Screening miRNAs Related to Fatty Acid Metabolism in Oil Palm Mesocarp and Preliminary Analysis of Functions
|Keywords||oil palm fatty acid metabolism miRNA qRT-PCR target geneprediction|
Oil palm(Elaeis guineensis Jacq.) is one of the most efficient oil production plant in the world. The main products are palm oil and palm kernel oil, and the former is widely used in food, chemical industry and other fields. However, the regulatory mechanism of fatty acid metabolism in mesocarp of oil palm are still need to been clarified. MicroRNA(miRNA), size of which is19-25nt, is a kind of endogenous non-coding small RNA, regulating gene expression through targeting gene complementary sequence. Most of miRNA played many important roles during the plant growth, disease resistance, as well as the transformation. In our study, miRNA related to development of oil palm were screened and further verify their functions.To obtain high quantity small RNA from mesocarp of oil palm, CTAB method was improved and then employed to isolate small RNA from the mesocarp of oil palm nuts of five development stages(Gl-G5). Then,13miRNAs were screened from our previous database of oil palm small RNA using bioinformatics analysis. Relative expression of each miRNA was determined by stem-loop quantitative real-time PCR (qRT-PCR) and further predicted and verified the targets of these miRNAs. The results indicated that the OD260/OD280value of these small RNA are between1.8and2.0, and the concentration are:289ng/μL(G1),364ng/μL(G2),476ng/μL(G3),213ng/μL(G4),390ng/μL(G5), respectively. Relative expression detected by qRT-PCR showed that13miRNAs were exhibited significantly different expression during the five development stages, specially, high expression level in the fourth stage (G4) and the fifth stage(G5). Among them, egu-miR395and egu-miR156are the most highest in G4phase, egu-miR528are the most highest in G5. Result of bioinformatics analysis of targets prediction showed that all most of these13miRNAs have more than one predicted targets. Among then,3predicted targets were selected randomly and used to verified its fidelity by5’-RACE method. Three predicting targets were identified that MADS box transcription factor、transcription factor AP2D23-like and transcription factor APETALA2were really the target genes of egu-miR5179、egu-miR172-1, respectively. Then, analysing the functions of these miRNA in fatty acid metabolism regulation in mesocarp of oil palm preliminarily.Above all, these results suggested that some miRNAs, screened through the five development stages of oil palm, expressed significantly during these phases and might be relative to fatty acid metabolism. As we known, some targets of these differently expressed miRNAs are involved in the fatty acid metabolism pathway, such as the transcription factor APETALA2, phosphatidate phosphate, and phospholipase D. This study may provide some clues to further clarify the molecular mechanism of fatty acid metabolism regulation in mesocarp of oil palm.