- 學位論文
Characterization of the Scutellaria barbata glycosyltransferase gene and its promoter
半枝蓮轉醣基因的選殖應用與其啟動子的調控
摘要
Unexpected efficacy of medicinal plants may be attributed to the perceived stimuli from the environment, including light, temperature, water, nutrients, pathogen attack and wounding that will result in various compositions of its secondary metabolites. To date, little is known about the regulation of secondary metabolite biosynthesis in medicinal plants. The emerging progress on genomics, proteomics, transcriptomics and metabolomics has an enormous potential for improvement and application of medicinal plants through mass propagation, metabolic engineering and understanding of the regulation of secondary metabolism in medicinal plants. Scutellaria species, belonging to the family Lamiaceae, are excellent model medicinal plants for studying the biochemistry and genetic regulation of secondary metabolism since they have a relatively small genome size and their chemical profiles were extensively documented. S. barbata D. Don is a popular traditional medicinal herb listed in the Chinese Pharmacopoeia, which is commonly used in the treatment of tumors, hepatitis, cirrhosis, and other diseases. The main constituents of this herb were flavonoid glycosides, especially baicalin and scutellarin. Glycosylation is a key and the last step modification of plant natural products that affect the stability, water solubility and bioactivity. In the first part of this thesis, a glycosyltransferase gene SbUGT from Scutellaria barbata and its promoter were cloned, and the expression of SbUGT responds to abiotic stimuli was elucidated. The SbUGT was characterized as a flavonoid glucosyltransferase using biotransformation. Serial deletions of the promoter region identified a 44-bp fragment that is important for gene expression in an inducible and organ-specific manner, which provides a potential transgene expression tool for practical applications. Medicinal plants have long been used as traditional Chinese drugs for treating many diseases, whereas the authenticity and efficacy of material medica remain to be concerned. Many adulterants with similar morphology but different efficacy appear in the market, which will cause unexpected severe side-effects if not properly used. Therefore, identification of genuine raw materials plays a crucial role for safe and therapeutic benefits in treating diseases. The second part of this thesis describes the authentication of medicinal herbs using PCR-amplified ITS2 with specific primers. Sets of designed primers led to accurate PCR product of the specific DNA barcoding region in ITS2 was validated with 55 medicinal materials belonging to 48 families, which could be used as DNA barcode for authentication.
並列摘要
The conversion of flavonoid aglycones to their glycosides by plant glycosyltransferase may affect a wide range of outcomes, including stability, solubility and bioavailability. Scutellaria barbata, rich in flavonoid glycosides, is widely used as a traditional Chinese herbal medicine. In the first part of this thesis, a flavonoid glycosyltransferase cDNA (SbUGT) and its promoter from S. barbata were cloned and characterized as a flavonoid glycosyltransferase using whole-cell biotransformation. Fragments of different lengths of the 5'-flanking region of the SbUGT gene were fused to the -glucuronidase (GUS) gene and analyzed with transgenic Arabidopsis plants using histochemical and fluorometric assays. GUS activity in transgenic plants carrying the SbP-850U construct (850 to +86 relative to the transcription start site) displayed the highest level and was enhanced by salt and methyl jasmonate (MeJA), similar to the expression patterns of the endogenous SbUGT. GUS activity disappeared when the promoter was deleted to 98, and deletion analyses indicated the existence of positive and negative regulatory element(s). Unexpectedly, plants carrying the construct SbP-102U (102 to +86) exhibited strong GUS activity exclusively in the roots. Our experiments revealed that the specific expression is mediated by different promoter regions and the unique region driving root-preferred expression can be used as a root-specific promoter.
參考文獻
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