【作者】 吴涛；

【导师】 李德全；

【作者基本信息】 山东农业大学 ， 植物学， 2007， 硕士

【摘要】 近年来,在植物中分离到大量MAPKs,并在其作用机理研究方面取得了长足进展。植物中MAPKs的研究主要集中在双子叶模式植物(如拟南芥、烟草和紫花苜蓿等),关于玉米MAPK方面的研究很少。本论文以玉米Zheng958为试材,研究玉米中已知的MAPK基因及相关基因在不同逆境及信号物质处理下的表达情况;对胁迫诱导型基因ZmOSMAPK1制备抗体,对其在不同逆境处理下蛋白质的表达情况进行分析;探讨转ZmOSMAPK1基因拟南芥和烟草的抗逆性。同时,成功构建了玉米幼苗根系cDNA文库。主要结果如下:1、干旱、高盐及低温胁迫下玉米根系MAPKs的表达利用RT-PCR研究干旱、高盐及低温胁迫下玉米幼苗根系中MAPKs的表达情况。结果发现,只有ZmOSMAPK1在对照中未见表达,而其余基因都存在或高或低的本底水平的表达。ZmOSMAPK1在PEG和NaCl胁迫下的表达有较大差异。对两种处理下不同时间段ZmOSMAPK1的表达情况进行研究,结果显示,处理5小时内,NaCl处理下ZmOSMAPK1的表达量高于PEG处理该基因的表达;5小时后表现出相反的表达模式。我们认为ZmOSMAPK1参与PEG和NaCl介导的MAPK信号途径中,但主要参与和干旱胁迫相关的信号途径。4℃低温下MAPKs表达高于4℃低温黑暗处理,推测在MAPKs级联信号途径中,光照可能作为一种刺激信号,对信号途径中基因表达具有调控作用。2、植物激素及信号物质作用下玉米根系MAPKs的表达应用植物激素GA和信号物质SA、CaCl2处理玉米幼苗根系,同一MAPK在上述三种处理下的表达情况基本一致,而另外一种植物激素ABA则不同。我们分析认为,植物激素GA和信号物质SA、CaCl2处理后会引起MAPKs级联途径中各组分表达量提高,而ABA处理可能与PEG和NaCl胁迫处理一致。3、机械损伤及紫外线照射对玉米根系MAPKs表达的影响机械损伤能诱导玉米幼苗根系中MAPKs不同程度的表达,紫外线照射下MAPKs的表达情况没有规律性。这说明玉米中的MAPKs参与机械损伤和紫外线介导的信号途径之中。4、ZmOSMAPK1基因在E.coli中的表达、多克隆抗体制备及ZmOSMAPK1蛋白在不同胁迫下的表达特性将ZmOSMAPK1编码区克隆到原核表达载体pET30a-c(+),在BL21中表达了部分融合蛋白。纯化融合蛋白免疫小白鼠,制备了具有高特异性的多克隆抗体,抗体滴度高达1∶100000以上。Western blot研究不同处理下ZmOSMAPK1蛋白质的表达情况。结果发现,ZmOSMAPK1在各处理下都有不同程度的表达,与ZmOSMAPK1在转录水平上的表达模式基本相似。表明该基因在转录水平和翻译水平上保持一致。5、转ZmOSMAPK1基因拟南芥和烟草的抗性分析将ZmOSMAPK1基因分别转化拟南芥和烟草。研究发现,转基因拟南芥对干旱和盐胁迫有较高的耐性,转基因烟草对4℃低温有较高的耐性。表明ZmOSMAPK1基因在玉米根系响应外界逆境上有重要作用。6、玉米幼苗根系cDNA文库的构建以不同浓度PEG和不同时间处理的玉米幼苗根系为材料,成功构建了cDNA文库,并对其进行初步筛选。结果显示文库容量较高,插入片断较大。更多还原

【Abstract】 Up to date, a number of MAPKs have been isolated from different plants, and considerable progresses have been made in understanding plant MAPKs. Current research of plant MAPK mostly foucs on dicot plant spe-cies (such as Arabidopsis, tobacco, and alfafa etc.), but relatively less study on Zea mays. In this paper, Zea mays (Zheng 958) seedlings are treated with different environmental stresses and signal substances, and expression pat-tern of MAPKs in Zea mays are analyzed under these conditions. The anti-body is made according to induce-expressed ZmOSMAPK1. Stress tolerance of transgenic Arobidopsis and transgenic tobacco are also investigated with different treatments. Meanwhile, the cDNA library of Zea mays roots treated with PEG is also constructed. The main results are as follows: 1 Expression analysis of MAPKs in Zea mays roots with treatments of drought, salt and low-temperatureExpression analysis of MAPKs in Zea mays with treatments of drought, salt and low-temperature were investigated using RT-PCR. The results indicate that only ZmOSMAPK1 has no expression only in the control, while other MAPKs have different combinational expression level. Sig-nificant differences exist in the expression level of ZmOSMAPK1 with treatments of PEG and NaCl. A detailed time course experiment with treatments of PEG and NaCl was conducted, and the results demon-strated that the expression level of ZmOSMAPK1 in Zea mays roots treated with NaCl is higher than that with PEG in the first 5 hours but reversed in the later time course. These indicate that ZmOSMAPK1 is involved in the signal pathway induced by PEG and NaCl respectively and mainly related with the signal pathway induced by drought. The ex-pression level of MAPKs at 4℃in light is higher than 4℃that in dark-ness. The result indicated that light might be a stimulus in the MAPK cascades and has effect on the regulation of gene expression. 2 Expression analysis of MAPKs in Zea mays roots with treatments of plant hormones and signal substancesThe expression pattern of a MAPK in Zea mays roots was generally similar with treatments of plant hormones GA and signal substances CaCl2 and SA, except ABA. The results indicated that the expression level of MAPK cascades components would be up-regulated with treatments of GA, CaCl2 and SA, while the effect of ABA might be similar to PEG and NaCl.3 Influence of mechanical injury and UV-irradiation on MAPKs expres-sion in Zea mays rootsMAPKs in Zea mays roots could be induced by mechanical injury at different level. Results also showed that there are no rules in the expres-sion pattern of MAPKs after UV-irradiation. These indicated that MAPKs in Zea mays roots were involved in the signal transduction in-duced by mechanical injury and UV-irradiation.4 Expression of ZmOSMAPK1 gene in E.coli, preparation of polyclone antibody and expression analysis of ZmOSMAPK1 protein with differ-ent stressesAfter cloning the encoding region of ZmOSMAPK1 into prokaryotic ex-pression vector pET-30a-c(+), we obtained the soluble fusion proteins in BL21 strain. Special polyclone antibody was prepared by injecting fu-sion proteins into immune mice and the titer reached up to 1:100000. Western blot analysis demonstrated that ZmOSMAPK1 could be de-tected in different levels with different stresses, and the expression pat-tern was accordant to that in transcriptional levels. These indicated that the expression of ZmOSMAPK1 gene was consistent in transcriptional and translational level.5 Tolerance analysis of transgenic Arabidopsis and transgenic tobacco The ZmOSMAPK1 gene was transformed into Arabidopsis and tobacco. The results showed that the transgenic Arabidopsis had ability of bear- ing drought and high salinity. And the transgenic Arabidopsis showed increased ability of bearing low-temperature. These results indicated that ZmOSMAPK1 gene played important roles in response to environ-mental stresses.6 Construction of the cDNA library of Zea mays roots The cDNA library was constructed with Zea mays roots which were treated with different concentrations of PEG for different time. Some of the screening results show that the capacity of the library was rather big for us to screen, and the inserted segments were relative large.更多还原