Cloning, Expression Vector And Genetic Transformation Of THM27 Gene In Tomato

The MYB transcription factor is the largest TF family known in plants, play key roles in the regulation of the gene expression of flavonoid biosynthesis-related multiple enzymay at the transcriptional level, which is an important way of regulating growth, development and physiological metabolism of plants. The common feature of MYB transcription factors is the highly conserved DNA-binding domain (MYB domain), which participate in the regulation of plant development and metabolism. Tomato is one of the most important vegetable crop world-wide, but also a significant source of flavonoid. For the improvement of the quality of tomato and increasing flavonoid content in tomato fruit, studing in MYB transcription factor is essential.As a MYB transcription factor, THM27 gene can regulate the biosynthesis of flavonoids. Overlapping PCR technique can be used in vitro recombination effectively, it’s does not require enzyme digestion and ligase treatment. By this technology, the fusion of target-gene and promoter/terminator, and the transformation of expression vector can be realized quickly. This research selected tomato as the study materials, cloned myb transcription factor superfamily member --- THM27 by Nasted PCR; cloned tomato fruit specific promoter(FQE2A11) from tomato DNA genome; and amplified CMV35S promoter and NOS terminator from the pCAMBIA1302 plasmid. CMV35S promoter-THM27-NOS terminator combination element was constructed by overlapping PCR, and constructed to binary expression vector(pCAMBIA1302). The recombinant plasmid was transformed by Agrobacterium-mediated EHA105 into tomato cotyledons, established the tomato THM27 transgenic transformation system, successfully obtained transgenic tomato seedings. This study lay a solid foundation for study the MYB transcription factors regulationg the flavonoid synthesis by liquid chromatography.