Li Xiaohui, Ding Xuezhi, Xia Liqiu, Sun Yunjun, Yuan Can, Yin Jia
Key Laboratory of Microbial Molecular Biology of Hunan Province, College of Life Science, Hunan Normal University, Changsha 410081, China.
ScientificWorldJournal. 2012;2012:798739. doi: 10.1100/2012/798739. Epub 2012 Apr 29.
The growth process of Bacillus thuringiensis Bt4.0718 strain was studied using proteomic technologies. The proteins of Bt whole cells at three phases-middle vegetative, early sporulation, and late sporulation-were extracted with lysis buffer, followed with separation by 2-DE and identified by MALDI-TOF/TOF MS. Bioactive factors such as insecticidal crystal proteins (ICPs) including Cry1Ac(3), Cry2Aa, and BTRX28, immune inhibitor (InhA), and InhA precursor were identified. InhA started to express at the middle vegetative phase, suggesting its contribution to the survival of Bt in the host body. At the early sporulation phase, ICPs started their expression. CotJC, OppA, ORF1, and SpoIVA related to the formation of crystals and spores were identified, the expression characteristics of which ensured the stable formation of crystals and spores. This study provides an important foundation for further exploration of the stable expression of ICPs, the smooth formation of crystals, and the construction of recombinant strains.
利用蛋白质组学技术研究了苏云金芽孢杆菌Bt4.0718菌株的生长过程。用裂解缓冲液提取了该菌株在对数中期、芽孢形成前期和芽孢形成后期三个阶段的全细胞蛋白质,随后通过双向电泳进行分离,并用基质辅助激光解吸电离飞行时间串联质谱进行鉴定。鉴定出了多种生物活性因子,如杀虫晶体蛋白(ICPs),包括Cry1Ac(3)、Cry2Aa和BTRX28、免疫抑制剂(InhA)以及InhA前体。InhA在对数中期开始表达,表明其对苏云金芽孢杆菌在宿主体内的存活有贡献。在芽孢形成前期,ICPs开始表达。鉴定出了与晶体和芽孢形成相关的CotJC、OppA、ORF1和SpoIVA,它们的表达特征确保了晶体和芽孢的稳定形成。该研究为进一步探索ICPs的稳定表达、晶体的顺利形成以及重组菌株的构建提供了重要基础。