Abstract: It is known that the threonine at position 1 084 of the third coiled-coil (CC3) of the new member of the tobacco-driven family NtTkr is crucial for the binding of the target protein. Multiple candidate proteins interacting with NtTkr were obtained by yeast two-hybrid screening of NtTkr tail. In order to determine the importance of T₁₀₈₄ deletion or replacement mutation between NtTkr and target protein vitro, first the pBI121-NtTkr plasmid was take as a template to obtain the tobacco NtTkr tail T₁₀₈₄ deletion and replacement tail by overlap extension PCR, and cloned T_1084d,T1084A into pUC19, by the blue white spot screening, Sma Ⅰ-BamH Ⅰ double enzyme cuting the identification and gene sequencing, geting the right T₁₀₈₄ deletion and replacement NtTkr tail; Then restructured pUC19-T_1084d, pUC19-T1084A and pMXB10 with Not Ⅰ-Nde Ⅰ double enzyme, the target fragment and the carrier fragment are recovered and connected, and the ligation product transforms DH5α. The recombinant was screened to Not Ⅰ-Nde Ⅰ double enzyme identification, managed to build the required pMXB10-NtTkr-T1084A and pMXB10-NtTkr-T_1084d prokaryotic expression vector; Finally, pMXB10-NtTkr-T1084A and pMXB10 -NtTkr-T_1084dd were transferred into BL21 (DE3), and the protein expression was detected by 12% SDS-PAGE after the induction of 0.05 and 0.06 mmol/L IPTG concentrations, respectively NtTkr-T1084A-1320 of about 77 ku and NtTkr -T_1084d-1317 of 76.2 ku were highly expressed.

Key words: Tbacco, NtTkR, Deletion and substitution mutations, Vector construction, Induced expression

CLC Number: 

• S188.2

• Q291