利用番茄cDNA酵母双杂交文库筛选Pti4互作蛋白
|
摘要
以番茄的根、叶、花及不同发育时期的果实提取RNA,构建酵母双杂交文库。经检测,文库容量为1.1×10~7 CFU,阳性率大于95%,插入片段平均长度大于1 000 bp,可用于互作蛋白的筛选。依据番茄抗病途径相关基因Pti4编码序列设计引物,构建重组诱饵载体并转化酵母菌,筛选与Pti4相互作用的蛋白质。经初步检测获得6个可能与Pti4互作的转录因子,为进一步研究番茄Pti基因的作用机制提供了基础。
RNA was extracted from tomato roots,leaves,flowers and fruits at different development stages to construct yeast two-hybrid library.The detection showed 1.1×10~7 CFU of total capacity of the library,and the positive rate was greater than 95% with average length of inserted fragments longer than 1 000 bp.According to the coding sequence of tomato disease-resistant Pti4 gene,primers were designed and the recombinant bait vector was constructed,then transformed into yeast to screen the proteins interacting with Pti4.The preliminary screening indicated that six transcription factors might interact with Pti4,which provided a basis for further investigation on the mechanism of Pti genes in tomato.
RNA was extracted from tomato roots,leaves,flowers and fruits at different development stages to construct yeast two-hybrid library.The detection showed 1.1×10~7 CFU of total capacity of the library,and the positive rate was greater than 95% with average length of inserted fragments longer than 1 000 bp.According to the coding sequence of tomato disease-resistant Pti4 gene,primers were designed and the recombinant bait vector was constructed,then transformed into yeast to screen the proteins interacting with Pti4.The preliminary screening indicated that six transcription factors might interact with Pti4,which provided a basis for further investigation on the mechanism of Pti genes in tomato.
引文
[1] 蔡义勇,巩振辉.番茄细菌性斑点病的发生和防治[J].西北园艺(蔬菜专刊),2007(6):21.
[2] 许志刚,沈秀萍,胡白石.番茄上两种细菌性病害的诊断与防治[J].植物检疫,2007,21(1):53-56.
[3] ZHOU J M,TANG X Y,MARTIN G B.The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes[J].The EMBO Journal,1997,16(11):3207-3218.
[4] TANG X Y,XIE M T,KIM Y J,et al.Overexpression of Pto activates defense responses and confers broad resistance[J].The plant cell,1999,11(1):15-29.
[5] BOGDANOVE A J.Pto update:Recent progress on an ancient plant defence response signalling pathway[J].Molecular plant pathology,2002,3(4):283-288.
[6] GU Y Q,WILDERMUTH M C,CHAKRAVARTHY S,et al.Tomato transcription factors Pti4,Pti5,and Pti6 activate defense responses when expressed in Arabidopsis[J].The plant cell,2002,14(4):817-831.
[7] RAJAGOPALA S V,UETZ P.Analysis of protein-protein interactions using array-based yeast two-hybrid screens[J].Methods in molecular biology,2009,548:223-245.
[8] ZHOU J M,TANG X Y,MARTIN G B.The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes [J].The EMBO Journal,1997,16(11):3207-3218.
[9] BOGDANOVE A J.Pto update:Recent progress on an ancient plant defence response signalling pathway [J].Molecular plant pathology,2002,3(4):283-288.
[10] SUZUKI K,SUZUKI N,OHME-TAKAGI M,et al.Immediate early induction of mRNAs for ethylene-responsive transcription factors in tobacco leaf strips after cutting [J].Plant journal,1998,15(9):657-665.
[11] SOLANO R,STEPANOVA A,CHAO Q,et al.Nuclear events in ethylene signaling:A transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1 [J].Genes and development,1998,12(23):3703-3714.
[12] GOLEMIS E A,SEREBRIISKII I,FINLEY R L,et al.Interaction trap/two-hybrid system to identify interacting proteins[J].Current protocols in protein science,2001-05[2019-01-20].http://www.doc.88.com/p-9763188181594.html.doi:10.1002/0471143030.cb1703s08.
[13] TONG Z,WANG D,SUN Y,et al.Comparative proteomics of rubber latex revealed multiple protein species of REF/SRPP family respond diversely to ethylene stimulation among different rubber tree clones [J].International journal of molecular sciences,2017,18(5):958-972.
[14] ARLANI P,VANDELLE E,WONG D,et al.Comprehensive workflow for the genome-wide identification and expression meta-analysis of the ATL E3 ubiquitin ligase gene family in grapevine [J].Journal of visualized experiments,2017,19(130):3874-3892.
[15] ECCO G,IMBEAULT M,TRONO D.KRAB zinc finger proteins [J].Development,2017,144(15):2719-2729.
[16] KIM S W,HO S C,HONG S J,et al.A Novel mechanism of thyroid hormone-dependent negative regulation by thyroid hormone receptor,nuclear receptor corepressor (NCoR),and GAGA-binding factor on the rat CD44 promoter [J].Journal of biological chemistry,2005,280(15):14545-14555.
[17] ZHAN X Q,WANG B S,LI H J,et al.Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis [J].Proceedings of the national academy of sciences of the United States of America,2012,109(44):18198-18203.
[18] 杨金丽,赵小明,尹恒,等.酵母双杂交筛选OIPK相互作用蛋白[J].应用与环境生物学报,2010,16(4):474-477.
[19] PARRISH J R,GULYAS K D,FINLEY R L.Yeast two-hybrid contributions to interactome mapping [J].Current opinion in biotechnology,2006,17(4):387-393.
[20] IZUMI K M.The yeast two-hybrid assay to identify interacting proteins [J].Methods in molecular biology,2001,174:249-258.
[21] 雷海英,白凤麟,段永红,等.玉米酵母双杂交cDNA文库的构建及ZmCEN互作蛋白的筛选[J].西北植物学报,2018,38(4):598-606.
[22] 赵倩倩,周晓今,陈茹梅.玉米籽粒酵母双杂交cDNA文库的构建及ZmSCL1互作因子筛选[J].生物技术通报,2017,33(12):99-107.
[23] MEHLA J,CAUFIELD J H ,UETZ P.The yeast two-hybrid system:A tool for mapping protein-protein interactions[J].Cold spring harbor protocols,2015(5):425-430.
[2] 许志刚,沈秀萍,胡白石.番茄上两种细菌性病害的诊断与防治[J].植物检疫,2007,21(1):53-56.
[3] ZHOU J M,TANG X Y,MARTIN G B.The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes[J].The EMBO Journal,1997,16(11):3207-3218.
[4] TANG X Y,XIE M T,KIM Y J,et al.Overexpression of Pto activates defense responses and confers broad resistance[J].The plant cell,1999,11(1):15-29.
[5] BOGDANOVE A J.Pto update:Recent progress on an ancient plant defence response signalling pathway[J].Molecular plant pathology,2002,3(4):283-288.
[6] GU Y Q,WILDERMUTH M C,CHAKRAVARTHY S,et al.Tomato transcription factors Pti4,Pti5,and Pti6 activate defense responses when expressed in Arabidopsis[J].The plant cell,2002,14(4):817-831.
[7] RAJAGOPALA S V,UETZ P.Analysis of protein-protein interactions using array-based yeast two-hybrid screens[J].Methods in molecular biology,2009,548:223-245.
[8] ZHOU J M,TANG X Y,MARTIN G B.The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes [J].The EMBO Journal,1997,16(11):3207-3218.
[9] BOGDANOVE A J.Pto update:Recent progress on an ancient plant defence response signalling pathway [J].Molecular plant pathology,2002,3(4):283-288.
[10] SUZUKI K,SUZUKI N,OHME-TAKAGI M,et al.Immediate early induction of mRNAs for ethylene-responsive transcription factors in tobacco leaf strips after cutting [J].Plant journal,1998,15(9):657-665.
[11] SOLANO R,STEPANOVA A,CHAO Q,et al.Nuclear events in ethylene signaling:A transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1 [J].Genes and development,1998,12(23):3703-3714.
[12] GOLEMIS E A,SEREBRIISKII I,FINLEY R L,et al.Interaction trap/two-hybrid system to identify interacting proteins[J].Current protocols in protein science,2001-05[2019-01-20].http://www.doc.88.com/p-9763188181594.html.doi:10.1002/0471143030.cb1703s08.
[13] TONG Z,WANG D,SUN Y,et al.Comparative proteomics of rubber latex revealed multiple protein species of REF/SRPP family respond diversely to ethylene stimulation among different rubber tree clones [J].International journal of molecular sciences,2017,18(5):958-972.
[14] ARLANI P,VANDELLE E,WONG D,et al.Comprehensive workflow for the genome-wide identification and expression meta-analysis of the ATL E3 ubiquitin ligase gene family in grapevine [J].Journal of visualized experiments,2017,19(130):3874-3892.
[15] ECCO G,IMBEAULT M,TRONO D.KRAB zinc finger proteins [J].Development,2017,144(15):2719-2729.
[16] KIM S W,HO S C,HONG S J,et al.A Novel mechanism of thyroid hormone-dependent negative regulation by thyroid hormone receptor,nuclear receptor corepressor (NCoR),and GAGA-binding factor on the rat CD44 promoter [J].Journal of biological chemistry,2005,280(15):14545-14555.
[17] ZHAN X Q,WANG B S,LI H J,et al.Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis [J].Proceedings of the national academy of sciences of the United States of America,2012,109(44):18198-18203.
[18] 杨金丽,赵小明,尹恒,等.酵母双杂交筛选OIPK相互作用蛋白[J].应用与环境生物学报,2010,16(4):474-477.
[19] PARRISH J R,GULYAS K D,FINLEY R L.Yeast two-hybrid contributions to interactome mapping [J].Current opinion in biotechnology,2006,17(4):387-393.
[20] IZUMI K M.The yeast two-hybrid assay to identify interacting proteins [J].Methods in molecular biology,2001,174:249-258.
[21] 雷海英,白凤麟,段永红,等.玉米酵母双杂交cDNA文库的构建及ZmCEN互作蛋白的筛选[J].西北植物学报,2018,38(4):598-606.
[22] 赵倩倩,周晓今,陈茹梅.玉米籽粒酵母双杂交cDNA文库的构建及ZmSCL1互作因子筛选[J].生物技术通报,2017,33(12):99-107.
[23] MEHLA J,CAUFIELD J H ,UETZ P.The yeast two-hybrid system:A tool for mapping protein-protein interactions[J].Cold spring harbor protocols,2015(5):425-430.