Regulation Of Anthocyanin Metabolic Pathway Of Rice By Transgenic Approach

Anthocyanin is a kind of the secondary metabolites in plant, which exists in form of glucoside in vacuole of cells and endows plant beautiful color. It plays a crucial role in protection against UV-light and phytopathogens during plant development. Anthocyanin is the strongest known free radical scavenger, shows strong nutrition and health-promoting functions from numerious studies, such as promoting visual acuity, inhibiting lipid peroxidation, preventing blood board gathered, maintaining normal permeability of the blood vessels, hindering obesity and diabetes, protecting certain cancers, radiation and mutations diseases.The genetics study and analysis of anthocyanin accumulation in rice has been performed for more than half a century. Due to domestication and cultivation, the morden major cultivars of rice in the world are white seeds. And most of anthocyanin accumulate in seeds coat in other colored cultivars, which may loss during food preparation dut to its physical solubility. In this thesis, we transformed two transcription factors, Delila and Rosea, which regulate anthocyanin synthesis in Antirrhinum majus, into rice genome. We anticipate that anthocyanin metabolism and synthesis in rice could be influenced by exogenous genes, which is a novel way of genetic improvement of anthocyanin in crops.The research mainly includes the following work:1. To study the function of two transcription factors--Delila and Rosea, three over-expression vectors, pCAMBIA1300-Rosea, pROK2-Delila and pCAMBIA1300-Rosea–Delila, were constructed by inserted them into binary vector driven with 35S promoter. These vectors were then transformed into the embryonic callus tissue of wild rice (Nipponbare) through Agrobacterium-mediated method. Through co-culture, resistance screening, pre-differentiation, redifferentiation and rooting-culture, 20,25,40 transgenic lines were generated respectively. 60 lines were confirmed as postive lines by molecular analysis .2. Exogenous genes have stably expressed in transgenic plants confirmed by RT-PCR and Northern Blot. The expression levels of some key structure genes of the pathway of anthocyanin synthesis including CHS, F3H, DFR, ANS and UFGT were analyzed by semi-quantitative PCR in the transgenic plants. The results indicated that the expression level of DFR in transgenic plants was increased in various degrees.3. The relative total content of rice anthocyanin was determinated by HPLC. The results show that the relative total content of anthocyanin in R21-3 line and RD33-5 line are 8.85% and 11.4% higher than that in wild type, respectively. However, the relative total content of anthocyanin in D17-8 line was 16.2% lower than that in wild type. The ingredients and contents of anthocyanin and flavonoids in transgenic plants are almost the same as wild type by HPLC analysis.4. DFR gene was cloned from black and wild type rice respectively. Sequence analysis showed that there was a stop codon TAG mutantion in the second exon of the coding sequence in wide type rice, while a codon TCG was in the same position in black rice. As the significant function of DFR in the pathway of anthocyanin synthesis, we constructed the overexpression vector of DFR for the future study.5. The rice GluC promoter was also cloned for specific expression of DFR in endosperm for future study.