The Studies On Transgenic HBMP-3m Gene In Rape (Brassica Napus L.)

Human bone morphogenetic protein 3 is a member of TGF-β superfamily. It can induce the differentiation of cartilage and bone tissue in mesenchymal cell, and is important to bone self-repairment and bone development during embryo morphogenesis. In addition, some other biological activities of hBMP-3 have also been found, such as inducing development of embryo and stimulating differentiation of neural and blood cells. Therefore, there is a great prospect in the use of hBMP-3. There is trace content of hBMP-3 in human body. It has been expressed in the expression system of eukaryotes and prokaryotes respectively, but its application is restricted because of defects in the process and modification after translation in prokaryotic cells and higher costs and lower yields existed in eukaryotic expression system. With the successful expression of exogenous gene in plant, the advantage of plant expression system is becoming a highlight increasingly. Therefore, in this paper, the recombinant plant expression vector pCAMBIA1300-hBMP-3m which had been constructed was introduces into Agrobacterium tumefaciens LBA4404. The system of high frequency regeneration and transformation of rape hypocotyls were set up. Subsequently the hBMP-3m gene was transformed into rape through Agrobacterium-mediated system. And the transgenic plants were confirmed by a series of examinations. The results were as follows:1. Introduction of pCAMBIA1300-hBMP-3m plasmid into Agrobacterium tumefaciens. The plasmid pCAMBIA1300-hBMP-3m carried hBMP-3m gene was introduced into Agrobacterium tumefaciens by electroporation. By screening in the medium containing kanamycin, PCR test and enzyme cutting, it was confirmed that the result was successful.2. Regeneration system of plants.The regeneration system of rape was established. The adventitious hypocotyls were induced from explants of rape based on MS basal medium supplemented with MS+6-BA5.0mg/L+NAA0.2mg/L. Then the regeneration plants were rooted on MS+IBA0.3mg/L. 3. Transformation and selection of plants. Hypocotyl explants had been pre-cultured for 2 days, then immersed in Agrobacterium suspension for less 3 minutes. The co-cultivation had been carried out for 2 days. After that , the transformants were obtained by transferring explants to selection medium containing Hyg 25mg/L and rooting medium containing Hyg 25mg/L. 4. Detection of transgenic plants.The PCR assay and Southern blot analysis of hyg-resistant plants showed that the target gene had been integrated into rape.