【作者】 吴丹丹；

【导师】 喻子牛； 李明顺；

【作者基本信息】 华中农业大学 ， 微生物学， 2011， 博士

【摘要】 作为目前世界上成功商品化的生物杀虫剂,苏云金芽胞杆菌(Bacillus thuringiensis, Bt)已被使用了近50年时间,其在细胞内形成的杀虫晶体蛋白(Insecidal crystal proteins, ICPs)对多种农业害虫和生活害虫具有防治效果。苏云金芽胞杆菌YBT-1520是本室从土壤中分离到的一株对棉龄虫、小菜蛾等鳞翅目害虫具有高毒力的菌株(专利号ZL 95 106749.4)。本研究在生理学基础上结合高通量蛋白质组学技术对菌株YBT-1520在42℃热胁迫下胞内蛋白的变化进行了系统性地描述,对其主要生理特性的变化机理进行了探讨,对其热胁迫下的生存机制进行了揭示。热胁迫下Bt胞内蛋白主要发生了如下变化：(1)Ⅰ型和Ⅲ型热应激蛋白DnaK、GroEL、GrpE、ClpC在长时间热胁迫后仍处于诱导表达状态；(2)长时间热胁迫亦造成了BDH1、GuaB、GST非典型热应激蛋白的诱导表达,推测它们可能为长时间热胁迫的特异效应分子；(3)胞内83%的差异蛋白呈下调趋势,包括毒素蛋白、潜在毒素蛋白、蛋白酶以和部分基础代谢酶。蛋白的下调可能缘于胞内ClpC蛋白酶的水解,亦可能缘于细胞的严谨反应；(4)在热胁迫后期,约50%的下调蛋白恢复至正常表达量,表明此时的热胁迫细胞具有了一定的热适应性。热胁迫下Bt主要生理特性发生了如下变化：(1)ICPs和其它潜在毒力因子的表达被阻断或显著下调；(2)细胞的粘附性和运动性丧失；(3)芽胞不能形成；(4)细胞持续性地积累PHB。长时间热胁迫下Bt的生存策略如下。策略一：诱导非典型热应激蛋白以补充典型热应激蛋白的消退。非典型热应激蛋白分别参与了PHB的合成、严谨反应信号分子的合成、有毒亲电物质的消除。策略二：代谢调控。细胞将代谢维持在较低水平,以减缓代谢可能对细胞造成的氧化压力或其它负担。对代谢的调控可能是通过胞内水解酶或／和严谨反应实现。策略三：持续积累PHB。细胞在长时间热胁迫下通过代谢调整,持续积累PHB。借助PHB的抗逆性,提高细胞对热胁迫的耐受性。更多还原

【Abstract】 Bacillus thuringiensis (Bt) has been widely used for 50 years as a safe biopesticide for controlling agricultural and sanitary insect pests because of its insecticidal crystal proteins (ICPs). The strain used in this study was B. ihuringiensis subsp. kurstaki YBT-1520, which demonstrates high insecticidal activities against lepidopteran larvae. YBT-1520 was originally isolated and characterized in our laboratory. In this study a proteomic approach conbined with physiological investigation was used to describe the proteomic and physiological changes and to reveal the survival strategies of Bt YBT-1520 under a long-term heat stress condition (42℃).The changes of the intracellular proteome under heat stress were as follows:(1) some of the heat shock proteins of class I and II (DnaK, GroEL, GrpE and ClpC) were still induced under long-term heat stress; (2) three non-classical heat shock proteins (BDH1, GuaB and GST) were also induced under long-term heat stress, they could be regarded as the specific factors of long-term heat stress; (3) 83% of the differentially expressed intracellular proteins decreased including toxin proteins, potential toxin proteins, proteases and enzymes involved in the basic metabolic pathways. All these decrease proteins were probably due to the proteolysis process of ClpC and/or the stringent responses triggered by heat stress; (4) about 50% of the decreased proteins were recovered at the late stage of heat stress condition, indicating that the heat stressed cells gained the stresss adaptive ability somehow.Heat stress mainly influenced the characteristics of YBT-1520 on four aspects:(1) the ability of synthesis of ICPs and other potential pathogenic factors were lost or significantly decreased; (2) cell adhesion and motility were also lost; (3) the cells did not sporulate, (4) cells kept accumulating poly-β-hydroxybutyrate (PHB).The survival strategies of YBT-1520 under long-term heat stress were as follows. The first strategy:the induction of non-classical heat shock proteins was a kind of supplementary for the fadeaway of classical heat induced proteins. The second one: metabolic adjustment. Cells down-regulated metabolic enzymes to reduce the oxidative stress or other metabolic burden. This regulation was probably due to the proteolysis process and/or the stringent responses. The third strategy:PHB accumulation. Bacterium adjusted the metabolism of PHB and kept accumulating PHB. These strategies would help cells to gain more tolerance to heat stress.更多还原