极端嗜酸热古菌Acidianus manzaensis胞外硫活化蛋白质基因的筛选及鉴定

生物技术通报 ›› 2016, Vol. 32 ›› Issue (12): 143-151.

极端嗜酸热古菌Acidianus manzaensis胞外硫活化蛋白质基因的筛选及鉴定

马亚龙¹,刘红昌¹,夏金兰¹，²，杨云¹,聂珍媛¹，²

1. 中南大学资源加工与生物工程学院，长沙 410083；
2. 中南大学教育部生物冶金重点实验室，长沙 410083

收稿日期:2016-08-08 出版日期:2016-12-25 发布日期:2016-12-07
作者简介:马亚龙，男，研究方向：生物冶金；E-?mail：yalong_ma@csu.edu.cn
基金资助:
国家自然科学基金项目（51274257）

Screening and Identification of Extracellular Protein Genes Relevant to Sulfur Activation of Extremely Thermoacidophilic Archaea Acidianus manzaensis

MA Ya-long¹,LIU Hong-chang¹,XIA Jin-lan¹，²,YANG Yun ¹,NIE Zhen-yuan¹，²

1. School of Minerals Processing and Bioengineering，Central South University，Changsha 410083；
2. Key Laboratory of Biometallurgy of Ministry of Education，Central South University，Changsha 410083

Received:2016-08-08 Published:2016-12-25 Online:2016-12-07

摘要/Abstract

摘要： 以极端嗜酸热古菌（Acidianus manzaensis）为研究对象，基于比较蛋白质组学的研究方法筛选和鉴定了13个A. manzaensis胞外与硫活化相关的蛋白质基因，并从转录水平对其进行了验证。首先通过80℃缓慢摇动水浴30 min分别对A. manzaensis在单质硫（S0）和亚铁（Fe2+）为能源底物进行生长时的胞外蛋白质进行提取，并用双向电泳（2-DE）进行分离，然后选取在S0底物下差异表达的蛋白质斑点进行串联飞行时间质谱（MALDI-TOF/TOF）鉴定和生物信息学分析及功能预测，最后用实时定量PCR（RT-qPCR）对筛选得到的胞外S0活化相关蛋白基因进行转录水平的验证；最终获得了13个极端嗜酸热古菌A. manzaensis胞外活化S0相关的蛋白质基因。筛选得到的蛋白质中一半以上含有较多的半胱氨酸残基（Cys），说明胞外富含巯基（-SH）的蛋白参与了S0活化；其中谷氧还蛋白（glutaredoxin）和FAD键合氧化酶均含有-CXXC-结构域，且两种蛋白质的基因表达量较高，说明含有-CXXC-结构域的蛋白质在极端嗜酸热古菌A. manzaensis活化S0的过程中起重要的作用。

关键词: Acidianus manzaensis, 比较蛋白质组学, 胞外蛋白质, 硫活化

Abstract: This study focused on extremely thermoacidophilic archaea Acidianus manzaensis，13 extracellular protein genes relevant to sulfur activation were screened and identified based on comparative proteomics，and their transcription levels were verified. First of all，extracellular proteins of A. manzaensis growing on elemental sulfur（S0）and ferrous（Fe2+）were extracted by slowly shaking water bath at 80℃ for 30 min，respectively. The proteins were separated by two-dimensional electrophoresis（2-DE）and differentially expressed spots under S0 substrate were selected. The selected spots were identified by tandem time of flight mass spectrometry（MALDI-TOF/TOF），and then analyzed using bioinformatics and functional prediction methods. Finally, the genes of the screened extracellular proteins associated with S0 activation were verified by real-time quantitative PCR（RT-qPCR）at transcription levels，and 13 relevant genes were acquired. More than half of the screened proteins contained plenty of cysteine residues（Cys），meaning thiol group（-SH）rich extracellular proteins participated in the process of S0 activation. Especially，both glutaredoxin and FAD-linked oxidase contained -CXXC- domain，and their corresponding genes were highly expressed，indicating that the proteins with -CXXC- domain play an important role during the S0 activation process of extremely thermoacidophilic archaea A. manzaensis.

Key words: Acidianus manzaensis, comparative proteomics, extracellular protein, sulfur activation

中图分类号:

10.13560/j.cnki.biotech.bull.1985.2016.12.023

马亚龙,刘红昌,夏金兰,杨云,聂珍媛，. 极端嗜酸热古菌Acidianus manzaensis胞外硫活化蛋白质基因的筛选及鉴定[J]. 生物技术通报, 2016, 32(12): 143-151.

MA Ya-long,LIU Hong-chang,XIA Jin-lan，YANG Yun ,NIE Zhen-yuan,. Screening and Identification of Extracellular Protein Genes Relevant to Sulfur Activation of Extremely Thermoacidophilic Archaea Acidianus manzaensis[J]. Biotechnology Bulletin, 2016, 32(12): 143-151.

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