【作者】 黄爱霞；

【导师】 佘小平；

【作者基本信息】 陕西师范大学 ， 植物学， 2004， 硕士

【摘要】 在哺乳动物中，已证明一氧化氮参与了神经传递、血管舒张、平滑肌松弛等过程。尽管一氧化氮研究在植物方面开始较晚，但最近10年已经证明NO参与了许多重要的植物生理过程，如生长、发育、病原体防御反应、程序性细胞死亡和胁迫抗性等。前人曾证明NO参与IAA诱导的黄瓜下胚轴不定根发生，但其研究未涉及不定根发生过程中NO的时间和空间变化以及NO的来源等问题，本文以绿豆下胚轴为材料，从不同角度研究了NO在不定根发生中的作用以及IBA、NAA诱导不定根发生的机制，并对不定根发生过程中NO的来源进行了探讨。结果如下： 1．NO供体SNP可有效促进插条生根，其最适浓度为300μmol／L，浓度高时则具有伤害效应。SNP处理时间、苗龄及插条处理位置都影响生根效果。从5天龄幼苗子叶节下6cm处切取的插条，以300μmol／LSNP处理24小时生根效果最好。SNP与IBA或NAA混合处理比各自单独处理的生根效果好。NO清除剂c-PTIO和动物NOS抑制剂L-NAME单独或与IBA和NAA混合处理明显抑制插条生根，表明内源NO在不定根形成中可能起着重要作用，其来源与NOS有关。 2．利用NO特异的荧光探针对对照及IBA和NAA处理插条生根过程中内源NO的时空变化进行了检测。结果表明，无论对照还是IBA或NAA处理插条，伴随着根原基的发育，插条基部2mm区域NO荧光从无到有逐渐增强，36h主要分布于维管组织之间不定根发生区域，48h后则主要分布于根原基前端分生组织中。非生根区域无NO荧光。对照与IBA或NAA处理插条基部2mm-5mm区域NO产生及分布则完全不同，在实验期间对照插条该区域无NO产生，而IBA或NAA处理插条该区域NO产生、分布及随时间变化趋势与对照插条基部2mm区域基本一致。NO清除剂c-PTIO抑制所有处理插条的根原基形成。这些结果显示，内源NO在不定根形成中起重要作用，而且IBA和NAA诱导的不定根发生也是由NO介导的。 3．NADPH—黄递酶为NOS标志酶，本文以该酶组织化学法对不定根发生过程中的类似动物NOS活性进行了检测。结果表明，0—60h，无论对照还是IBA或NAA处理插条，伴随着根原基的发生和发育，插条基部2mm区域NADPH—黄递酶活性逐渐增强，且集中分布在维管组织之间根原基发生区域及正在形成的根原基中，其它非生根区域基本无阳性反应。在根原基中较强阳性也集中分布于分生组织中，这与NO的分布一致。与对照相比，相同时间IBA或NAA处理的插条NADPH一黄递酶阳性反应略强。对照与工BA或NAA处理插条基部Zlnm一Slnln区域NADPH一黄递酶分布也完全不同，对照插条该区域NADPH一黄递酶活性较弱，而工BA或NAA处理插条该区域NADPH一黄递酶活性、分布及随时间变化趋势与插条基部Zllun区域基本一致。L一NAME既抑制所有处理插条的根原基的形成，也降低NO荧光和NADPH一黄递酶活性。以上结果表明植物中也存在类似于动物的NOS，而且该酶以及它催化产生的NO在不定根发生过程中起着重要作用。更多还原

【Abstract】 Several roles have been described for NO in animals, where it takes part in neurotransmission, vasorelaxation, smooth muscle relaxation, and immunoregulation of pathophysiological processes. Even though NO research in plants is not as advanced as in animals, in the last decade No was proved to participate in many key plant physiological process such as growth, development, pathogen defense reaction, programmed cell death, and stress tolerance. Though NO mediation of the adventitious rooting process induced by auxins had been demonstrated, the time and space changes of NO and the source of NO accompanied adventitious root generation and development are unknown. In this study, mung bean hypocotyls were used as materials, and the role and the source of NO of the cuttings treated by H2^O, IBA or NAA during the adventitious root formation were studied from several aspects. The results were as follows:1. The cuttings were treated with NO-associated reagent. The results showed that NO donor-SNP significantly enhance the adventitious rooting. 300μmol/L SNP proved an optimal concentration for the initiation of organogenesis. The treatment time of SNP, the ages and the position of the cuttings also influenced the rooting. The hypocotyls of 5 days old seedlings was excised 6 cm below the cotyledonary node and then treated with 300μmol/L SNP by 24h had the optimal rooting effect. SNP obviously enhanced the effects of IBA or NAA in stimulating rooting. C-PTIO, NO specific scavenger, or NOS inhibitor L-NAME treted alone or plus IBA or NAA resulted in an inhibitory effect. It’s indicated that endogenous NO appears to play a key role in the generation and development of adventitious roots, and the production of NO in this process maybe catalyzed by NOS.2. The temporal and spatial change of NO of the cuttings treated by H2O, IBA or NAA during the generation and the development of adventitious roots of mung bean hypocotyl cuttings were detected by NO-sensitive fluorescence probe DAF-2DA. Theresults indicated that, with the development of root primordium, the fluorescence of NO appeared and increased gradually in 2mm region of hypocotyl basal part in all treatments. The green fluorescence of NO was detected in the region between the vascular tissues of the hypocotyls at 36h after cutting. The cytosol of DAF-2DA-positive cells stained uniformly for NO. With the development and elongation of root primordium, the number of DAF-2DA-positive cells increased gradually, and the root apical meristem cells show intense fluorescence. No NO was observed in non-rooting areas. In 2mm-5mm region of hypocotyl basal part, the generation and distribution of NO of the cuttings treated by IB A or NAA were different with that of treated by H2O. No NO was detected in control. The generation, distribution and change of NO of cuttings treated by IBA or NAA in this region were in accordance with that of in 2mm region of hypocotyl basal part. C-PTIO, the specific NO scavenger, suppressed the fluorescence and inhibited the formation of root primordial clearly. The results strongly argue that that endogenous NO appears to play a key role in the adventitious rooting process. It also suggested that NO could also mediate the IBA and NAA response during the rooting process in mung bean.3. NOS-like enzyme activity was detected during adventitious root formation by NADPH-diaphorase histochemical method. With the origination and development of adventitious roots, not in control but in the cuttings treated by IBA or NAA, the activity of NADPH-diaphorase and the number of cell expressing NADPH-diaphorase-positive increased rapidly in the forming root in 2mm region of hypocotyl basal part. The non-rooting areas haven’t positive cells. In accordance with the distribution of NO, intense staining mainly distributed in root meristem. The NADPH-diaphorase activity of the cuttings treated by IBA or NAA were more intense than the control in the same time. In 2mm-5mm region of hypocotyl basal part, the distribution of NADPH-diaphorase of the cuttings treated by IBA or更多还原