Genetic Transformation Of Sweet Protein Brazzein In Tomato

Sweet protein Brazzein is one of the natural protein sweeteners. Brazzein has low calorie and intrinsic sweetness 500-2000 times over sucrose and will not cause weight gain, tooth decay, and the sugar diabetes that people suffered frequently when taking sugar. We aimed in the present study at improving tomato sweetness without increasing the sugar content by introducing Brazzein gene into tomato through transgenic technique. We cloned a fruit specific expression promoter E8P1 and constructed a vector which expresses a synthesized Brazzein gene in tomato fruit. Transgenic materials were obtained in two tomato varieties Zhongshu No. 4 and Lichun.1 , Development of an efficient tomato transformation system :We used leaf disc and agrobacterium mediated transformation system in tomato. The binary vector pBI121 was used as a basic expression vector to make constructs. We selected proper medium and determined 100mg/l kanamycine being the effective selection antibiotic concentration in transforming tomato. Tomato varieties, Meiwei Yingtao, Zhongshu No4 and Li Chun were successfully transformed with different constructs. Maintaining 100 mg/l kanamycine in the whole process of transformation reduce significantly the escapes and increase the efficiency to detect positive transformants. In the present case, 90% of all the rooted plants were detected positive.2, Cloning and function analysis of a fruit specific promoter-E8PlWe cloned from the tomato variety Hongmanao 213 the 1121bp promoter E8P1 and made the GUS expressing construct E8P1::GUS. The construct were successfully transformed into tomato. PCR testing of the genomic DNA and one step RT-PCR verified the integration and the expression of the GUS gene. GUS transcription was detected only in the mature fruits, and expression was at a higher level than that of 35S::GUS transformed plants in fruit. Histochemical dying of the GUS activity also indicated that E8P1::GUS transgenic tomato expressed GUS specifically in mature fruit, The data proved that E8P1 could serve as an efficient promoter for expressing genes in tomato fruit.3 , Synthesizing of Brazzein gene and its transformation in tomatoWe synthesized by PCR a 162bp Brazzein gene using its coding sequence and cloned the PCR fragment into a T vector. We constructed two plant expression vectors pE8Pl.B and pBI121.B for the fruit specific and constitutive expression respectively. We confirmed by RT-PCR that transgenic tomatoes of the two constructs expressed Brazzein gene at the transcriptional level. Sensory test indicated transgenic tomato fruits were obviously sweeter than that of non-transgenic control while maintaining the same sugar content.