Research On Agrobacterium Tumefaciens-Mediskted Transformation Of Gene3α-HSD To Chewing Cane

With the rapid development of human economy, lots of waste gas, waste water and wastematerials were discharged into the environment, which result in more and more severeenvironmental pollution.3α-HSD gene is a key enzyme3α-hydroxysteroid dehydrogenase/carbonyl reductase (3a-HSD/CR) gene from Comamonas testosteroni, which has very gooddegradation function for the steroid compounds of environmental pollutants. C. testosteronitherefore play a significant role in the bioremediation of these stablely hormonally activecompounds in the environment. In the study, the plant expressing vector of UBI-HSD wasconstructed. The callus of NiDeng and FuAn were transformed by the Agrobacterium-mediatedway and the transgenic chewing cane individual plants were got.The positive plants wereanalyzed in molecular, protein and function.The results were as follows:1. The chewing cane plant tissue culture regeneration system is optimized.2mg/L2,4-D isappropriate to the BMMY,1mg/L NAA,2mg/L6-BA is optimum to the differential medium,1.5mg/L NAA is suitable to the rooting medium.2. Hpt resistance screening test showed that the sensitivity of Hpt exists in certain differencein different culture stages. The Hpt sensitivity to the differentiation and rooting of tissue culture ishigher than the callus.50mg/L Hpt is optimum to the BMMY,30mg/L Hpt is appropriate to theplants differentiation and rooting.3.3α-HSD gene was transformed into the chewing cane by Agrobacterium-mediatedway.The results of PCR, PCR-Southern bolt, GUS staining showed that3α-HSD gene had beentransformed into the chewing cane genome succesfully.6Ning-De and2Fu-An transgenic plantswere obtained among the62plants.4. The result of enzyme-linked immunity assay (ELISA) analysis showed that the3α-HSDprotein was successfully expressed in the transgenic chewing cane. The protein express quantitywas higher than the control.5. The cholesterol degradation test showed that the transgenic plants average rate ofcholesterol degradation was80%, which was25%higher than the control.