Genetic Transformation Of Thompson Seedless With A Stilbene Synthase Gene VqSTS6 From Vitis Quinquangularis

Stilbene synthase(STS) was a key enzyme in the biosynthesis of resveratrol. Resveratrol, considered as phytoalexin and antitoxin, was found to have anti-inflammatory, antiplatelet, anticarcinogenic, antifungal, and antibacterial activities. In previous studies, compared the resveratrol content of thirteen wild Chinese grapes and four Vitis vinifera. grape varieties, our research group had verified that resveratrol contents in cv. Danfeng-2 reached 6 μg/g. Moreover, the expression of gene VqSTS6 from Danfeng-2 was markedly higher than the other VqSTS genes in the six tissues by Real-time PCR test. In this research, embryogenic cultures, initiated from anthers and flower buds on medium PIV, were used for transformation via Agrobacterium tumefaciens. Transgenic plants were successfully obtained from cv. Thompson Seedless with the gene VqSTS6, which was cloned from Vitis quinquangularis cv. Danfeng-2,and the expression of the gene VqSTS6 was investigated. In addition, Agrobacterium-mediated transient assays of leaves and somatic embryos in different condition was performed, and the influencing factors were optimized. The specific contents and achievements are as follows:1. Anthers and flower buds of Vitis vinifera L.cv.Thompson Seedless were used as the explants and inoculated on medium PIV(which contain 60g/L sucrose, 1 mg/L2,4-D, 2 mg/L 6-BA, 0.1 g/L Inositol and 1g/L active chlorite), and subcultured until embryonic callus were induced. Conditions for keeping the activity of embryonic callus were optimized. Improved X6 medium(which contain 60g/L sucrose and 1g/L active chlorite) with 0.2 mg/L of the 2, 4-D could reform embryonic callus brown necrotic.2. Vectors with gene VqSTS6 cloned from cv. Danfeng-2 were transferred into Thompson Seedless via Agrobacterium tumefaciens. After cocultivation,embryonic callus were cultured on the selection mediumto produce the resistance lines. The resistance lines were detected by means of molecular technique,which showed that gene VqSTS6 wasintegrated into Thompson Seedless. 35 transgenic lines with gene VqSTS6 were obtaied.3. In order to validate STS expression profiles, the steady-state transcript levels of 6 related genes were analyzed with transgenic lines using qRT-PCR. Among them, STS and RSGT were upregulated,PAL and MYB14 were downregulated. However, MYB15 and CHS transcripts could not be compared with these two levels in controls.4. The accumulation of stilbene in transgenic strains were detected by high performance liquid chromatography(HPLC).Compared with the no-transformed control, ε-Viniferin, pterostilbene, trans-resveratrol and trans-piceid accumulation were significantly increased in transgenic lines.The accumulation of resveratrol is significantly higher than control in three transgenic plants,which is up to 108.94 μg/g Fw, 80.04 μg/g Fw and 77.90 μg/g Fw.5. To investigate the resistance to pathogens, transgenic strains and non-transgenic strains were inoculated with Erysiphe necator. The secondary hyphae grew on transgenic plants was significantly lower than the non-transformed control at 72 hpi. At 168 hpi, the infection led to the formation of colonies with dense conidiophores on non-transformed leaves but only small colonies with conidiophores on transgenic leaves. Furthermore, the number of conidiophores sporulated on transgenic lines were lower than on non-transformed control lines. To sum up, over-expression of VqSTS6 in the transgenic lines conferred a lower susceptibility to Erysiphe necator. This study confirmed that VqSTS6 was involved in the resveratrol synthesis pathway in transgenic grapes.6. Agrobacterium-mediated transient assays of leaves and somatic embryo in different condition was performed. The efficiency of transformation was confirmed by GUS staining, showed that the intensity of GUS stainings on somatic embryos were obviously higher than leaves. To select the optimum conditions, growth of somatic embryos, concentration of Agrobacterium cultures, time of agro-infiltration and time interval of GUS staining were Investigated. Result proved that somatic embryos that were just differentiated were the best tissue culture. The optimum conditions were concentration of Agrobacterium cultures OD600=1.0, somatic embryos were dipped in the agrobacterial suspension for 10 min, and GUS staining was performed 72 h after infection.