普通烟草ẞ-半乳糖苷酶基因 (NtBGAL) 的 生信分析及其在不同组织及原核诱导的表达.

[Objective] The expression pattern and protein characteristics of the B-galactosidase gene NIBGAL from Nicotiana tabacum were explored to provide a basis for the future development of tobacco as a versatile platform material. (Method) Through comparing the homologous between the gene sequences of N. tabacum K326 and NbBGALI gene of N. benthamiana, NIBGAL gene was cloned through PCR by taking cDNA of K326 as the template. Subsequently, bioinformatics tools were used to analyze its gene structure and protein physicochemical properties. The gene expression pattern in different tobacco tissues (roots, stems, leaves and flowers) was examined using qRT-PCR. Finally, the research on prokaryotic expression, protein purification and protein characterization of the gene was carried out. (Result) The cloned tobacco NIBGAL gene obtained in the study exhibited the highest homology with the NbBGALI gene in N. benthamiana, and both possessed identical structural domains (Motif). The gene was located on chromosome 9, and it contained 18 introns. The mRNA length of the gene was 6649 bp. The CDS was 2541 bp encoding 846 amino acids. The enzyme belonged to the GH35 family. The NtBGAL protein had a molecular weight of 92.44 kDa, a theoretical isoelectric point (pl) of 6.8, GRAVY score of -0.222, and was localized on the cell wall. There was a significant difference in gene expression levels of NIBGAL in different tissues of N. tabacum, followed by flower > leaf > stem > root. The prokaryotic expression was performed at 37 °C, NtBGAL protein had obvious expression, and the higher purified NIBGAL protein was obtained by purification method of inclusion body. The optimal temperature for the enzymatic reaction of NtBGAL protein was 30-40 °C and the optimal pH was 4.0-6.5. NIBGAL enzyme activity was enhanced at Mn2+ concentrations ranging from 0.05 to 0.15 mmol/L. [Conclusion] The study clarifies the tissue expression pattern and protein characterization of the NIBGAL gene, which lays the foundation for further modification of the NIBGAL gene to produce humanized glycoproteins in N. tabacum. [ABSTRACT FROM AUTHOR]