菌菌株SCG1为例，当菌株SCG1的细胞浓度OD_600nm<0.55时，经苏云金芽胞杆菌菌株4D1培养物处理后感染马铃薯，在24小时内，可完全抑制软腐病斑，但随着培养时间的延长，其抑制作用逐渐减弱。当SCG1的细胞浓度OD_600nm>0.55时，4D1培养物不能完全抑制病原菌的致病性。由此推测，苏云金芽胞杆菌影响病原菌致病性的原因在于降低了病原菌细胞内AHL类信号分子的浓度，使其达不到激活毒素基因表达的水平。因此，利用Aii蛋白的活性，构建出转基因抗病植物，或提高苏云金芽胞杆菌发酵液Aii蛋白产量，研制出既杀虫又抗病的制剂，可在植物病虫害的防治上开辟出新的途径。 2．绿色荧光蛋白基因在苏云金芽胞杆菌中的表达 绿色荧光蛋白基因是一种容易检测的报告基因，可用于苏云金芽胞杆菌制剂施用后的环境监测，杀虫机理等方面的研究。 本研究利用苏云金芽胞杆菌cry3A启动子和BtⅠ-BtⅡ启动子，分别与gfp基因构成融合基因，以调控加基因在苏云金芽胞布「菌中的表达。将重组质粒pGI了PExp八(含cI，y了A]，]’（，召加融合基因）和pGFPExpB（含Bll-Blljl，。一肺融合基因）分别分入大肠杆菌和苏云金芽胞杆菌后发现，Btl-Btlj启动子不仅在苏云金芽胞杆菌中可驱动加基因表达出较强的绿色荧光，在大肠杆菌中也具有极强的启动墓因表达的能力。而cry了A启动子不能启动加基因在大肠杆菌中表达，在苏云金芽胞丰「菌中启动加基因表达的荧光强度也较弱。ILT一PCR实验从转录水平揭示了c理3A启动子驱动加基因在芽胞形成前期的转录水平较高，而Bll-Blll启动子驱动动，基因在芽胞形成前期和后期表现出两次转录高峰。而荧光检测结果表明，苏云金芽胞杆菌重组菌株分别在营养生长期和芽胞形成前期表现出较强的绿色荧光，表明脚基因在苏云金芽胞杆菌中的表达，还受其它调控系统或蛋白酶活性的影响。 本研究首次获得了表达GFP蛋白的苏云金芽胞杆菌重组菌株，在国内外文献中尚无报道。3.苏云金芽胞杆菌质粒pBMB9741的研究 苏云金芽胞杆菌含有丰富的质粒。由于检测手段的限制，许多质粒的功能和特性尚不清楚。通过克隆质粒分子，并对其进行DNA序列测定，在此基础上分析和研究其携带的基因，既可了解质粒的功能和特性，又也可为苏云金芽胞杆菌构建工程菌提供合适的载体。 本研究在构建苏云金芽胞杆菌质粒克隆载体pBMBI 105的基础上，克隆了菌株YBT一1 520的质粒，获得6.6kb的DNA片段。对其进行了限制性酶切图谱分析。经DNA全序列测定，发现该片段由6578个碱基组成，G+C百分比为32.4%。通过PCR确定该片段是一个完整的质粒分子，命名为pBMB9741。该质粒的DNA序列已被GenBank不11 Genome收录，序列号分别为AF 202532不11 NC 001272。 序列分析表明，该质粒包含两个开放阅读框架:口lfj（IO44bPs）和。嘴（1212bPs）。经BLAsT搜索，口rfI编码的蛋白质ORFI与许多细菌编码的复制蛋白同源。通过亚克隆和DNA缺失实验，确定orf]包含在与复制功能有关的2.2kb区段内，因此推断ol:fj基因编码的是该质粒的复制蛋白。BLAsT搜索结果表明，0嘴编码的蛋白质与一些细菌的重组蛋白或诱动蛋白同源。 对质粒pBMB9741在苏云金芽胞杆菌野生型菌株，以及不同亚种无质粒突变菌株和蜡状芽胞杆菌中的遗传稳定性进行了检测，发现该质粒在无抗性选择压力下，既使经过200个世代，仍可稳定遗传。表明该质粒经改造后，可用作苏云金芽胞杆菌构建工程菌的载体。更多还原

【Abstract】 Three kinds of works were described in this thesis with Bacillus thuringiensis as major material.1. Autoinducer inactivity protein Aii in B. thuringiensis strains and its effect on pathogenicity of plant-pathogen bacteriaAutoinducer, especially AHL molecule, is a kind of cell-to-cell communication molecule that has extensive distribution in gram-negative plant pathogen bacteria. It has direct or indirect relationship with the pathogenicity. A chemical or bacterium that inhibits the activity of autoinducer will affect the pathogenicity.In this project, based on a great quantity of screening experiments, B. thuringiensis and B.cereus strains’ culture was found containing a kind of protein which inhibits autoinducer activity. This protein was named Aii and its encoding gene was named aii. Detection of distribution of aii gene and Aii protein in Bacillus species and B.thuringiensis subspecies indicated that except for B.thuringiensis and B.cereus strains, B.subtilis and B.sphaericus strains had lower Aii activity, but B.megalerium strain had no activity at all. PCR amplification results indicated that aii gene could be found in B.thuringiensis, B.cereus, B.subtilis and B.sphaericus strains, but not in B.megaterium. According to the Southern hybridization results, 43 Bacillus strains were divided into several groups, 22 strains were chosen randomly from all these groups for sequencing of aii gene. It was found that the nucleotide acid sequence of aii genes showed 85.4%-100% homology and amino acid sequence of Aii proteins showed 8 8.1%-100% homology.Further study showed that Aii protein was a kind of non-secreted protein, and aii gene was located on the chromosome of B.thuringiensis or B.cereus. Expression of aii gene expanded from vegetative stage to early stage of sporalation. Aii protein not only inhibited the activity of various purified AHL molecules but also inhibited the activity of AHL molecules in bacterial culture. Using Erwinia carotovora strain SCG1 as pathogen, the effect of B. thuringiensis strain 4D1 culture was detected on the rot soft disease. The results indicated that 4D1 culture inhibited the pathogenicity of SCG1 in 24 hours when the cell density of SCG1 was lower than ODeoonm=0.55, but when the incubation time after infection was longer than 24 hours, the effect decreased. However, 4D1 culture showed a little of inhibitory activity when the cell density of SCG1 was higher than OD600nm=0.55. All these results suggested that the cause of 4D1 culture inhibiting thepathogenicity of SCG1 is the decreased concentration of AHL molecule in SCG1 cells, when presenting Aii protein in 4D1 cells, and then not inducing the expression of virulence genes of SCG 1 at low concentration of AHL molecules.A new strategy in controlling plant pathogen bacteria based on inhibiting the activity of autoinducer molecules could be developed. Construction of transgenic plants which express Aii protein or development of new B.lhitringiensis products which produce high yield of Aii protein will be very useful in plant disease control.2. Expression of gfp gene in B.thuringiensis strainGreen fluorescent protein gene is a very useful report gene which could be detected easily. It can be used in the detection of B. thuringiensis environmental release and study of the insecticidal mechanism.In this study, two kinds of specific promoter of B.thuringiensis: cry 3A promoter and Btl-Btll promoter were chosen to construct fusion genes to drive the expression of gfp gene in B.thuringiensis strain. Nucleotide acid sequence analysis indicates that two construts were correct. Recombinant plasmids pGFPExpA that containing cry3Apro-gfp fusion gene and pGFPExpB that containing Btl-BtHpro-gfp fusion gene were transformed into E.coli and B.thuringiensis plasmidless strains, respectively. The Btl-Btll promoter was found to drive gfp gene expression strong green fluorescence in not only B.thuringiensis but also E.coli strain. However, cry3A promoter could not drive gfp gene expression in E.coli, and the expression i更多还原