Cloning and function analysis of VvMADS46 in Red Globe and Thompson Seedless for seedless regulation

Abstract: 【Objective】Grapevine (Vitis vinifera L.) as one of the most economically important fruit crop is widely cultivated all over the world. Seedlessness of the fruit is one of the most desired traits for table grape consumers. The MADS-box genes, which generally comprise large families, encoded transcription factors (TFs) with a highly conserved MADS domain, and played diverse roles in plant embryo develop- ment, flowering time, floral meristem, and fruit ripening regulation. In this study, VvMADS46 was ex- pressed in the different grape cultivated species tissues and ovule developmental stages. Gene cloning, sequence alignment and overexpression in the Micro-Tom tomato were analyzed for VvMADS46 gene related grape seedless function identification, in order to provide foundation for further research ofVvMADS46 participating in regulation of seedless grapes.【Methods】Grape tissues and organs, includ- ing roots, stems, leaves, tendrils, inflorescences, flowers in the fully opening stage and fruits on 33 days after flowering (DAF) were harvested from the seedless grape Thompson Seedless and the seeded grape  Red Globe, respectively. Ovules were collected from the two seeded grape cultivars (Kyoho and Red Globe) and the two seedless grape cultivars (Thompson Seedless and Flame Seedless) on 33, 36, 39 and 42 DAF. Total RNA extraction and first strand cDNA synthesis were accomplished using EZNA Plant RNA Kit (R6827-01, Omega Bio-tek, USA) and PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China), respectively. VvMADS46 expression in the different grape tissues and or- gans was made by quantitative real-time PCR. VvMADS46 cDNA sequences, alignment and phylogenet- ic tree were analyzed by homologous clone. The sub-cellular localization and the overexpression ofVvMADS46 were analyzed in the onion and the model plant Micro-Tom tomato.【Results】VvMADS46was expressed in all the different tissues and organs of the Thompson Seedless and the Red Globe. Obviously, there was no significant difference in the roots, stems, leaves and tendrils between the seedless and seeded grapes, but the expression of VvMADS46 in the inflorescences, flowers and fruits were sig- nificantly higher than those in four vegetative growth organs, also higher in the Thompson Seedless than that in the Red Globe. In four researched grape ovule development stages, the expression levels ofVvMADS46 on 33 and 36 DAF were higher than those on 39 and 42 DAF. Additionally, the expression levels of VvMADS46 in the seedless grape Thompson Seedless and Flame Seedless were significantly higher than those the in seeded grape Red Globe and Kyoho in all these four stages. The full-length cD- NA sequence of VvMADS46 was 681 bp, encoding 226 amino acids, with a complete MADS conserva- tive structure. The sequence alignment showed that mRNA sequences and amino acid sequences ofVvMADS46 cDNA in the Thompson Seedless and Red Globe were similar to the Pinot Noir (cv. PN40024), with 99.8% and 99.1% similarity, respectively. According to the homologous alignment, 8 AP3 proteins (AtAP3, AeAP3, CmAP3, LjAP3, LrAP3, PhAP3, AcAP3 and PtAP3) were screened from the NCBI database, the phylogenetic tree was constructed using MEGA 5.0. Additionally, the sub- cellular localization vector PBI221-VvMADS46-GFP and the overexpression vector pCAMBIA2300-VvMADS46 were constructed, following Agrobacterium tumefaciens mediated infection in the onion sur- face cells and transformation into the Micro-Tom tomatoes, respectively. GFP signaling was observed by the inverted fluorescence microscope and localized in the grape nucleus. The growth and develop- ment phenomenon of the overexpression tomatoes, the empty vector transgenic tomatoes and the wild type tomato lines were identified and compared on more than 30 DAF with the same condition. The empty vector transgenic tomatoes and the wild type tomato lines had no significantly growth phenome- non difference, however, the height of the VvMADS46 overexpression tomatoes were significantly short- er than that of the empty vector transgenic tomatoes and the wild type. Moreover, the fruit size and seed number of the empty vector transgenic tomato lines and the wild type tomato fruits showed no signifi- cantly difference, but the fruit size of the VvMADS46 overexpression tomatoes was significantly smaller than that of the empty vector transgenic tomatoes and the wild type, also the seed number was signifi- cantly decreased, with nearly no healthy and plump seeds in the VvMADS46 overexpression tomato lines. All these researches suggested that a possible function of VvMADS46 in development and forma- tion of seedless fruits in grape.【Conclusion】VvMADS46 was a reproductive growth organ induction gene in grapevine, and possibly played an important role in grape seedless character regulation through inhibiting the ovule development.