RNA干扰UGP基因表达对三角褐指藻碳流分配的影响

Effectof RNA Interference of UGP-Glucose Pyrophosphorylase Gene Expression Oncarbon Allocation in Phaeodactylum tricornutum

为深入研究RNA干扰尿苷二磷酸葡萄糖焦磷酸化酶(UGPase)基因的表达对三角褐指藻(Phaeodactylum tricornutum Bohlin)碳流分配的影响,本研究以RNA干扰UGP表达的三角褐指藻为对象,测定了野生和转基因藻株的叶绿素荧光参数和生物量,分析了它们的脂肪酸组成和金藻昆布多糖、总脂及蛋白质的含量。研究显示,转基因藻株的最大光能转化效率(Fv/Fm)从指数生长后期开始显著低于野生型,但野生和转基因藻株的终生物量无明显差别。转基因藻株(AS、IR)的C16与C18之和分别占总脂肪酸含量的66.90%和68.89%,比野生藻株分别提高了19.40%和22.97%,说明抑制UGP表达改变了三角褐指藻的脂肪酸组成。转基因藻株的金藻昆布多糖、总脂及蛋白质含量与野生型藻株差别明显:藻株AS的金藻昆布多糖含量降低25.70%,蛋白质含量上升3.80%,总脂含量提高了13.2%;藻株IR的金藻昆布多糖含量降低40.70%,蛋白质含量上升8.30%,总脂含量增加了25.20%。研究结果表明,RNA干扰UGP的表达改变了三角褐指藻的脂肪酸组成,使得在最终生物量无明显减少的情况下,光合作用固定的碳更多转向油脂合成。本研究进一步明确了RNA干扰UGP的表达对三角褐指藻碳流分配的影响机制,为通过基因工程改造藻种提高微藻的生物柴油生产潜力提供了新思路。

Diatom are unicellular, photosynthetic and eukaryotic microalgae, which are responsible for around 40% of the total carbon fixation in oceans. Chrysolaminaran is the main energy storage polysaccharide of diatom. To date, the biosynthetic pathway of chrysolaminaran has not been fully elucidated in Phaeodactylumtricornutum. This species has a fully sequenced genome and a perfect transgenic platform, making it feasible to exogenously trigger RNA-mediated silencing in this alga. UDP-glucose pyrophosphorylase （UGPase） catalyzes the reversible production of UDP-glucose and pyrophosphate （PPi） from glucose-1-phosphate （G-1-P） and UTP. It is a key enzyme in carbohydrate metabolism, playing an important role in carbon partitioning. In order to study the mechanism of UGPase in regulating carbon partitioning, the contents of chrysolaminarin, protein and total lipid were measured in transgenicP, tricornuturnwith the expression UGP genesuppressed, and compared with those of the wild. The measured and compared included also chlorophyll fluorescence parameter and fatty acid composition. Our results showed that maximal photochemical efficiency （Fv/Fm） of transgenic P. tricornutum was significantly lower than that of the wild after the eighth day of cultivation, but their biomass was not significantly different. The C16 and C18 series contents of the two transgenic strains （AS and IR） accounted for 66.9% and 68. 89% of the total, increased by 19. 40% and 22. 97% in comparison with those of the wild, respectively. Our findings indicated that suppression of UGP gene changed the fatty acid metabolism ofP. tricornutum. Chrysolaminarin, protein and total lipid of transgenic strains （AS and IR） were significantly different from those of the wild. The content of chrysolaminarin decreased by 25.70%, and the contents of protein and total lipid increased by 3.80% and 13.20% in transgenic strain AS. Nevertheless, the content of chrysolaminarin decreased by 40. 70%, the contents of protein and total lipid increased by 8.30% and 25. 2% in IR. The results of the study showed that RNA interference on UGPgene expression had altered carbon allocation and fatty acid composition ofP. tricornutum. Biomass changed little; however more carbon captured during photosynthesis had turned to lipid synthesis. This study has further revealed the carbon allocation mechanism of RNA interference on UGP gene expression in P. tricornutum, which will provide a novel avenue for improving the total lipid in microalgal cellsand lay the foundation for a complete understanding of the chrysolaminarin biosynthesis pathway.