[Objective] The aim was to study the cDNA cloning of SMAT gene from Chimonanthus praecox and its expression. [Method] A novel cDNA was cloned by PT-PCR using the total RNA as template, and amplified to the desirable s...[Objective] The aim was to study the cDNA cloning of SMAT gene from Chimonanthus praecox and its expression. [Method] A novel cDNA was cloned by PT-PCR using the total RNA as template, and amplified to the desirable size. The RT-PCR products were reclaimed and transformed into E. coli DH 5o together with the PMD18-T vector after ligating by T-A cloning. Identified by colony PCR and EcoRI and Notl digestion, the recombinant plasmid with target gene was screened out and conducted the sequence analysis. [Result] Results of the sequence analysis showed that the ORF fragment of the SAMT cDNA was successfully cloned from Chimonanthus praecox gene, with the length of 1 196 bp and encoding 380 amino acids fragment which shared 99.2% homology to that of previously reported SAMT cDNA from Chimonanthus praecox （ABU88887）. The SAMT cDNA fragment was sub-cloned into the prokaryotic expression vector PGEX1-4T-1, and the obtained re- combinant plasmid was named PGSAMT. After inducting by 0.01mol/L IPTG, the re- sult of the SDS-PAGE analysis showed that the molecular weight of the fusion ex- pression SAMT protein was about 66 kDa, which was close to the predicted fusion protein derived from the 26 kDa GST band and 42.3 kDa SAMT gene of Chimo- nanthus praecox encoded protein. [Conclusion] This study successfully cloned and expressed the SAMT gene of Chimonanthus praecox.展开更多
文摘[Objective] The aim was to study the cDNA cloning of SMAT gene from Chimonanthus praecox and its expression. [Method] A novel cDNA was cloned by PT-PCR using the total RNA as template, and amplified to the desirable size. The RT-PCR products were reclaimed and transformed into E. coli DH 5o together with the PMD18-T vector after ligating by T-A cloning. Identified by colony PCR and EcoRI and Notl digestion, the recombinant plasmid with target gene was screened out and conducted the sequence analysis. [Result] Results of the sequence analysis showed that the ORF fragment of the SAMT cDNA was successfully cloned from Chimonanthus praecox gene, with the length of 1 196 bp and encoding 380 amino acids fragment which shared 99.2% homology to that of previously reported SAMT cDNA from Chimonanthus praecox （ABU88887）. The SAMT cDNA fragment was sub-cloned into the prokaryotic expression vector PGEX1-4T-1, and the obtained re- combinant plasmid was named PGSAMT. After inducting by 0.01mol/L IPTG, the re- sult of the SDS-PAGE analysis showed that the molecular weight of the fusion ex- pression SAMT protein was about 66 kDa, which was close to the predicted fusion protein derived from the 26 kDa GST band and 42.3 kDa SAMT gene of Chimo- nanthus praecox encoded protein. [Conclusion] This study successfully cloned and expressed the SAMT gene of Chimonanthus praecox.
