【作者】 高俊杰；

【导师】 于贤昌；

【作者基本信息】 山东农业大学 ， 设施农业科学与工程， 2008， 博士

【摘要】 黄瓜(Cucumis sativus L.)是我国北方设施栽培的主要作物之一,但连年种植引起的土壤次生盐渍化已严重影响到黄瓜的产量和品质。黄瓜属于典型的冷敏植物,低于10℃就会受到冷害。我国北方冬春季节气候寒冷,而没有加温设施的日光温室,若遇连续阴冷天气,室内最低气温常常会降到5℃左右,易使黄瓜受到低温伤害,造成黄瓜减产甚至绝产。克服连作盐害和低温冷害是当前日光温室黄瓜生产中亟待解决的主要问题。利用耐盐和抗冷砧木进行黄瓜的嫁接栽培是克服设施土壤次生盐渍化和提高植物抗冷性的一条有效途径。以黑籽南瓜为砧木的嫁接黄瓜幼苗叶片抗冷性可增强约3℃。同时,黄瓜的抗盐性明显增强,嫁接黄瓜叶片SOD、POD、CAT和APX酶活性均显著高于自根黄瓜等。嫁接黄瓜叶片超氧化物歧化酶(SOD)等抗氧化酶活性的明显增强与抗氧化物质抗坏血酸(AsA)等含量的显著增加是其抗逆性增强的主要原因之一。为了弄清嫁接提高黄瓜抗冷和耐盐的分子机制,本试验首先从黄瓜叶片克隆了Cu/Zn-SOD、Mn-SOD、CAT、GalLDH、APX、DHAR和GR基因的中间片段,然后以黑籽南瓜为砧木的嫁接黄瓜和自根黄瓜为试材,从基因转录水平分别研究了低温胁迫下和NaCl胁迫下两者叶片相关基因的表达与其抗氧化酶活性及AsA、GSH、H202和MDA等物质含量变化之间的关系,结果表明:1、从黄瓜叶片中分别克隆了Cu/Zn-SOD、Mn-SOD、CAT、GalLDH、APX、DHAR和GR基因的中间片段,其中,Mn-SOD、GalLDH、DHAR和GR基因的中间片段在黄瓜中均为首次克隆。将克隆基因经同源性分析,并在GeneBank中进行注册,其注册号分别为EF121763, EF203086, EF468517, EF468516, EF468515, EF426538和EF530128。2、低温胁迫下,嫁接与自根黄瓜叶片Cu/Zn-SOD和Mn-SOD基因mRNA相对表达量的变化分别与其Cu/Zn-SOD和Mn-SOD活性的变化相吻合, CAT基因mRNA相对表达量的变化与其CAT活性的变化并不一致。嫁接黄瓜叶片SOD基因家族中Cu/Zn-SOD和Mn-SOD基因mRNA的上调是其低温胁迫下SOD活性高于自根黄瓜叶片的决定因素;而低温胁迫下嫁接与自根黄瓜叶片CAT活性差异不大, CAT不是嫁接黄瓜抗冷性增强的主要因素。3、低温胁迫下,嫁接黄瓜叶片DHAR和GR基因mRNA的相对表达量均大于自根黄瓜,GalLDH和APX基因mRNA的相对表达量与自根黄瓜差异不大。嫁接黄瓜叶片GalLDH、DHAR、APX和GR活性均高于自根黄瓜,AsA和GSH含量与AsA/DHA和GSH/GSSG比值也均高于自根黄瓜,DHA、GSSG和H2O2含量均低于自根黄瓜。嫁接黄瓜较高的AsA和GSH含量与AsA/DHA和GSH/GSSG比值是其抗冷性强于自根黄瓜的重要因素,而低温胁迫下嫁接黄瓜叶片DHAR和GR基因mRNA的上调引起的DHAR和GR活性的增强是嫁接黄瓜AsA和GSH含量较自根黄瓜高的重要原因。4、NaCl胁迫条件下,嫁接与自根黄瓜叶片Cu/Zn-SOD、Mn-SOD和CAT基因mRNA的相对表达量均高于自根黄瓜,SOD、Cu/Zn-SOD、Mn-SOD和CAT活性也均高于自根黄瓜。嫁接黄瓜叶片Cu/Zn-SOD、Mn-SOD和CAT基因mRNA的上调是其维持较高Cu/Zn-SOD、Mn-SOD和CAT活性的重要原因;但是随NaCl胁迫时间的延长,嫁接和自根黄瓜叶片Cu/Zn-SOD、Mn-SOD和CAT mRNA的相对表达量均呈上升趋势,但其酶活性变化并不完全一致,说明还有其它因素也参与了SOD和CAT活性的调控;嫁接黄瓜叶片MDA含量和电解质渗漏率均低于自根黄瓜,嫁接黄瓜具有较高的活性氧清除系统,可以减少活性氧物质的危害,提高其耐盐性。5、NaCl胁迫下,嫁接黄瓜叶片GalLDH、DHAR和APX基因mRNA相对表达量均大于自根黄瓜,GalLDH、DHAR、APX和GR活性均高于自根黄瓜,GR基因mRNA相对表达量与自根黄瓜相比差异不大,但其AsA和GSH含量与AsA/DHA和GSH/GSSG比值均高于自根黄瓜,DHA、GSSG和H2O2含量均低于自根黄瓜。嫁接黄瓜叶片较高的AsA和GSH含量是嫁接黄瓜较自根黄瓜耐盐性强的主要原因,嫁接黄瓜叶片较高的GalLDH、APX、DHAR及GR等抗氧化酶活性是其AsA和GSH含量较自根黄瓜高的主要因素,而嫁接黄瓜叶片GalLDH、DHAR和APX基因mRNA的上调是其维持较高GalLDH、APX和DHAR活性的重要原因,在NaCl胁迫下嫁接黄瓜叶片具有高于自根黄瓜的GR活性并非GR基因mRNA的上调所致。更多还原

【Abstract】 Cucumber(Cucumis sativus L.)is one of the main crops planted in facilities in North of China. However the yield and quality of cucumber was severely affected by the soil salinity accumulating resulted from planting year after year. Cucumber is cold-sensitive, and could be damaged when the temperature belows 10℃. In North of China, it often drops to about 5℃in greenhouse in winter and spring. The low temperature could make cucumber hurt, result in reduction of cucumber or even no yield. It is urgent to resolve salt injury and chilling damage in current greenhouse cucumber production.It is an effective way that using rootstock for grafting cucumber cultivation to overcome the soil salinization and improve the cold resistance of the plant. The cold resistance of grafted cucumber’s leaves can be enhanced by 3℃. Meanwhile, the salt tolerance of cucumber significantly enhanced. The activities of SOD, POD, CAT and APX of the grafted cucumber leaves are higher than that of own-rooted cucumber. The increase of activities of antioxidant enzymes and the content of AsA is the main reason that the grafted cucumber increase its resistance.In order to identify the molecular mechanism of salt-tolerance and cold resistance of grafted cucumber, firstly the middle region of Cu / Zn-SOD, Mn-SOD, CAT, GalLDH, APX, DHAR and GR gene in cucumber leaves was cloned in this experiment. Then taking grafted and own-root cucumber (Junlv No.3) as trial material, the changes of expressions of genes, the activities of antioxidant enzyme, and the content AsA, GSH, H202 and MDA of grafted cucumber and own-root under NaCl stress and the low-temperature stress were analyzed.The results showed that:1. Cu/Zn-SOD,Mn-SOD,CAT,GalLDH,APX,DHAR and GR genes in cucumber leaves was cloned respectively. And the middle region of Mn-SOD,GalLDH,DHAR and GR genes were firstly cloned from cucumber leaves. Through analyzing the homology of the cloned gene, it was registered in gene bank, the number are EF121763, EF203086, EF468517, EF468516, EF468515, EF426538 and EF530128 respectively.2. The changes of activities of Cu/Zn-SOD and Mn-SOD correspond to the changes of their expression under low temperature stress, but the changes of activities of CAT were not the same as that of mRNA. SOD activity of grafted cucumber is higher than that of own-root cucumber under low-temperature stress. The key factor is that Cu/Zn-SOD and Mn-SOD mRNA in grafted cucumber leaves increases. And no significant change of CAT activities was observed between grafted and own-root cucumbers under low temperature stress. Thus CAT is not the main factor that enhanced the cold resistance of grafted cucumber.3. Under low temperature stress, the relative expression of DHAR, GR mRNA were all higher in leaves of grafted cucumber than that of own-root cucumber, while no significant change of the relative expression of GalLDH, APX mRNA was observed between them. The content of AsA, GSH and the ratio of AsA/DHA, GSH/GSSG were all higher in grafted cucumber than that of own-root cucumber, while the content of DHA, GSSG and H2O2 were all lower. Which demonstrates the higher content of AsA,GSH in the grafted cucumber plants enhance their chilling tolerances. And the higher relative expression of DHAR, GR mRNA resulted in the higher activities of DHAR, GR in grafted cucumber leaves, which resulted in the higher content of ASA and GSH in grafted cucumber leaves.4. Under NaCl stress,the relative expression of Cu/Zn-SOD, Mn-SOD and CAT mRNA were all higher in leaves of grafted cucumber than that of own-root cucumber, and the activities of SOD, Cu/Zn-SOD, Mn-SOD and CAT in leaves of grafted cucumber were higher too. It could be concluded that the higher relative expression of Cu/Zn-SOD, Mn-SOD and CAT mRNA resulted in the higher activities of SOD, Cu/Zn-SOD, Mn-SOD and CAT in grafted cucumber leaves. With NaCl stress time prolonging, the relative expression of Cu/Zn-SOD, Mn-SOD and CAT mRNA both increased in grafted and own-root cucumber leaves, but the changes of the activities of Cu/Zn-SOD, Mn-SOD and CAT were not the same as that in mRNA, indicting that the activities of SOD and CAT were also regulated by other factors. The MDA content and electrolytic leakage in grafted cucumber plants was lower than that of in own-root cucumber plants, which demonstrates the grafted cucumber plants have higher scavenging metabolism that can decrease the oxidative damage and increase their salt tolerances.5. Under NaCl stress, the activities of GalLDH, DHAR, APX, GR were all higher in leaves of grafted cucumber than those of own-root cucumber. The relative expression of GalLDH, DHAR and APX mRNA were all higher in leaves of grafted cucumber than those of own-root cucumber, while no significant change of the relative expression of GR mRNA was observed between grafted and own-root cucumbers. The content of AsA, GSH and the ratio of AsA/DHA, GSH/GSSG in leaves of grafted were all higher than that of own-root cucumber, while the content of DHA, GSSG and H2O2 were lower. This demonstrates the higher content of AsA, GSH in the grafted cucumber plants enhance their salt tolerances. And the higher content of AsA and GSH in the grafted cucumber plants was resulted from the higher activities of GalLDH, APX, DHAR and GR. The higher activities of GalLDH, APX and DHAR was regulated by the higher relative expression of GalLDH, APX and DHAR mRNA. Under NaCl stress, the activities of GR were also regulated by other factors.更多还原