Research On3α-HSD/CR-aiiA Gene Co-Transformation Of Rice By Agrobacterium Tumefaciens Mediated Method

With the development of human activities, a large number of chemicals are entered people'sproductional and living environment, causing serious pollutions. Gram-negative bacteriumComamonas testosteroni is widely distributed in nature, which has the ability to utilize steroids(testosterone, bile acid) as sole carbon and energy sources and degrade the substrate completely.3a-Hydroxysteroid dehydrogenase/carbonyl reductase (3a-HSD/CR) from Comamonastestosteroni is a key enzyme involved in the degradation of the stablely hormonally activecompounds, which is very important in the bioremediation of pollutants. The Bt aiiA gene encodedan enzyme involved in the degradation of N-acyl-homoserine lactones (AHLs) which decrease thevirulence of bacterial pathogens.In this study, eukaryotic double gene expression vector was constructed based on theprokaryotic double gene expression vector and then introduced into Agrobacterium tumefaciens toobtain transgenic rice. Then detect the fusion protein expression and biological activity of thegenetically modified rice were detected. Research methods and findings were as follows:1. The eukaryotic expression vectors containing fusion gene3α-HSD/CR-aiiA wereconstructed and named pTCK303-3α-HSD/CR-aiiA. The results of restriction endonucleasedigestion and sequencing showed that the vectors were successfully constructed.2. With the help of NB+2.0mg/L2.4-D culture medium, Minghui86and Nipponbare callusreceptor system were induced. Fusion gene3α-HSD/CR-aiiA was transferred to the two kinds ofrice callus receptors respectively by agrobacterium mediated method. The results of PCR andGUS histochemical staining showed that transgenic rice plants contaning these genes weresuccessfully obtained.3. The results of ELISA showed that the3α-HSD/CR protein was existed in the transgenicrice plants and the content of target proteins were more than the control.4. The leaves from the transgenic rice plants were inoculated Erwinia carotovora, and theresults showed that AiiA proteins were expressed in transgenic rice leaves.5. The results of ELISA and Erwinia carotovora demonstrated that the fusion gene3α-HSD/CR-aiiA were expressed in the transgenic rice leaves.