模式蓝细菌Synechocystis PCC 6803中响应高盐胁迫的假定蛋白基因功能解析

The genome of model cyanobacterium, Synechocystis sp. PCC 6803, encodes 46% hypothetical proteins. The large proportion of unknown genes become one of the great challenges for both cyanobacteria genomic research and further understanding cellular metabolism and protein of microorganisms.This proposal aims to explore the characterization of hypothetical proteins responsive to salt stress in Synechocystis PCC 6803. In the study, preliminary efforts will: i) systematically assess authenticity of the hypothetical proteins in the Synechocystis genome under salt stress condition using a quantitative proteomics approach with isobaric tag for relative and absolute quantification (iTRAQ) technique and liquid chromatography–tandem mass spectrometry (LC–MS/MS); ii) establish gene expression networks of Synechocystis using proteomic and transcriptomic data collected from salt stress condition using a Weighted Gene Co-expression Network Analysis (WGCNA) approach, so that the possible relationship of hypothetical proteins with functionally known proteins and salt stress can be inferred; iii) identify the functions of 3-5 selected core hypothetical proteins with connections with more than 10 genes in salt stress correlated functional modules detected in WGCNA,using high throughput gene knockout and konckin techniques previously developed in our lab, which may represent important core stress response in Synechocystis; The analysis will result in functional assignments to stress-responsive core hypothetical proteins in the Synechocystis genome. The study will provide an improved annotation for Synechocystis PCC 6803, and also demonstrat that high-throughput methodologies, such as mass spectrometry-based proteomics and transcriptional networks using transcriptome datasets, could be good supplementations in deciphering function of hypothetical proteins.

模式蓝细菌Synechocystis PCC 6803基因组中有高达46%的基因编码未注释功能的假定蛋白(HP), 如此大量的功能不明HP成为对蓝细菌进行深入理论研究和应用开发的严重障碍。采用传统实验手段确定HP功能会消耗大量的人力和时间，而新开发的权重基因调控网络分析方法（WGCNA）被证实能更高效地用于基因调控网络构建、功能模块抽提及核心基因鉴定。基于此，本申请提出模式蓝细菌Synechocystis HP基因功能解析的新策略：以常见的高盐胁迫处理蓝细菌，获取蛋白组和转录组数据，利用WGCNA方法构建基因调控网络，筛选出高盐胁迫响应相关HP基因；并结合基因敲除或过表达突变株的表型分析鉴定高盐胁迫响应的核心HP的功能。研究结果有助于阐明Synechocystis PCC 6803适应高盐胁迫的机制，也为其它蓝细菌中众多HP基因的功能解析提供可借鉴的新思路及有效解决途径。

模式蓝细菌Synechocystis sp. PCC6803是最早完成基因组测序的单细胞蓝细菌，而且含有多种生物活性物质，然而其大量功能不明的假定蛋白（HP）是对蓝细菌Synechocystis进行应用开发和深入理论研究的严重障碍。本项目首先以高盐胁迫处理蓝细菌Synechocystis PCC 6803，对其进行了长达303天的高盐（3%）实验室驯化，成功获得了多株耐受高盐的Synechocystis PCC 6803驯化株，这些驯化株不仅为探究其耐盐及逆境机制提供实验材料，而且能为用海水培养基培养Synechocystis PCC 6803奠定基础，预期在大规模培养时可节约大量淡水。对这些高盐驯化株的代谢组和转录组进行了检测，并用多种生物信息学手段对代谢组和转录组数据进行了分析，构建了代谢物组成模块，鉴定了核心基因，分析了这些驯化株具不同适应高盐胁迫的机制。.进一步研究发现，和高盐胁迫实验室驯化能提高耐盐能力不同，缺氮实验室驯化不能提高Synechocystis PCC 6803的耐缺氮能力。生物抗胁迫能力与DNA甲基化修饰息息相关，但相关的研究尤其是在Synechocystis PCC 6803中的研究甚少。本项目利用全基因组亚硫酸盐测序（WGBS）技术首先研究了缺氮胁迫或丁醇胁迫对Synechocystis PCC 6803 DNA甲基化修饰模式的影响，发现它处于缺氮胁迫时的DNA甲基化修饰模式与正常培养时不同，并且由缺氮胁迫诱导的DNA甲基化修饰模式可在氮源恢复培养后继续保留，胁迫诱导的DNA甲基化修饰模式能跨代遗传。此外，通过对Synechocystis PCC 6803中三个DNA甲基化酶的敲除以及突变株的表型分析，分析了DNA甲基化与SynechocystisDNA重组、基因表达、光合作用等重要生理过程之间的关联，用权重基因调控网络分析方法（WGCNA）初步构建了基因调控网络，筛选出部分与高盐或缺氮胁迫响应相关的HP基因，但未能分离出HP相应的纯合缺失突变株。该结果将为Synechocystis PCC 6803中DNA甲基化及HP蛋白的深入解析提供基础，有助于我们理解Synechocystis中胁迫响应、转录调控等关键生理过程，并为Synechocystis的定向分子改造提供新的思路，也为其它蓝细菌中众多HP基因的功能解析提供参考。