【作者】 李婷婷；

【导师】 李春喜；

【作者基本信息】 河南师范大学 ， 植物学， 2012， 硕士

【摘要】 低温是影响作物生长发育的重要环境因子之一，作物如何应对低温一直是世界性的研究课题。本文采用溶液培养法，选用两种不同冬春性的小麦品种（弱春性品种偃展4110，半冬性品种周麦18）作为试验材料，研究了不同浓度外源一氧化氮（NO）供体硝普钠（SNP）对4°C低温胁迫0h、24h、48h和96h，两种小麦幼苗的相对含水量（RWC）、叶绿素、可溶性糖、果聚糖等的含量以及果聚糖合成酶（FBEs）活性的影响，并应用荧光定量PCR（qRT-PCR）方法，探索两种小麦幼叶中FBEs基因表达的动态变化趋势。力求通过在低温胁迫下NO对小麦果聚糖合成代谢的生理及分子机理研究，为缓解小麦低温冷害研究提供一定的理论依据，为我国的粮食安全和食品安全做出一定的努力。本研究的主要结果如下：1.低温胁迫下，两种小麦幼叶中的可溶性糖含量、叶绿素a／b值均表现不同程度的上升，叶绿素a和b、类胡萝卜素含量呈现下降趋势，周麦18中RWC明显下降。外源NO能够诱导低温胁迫下两种小麦幼叶果糖、葡萄糖、低聚果糖（DP3）、可溶性总糖、叶绿素a和b以及类胡萝卜素的含量，使周麦18中蔗糖和高聚果糖（DP≥4）的含量出现一定的上升，并降低两种小麦幼叶的RWC，从而保证幼苗的正常生长。2.低温胁迫下两种小麦幼叶中果聚糖（DP3和DP≥4）含量均有一定程度的上升。低浓度(0.075mmol·L^-1)NO能够诱导两种小麦中果聚糖含量；而高浓度(0.125mmol·L^-1)NO也能够显著诱导周麦18中果聚糖含量，却对偃展4110果聚糖含量表现出一定的抑制效应。说明适宜浓度的外源NO能够诱导低温胁迫下两种小麦幼叶中果聚糖含量，以抵抗外界低温环境，从而减轻低温损伤。3.低温胁迫下两种小麦幼叶中果聚糖：果聚糖－1－果糖基转移酶（1-FFT）活性均保持在很低的水平，显著低于FS（蔗糖：蔗糖－1－果糖基转移酶（1-SST）和蔗糖：果聚糖－6－果糖基转移酶（6-SFT））酶活性。高浓度NO大大诱导周麦18中FFT活性，以及偃展4110中FS活性，说明半冬性小麦品种的FFT酶活性以及弱春性小麦品种的FS酶活性受到高浓度NO的诱导，从而促使幼苗叶片中果聚糖的大量积累以抵制低温胁迫所造成的伤害。4.低温胁迫下两种小麦幼叶中FBEs基因的表达在转录水平得到提高。外源NO的施加也能够在此水平上对FBEs基因的表达量进行调节，主要表现在：除了周麦18中1-SST和6-SFT基因表达量受高浓度NO抑制外，1-FFT基因以及偃展4110中三种FBEs基因表达量均受到外源NO的诱导。说明外源NO能够在转录水平上对FBEs基因的表达量进行调节，适宜浓度的外源NO能够诱导低温胁迫下两种小麦幼叶中FBEs基因的表达。5.比较两个小麦品种，半冬性品种周麦18中，外源NO一方面诱导蔗糖的合成，为果聚糖的合成提供丰富的底物，另一方面诱导FFT基因大量表达，使FFT酶活性大大增加，高聚果糖得以在植株体内大量积累，从而增强植株对外界不良环境的抵抗力；而在弱春性品种中，蔗糖的合成却并未发现受外源NO的诱导，体内果聚糖自行合成的能力较差，却可以明显受低温诱导，且均表现与蔗糖水平显著相关。由此推断，果聚糖的合成可能取决于植物体内高水平的蔗糖浓度。目前关于外源NO通过诱导小麦体内果聚糖积累从而缓解植株冷害损伤的报道较少。本研究结论证明，施加适宜浓度的外源NO，可以通过诱导植株体内FBEs基因的表达，从而促进FBEs酶的活性，使果聚糖在幼叶中积累大量，保护幼苗少受或不受低温胁迫的伤害，提高冬小麦的抗寒性。更多还原

【Abstract】 Low temperature is one of the most important environmental factors affecting plant growth anddevelopment, and the adaptability of plants to low temperature has been a worldwide research project. Thisstudy was to investigate the effect of exogenous nitric oxide （NO） on fructan anabolism and fructanbiosynthesic enzymes （FBEs） expression in seedlings leaves of two wheat （Triticum aestivum L.） cultivars,semi-winter wheat Zhoumai18and weak spring wheat Yanzhan4110, under4°C. The seedlings of twowheat cultivars were subjected to different concentrations of sodium nitroprussiate （SNP） for0,24,48, and96h. By the research on the molecular mechanism of NO effect on wheat resisting low temperature stress,we strive hard to make certain efforts to resolve this important issue of wheat chilling injuries, providing atheoretical basis for food security. The main findings are as follows:1. To chilling stress, the soluble sugar content and chlorophyll a/b ratio showed the certain extent risein seedlings leaves of the two wheat cultivars, chlorophyll a, b and carotenoid contents showed a downwardtrend, and RWC in Zhoumai18declined significantly. Exogenous NO could induce fructose, glucose, lowDP fructan, total soluble sugar, chlorophyll a, b and carotenoids contents in the two wheat cultivars, andsucrose and high DP fructan contents in Zhoumai18, and brought RWC down in the two wheat cultivars.2. To chilling stress, fructans （including DP3and DP≥4） content have a certain degree of increase inseedlings leaves of the two wheat cultivars. Low concentration (0.075mmol·L^-1) of NO could inducefructans content in the two wheat cultivars; high concentration (0.125mmol·L^-1) of NO could also inducefructans content significantly in Zhoumai18, but showed some toxic effects to Yanzhan4110. It could bedemonstrated that the appropriate concentration of exogenous NO could induce fructans content in the twowheat cultivars under low temperature stress, resisting external low-temperature environment to reduce lowtemperature injury.3. To chilling stress, fructan: fructan1-fructosyltransferase （1-FFT, EC:2.4.1.100） activity remained ata significantly lower level than FS （including sucrose: sucrose1-fructosyltransferase （1-SST, EC:2.4.1.99）and sucrose: fructan6-fructosyltransferase （6-SFT, EC:2.4.1.10）） activity in seedlings leaves of the twowheat cultivars. High concentration of NO significantly induced FFT activity in Zhoumai18and FS activity in Yanzhan4110. It could be demonstrated that FFT activity in semi-winter wheat cultivar and FSactivity in weak spring wheat cultivar could be induced by high concentration of NO, contributing tofructan accumulation in seedling leaves to reduce low temperature injury.4. To chilling stress, FBEs gene expression at the transcriptional level was improved in seedlingsleaves of the two wheat cultivars. Exogenous NO could regulate the gene expression at this level infollowing ways: except that1-SST and6-SFT gene expression in Zhoumai18were inhibited by highconcentration of NO,1-FFT and Yanzhan4110’ three FBEs gene expression were all induced by exogenousNO. It could be demonstrated that exogenous NO could take part in regulate FBEs gene expression at thetranscriptional level, and the appropriate concentration of exogenous NO could induce FBEs geneexpression in the two wheat cultivars under low temperature stress.5. In Zhoumai18, exogenous NO induced the synthesis of sucrose which provided rich substrate forfructan synthesis, and induced FFT gene expression to promote FFT enzyme activity greatly, so that highDP fructans accumulated abundantly in plant to enhance the resistance to external adverse environment.However in Yanzhan4110, the ability of fructans generated internally was poor. Sucrose was not inducedby exogenous NO but low temperature, and was significantly associated with fructans content. Byinference, the synthesis of fructans may depend on high concentration of sucrose in wheat.There has been less reports on exogenous NO reduces chilling injuries by inducing the accumulationof fructans in wheat. It can be concluded from this study that the appropriate concentrations of exogenousNO will induce FBEs gene expression in plants to promote the activities of FBEs. Thus large amounts offructans are accumulated in young leaves, to make seedlings less susceptible to low temperature stress, andto improve the cold resistance of winter wheat.更多还原