油莎豆块茎油脂积累相关基因CeWRI1的克隆与功能分析

起源于非洲的油莎豆是迄今唯一已知在块茎中高水平积累油脂的新型草本油料作物。基于我国当前食用植物油和生物柴油原料供给紧张的局面,挖掘参与油莎豆块茎油脂积累的关键基因具有重要的理论意义和应用价值。WRI1（WRINKLED1）隶属于AP2/ERF转录因子家族,是一类被证实在油料种子发育过程中控制碳源由糖向油分配的关键基因。本研究采用RT-PCR技术从油莎豆的块茎中分离到一个WRI1同源基因（CeWRI1）,该基因的编码区为1116 bp,预测编码371 AA,其理论分子量为41.58 kDa,等电点为5.76,总平均疏水指数为-0.750,不稳定系数为60.75,细胞核定位,这与其转录调控功能是一致的。与拟南芥WRI1类似,序列分析显示CeWRI1含有2个保守的AP2结构域（PF00847）、1个VYL基序和1个14-3-3/BPM结合基序;相比AP2结构域和N端,其C端序列的变异较大,但包含与蛋白降解相关的PEST基序。qRT-PCR分析显示,CeWRI1在叶片、叶鞘、根、匍匐茎和块茎等主要组织中都有表达,且其在起始期、膨大初期、膨大中期、膨大晚期和成熟期等不同发育时期块茎中呈现先降后升的J型表达趋势,表达丰度最高的为成熟期,最低是膨大中期,这与油脂的积累模式大体一致。在烟草中的功能分析显示,CeWRI1的异源瞬时过表达可显著提高叶片的油脂（甘油三酯）含量,这进一步证实该基因具有油脂调控功能。上述结果表明CeWRI1是调控油莎豆块茎高水平积累油脂的关键基因之一,这不仅为进一步揭示油莎豆块茎油脂积累的调控机制奠定了坚实的基础,也为后期的品种改良提供了宝贵的基因资源。

Cloning and Functional Characterization of CeWRI1, a Gene Involved in Oil Accumulation from Tigernut (Cyperus esculentus L.) Tubers

Tigernut (Cyperus esculentus L.), which originates from Africa, is an herbaceous oil crop uniquely accumulating high level of oil in underground tubers. In view of the shortage of edible vegetable oil and biodiesel in China currently, it is of great significance to explore key genes involved in tuber oil accumulation. WRINKLED1 (WRI1), which encodes a transcription factor belonging to the AP2/ERF family, has been proven to be a key factor controlling carbon distribution from sugar toward oil during seed development of oil crops. In this study, a gene named CeWRI1 that is homologous to Arabidopsis thaliana WRI1 (AtWRI1) was successfully isolated from tigernut tubers by using the reverse transcription polymerase chain reaction (RT-PCR) technique. The gene, of an open reading frame 1116 bp, was predicted to encode 371 amino acids with the theoretical molecular weight (MW) of 41.58 kDa, the isoelectric point (pI) of 5.76, the grand average of hydropathicity (GRAVY) of -0.750, and the instability index (II) of 60.75, implying its hydrophilic and instable features. Subcellular localization analysis suggests that the CeWRI1 protein is located in the nucleus, corresponding to its transcriptional regulatory function as a transcription factor. As observed in AtWRI1, sequence analyses show that CeWRI1 harbors several conserved structural features, i.e. two AP2 domains (PF00847), one VYL motif, and one 14-3-3/BPM-binding motif. Compared with the N terminus and AP2 domains, the C-terminal sequences of CeWRI1 are relatively variable, though a PEST motif associated with protein degradation was found. Quantitative real-time PCR (qRT-PCR) analysis reveals that CeWRI1 is expressed in all tested tissues, i.e. leaf blade, sheath, root, rhizome, and tuber; during various stages of developmental tuber, i.e. initial, early swelling, middle swelling, late swelling, and mature stages, a typical J-shape expression pattern was observed, peaking at the mature stage and minimizing at the middle swelling stage, which is generally consistent with the accumulation pattern of oil. Transient over-expression of CeWRI1 in tobacco (Nicotiana tabacum) leaves significantly increased the triacylglycerol (TAG) content, supporting its role in oil accumulation. Taken together, our data suggests that CeWRI1 is most likely to be one of the key genes controlling high oil accumulation in tigernut tubers, which would not only lay a solid foundation for further uncovering the regulatory mechanism of tuber oil accumulation, but also provide a valuable resource for genetic improvement in tigernut and species beyond.