基于基因组和转录组分析冷藏大黄鱼特定腐败菌腐败希瓦氏菌的主要致腐机理

Large yellow croaker (Pseudosciaena crocea), as an economic food fish, has been one of the most widely marine cultured species in China. Due to active and aggressive characters, large yellow croaker is highly susceptible to die during transport, so it is commonly sold under low-temperature stored condition. Shewanella putrefaciens, the specific spoilage organism of large yellow croaker during low-temperature storage, will cause deterioration of fish quality and produce smell and odor. However, little information is available on the genome and spoilage mechanism of this bacterium. Several strains of S. putrefaciens with different spoilage capability were isolated during the fish storage, and two strains with the strongest or weakest spoilage capacity were selected as research objects. Then they will be subjected to whole genome sequence analysis to produce detailed maps of genome. Comparative genome analysis will reveal specific genomic regions that are potentially implicated in spoilage capability. In addition, whole transcriptome RNA-Seq analysis and real-time quantitative PCR method will be used to identify genes important for characteristic phenotypes of S. putrefaciens such as those involved in reducing trimethylamine N-oxide to nauseous trimethylamine, and finally to explore the main spoilage mechanism of this specific spoilage organism. These findings will not only lay the foundation for basic biological research of S. putrefaciens, but also provide the strong support for the development of preservation technology of large yellow croaker, and facilitate the understanding of the mechanism of food spoilage bacteria in food spoilage process.

大黄鱼是我国近海重要的经济鱼类，鱼在运输过程中极易死亡，因此市售大黄鱼常以冷藏方式贮运。大黄鱼在冷藏过程中的特定腐败菌是腐败希瓦氏菌，该菌是导致冷藏大黄鱼品质劣变、产生腐败臭味和异味的重要原因；但是目前缺乏腐败希瓦氏菌的基因组信息和明确的致腐机理。本项目从冷藏大黄鱼保鲜过程中分离到多株具有不同致腐能力的腐败希瓦氏菌，并以其中具有最强和最弱致腐能力的2株菌作为研究对象，在通过全基因组测序得到它们的基因组精细图基础上，利用比较基因组学分析不同腐败希瓦氏菌在致腐能力上的差异原因，进而采用RNA-Seq测序和实时荧光定量PCR技术，探明这2株菌在不同生长时序的转录组以及致腐因子和差异表达基因的转录本、代谢途径等信息，开展腐败希瓦氏菌致腐机理的研究，这不仅为腐败希瓦氏菌的基础生物学研究奠定基础，以及为发展大黄鱼低温保鲜技术提供有力的支撑，而且还将大大推动对食品腐败菌在食品腐败过程中致腐机理的认识。

本项目从冷藏大黄鱼保鲜过程中分离到多株具有不同致腐能力的腐败希瓦氏菌，其中S18株和W9株分别具有最强和最弱的导致冷藏大黄鱼腐败能力的细菌。基于16r RNA基因所构建的系统发育树表明，所分离出的这2株细菌均为希瓦氏菌。提取这2株细菌的总DNA，并构建150bp和2kb的文库，接着采用pair end和mate pair测序方式分别对这些基因组文库进行测序，所得reads经组装拼接后分别得到希瓦氏菌S18株和W9株的全基因组序列。在对全基因组内的功能基因进行注释后，构建这2株细菌各自的本地数据库，接着根据目前已报道的TMAO还原酶操纵子序列比对至已构建的数据库中，鉴定出S18株和W9株内均存在TMAO还原酶操纵子。.为分析S18和W9这2株细菌在腐败能力差异方面的分子机制，将它们在营养肉汤中增菌培养，分别在延滞期、对数生长期、稳定期这3个不同的生长阶段收集菌液，共6个样本。接着，提取这6个样本的总RNA，并进一步的富集mRNA，然后用RNA-Seq进行转录组测序，所得reads比对至拼接好的基因组，得到这2株细菌在不同生长阶段的差异表达基因。虽然，单纯比较差异表达基因无法有效筛选出希瓦氏菌内与腐败能力相关的基因，但是，对所有差异表达上调基因进行KEGG代谢通路分析，发现强腐败能力的S18株内参与核黄素、氨基酸、丙酮酸代谢、及TCA等代谢途径的表达要高于弱腐败能力的W9株，这说明希瓦氏菌的腐败能力可能主要与细胞内能量代谢有关，腐败能力越强，细胞内的能量代谢越旺盛。尽管弱腐败能力W9株内也鉴定到完整的TMAO还原酶操纵子，但其体内的能量代谢水平要弱于强腐败能力S18株；说明希瓦氏菌的强腐败能力并不是单纯由TMAO还原酶操纵子决定，而是由细胞的整体代谢水平决定，即希瓦氏菌促进水产品腐败是一个多基因调控的复杂的生化事件，其中TMAO还原酶操纵子是细菌腐败能力的充分不必要条件。