Over-expressing One New Tobacco Glycosyltransferase And Its Related Research

Glycosyltransferases (GTs) are found in all living organisms, prokaryotes, eukaryotes, Archaea and viruses. They catalyze the transfer of glycoside from activated donor to specific acceptor. GTs play a key part in the hormone level regulation, against biotic stress, detoxification of biotic toxins and xenotoxins, and cellular secondary metabolism. To date, all the known glycosyltransferases have been classified into 91 families. As their potential economical values and the necessity of their function in plant life cycle, GTs have raised great interest.Our laboratory previously cloned a novel tobacco GT gene, which induced by SA and MeJA. To further investigate its role in plant, the studies on its morphological effect by over-expression of this gene in transgenic tobacco plant, its sub-localization by method of GT-GFP fused protein, and its prokaryotic expression in E.coli were carried out. Main results are as follows.The conservative domain analysis by comparing the novel GT protein with the NCBI and Prosite databases showed that it belongs to UDP- Glycosyltransferase in GT 28 family. There was one transmembrane region between 100-150 aa in the N terminal of novel GT protein predicted by TMPred method. Using the novel GT sequence and the sequences from CAZy database, there were two phylogenetic trees constructed, one with plant glycosyltransferases from GT 28 family, and the other with glycosyltransferases only from tobacco databases. The results showed that this GT was classified into a new cluster in the two trees.Utilizing the plant expressing vector, pCAMBIA1305.1, the GT over-expression vector under the control of CaMV 35S promoter was constructed. Using Agrobacterium-mediated leaf-disk method, its genetic transformation was carried out. After PCR and RT-PCR detection of transgenic tobacco plants by the specific primers, about 40% of positive transgenic plants were selected. Real-time quantitative PCR analysis further showed that the GT expression in transgenic tobacco plants was 5-23 times up-regulated compared with the control. The flanking sequence analysis on GT gene insertion site in one of transgenic plants amplified by TAIL-PCR showed that there is no significant homology with the known gene sequences in NCBI database, indicating that the phenotype of the transgenic plants should be caused by the up-regulated GT expression.Utilizing the vector pCAMBIA132, the GT-GFP fusion protein expression vector under the control of CaMV 35S promoter for GT sub-cellular localization was constructed. Some transgenic tobacco plants were obtained by method of Agrobacterium-mediated transformation and the transcription of the target gene was then conformed by RT-PCR. Fluorescence microscopic observation on plasm-wall separated cell from the root tip of the transgenic tobacco plants showed that the GT-GFP fused protein was mainly localized in the plasm membrane.Using the vector pTYB1, the IPTG induced GT expression recombinant plasmid, pT, was constructed. After its expression in E.coli strain ER2566, the different fractions from crude cell extract were analyzed by SDS-PAGE. The result showed that the protein with its size expected was detected only in the fraction of the deposit, suggesting that the GT protein in E.coli might existed as a form of inclusion body.