基于iTRAQ方法分析副溶血弧菌活的非可培养状态的差异表达上调蛋白
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摘要
采用低温、寡营养和食品防腐剂处理诱导副溶血弧菌进入活的非可培养状态(viable but nonculturable state,VBNC),并利用同位素标记相对与绝对定量(isobaric rags for relative and absolute quantitation,i TRAQ)蛋白组学技术分析处于VBNC状态和对数生长期的副溶血弧菌的差异表达蛋白,结合液质联用技术对蛋白进行鉴定,对差异蛋白进行GO富集分析和Pathway通路分析。结果表明,副溶血弧菌于山梨酸钾浓度为10 mmol/L的3%Na Cl寡营养培养基中,4℃条件下40~45 d即可进入到VBNC状态。通过i TRAQ蛋白组学技术共鉴定到135880个蛋白,其中定量的蛋白有1088个;在VBNC状态显著下调蛋白36个,上调蛋白15个。差异表达的上调蛋白主要是外膜蛋白、转运蛋白、铁蛋白和其他参与代谢的关键蛋白,表明VBNC状态的副溶血弧菌根据环境应激提高这些蛋白的表达以保持活性。该研究结果为更深入探讨VBNC的分子形成机制提供了理论基础。
Vibrio parahaemolyticus was induced into viable but nonculturable(VBNC) state by food preservative at low temperature and oligotrophic condition. The aim of this study is to screen differentially expressed proteins of VBNC state and exponential phase of V. parahaemolyticus using isobaric rags for relative and absolute quantitation(i TRAQ) combined with LC/MS. Functional annotations of unigenes were analyzed using protein sequence similarity, KEGG pathway and GO analysis. The results showed that only 40~45 days V. parahaemolyticus could induce into VBNC state at 4 ℃ in seawater containing 10 mmol/L potassium sorbate. A total of 135,880 proteins, including 1,088 unique proteins were identified. Of the quantitatively different proteins under the VBNC state, 15 were significantly up-regulated and 36 were down-regulated. The up-regulated expressed proteins were mainly focused on outer membrane protein, transporter proteins, ferritin and other key components involved in metabolism. This study provided basis for understanding the mechanism of adaptation of the VBNC state of V. parahaemolyticus.
Vibrio parahaemolyticus was induced into viable but nonculturable(VBNC) state by food preservative at low temperature and oligotrophic condition. The aim of this study is to screen differentially expressed proteins of VBNC state and exponential phase of V. parahaemolyticus using isobaric rags for relative and absolute quantitation(i TRAQ) combined with LC/MS. Functional annotations of unigenes were analyzed using protein sequence similarity, KEGG pathway and GO analysis. The results showed that only 40~45 days V. parahaemolyticus could induce into VBNC state at 4 ℃ in seawater containing 10 mmol/L potassium sorbate. A total of 135,880 proteins, including 1,088 unique proteins were identified. Of the quantitatively different proteins under the VBNC state, 15 were significantly up-regulated and 36 were down-regulated. The up-regulated expressed proteins were mainly focused on outer membrane protein, transporter proteins, ferritin and other key components involved in metabolism. This study provided basis for understanding the mechanism of adaptation of the VBNC state of V. parahaemolyticus.
引文
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[2]Ross P L,Huang Y L N,Marchese J N,et al.Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents[J].Molecular&Cellular Proteomics,2004,3(12):1154-1169
[3]Oliver JD.The viable but nonculturable state in bacteria[J].Microbiol.,2005,43:93-100
[4]Oliver JD.Recent findings on the viable but nonculturable state in pathogenic bacteria[J].FEMS Microbiol.Rev.,2010,34:415-425
[5]Drake S L,Depaola A,Jaykus L.An overview of Vibrio vulnificus and Vibrio parahaemolyticus[J].Comprehensive Reviews in Food Science and Food Safety,2007,6(4):120-144
[6]窦勇,胡佩红,顾鹏程.副溶血弧菌TDH毒素及其检测方法研究进展[J].现代食品科技,2013,29(1):215-218DOU Yong,HU Pei-hong,GU Peng-cheng.Research progress in Vibrio parahaemolyticus TDH toxin and detection methods[J].Morden Food Science and Technology,2013,29(1):215-218
[7]李青,钟青萍,王丽.EMA结合PCR技术有效鉴别副溶血弧菌死活细胞[J].华南农业大学学报,2011,32(1):108-111LI Qing,ZHONG Qing-ping,WANG Li.Use of ethidium monoazide-polymerase chain reaction(EMA-PCR)for differentiation of the viable and dead cells of Vibrio parahaemolyticus[J].Journal of South China Agricultural University,2011,32(1):108-111
[8]Chiu S,Chen S,Wong H.Localization and expression of Mre B in Vibrio parahaemolyticus under different stresses[J].Applied and Environmental Microbiology,2008,74(22):7016-7022
[9]Um,Kong,Lee,et al.Altered gene expression and intracellular changes of the viable but nonculturable state in Ralstonia solanacearum by copper treatment[J].Plant Pathology Journal,2013.29(4):374-385
[10]Lai C,Chen S,Lin I H,et al.Change of protein profiles in the induction of the viable but nonculturable state of Vibrio parahaemolyticus[J].International Journal of Food Microbiology,2009,135(2):118-124
[11]董妍玲,潘学武.细菌内依赖Ton B的外膜铁转运体的研究进展[J].生物技术通报,2012,1:23-29DONG Yan-ling,PAN Xue-wu.Progress of Ton B-dependent transporters for iron on outer membrane in bacteria[J].Biotechnology Bulletin,2012,1:23-29
[12]Monahan L G,Hajduk I V,Blaber S P,et al.Coordinating bacterial cell division with nutrient availability:a role for glycolysis[J].Mbio,2014,5(3):e00935-14
[13]Jia J,Li Z,Cao J,et al.Proteomic analysis of protein expression in the induction of the viable but nonculturable state of Vibrio harveyi SF1[J].Current Microbiology,2013,67(4):442-447