酵母双杂交系统筛选马铃薯StLURP1基因的互作蛋白
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摘要
寄生霜霉菌响应迟发上调基因1(late up-regulated in response to Hyaloperonospora parasitica 1,LURP1)在植物抵御致病菌寄生霜霉菌(Hyaloperonospora parasitica, Hp)的反应和抗逆中起到关键作用。为了阐明马铃薯(Solanum tuberosum)LURP1基因的生物学功能并明确其在蛋白互作网络中的作用,本研究利用同源重组的定向克隆方式构建了其诱饵载体p GBKT7-StLURP1,并通过酵母(Saccharomyces cerevisiae)双杂交系统筛选马铃薯c DNA文库。结果表明,双杂筛选的阳性率约为70%,最终获得88个阳性克隆,经测序鉴定出12种含完整开放阅读框的StLURP1互作蛋白,互作的真实性通过回转实验进行验证。对互作蛋白进行Blast比对和基因本体注释(Gene Ontology, GO)分析,结果显示,StLURP1与甘油磷酸二酯酶、富脯氨酸蛋白、多酚氧化酶、肌动蛋白解聚因子、重金属离子转运蛋白及DnaJ家族蛋白等多种蛋白相互作用,可能参与植物脂类代谢、生物与非生物胁迫响应、肌动蛋白解聚、重金属离子解毒及蛋白质折叠等生物过程。本研究为进一步研究StLURP1基因的生物学功能与作用提供了理论依据。
The late up-regulated in response to Hyaloperonospora parasitica gene 1(LURP1) plays a key role in the response against pathogenic oomycete H. parasitica and resistance to stress in plants. In order to elucidate the biological function of the potato(Solanum tuberosum) LURP1 gene and to clarify its position in the protein interaction network, the bait vector p GBKT7-StLURP1 was constructed by homologous recombination and used to screen the potato cDNA library via the yeast(Saccharomyces cerevisiae) two-hybrid(Y2 H) system in this research. The result showed that the positive rate of Y2 H library screening was about70%, and finally 88 positive clones were yielded, which was corresponded to 12 StLURP1 interacting proteins with complete ORF. The authenticity of the interactions was verified by retest. Blast alignment and Gene ontology(GO) annotation analysis demonstrated that StLURP1 could interact with the proteins such as DnaJ family protein, oxidoreductase/transcriptional repressor, glycerophosphodiester phosphodiesterase(GPGD), polyphenoloxidase, proline-rich protein, heavy metal transport/detoxification domain-containing protein, actin depolymerizing factor(ADF), nonspecific serine/threonine or tyrosine protein kinase, et al. The result indicated that StLURP1 may be involved in multiple biological processes, including lipid metabolism,biotic and abiotic stress responses, actin depolymerizing, heavy metal ions transport, protein folding and other biological processes. The present study would provide a theoretical foundation for further studying of biological function and role of StLURP1 gene.
The late up-regulated in response to Hyaloperonospora parasitica gene 1(LURP1) plays a key role in the response against pathogenic oomycete H. parasitica and resistance to stress in plants. In order to elucidate the biological function of the potato(Solanum tuberosum) LURP1 gene and to clarify its position in the protein interaction network, the bait vector p GBKT7-StLURP1 was constructed by homologous recombination and used to screen the potato cDNA library via the yeast(Saccharomyces cerevisiae) two-hybrid(Y2 H) system in this research. The result showed that the positive rate of Y2 H library screening was about70%, and finally 88 positive clones were yielded, which was corresponded to 12 StLURP1 interacting proteins with complete ORF. The authenticity of the interactions was verified by retest. Blast alignment and Gene ontology(GO) annotation analysis demonstrated that StLURP1 could interact with the proteins such as DnaJ family protein, oxidoreductase/transcriptional repressor, glycerophosphodiester phosphodiesterase(GPGD), polyphenoloxidase, proline-rich protein, heavy metal transport/detoxification domain-containing protein, actin depolymerizing factor(ADF), nonspecific serine/threonine or tyrosine protein kinase, et al. The result indicated that StLURP1 may be involved in multiple biological processes, including lipid metabolism,biotic and abiotic stress responses, actin depolymerizing, heavy metal ions transport, protein folding and other biological processes. The present study would provide a theoretical foundation for further studying of biological function and role of StLURP1 gene.
引文
段晓娜,陈宗懋,任炳忠,等.2011.植物多酚氧化酶的诱导[J].吉林师范大学学报(自然科学版),2:90-95.(Duan X N,Chen Z M,Ren B Z,et al.2011.Inducement of plant polyphenol oxidase[J].Journal of Jilin Normal University(Natural Science Edition),2:90-95.)
韩青,陈瑞,杨野,等.2015.植物富含脯氨酸蛋白的研究进展[J].植物生理学报,51(8):1179-1184.(Han Q,Chen R,Yang Y,et al.2015.Progress on plant proline-rich protein[J].Plant Physiology Journal,51(8):1179-1184.
金枫,王翠,林海建,等.2010.植物重金属转运蛋白研究进展[J].应用生态学报,21(7):1875-1882.(Jin F,Wang C,Lin H J,et al.2010.Heavy metal-transport proteins in plants:A review[J].Chinese Journal of Applied Ecology,21(7):1875-1882.)
梁丽娜.2017.干旱胁迫下马铃薯酵母cDNA文库构建及ERF转录因子筛选[D].硕士学位论文,甘肃农业大学,导师:张宁.pp.21-30.(Liang L N.2017.Yeast cD-NA library construction and ERF transcription factor screening of potato(Solanum tuberosum L.)under drought stress[D].Thesis for M.S.,Gansu Agricultural University,Supervisor:Zhang N.pp.21-30.)
马凯,胡红霞,于婧,等.2015.双酶切和同源重组方法构建pMIR-reporter载体的比较[J].中国病原生物学杂志,10(6):495-499.(Ma K,Hu H X,Yu J,et al.2015.Comparison of the construction of a p MIR-reporter vector using conventional double enzyme restriction and recombination[J].Journal of Pathogen Biology,10(6):495-499.)
王曼玲,胡中立,周明全,等.2005.植物多酚氧化酶的研究进展[J].植物学通报,22(2):215-222.(Wang M L,Hu Z L,Zhou M Q,et al.2005.Advances in research of polyphenol oxidase in plants[J].Chinese Bulletin of Botany,22(2):215-222.)
王楠,吕香玲,李亮,等.2010.植物肌动蛋白解聚因子ADF的研究进展[J].西北植物学报,35(11):2349-2354.(Wang N,Lv X L,Li L,et al.2015.Research progress of ADF/cofilin in plant[J].Acta Botanica Boreali-Occidentalia Sinica,35(11):2349-2354.)
徐文琳,廖志勇,王春丽,等.2003.酵母双杂交相关方法的改良及应用[J].生物技术通讯,14(5):372-37.(Xu WL,Liao Z Y,Wang C L,et al.2003.The improvements of some methods involved in yeast two-hybrid experiments[J].Letters in Biotechnology,14(5):372-37.)
张宏磊,杨志辉,胡珍珠,等.2013.致病疫霉基因组微卫星标记开发[J].农业生物技术学报,21(9):1110-1118.(Zhang H L,Yang Z H,Hu Z Z,et al.2013.Development of genomic microsatellite marker for Phytophthora infestans[J].Chinese Journal of Agricultural Biotechnology,21(9):1110-1118.)
张晓光,药立波,苏成芝.2001.酵母双杂交系统及其应用[J].生命科学,13(5):228-231.(Zhang X G,Yao L B,Su C Z.2001.Yeast two-hybrid system and its applications[J].Chinese Bulletin of Life Sciences,13(5):228-231.)
Bricchi I,Bertea C M,Occhipinti A,et al.2012.Dynamics of membrane potential variation and gene expression induced by Spodoptera littoralis,Myzus persicae,and Pseu?domonas syringae in Arabidopsis[J].PLoS One,7(10):e46673.
Caillaud M C,Asai S,Rallapalli G,et al.2013.A downy mildew effector attenuates salicylic acid-triggered immunity in Arabidopsis by interacting with the host mediator complex[J].PLoS Biology,11(12):e1001732.
Capecchi M R.1989.Altering the genome by homologous recombination[J].Science,244(4910):1288-92.
Cheng Y X,Zhou W B,Ibrahim N,et al.2011.Characterization of the Arabidopsis glycerophospho diester phosphodiesterase(GDPD)family reveals a role of the plastidlocalized AtGDPD1 in maintaining cellular phosphate homeostasis under phosphate starvation[J].The Plant Journal,66:781-795.
Coker T L R,Cevik V,Beynon J L,et al.2015.Spatial dissection of the Arabidopsis thaliana transcriptional response to downy mildew using fluorescence activated cell sorting[J].Frontiers in Plant Science,6:527.
Fry W.2008.Phytophthora infestans:The plant(and R gene)destroyer[J].Molecular Plant Pathology,9(3):385-402.
Gupta A,Senthil-Kumar M.2017.Transcriptome changes in Arabidopsis thaliana infected with Pseudomonas syrin?gae during drought recovery[J].Scientific Report,7:9124.
Knoth C,Eulgem T.2008.The oomycete response gene LURP1 is required for defenseagainst Hyaloperonospora parasitica in Arabidopsis thaliana[J].The Plant Journal,55:53-64.
Lee M H,Jeon H S,Kim H G,et al.2017.An Arabidopsis NAC transcription factor NAC4 promotes pathogen-induced cell death under negative regulation by microR-NA164[J].New Phytologist,214:343-360.
Qian D,Zhang Z,He J X,et al.2019.Arabidopsis ADF5 promotes stomatal closure by regulating actin cytoskeleton remodeling in response to ABA and drought stress[J].Journal of Experimental Botany,70(2):435-446.
Shao M R,Kumar S,Laurie J D,et al.2017.Stress-responsive pathways and small RNA changes distinguish variable developmental phenotypes caused by MSH1 loss[J].BMC Plant Biology,17:47.
Shinwari Z K,Nakashima K,Miura S,et al.1998.An Arabi?dopsis gene family encoding DRE/CRT binding proteins involved in low-temperature-responsive gene expression[J].Biochemical and biophysical research communications,250(1):61-70.
Walsh P,Bursac D,Law YC,et al.2004.The J-protein family:Modulating protein assembly,disassembly and translocation[J].EMBO Reports,5:567-571.
韩青,陈瑞,杨野,等.2015.植物富含脯氨酸蛋白的研究进展[J].植物生理学报,51(8):1179-1184.(Han Q,Chen R,Yang Y,et al.2015.Progress on plant proline-rich protein[J].Plant Physiology Journal,51(8):1179-1184.
金枫,王翠,林海建,等.2010.植物重金属转运蛋白研究进展[J].应用生态学报,21(7):1875-1882.(Jin F,Wang C,Lin H J,et al.2010.Heavy metal-transport proteins in plants:A review[J].Chinese Journal of Applied Ecology,21(7):1875-1882.)
梁丽娜.2017.干旱胁迫下马铃薯酵母cDNA文库构建及ERF转录因子筛选[D].硕士学位论文,甘肃农业大学,导师:张宁.pp.21-30.(Liang L N.2017.Yeast cD-NA library construction and ERF transcription factor screening of potato(Solanum tuberosum L.)under drought stress[D].Thesis for M.S.,Gansu Agricultural University,Supervisor:Zhang N.pp.21-30.)
马凯,胡红霞,于婧,等.2015.双酶切和同源重组方法构建pMIR-reporter载体的比较[J].中国病原生物学杂志,10(6):495-499.(Ma K,Hu H X,Yu J,et al.2015.Comparison of the construction of a p MIR-reporter vector using conventional double enzyme restriction and recombination[J].Journal of Pathogen Biology,10(6):495-499.)
王曼玲,胡中立,周明全,等.2005.植物多酚氧化酶的研究进展[J].植物学通报,22(2):215-222.(Wang M L,Hu Z L,Zhou M Q,et al.2005.Advances in research of polyphenol oxidase in plants[J].Chinese Bulletin of Botany,22(2):215-222.)
王楠,吕香玲,李亮,等.2010.植物肌动蛋白解聚因子ADF的研究进展[J].西北植物学报,35(11):2349-2354.(Wang N,Lv X L,Li L,et al.2015.Research progress of ADF/cofilin in plant[J].Acta Botanica Boreali-Occidentalia Sinica,35(11):2349-2354.)
徐文琳,廖志勇,王春丽,等.2003.酵母双杂交相关方法的改良及应用[J].生物技术通讯,14(5):372-37.(Xu WL,Liao Z Y,Wang C L,et al.2003.The improvements of some methods involved in yeast two-hybrid experiments[J].Letters in Biotechnology,14(5):372-37.)
张宏磊,杨志辉,胡珍珠,等.2013.致病疫霉基因组微卫星标记开发[J].农业生物技术学报,21(9):1110-1118.(Zhang H L,Yang Z H,Hu Z Z,et al.2013.Development of genomic microsatellite marker for Phytophthora infestans[J].Chinese Journal of Agricultural Biotechnology,21(9):1110-1118.)
张晓光,药立波,苏成芝.2001.酵母双杂交系统及其应用[J].生命科学,13(5):228-231.(Zhang X G,Yao L B,Su C Z.2001.Yeast two-hybrid system and its applications[J].Chinese Bulletin of Life Sciences,13(5):228-231.)
Bricchi I,Bertea C M,Occhipinti A,et al.2012.Dynamics of membrane potential variation and gene expression induced by Spodoptera littoralis,Myzus persicae,and Pseu?domonas syringae in Arabidopsis[J].PLoS One,7(10):e46673.
Caillaud M C,Asai S,Rallapalli G,et al.2013.A downy mildew effector attenuates salicylic acid-triggered immunity in Arabidopsis by interacting with the host mediator complex[J].PLoS Biology,11(12):e1001732.
Capecchi M R.1989.Altering the genome by homologous recombination[J].Science,244(4910):1288-92.
Cheng Y X,Zhou W B,Ibrahim N,et al.2011.Characterization of the Arabidopsis glycerophospho diester phosphodiesterase(GDPD)family reveals a role of the plastidlocalized AtGDPD1 in maintaining cellular phosphate homeostasis under phosphate starvation[J].The Plant Journal,66:781-795.
Coker T L R,Cevik V,Beynon J L,et al.2015.Spatial dissection of the Arabidopsis thaliana transcriptional response to downy mildew using fluorescence activated cell sorting[J].Frontiers in Plant Science,6:527.
Fry W.2008.Phytophthora infestans:The plant(and R gene)destroyer[J].Molecular Plant Pathology,9(3):385-402.
Gupta A,Senthil-Kumar M.2017.Transcriptome changes in Arabidopsis thaliana infected with Pseudomonas syrin?gae during drought recovery[J].Scientific Report,7:9124.
Knoth C,Eulgem T.2008.The oomycete response gene LURP1 is required for defenseagainst Hyaloperonospora parasitica in Arabidopsis thaliana[J].The Plant Journal,55:53-64.
Lee M H,Jeon H S,Kim H G,et al.2017.An Arabidopsis NAC transcription factor NAC4 promotes pathogen-induced cell death under negative regulation by microR-NA164[J].New Phytologist,214:343-360.
Qian D,Zhang Z,He J X,et al.2019.Arabidopsis ADF5 promotes stomatal closure by regulating actin cytoskeleton remodeling in response to ABA and drought stress[J].Journal of Experimental Botany,70(2):435-446.
Shao M R,Kumar S,Laurie J D,et al.2017.Stress-responsive pathways and small RNA changes distinguish variable developmental phenotypes caused by MSH1 loss[J].BMC Plant Biology,17:47.
Shinwari Z K,Nakashima K,Miura S,et al.1998.An Arabi?dopsis gene family encoding DRE/CRT binding proteins involved in low-temperature-responsive gene expression[J].Biochemical and biophysical research communications,250(1):61-70.
Walsh P,Bursac D,Law YC,et al.2004.The J-protein family:Modulating protein assembly,disassembly and translocation[J].EMBO Reports,5:567-571.