Studies On Transgenic HBMP-3m Gene In Tobacco

Human bone morphogenetic protein 3 is a member of TGF- P 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. At present it has been tried to use to cure some fracture and bone coloboma. BMP-3 is a secretory protein that is synthesized as a single peptide-containing precursor, then the precursor can be post-translationally modified to form the mature protein BMP-3. Human BMP-3 cDNA encodes a whole peptide of 472 amino acid residues . According to the common endopeptidase site RXXR of BMPs precursor, the possible mature peptide is presumably at the C-terminal 127 amino acid region.. 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 become a highlight increasingly. According to this, based on the expression vector of hBMP-3 mature peptide constructed by Dang Yong-jun,we introduced the plasmid pCAMBIA-hBMP-3m directly to the strain LBA4404 by freeze-thaw method and set up the system of high frequency regeneration and transformation of tobacco and obtain transgenic tobacco.On the basis of tobacco NC89 seed, we cultivated aseptic seedling from culture medium MS with additional 20g/L sucrose. After cultivation of three or four generation, we used leafdisk as explant to germinate in different culture media. After analysis of the influence of callus differentiation and shoot regeneration under different concentration of Hygromycin (Hyg) , 25mg/L Hyg was considered as chosen pressure of tobacco regenaration. Using NC89 aseptic seedling leaf as explant, soaked tobacco by Agrobacterium tumefaciens with hBMP-3m gene, we studied the genetic transformation and obtained Hyg fastness-plant after induced filtrating differentiation and rooted cultivating . Two pairs of premers were formed by hBMP-3m gene both sides sequence, 35S promoter sequence and hBMP-3m end sequence , which aimed at examing the fastness-plant general DNA.The general proteins of tobacco were extracted and examed by SDS-PAGE and Western Blot. The main results are as follows:1 Introduce hBMP-3m plant expression vector pCAMBIA-hBMP-3m to Agrobacteriumtumefaciens by way of ice-thaw-media.2 Through the ladder experiment of selecting transformer concentration by Hyg, define the proper concentration as 25mg/L Hyg to transform inconstant shoot.3 Construct a genetic transform system with NC89 aseptic seeding leafdisk as explant and obtain Hyg fastness-plant by introducing hBMP-3m gene into tobacco by way of Agrobacterium tumefaciens mediation.4 Part of fastness-plant has combined hBMP-3m gene with tobacco gene ,and for the first time, obtain tobacco transgenic plant with hBMP-3m gene.5 The result of Western Blot showed that there were trace BMP in transgenic tobacco.