油棕（Elaeis guineensis）中果皮发育过程中miRNA的表达动态分析

以CTAB法提取油棕（Elaeis guineensis）中果皮5个不同发育时期（G1～G5）的小RNA。从前期研究获得的油棕小RNA测序数据库中筛选12个候选miRNA,实时荧光定量PCR法（qRT-PCR）检测其在果实发育过程中的表达量变化,并进一步对显著差异表达的miRNA进行靶基因预测。结果表明：中果皮5个不同发育时期小RNA的OD260/OD280比值在1.7～2.0之间;浓度分别是289、364、476、213、390 ng/μL;qRT-PCR检测结果显示,12个候选miRNA在5个发育时期均显著性差异表达,特别是在中果皮发育第4个时期（G4）和第5个时期（G5）表达量极显著增高,其中miR395和miR156在第4个时期表达量最高;miR395和miR528在发育第5时期表达量最高;靶基因预测结果显示差异表达的部分miRNA,其靶基因可能参与了脂肪酸代谢通路,如磷脂酸磷酸脂酶和磷脂酶D。本研究筛选的与脂肪酸代谢相关的miRNA为今后油棕脂肪酸代谢调控通路研究提供了可能的线索。

Dynamic Expression Analysis of miRNAs during the Development Process of Oil Palm Mesocarp

We obtained high quantity small RNA from the mesocarp of oil palm（Elaeis guineensis） and screened fatty acid biosynthesis related miRNAs.The extracted method of CTAB was improved and then employed to isolate small RNA from the mesocarp of oil palm nuts at five development stages（G1-G5）.Twelve miRNAs were screened from our previous database of oil palm small RNA using bioinformatics analysis.Relative expression of each miRNA was determined by stem-loop real-time quantitative PCR（qPCR） and further predicted the targets of these miRNAs.The results indicated that the OD260/OD280 values of the small RNA were between 1.7 and 2.0,and the concentrations of G1 to G5 were 289 ng/μL,364 ng/μL,476 ng/μL,213 ng/μL,and 390 ng/μL,respectively.Relative expression detected by qPCR showed that the twelve miRNAs exhibited significantly different expression during the five development stages,especially high expression level in the fourth stage（G4） and fifth stage（G5）.Among them,miR395 and miR156 were highest in the G4 phase and miR395 and miR528 were highest in the G5 phase.Target prediction suggested that some targets of these differently expressed miRNAs were involved in the fatty acid metabolism pathway,such as phosphatidate,phosphatase and phospholipase D.In this study,high quantity small RNA was isolated by the improved CTAB method.qPCR further determined that the twelve candidate miRNAs were differently expressed during the five development stages,of which five miRNAs may be involved in the regulatory pathway of fatty acid metabolism.