【作者】 丁海东；

【导师】 蒋明义；

【作者基本信息】 南京农业大学 ， 植物学， 2009， 博士

【摘要】 脱落酸(abscisic acid,ABA)是一种重要的植物激素,在植物生长、发育以及对环境的适应中起着重要的信号分子作用。促分裂原活化蛋白激酶(Mitogen-Activated Protein Kinase,MAPK)是真核细胞中普遍存在的信号组分,由逆境胁迫、细胞因子、植物激素、生长因子等诱导,并在植物信号中发挥重要的作用。已知许多蛋白激酶参与了ABA信号途径,MAPK也能够作为ABA信号的下游组分,发挥调节作用。因此,鉴定ABA信号途径中相关的MAPK,对于揭示ABA信号的细胞和分子机理具有重要意义。本实验室在研究ABA和H2O2活化玉米(Zea mays)叶片的MAPK时,已经发现了一种分子量为46 kDa的MAPK(p46MAPK)参与ABA诱导的抗氧化防护,但是我们不知道它是一个什么类型的MAPK,与其它植物MAPK的同源性怎么样。澄清它的性质对于进一步的分子生物学研究至关重要。因此,我们首先对p46MAPK进行分离纯化,其次进行质谱分析并确定类型,最后进行相关特性研究。结果如下：1.p46MAPK纯化：髓鞘碱性蛋白(myelin basic protein, MBP)是MAPK的最适底物。我们以MBP为底物,采用溶液激酶分析和凝胶激酶分析(in-gel kinase assay)相结合的方法对纯化过程中的目的蛋白进行跟踪鉴定,并在4℃条件下完成所有纯化步骤。叶片提取上清液先经30%饱和度硫酸铵沉淀,100,000g超速离心,上清经80 mlHiPrep 26/10 (Sephdex G-25F)脱盐柱脱盐后,依次进行40 ml Q sepharose FF阴离子柱、15 ml Pheny sepharose FF疏水柱、6 ml Resource Q阴离子柱、1 ml Mono Q阴离子柱、3.5 ml poly-L-lysine-agarose亲和柱和120 ml Hiload 16/60Superdex 75pg凝胶过滤柱和超滤管浓缩.经过上述步骤,最终经SDS-PAGE电泳,银染检测45 kDa maker处有单一条带,凝胶激酶实验显示能够磷酸化MBP底物。对照与ABA处理的样品同时经Mono Q纯化,进一步证明部分纯化的蛋白为ABA诱导的p46MAPK。部分纯化蛋白的纯化倍数大于10000,回收率为1.1%,激酶活性为20,420pmol min-1mg-1。2. p46MAPK鉴定：质谱已成为鉴定蛋白质的最重要技术平台之一。将目的蛋白胶切下,送北京军事医学科学院国家生物医学分析中心进行基质辅助激光解析电离串联飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry, MALDI-TOF-TOF-MS)鉴定。利用BioTools3.0分析软件在http: //www.matrixscience. com的NCBI绿色植物数据库中进行分析。结果显示,p46MAPK与玉米中已知序列的ZmMPK5 (gi|4239889)序列覆盖达32%,匹配得分207,匹配显著(得分大于69为显著,P<0.05)。选取肽段(m/z 1779.841)进行MS/MS鉴定,序列为TTSETDFMTEYWTR,对应于ZmMPK5的218-232处肽段。为进一步证明该结果,ZmMPK5的多克隆抗体被制备。免疫共沉淀凝胶激酶的结果显示ZmMPK5为ABA诱导的p46MAPK。因此,p46MAPK即为ZmMPK5。3.ZmMPK5生物信息学分析：利用生物信息学分析ZmMPK5的分子量、等电点、细胞定位等。利用NCBI中BLAST软件,与植物中其他MAPK进行了比对,并构建了基因进化树。结果发现,ZmMPK5分子量为44.9,等电点为5.39,在线软件程序SubLoc v1.0 (http://www.bioinfo.tsinghua.edu.cn/SubLoc/)将其定位在细胞核中,属于MAPK家族的A族,与小麦TaMAPK、水稻OsSIPK和OsMPK6亲缘关系最近,与苜蓿MMK1、拟南AtMPK6、欧芹PcMPK6、番茄LeMPK2、马铃薯StMPK1和烟草SIPK亲缘关系其次,而与玉米中已知的其他MAPK蛋白亲缘关系相对较远。4.ZmMPK5相关特性分析：ZmMPK5的凝胶层析分子量为45.74 kDa。在温度(20-50℃)、MgCl2浓度(2.5-15 mM)和pH(5.0-9.0)情况下,该激酶都具有活性。理想反应条件为30℃、pH 8.0和10mMMgCl2.MBP和ATP的Km值分别为0.13μgμL-1和23μM。与histone和casein相比,MBP是ZmMPK5的首选底物。将磷酸化的MBP水解进行薄层层析,其磷酸化位点发生在苏氨酸上,而丝氨酸和酪氨酸未见磷酸化.在ZmMPK5:YFP表达的玉米叶片原生质体中,ZmMPK5存在于细胞质和细胞核中,主要定位在细胞核中。不同的环境胁迫因子如低温、干旱、NaCl、CdCl2、伤害、UV和激素(SA, ETH)均能够诱导ZmMPK5转录和ZmMPK5激酶活性,而ZmMPK5蛋白水平上未见明显的变化。更多还原

【Abstract】 Abscisic acid (ABA), one of the most important phytohormones, plays an important role as a signaling molecule in plant growth, development and adaptation to the environment. Mitogen-activated protein kinase (MAPK) signaling is prevalent among eukaryotic cells, which is induced by stresses, cytokines, plant hormones, growth factors and so on, playing a vital role in plant stress signal. Many studies have shown that ABA signal transduction is associated with the MAPK cascade pathway. MAPK is one of crucial downstream components in the signal transduction pathway of ABA. Therefore, it is important to identify MAPKs in ABA signal transduction and networks to reveal the cellular and molecular mechanism of ABA signalIn previous studies, our lab reported that a 46 kDa MAPK (p46MAPK) was involved in ABA-induced antioxidant defense in leaves of maize (Zea mays) plants (Zhang et al.,2006 Plant physiology; 2007 New phytologist). However, the identity of the p46MAPK in maize is not clear, and the alignment to other MAPKs is not known. It is important to reveal the identity and characterization for further research at molecular level. Firstly, we isolated and purified p46MAPK. Secondly, the identity of p46MAPK was identified by MS and bioinformatics assay. Lastly, the characterization of p46MAPK was studied.1. Purification of p46MAPK. In this study, partial purification of p46 MAPK was performed by monitoring its activity with an in-gel kinase assay and in-solution kinase assay using myelin basic protein (MBP), which is the best fit substrate for MAPKs. All the steps were carried out at 4℃unless stated otherwise. All chromatographic runs were carried out on an AKTA Purifier 100 system (GE Healthcare) and AKTA Prime System (GE Healthcare). The p46MAPK induced by ABA was partially purified by the 30%(NH4)2SO4 precipitation followed by 100,000g ultracentrifugation and chromatography on 40 ml Q-Sepharose FF,15 ml Phenyl-Sepharose FF,6 ml Resource Q,1 ml Mono QTM 5/50 GL, 3.5 ml Poly-L-lysine-agarose, and 120 ml Superdex 75 prep grade columns. After SDS-PAGE analysis and sliver staining, the enzymes from Superdex 75 prep grade column showed Only 45.4 kDa protein band is near to molecular weight of p46MAPK. And only one band of kinase activity was detected by in-gel kinase assays with MBP as a substrate. These results suggest that this polypeptide was the 46 kDa ABA-activated MAPK. We also purified the kinase from the ABA-treated maize leaves and water treated maize leaves at the same time, which further determined the purified kinase was the ABA-induced p46MAPK. Taking the activity at the first purification step as 100%, the kinase was purified more than 10,000 fold, with an overall yield of 1.1. The specific activity of the kinase was 20,420 pmol min mg-1.2. Identification of p46MAPK. Development in mass spectrometry (MS) technology has dramatically accelerated the application of proteomics in recent years. Biological mass spectrometry has become one of the most important technology platforms for protein identification. It is an important part of continuing study on isolated and purified protein. The partial purified protein was excised and sent to the National Center of Biomedical Analysis, Academy of Military Medical Sciences (Beijing, China) for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-TOF-MS) analyses. Proteins were identified using MS/MS ion search of Mascot search engine (http://www.matrixscience.com, Matrix Science, London, England) and Viridiplantae (Green Plants) protein database (NCBI,20071116). It was identified that the p46MAPK was ZmMPK5 (gi|4239889) in maize. The search yielded a top score of 207 (protein scores greater than 69 are significant; P<0.05). Furthermore, the selected tryptic peptide (m/z 1779.841) sequenced by MS/MS revealed an amino acid sequence of TTSETDFMTEYVVTR, corresponding to residues 218-232 of ZmMPK5. In order to further confirm the result, the polyclonal antibody that recognizes the C-terminal region of ZmMPK5 was raised in rabbits, and the immunoprecipitation kinase assay confirmed the results. These results clearly indicate that the ABA-activated p46MAPK is ZmMPK5. 3. Bioinformatics assay of ZmMPK5. The related characterizations of ZmMPK5 is analyzed by bioinformatics assay, including molecular weight, isoelectric point, protein subcellular localization, alignment and phylogenetic trees analysis of the ZmMPK5 with other homologous MAPKs. The nominal mass of the kinase and isoelectric point are 44.9 kD and 5.39, respectively. ZmMPK5 is localized in the nucleus by SubLoc v1.0 database. Based on the sequence alignment a phylogenetic tree was constructed, which indicates that ZmMPK5 can be grouped into subgroup A, and ZmMPK5 is most homologous to OsMPK6, AtMPK6, NtSIPK and StMPK1. Furthermore, the similarity with the function of the protein known before is analyzed, and its function is also speculated.4. Characterization of ZmMPK5. Using of biochemistry, pharmacology, cell biology and other experimental methods, we studied the related characterization of p46MAPK (ZmMPK5). The results show that the molecular mass of the kinase is found to be 45.74 kDa by gel-filtration. The kinase showes activity in the range of 20 to 50℃in temperature, 2.5 to 15 mM MgCl2, and broad pH 5.0 to 9.0, with optimal activity at pH 8.0,35℃, and 10 mM MgCl2. For this kinase, the Km for myelin basic protein (MBP) substrate and ATP are 0.13μgμL-1 and 23μM, respectively. MBP is the preferred substrate, of which the threonine residue is phosphorylated. Immunoprecipitation gel kinase analysis further confirm that ABA-and H2O2-induced 46 kD MAPK is ZmMPK5.100μM SA,100μM ETH,10% PEG, 250 mM NaCl,500μM CdCl2, low temperature, wounding and UV are able to mediate the expression of ZmMPK5 and activation of ZmMPK5.In ZmMPK5:YFP-expressing Zea mays protoplasts, it was localized predominantly in the nucleus. Successful identification and related analysis of the nature of p46MAPK enable us to further define the function of the ZmMPK5 in ABA signal.更多还原