基于蛋白组学和代谢组学技术解析腐败希瓦氏菌净化水产品加工废水的生物学机制  

The Mechanism Research on Shewanella putrefaciens in Aquatic Products Processing Wastewater Basing on Proteomics and Metabolomics

作　　者：何伟娜[1] 王中华[1] 张迪骏[1] 陈丽萍[1] 周君[1] 苏秀榕[1] 

机构地区：[1]宁波大学海洋学院,浙江宁波315211

出　　处：《中国食品学报》2016年第5期136-146,共11页Journal of Chinese Institute Of Food Science and Technology

基　　金：海洋公益性行业专业专项资助项目(201105007);浙江省重大科技攻关项目(2006C13089);宁波市科技局资助项目(2008C50027)

摘　　要：目的:利用蛋白组学、代谢组学和实时荧光定量PCR技术研究腐败希瓦氏菌净化水产品加工废水的生物学机制,挖掘相关功能蛋白,为食品加工废水治理提供理论依据。方法:以淡水培养为对照,宁波象山石浦水产品加工园区排污口处废水培养为试验组,应用双向电泳(2-DE)技术筛选2种培养的腐败希瓦氏菌功能蛋白;利用实时荧光定量PCR(q RT-PCR)技术验证基因表达情况;核磁共振(NMR)技术鉴定菌体胞内代谢产物。结果:双向电泳筛选出31个差异蛋白,表达差异1.5倍以上的蛋白22个,最大差异比率为12.1。采用MALDITOF-MS-MS技术进行蛋白定性,GO和KEGG分析,其差异蛋白涉及铁代谢、ABC转运系统、抗生素合成、硫代谢、谷胱甘肽合成等。选取11个基因进行q RT-PCR,7个基因与蛋白表达水平一致,4个基因呈现负相关。NMR检出10类59种胞内代谢产物,其中腐胺、乙酸盐等8种代谢物的表达差异较大。结论:与净化水产品加工废水相关的功能蛋白2个,即半胱氨酸合酶和异柠檬酸脱氢酶。这两种酶在降解无机硫、重金属解毒、清除自由基,抵御氧化性损伤等方面发挥关键的作用,有望将其应用于废水治理。Objective： The method of combination of differential proteomics, metabolomics, and real-time fluorescent quantitative PCR techniques has utilized to investigate the wastewater purification mechanism of Shewanella putrefaciens under aquatic products processing wastewater surroundings and excavate related functional protein as well as providing theoretical basis for food processing wastewater treatment. Methods： With freshwater culture medium for comparison, taking samples from Xiangshan Shipu aquatic products processing wastewater as bacteria culture environment, then applying proteomics technology to filtrate and authenticate related protein, RT-PCR to validate gene expression; NMR technology to identify bacteria intracellular metabolites. Results show that 38 different proteins were filtrated by 2-DE, the expression difference of 22 proteins were more than 1.5 times and the largest express difference ratio was 12.1. They were identified by MALDI-TOF-MS-MS, refering to in iron metabolism, ABC transit system, antibiotic biosynthesis, sulfur metabolism, glutathione biosynthesis etc. 11 candidate genes in m RNA expression level were researched by q RT-PCR technology, the results show that 7 genes presented the consistency in protein expression levels and genes, and the remaining were negatively correlated. 59 different metabolites of 10 classes is detected by Nuclear magnetic resonance（NMR）, which putrescine, Acetate etc shows the biggest difference in two groups. Conclusion： Exploring related 2 functional proteins relating to purify waste water, the cysteine synthase and isocitrate dehydrogenase, they plays a key role in harmful inorganic sulfur degradation, heavy metal detoxification, scavenging free radicals, resisting oxidation damage and so on. So they are hopeful for aquatic products processing wastewater governance.

关 键 词：腐败希瓦氏菌 蛋白组学 代谢组学 实时荧光定量PCR 水产加工废水 

分 类 号：X792[环境科学与工程—环境工程]