冷藏大黄鱼特定腐败菌Shewanella致腐性差异的分子机制研究

Large yellow croaker, Pseudosciaena crocea, is commercially important due to the good taste and high nutrition, and is cultivated in marine net-cages in southeastern China with large yields. However, spoilage of large yellow croaker often happens during the storage, production and commodity circulation leading to loss rate of 16.5%, which causes a large economic loss. Microorganisms are the major cause of spoilage of most seafood products. Only a few members of the microbial community, the specific spoilage organisms (SSOs), give rise to the offensive off-flavours and toxic compounds associated with seafood spoilage. However, molecular mechanism of spoilage abilities in different Shewanella strains isolated from seafood is poorly understood. Therefore, we aim to sequence the whole genome of Shewanella isolates, and analyze the spoilage related genes to find out the relationship of spoilage capability and genetic differences; detect the diverse signal molecules and their regulation to spoilage capability; characterize quorum sensing (QS) transcriptional regulators and their regulation to spoilage related genes as well as spoilage capability; analyze the differential proteomics of typical Shewanella isolates, and finally clarify the molecular mechanism of differential spoilage abilities in Shewanella strains. Our results may have important implications for understanding the genetics, QS systems and proteomics associated with spoilage potential in SSOs, which may benefit for development of new techniques in seafood preservation.

大黄鱼（Pseudosciaena crocea）是我国具有重要商业价值的海洋鱼类，在贮藏、加工及流通过程中易腐败变质，损失率可高达16.5%。研究证实，作为特定腐败菌（SSO）的腐败希瓦氏菌（Shewanella）是引起冷藏大黄鱼腐败变质的主要原因，进一步探究发现不同Shewanella分离株间存在致腐能力明显差异，但对其致腐性差异的分子机制尚不明确。鉴于此，本项目通过全基因组学分析，解析致腐性与遗传背景差异的关系并挖掘腐败相关基因；探明群体感应信号分子的多样性及其与致腐能力的相关性；比较研究关键调控因子对其致腐相关基因及致腐能力调节的差异；结合差异蛋白质组学方法从蛋白分子层面进一步剖析分离株致腐性的差异。本项目旨在探究大黄鱼特定腐败菌Shewanella致腐性差异的分子机制，充实和完善食品贮藏与保鲜中腐败微生物作用的相关理论，为研发海洋鱼类保鲜新技术提供借鉴。

本研究通过对S. baltica分离株进行全基因组序列测定与解析，发现致腐能力最强菌株S. baltica 073与最弱菌株S. baltica 070的氧化三甲胺还原酶（TorT）基因、半胱氨酸合成酶B和硫氧还原蛋白还原酶基因的关键位点均存在一定的差异。通过与腐败表型拟合分析，发现接种不同希瓦氏菌株的无菌鱼汁中腐败指标TVB-N值与基因差异率的拟合，说明两者线性关系良好。接种实验表明群体感应信号分子cyclo-(L-Pro-L-Leu)和cyclo-(L-Pro-L-Pro)与腐胺形成均呈现显著正相关性，可向正调控腐胺的生成。通过外源添加合成信号分子AHLs，发现AHLs能显著促进S. baltica生物被膜形成以及致腐能力。通过体外结合实验、基因敲除以及转录组测序分析，挖掘了LuxR01和LuxR02受体蛋白，揭示了LuxR家族蛋白与DKPs的互作关系，从分子水平阐明了基于LuxR01和LuxR02的QS系统对S. baltica腐败能力的调控机制。最后通过差异蛋白组学，挖掘出DGC和PDE双活性蛋白Sbal_3235，通过基因敲除和回补技术，阐明Sbal_3235蛋白通过调控c-di-GMP信号的代谢从而影响Shewanella菌株的致腐性，全面解析了Shewanella菌株致腐性差异的分子机制。本项目有助于形成食品贮藏过程中腐败微生物致腐机制的理论，为研发海产养殖鱼类源头保鲜新技术提供借鉴和支撑，对提高养殖鱼类鲜度和品质具有重要科学意义和应用价值。