【作者】 林少航；

【导师】 白吉刚；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 土壤盐渍化是一个世界性的资源环境和生态问题。因此,植物耐盐性研究具有重要的意义。本实验以温室型黄瓜品种“春光2号”为材料,报告了外源甲基紫精缓解植株的盐胁迫,研究了外源甲基紫精对盐胁迫下黄瓜叶片抗氧化酶与DNA甲基化的影响,分离了盐胁迫下外源甲基紫精诱导的甲基化差异片段,然后对这些差异片段进行克隆、测序和序列的生物信息学分析。本文从抗氧化酶、整个基因组水平上初步揭示外源甲基紫精缓解盐胁迫的机制,为利用甲基化差异基因打下基础,为外源甲基紫精在黄瓜耐盐生产中的应用提供依据,并为其它外源物质缓解植物逆境胁迫的机制研究提供参考。实验在黄瓜幼苗长至两叶一心时进行,用10μM甲基紫精(PQ)浇灌黄瓜幼苗后于100μmol m-2 s-1光照下处理1 h,然后分别用水和营养液冲洗植株各6次,将黄瓜幼苗在暗处放置24 h后浇灌100mM NaCl,进行盐处理。盐胁迫处理两天后取第二片真叶,测定其超氧化物歧化酶(SOD,EC 1.15.1.1)、愈创木酚过氧化物酶(GPX,EC 1.11.1.7)、过氧化氢酶(CAT,EC 1.11.1.6)、谷胱甘肽还原酶(GR,EC 1.6.4.2)、谷胱甘肽过氧化物酶(GSH-Px, EC 1.11.1.9)、脱氢抗坏血酸还原酶(DHAR, EC 1.8.5.1)、单脱氢抗坏血酸还原酶(MDHAR, EC 1.6.5.4)和抗坏血酸过氧化物酶(APX, EC 1.11.1.11)的活性,检测叶片中丙二醛(MDA)、内源H2O2、抗坏血酸(AsA)还原型谷胱甘肽(GSH)和超氧阴离子(O2.-)的水平。另外,实验提取黄瓜叶片基因组DNA,利用甲基化敏感扩增多态性(MSAP)技术研究黄瓜叶片基因组DNA甲基化的水平和状态变化,对盐胁迫下甲基紫精预处理诱导的DNA甲基化差异片段进行回收,克隆和测序分析。主要结果如下:1、甲基紫精预处理对盐胁迫下黄瓜叶片抗氧化酶的影响盐胁迫严重抑制了黄瓜幼苗的生长,使地上部和根系干重均显著下降;PQ处理后再进行盐胁迫的黄瓜幼苗中,地上部和根系干重明显上升,PQ处理缓解了盐胁迫对黄瓜生长的抑制作用。PQ处理提高了盐胁迫下黄瓜叶片抗氧化酶SOD、GPX、CAT、APX、GSH-Px、DHAR、MDHAR和GR的活性,增加了盐胁迫下抗氧化物AsA和GSH的含量、还原型AsA与氧化型AsA比值以及GSH与GSSG的比值,同时显著降低了盐胁迫下黄瓜叶片MDA的含量、O2.-生成速率以及H2O2的含量,缓解了盐胁迫下ROS对黄瓜叶片的伤害。因此,我们推断,盐胁迫下PQ处理通过提高黄瓜幼苗抗氧化酶的活性和抗氧化物的含量,降低活性氧的含量,进而缓解了盐胁迫诱导的膜脂过氧化损伤,减轻了盐胁迫对黄瓜幼苗生长的抑制,提高了黄瓜幼苗的耐盐性。2、甲基紫精预处理对盐胁迫下黄瓜叶片DNA甲基化的影响利用32对引物对黄瓜叶片基因组DNA甲基化进行了MSAP分析:共扩增出1324条带,对照、PQ预处理、salt处理以及PQ+salt处理的总甲基化比率分别为19.8%、19.9%、21.5%和20.4%。在盐胁迫下,黄瓜植株DNA甲基化水平发生改变;外源甲基紫精预处理黄瓜幼苗后,能诱导盐胁迫下黄瓜DNA发生去甲基化。对部分甲基化差异片段回收、克隆、测序及序列比对后,发现6条片段与植物响应逆境胁迫的基因序列具有较高的同源性。更多还原

【Abstract】 Salt tolerance of plants is of great significance as soil salinization is one of the most important global environmental and ecological problems. Cucumber （Cucumis sativus L.） cv. Chunguang no. 2 was cultivated to study whether exogenous paraquat （PQ） could protect plants from salt stress and whether the protection was associated with the increasing of antioxidant enzymes activities and the changes of DNA methylation. We also aimed to clone the methylation polymorphic fragments and analyze their sequence by using bioinformatics. Our work might help to elucidate the mechanisms of salt stress mitigated by PQ on the sides of antioxidant enzymes and DNA methylation. It also could lay the foundations for applying methylation polymorphic genes and exogenous PQ to salt-resisting cultivation of cucumbers and give references to studying the mechanisms of stress mitigated by other substances.At the two-leaf stage, cucumber seedlings were selected and pretreated with 10μM PQ under moderate light （100μmol m-2 s-1） for 1 h. After rinsed 6 times with water and nutrient solution, cucumber plants were kept in darkness for 24 h. Then, the cucumbers were watered with 100 mM salt for 2 d. Samples of the second leaf was used to determine the activities of antioxidants such as superoxide dismutase （SOD, EC 1.15.1.1）, guaiacol peroxidase （GPX, EC 1.11.1.7）, ascorbate peroxidase （APX, EC 1.11.1.11）, catalase （CAT, EC 1.11.1.6）, dehydrateascorbate reducatase （DHAR, EC 1.8.5.1）, monodehydroascorbate reductase （MDHAR, EC 1.6.5.4）, glutathione reductase （GR, EC 1.6.4.2）, ascorbic acid （AsA） and reduced glutathione （GSH）. The leaf samples were also used to assay the cntents of malondialdehyde （MDA）, hydrogen peroxide （H₂O₂） and superoxide anion （O₂.-）. After extracting genomic DNA from the second leaves, DNA methylation was surveyed by using the methylation-sensitive amplified polymorphism （MSAP） method. Then, DNA methylation polymorphic fragments were isolated, cloned and sequenced. Subsequently, homology search and sequence analysis were performed at the public database NCBI. The main results of this study were listed as the following:1. Effects of paraquat on antioxidant enzymes in salt-stressed cucumber leavesThe growth of cucumber seedlings was inhibited observably by salt stress. The dry weights of shoot and root were significantly decreased under salt stress, while they were increased in PQ-pretreated stressed plants. So PQ pretreatment alleviated the inhibition of salt stress on the growth of cucumber seedlings. Under salt conditions, PQ pretreatment relieved oxidative stress as observed by the decreases in levels of MDA, H₂O₂ and O₂.-, which correlated with the increase in antioxidant defenses. Compared to the salt treatment, PQ pretreatment increased the activities of antioxidants such as SOD, GPX, CAT, APX, GSH-Px, DHAR, MDHAR, GR, ASA and GSH and also enhanced the ratios of reduced ASA/oxidized ASA and GSH/GSSG. Therefore, we proposed that the PQ pretreatment increased the activities of antioxidant enzymes under conditions of salt and thereby decreased the levels of ROS and improved the salt tolerance of cucumber seedlings.2. Effects of paraquat on DNA methylation in salt stressed cucumber leavesThirty-two pairs of primers were used for MSAP analysis. Total of 1324 fragments were amplified, and the level of global DNA methylation in four treatments （control, PQ pretreatment, salt and PQ+salt treatments） were 19.8%, 19.9%, 21.5% and 20.4%, respectively. The levels of DNA methylation in cucumber plants were obviously changed under drought stress, while PQ pretreatment directionally induced the demethylation. After part of the methylated DNA fragments were cloned and sequenced, six fragments were found to be homologous to the functional protein, indicating that the fragments were involved in the process where PQ improved the salt tolerance of cucumber seedlings.更多还原