Ectopic Expression Of MicroRNA5179 From Oil Palm(Elaeis Guineensis Jacq.) Alters Secondary Metabolites Biosynthesis And Glycerolipid Accumulation In Arabidopsis Thaliana

Oil palm(Elaeis guineensis Jacg.),which is a perennial monocotyledonous plant belonging to the Arecaceae palm family,is the most productive oil-bearing crop in tropical area.In China,oil palm distrubuted wildly in Hainan province,which is most important cash crop in that area.However,there were rarely reports about molecular regulation mechanism of microRNA in oil palm.MicroRNAs(miRNAs)are small,endogenous,non-coding RNAs that regulate eukaryotic gene expression post-transcriptionally.Plant miRNAs are vital components of the translation control system that regulates plant development and reproduction.By the previous studies of deep sequencing of oil palm mesocarp,we studied Eg-miR5179 expression level during 5 developmental stages,results showed that significant high expression in phase 5.These results are consistent with the accumulation of lipid in oil palm mesocarp.To analyze the function of Eg-miR5179 in Arabidopsis,the research contents are as follow:1.Generating an amiRNA vector(Eg-miR5179)based on the structure of Arabidopsis miR319a precursor(pre-miR319a).The Arabidopsis thaliana pre-miR319a sequence was used as backbone for the construction of amiRNA expression vectors.Through oligonucleotide-directed mutagenesis,the natural miR319 and miR319*sequences were replaced with synthetic sequences.The binary vectors were then transformed into Agrobacterium tumefaciens strain EHA105.Arabidopsis transformation was conducted by using the floral dip method.T1 generation Arabidopsis seeds were selected on 1/2 MS medium supplemented with 50 mg/mL hygromycin to obtain the transgenic plants.Real-time fluorescent quantitative PCR analysis indicated that the Eg-miR5179 expression level was most abundant in leaf(>3000 fold)while in flower and silique were 2-10 fold.Meanwhile,we observed the flowering time and its structure due to previous studies in other microRNAs were related to morphologic development in flower.The results exhibited transgenic plants have advanced flowering time than wild plants while there are no differences in flower structure between them.2.To further understand the Eg-miR5179 impact on growth and development in Arabidopsis,we use RNA-Seq to cluster the differential genes in three tissues of transgenic plants.Differential genes in leaf tissue are much more than flower and silique,and most of them are down-regulated genes.Through GO and KEGG pathway analysis,we found that enrichment pathway of differential genes are mainly concentrated in secondary metabolism and oil synthesis.3.In order to explain bioinformatics results we examined mechanism action of Eg-miR5179 via cleavage validation in Arabidopsis.Predicting Eg-miR5179 target gene online and then testing by 5'RACE.Additionally,Real-time PCR analysis of target gene NDT1 indicated a noticeable declined in leaf and silique.Meanwhile,content of NAD+ and NADH in transgenic Arabidopsis were lower than wild plants.These results suggest that NAD+ Transporter 1(NDT1)was cleavaged by Eg-miR5179 in Arabidopsis,thus decline of NDT1 led to obvious influence on metabolism pathway which involved in NAD/NADH regulation.4.Down-regulated gene NDT1 may have some effects on Arabidopsis growth and metabolism.We detected the content of starch,oil and flavonoids in transgenic Arabidopsis.The results revealed that starch content decreased significantly,but an increasing in oil content,as well as dramatically lacking in flavonoids substances.In conclusion,Eg-miR5179-mediated cleavage of the NDT1 gene regulated Arabidopsis growth and development has effect on secondary metabolism and oil accumulation.Molecular mechanisms of Eg-miR5179 provides theoretical basis for improving oil palm furit development and oil proprety.