The Cloning And Characterization Of Sly-miT166and Its Target SlREV On Fruit Formation In Tomato

microRNAs (miRNA) are a class of endogenous small non-coding RNA with20-24nucleotides in length. They precisely regulate organism growth and developmentby down-regulating or inhibiting their targets at post-transcriptional level. In plants,miRNAs largely play critical roles in vegetative and reproductive growth anddevelopment. Such as miR166, a conserved miRNA in plants, which has been showedboardly involving in root meristem development, stem growth, leaf polarity and floralmeristem growth. However, the mechanism of its role in fruit formation is barely knownso far.In our study, tomato was considered as a model plant for research due to itscharacteristics of flesh fruit formation, growth, development and ripening that lacking inArabidopsis. The research work focus on how Sly-miR166and its targets affect flowerdevelopment and fruit formation, wich may provide evidence for elucidating thesignificant roles of miR166in floral identity, fruit set, fruit growth and development.Main results were listed below:1. There are probably9gene loci of miR166in tomato genome throughbioinformatic analysis. We cloned one precusor, sly-pre-miR166b for further analysis.6target genes belonging to HD-ZIP III subfamily were validated to be cleaved uniformlyby miR166by5’ RACE RT-PCR, including Solyc11g069470, Solyc08g066500,Solyc12g044410, Solyc03g120910, Solyc02g024070and Solyc02g069830.2. After analyzing the expression pattern of Sly-miR166and6target genes, wefound that Sly-miR166mainly accumulated in root, leaf, flower and fruit, whereas all oftargets highly enriched in root and stem, moderatly expressed in leaf, flower andimmature fruit, and almost cann’t be detected in mature fruits. However, transcript levelof all targets and Sly-miR166in stem, leaf and fruit were complementary to each other,suggesting they may play significant roles in these tissues.3. Fruit-specific expression of sly-pre-miR166b in tomato displayed importantphenotype of altered carpel development pattern, which leading to fruit growing out offruit and parthenocarpy partialy. Quantative RT-PCR showed that transcript of maturemiR166was increased in transgenic plants where all of targets mRNA were decresed,leading to the restricted expression of TAG1, which controled the development oftomato carpel directly and floral determinancy. Once the indeterminancy happened, contionous floral organ will be formed and developed. Meanwhile, some transcriptsrelated to auxin signal and ovule development also changed, which may explain thereason of seed defect and parthenocarpy of transgenic tomato.4. We over-expressed one of the target genes, SlREV, and its miR166-resistantversion—SlREVRisin tomato.by Agrobacterium-guided transformation. By comparativeanalysis, we found that overexpression of SlREVRiscaused alteration of leaf polarity andvasculature pattern, ectopic formation of floral tissues on pedicel abscission zone,ectopic carpel formation and fruit fusion, all of which were hardly observed in35S::SlREV plants except curly leaves. It is suggested that SlREV executes its possiblepleiotropy in stem, flowers and fruits, mainly at the post-transcriptional level bySly-miR166.5. Considering the phenotypes of contionous buds forming and surrounding pedicelabscission zones, quantative RT-PCR were directed by samlpling the specific zone oftransgenic plants and WT. Results indicated that SlREV probably direct BLIND,GOBLET and LeWUS to maintain meristem, regulate the formation of lateral organs andorgan boundary.