烟草NtTkr尾部T_(1084)缺失和替换突变原核表达载体的构建及诱导表达
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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(T_(1084))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T_(1084)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T_(1084)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。
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 T_(1084) 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 T_(1084) 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 T_(1084) 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.
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 T_(1084) 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 T_(1084) 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 T_(1084) 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.
引文
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[15] 黄申,霍梦杰,钱玉梅,魏涛,贾春晓,何春雨,杨峰,樊新顺,毛多斌.西柏三烯-4,6-二醇降解菌的筛选鉴定及其在再造烟叶中的应用[J].河南农业科学,2018,47(10):137-142,148.doi:10.15933/j.cnki.1004-3268.2018.10.025.Huang S,Huo M J,Qian Y M,Wei T,Jia C X,He C Y,Yang F,Fan X S,Mao D B.Screening and identification of a cembratriene-4,6-diol degradating strain and its application in reconstituted tobacco [J].Journal of Henan Agricultural Sciences,2018,47(10):137-142,148.
[16] 李芬,田树娟,许森,武玉光,孔艳,张帅,王艳芳.抗人Elp3的N末端抗体制备及在染色质免疫沉淀中的应用[J].中国生物化学与分子生物学报,2009,25(10):919-925.doi:10.13865/j.cnki.cjbmb.2009.10.005.Li F,Tian S J,Xu S,Wu Y G,Kong Y,Zhang S,Wang Y F.Preparation of polyclonal antibody against n-terminal of human Elp3and its application in chromatin immunoprecipitation[J].Chinese Journal of Biochemistry and Molecular Biology,2009,25(10):919-925.
[17] 连凯琪,周玲玲,张明亮,王英杰,张慢,宋玉伟.新发猪圆环病毒3型Cap蛋白的原核表达和纯化[J].河南农业科学,2018,47(11):120-123,129.doi:10.15933/j.cnki.1004-3268.2018.11.022.
[18] Zhao B,Lu W,Yang L,Zhang B,Wang L,Yang,S.Cloning and characterization of the genes for biosynthesis of the compatible solute ectoine in the moderately halophilic bacterium Halobacillus dabanensis D-8(T)[J].Current Microbiology,2006,53(3):183-188.doi:10.1007/s00284-005-0396-0.
[2] 李芬,吴秀丽,郭君丽.新基因NtTkr及其同源物LeTKR的生物信息学分析[J].河南师范大学学报(自然科学版),2014,42(5):110-115.doi:10.16366/j.cnki.1000-2367.2014.05.011.Li F,Wu X L,Guo J L.Bioinformatics analysis of the new gene NtTkr and its counterpart LeTKR [J].Journal of Henan Normal University(Natural Science Edition),2014,42(5):110-115.
[3] 李芬,任向波,腾飞,吴秀丽,田树娟.烟草新驱动蛋白NtTKR过表达对酵母生长的影响[J].河南师范大学学报(自然科学版),2014,42(6):97-102.doi:10.16366/j.cnki.1000-2367.2014.06.022.Li F,Ren X B,Teng F,Wu X L,Tian S J.Effect of new driver protein NtTKR overexpression on yeast growth[J].Journal of Henan Normal University(Natural Science Edition),2014,42(6):97-102.
[4] Vale R,Reese T,Sheetz M.Identification of a novel force-generating protein,kinesin,involved in microtubule-based motility[J].Cell,1985,42(1):39-50.doi:10.1016/S0092-8674(85)80099-4.
[5] Sablin E P,Kull F J,Cooke R.Crystal structure of the motor domain of the kinesin-related motor ncd[J].Nature,1996,380(6574):555-559.doi:10.1038/380555a0.
[6] Asbury C L,Fehr A N,Block S M.Kinesin moves by an asymmetric hand-over-hand mechanism [J].Science,2003,302:2130-2134.doi :10.1126/science.1092985.
[7] Yildiz A,Tomishige M,Vale R D,Selvin P.Kinesin walks hand-over-hand[J].Science,2004,303:676-678.doi:10.1126/science.1093753.
[8] Vale R D,Fletterick R J.The design plan of kinesin motors[J].Annu Rev Cell Dev Biol,1997,13:745-777.doi :10.1146/annurev.cellbio.13.1.745.
[9] Khazak V I,Golemis E A,Weber L.Development of a yeast two-hybrid screen selection of human Ras-Raf protein interaction inhibitors [J].Methods Mol Biol,2005,310:253-271.doi :10.1007/978-1-59259-948-618.
[10] Zheng Y,Tan X,Pyczek J,Nolte j,Engel W.Generation and characterization of yeast two-hybrid cDNA libraries derived from two distinct mouse pluripotent cell types[J].Mol Biotechnol,2013,54(2):228-237.doi :10.1007/s12033-012-9561-4.
[11] Watanabe T,Ito Y,Yamada T,Hashimoto M,Tanaka H.The roles of the c-terminal domain and type iii domains of chitinase a1 from bacillus circulans wl-12 in chitin degradation [J].Journal of Bacteriology,1994,176(15),4465-4472.doi:10.1128/jb.176.15.4465-4472.1994.
[12] 童文艳,徐林娜,乔慧聪,李芬.烟草H2A的序列分析、原核表达载体构建及诱导表达[J].安徽农业科学,2018,46(10):94-96,108.doi:10.3969/j.issn.0517-6611.2018.10.027.Tong W Y,Xu L N,Qiao H C,Li F.Sequence analysis,construction and induced expression of prokaryotic expression vector of tobacco H2A gene[J].Anhui Agric Sci,2018,46(10):94-96,108.
[13] Chong S,Mersha F B,Comb D G,Scott M E,Landry D,Vence L M.Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element[J].Gene,1997,192(2):271-281.doi:10.1109/84.825779.
[14] 李芬,田树娟,张帅,孔艳,王艳芳.酵母组蛋白乙酰转移酶Elp3多克隆抗体的制备、鉴定及应用[J].生物工程学报,2009,25(8):1261-1266.doi:10.3321/j.issn:1000-3061.2009.08.020.Li F,Tian S J,Zhang S,Kong Y,Wang Y F.Identification and application of yeast histone acetyltransferases Elp3 polyclonal antibody [J].Chinese Journal of Biotechnology,2009,25(8):1261-1266.
[15] 黄申,霍梦杰,钱玉梅,魏涛,贾春晓,何春雨,杨峰,樊新顺,毛多斌.西柏三烯-4,6-二醇降解菌的筛选鉴定及其在再造烟叶中的应用[J].河南农业科学,2018,47(10):137-142,148.doi:10.15933/j.cnki.1004-3268.2018.10.025.Huang S,Huo M J,Qian Y M,Wei T,Jia C X,He C Y,Yang F,Fan X S,Mao D B.Screening and identification of a cembratriene-4,6-diol degradating strain and its application in reconstituted tobacco [J].Journal of Henan Agricultural Sciences,2018,47(10):137-142,148.
[16] 李芬,田树娟,许森,武玉光,孔艳,张帅,王艳芳.抗人Elp3的N末端抗体制备及在染色质免疫沉淀中的应用[J].中国生物化学与分子生物学报,2009,25(10):919-925.doi:10.13865/j.cnki.cjbmb.2009.10.005.Li F,Tian S J,Xu S,Wu Y G,Kong Y,Zhang S,Wang Y F.Preparation of polyclonal antibody against n-terminal of human Elp3and its application in chromatin immunoprecipitation[J].Chinese Journal of Biochemistry and Molecular Biology,2009,25(10):919-925.
[17] 连凯琪,周玲玲,张明亮,王英杰,张慢,宋玉伟.新发猪圆环病毒3型Cap蛋白的原核表达和纯化[J].河南农业科学,2018,47(11):120-123,129.doi:10.15933/j.cnki.1004-3268.2018.11.022.
[18] Zhao B,Lu W,Yang L,Zhang B,Wang L,Yang,S.Cloning and characterization of the genes for biosynthesis of the compatible solute ectoine in the moderately halophilic bacterium Halobacillus dabanensis D-8(T)[J].Current Microbiology,2006,53(3):183-188.doi:10.1007/s00284-005-0396-0.
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