Construction And Application Of Transient Expression System To Validate The Target Genes Of The MicroRNAs In Rice

MicroRNAs(miRNAs)are a class of endogenous small regulatory RNA molecules,which play a key role in the regulation of gene expression through cleavage target mRNAs and inhibiting translation initiation in most eukaryotic cells.MiRNAs regulate diverse biological process,such as growth and development,stress response,etc.With the rapid development of sequencing technology,a large number of new miRNAs have been discovered,and 38589 miRNAs have been registered in miRNA database.However,the biological functions of most miRNAs and their target genes still need to be further characterized.To study the role of miRNA,the key is to study the regulation of target genes by miRNA.The common methods for the validation of miRNAs target genes include Northern blot,Western blot,Degradation Group Sequencing,MirTrap,RACE(Rapid amplication of cDNA ends)and luciferase reporting system.During prediction of candidate targets of miRNAs by bioinformatics,different methods or different parameters usually result into the different target or different number of genes.Which indicates that the prediction results involve in false positive or negative potential targets.So,it is necessary to validate predicted targets by experimental methods.Owing to that the common methods for validation of targets are complicated,time-consuming,it is necessary to establish an accurate,quickly and simple methods for validation of miRNAs targets.It is a fast and convenient method for validate miRNAs targets to integrate transient expressions and luciferase reporter system.Using the Agrobacterium-mediated transient expression in tobacco,combined with the transient expression in rice protoplast,we established a optimized validation methods by systematic study the dynamic expression pattern of miRNAs,and the optimized detection time.The main results are as follows:(1)Constructed miR169 o expression vector,target gene LOC_Os03g48970.1 fusion with luciferase gene(named as LUC-48970),mutation LOC_Os03g48970.1 fusion with luciferase gene(named as LUC-48970m3).Compared to that in co-transformation miR169 o plus empty LUC,miR169 o plus LUC-48970m3,the LUC activity in co-transformation miR169 o plus LUC-48970 is significantly weaker.It suggested that the system can be used for validation of miRNAs targets.(2)Detected LUC activity at 12 h,18h,24 h and 36 h respectively after the conversion of protoplast,the results showed that at 24 h,LUC had the highest activity.In addition,the expression pattern of miR169 o was detected by stem-loop qRT-PCR.Generally,the expression level of miR169 o was positively correlated with the detection time course,with the lowest level at 12 h and the highest level at 36 h.And at 24 h after co-transformation,the expression level of miR169 o was regulated up to 10 folds.Combined the miRNAs expression level and LUC activity after co-transformation,we suggested that 24h-36 h after co-transformation are the optimum detection time for the transient expression system in rice protoplast.(3)Construction the binary expression vector of miR169 o,LUC-48970,LUC-48970m3,and transformation into Agrabacterium tumefaciens GV3101,respectively.Co-transform miRNA and candidate target gene in tobacco by co-infiltration the bacteria with different plasmid in tobacco leaves with a no-needle spring.The results showed that target gene 48970 can be repressed by miR169 o specifically.The activity of LUC was detected by 48 h,72h and 96 h,and the activity of LUC was found to be decreased after the peak of LUC activity.Therefore,72 h after inoculation was the best detection point.In addition,the expression pattern of miR169 o was detected by stem-loop qRT-PCR.The expression level of miR169 o was lowest at 24 h and regulated up to 20 folds at 48 h.Together with miRNAs expression level and LUC activity after co-infiltration,we suggested that 48h-72 h after co-transformation are the optimum detection time for the transient expression system in tobacco..(4)Using the built rice protoplast transient expression system and tobacco transient expression system,we tested the miR812 g predicted target genes LOC_Os02g07960.4 and LOC_Os04g47140.1,miR5076 predicted target gene LOC_Os02g27594.2,miR818 a predicted target gene LOC_Os09g36320.1,miR169 o predicted target gene LOC_Os02g53620.The results suggested that in addition to 53620 can be miR169 o target genes,other miRNAs and their predicted target genes may not exist shear or inhibit relationship.This item established the transient expression system for validation of rice miRNAs target genes through optimization miRNAs and luciferase reporter gene expression.The system provides an easy,fast,and almost in vivo method to validate rice miRNAs,and thus will lay a foundation for studying the miRNA biological function.