Wei Li, Xin Yi, Wang Qintao, Yang Juan, Hu Hanhua, Xu Jian
Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Plant J. 2017 Mar;89(6):1236-1250. doi: 10.1111/tpj.13411. Epub 2017 Feb 11.
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 Nannochloropsis 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 were 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 were 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 the CA inhibition likely partially abrogated the mechanism that normally deactivates CCM under a high level of dissolved CO . 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 the 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.
微藻是生物柴油等可再生燃料的理想原料，但反向遗传学工具的匮乏和低效阻碍了工业产油菌株的开发。在此，我们为海洋微拟球藻建立了一种基于RNA干扰的高效靶向基因敲低方法，海洋微拟球藻是工业微藻产油新兴的模式生物。该方法在海洋微拟球藻IMET1菌株中的成功率达到40%-80%。当通过RNA干扰使一种碳酸酐酶（CA）的转录水平被抑制62%-83%时，突变细胞在pH 6.0时的光合放氧（POE）速率比野生型（WT）高68%-100%，在pH 8.2时与WT相当，但在pH 9.0时比WT低39%-45%。此外，突变体的POE速率与培养pH的升高呈负相关，这与WT正好相反。因此，揭示了一种对pH稳态高度敏感的动态碳浓缩机制（CCM），其中CA抑制可能部分消除了在高溶解CO水平下通常使CCM失活的机制。将该方法扩展到另一个测序的海洋微拟球藻CCMP 1779菌株，表现出类似的性能。最后，McrBC-PCR随后进行亚硫酸氢盐测序表明，基因敲低是由靶向基因编码区的CG、CHG和CHH类型的DNA甲基化介导的。这种反向遗传学方法的效率、稳健性和普遍适用性表明了在工业微藻中基于RNA干扰进行大规模基因功能筛选的可能性。
