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

作　　者：施旭骏[1] 赵超[1] 牛辰[1] 高谦[1] 

机构地区：[1]复旦大学基础医学院,教育部/卫生部医学分子病毒学重点实验室,上海200032

出　　处：《微生物学报》2016年第9期1496-1503,共8页Acta Microbiologica Sinica

基　　金：国家自然科学基金(81271790,81201256)~~

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,i TRAQ试剂标记后进行质谱鉴定和定量分析,并利用Uni Prot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Des A3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i TRAQ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。[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 ofM. 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 ofmkl gene in mycobacterial pathogenesis.

关 键 词：分枝杆菌 定量蛋白质组学 ITRAQ mkl基因 

分 类 号：R378.911[医药卫生—病原生物学]