玉米促丝裂原活化蛋白激酶家族基因的电子克隆及生物信息学分析
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摘要
本研究利用资源丰富的玉米EST数据库，基因组数据库和蛋白质数据库，采用基于EST及蛋白质数据库的电子克隆的方法，预测可能存在的玉米MAPK家族基因。凭借众多生物信息学工具对预测得到的玉米MAPK进行基本的生物信息学分析，并在实验室内克隆得到ZmMPK5，ZmMPK6，ZmMPK9三个玉米MAPK基因。本研究的结果如下：
（1）利用水稻的MAPK序列和玉米Maize GDB数据库，采用基于EST的电子克隆的方法获得了11个玉米MAPK基因，通过对玉米cDNA数据库的同源检索得到了4个玉米MAPKs基因，运用隐马尔科夫模型检索玉米蛋白质数据库得到了6个玉米MAPKs基因，最终预测得到21个玉米MAPKs基因；利用玉米MAPK与水稻及拟南芥MAPK的同源进化关系绘制系统进化树，将预测到的玉米MAPK归类并重新命名为ZmMPK1-ZmMPK21。
（2）运用网络上丰富的蛋白质结构和功能预测工具对预测得到的21个玉米MAPKs进行一级结构、二级结构、三维结构和结构域分析。使用的软件工具包括ProtParam分析蛋白质的理化性质；使用ProtScale分析蛋白质的亲疏水性；使用TMpred预测蛋白质的跨膜结构域；使用COILS预测卷曲螺旋；使用TargetP对蛋白质进行亚细胞定位；使用Pfam和PlantsP分析蛋白质功能结构域；使用SWISS-MODEL分析蛋白质的三维结构。结果显示ZmMPK7，ZmMPK8，ZmMPK9，ZmMPK10，ZmMPK11，ZmMPK12，ZmMPK14，ZmMPK21的稳定性较强，其余ZmMPK的稳定性较差；所有ZmMPK均显示较强的亲水性，无跨膜结构，卷曲螺旋较少；ZmMPK14，ZmMPK17，ZmMPK18，ZmMPK20可能位于线粒体内，其余ZmMPK处于胞质内，基本符合大多数MAPK位于核周区的特点；初步展示了19个ZmMPK的三维结构。
（3）根据预测的玉米MAPK家族基因设计PCR引物，以玉米总mRNA反转录得到的cDNA为模板，扩增得到了ZmMPK5，ZmMPK6，ZmMPK9三个玉米MAPK基因以及ZmMPK2，ZmMPK3，ZmMPK4，ZmMPK5，ZmMPK17，ZmMPK18，ZmMPK20的特异性片段，并构建了RNAi植物表达载体，为后续研究打下了基础。
Based on EST (expressed sequence tag) database and maize genome database, wepredicted maize mitogen-activated protein kinase (MAPK) gene family, using an in silicocloning method. The putative MAPK genes were identified with the aid of various types ofbioinformatic software and designated to be ZmMPKs (Zea mays MAPK). Subsequently, threeZmMPKs were cloned, i.e. ZmMPK5, ZmMPK6, and ZmMPK9. The main results of this studywere:
(1) Based on OsMPK (Oryza sativa MAPK) genes and Maize Genetics and GenomicsDatabase (Maize GDB), 11 putative ZmMPKs were predicted through in silico analysis ofESTs; 3 putative ZmMPKs were found through searching in cDNA database and 6 putativeZmMPKs were found in maize protein database using HMMer 2.3.2. According to theevolutionary relationship in OsMPKs, AtMPKs (Arabidopsis thaliana MAPK) and ZmMPKs,the cladogram of 58 MAPK genes from the three species were drawn classifying 21 putativeZmMPKs which were renamed to be ZmMPK1 through ZmMPK21.
(2) The 21 putative ZmMPKs were analysed by various types of bioinformatic software:ProtParam for analyzing protein physico-chemical property; ProtScale for analyzinghydrophilicity and hydrophobicity; TMpred (Prediction of Transmembrane Regions andOrientation) for analyzing transmembrane regions; COILS for analyzing coiled-coils; TargetPfor the subcellular location; Pfam and PlantsP for analyzing the protein functional domains;and the SWISS-MODEL for making homology modeling. The results showed that theproteins of ZmMPK7, ZmMPK8, ZmMPK9, ZmMPK10, ZmMPK11, ZmMPK12,ZmMPK21 protein were stable, while the rest of other putative ZmMPKs were unstable. Allputative ZmMPKs were found to be hydrophilic with no transmembrane region and fewcoiled-coils; ZmMPK14, ZmMPK17, ZmMPK18 and ZmMPK20 are likely localized insideof mitochondrion, while the others ZmMPKs were in the cytoplasm. This characteristics wascoincided with MAPKs from other species. Furthermore, the 3D structure of 19 putativeMAPKs was illustrated.
(3) According to the sequences of the putative ZmMPKs, the PCR primers weredesigned and 3 ZmMPK genes, i.e. ZmMPK5, ZmMPK6, and ZmMPK9 were clonedsuccessfully，the special fragment of ZmMPK2，ZmMPK3，ZmMPK4，ZmMPK5，ZmMPK17，ZmMPK18 and ZmMPK20 were also cloned, and RNAi vectors were contructed.
（1）利用水稻的MAPK序列和玉米Maize GDB数据库，采用基于EST的电子克隆的方法获得了11个玉米MAPK基因，通过对玉米cDNA数据库的同源检索得到了4个玉米MAPKs基因，运用隐马尔科夫模型检索玉米蛋白质数据库得到了6个玉米MAPKs基因，最终预测得到21个玉米MAPKs基因；利用玉米MAPK与水稻及拟南芥MAPK的同源进化关系绘制系统进化树，将预测到的玉米MAPK归类并重新命名为ZmMPK1-ZmMPK21。
（2）运用网络上丰富的蛋白质结构和功能预测工具对预测得到的21个玉米MAPKs进行一级结构、二级结构、三维结构和结构域分析。使用的软件工具包括ProtParam分析蛋白质的理化性质；使用ProtScale分析蛋白质的亲疏水性；使用TMpred预测蛋白质的跨膜结构域；使用COILS预测卷曲螺旋；使用TargetP对蛋白质进行亚细胞定位；使用Pfam和PlantsP分析蛋白质功能结构域；使用SWISS-MODEL分析蛋白质的三维结构。结果显示ZmMPK7，ZmMPK8，ZmMPK9，ZmMPK10，ZmMPK11，ZmMPK12，ZmMPK14，ZmMPK21的稳定性较强，其余ZmMPK的稳定性较差；所有ZmMPK均显示较强的亲水性，无跨膜结构，卷曲螺旋较少；ZmMPK14，ZmMPK17，ZmMPK18，ZmMPK20可能位于线粒体内，其余ZmMPK处于胞质内，基本符合大多数MAPK位于核周区的特点；初步展示了19个ZmMPK的三维结构。
（3）根据预测的玉米MAPK家族基因设计PCR引物，以玉米总mRNA反转录得到的cDNA为模板，扩增得到了ZmMPK5，ZmMPK6，ZmMPK9三个玉米MAPK基因以及ZmMPK2，ZmMPK3，ZmMPK4，ZmMPK5，ZmMPK17，ZmMPK18，ZmMPK20的特异性片段，并构建了RNAi植物表达载体，为后续研究打下了基础。
Based on EST (expressed sequence tag) database and maize genome database, wepredicted maize mitogen-activated protein kinase (MAPK) gene family, using an in silicocloning method. The putative MAPK genes were identified with the aid of various types ofbioinformatic software and designated to be ZmMPKs (Zea mays MAPK). Subsequently, threeZmMPKs were cloned, i.e. ZmMPK5, ZmMPK6, and ZmMPK9. The main results of this studywere:
(1) Based on OsMPK (Oryza sativa MAPK) genes and Maize Genetics and GenomicsDatabase (Maize GDB), 11 putative ZmMPKs were predicted through in silico analysis ofESTs; 3 putative ZmMPKs were found through searching in cDNA database and 6 putativeZmMPKs were found in maize protein database using HMMer 2.3.2. According to theevolutionary relationship in OsMPKs, AtMPKs (Arabidopsis thaliana MAPK) and ZmMPKs,the cladogram of 58 MAPK genes from the three species were drawn classifying 21 putativeZmMPKs which were renamed to be ZmMPK1 through ZmMPK21.
(2) The 21 putative ZmMPKs were analysed by various types of bioinformatic software:ProtParam for analyzing protein physico-chemical property; ProtScale for analyzinghydrophilicity and hydrophobicity; TMpred (Prediction of Transmembrane Regions andOrientation) for analyzing transmembrane regions; COILS for analyzing coiled-coils; TargetPfor the subcellular location; Pfam and PlantsP for analyzing the protein functional domains;and the SWISS-MODEL for making homology modeling. The results showed that theproteins of ZmMPK7, ZmMPK8, ZmMPK9, ZmMPK10, ZmMPK11, ZmMPK12,ZmMPK21 protein were stable, while the rest of other putative ZmMPKs were unstable. Allputative ZmMPKs were found to be hydrophilic with no transmembrane region and fewcoiled-coils; ZmMPK14, ZmMPK17, ZmMPK18 and ZmMPK20 are likely localized insideof mitochondrion, while the others ZmMPKs were in the cytoplasm. This characteristics wascoincided with MAPKs from other species. Furthermore, the 3D structure of 19 putativeMAPKs was illustrated.
(3) According to the sequences of the putative ZmMPKs, the PCR primers weredesigned and 3 ZmMPK genes, i.e. ZmMPK5, ZmMPK6, and ZmMPK9 were clonedsuccessfully，the special fragment of ZmMPK2，ZmMPK3，ZmMPK4，ZmMPK5，ZmMPK17，ZmMPK18 and ZmMPK20 were also cloned, and RNAi vectors were contructed.
引文
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[7] Louis-Philippe Hamel，Marie-Claude Nicole，Somrudee Sritubtim，et al．Ancient signals：comparative genomics of plant MAPK and MAPKK gene families[J]．TRENDS in PlantScience，2006，11(4)：192-198．
[8] Reyna S Nathan，Yang Yinong．Molecular Analysis of the Rice MAP Kinase Gene Familyin Relation to Magnaporthe grisea Infection[J]．MPMI，2006，19 (5)：530–540．
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[19] Fu SF，Chou WC，Huang DD，et al．Transcriptional regulation of a ricemitogen-activated protein kinase gene，OsMAPK4，in response to environmental stresses[J]．Plant CellPhysiol，2002，43：958?963．
[20] Agrawal GK，Agrawal SK，Shibato J，et al．Novel rice MAP kinases OsMSRMK3 andOsWJUMK1 involved in encountering diverse environmental stresses and developmentalregulation[J]．Biochem Biophys Res Commun，2003，300：775?783．
[21] Huang HJ，Fu SF，Tai YH，et al．Expression of Oryza sativa MAP kinase gene isevelopmentally regulated and stress-responsive[J]．Physiol Plant，2002，114：572-580．
[22] Song FM，Goodman RM．OsBIMK1，a rice MAP kinase gene involved in diseaseresistance responses[J]．Planta，2002，215：997?1005．
[23] Wen JQ，Oono K，Imai R．Two novel mitogen-activated protein signaling components，OsMEK1 and OsMAP1，are involved in a moderate low-temperature signaling pathway inrice[J]．Plant Physiol，2002，129：1880?1891．
[24] Agrawal GK，Rakwal R，Iwahashi H．Isolation of novel rice (Oryza sativa L.) multiplestress responsive MAP kinase gene,OsMSRMK2，whose mRNA accumulates rapidly inresponse to environmental cues[J]．Biochem Biophys Res Commun，2002，294：1009?1016．
[25] Xiong L，Yang Y．Disease resistance and abiotic stress tolerance in rice are inverselymodulated by an abscisic acid-inducible mitogen-activated protein kinase[J]．Plant Cell，2003，15：745?759．
[26] Yeh CM，Hung WC，Huang HJ．Copper treatment activates mitogen-activated proteinkinase signalling in rice[J]．Physiol Plant，2003，119：392?399．
[27] Jeong MJ，Lee SK，Kim BG，et al．A rice (Oryza sativa L.) MAP kinase gene，OsMAPK44，is involved in response to abiotic stresses[J]．Plant Cell Tiss OrganCult，2006，85：151?160．
[28] He C，Fong SH，Yang D，et al．BWMK1，a novel MAP kinase induced by fungalinfection and mechanical wounding in rice[J]．Mol Plant Microbe In，1999，12：1064?1073．
[29] Agrawal GK，Tamogami S，Iwahashi H，et al．Transient regulation of jasmonicacid-inducible rice MAP kinase gene (OsBWMK1) by diverse biotic and abioticStresses[J]．Plant Physiol Biochem，2003，41：355?361．
[30] Cheong YH，Moon BC，Kim JK，et al．BWMK1，a rice mitogen-activated proteinkinase，locates in the nucleus and mediates pathogenesis-related gene expression by activationof a transcription factor[J]．Plant Physiol，2003，132：1961?1972．
[31] Ning J，Yuan B，Xie KB，et al．Isolation and identification of SA and JA inducibleprotein kinase gene OsSJMK1 in rice[J]．Yi Chuan Xue Bao，2006，33：625?633．
[32] Berberich T，Sano H，Kusano T．Involvement of a MAP kinase，ZmMPK5，in senescenceand recovery from low-temperature stress in maize[J]．Mol Gen Genet，1999，262：534-542．
[33] Lalle Marco，Visconti Sabina，Marra Mauro．ZmMPK6，a novel maize MAP kinase thatinteracts with 14-3-3 proteins[J]．Plant Molecular Biology，2005，59：713–722．
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[2] Ray L Bryan，Sturgill W Thomas．Insulin-stimulated microtubule-associated proteinkinase is phosphorylated on tyrosine and threonine in vivo[J]．Proc Natl Acad Sci，1988，85：3753-3757．
[3] Wilsbacher J L，Goldsmith EJ，Cobb M H．Phosphorylation of MAP kinases byMAP/ERK involves multiple regions of MAP kinases[J]．J Biol Chem，1999，74(24)：16988-16994．
[4] M Camps，A Nichols，S Arkinstall．Dual specificity phosphatases：a gene family forcontrol of MAP kinase function[J]．The FASEB Journal，1999，14：6-16．
[5] Jiang Y，Li Z，Schwarz E M，et al．Structure-function studies of p38 mitogen activatedprotein kinase．Loop 12 influences substrate specificity and autophosphorylation，but notupstream kinase selection[J]．J Biol Chem，1997，272：11096-11102．
[6] Dufresne S D，Bjorbek C，El-Haschimi K．Altered extracellular signal-regulated kinasesignaling and glycogen metabolismin skeletal muscle from p90 ribosomal S6 kinase 2knockout mice[J]．Mol Cellular Boil，2001，21 (1)：81-87．
[7] Louis-Philippe Hamel，Marie-Claude Nicole，Somrudee Sritubtim，et al．Ancient signals：comparative genomics of plant MAPK and MAPKK gene families[J]．TRENDS in PlantScience，2006，11(4)：192-198．
[8] Reyna S Nathan，Yang Yinong．Molecular Analysis of the Rice MAP Kinase Gene Familyin Relation to Magnaporthe grisea Infection[J]．MPMI，2006，19 (5)：530–540．
[9] J English，G Pearson，J Wilsbacher，et al．New insights into the control of MAP kinasepathways [J]．Exp Cell Res，1999，253 (1)：255-270．
[10] Ichimura Kazuya，et al．Mitogen-activated protein kinase cascades in plants：a newnomenclature[J]．TRENDS in Plant Science，2002，7(7)：301-308．
[11] Tena Guillaume，Asai Tsuneaki，Chiu Wan-Ling，et al．Plant mitogen-activated proteinkinase signaling cascades[J]．Plant Biology，2001，4：392–400．
[12] Yoo Sang-Dong，Cho Young-Hee，Tena Guillaume，et al．Dual control of nuclear EIN3by bifurcate MAPK cascades in C2H4 signalling[J]．Nature，2008，451：789-796．
[13] Shan Libo，He Ping，Sheen Jen．Intercepting Host MAPK Signaling Cascadesby Bacterial Type III Effectors[J]．Cell Host & Microbe，2007，4: 167-174．
[14] Yoo Sang-Dong，Cho Younghee，Sheen Jen．Emerging connections in the ethylenesignaling network[J]．Cell，2009，2：1-10．
[15] Zhang Shuqun，Klessig F Daniel．MAPK cascades in plant defense signaling[J]．TRENDS in Plant Science，2001，6(11)：520-527．
[16] Asai Tsuneaki，Tena Guillaume，Plotnikova Joulia，et al．MAP kinase signalling cascadein Arabidopsis innate immunity [J]．Nature，2002，415：977-983．
[17] Sheen Jen，He Ping，Shan Libo，et al．Signaling specificity and complexity of MAPKcascades in plant innate immunity[J]．BIOLOGY OF PLANT-MICROBE INTERACTIONS，2008，6: 1-10．
[18] Lieberherr D，Thao NP，Nakashima A，et al．A sphingolipid elicitor-induciblemitogenactivated protein kinase is regulated by the small GTPase OsRac1 and heterotrimericG-protein in rice[J]．Plant Physiol，2005，138：1644-1652．
[19] Fu SF，Chou WC，Huang DD，et al．Transcriptional regulation of a ricemitogen-activated protein kinase gene，OsMAPK4，in response to environmental stresses[J]．Plant CellPhysiol，2002，43：958?963．
[20] Agrawal GK，Agrawal SK，Shibato J，et al．Novel rice MAP kinases OsMSRMK3 andOsWJUMK1 involved in encountering diverse environmental stresses and developmentalregulation[J]．Biochem Biophys Res Commun，2003，300：775?783．
[21] Huang HJ，Fu SF，Tai YH，et al．Expression of Oryza sativa MAP kinase gene isevelopmentally regulated and stress-responsive[J]．Physiol Plant，2002，114：572-580．
[22] Song FM，Goodman RM．OsBIMK1，a rice MAP kinase gene involved in diseaseresistance responses[J]．Planta，2002，215：997?1005．
[23] Wen JQ，Oono K，Imai R．Two novel mitogen-activated protein signaling components，OsMEK1 and OsMAP1，are involved in a moderate low-temperature signaling pathway inrice[J]．Plant Physiol，2002，129：1880?1891．
[24] Agrawal GK，Rakwal R，Iwahashi H．Isolation of novel rice (Oryza sativa L.) multiplestress responsive MAP kinase gene,OsMSRMK2，whose mRNA accumulates rapidly inresponse to environmental cues[J]．Biochem Biophys Res Commun，2002，294：1009?1016．
[25] Xiong L，Yang Y．Disease resistance and abiotic stress tolerance in rice are inverselymodulated by an abscisic acid-inducible mitogen-activated protein kinase[J]．Plant Cell，2003，15：745?759．
[26] Yeh CM，Hung WC，Huang HJ．Copper treatment activates mitogen-activated proteinkinase signalling in rice[J]．Physiol Plant，2003，119：392?399．
[27] Jeong MJ，Lee SK，Kim BG，et al．A rice (Oryza sativa L.) MAP kinase gene，OsMAPK44，is involved in response to abiotic stresses[J]．Plant Cell Tiss OrganCult，2006，85：151?160．
[28] He C，Fong SH，Yang D，et al．BWMK1，a novel MAP kinase induced by fungalinfection and mechanical wounding in rice[J]．Mol Plant Microbe In，1999，12：1064?1073．
[29] Agrawal GK，Tamogami S，Iwahashi H，et al．Transient regulation of jasmonicacid-inducible rice MAP kinase gene (OsBWMK1) by diverse biotic and abioticStresses[J]．Plant Physiol Biochem，2003，41：355?361．
[30] Cheong YH，Moon BC，Kim JK，et al．BWMK1，a rice mitogen-activated proteinkinase，locates in the nucleus and mediates pathogenesis-related gene expression by activationof a transcription factor[J]．Plant Physiol，2003，132：1961?1972．
[31] Ning J，Yuan B，Xie KB，et al．Isolation and identification of SA and JA inducibleprotein kinase gene OsSJMK1 in rice[J]．Yi Chuan Xue Bao，2006，33：625?633．
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