【摘要】 已知烟草驱动家族新成员NtTkr尾部第3个卷曲螺旋（Coiled-coil, CC3）第1 084位苏氨酸(T₁₀₈₄)对靶蛋白的结合至关重要,通过对NtTkr尾部的酵母双杂交筛选得到多个与Ntkr互作的候选蛋白。为确定T₁₀₈₄缺失(T_1084d)或替换（T1084A）突变对NtTkr与这些候选蛋白体外互作的重要性,首先以pBI121-NtTkr为模板,通过重叠延伸PCR扩增得到烟草NtTkr尾部T_1084d、T1084A片段并将其克隆入质粒pUC19,经蓝白斑筛选、SmaⅠ-BamHⅠ双酶切鉴定后送去测序,获得正确的T₁₀₈₄缺失和替换的NtTkr尾部;然后将重组的pUC19-T_1084d、pUC19-T1084A与pMXB10进行NotⅠ-NdeⅠ双酶切,回收目的片段和载体片段并连接,连接产物转化大肠杆菌感受态DH5α,筛选得到重组子,进行NotⅠ-NdeⅠ双酶切鉴定,最终成功构建pMXB10-NtTkr-T1084A和pMXB10-NtTkr-T_1084d原核表达载体;最后将原核表达载体pMXB10-T_1084d和pMXB10-T1084A转入BL21（DE3）中,分别经0.05,0.06 mmol/L IPTG浓度诱导,12%SDS-PAGE电泳检测蛋白质表达情况,结果证明获得了NtTkr-T_1084d-1317和NtTkr-T1084A-1320的成功表达,且IPTG浓度大于0.06 mmol/L时,均可高效表达约77 ku的NtTkr-T1084A-1320和76.2 ku的NtTkr-T_1084d-1317蛋白量。更多还原

【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_{1084 d},T1084 A 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_{1084 d}, pUC19-T1084 A 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-T1084 A and pMXB10-NtTkr-T_{1084 d} prokaryotic expression vector; Finally, pMXB10-NtTkr-T1084 A and pMXB10-NtTkr-T_{1084 d}d 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-T1084 A-1320 of about 77 ku and NtTkr-T_{1084 d}-1317 of 76.2 ku were highly expressed.更多还原