油棕甾醇合成关键酶基因DWF5的克隆及表达分析

DWARF5（DWF5）基因,编码7-脱氢胆固醇还原酶（7-dehydrocholesterol reductase）,是甾醇合成的关键酶,通过调控甾醇的合成参与植物油体（oil bodies,OBS）的形成。为了探究DWF5基因对油棕果肉中油体发育的影响,以‘热油4号’为材料,提取油棕果肉的RNA,以反转录的cDNA为模板进行基因的全长克隆,获得2个EgDWF5基因并分别命名为EgDWF5-1和EgDWF5-2。运用生物信息学的方法对其理化性质、结构特点和亲缘关系等进行分析预测,利用实时荧光定量PCR对其在油棕的根、茎、叶、花及花后不同时期的果实进行表达水平分析。结果表明：EgDWF5-1和EgDWF5-2基因编码的氨基酸数量分别为434个和374个,相对分子质量为49.71 kDa和42.68 kDa,等电点为8.61和8.69,其蛋白不稳定指数为38.60和38.13,脂肪族指数为98.80和96.68,总平均亲水性为0.347和0.313,均具有疏水性。EgDWF5与水稻和玉米亲缘关系最近,与其他物种关系较远。qRT-PCR分析表明,EgDWF5基因在油棕的各个器官均有表达,其中EgDWF5-1基因在果肉中高表达,显著高于根、叶、花等部位,其表达趋势为花后12~18周升高,至20周时达到最高后表达量逐渐降低,这与油棕果肉中油体的发育趋势基本一致。EgDWF5-2基因在根和茎中高表达而在果肉中表达量低,其果肉中表达趋势为花后12~18周逐渐降低。因此,推测EgDWF5-1参与调控油棕的油体发育,EgDWF5-2未参与该调控而可能在油棕的生长发育中发挥功能。本研究为进一步探索EgDWF5基因调控油棕中油体的形成机制奠定基础。

Cloning and Expression Analysis of DWF5 Genes Involved in the Sterol Synthesis in Oil Palm

DWARF5 (DWF5) encodes 7-dehydrocholesterol reductase, which is a key enzyme in the synthesis of sterols and participates in the formation of plant oil bodies (OBS) by regulating the synthesis pathway of sterols. The cultivar, ‘Reyou No. 4’, was taken as the experimental material to explore the effects of DWF5 gene on the development of OBS in the mesocarp of oil palm. After extracting RNA from mesocarp, the reverse transcription cDNA was used as a template to conduct full-length cloning of genes. Two EgDWF5 genes obtained were named EgDWF5-1 and EgDWF5-2. The bioinformatics method was applied to analyze and predict physicochemical properties, structural characteristics, and genetic relationship, etc.. The expression levels of the genes in roots, stems, leaves, flowers, and fruits at different stages after flowering were analyzed by quantitative real-time PCR. The results showed that the number of amino acids encoded by the EgDWF5-1 and EgDWF5-2 gene was 434 and 374, respectively, the relative molecular masse was 49.71 kDa and 42.68 kDa, the isoelectric points was 8.61 and 8.69, the protein instability indexes was 38.60 and 38.13, and the aliphatic index was 98.80 and 96.68. The gravy was 0.347 and 0.313. Both proteins were hydrophobic. EgDWF5 has the most closed genetic relationship with rice and maize, but less related to other species. The qRT-PCR analysis showed that the two EgDWF5 genes were expressed in all organs of oil palm, among which EgDWF5-1 was highly expressed in mesocarp, which was significantly higher than that in roots, leaves, flowers and other parts. The expression trend of EgDWF5-1 gene increased from 12 to 18 weeks after flowering and reached the peak at 20 weeks after flowering, and then gradually decreased, which was consistent with the synthesis trend of the OBS in the oil palm mesocarp. EgDWF5-2 gene was highly expressed in roots and stems, but the expression level of it was low in mesocarp, and its expression trend in mesocarp gradually decreased from 12 to 18 weeks after flowering. Therefore, it is speculated that EgDWF5-1 is involved in the regulation of development of OBS in oil palm, while EgDWF5-2 is not involved in this regulation and may play a role in the growth and development of oil palm. This study would lay a foundation for further exploring the mechanism of EgDWF5 regulating OBS formation in oil palm.