Protokaryotic Expression Analysis Of ORF216 Protein In Rice And SM-NGT1 Protein In Tobacco

Plant cytoplasmic male sterility (CMS) is maternally inherited with characteristic of abnormal male organ but normal female organ. In rice, CMS can be categorized into three main types of wild abortive (WA), Chinsurah Boro (BT) and Honglian (HL) based on their distinctive cytological and genetic character.Glycosylation reaction, catalyzed by glycosyltransferases (GTs), has an important biological function in both prokaryotes and eukaryotes. GTs catalyze the transfer of glycoside from activated donor (ribonucleoside diphosphate glucose, generally) to specific acceptor, which can be glucose, lipids, proteins and nucleic acid. In plants, GTs play an important role in many aspects, such as regulating the secondary metabolism, the activity of the hormone levels, the detoxication, and involved in biosynthesis of the cell wall.A HL-CMS-associated gene, orf216, and a tobacco glycosyltransferase gene, sm-Ngt1, were cloned by our laboratory previously. In this article, the two target genes were cloned into the prokaryotic expression vector for efficient expression in E. coli, and the proteins were purified in order to prepare the polyclonal antibodies. By method of Western Blotting, the two gene expressions in rice and tobacco plants were analyzed. The main results are as follows.1. The gene, orf216, was obtained by PCR with the specific PCR primers. The recombinant prokaryotic expression vector pTYB1-orf216 was constructed by inserting the orf216 gene into a prokaryotic expression vector pTYB1 with the intein tag. The recombinant plasmid pTYB1-orf216 was then transformed into E.coli ER2566 competent cells. To achieve the high expression of orf216 gene, the expression condition was optimized. The result showed that the optimized condition for inducing protein expression was at 15℃for 15 hours with 0.8 mmol/L IPTG (isopropy-β-D-thiogalactoside). By SDS-PAGE, an approximately 85 kD target protein was observed in the soluble part of isolates. Therefore, the target protein was further isolated and purified through affinity chromatography, and polyclonal 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-PAGE, 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.