| Summary: | 碩士 === 元智大學 === 生物科技與工程研究所 === 104 === When recombinant glycoproteins are expressed in rice cells, they will be added plant-specific xylose and fucose on glycoproteins. It may lead to allergic reactions in some patients, thereby affect the application in plant recombinant protein expression system. We previously applied RNA interference (RNAi) to known down plant specific glycotransferases, controlled by either constitutive ubiquitin promoter and or inducible α-Amy3 promoter to reduce β-1,2-xylosyltransferase (XT) and α-1,3-fucosyl transferase (FT) for humanization of plant glycosylation system. This study analyzed ubiquitin (UT)and α-Amy3 promoter(AT) regulated reduction of plant specific glycosyltransferase gene expression during rice seed germination. Compare to WT, UT and AT were late in germination, and the amylolytic enzyme activity was low in UT, whereas AT and WT were similar after seed germination for 3-5 days, implying that inhibition of plant specific glycan may affect the enzyme activity of the only glycoprotein in α-amlylase family, αAmy7. For application of recombinant glycoprotein in rice cells, α-Amy3 promoter and its signal peptide was fused to Bone morphogenetic protein 2 (BMP2), which functions in bone cell induction. BMP2 monomer formed by 114 amino acids, and glycosylation increases the molecular weight from 18kDa to 22kDa. Mature and active form BMP2 is dimer linked by disulfide bond, and molecular weight of BMP2 dimer is predicted as 30 kDa. In this study, 16 putative calli containing BMP2 gene, and 11 lines showed BMP2 mRNA. We used western blot to analyze seven suspension cell line treated in 1 / 10X PBS for three days for a better yield than sugar free medium,and observed two bands around 18 and 38kDa by BMP2 polyclonal antibodies. A highest BMP2 protein expression line generated in this study was BB15 among six BMP2 transgenic plants.
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