Isolation and Regulation Analysis of Sly-miR167 Promoters in Tomato
|Keywords||Sly-miRNA Tomato Promoter GUS Plant hormones|
Tomato Sly-miR167 in plants, has an important function in the fruit and fruit development process, and there are three loci in the tomato genome. MiRNA existence of multiple loci is an important regulation of miRNA expression may have important physiological significance. Sly-miR167 In tomato there are three loci, respectively having a different promoter, Sly-miR167 promoter tissue specificity and temporal specificity and exogenous stimulus conditions, response characteristics, and to be able to clarify miRNA multi-locus phenomenon and Sly-miR167 expression regulation mechanism to lay the foundation. Sly-miR167 three loci promoter fragment This study is separated from the tomato genome, were constructed and GUS gene fusion expression vector, transforming tomato, to study the tissue specificity and temporal specificity of its expression and the use of exogenous The hormone treatment, study their response characteristics of the hormone. The main findings are as follows: (1) Sly-miR167 locus promoter fragment cloned by PCR technique using bioinformatics methods to analyze the promoter sequence. The results show that the three promoter characteristic conserved sequence containing a plant promoter, such as the TATA-box, the CAAT-box. Further, Sly-miR167a promoter containing two auxin responsive element (AuxRE), three ethylene response element (ERE), three gibberellin-responsive element (GARE); The Sly-miR167b start seeds contain two EREs a TGA--element (AuxRE); the Sly-miR167c promoter fragment containing four GARE. (2) of the amplified fragments of the promoter were constructed to plant expression vector pMDC162, fused with the GUS gene, and then were transferred to Agrobacterium tumefaciens strain GV3101. (3) by Agrobacterium-mediated transformation of tomato, hygromycin (20 mg / L) selection pressure for resistant plants. Promoter-GUS fusion gene was integrated into the tomato genome by GUS staining, Sly-miR167 start successful positive transgenic tomato plants. (4) GUS staining showed that the transgenic seedlings, Sly-miR167a and Sly-miR167b, promoter can drive the GUS gene in the roots, stems and leaves of the expression, and the highest expression in leaves, stems, followed by roots weak. The the Sly-miR167c promoter in transgenic plants, GUS gene is expressed only in the leaves and stems, roots, GUS activity is not detected. (5) GUS staining results showed that the three promoter can drive GUS gene expression in various parts of the flower, but the different periods of both temporal and spatial specificity: Sly-miR167a promoter-GUS in transgenic plants flowers and fruits of different periods. 2 d before flowering and anthesis expressed strong 4 d after flowering expression weakened and petals undetectable; Sly-miR167b and the Sly-miR167c promoter in transgenic plants, GUS gene expressed strong two days before flowering, flowering day and 4 d after flowering, gradually weakened. At different stages of fruit development, the three promoters also exhibit spatial and temporal specificity: the Sly-miR167a and Sly-miR167b promoter in young fruit, green mature stage and broken color period expressed strong mature expression weakened; Sly-miR167c The strong promoter expression in young fruit and mature green stage, breaking color and ripening stages lowly. (6) of the transgenic plants were exogenous auxin (NAA), gibberellin (GA) and ethephon treatment, Sly-miR167a promoter in transgenic plants after treatment, GUS gene expression were raised; Sly-miR167b the the promoter transformants were handled by the NAA and GA GUS gene expression were significantly up-regulation; Sly-miR167c promoter GA and ethephon treatment, the GUS gene expression was significantly upregulated in transgenic plants.