【作者】 宗晓娟；

【导师】 李德全；

【作者基本信息】 山东农业大学 ， 植物学， 2009， 博士

【摘要】 MAPK级联途径是存在于真核生物中进化上保守的信号通路,在细胞信号的转换和放大过程中起重要作用。MAPK级联途径由三个成员组成,分别是MAPK、MAPKK （MAPK kinase）及MAPKKK（MAPKK kinase）,此三个信号组分按照MAPKKK-MAPKK-MAPK的方式依次磷酸化将外源信号级联放大向下传递。植物中的MAPK通常分为四组,即A, B, C, D。关于A, B组MAPK的报道较多,研究较为详细。关于C组MAPK的分析较为欠缺。但近年研究发现,C组MAPK同样参与介导环境胁迫及信号分子（如ABA, H₂O₂等）的传递。由于不同物种中C组MAPK编码基因相继报道,所以有理由推测不同物种间C组MAPK调控的信号转导通路可能存在某些相似性。玉米作为一种重要的禾本科粮食作物,其C组MAPK基因迄今未见报道。ABA,H2O2是植物逆境信号转导中重要的信号分子, C组MAPK是否介导这两种信号的传递?与抗氧化系统关系如何?这一问题亟待回答。本研究从玉米栽培种鲁单50幼苗中分离到一个C组MAPK基因,命名为ZmMPK7,对其序列特征、表达模式、以及过表达转基因烟草的抗逆生理功能做了初步研究。主要结果如下:1. GenBank中玉米MAPK基因信息的收集及整理根据拟南芥20个MAPK的基因序列,逐一在GenBank中比对,共得到39条玉米序列的信息。其中许多基因序列存在重复提交的现象。经分析后共保留下12条特异的基因全序列,它们都具有植物MAPK的序列特征,分属于四组,即A, B, C, D。其中C,D组成员占大多数,由此推测C,D组MAPK的多样性可能与其介导信号转导的特异性有关。2.玉米C组MAPK基因ZmMPK7的分离及序列分析根据玉米MAPK基因信息分析结果,设计特异引物从玉米幼苗中特异性扩增DNA片段。该扩增片段包含一个1113bp的ORF,编码370个氨基酸,预测蛋白质分子量为42.5kD,命名为ZmMPK7。序列分析显示,ZmMPK7包含11个植物MAPK保守的Ser/Thr蛋白激酶亚区以及一个TEY基序。进化树分析显示其与水稻OsMSRMK3,拟南芥ATMPK1、ATMPK2、ATMPK7、ATMPK14,烟草NtNTF3及豌豆PsMPK2的同源性较高,属于C组MAPK。3. ZmMPK7的表达分析为了研究ZmMPK7对不同胁迫刺激的响应模式,我们首先进行Northern blot分析。ZmMPK7具有较高的本底水平表达,100μM ABA或1.5 mM H₂O₂明显诱导该基因的表达,而PEG-6000及4oC低温处理未能检测到该基因的诱导表达。ABA及H2O2对该基因的诱导呈现不同的时间效应,并且依赖于Ca²⁺的存在。Imidazole、Tiron及DMTU为ROS效应剂,其中Imidazole是NADPH oxidase的抑制剂,而Tiron及DMTU是细胞内源ROS淬灭剂。使用以上药剂预处理玉米幼苗,半定量RT-PCR分析显示不同程度地抑制了ABA对ZmMPK7的诱导表达。正常生长状态下的玉米幼苗,ZmMPK7的本底表达呈现稳态水平,未检测到节律周期性。4.过表达ZmMPK7的转基因烟草在渗透胁迫下抗氧化能力增强为进一步研究ZmMPK7在介导ABA、H2O2信号转导过程中的详细功能,本研究构建该基因的正义过表达载体,农杆菌介导法转化烟草,挑取T3代纯合阳性转基因株系及野生型烟草进行生理分析。发现渗透胁迫下转基因烟草各株系内H2O2及丙二醛（MDA）积累量较野生型烟草少。以PEG-6000模拟渗透胁迫处理烟草,超氧化物歧化酶（SOD）、过氧化氢酶（CAT）的总酶活在转基因烟草和野生型烟草中未检测到明显差异,而过表达ZmMPK7显著增强了转基因烟草的过氧化物酶（POD）及抗坏血酸过氧化物酶（APX）总酶活。转基因烟草各株系的总抗坏血酸（AsA）及脱氢抗坏血酸（DAsA）含量明显高于野生型烟草。根据以上证据有理由推测过表达ZmMPK7的转基因烟草抗氧化系统得以改善,渗透胁迫下受到的氧化伤害较轻。此外,利用不同浓度的甲基紫精（MV）模拟氧化胁迫,分析烟草幼苗生长受到的影响。结果显示转基因各株系在较高或较低浓度的MV处理培养基中,均表现为较好的长势。该证据再次说明过表达ZmMPK7的转基因烟草在耐受氧化胁迫方面的优势。5. ZmMPK7定位于细胞核生物信息学软件预测ZmMPK7细胞核内定位的几率较高,瞬时转化ZmMPK7-GFP于洋葱表皮细胞的实验验证了该推测,即ZmMPK7为细胞核定位。6.玉米B组MAPKK基因ZmMKK3的分离及序列分析按照前文相似的分析方式,对GenBank中关于玉米MAPKK推测的基因信息收集整理,根据整理结果设计特异引物,从玉米幼苗中分离到一段DNA序列,该序列具有植物MAPKK的序列特征,包含有1572bp的ORF,编码523个氨基酸,将其命名为ZmMKK3。序列分析显示ZmMKK3含有所有植物MAPKK的保守氨基酸亚区以及磷酸化一致序列S/TxxxxxS/T。进化树分析显示与水稻OsMKK3、拟南芥AtMKK3同源性最高,同属于B组MAPKK。由于已有文献证明拟南芥AtMKK3是C组MAPK的上游激酶,与拟南芥AtMPK7直接互作。所以根据序列同源性分析,我们推测玉米ZmMKK3与ZmMPK7可能也存在互作关系。此推测尚需进一步验证。更多还原

【Abstract】 The MAPK cascade is an evolutionarily conserved signaling pathway among eukaryotic organisms and plays an important role in the conversion and amplification of extracellular stimuli. The MAPK cascades are composed of MAPK, MAPKK （MAPK kinase） and MAPKKK （MAPKK kinase）. They transfer signals through phosphorylation of MAPKKK-MAPKK-MAPK in turn. In plant, MAPKs are classified into four major groups designated A-D according to their sequence homology. Many MAPKs belonging to groups A and B have been studied. Little information, however, is available for group C MAPKs, although recent studies have demonstrated that some group C members are in response to environmental stress and hormonal stimuli, including ABA and H2O2. Since the responses of some group C MAPKs in different species have been characterized, there is evidence that they may have similar functions across species. However, no data regarding group C MAPKs from maize are currently available. There are also no available data that detail how they mediate ABA or H2O2 signaling. In particular, the relationship between group C MAPKs and antioxidant defense systems warrants further research. In the present study, a series of studies based on the materials of Ludan50 seedlings （Zea mays L.） have been conducted on the ZmMPK7 isolation, sequence comparison, expression analysis and further functional studies on the over-expressing ZmMPK7 transgenic tobacco. The main results are as follows:1. Collecting and analyzing MAPK genes information of Zea mays from GenBankAccording to the sequences of the 20 Arabidopsis MAPK genes, 39 pieces of information on gene accession have been identified from GenBank. Among them, some are repeated in completed sequences or in partial sequences. After the analysis, 12 specific gene completed sequences were reserved and can be classified into four groups, A-D. Most of the members belong to the Group C and D, which suggests that the diversity of these MAPKs may related to the specific activation of signal transduction.2. Isolation and characterization of a group C MAPK gene ZmMPK7 from Zea mays According to the sequences analysis about the MAPK genes information in maize, the specific primers were designed and a DNA fragment was amplified from the maize seedlings. The isolated fragment of the MAPK gene from maize contains a 1113 bp ORF, which encodes a protein of 370 amino acids with a calculated molecular weight of about 42.5 kDa. An alignment of the amino acid sequences of ZmMPK7 and MAPKs from other plants shows that the ZmMPK7 protein contains all 11 subdomains of protein kinases with serine/threonine specificity and the TEY motif. A phylogenetic tree analysis indicates that it is most homologous to Oryza sativa OsMSRMK3, Arabidopsis thaliana ATMPK1, ATMPK2, ATMPK7 and ATMPK14, Nicotiana tabacum NtNTF3, and Pisum sativum PsMPK2, all of which are classified as group C MAPKs.3. Expression analysis of ZmMPK7 in Zea maysIn order to identify the stressors that elicit a response from ZmMPK7, Northern blot analysis was carried out firstly. The ZmMPK7 gene had a relatively high level of basal expression. An increase in the expression of ZmMPK7 was detected in samples treated with 100μM ABA or 1.5 mM H₂O₂, but the samples exposed to 15% PEG-6000 or 4oC demonstrated no obvious increase in ZmMPK7 expression. ABA and H₂O₂-induced expression of ZmMPK7 was time-dependent in a Ca²⁺-dependent way. Semi-quantitative RT-PCR analysis indicated that several ROS manipulators, including imidazole, an inhibitor of NADPH oxidase, and the H2O2 scavengers Tiron and dimethylthiourea （DMTU） may inhibite the upregulation caused by ABA to varying extents. The basal ZmMPK7 mRNA level was relatively stable across the photoperiods and no rhythmic expression is detected in maize seedlings.4. Over-expressing ZmMPK7 in Nicotiana tabacum provides protection against ROS-mediated injury under osmotic stressTo further test the roles of ZmMPK7 in ABA and H2O2-mediated signaling, ZmMPK7 was over-expressed in transgenic tobacco plants under control of the CaMV 35S promoter. Homozygous T3 seeds of transgenic tobacco and wild-type lines were selected for the physiological analysis. It revealed that ZmMPK7-overexpressing tobacco plants showed less H2O2 accumulation and less MDA content compared with wild-type lines under osmotic stress treatment. After PEG-6000 treatment, no obvious differences in total SOD and CAT activities were detected between wild-type and transgenic tobacco plants. However, differences were detected in total peroxidase （POD） and ascorbic acid peroxidase （APX） activities. In the ZmMPK7-overexpressing tobacco lines, total ascorbic acid and dehydroascorbic acid contents were detected higher than those in wild-type tobacco. It is reasonable to speculate that the enhanced antioxidant system in transgenic tobacco plants may contribute to the protection against ROS-mediated injury under osmotic stress. Moreover, Methyl viologen （MV） in different concentration was used to estimate the effects of the oxidative stress on the growth of the wild-type and transgenic tobacco plants. It showed that the transgenic tobacco lines grew better under both high and low MV concentrations. This suggests again the advantages of anti-oxidative characterization in the ZmMPK7-overexpressing tobacco lines.5. ZmMPK7 is localized in the nucleusBioinformatics analysis showed it occupied the nucleus localization characterization. Transient expression of ZmMPK7-GFP fusion gene in onion （Allium cepa） epidermal cells confirmed that ZmMPK7 is a nuclear-localized protein.6. Isolation and characterization of a MAPKK gene ZmMKK3 in Zea maysSimilar analysis about the putative sequence information of maize MAPKK in GenBank was conducted. We isolated a specific DNA fragment from maize seedlings, which was speculated to encode a MAPKK. The isolated fragment contains a 1572 bp ORF, which encodes a protein of 523 amino acids and it was designated as ZmMKK3. An alignment of the amino acid sequences of ZmMKK3 and MAPKKs from other plants shows that the ZmMKK3 protein contains all the subdomains of the plant MAPKKs and the consensus sequence S/TxxxxxS/T. A phylogenetic tree analysis indicates that it is most homologous to Oryza sativa OsMKK3 and Arabidopsis thaliana AtMKK3, all of which are classified as group B MAPKKs. It has been indicated in a previous report that the group B MAPKK member, AtMKK3, is upstream of AtMPK7. So we speculated that ZmMKK3 may also interact with ZmMPK7. Further confirmation is required.更多还原