摘要
苏云金芽胞杆菌(Bacillus thuringiensis, Bt)是一种产生杀虫晶体蛋白的革兰氏阳性芽胞杆菌。它产生的杀虫晶体蛋白在国内外的各种害虫防治上得到了广泛应用。在研体内杀虫晶体蛋白能够积累到细胞干重的20-30%。这种芽胞杆菌体内大量表达蛋白并形成晶体的机制受到研究者的广泛关注。虽然杀虫晶体蛋白在转录调控上的机制研究的比较透彻,但是在翻译以及翻译后修饰的研究上还很欠缺。为了更加清楚地了解这个机制,我们使用了蛋白质组学方法来研究Bt杀虫晶体蛋白在翻译水平的调控与细胞代谢之间的关系。本研究通过2-DE和MALDI-TOF-MS方法建立了苏云金芽胞杆菌YBT-1520在合成培养基不同生长时期的比较蛋白质组研究。首先我们对不同pH值范围(pH4-7、pH5-8、pH3-10、pH7-11)的苏云金芽胞杆菌全细胞蛋白的双向电泳条件进行了摸索,发现pH4-7为最适蛋白分离pH范围。使用pH4-7的双向电泳分离条件,我们完成了苏云金芽胞杆菌YBT-1520在合成培养基中生长的三个时间点：对数中期(17h)、稳定期前期(20h)和稳定期后期(26h)全细胞蛋白的双向电泳分离并进行了考马斯亮蓝G-250染色。双向电泳胶上分离的蛋白数量为800-900之间,同时对三个不同生长期分离的蛋白胶进行比较分析发现不同生长期间表达差异超过2倍的蛋白在120-205之间。选取101个高丰度差异表达蛋白进行了MALDI-TOF-MS的质谱鉴定,有72个蛋白质得到了鉴定。对这些鉴定蛋白进行功能分类发现80%的蛋白都在与代谢相关。而对不同功能分类蛋白总表达量分析发现氨基酸代谢和蛋白翻译合成相关蛋白总表达量变化最为显著。接下来我们着重分析了Bt不同生长期细胞内代谢变化和杀虫晶体蛋白翻译调控之间的关系。并通过实时荧光定量PCR方法研究了糖酵解和TCA循环的关键酶基因的转录水平。通过分析我们发现从对数期到稳定期细胞内代谢途径发生了明显的改变：1)糖酵解途径中间产物向磷酸戊糖途径和丝氨酸合成途径转移；2)TCA循环受到了严重抑制；3)PHB被认为是重要的碳源和能量的载体；4)蛋白酶表达量的上升和氨基酸合成途径的抑制暗示着细胞内有大量的蛋白质降解为氨基酸,为杀虫晶体蛋白的合成提供前体；5)翻译相关因子：EF-Tu、GroEL和GatB表现出上调而核糖体抑制因子YfiA表现出下调,这说明了细胞在翻译水平上积极的响应杀虫晶体蛋白的合成。本研究首次建立了苏云金芽胞杆菌YBT-1520在合成培养基中生长的不同生长期的比较蛋白质组研究。我们发现B,细胞代谢在不同生长期发生了显著的改变,这些改变为杀虫晶体蛋白的大量合成提供了物质和能量基础。同时翻译相关因子的表达变化也说明了研细胞为杀虫晶体蛋白的大量合成进行了翻译水平的调控。这些研究结果给研杀虫晶体蛋白高表达机制提供了新的可操纵和调控的位点,为今后杀虫晶体蛋白的更高量表达和异源蛋白的高表达提供了新的理论基础。
Bacillus thuringiensis is a Gram-positive endospore forming bacterium that produces insecticidal crystal proteins (ICPs) which are principal bio-pesticides used in agricultural and sanitary pest controls. ICPs could accumulate and account for up to20to30%of the dry weight of sporulated cells. Although the transcription studies of ICPs' genes were performed a lot, very little is known about the translational regulation of ICPs' synthesis. The comparative proteomic analysis of B. thuringiensis strain YBT-1520that grew in a minimal medium was performed to understand the translational regulation of ICPs synthesis. The different pH gradient of2-DE gels included pH4-7, pH5-8, pH3-10, and pH7-11were tested before the formal experiment. The pH4-7gradient was observed well separation of whole cell protein in YBT-1520. Cell samples for protein and for total RNA isolation were collected at the time points of17h,20h and26h with corresponding OD600at0.284,0.522and0.724respectively. The three replication pH4-7gradient2-DE gels for differrent growth phases of YBT-1520were constructed. Eight to nine hundreds protein spots could be separated in pH4-7gradient2-DE gels which were stained by coomassie brilliant blue G-250. Two hundreds and four proteins changed at least two folds in spot volumes on2-DE gels between mid-log phase and stationary phase. Among them,101proteins were chosen for MALDI-TOF-MS analysis, and72of which were further identified by the MASCOT Peptide Mass Fingerprinting (PMF) search. The transcription levels of carbohydrate metabolism related genes were also studied by real-time RT-PCR. Significant changes in carbohydrate, fatty acid, acetyl-CoA, and amino acid metabolisms in the different phases were revealed in this study. Tricarboxylic acid cycle (TCA cycle), the part of glycolysis and the synthesis of most amino acids were depressed from log to stationary phases. Acetyl-CoA mainly entered into TCA cycle, PHB, fatty acid, and acetate synthesis in log phase but only converted to fatty acid in stationary phase. Metabolic changes indicated that the amino acid precursors of ICPs were mainly oBtained from the protein degradation in vivo. The energy produced from fatty acid oxidation and glycolysis in stationary phase could meet the need of ICPs synthesis. The expression changes of EF-Tu, YfiA, GroEL and GatB suggested the direct translational regulation of ICPs'synthesis in B. thuringiensis. This work was the first comparative proteomic study of B. thuringiensis grew in defined medium at different growth phases. The research contributes to better understand the relationship between metabolic changes and ICPs synthesis in B. thuringiensis. The analysis of metabolic changes provides a new sight in the translational regulation of ICPs'synthesis.
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