【作者】 孙军利；

【导师】 郁松林；

【作者基本信息】 石河子大学 ， 作物栽培学与耕作学， 2015， 博士

【摘要】 为了明确外源水杨酸(salicylic acid,SA)对高温胁迫下葡萄幼苗耐热性和膜脂过氧化以及抗坏血酸-谷胱甘(ascorbate-glutathione,As A-GSH)循环代谢的影响;探明不同底物、不同处理时间、不同金属离子及浓度以及不同腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)浓度对蛋白激酶活性的影响;探明高温和外源SA诱导的蛋白激酶类型。本试验以两年生克瑞森无核葡萄苗为试验材料,探讨了外源SA对高温胁迫下葡萄幼苗生理特性及其诱导的蛋白激酶性质。主要的研究内容和研究结果如下:1.高温胁迫下,葡萄幼苗叶片内丙二醛含量和电解质渗透率随着胁迫时间的延长而增加。喷施外源SA可以减少高温胁迫下丙二醛含量和电解质渗透率的增加量,特别是胁迫时间超过60min时,外源SA能够显著地降低丙二醛含量和电解质渗透率的增加量,从而有效保护了细胞膜的完整性;外源SA在高温胁迫下能够维持较高的超氧化物歧化酶(superoxidase,SOD)、过氧化物酶(peroxidase,POD)、过氧化氢酶(catalase,CAT)活性,并且随着胁迫时间的延长(>120min)外源SA可以显著提高SOD、POD、CAT等细胞保护酶活性的增加量,增强了清除细胞内活性氧和自由基的能力;葡萄幼苗叶片的过氧化氢含量和超氧阴离子产生速率随着胁迫时间的延长而增加,叶片喷施外源SA能够显著减少过氧化氢含量和超氧阴离子产生速率,降低了膜脂过氧化程度,提高了葡萄幼苗的耐热性。2.高温胁迫条件下,葡萄幼苗叶片内的游离脯氨酸含量随着胁迫时间的延长而增加,可溶性蛋白含量随着胁迫时间的延长出现先增加后降低的变化趋势,在胁迫60 min时可溶性蛋白含量增加到最大。高温胁迫和外源SA均可以诱导刺激葡萄叶片蛋白激酶的活性,蛋白酶活性也出现了随着胁迫时间的延长先增加后降低的变化趋势,且蛋白激酶活性的变化与可溶性蛋白含量变化具有高度的一致性,即胁迫60 min时可溶性蛋白含量增加到最大时,蛋白激酶活性也同时增高到最大值。3.高温胁迫下,葡萄幼苗叶片内抗坏血酸(ascorbate,As A)在胁迫初期下降,高温抑制了As A的合成速度,As A含量有所下降。随后可能由于是葡萄对高温的适应性反应,As A含量升高,胁迫时长达到120min后As A含量又显著降低,其可能原因是长时间的高温加剧了活性氧代谢失衡,产生大量的自由基,As A作为抗氧化剂参与了自由基的猝灭,因此其含量降低。随着胁迫时间延长增加,外源SA可以促进还原型谷胱甘肽(glutathione,GSH)含量的积累,降低氧化型谷胱甘肽(oxidativeglutathione,GSSG)的含量。高温胁迫下喷施150μmol/LSA可以促进葡萄幼苗As A-GSH循环系统中As A、GSH含量的积累量,外源SA可以维持较高的As A-GSH循环系统中抗坏血酸过氧化物酶(ascorbic acid peroxidase,APX)、脱氢抗坏血酸还原酶(dehydroascorbate reductase,DHAR)、单脱氢抗坏血酸还原酶(monodehydroascorbate reductase,MDHAR)、谷胱甘肽还原酶(glutathionereductase,GR)活性,降低较长时间高温胁迫(>240min)下脱氢抗坏血酸(DHA)的含量,促进As A-GSH循环的快速有效的运转,降低植株的氧化伤害,缓解高温胁迫对葡萄幼苗的伤害作用。4.高温和外源SA诱导的葡萄幼苗叶片中存在着能以髓鞘碱性蛋白(myelin basic protein,MBP)为底物,促进蛋白质的磷酸化反应的蛋白激酶;建立了葡萄高温胁迫下蛋白质磷酸化反应实验体系:以MBP为磷酸化底物,反应时间以10min为最好;ATP适宜浓度为50μmol/L时,Mg2+对于激活SA预处理和高温胁迫诱导产生的蛋白激酶活性发挥具有重要作用,其浓度并非越高越好,但其适宜的浓度需要限定在一定范围,蛋白激酶活性会随着Mg2+浓度过大反而下降,最佳的Mg2+浓度以5.0mmol/L为宜。5.利用生物化学手段,采用专一作用底物MBP,利用蛋白激酶抑制剂W7和PD98059以及专一性蛋白磷酸酶YOP及其抑制剂Na3VO4,对高温胁迫和外源SA诱导的蛋白激酶性质进行了探索,初步推断出高温胁迫和外源SA处理条件下都可以诱导以MBP为磷酸化底物的蛋白激酶,且这两者诱导的激酶同属MAPK类型。更多还原

【Abstract】 This trail was in order to define the influence of the exogenous SA on grape seedlings heat resistanceand membrane lipid peroxidation under high temperature stress(HTS) and the influence of(ascorbateglutathione, As A-GSH) circulation metabolism, find out the influence of different substrates, differentprocessing time, different concentration of different metal ions and different concentration of ATP onprotein kinase activity, certain the types of protein kinase induced by high temperature and exogenoussalicylic acid. In this trail, two-year-old vitis vinifera L. Crinson seedless grape young plants were chosento explore the effects of exogenous SA on HTS by studying the physiological characteristics and thecharacter of protein kinase induced by HTS and exogenous SA. Got the following results:1.The contents of malondialdehyde(MDA) and electrolyte permeability of grape leaves increased withthe extension of time under HTS, the augmenter of contents of MDA and the electrolyte permeability canbe increased by spraying exogenous SA on grapes under HTS, especially HTS for more than 60 min, theaugmenter of MDA contents and electrolyte permeability could be significantly increased by exogenous SA,thus effectively protecting the integrity of the cell membrane; It could maintain higher activity ofsuperoxidase(SOD), peroxidase(POD), catalase(CAT) by spraying exogenous SA under HTS, and longerduration of stress(> 120 min) can significantly improve the augmenter of activity of SOD, POD, CAT,enhances the ability of removing active oxygen and free radicals in cells; the content of hydrogen peroxideand superoxide anion producing rate of grape leaves increased with the extension of time. It wassignificantly reduced by spraying exogenous SA on the grape leaves under high temperature, so the level ofmembrane lipid peroxidation was reduced, the heat resistance of grape seedlings was increased.2.The contents of free proline in grape leaves increased with the extension of time under HTS, thecontent of soluble protein with the extension of stress time appears to increase the change trend of lower,after 60 min in stress when the content of soluble protein increased to the maximum. High temperaturestress and exogenous salicylic acid can induce stimulus the activity of protein kinase of grape leaves, theactivity of protein kinase increased at first,then decreased with the stress change trend, and the change ofprotein kinase activity and soluble protein content changes with high consistency, namely stress 60 minsoluble protein content increased to the maximum, protein kinase activity has increased to the maximum atthe same time.3. The synthesis of ascorbate(As A) speed was inhibited at the beginning under high temperature,thecontents of As A in grape leaves was declined under HTS. That might be due to adaptability to hightemperature reaction of grape leaves, the As A contents increased later. the stress time over 120 min,theAs A contents decreased significantly. The possible reason was that long time high temperature sharplychanged the active oxygen metabolism imbalance, produced a large number of free radicals, As A asantioxidants in the quenching free radicals, so its content was lower. With the increase of prolonged stress,exogenous SA could induce accumulation of glutathione(GSH) content, reduce the content of oxidativeglutathione(GSSG). Spraying under high temperature stress with 150 μmol/L SA could promote As A andGSH in the circulatory system of As A and GSH content accumulation, and exogenous SA could maintainhigher As A and GSH in the circulatory system ascorbic acid peroxidase(APX), dehydroascorbate reductase(DHAR), monodehydroascorbate reductase(MDHAR), glutathione reductase(GR) activity, reduced theHTS for a long time(> 240 min) dehydrogenation under the contents of ascorbic acid(DHA), promoted therapid effective operation of As A and GSH cycle, reduce the oxidative damage of plants, alleviated injury ofgrape seedlings under high temperature.4.The high temperature and exogenous SA could induce a kind of protein kinase that could exist in themyelin basic protein(MBP) as substrates, promoted protein kinase protein phosphorylation reaction;established experimental conditions to protein phosphorylation under HTS and exogenous SA treatment,heat treatment, and cross processing of protein phosphorylation reaction experiment system wasphosphorylated substrates with MBP, reaction time was best for 10 min; ATP suitable concentration was50μmol/L. The Mg2+ for activation pretreatment and HTS induced protein kinase activity played animportant role, and the concentration was not the higher the better. But the appropriate concentration needsto be qualified in a certain range, protein kinase activity declined with Mg2+ concentration was too high, thebest concentration of Mg2+ tendency at 5.0mmol/ L was advisable.5.Using the biochemical method, we explored the nature of the protein kinase induced by HTS andexogenous SA by using the single substrate MBP, protein kinase inhibitors W7 and PD98059, specificity ofprotein phosphatase YOP and its inhibitor Na3VO4. And it preliminarily deduced that a kind of proteinkinase could be induced by exogenous SA treatment under HTS; phosphorylation reaction took MBP as thesubstrate, and both the induction of MAPK kinase were homogeneous type.更多还原