基于转录组分析的小球藻同步生长与产油分子基础研究

All the scale-up cultivation and lipid regulation researches of oleaginous microalgae are based on batch cultivation strategies up to now. The cell growth and lipid accumulation are divided in two stages in batch culture, which results in lower total lipid productivity. The applicant laboratory have established chemostat cultures for continuous concurrent production of biomass and lipids in Chlorella pyrenoidosa XQ-20044 successfully for the first time, by regulate the threshold values of nitrogen dilution rate (supply rate). Continuous lipid production and high lipid productivity were realized in this developed cultivation system. It is very interesting and unclear that the molecular basis of steady-state in algal cells, which is also the key technique of continuous cultivation strategy. Based on above-mentioned, this study will analyze C. pyrenoidosa XQ-20044 cells which in steady-state using comprehensive methods, including transcriptome, gene clone and RT-qPCR techniques, etc., to explore the molecular basis of simultaneous microalgae growth and lipid production, find the coordination and balance model of lipid synthesis pathway with cell growth and other metabolic pathways. The results will increase our understanding of continuous cultivation deeply, and improve the lipid regulation theory of oleaginous microalgae.

目前，产油微藻培养及油脂调控研究都是基于分批培养方式，微藻的生长和产油在两个阶段分别进行，限制了微藻产油的潜力。申请人所在团队利用连续培养方式，通过控制氮源的“比输入速率”，首次使小球藻Chlorella pyrenoidosa XQ-20044达到并维持同步生长与产油的稳态，大幅提高了油脂产率。“藻细胞同步生长与产油的分子基础”这一问题的阐明将为微藻产油调控机理研究开辟一条新途径。本申请拟以C. pyrenoidosa XQ-20044为材料，通过比较三种典型的稳态，即同步高生长高油脂积累、低生长高油脂积累和高生长低油脂积累的转录组，从转录水平阐释油脂合成与生长及其它代谢的平衡机制。在此基础上，对稳态建成过程中高度相关基因的表达模式和相应的生理生化响应进行比较，探索同步生长与产油的分子机制，为深入研究微藻油脂调控机制奠定基础，也为建立高效的微藻产油模式提供理论依据。

以处于不同细胞稳态的小球藻Chlorella pyrenoidosa XQ-20044为材料,本研究采用转录组RNA-seq、microRNA (miRNA)测序、荧光定量PCR等技术,较为系统的阐明了藻细胞同步生长与产油的分子机理。首先,分别通过0.24,0.96和1.72 d-1的稀释速率恒化培养,成功建立了低生长高油脂积累(L)、同步高生长高油脂积累(M)和高生长低油脂积累(H) 3种典型稳态。M稳态具有最高的油脂产率(135.6 mg L-1d-1),在稳态的建立过程中能够保持较高的光合速率,脂类合成限速基因DGAT表达量显著上调8倍左右。ACCase和AGPase在3种稳态建立和维持过程中,表达量无显著变化。PEPC的表达量在L稳态中上调3倍,在M和H稳态中无显著变化。在此基础上,对上述3种稳态进行了UMI-绝对定量转录组测序和分析。结果显示M稳态相对于L和H稳态,差异表达基因主要集中在脂类合成、光合作用、氮代谢等代谢通路。M稳态氮利用相关基因(NR、NT)具有最高的表达量,脂类合成等通路基因的表达量显著高于H稳态低于L稳态,光合作用等生长相关基因表达量显著高于L稳态低于H稳态。进一步对3种稳态miRNA测序和分析显示,M和L、H稳态的17个差异表达miRNA靶基因主要集中在脂肪酸代谢和光合作用途径。6个miRNA, 即MIR2916，MIR11514，MIR857，MIR5139，MIR2916，MIR9560b与C.pyrenoidosa XQ-20044同步生长与产油状态的维持和调控密切相关。基于此,我们认为C. pyrenoidosa XQ-20044同步高生长高油脂积累在机制上可以被解释为:藻细胞受到一定程度的氮胁迫，细胞在转录水平上调了脂类合成相关基因，促进了脂质的合成与积累，同时光合作用等生长相关通路受抑制程度较低，能够保持相对较高的生长速率。此外,在项目资助下,我们对具规模化潜力的产油小球藻Chlorella sp. GA1进行了基因组测序,首次阐明了微藻进化过程中的全基因组加倍事件;以Graesiellasp. WBG-1为材料,在微藻产油与固碳一体化方面进行了探索研究。本项目研究结果不仅是对微藻同步生长与产油模式分子机制的阐明和进一步深入研究的基础，也是对目前微藻油脂积累调控机理新的认识与补充。