Transformation Of Anti-β-Lyc To Tomato

Lycopene not only affects the fruit color of tomato, but also has strong functions of antioxidant, anticancer and preventive cancer. Generally, the tomato fruit is one of main resources for people to obtain lycopene. However, the lycopene content in commen tomato variety fruit is not so high, therefore, it becomes very important and necessary to improve the lycopene content to improve the quality of tomato fruit, promote peasant's income and increase tomato processing enterprise's benefit. It is possible to regulate lycopene metabolism in tomato depending on the reasearh of synthesis and degradation pathway of lycopene and its related enzyme and genes. In this study, commen tomato cultivers TTI1117A and Ling-guang No.3 were used as materials, on the basis of optimization of regeneration system and genetic transformation system of tomato, the Anti-LYC-b 5′gene was introduced into the two tomato variety plants by Agrobacterium tumefaciens to improve lycopene content in tomato through the inhibition of lycopene degradation. The main results were as follows:1. High frequency regeneration systems of TTI1117A and Ling-guang No.3 were established.Tomato cultivars, TTI1117A, Ling-guang NO.3 were used to study the effect of different concentration combinations of 6-BA and IAA on the plant regeneration by cotyledon and hypocotyl culture.It were found that: The optimum shoot inducing medium of cotyledon and hypocotyl of TTI1117A and Ling-guang No.3 were MS+6-BA 2.0mg/L +IAA 0.1mg/L, The optimum rooting medium for both the cultivars was MS+IAA0.2mg/L.With the same optimum shoot inducing medium, the effect of different genotypes and explants on regeneration of tomato was studied, the results showed that: there were significant difference on differentiation rate between different genotypes with the same explant, the differentiation rate of TTI1117A was higher than Ling-guang No.3; there were also significant difference on differentiation rate among explants: cotyledon was higher than hypocotyl and cotyledonary node.2. High frequency transformation systems of TTI1117A and Ling-guang No.3 were establishedThe sensitivity of cotyledon explant and adventitious bud to Kan were studied on the optimum shoot inducing and adventitious bud regeneration medium respectively.The results showed that: 60mg/L of Kan be used during bud differentiating stage, and 30mg/L of Kan during root-inducing stage were deal for resistance selection. Resistance selection was carried out both in bud differentiating stage and root-inducing stage.The time of preculture, concentrations of Agrobacterium tumefaciens, the days of co- culture, the time of being infected on the optimum culture medium and concentration of cef were studied. The high-frequency transformation systems of TTI1117A and Ling-guang No.3 were established as follows: the cotyledon explant precuture in darkness for 48h, then diluted Agrobacterium tumefaciens (incLude Anti-LYC-b 5′+GUS /pBINPLUS) with (MS) liquid medium, dipped for 10min in GV3101 suspension (OD600=0.5), co-cuLtured with Agrobacterium for 36h, and 500 mg/L cef was used to restrain bacteria grow as well as reduce the ingury to the explant.3. Transgenic plants of tomato were obtained.Take cotyledon explant as transformation recepter, the Anti-LYC-b 5′gene was introduced into the two tomato variety plants by high efficient Agrobacterium tumefaciens transgenic system, 9 TTI1117A and 26 Ling-guang No.3 with Kan-resistance were obtained, GUS detection showed that there were 1 positive plant of TTI1117A and 4 positive plant of Ling-guang No.3, positive plant rate was 11.1% and 15.3% respectively, these gus detection positive plants were assayed by PCR and Nos.Ter PCR all of them were positive plants, which proved that target genes had been intergrated into TTI1117A and Ling-guang No.3 genome. RT-PCR detection showed that anti-lyc-b 5′had been expressed on RNA level.4. Preliminary detection of lycopene content of transgenic tomato.Lycopene and carotene assay of leaf of transgenic tomato showed that, lycopene content of leaf of transgenic tomato was higher than untransgenic plant, and the diffenence was very significant. And also the lycopene content was higher than carotene content of leaf of transgenic plant, carotene content of leaf of untransgenic tomato was higher than lycopene content.These showed that anti-lyc-b5′had been expressed and it had inhibit the expression of endogenous genes of LYC-b.