Analysis And Optimization Of The Functional Regulatory Elements Of Trans-acting SiRNA Gene TAS3a

Artificial microRNA and syn-tasiRNAs have been used extensively for gene functional analysis in plants. Since a single tasiRNA gene (TAS) can produce multiple functional syn-tasiRNAs simultaneously, so the TAS genes possess the potential of interfering multiple target genes at the same time. However, TAS-based interference efficiency needs to be improved. In this study, we analysed the limiting factors for the generation of tasiRNAs by TAS3a in Arabidopsis thaliana, demonstrating miR390, among others, is the limiting factor. We then obtained optimized synthetic TAS3a genes useful for functional research of gene of interest in plants.Using the XVE inducible multiple-gene expression system, we analysed the roles of some factors involved in tasiRNA biosynthesis in the formation of synthetic tasiRNAs derived from overexpressed TAS3a. The factors we analysed include miR390, AG07, DRB4and SGS3. The results indicated that when induced for overexpressing, it was miR390a rather than AG07, DRB4and SGS3that greatly enhanced the efficiency of interfering target gene PDS as well as the expression level of silencing reporter syn-tasiRNAs/PDS. Moreover, time-course analysis demonstrated that the expression levels of silencing reporter syn-tasiRNAs/PDS increased along with the increased expression levels of miR390, and the expression levels of target gene PDS decreased accordingly. These results suggested that miR390was the limiting factor for the generation of tasiRNAs by overexpressed TAS3a.To improve the expression level and the in-phased generation of tasiRNAs were the two important aspects of strengthening the effectiveness of tasiRNAs. The longer are the functional regions of TAS3a for the generation of tasiRNAs, the higher is the probability of producing non-in-phased and useless tasiRNAs, and thus the more serious are off-target effects. To reduce off-target effects dervied from non-in-phased tasiRNAs, we shortened TAS3a, and demonstrated that shorten form of TAS3a performed better than the full-length gene.By inserting miR390a into the intron of the shortened TAS3a, we obtained TAS3a-miR390a fusion gene. Comparison of the fusion gene with the corresponding shortened TAS3a found that the interference efficiency of the former was better than that of the latter. We aslo demonstrated that TAS3a-miR390a fusion gene could be used for efficiently interfering some target genes of TCP gene family. These results suggested that as an optimized TAS3a, TAS3a-miR390a fusion gene can be used for interfering one or more target genes in replace of original TAS3a.Although the shortend forms of TAS3a have higher interference efficiency and specificity, the overly shortened TAS3a may influence the generation of tasiRNAs. We investigated this possibility by analysing shorter forms of the TAS3a. The results showed that when the region between the two miR390target sites was shortened to harbor only two, rather than one, effective21-nt tasiRNAs, the shortened form of TAS3a still have obvious interference to target gene PDS, suggesting that the TAS3a harboring only two21nt tasiRNAs is the shortest form. To be able to produce more effective syn-tasiRNAs, we substituted miR173target site for3’-miR390target site in TAS3a, and inserted the miR390a-miR173fusion gene into the intron of TAS3a, resulting in the generation of TAS3a, TAS1c, miR390a and miR173four-gene fused TAS. In the fusion gene, one region to generate tasiRNAs locates between the miR390and miR173target sites, and another region locates behind the miR173target site. Functional analysis of the fusion gene showed that both regions have strong interference effect on target genes, suggesting that two tasiRNA-generating mechanisms from TAS3a and TAS1c, respectively, worked efficiently in the fusion gene and miR390and miR173enhanced the function of the two TAS genes. Furethermore, functional analysis of the fusion gene demonstrated again that the shortest form of TAS3a was one harboring only two21nt tasiRNAs.In brief, we analysed the factors influencing the efficiency of the TAS3a-generated tasiRNAs, discovered that miR390was the limiting factor, and the length requirement for the region between the two miR390target sites of functional TAS3a must habor at leat two21nt tasiRNAs. Based on these observations, we generated the optimized TAS3a, including TAS3a-miR390a two-gene fused TAS and TAS3a-TAS1c-miR390a-miR173four-gene fused TAS, and demonstrated that these optimized TAS genes will be able to be used as a method for efficiently interfering multiple taget genes for functional research, especially when target genes are lethal or influencing seed germination, plant growth and development.