利用转移PCR技术构建玉米SnRK2基因家族表达载体
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摘要
在构建玉米蔗糖非酵解型蛋白激酶2(SnRK2)基因家族表达载体的过程中,因为表达载体可供选择的多克隆位点较少,且有些还与目的基因同源,所以采用转移PCR(T-PCR)扩增技术替代通常的酶切连接方法。为避免错误连接或未连接的第一轮T-PCR中间产物对退火温度不同的第二轮扩增循环产生干扰,本研究对T-PCR接头引物3′-端和5′-端的两段序列及其退火温度、引物、供体质粒和目标质粒模板的浓度,特别是温度循环程序进行设计和筛选,并用扩增构建的重组载体转化大肠杆菌感受态细胞,筛选阳性克隆,进行菌液PCR和测序验证。结果表明,在两条引物3′-端序列理论退火温度相差不大的情况下,T-PCR第一轮扩增的退火温度以低于或接近其中较低的理论退火温度为宜。但在两条引物3′-端序列理论退火温度相差较大的情况下,则应高于其中较低的理论退火温度。T-PCR第二轮扩增的退火温度,适当低于两条引物理论退火温度的平均值。按优化的温度循环程序,从供体质粒pMD19-T扩增ZmSnRK2基因家族8个成员的编码序列,并整合到目标载体pHBT95启动子下游特异位点,重组率达60%以上。综上表明,T-PCR对没有适用多克隆位点的载体构建的实用性较强。本研究通过优化的温度循环程序为表达或诱变载体构建提供了一定的理论参考。
For construction of expression vectors of sucrose non-fermenting protein kinase 2(SnRK2) gene family, transfer-PCR(T-PCR) technique was applied instead of the usual ligation-dependent method, because of the unavailable multiclonal sites of the vector and their homology to the target genes. In order to avoid the interference of mis-ligated or un-ligated intermediates amplified in the first round of T-PCR on the second round of T-PCR with different annealing temperatures, the 3′-and 5′-end sequences of the adaptor primers, their annealing temperatures, the concentration of the primers, the template concentration of the donor and the target plasmids, and especially the temperature cycles were carefully designed and screened. The recombined vectors were transformed into competent cells of Escherichia coli. The positive clones were screened, and identified by bacterial PCR and sequencing. The results showed that the annealing temperature of the first round of T-PCR amplification should be lower than or close to the lower theoretical annealing temperature, in case the theoretical annealing temperatures of the 3′-end sequences of the two primers were not very different. However, the annealing temperature should be higher than the lower theoretical annealing temperature, in case the theoretical annealing temperatures of the two primers were different greatly. The annealing temperature of the second round of T-PCR amplification should be lower than the average of the theoretical annealing temperatures of the two primers. According to the optimized temperature cycle, the coding sequences of eight members of the ZmSnRK2 gene family were amplified from the donor plasmids pMD19-T, and successfully integrated into the specific site downstream of the promoter of the target vectors pHBT95 with recombination rate of more than 60%. The result indicated the practicability of T-PCR for vector construction without available multiclonal sites. The optimized temperature cycle provided reference for construction of expression or mutagenesis vectors.
For construction of expression vectors of sucrose non-fermenting protein kinase 2(SnRK2) gene family, transfer-PCR(T-PCR) technique was applied instead of the usual ligation-dependent method, because of the unavailable multiclonal sites of the vector and their homology to the target genes. In order to avoid the interference of mis-ligated or un-ligated intermediates amplified in the first round of T-PCR on the second round of T-PCR with different annealing temperatures, the 3′-and 5′-end sequences of the adaptor primers, their annealing temperatures, the concentration of the primers, the template concentration of the donor and the target plasmids, and especially the temperature cycles were carefully designed and screened. The recombined vectors were transformed into competent cells of Escherichia coli. The positive clones were screened, and identified by bacterial PCR and sequencing. The results showed that the annealing temperature of the first round of T-PCR amplification should be lower than or close to the lower theoretical annealing temperature, in case the theoretical annealing temperatures of the 3′-end sequences of the two primers were not very different. However, the annealing temperature should be higher than the lower theoretical annealing temperature, in case the theoretical annealing temperatures of the two primers were different greatly. The annealing temperature of the second round of T-PCR amplification should be lower than the average of the theoretical annealing temperatures of the two primers. According to the optimized temperature cycle, the coding sequences of eight members of the ZmSnRK2 gene family were amplified from the donor plasmids pMD19-T, and successfully integrated into the specific site downstream of the promoter of the target vectors pHBT95 with recombination rate of more than 60%. The result indicated the practicability of T-PCR for vector construction without available multiclonal sites. The optimized temperature cycle provided reference for construction of expression or mutagenesis vectors.
引文
[1] Wang K.Agrobacterium Protocols [M].Second Edition.New Jersey:Humana Press Inc.,2006:185-199
[2] Wang K,Frame B.Biolistic gun-mediated maize genetic transformation [J].Methods in Molecular Biology,2009,526:29-45
[3] Char S N,Unger-Wallace E,Frame B,Briggs S A,Main M,Spalding M H,Vollbrecht E,Wang K,Yang B.Heritable site-specific mutagenesis using TALENs in maize [J].Plant Biotechnology Journal,2015,13(7):1002-1010
[4] Char S N,Neelakandan A K,Nahampun H,Frame B,Main M,Spalding M H,Becraft P W,Meyers B C,Walbot Ⅴ,Wang K,Yang B.An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize [J].Plant Biotechnology Journal,2017,15(2):257-268
[5] Cregg J M,Cereghino J L,Shi J,Higgins D R.Recombinant protein expression in Pichia pastoris [J].Molecular Biotechnology,2000,16(1):23-52
[6] Primrose S B,Twyman R M.Principles of Gene Manipulation and Genomics [M].Seventh Edition.Carlton:Blackwell Publishing,2006:1-440
[7] Graslund S,Nordlund P,Weigelt J,Graslund S,Nordlund P,Weigelt J,Hallberg B M,Bray J,Gileadi O,Knapp S,Oppermann U,Arrowsmith C,Hui R,Ming J,dhe-Paganon S,Park H W,Savchenko A,Yee A,Edwards A,Vincentelli R,Cambillau C,Kim R,Kim S H,Rao Z,Shi Y,Terwilliger T C,Kim C Y,Hung L W,Waldo G S,Peleg Y,Albeck S,Unger T,Dym O,Prilusky J,Sussman J L,Stevens R C,Lesley S A,Wilson Ⅰ A,Joachimiak A,Collart F,Dementieva Ⅰ,Donnelly M Ⅰ,Eschenfeldt W H,Kim Y,Stols L,Wu R,Zhou M,Burley S K,Emtage J S,Sauder J M,Thompson D,Bain K,Luz J,Gheyi T,Zhang F,Atwell S,Almo S C,Bonanno J B,Fiser A,Swaminathan S,Studier F W,Chance M R,Sali A,Acton T B,Xiao R,Zhao L,Ma L C,Hunt J F,Tong L,Cunningham K,Inouye M,Anderson S,Janjua H,Shastry R,Ho C K,Wang D,Wang H,Jiang M,Montelione G T,Stuart D Ⅰ,Owens R J,Daenke S,Schutz A,Heinemann U,Yokoyama S,Bussow K,Gunsalus K C.Protein production and purification [J].Nature Methods,2008,5(2):135-146
[8] Green M R,Sambrook J.Molecular cloning:A laboratory manual [J].Analytical Biochemistry,2000,186(1):182-183
[9] Hartley J L,Temple G F,Brasch M A.DNA cloning using in vitro site-specific recombination [J].Genome Research,2000,10(11):1788-1795
[10] Benoit R M,Wilhelm R N,Scherer-Becker D,Ostermeier C.An improved method for fast,robust,and seamless integration of DNA fragments into multiple plasmids [J].Protein Expression and Purification,2006,45(1):66-71
[11] Li M Z,Elledge S J.Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC [J].Nature Methods,2007,4(3):251-256
[12] Aslanidis C,de Jong P J.Ligation-independent cloning of PCR products (LICPCR) [J].Nucleic Acids Research,1990,18(20):6069-6074
[13] Tillett D,Neilan B A.Enzyme-free cloning:A rapid method to clone PCR products independent of vector restriction enzyme sites [J].Nucleic Acids Research,1999,27(19):e26
[14] Neilan B A,Tillett D.Enzyme-free cloning of PCR products and fusion protein expression [J].Methods in Molecular Biology,2002,192:125-132
[15] Gileadi O,Burgess-Brown N A,Colebrook S M,Berridge G,Savitsky P,Smee C E,Loppnau P,Johansson C,Salah E,Pantic NH.High throughput production of recombinant human proteins for crystallography [J].Methods in Molecular Biology,2008,426:221-246
[16] Chen G J,Qiu N,Karrer C,Caspers P,Page M G.Restriction site-free insertion of PCR products directionally into vectors [J].Biotechniques,2000,28(3):498-500,504-505
[17] Geiser M,Cebe R,Drewello D,Schmitz R.Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase [J].Biotechniques,2001,31(1):88-90
[18] Miyazaki K,Takenouchi M.Creating random mutagenesis libraries using megaprimer PCR of whole plasmid [J].Biotechniques,2002,33(5):1033-1034,1036-1038
[19] Ent F V D,Lowe J.RF cloning:A restriction-free method for inserting target genes into plasmids [J].Journal of Biochemical and Biophysical Methods,2006,67(1):67-74
[20] Unger T,Jacobovitch Y,Dantes A,Bernheim R,Peleg Y.Applications of the restriction free (RF) cloning procedure for molecular manipulations and protein expression [J].Journal of Structural Biology,2010,172(1):34-44
[21] Erijman A,Dantes A,Bernheim R,Shifman J M,Peleg Y.Transfer-PCR (TPCR):A highway for DNA cloning and protein engineering [J].Journal of Structural Biology,2011,175(2):171-177
[22] Erijman A,Shifman J M,Peleg Y.A single-tube assembly of DNA using the transfer-PCR (TPCR) platform [J].Methods in Molecular Biology,2014,1116:89-101
[23] Cohen I,Wiener R,Reiss Y,Ravid T.Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases [J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(7):625-632
[24] Mishra N K,Habeck M,Kirchner C,Haviv H,Peleg Y,Eisenstein M,Apell H J,Karlish S J.Molecular mechanisms and kinetic effects of FXYD1 and phosphomimetic mutants on purified human Na,K-ATPase [J].The Journal of Biological Chemistry,2015,290(48):28746-28759
[25] 陶怡,王盈阁,李鸿杰,李晚忱,付凤玲.植物脱落酸信号转导途径的上游信使[J].核农学报,2016,30(9):1722-1730
[26] Wang Y G,Fu F L,Yu H Q,Hu T,Zhang Y Y,Tao Y,Zhu J K,Zhao Y,Li W C.Interaction network of core ABA signaling components in maize [J].Plant Molecular Biology,2018,96(3):245-263
[27] 陶怡.蔗糖非酵解型蛋白激酶2及其底物蛋白在玉米脱落酸信号转导中的作用[D].成都:四川农业大学,2017:1-52
[28] Niyazi M,Niyazi I,Belka C.Counting colonies of clonogenic assays by using densitometric software [J].Radiation Oncology,2007,2:4
[2] Wang K,Frame B.Biolistic gun-mediated maize genetic transformation [J].Methods in Molecular Biology,2009,526:29-45
[3] Char S N,Unger-Wallace E,Frame B,Briggs S A,Main M,Spalding M H,Vollbrecht E,Wang K,Yang B.Heritable site-specific mutagenesis using TALENs in maize [J].Plant Biotechnology Journal,2015,13(7):1002-1010
[4] Char S N,Neelakandan A K,Nahampun H,Frame B,Main M,Spalding M H,Becraft P W,Meyers B C,Walbot Ⅴ,Wang K,Yang B.An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize [J].Plant Biotechnology Journal,2017,15(2):257-268
[5] Cregg J M,Cereghino J L,Shi J,Higgins D R.Recombinant protein expression in Pichia pastoris [J].Molecular Biotechnology,2000,16(1):23-52
[6] Primrose S B,Twyman R M.Principles of Gene Manipulation and Genomics [M].Seventh Edition.Carlton:Blackwell Publishing,2006:1-440
[7] Graslund S,Nordlund P,Weigelt J,Graslund S,Nordlund P,Weigelt J,Hallberg B M,Bray J,Gileadi O,Knapp S,Oppermann U,Arrowsmith C,Hui R,Ming J,dhe-Paganon S,Park H W,Savchenko A,Yee A,Edwards A,Vincentelli R,Cambillau C,Kim R,Kim S H,Rao Z,Shi Y,Terwilliger T C,Kim C Y,Hung L W,Waldo G S,Peleg Y,Albeck S,Unger T,Dym O,Prilusky J,Sussman J L,Stevens R C,Lesley S A,Wilson Ⅰ A,Joachimiak A,Collart F,Dementieva Ⅰ,Donnelly M Ⅰ,Eschenfeldt W H,Kim Y,Stols L,Wu R,Zhou M,Burley S K,Emtage J S,Sauder J M,Thompson D,Bain K,Luz J,Gheyi T,Zhang F,Atwell S,Almo S C,Bonanno J B,Fiser A,Swaminathan S,Studier F W,Chance M R,Sali A,Acton T B,Xiao R,Zhao L,Ma L C,Hunt J F,Tong L,Cunningham K,Inouye M,Anderson S,Janjua H,Shastry R,Ho C K,Wang D,Wang H,Jiang M,Montelione G T,Stuart D Ⅰ,Owens R J,Daenke S,Schutz A,Heinemann U,Yokoyama S,Bussow K,Gunsalus K C.Protein production and purification [J].Nature Methods,2008,5(2):135-146
[8] Green M R,Sambrook J.Molecular cloning:A laboratory manual [J].Analytical Biochemistry,2000,186(1):182-183
[9] Hartley J L,Temple G F,Brasch M A.DNA cloning using in vitro site-specific recombination [J].Genome Research,2000,10(11):1788-1795
[10] Benoit R M,Wilhelm R N,Scherer-Becker D,Ostermeier C.An improved method for fast,robust,and seamless integration of DNA fragments into multiple plasmids [J].Protein Expression and Purification,2006,45(1):66-71
[11] Li M Z,Elledge S J.Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC [J].Nature Methods,2007,4(3):251-256
[12] Aslanidis C,de Jong P J.Ligation-independent cloning of PCR products (LICPCR) [J].Nucleic Acids Research,1990,18(20):6069-6074
[13] Tillett D,Neilan B A.Enzyme-free cloning:A rapid method to clone PCR products independent of vector restriction enzyme sites [J].Nucleic Acids Research,1999,27(19):e26
[14] Neilan B A,Tillett D.Enzyme-free cloning of PCR products and fusion protein expression [J].Methods in Molecular Biology,2002,192:125-132
[15] Gileadi O,Burgess-Brown N A,Colebrook S M,Berridge G,Savitsky P,Smee C E,Loppnau P,Johansson C,Salah E,Pantic NH.High throughput production of recombinant human proteins for crystallography [J].Methods in Molecular Biology,2008,426:221-246
[16] Chen G J,Qiu N,Karrer C,Caspers P,Page M G.Restriction site-free insertion of PCR products directionally into vectors [J].Biotechniques,2000,28(3):498-500,504-505
[17] Geiser M,Cebe R,Drewello D,Schmitz R.Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase [J].Biotechniques,2001,31(1):88-90
[18] Miyazaki K,Takenouchi M.Creating random mutagenesis libraries using megaprimer PCR of whole plasmid [J].Biotechniques,2002,33(5):1033-1034,1036-1038
[19] Ent F V D,Lowe J.RF cloning:A restriction-free method for inserting target genes into plasmids [J].Journal of Biochemical and Biophysical Methods,2006,67(1):67-74
[20] Unger T,Jacobovitch Y,Dantes A,Bernheim R,Peleg Y.Applications of the restriction free (RF) cloning procedure for molecular manipulations and protein expression [J].Journal of Structural Biology,2010,172(1):34-44
[21] Erijman A,Dantes A,Bernheim R,Shifman J M,Peleg Y.Transfer-PCR (TPCR):A highway for DNA cloning and protein engineering [J].Journal of Structural Biology,2011,175(2):171-177
[22] Erijman A,Shifman J M,Peleg Y.A single-tube assembly of DNA using the transfer-PCR (TPCR) platform [J].Methods in Molecular Biology,2014,1116:89-101
[23] Cohen I,Wiener R,Reiss Y,Ravid T.Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases [J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(7):625-632
[24] Mishra N K,Habeck M,Kirchner C,Haviv H,Peleg Y,Eisenstein M,Apell H J,Karlish S J.Molecular mechanisms and kinetic effects of FXYD1 and phosphomimetic mutants on purified human Na,K-ATPase [J].The Journal of Biological Chemistry,2015,290(48):28746-28759
[25] 陶怡,王盈阁,李鸿杰,李晚忱,付凤玲.植物脱落酸信号转导途径的上游信使[J].核农学报,2016,30(9):1722-1730
[26] Wang Y G,Fu F L,Yu H Q,Hu T,Zhang Y Y,Tao Y,Zhu J K,Zhao Y,Li W C.Interaction network of core ABA signaling components in maize [J].Plant Molecular Biology,2018,96(3):245-263
[27] 陶怡.蔗糖非酵解型蛋白激酶2及其底物蛋白在玉米脱落酸信号转导中的作用[D].成都:四川农业大学,2017:1-52
[28] Niyazi M,Niyazi I,Belka C.Counting colonies of clonogenic assays by using densitometric software [J].Radiation Oncology,2007,2:4