Cloning And Functional Analysis Of Fruit Shape-Related Gene In Grape

Grape(Vitis vinifera) belongs to Vitaceae. As an important deciduous fruit tree, which is cultivated in most parts of China. Table-grape accounted for more than 80% of total output of grape. With the improvement of people consuming need, producers and researchers ensure the increasing of output, all the while developing high-quality products which are new, extraordinary and special. Fruit shape is an important part of the appearance of fruit quality, which is mostly quantitative trait controlled by multiple genes. Using the traditional way to breed cost a large scale to grow and a long time to screen. Studying the heredity rule and achieving the overall genetic control of fruit shape will be an important direction for fruit shape research.In this study, two genes associated with fruit shape VvGW2 and VvOVATE were cloned from the grape cultivars ’Manicure Finger’ and ’Ruby Seedless’ respectively. We study these genes by bioinformatics analysis, subcellular localization and qRT-PCR analysis. We analyzed gene function through overexpressing in transgenic tomato plants. The main findings are as follows:1 Search the grape sequence in NCBI which is the highest homology with the rice grain shape gene OsGW2. The sequence of VvGW2 cDNA was isolated using RT-PCR and RACE, bioinformatics software was used to analyzed the VvGW2 gene structure characteristics. Quantitative real-time PCR (qRT-PCR) analysis the expression level of ’Manicure Finge’in different development stage and the expression level of different cultivars at anthesis. The sequence of’Manicure Finger’GW2 was isolated using RT-PCR and RACE technologies, named VvGW2. The VvGW2 gene contains 1272bp ORF, encoding 423 amino acids which has a RING domain. RING-type belongs to C5HC2. Protein molecular weight is 46.96KD, isoelectric point is 4.68. Compared with other sequences of GW2 in GenBank, the deduced amino acid sequence of WGW2 shared 47-53% in homology. Subcellular localization results showed that VvGW2 protein was localized in cell cytoplasm. Quantitative real-time PCR analysis showed that VvGW2 gene expressed in each stage of’Manicure Finger’, the highest expression was exhibited at anthesis. The expression level of VvGW2 gene was various in different cultivars, the highest expression was exhibited at ’Manicure Finger’, while the lowest expression is exhibited at round shape grape. The abnormal expression of VvGW2 in ’Manicure Finger’ and different cultivars might be related to fruit shape.2 Search the grape sequence in NCBI which is highest homology with tomato fruit shape gene OVATE, and a sequence of ’Ruby Seedless’ OVATE was isolated using RT-PCR and RACE strategies. The VvOVATE gene contains an open reading frame of 1083bp encoding 360 amino acids which have an OVATE domain. Compared with other sequences of OVATE in GenBank, the deduced amino acid sequence of VvOVATE shared 40-70% in homology. Subcellular localization results showed that the OVATE protein was localized in cell nucleus. Real-time PCR analysis showed that OVATE gene expressed in all stages, the highest expression in ’Ruby Seedless’ is exhibited at 30 days after anthesis, however ’Manicure Finger’is exhibited at anthesis. The quantity and timing difference of VvOVATE expression in two cultivars may affect fruit shape.3 Plant expression vector PBI121-VvOVATE was successfully constructed. Transgenic tomatoes were obtained through the method of agrobacterium mediated transformation system. Nine transgenic positive lines were strained using GUS staining. Seven positive lines were detected by PCR, indicating that VvOVATE gene was integrated into tomato genome. Three lines were detected target gene using RT-PCR amplification, indicating VvOVATE in transgenic plants can successfully transcribed; And finally we got three lines of transgenic plants. Expression analysis of transgenic tomatoes showed that VvOVATE had high expression during the stage from blooming to coloring. The highest level occurred in young fruit period, while the expression was lower before anthesis and after coloring. The observe of phenotype showed that changes occurred in vegetative organs and floral organs. The transgenic tomatoes had dwarf plant, small and round leaves; Flowers and fruits became smaller. These results suggested that OVATE may be a suppressor of plant growth.