Key Laboratory of Biofuels, Key Laboratory of Shandong Energy Biological Genetic Resources, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Int J Mol Sci. 2020 Mar 19;21(6):2106. doi: 10.3390/ijms21062106.
Eukaryotic filamentous yellow-green algae from the genus are considered to be excellent candidates for biofuels and value-added products, owing to their ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids. To elucidate the molecular mechanism of fatty acids and/or establish the organism as a model strain, the development of genetic methods is important. Towards this goal, here, we constructed a genetic transformation method to introduce exogenous genes for the first time into the eukaryotic filamentous alga via particle bombardment. In this study, we constructed pSimple-- and pEASY-- plasmids in which the green fluorescence protein () gene and the neomycin phosphotransferase Ⅱ-encoding G418-resistant gene () were flanked by the -derived tubulin gene () promoter and terminator, respectively. The two plasmids were introduced into cells through particle-gun bombardment under various test conditions. By combining agar and liquid selecting methods to exclude the pseudotransformants under long-term antibiotic treatment, plasmids pSimple- and pEASY- were successfully transformed into the genome of , which was verified using green fluorescence detection and the polymerase chain reaction, respectively. These results suggest new possibilities for efficient genetic engineering of for future genetic improvement.
真核丝状黄绿藻属的藻类被认为是生物燃料和高附加值产品的优秀候选者，因为它们能够在自养、混合营养和异养条件下生长，并合成大量的脂肪酸，特别是不饱和脂肪酸。为了阐明脂肪酸的分子机制或建立该生物作为模式菌株，开发遗传方法很重要。为此，我们首次通过粒子轰击构建了一种将外源基因引入真核丝状藻的遗传转化方法。在这项研究中，我们构建了 pSimple- 和 pEASY- 质粒，其中绿色荧光蛋白（GFP）基因和新霉素磷酸转移酶Ⅱ编码的 G418 抗性基因（G418R）分别被 - 衍生的微管蛋白基因（TUB）启动子和终止子侧翼。这两个质粒通过粒子枪轰击在各种测试条件下被引入到细胞中。通过结合琼脂和液体选择方法，在长期抗生素处理下排除假转化体，我们成功地将 pSimple- 和 pEASY- 质粒转化到的基因组中，这分别通过绿色荧光检测和聚合酶链反应得到了验证。这些结果为未来的遗传改良提供了对进行高效遗传工程的新可能性。
