An effective artificial microRNA vector based on Fv-miR166 precursor from strawberry

摘要

Artificial microRNAs (amiRNAs) are single-stranded, approximately 21 nt long, and designed by replacing the mature miRNA sequences of duplex within pre-miRNAs. They are processed via small RNA biogenesis and silencing machinery and deregulate target expression. AmiRNA became an important strategy to study gene function, mostly focused on knocking down the endogenous mRNAs in plants, a few used in function of endogenous miRNA. Furthermore, choosing a suitable miRNA precursor backbone used to express amiRNA is vital foundation. The published papers exhibited effective amiRNA vectors most on the natural miRNA precursors of Arabidopsis and rice. In this study, we firstly generated a simple amiRNA vector based on miR166 precursor of Fragaria vesca by annealing of eight synthetic oligonucleotides, in which mature miR166 sequence were replaced by amiR-GUS, amiR-GFP, amiR-NE and amiR390, respectively. Next, we cloned the amiR-GUS, amiR-GFP, amiR-NE and amiR390 into the plant expression vector pRI 101-AN with a CaMV 35S promoter, producing the pAMIR-GUS, pAMIR-GFP, pAMIR-NE and pAMIR390 vectors. The transient transformation of strawberry fruit showed that amiR-GUS and amiR-GFP were produced based on Fv-miR166 precursor, resulting in the obvious low expression levels of GUS and GFP. The qRT-PCR results suggested that amiR-NE could also be processed into mature amiRNA from the Fv-miR166 backbone. amiR390 was effectively targeting the endogenous TAS3 gene for increasing ta-siRNA3 biosynthesis in tobacco. Additionally, in over-expression tobacco plants of amiR390, the obvious phenotype changes were found that the ratio of average leaves length divided by width became smaller and the juvenile-to-adult phase transition phenotypes were delayed about ten days. The results showed that we have developed an effectively amiRNA backbone based on miRNA precursor of F. vesca. This is the first paper about strawberry amiRNA backbone.