水稻orf216蛋白和烟草sm-ngt1蛋白的原核表达研究-prokaryotic expression of rice orf 216 protein and tobacco sm - ng t1 protein.docxVIP

水稻 水稻 ORF216 蛋白和烟草 SM-NGT1 蛋白的原核表达研究 中南民族大学硕士学位论文 中南民族大学硕士学位论文 IV IV V V antibody was prepared after N-terminal sequence analysis of the protein by Liquid-MS. The results of Western Blotting analysis on proteins isolated from rice tissues indicated that the prepared polyclonal antibody was with high specificity. 2. The sm-Ngt1 gene was obtained by PCR using the specific PCR primers. The recombinant prokaryotic expression vectors pET-smNgt1, pTYB1-smNgt1 and pGEX-smNgt1 were constructed by inserting the sm-Ngt1 gene into expression vectors pET-30a with the 6×his tag, pTYB1 with the intein tag and pGEX-6P-1 with the GST tag, respectively. Three recombinant prokaryotic expression vectors were transformed into E.coli BL21, ER2566 and BL21 competent cells, respectively. And the expression conditions were optimized. It was showed that the optimized protein expression conditions were in 0.8 mmol/L IPTG at 15 ℃ for 20 hours for pET-smNgt1, 0.3 mmol/L IPTG at 15 ℃ for 15 hours for pTYB1-smNgt1 and 0.2 mmol/L IPTG at 37 ℃ for 3 hours for pGEX-smNgt1 in E. coli, respectively. By SDS, the target protein with an approximately 55 kD, 100 kD and 80 kD were present only in the insoluble isolates as inclusion bodies. The inclusion bodies were dissolved in denaturant, and then renatured by dialysis. The polyclonal antibody was prepared after N-terminal sequence analysis of the protein by Liquid-MS. The results of Western Blotting indicated that the prepared polyclonal antibody was with high specificity. Key words: rice; tobacco; cytoplasmic male sterile; glycosyltransferases; protokaryotic expression; antibody 英文缩略语及英文对照 Amp bp CMS cpDNA ddH2O Ampicillin Base pair Cytoplasmic male sterility chloroplast DNA Double distilled water 氨苄青霉素 碱基对 细胞质雄性不育 叶绿体 DNA 双蒸水 dNTPs Deoxyribonucleoside triphosphate 脱氧核糖核苷三磷酸(4 种) DTT Dithiothreitol 二巯苏糖醇 E.coli Eschericia coli 大肠杆菌 EB Ethdium bromide 溴化乙啶 EDTA Didodium ethylenediaminetetra-acetate 乙二胺四乙酸 mtDNA GT mitochondrial DNA Gly