| The dihydroflavonol 4-reductase(DFR) is the key enzyme of anthocyanin synthesis pathway in plants, it plays an important role in the ornament and impact on the color of flower and fruit. According to earlier stage research in our laboratory, it had function of increase production in Zea mays, and the effects on the enzyme for the production has no report.Therefore, this experiment with DFR gene as the research object, with Oryza sativa as experiment material, using bioinformatics related technology for system analysis of DFR gene family, digging their characteristics, origin and evolution, biological function and expression pattern, on the basis of relevant information and use the sequence in Zea mays as reference, cloning the DFR sequence in Jijing 88, construct overexpression vector, by using agrobacterium mediated transformation of Oryza sativa genetic transformation technology, verify the gene’s function further, the results as follows:1. There are at least 55 members of the DFR gene family in the Oryza sativa Japonica Group. They belonged to dehydrogenase and restore enzymes. It originated later than the differentiation between monocotyledon and dicotyledon, it’s obvious participating in diverse metabolic pathways and functional differentiation.2. This study analyzed the Oryza sativa DFR gene family expression patterns in different tissue, development stage and different hormone treatment, the results founded that some of the members are expressed in a tissue-specific and developmental-specific fashion. To express quantity data to construct the interactions of network analysis, can determine the interaction mode and influence between the members of the family.3. Discussed the number of transgenerational effect on callus. Researches showed that, with the increase of the number of transgenerational times, the results showed a sharp decline in the number of callus differentiation rate and regenerative rate, differentiation rate fell to 2.6% from 85.3% original, regeneration rate from 22.3% to 1.25%; The soluble protein content had no effect; Soluble sugar content decreased steadily; The influence of the peroxidase activity was in more complex, presents a trend that climb up and then decline; With the increase of transgenerational number, callus cells had transition from close structure, rich contents, small vacuoles, arranged a tidy state to a loose structure, volume relatively large, containing a large vacuole, almost no significant organelles gradually.This showed that cell change from embryonic callus callus into non-embryonic callus with transgenerational times increase gradually.4. Construct the DFR gene overexpression vector and genetic transformation, to obtain resistance callus;5. To use the Oryza sativa root, stem, leaf, callus, resistant callus as material conducted a study of qPCR. The results showed that, use callus as reference, the DFR gene expression quantity of resistant callus was 9.6 times of callus, DFR gene expression in resistant callus that amount raised obviously. In not genetically modified samples, expressed in differentiated group higher than that of undifferentiated tissue, relative expression quantity in the leaf is the highest, it is 217.6 times than in callus; Followed by the root, the expression is 136.3 times than in callus; Finally as the stem, it is 124.9 times than in callus.This indicated that the gene involved in specific biological processes.This study to further excavate the function of the known genes provides an important tool and method, for application of bioinformatics in crop breeding and germplasm innovation provides the beneficial reference. |
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