海洋酸化胁迫下三角褐指藻固碳途径中的差异表达基因研究

The results of meta-analysis showed that ocean acidification (OA) has significant effects on the process of algal growth and metabolism. To explore the molecular response mechanism of algae to OA, Phaeodactylum tricornutum was used as raw material, and digital gene expression profiling (DGE) technology was first performed to study the gene expression of P. tricornutum under the stress of OA. Sequence information of 1185 differentially expressed genes were obtained from the DGE library..Based on the above results and databases, methods of sequence alignment and bioinformatics analysis were further employed to mainly screen differentially expressed genes involved in the carbon fixation pathway of P. tricornutum under the stress of OA. And their expression patterns were studied by qRT-PCR under different stress conditions to validate and identify these genes for the carbon fixation of P. tricornutum. Furtherly, RACE or PCR technology was used to isolate 3-5 differentially expressed genes, which were subcloned into the expression vector (pFCP-gfp/CaMV-bar) with Xho I and Spe I digestion to replace the gfp gene, respectively. Subsequently, P. tricornutum was transformed via microparticle bombardment using the biolistic PDS-1000/He gene gun to improve its carbon fixation performance and resistant to elevated pCO2. After screening with basta, transgenic lines were selected to detect and analyze their characteristics of carbon fixation using the methods of Clark-type oxygen electrode and total carbon determination. At last, characteristics of carbon fixation between transgenic and control lines were compared and studied to confirm the biological functions of the differentially expressed genes..The results of this study may not only help to understand the molecular response mechanism of P. tricornutum and other algae to OA, but also provide a theoretical basis for further artificial regulation of algal carbon fixation and supply materials or new thoughts to our work of energy saving and environmental protection.

Meta分析表明海洋酸化显著影响藻类的生长代谢过程。为从分子水平上揭示藻类对海洋酸化的响应机制，申请人以三角褐指藻（Phaeodactylum tricornutum）为材料，首次利用数字基因表达谱技术分析了海洋酸化胁迫下P. tricornutum的差异表达基因，共获得1185个差异表达基因。.在此基础上，本项目拟通过序列比对和生物信息学分析，筛选、鉴定海洋酸化胁迫下P. tricornutum固碳途径中的差异表达基因，并通过研究其时相表达特征及P. tricornutum固碳量的变化规律，明确这些差异表达基因。然后，选取3-5个差异表达基因，构建转基因载体，采用基因枪法转化P. tricornutum，经筛选获得转基因藻株，分析其固碳特性，并鉴定基因功能。本研究有助于系统解析P. tricornutum对海洋酸化的分子响应机制，为人工调控藻类固碳奠定基础，也有助于其它藻类开展相关研究。

本项目利用数字基因表达谱技术对海洋酸化胁迫下三角褐指藻（Phaeodactylum tricornutum）中的差异表达基因进行分析，共获得差异表达基因1185个（727个为上调表达基因，458个为下调表达基因），其中，358个基因（36.0%）归为分子功能，462个基因（46.5%）归为生物进程，174个基因（17.5%）归为细胞组分。.聚类分析结果表明：P. tricornutum固碳途径中上调表达的差异基因主要有：参与C4途径的PPDK基因和PEPCK基因等，参与C2途径的SHMT基因和GK基因等，参与Calvin循环的Rbc L基因、GAPDH基因和TRA基因等，以及与碳转运相关的基因，如糖基转移酶基因、寡糖基转移酶基因、海藻糖6-磷酸-磷酸酶基因等；下调表达的差异基因主要集中在CA基因以及部分与能量代谢相关的基因上。利用qRT-PCR技术验证了海洋酸化胁迫下PtCA1、PPDK、PGK、Rbc L、GK和CoA等基因的表达情况。研究发现：除PtCA1和CoA基因的表达发生下调外，其它基因的表达均上调，其中PGK和Rbc L基因的上调表达幅度最高，分别为对照组的1.7倍和1.4倍。.利用RACE技术和电子克隆等手段，成功克隆了P. tricornutum的PtCA1、PPDK、PGK、SHMT、Rbc L、GK、CoA等基因的全长序列。利用基因枪法将PtCA1、PPDK、CoA等3个基因转入P. tricornutum中，经草丁膦筛选获得转基因P. tricornutum藻株。与对照组相比，转PtCA1和PPDK P. tricornutum的固碳速率提高了5.68和7.32%；而转CoA P. tricornutum的固碳速率却下降了2.35%。此外，转基因P. tricornutum的生理生化特性与原始藻株间没有显著差别，表明转基因操作未对P. tricornutum产生显著影响，但转基因藻株的固碳能力在培养200代后会有所降低，表明该转基因操作的稳定性还有待进一步提高。.在本项目的资助下，共发表学术论文11篇，其中SCI论文2篇，申报专利4件（授权专利1件），获得科研奖励3项，基本达到本项目预定的研究目标。