Heterologous Expression Of External Plant Sweet Protein Thaumatin Ⅱ In Marker-free Trangenic Tomato

Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops in the world. With the development of genetics and whole tomato genome sequencing from International Solanaceae Genomics Project (SOL), tomato has become a good model system to study transgenic plants. Plant sweet proteins have the characteristics with high and pure sweetness, low calorie, good taste and non-toxic. It can also be digested into various amino acids necessary for human body. As a bioreactor, there are tremendous commercial potentials for tomato:first, it is easy to be transformed by heterologous genes; second, if transformated successfully, it is possible to be increased on sweetness and fruit quality; finally, we can manufacture Thaumatin II sweet proteins in high-volume, low-cost with tomato. In the present study, we conducted an expression vector containing sweet protein ThaumatinⅡgene, and transferred it into tomato by Agrobacterium-mediated transformation. The results are as follows:1. We first established an efficient tomato regeneration system by screening differentiation and rooting medium. The results showed that regeneration frequency was the highest with 0.2mg/L ZT and 1.0mg/L IAA, and rooting was the best with 0.05mg/LNAA;2. Using sterile tomato cotyledons as explants and kanamycin-resistant NTPⅡas a marker gene, the ThaumatinⅡgene which encodes a plant sweet protein from Thaumatococcus daniellii has been introduced into the tomato genome by Agrobacterium-mediated transformation. As a result,10 independent tomato transgenic lines were acquired;3. The introduction of the foreign gene was confirmed by PCR for the expected specific bands. Furthermore, transgenic line Thau-10 was shown by two copies of integration on Southern blotting analysis; 4. Analyzing the segregation ratio of T1 seeds on 1/2 MS medium containing 200 mg/L Kan, and the results showed that the Kan-resistant and susceptible ratio of Thau-2 and Thau-4 lines were close to 3:1, indicating that these transgenic lines were inserted by single copy;5. Using sterile To generation euphllas and T1 generation tomato cotyledons and hypocotyls as explants andβ-estradiol of different concentration as inducer, we got the result suggesting that concentration ofβ-estradiol at 4μM was fittest, we observed the generation of 10 adventitious shoots from 60 explants preliminary. Furthermore, the external gene was detected to remove by PCR molecular analysis in 1 differential tomato plants.We have accomplished the first four tasks mentioned above, and the identification of induced shoots is in progess.