应用iTRAQ定量蛋白质组学研究海分枝杆菌mkl的基因功能

Quantitative proteomic analysis of mkl gene function in Mycobacterium marinum using iTRAQ

摘要:

[目的]通过分离海分枝杆菌野生株和mkl突变株的全菌蛋白，并进行差异蛋白质组分析，以期为探索分枝杆菌重要毒力基因mkl的功能提供新思路。[方法]以海分枝杆菌野生株和mkl突变株为研究材料，提取全菌蛋白，iTRAQ试剂标记后进行质谱鉴定和定量分析，并利用UniProt数据库对差异蛋白进行生物信息学分析。[结果]共鉴定出在野生株和mkl突变株中差异表达蛋白566个，其中在突变株中上调表达蛋白232个（比值≥1.4），下调表达蛋白334个（比值≤0.7）。生物信息学预测这些蛋白主要参与细菌脂质代谢、细胞壁和细胞进程、中间代谢、呼吸作用等生物学功能。其中DesA3下调最显著，其功能为脂肪酸去饱和酶，与油酸合成相关，进一步验证发现mkl突变株在不含油酸的固体培养基中生长受限，提示mkl可能在油酸的生物合成通路中发挥功能。[结论]通过iTRAQ分析了海分枝杆菌mkl突变株和野生株的差异表达蛋白谱，发现可能影响分枝杆菌油酸、脂质等合成代谢通路，为进一步研究mkl基因在分枝杆菌致病中发挥作用的相关机制奠定了基础。

Abstract:

[Objective] To identify differentially expressed proteins in Mycobacterium marinum wild-type (WT) and mkl::Tn mutant strains, and provide new clues for exploring the functions of mkl gene.[Methods] Cellular proteins were extracted from cultures of M. marinum WT and mkl::Tn strains, and labelled with isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex. Differentially expressed proteins were identified with LC-MS/MS and subjected to biological information analysis.[Results] A total of 566 differentially expressed proteins were revealed, among which 232 proteins were up-regulated (ratio≥1.4) and 334 proteins were down-regulated (ratio≤0.7). These proteins are mainly associated with lipid metabolism, cell wall and cell processes, intermediary metabolism and respiration, and hypothetical proteins. The most down-regulated protein DesA3, is a fatty acid desaturase and involved in the synthesis of oleic acid. Further experiments showed that the growth of mkl::Tn strain was attenuated on 7H10-ADC agar plate without oleic acid, suggesting that mkl may play a role in the biosynthesis of oleic acid.[Conclusion] Differentially expressed proteins were identified in M. marinum mkl::Tn compared to WT, and these results shed light on the mechanisms of mkl gene in mycobacterial pathogenesis.