Acidianus manzaensis菌硫氧化相关膜蛋白基因的筛选及鉴定  

Screening and Identification of Related Sulfur Oxidation Membrane Protein Gene of Acidianus manzaensis

作　　者：朱薇 马亚龙[1,2] 杨云 ZHU Wei;MA Yalong;YANG Yun(School of Science,Hunan University of Technology,Zhuzhou Hunan 412007,China;School of Minerals Processing and Bioengineering,Central South University,Changsha 410083,China)

机构地区：[1]湖南工业大学理学院,湖南株洲412000 [2]中南大学资源加工与生物工程学院,湖南长沙410083

出　　处：《包装学报》2018年第1期67-75,共9页Packaging Journal

基　　金：国家自然科学基金资助项目(51404104);湖南省自然科学基金资助项目(2015JJ3062);湖南省教育厅基金资助项目(14C0341)

摘　　要：基于比较蛋白质组学的方法,筛选和鉴定了嗜酸热古菌A.manzaensis中与硫氧化相关的膜蛋白。首先利用温度诱导Triton X-114两相分离萃取技术分别对以单质硫(S0)和亚铁(Fe2+)为能源底物生长的A.manzaensis菌的膜蛋白进行选择性分离,再用聚丙烯酰胺凝胶电泳和双向电泳对所提取的膜蛋白作进一步分析,最后利用基质辅助激光解吸附电离飞行时间串联质谱对以S0为能源底物下的差异表达膜蛋白斑点进行鉴定,并通过实时荧光定量PCR(RT-q PCR)技术进行初步验证。实验结果表明:A.manzaensis菌在2种不同能源底物下生长时所提取的膜蛋白存在明显的差异,并发现8个与S0氧化相关的膜蛋白,其中,一个未知功能的膜蛋白富含巯基(—SH)及—CXXC—功能域,该膜蛋白可能通过巯基对S0进行键和并生成Pr—SSnH(n≥2)化合物,以协助S0跨膜转运;实验还筛选得到了硫醌氧化还原酶、电子传递蛋白、转录激活蛋白和与细胞生长相关的蛋白,这些膜蛋白在嗜热古菌A.manzaensis氧化S0的过程中起重要作用。The related sulfur oxidation membrane protein genes of extremely thermophilic archaea Acidianus manzaensis were screened and identified based on comparative proteomics methods.Firstly,membrane proteins of A.manzaensis grown on two energy sources,elemental sulfur(S0)and ferrous ions(Fe2+)were selectively extracted and primitively validated by temperature-induced two-phase separation technique of Triton X-114and real time polymerase chain reaction(RT-PCR).The extracted membrane proteins were further analyzed by polyacrylamide gel electrophoresis(SDS-PAGE)and two-dimensional electrophoresis(2-DE).Finally the membrane proteins expressed spots under S0substrate were selected to identify by tandem time of flight mass spectrometry(MALDI-TOF/TOF)and then were verified by RT-qPCR.The results of the present study indicated that the membrane proteins extracted from the two energy sources were significantly different.One hypothetical protein was found rich in cysteine residues and had one-CXXC-functional motif in eight related sulfur oxidation membrane protein genes,which might play an important role in the sulfur transportation by forming the Pr-SSnH(n≥2)complex using the thiol groups(-SH)to bind the S0.Sulphide-quinone oxidoreductas,electrons transportation proteins,transcriptional regulation proteins and grown relevant proteins were also identified,these membrane proteins might play important roles with thermophilic archaea Acidianus manzaensis during S0oxidation process.

关 键 词：Acidianus manzaensis 硫氧化 膜蛋白 比较蛋白质组学 

分 类 号：Q93[生物学—微生物学]