MiR164is Required For Tomato Flower Initiation And Fruit Development

MicroRNAs (miRNAs), which are about21nucleotide RNAs, are ubiquitousregulators of gene expression in eukaryotic organisms. Some miRNAs act as importantendogenous regulators in plant organ formation and development. MiR164is aconservative miRNA family that exists in a variety of different plants. There are threecopies in Arabidopsis including miR164a, miR164b and miR164c. Previous studyshowed that miR164control embryogenesis, vegetable organ and flower developmentby repressing or cleavage the target gene NAC transcription factor family. Itaya et al（2008）found that miR164was abundance in tomato fruit, and we hypothesized thatmiR164may play an important role in fruit development. Up to date, there are fewreporters on fruit development regulated by miR164. Hence, it is important to clonemiR164and clear the relationship between miR164and fruit development and ripening.To investigate the function of miR164in fruit development and ripening, in thisstudy we cloned and identified the precursor of miR164, and its target gene NAM intomato, constructed overexpression vectors and transformed into tomato. The mainresults are as follows:（1）Isolation and identification of the precursor of miR164: cloning the precursorof miR164with the gene specific primer designed according to the genomic DNA andobtained a sequence of about500bp. Bioinformatic analysis showed that the candidatemiR164can fold into stable stem-loop structure.（2）Expression patterns of miR164in tomato: qPCR was applied in analysis ofmiR164expression level in all tissues and the results showed that miR164expressed inall tissues, but mainly in stem and fruit.（3）The isolation of miR164target gene NAM and NAM cleavage site mutation:we searched miR164target gene in tomato EST database, then designed primers andcloned a fragment of about1,315bp sequence.5′RACE analysis revealed that miR164cleavage NAM gene at the nucleotide complementary to the10th position of maturemiR164. So we concluded that NAM was a target gene of miR164. The large primermutational method was used to make the cleavage site mutation which creating4nucleotides mutants in the binding site, but the amino acid sequences were not change.（4）Expression patterns analysis of NAM in tomato: qPCR was applied in analysisof NAM expression level in all tissue and the result revealed NAM expressed high in all tissues, but more higher in stem.（5）The phenotypes of transgenic plants: the lines of miR164over-expressionexhibited delayed petal withering in flower development and finally parthenocarpicfruits. While miR164cosuppression and NAM overexpression lines lead to phenotypesincluding abnormal flowers with thick sepals, no petals and stamens, as well asparthenocarpic fruits. Dwarf plants, aberrant flowers with thick sepals and seedlessfruits were described in NAM-M overexpression lines（6）The expression levels of flower homeotic genes：in miR164overexpressionlines, expression levels of MADS-box transcriptional family genes including AP1, AP3,TPI, TAG and TM6were down-regulated, whereas they were up-regulated.in NAMoverexpression lines（7）The expression levels of NAC downstream genes：in miR164overexpressionlines, expression levels of these genes involving in KNOX, PTS, CUC2-LIKE, DBP andPDBP were down-regulated, whereas they were all up-regulated in NAMoverexpression lines.In conclusion, the phenotypes of tomato flower and fruit development wereregulated by miR164which plays an important role in flower initiation and fruitdevelopment.