Cloning And Functional Analysis Of Sly-miR393in Tomato

MicroRNAs (miRNAs) are endogenous small non-coding RNAs at length of21-24nt that posttranscriptional regulate the expression of its target genes throughmRNAs cleavage or translation repressing. In plant, miR393is a highly conservedmiRNA. It plays an important role in auxin signal pathway by targeting the auxinreceptor TIR1and three F-box family protein members (AFB1, AFB2and AFB3).This study aimed to identify and characterize Sly-miR393from tomato.Furthermore, Sly-miR393was overexpressed in transgenic tomato as an attempt tounderstand the regulation mechanisms of it in auxin signal pathway. The mainconclusions are as follows:1. The sequence of Sly-miR393with hairpin structure was found in tomato genomesequence through the computational approach. The Sly-miR393preceursor was clonedand its overexpression vectors were constructed, that is pLP35S-pre-SlymiR393.2. The transgenic tomato plants were obtained via Agrobacterium-mediatedtransformation. Overexpression of Sly-miR393was validated by RT-PCR in tomato.3. The expression patterns showed that Sly-miR393has strongest expression in stem,higher abundance in flower, moderate expression in roots, leaves, bud and weakerexpression in fruits.4. Three auxin receptor homologs SlTIR, SlTIR1-like1and SlAFB were found intomato ITAG2.3cDNA database. Sly-miR393guided-cleavage to SlTIR1, SlTIR1-like1and SlAFB transcripts was validated.5. The spatial and temporal expression patterns of target genes showed that therewas an inverse correlation between Sly-miR393and SlTIR1, SlTIR1-like1and SlAFBmRNA levels in the leaves and flower.6. The study results of Sly-miR393overexpression transgenic plants showed thetaproot of transgenic plants is shorter compared with wild-type; the auxin treatmentfound that transgenic plants had significantly lower sensitivity of lateral root formationon auxin than the wild-type, and overexpression Sly-miR393not only negative regulatedthe expression of three auxin receptor homologous genes, but also changed the level oftranscription of auxin response genes. So we speculated that these genes may directly orindirectly regulated by Sly-miR393, which contributed to the alteration of plant auxin sensitivity.