The Functional Evaluation Of Key Genes In Steviol Glycosides Biosynthetic Pathway

Steviol glycosides produced in Stevia rebaudiana Bertoni have a high sweetness, low calorie, with many hygienical functions and disinvolve in human metabolism. They have been regarded as the most promising substitute for sucrose with a wide range of applications and high economic value. In the present research, the functions of genes, and their encoding enzymes that involved in steviol glycoside biosynthesis pathway have been charactered. The key genes have been transfered into S. rebaudiana for regulation their expresses and biosynthesis. The research will be helpful for breeding new varieties of S. rebaudiana for cultivation in Ningxia area with high production and high quality of purposed steviol glycosides.The results are as follows:1. A novel key gene involved in steviol glycosides biosynthesis pathway has been isolated and charactered. The gene, encodings an uridine diphosphate glycosyltransferase (UGT), named SrUGT91D2m which catalyzed steviolmonoside adding another glucosyl at the C-2’position of the C-13-O-glucosyl to steviolbioside, has been first successfully isolated. Its full length of sequence has been determined and charactered.2. The key genes involved in steviol glycosides biosynthesis pathway have been constructed into an expressing vector. By using PCR amplification, enzyme digestion and ligation methods, the full length of the key genes including SrUGT76G1, SrUGT86C2 and SrUGT91D2m, have been constructed into a pKAFCR100 binary vector separately or together, and then transferred into Agrobacterium strain EHA105 for S. rebaudiana transfermation.3. An exogenous gene transformation system for S. rebaudiana has been established. Using the leaves as explant, the important factors affecting the transformation efficiency, such as the Agrobacterium strains, infection and co-cultivation media, infection time, explant treatments, as well as antibiotic kinds and concentrations in the callus inducement and selection period, have been investigated and optimized.4. The individual functions of the key genes, and their interrelationship have been identified. Using Agrobacterium-mediated transformation. The 3 kinds of key genes constructed in binary vector separately or together that described above, have been transferred into the leaves of S. rebaudiana. By comparison of the steviol glycoside contents from each kind of transgenic callus, the individual functions of the key genes, and their interrelationship have been identified.