莱茵衣藻E2泛素结合酶CrUBC23调控油脂积累

Ubiquitin-conjugating enzyme(E2)CrUBC23 regulates lipid accumulation in Chlamydomonas reinhardtii

【目的】为研究莱茵衣藻(Chlamydomonas reinhardtii)泛素结合酶(ubiquitin-conjugating enzymes,E2)CrUBC23在莱茵衣藻油脂代谢中的作用,为高产油微藻基因工程改良和揭示藻类油脂合成及代谢调控机理奠定基础。【方法】qRT-PCR分析莱茵衣藻在低氮、低磷胁迫下泛素结合酶CrUBC23表达情况;克隆CrUBC23同源基因干涉片段和全长基因,构建RNAi干涉载体和过量表达载体,转化莱茵衣藻并检测生物量和油脂含量;构建CrUBC23-GFP融合表达载体,用农杆菌浸染洋葱表皮细胞进行亚细胞定位。【结果】莱茵衣藻在低氮、低磷胁迫下CrUBC23基因表达量显著增加,增加幅度分别为正常培养的4.98–5.80倍和1.85–5.20倍。RNAi干扰结果显示,转基因藻细胞中性脂含量降低5.5%,总脂含量降低3.16%–17.6%。过量表达结果显示,转基因藻细胞中性脂含量增加8.8%,总脂含量增加4.51%–14.03%。【结论】CrUBC23正向调控莱茵衣藻油脂代谢,该基因定位于细胞核。

[Objective]To study the lipid metabolism mechanism regulated by Ubiquitin-conjugating Enzyme(E2)CrUBC23 in Chlamydomonas reinhardtii.[Methods]The mRNA levels of ubiquitin-conjugating enzyme CrUBC23 were analyzed by qRT-PCR under low nitrogen and low phosphorus conditions in C.reinhardtii.Fragments for RNAi interference and over-expression of CrUBC23 gene were cloned,and then the RNA interference vector and over-expression vector were constructed and transformed into C.reinhardtii.The biomass and lipid content of the transgenic strain was detected.The fusion expression vector CrUBC23-GFP was constructed also,and the subcellular localization of onion epidermal cells was carried out by using Agrobacterium tumefaciens.[Results]The level of RNA in C.reinhardtii were increased significantly under low nitrogen and low phosphorus conditions,with an increase of 4.98–5.80 times and 1.85–5.20 times as much as that in normal culture,respectively.RNA interference results showed that the neutral lipid content and total lipid content of transgenic algae decreased by 5.5%and 3.16%–17.6%.While,over-expression showed that the neutral lipid content increased by 8.8%and the total lipid content increased by 4.51%–14.03%.[Conclusion]CrUBC23 is a positive regulator in lipid metabolism,and may locate in the nucleus.