细胞壁相关的莱茵衣藻光合产氢代谢基因表达调控网络的转录组学研究

Chlamydomonas reinhardtii is the model species for study of photosynthetic hydrogen production of green algae. Its hydrogen production metabolism is a complex network-regulated progress involved by multiple bio-pathways. The hydrogen yield of a cell wall-deficient algal strain cc849 is 5-7 times less than that of strain cc124, which has normal cell wall and common ancestry of strain cc849 of C. reinhardtii. A mutant T1 with recovered hydrogen yield was screened from random insertion mutagenesis of the nuclear genome of strain cc849 with antibiotic selection marker, ble gene. Transcript is the key step for the gene expression and regulation. In this study, the new generation sequencing method will be used to sequence the transcriptome of above three species, which have clear genetic background but with significantly different hydrogen yield, at different hydrogen production phases and modern bioinformatics software will be used to compare the differentiation of kinetic transcription of different algal species at different hydrogen production phases. To reveal the mechanisms of low hydrogen yield of the cell wall-deficient strain cc49 as well as the high hydrogen yield of mutant T1, physiological phenotype and further biological molecular experiments will also be used to determine the biological function of the key differential expressed genes according to above transcriptome sequencing analysis and the mechanism of metabolic regulation network for biological hydrogen production of C. reinhardtii will be illustrated. The results in this wok will direct the creation of engineered algal strain of C. reinhardtii with high yield of hydrogen production in the future by using genetic engineering approaches.

莱茵衣藻是研究绿藻光合制氢技术的模式物种，其产氢代谢是多途径参与的复杂网络调控过程。细胞壁结构缺失型莱茵衣藻藻株cc849的产氢量比有正常细胞壁、且遗传背景起源相同的莱茵衣藻藻株cc124的低5-7倍；对藻株cc849细胞核随机插入抗生素基因ble进行诱变，筛选到一个产氢量恢复的突变株T1。转录是研究基因表达调控的关键步骤。本项目采用第二代高通量测序技术对上述三个遗传背景清晰、产氢量明显不同的莱茵衣藻藻株在不同产氢时期的转录组分别深度测序，利用现代生物信息学软件分析不同藻株在不同产氢条件下的动态转录差异，进一步结合生理表型和分子生物学实验分析差异表达基因的生物学功能，并对其所参与的信号通路进行网络整合，在转录水平上揭示细胞壁结构缺失型藻株产氢量低和突变株T1产氢量高的分子机理以及莱茵衣藻产氢代谢网络调控的分子机制，为利用基因工程手段构建高效工程藻株奠定基础。

莱茵衣藻在缺氧条件下启动产氢特殊代谢，以获得维持细胞生存的能量，是研究绿藻生物制氢技术和缺氧胁迫适应的模式物种，揭示其缺氧适应的分子机制和影响产氢代谢的关键因素，具有重要的应用价值和科学意义。本项目选取了三株遗传背景起源相同、产氢量明显不同、细胞壁关联的莱茵衣藻藻株为研究对象，采用第二代高通量测序技术，对三个藻株开展不同产氢时期的转录组学比较研究，系统而全面绘制了莱茵衣藻缺氧适应和产氢代谢的转录组学特征图谱，共得到15000多个unigene，鉴定了4436个差异表达基因，其中约80%基因参与了缺氧胁迫适应和产氢代谢，约39%基因在缺氧胁迫下显著下调表达，功能集中在光合作用、代谢物前体和能量生成、碳水化合物催化及代谢过程、含氮化合物代谢及生物合成过程、氧化磷酸化、离子跨膜运输、次生代谢、核苷酸代谢及生物合成过程、蛋白水解催化过程、铁硫簇结合和金属簇结合、细胞内结构、蛋白复合体、膜系统等功能；约38.9%基因为上调表达，其中与产氢代谢正相关的功能集中在核糖体生物合成、特定氨基酸和小分子生物合成过程、硫代谢、基因表达调控、细胞大分子生物合成过程、运输等功能；在缺氧胁迫后期上调表达脂肪酸降解、不饱和脂肪酸合成、蛋白泛素化降解等过程。.定位了突变株T1中ble基因插入位点为ITN7（Importin beta）基因内含子区。比较了三个藻株在不同产氢时期的差异基因表达特征和细胞壁代谢相关基因表达特征，并与产氢量进行相关性分析，揭示了在缺氧胁迫条件下莱茵衣藻通过糖酵解-TCA循环-乙醛酸循环-PPP途径、能否顺利调控代谢中间产物进入特定氨基酸合成代谢及脂肪酸代谢，使代谢产物和能量流向产氢代谢，是影响衣藻产氢量的重要因素，间接影响淀粉、细胞壁等存储碳源的利用。首次发现精氨酸甲基化和FtsJ家族甲基转移酶的表观遗传修饰在莱茵衣藻产氢代谢过程中发挥重要作用。.本研究结果为深入揭示细胞缺氧适应的分子机制、表观调控和利用基因工程手段构建高效工程藻株奠定了理论和实验基础。