马铃薯StERF5转录因子酵母双杂交诱饵载体的构建及鉴定
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摘要
为了筛选与马铃薯StERF5转录因子相互作用的宿主蛋白,构建其诱饵载体,本研究利用PCR方法扩增得到马铃薯StERF5基因的编码序列,克隆至载体p MD18-T,验证正确后插入到酵母双杂交系统诱饵表达载体pGBKT7,经双酶切、PCR反应及测序验证其正确后,利用PEG/LiAc法将构建好的重组诱饵载体pGBKT7-StERF5及空载体pGBKT7分别转化到酵母Y2HGlod感受态细胞中,检测其对酵母菌株是否有毒性作用和自激活活性。结果表明:扩增得到StERF5基因,成功构建了诱饵表达载体pGBKT7-StERF5,此诱饵载体对酵母宿主细胞无毒性且不具自主激活报告基因的功能。研究结果可为进一步利用酵母双杂交技术筛选与ERF5基因互作的宿主蛋白及功能研究提供科学依据。
In order to screen host proteins that interacts with potato transcription factor ERF5 and construct bait vector, the coding sequence of potato StERF5 gene was amplified by PCR, and cloned into vector pMD18-T. After verified correctly, the gene was inserted into yeast two-hybrid bait expression vector pGBKT7. After double enzyme digestion, PCR and sequencing identification, the recombinant vector pGBKT7-StERF5 and empty vector pGBKT7 was respectively transformed into yeast Y2 HGlod cells by PEG/LiAc method to test whether it had toxic activity and self-activation in yeast cells. The results showed that StERF5 gene was amplified and the bait expression vector pGBKT7-StERF5 was successfully constructed. The bait vector was non-toxic to yeast host cells and had no function of autonomously activating reporter gene. The result could provide scientific basis for further screening of host protein and function of ERF5 gene interaction with yeast two-hybrid technique.
In order to screen host proteins that interacts with potato transcription factor ERF5 and construct bait vector, the coding sequence of potato StERF5 gene was amplified by PCR, and cloned into vector pMD18-T. After verified correctly, the gene was inserted into yeast two-hybrid bait expression vector pGBKT7. After double enzyme digestion, PCR and sequencing identification, the recombinant vector pGBKT7-StERF5 and empty vector pGBKT7 was respectively transformed into yeast Y2 HGlod cells by PEG/LiAc method to test whether it had toxic activity and self-activation in yeast cells. The results showed that StERF5 gene was amplified and the bait expression vector pGBKT7-StERF5 was successfully constructed. The bait vector was non-toxic to yeast host cells and had no function of autonomously activating reporter gene. The result could provide scientific basis for further screening of host protein and function of ERF5 gene interaction with yeast two-hybrid technique.
引文
Bouaziz D.,Charfeddine M.,Jbir R.,Saidi M.N.,Pirrello J.,Charfeddine S.,Bouzayen M.,and Gargouri B.R.,2015,I-dentification and functional characterization of ten AP2/ERFgenes in potato,Plant Cell Tiss.Organ.Cult.,123(1):155-172
Charfeddine M.,Bouaziz D.,Charfeddine S.,Hammami A.,Ellouz O.N.,and Bouzid G.R.,2015,Overexpression of dehydration-responsive element-binding 1 protein(DREB1)in transgenic Solanum tuberosum enhances tolerance to biotic stress,Plant Biotechnol.Rep.,9(2):79-88
Cui L.C.,Feng K.W.,Wang M.X.,Deng P.C.,Song W.N.,and Nie X.J.,2016,Genome-wide identification,phylogeny and expression analysis of AP2/ERF transcription factors family in Brachypodium distachyon,BMC Genomics,17(1):636
Fujita M.,Fujita Y.,Noutoshi Y.,Takahashi F.,Narusaka Y.,Yamaguchi S.K.,and Shinozaki K.,2006,Crosstalk between abiotic and biotic stress responses:a current view from the points of convergence in the stress signaling networks,Curr.Opin.Plant Biol.,9(4):436-442
He W.H.,2014,Study on cloning and function analysis of ERF1gene in potato,Thesis for M.S.,Gansu Agricultural University,Supervisor:Si H.J.,pp.48(贺文辉,2014,马铃薯ERF1基因克隆及其功能研究,硕士学位论文,甘肃农业大学,导师:司怀军,pp.48)
Komivi D.,Wei X.,Li D.H.,Daniel F.,Zhang Y.X.,Wang L.H.,Yu J.Y.,Liao B.S.,Diaga D.,Ndiage C.,and Zhang X.R.,2016,Insight into the AP2/ERF transcription factor superfamily in sesame and expression profiling of DREB subfamily under drought stress,BMC Plant Biol.,16(1):171
Lata C.,Mishra A.K.,Muthamilarasan M.,Bonthala V.S.,Khan Y.,and Prasad M.,2014,Genome-wide investigation and expression profiling of AP2/ERF transcription factor superfamily in foxtail millet(Setaria italica L.),PLoS One,9(11):e113092
Li C.W.,Su R.C.,Cheng C.P.,Sanjaya,You S.J.,Hsieh T.H.,Chao T.C.,and Chan M.T.,2011,Tomato RAV transcription factor is a pivotal modulator involved in the AP2/EREBP-mediated defense pathway,Plant Physiol.,156(1):213-227
Mawlong I.,Ali K.,Srinivasan R.,Rai R.D.,and Tyagi A.,2015,Functional validation of a drought-responsive AP2/ERFfamily transcription factor-encoding gene from rice in A rabidopsis,Mol.Breeding,35(8):163
Mei Q.,Li S.,Liu P.,Xi L.,Wang S.X.,Meng Y.H.,Liu J.,Yang X.W.,Lu Y.P.,and Wang H.,2010,Construction and identification of a yeast two-hybrid bait vector and its effect on the growth of yeast cells and the self-activating function of reporter genes for screening of HPV18 E6-interacting protein,J.Huazhong Univ.Sci.Technol.,30(1):8-12
Mizoi J.,Shinozaki K.,and Yamaguchi S.K.,2012,AP2/ERFfamily transcription factors in plant abiotic stress responses,Biochim.Biophys.Acta,1819(2):86-96
Rehman S.,and Mahmood T.,2015,Functional role of DREBand ERF transcription factors:regulating stress-responsive network in plants,Acta Physiol.Plant,37(9):1-14
Sakuma Y.,Liu Q.,Dubouzet J.G.,Abe H.,Shinozaki K.,and Yamaguchi S.K.,2002,DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration-and cold-inducible gene expression,Biochem.Biophys.Res.Commun.,290(3):998-1009
Sohn K.H.,Lee S.C.,Jung H.W.,Hong J.K.,and Hwang B.K.,2006,Expression and functional roles of the pepper pathogen-induced transcription factor RAV1 in bacterial disease resistance,and drought and salt stress tolerance,Plant Mol.Biol.,61(6):897-915
Taketa S.,Amano S.,Tsujino Y.,Sato T,Saisho D.,Kakeda K.,Nomura M.,Suzuki T.,Matsumoto T.,Sato K.,Kanamori H.,Kawasaki S.,and Takeda K.,2008,Barley grain with adhering hulls is controlled by an ERF family transcription factor gene regulating a lipid biosynthesis pathway,Proc.Natl.Acad.Sci.USA,105(10):4062-4067
Zhang G.Y.,Chen M.,Chen X.P.,Xu Z.S.,Guan S.,Li L.C.,Li A.L.,Guo J.M.,Mao L.,and Ma Y.Z.,2008,Phylogeny,gene structures,and expression patterns of the ERF gene family in soybean(Glycine max L.),J.Exp.Bot.,59(15):4095-4107
Zhang H.,Zhu Y.Y.,Shao X.,Wang Y.,Li Y.Q.,and Guo W.D.,2015,Isolation and characterization of PpcCBF CBF/DREBtranscription factor cDNA of Chinese cherry(Prunus pseudocerasus),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):149-154(张弘,朱友银,邵姁,王月,李永强,郭卫东,2015,中国樱桃CBF/DREB转录因子PpcCBF的克隆与表达分析,基因组学与应用生物学,34(1):149-154)
Zhuang J.,Cai B.,Peng R.H.,Zhu B.,Jin X.F.,Xue Y.,Gao F.,Fu X.Y.,Tian Y.S.,Zhao W.,Qiao Y.S.,Zhang Z.,Xiong A.S.,and Yao Q.H.,2008,Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa,Biochem.Biophys.Res.Commun.,371(3):468-474
Zhuang J.,Jiang H.H.,Wang F.,Peng R.H.,Yao Q.H.,and Xiong A.S.,2013,A rice Os AP23,functioning as an AP2/ERFtranscription factor,reduces salt tolerance in transgenic Arabidopsis,Plant Mol.Biol.Rep.,(31):1336-1345
Charfeddine M.,Bouaziz D.,Charfeddine S.,Hammami A.,Ellouz O.N.,and Bouzid G.R.,2015,Overexpression of dehydration-responsive element-binding 1 protein(DREB1)in transgenic Solanum tuberosum enhances tolerance to biotic stress,Plant Biotechnol.Rep.,9(2):79-88
Cui L.C.,Feng K.W.,Wang M.X.,Deng P.C.,Song W.N.,and Nie X.J.,2016,Genome-wide identification,phylogeny and expression analysis of AP2/ERF transcription factors family in Brachypodium distachyon,BMC Genomics,17(1):636
Fujita M.,Fujita Y.,Noutoshi Y.,Takahashi F.,Narusaka Y.,Yamaguchi S.K.,and Shinozaki K.,2006,Crosstalk between abiotic and biotic stress responses:a current view from the points of convergence in the stress signaling networks,Curr.Opin.Plant Biol.,9(4):436-442
He W.H.,2014,Study on cloning and function analysis of ERF1gene in potato,Thesis for M.S.,Gansu Agricultural University,Supervisor:Si H.J.,pp.48(贺文辉,2014,马铃薯ERF1基因克隆及其功能研究,硕士学位论文,甘肃农业大学,导师:司怀军,pp.48)
Komivi D.,Wei X.,Li D.H.,Daniel F.,Zhang Y.X.,Wang L.H.,Yu J.Y.,Liao B.S.,Diaga D.,Ndiage C.,and Zhang X.R.,2016,Insight into the AP2/ERF transcription factor superfamily in sesame and expression profiling of DREB subfamily under drought stress,BMC Plant Biol.,16(1):171
Lata C.,Mishra A.K.,Muthamilarasan M.,Bonthala V.S.,Khan Y.,and Prasad M.,2014,Genome-wide investigation and expression profiling of AP2/ERF transcription factor superfamily in foxtail millet(Setaria italica L.),PLoS One,9(11):e113092
Li C.W.,Su R.C.,Cheng C.P.,Sanjaya,You S.J.,Hsieh T.H.,Chao T.C.,and Chan M.T.,2011,Tomato RAV transcription factor is a pivotal modulator involved in the AP2/EREBP-mediated defense pathway,Plant Physiol.,156(1):213-227
Mawlong I.,Ali K.,Srinivasan R.,Rai R.D.,and Tyagi A.,2015,Functional validation of a drought-responsive AP2/ERFfamily transcription factor-encoding gene from rice in A rabidopsis,Mol.Breeding,35(8):163
Mei Q.,Li S.,Liu P.,Xi L.,Wang S.X.,Meng Y.H.,Liu J.,Yang X.W.,Lu Y.P.,and Wang H.,2010,Construction and identification of a yeast two-hybrid bait vector and its effect on the growth of yeast cells and the self-activating function of reporter genes for screening of HPV18 E6-interacting protein,J.Huazhong Univ.Sci.Technol.,30(1):8-12
Mizoi J.,Shinozaki K.,and Yamaguchi S.K.,2012,AP2/ERFfamily transcription factors in plant abiotic stress responses,Biochim.Biophys.Acta,1819(2):86-96
Rehman S.,and Mahmood T.,2015,Functional role of DREBand ERF transcription factors:regulating stress-responsive network in plants,Acta Physiol.Plant,37(9):1-14
Sakuma Y.,Liu Q.,Dubouzet J.G.,Abe H.,Shinozaki K.,and Yamaguchi S.K.,2002,DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration-and cold-inducible gene expression,Biochem.Biophys.Res.Commun.,290(3):998-1009
Sohn K.H.,Lee S.C.,Jung H.W.,Hong J.K.,and Hwang B.K.,2006,Expression and functional roles of the pepper pathogen-induced transcription factor RAV1 in bacterial disease resistance,and drought and salt stress tolerance,Plant Mol.Biol.,61(6):897-915
Taketa S.,Amano S.,Tsujino Y.,Sato T,Saisho D.,Kakeda K.,Nomura M.,Suzuki T.,Matsumoto T.,Sato K.,Kanamori H.,Kawasaki S.,and Takeda K.,2008,Barley grain with adhering hulls is controlled by an ERF family transcription factor gene regulating a lipid biosynthesis pathway,Proc.Natl.Acad.Sci.USA,105(10):4062-4067
Zhang G.Y.,Chen M.,Chen X.P.,Xu Z.S.,Guan S.,Li L.C.,Li A.L.,Guo J.M.,Mao L.,and Ma Y.Z.,2008,Phylogeny,gene structures,and expression patterns of the ERF gene family in soybean(Glycine max L.),J.Exp.Bot.,59(15):4095-4107
Zhang H.,Zhu Y.Y.,Shao X.,Wang Y.,Li Y.Q.,and Guo W.D.,2015,Isolation and characterization of PpcCBF CBF/DREBtranscription factor cDNA of Chinese cherry(Prunus pseudocerasus),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):149-154(张弘,朱友银,邵姁,王月,李永强,郭卫东,2015,中国樱桃CBF/DREB转录因子PpcCBF的克隆与表达分析,基因组学与应用生物学,34(1):149-154)
Zhuang J.,Cai B.,Peng R.H.,Zhu B.,Jin X.F.,Xue Y.,Gao F.,Fu X.Y.,Tian Y.S.,Zhao W.,Qiao Y.S.,Zhang Z.,Xiong A.S.,and Yao Q.H.,2008,Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa,Biochem.Biophys.Res.Commun.,371(3):468-474
Zhuang J.,Jiang H.H.,Wang F.,Peng R.H.,Yao Q.H.,and Xiong A.S.,2013,A rice Os AP23,functioning as an AP2/ERFtranscription factor,reduces salt tolerance in transgenic Arabidopsis,Plant Mol.Biol.Rep.,(31):1336-1345
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