| Summary: | 碩士 === 國立中興大學 === 生命科學系 === 91 === Abstract
During the past century, the chemical, industrial and agricultural activities have used heavy metals in a variety of applications, and have released tremendous amount of heavy metals into the biosphere. Bioaccumulation of those heavy metals through food chain will threat people’s daily life. As a model system for genetically modify crops to avoid the bioaccumulation of heavy metal, we attempted to establish transgenic rices by introducing bacterial mercury resistance genes. Previous studies have demonstrated that merA encoding mercuric reductase and merB3 encoding organomercurial lyase, are the key enzymes for detoxification of mercurials in a Minamata Bay isolated Gram-positive bacterium Bacillus megaterium MB1. In this study , we added both of the bacteria and plant translational binding sites in front of merA and merB coding regions. And these gene fragments were cloned downstream the T7 promoter by the pGEM T-easy vector system.
This merA and merB3 was then cloned into plant transformation vectors under the control of actin promoter. The recombinant plasmids were introduced into rice callus through Agrobacterium-mediated transformation method. After selection by hygromycin medium, cells that were antibiotic resistance were able to continue proliferation. The hygromycin-resistance transgenic callus was confirmed by PCR and Southern hybridazation assays. The result has demonstrated that the transgenic callus contain the merA gene and was successfully integrated into the rice callus genome. Besides, the merA was then cloned into yeast expression vector and transformation to yeast. Integration of the merA gene into genome of recombinant strain P. pastoris was confirmation by PCR analysis.
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