【作者】 刘雪梅；

【导师】 丁学知；

【作者基本信息】 湖南师范大学 ， 微生物学， 2014， 硕士

【摘要】 苏云金芽胞杆菌(Bacillus thuringiensis, Bt) X022是从土壤中分离筛选到的一株产菱形伴胞晶体的菌株,经形态特征镜检和16SrRNA基因鉴定,初步确定属于苏云金芽胞杆菌库斯塔克亚种。SDS-PAGE检测显示Bt X022菌株晶体蛋白分子量大约为130kDa。采用二维液质联用质谱仪(2D-LC-MS/MS)分析Bt X022菌株的芽胞释放期全菌体蛋白,鉴定到该菌株表达Cry1Ca、CrylAc和Cry1Da等毒蛋白。利用刘飞等优化的Bt4.0718菌株的原培养基配方,进一步改进初始pH值、改善溶氧条件和添加适量Cu2+,使Bt X022菌株的130kDa毒蛋白产量得到了提高。SDS-PAGE结合生物医学实验专用影像分析软件MiBio分析显示,10-6mol/L的Cu2+使Bt X022菌株的130kDa晶体蛋白产量提高了21%左右,且最适合Bt X022菌株晶体蛋白产生的Cu2+浓度范围为10-6-10-4mol/L.毒素蛋白emPAI值半定量和qRT-PCR分析显示,Cu2+主要提高了Cry1Da和Cry1Ca的表达量。毒力生测结果显示,培养基中添加微量Cu2+显著增强了Bt X022菌株发酵液对甜菜夜蛾和棉铃虫幼虫的杀虫活性。检测Cu2+对Bt X022菌株发酵过程的生长曲线、pH变化和葡萄糖消耗的影响,结果显示Cu2+使Bt X022菌株稳定期延长、衰亡期菌体裂解加速；发酵过程pH回升加快、平台期pH值降低且平台期持续时间长；葡糖糖消耗情况无差异。为了进一步探索Cu2+提高BtX022新菌株毒蛋白产量及杀虫活性的机制,对原培养基和添加Cu2+培养基中芽胞释放期的Bt X022菌体全蛋白进行了2D-LC-MS/MS检测,蛋白功能分类及表达谱的比较分析显示Cu2+使环境信息加工过程蛋白和聚糖合成与代谢蛋白表达下调；emPAI1(?)半定量结合qRT-PCR分析发现Cu2+使PhaR、BDH、EF-G、 KASI Ⅱ ALDH等一些功能蛋白表达上调。综合分析Cu2+引起的发酵参数改变(稳定期延长、菌体裂解加速,pH回升加快)以及蛋白质组学变化,推测适量的Cu2+通过增加菌体内PhaR的表达量改变了Bt X022菌株的碳源流向：环境信息加工过程蛋白和聚糖合成与代谢蛋白表达下调,导致胞外多糖生物合成减少,从而菌体裂解加快；更多碳源用于合成胞内PHB, PHB储存物的增加使Bt X022菌株稳定期延长、pH回升加快,且稳定期可释放更多能量用于蛋白的翻译,最终增加了杀虫晶体蛋白的产量。更多还原

【Abstract】 Bacillus thuringiensis X022(Bt X022) is a novel strain isolated from the soil in China and it produces diamond parasporal crystals. Referring to the morphology under microscope and16S rRNA gene sequenced, we preliminarily determined that Bt X022belongs to Bacillus thuringiensis subsp.kurstaki. SDS-PAGE showed that the molecular weight of crystal proteins produced by Bt X022strain is about130kDa. The whole proteins of cells in spore release period revealed by2D-LC-MS/MS demonstrate that Bt X022mainly expresses3kinds of crystal proteins: Cry1Ca, CryaAc and Cry1Da. Based on the medium optimized for Bt4.0718by Liu Fei et al, we improved the initial pH and dissolved oxygen condition and also find that130kDa crystal proteins production was more efficient when an appropriate amount of Cu2+was added. SDS-PAGE and biomedical image analysis software MiBio showed that10"6mol/L Cu2+makes the production of130kDa crystal proteins increased by21%and the favorable concentration of Cu2+ranges from10-6to10-4mol/L. The emPAI semi-quantitative of ICPs and RT-qPCR showed that Cu2+mainly increases the expression of Cry1Da and Cry1Ca. Toxicity test showed that Cu2+made the toxicity of Bt X022fermentation broth to spodoptera exigua and helicoverpa armigera increased greatly.The influences of Cu2+on growth parameters of Bt X022strain was detected, which revealed that Cu2+resulted in a prolongated stationary phase, acceleration of cell lysis in decline phase; rapid pH rebound, lower pH value on the plateau and a prolonged pH plateau phase.To explore the reasons for the crystal proteins production increase caused by Cu2+, we performed two-dimensional-liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) to analyze the whole proteins of cells in spore release period of Bt X022strain which was cultivated in two different mudium (Cu:added10-6mol/L Cu2+; CK:the original medium, as control). Functional classification and comparative analysis of the proteome revealed that Cu2+caused the down-regulation of environmental information-processing proteins and glycan biosynthesis and metabolism proteins; emPAI semiquantitative comparative analysis and RT-qPCR found that Cu2+induced up-regulation of some functional proteins, such as PhaR, BDH, EF-G, KAS II, ALDH and et al.With comprehensive analysis of the changes of proteome and fermentation parameters (prolongated stationary phase, acceleration of cell lysis, rapid pH rebound) caused by adding Cu2+, we proposed that Cu2+increased the expression of PhaR and consequently changed the carbon flow in Bt X022strain. It caused the down-regulation of environmental information-processing proteins and glycan biosynthesis and metabolism proteins and thus reduced the production of extracellular polysaccharide and result in acceleration of cell lysis. More carbon sources can be used to synthetise intracellular PHB. The increase of PHB as storage material brings about prolongated stationary phase, rapid pH rebound and also releasing more energy for protein translation and ultimately raised the production of ICPs.更多还原