Bioinformatics analysis and expression in different tissues and prokaryotic induction of NtBGAL gene from Nicotiana tabacum
[Objective]The expression pattern and protein characteristics of the β-galactosidase gene NtBGAL from Nicotiana tabacum were explored to provide a basis for the future development of tobacco as a versatile platform material.[Method]Through comparing the homolo-gous between the gene sequences of N.tabacum K326 and NbBGAL1 gene of N.benthamiana,NtBGAL gene was cloned through PCR by tak-ing 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.Final-ly,the research on prokaryotic expression,protein purification and protein characterization of the gene was carried out.[Result]The cloned tobacco NtBGAL gene obtained in the study exhibited the highest homology with the NbBGAL1 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 mo-lecular weight of 92.44 kDa,a theoretical isoelectric point(pI)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 NtBGAL in different tissues of N.tabacum,followed by flower＞leaf＞stem＞root.The prokaryotic expression was performed at 37 ℃,NtBGAL protein had obvious expression,and the higher purified NtBGAL protein was obtained by purification method of inclusion body.The optimal temperature for the enzymatic reaction of NtBGAL protein was 30-40 ℃ and the optimal pH was 4.0-6.5.NtBGAL enzyme activity was enhanced at Mn2+concentrations ranging from 0.05 to 0.15 mmol/L.[Conclu-sion]The study clarifies the tissue expression pattern and protein characterization of the NtBGAL gene,which lays the foundation for further modification of the NtBGAL gene to produce humanized glycoproteins in N.tabacum.
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