The Plant Journal:中科院青岛能源所徐建研究组等建立工业产油微藻
2016年11月7日，国际植物学权威期刊《The plant Journal》在线发表了中国科学院青岛生物能源与过程研究所徐健研究组与中国科学院水生生物研究所胡汉华研究组合作的一篇研究论文，研究报道了研究组建立的工业产油微藻基因敲低技术。
微藻通过光合作用将二氧化碳、光和水转化为油脂，因此，作为一种潜在的清洁能源生产和二氧化碳高值化方案，工业产油微藻受到了广泛关注。然而，藻类高效遗传工具的匮乏，一直是工业产油微藻分子育种和光驱固碳合成生物技术的重要瓶颈之一。研究以微拟球藻为模式，率先建立了工业产油微藻基因敲低技术。
微拟球藻是一种可利用海水或淡水、在室外大规模培养的工业微藻，具有生长速度快、二氧化碳耐受能力强、强劲积累油脂和高值不饱和脂肪酸等优点，因此已经成为能源与经济微藻领域的研究模式藻种之一，也成为国内外许多微藻固碳示范工程的优先选择。然而反向遗传工程技术的匮乏从根本上阻碍了针对二氧化碳固定能力和产油效率等诸多工业性状的遗传改造。
单细胞中心博士后学者魏力带领的研究小组在海洋微拟球藻(Nannochloropsis oceanica IMET1)中，基于RNA干扰技术，通过设计靶基因的反向重复序列，形成特异茎环结构，实现了碳酸酐酶和碳酸氢盐转运蛋白等碳代谢相关基因的高效、特异性敲低，基因沉默株构建的成功率达到40%以上。通过重亚硫酸测序方法，发现在基因沉默株中，靶基因的特定区域展示出独特的甲基化规律，从而揭示了靶基因沉默的分子机制。研究人员还证明该技术对于另一藻株N. oceanica CCMP1779也同样高效，表明该技术具有通用性。
相关阅读：Plant Journal:中科院青岛能源所徐健研究组建立工业产油微藻基因组编辑技术
工业产油微藻基因敲低技术
原文链接：
RNAi-based targeted gene-knockdown in the model oleaginous microalgaeNannochloropsis oceanica
原文摘要：
Microalgae are promising feedstock for renewable fuels such as biodiesel, yet development of industrial oleaginous strains has been hindered by the paucity and inefficiency of reverse genetics tools. Here we established an efficient RNAi-based targeted gene knockdown method for Nannochloropsis spp., which are emerging model organisms for industrial microalgal oil production. The method achieved a 40~80% success rate in N. oceanica strain IMET1. When transcript level of one carbonic anhydrase (CA) was inhibited by 62~83% via RNAi, mutant cells exhibited photosynthetic oxygen evolution (POE) rates that are 68~100% higher than wild type (WT) at pH 6.0, equivalent to WT at pH 8.2, yet 39~45% lower than WT at pH 9.0. Moreover, the mutant POE rates are negatively correlated with the increase of culture pH, an exact opposite of WT. Thus a dynamic carbon concentration mechanism (CCM) that is highly sensitive to pH homeostasis was revealed, where theCA inhibition likely partially abrogated the mechanism that normally deactivates CCM under high level of dissolved CO2. Extension of the method to another sequenced N. oceanica strain of CCMP 1779 demonstrated comparable performance. Finally, McrBC-PCR followed by bisulfite sequencing revealed that the gene knockdown is mediated by the CG, CHG and CHH types of DNA methylation at coding region of the targeted gene. The efficiency, robustness and general applicability of this reverse genetics approach suggested the possibility of large-scale RNAi-based gene function screening in industrial microalgae.
作者：徐建
