【作者】 张凤芝；

【导师】 苏国兴；

【作者基本信息】 苏州大学 ， 植物学， 2011， 硕士

【摘要】 精胺(Spm)、亚精胺(Spd)和它们的二胺前体腐胺(Put)是小分子脂肪族多聚阳离子,广泛存在于生物有机体中,在植物中介导了多种生理生化过程。一氧化氮(NO)是一种高活性生理活性物质,作为信号分子在植物根系等生长发育过程中起重要作用。我们前期的工作表明,多胺和NO对植物种子萌发及幼苗的生长都有促进作用,对植物侧根发生也有促进作用,且多胺可以诱导植物多种组织产生NO。那么,经多胺和NO处理后,在促进种子萌发过程中,种子贮藏物质和相应水解酶活性有怎样的变化?国内外鲜见相关文献报道。在本研究中,我们选用正常莴苣(Lactuca sativa L.)种子(挂丝红)为材料,研究多胺和一氧化氮促进莴苣种子萌发时的生理生化变化。结果如下:低浓度的外源多胺对莴苣种子发萌都有一定的促进作用,其中以0.5 mM Spd效果最佳,其次是Spm,Put作用较小。时间进程曲线研究表明,48 h后多胺这种促早萌作用逐渐消失。0-0.1 mM SNP可以明显促进莴苣种子的萌发,0.1 mM效果最佳,SNP浓度大于0.2 mM时对莴苣种子萌发有抑制作用。时间进程曲线研究表明,在种子萌发前36 h, 0.1 mM SNP对种子萌发的促进作用最为显著,48 h后多胺这种促早萌作用逐渐消失。种子内含物质变化的时间进程曲线发现,随着莴苣种子的萌发,蛋白质、粗脂肪和淀粉含量逐渐下降,而可溶性糖含量逐渐增加。用0.5 mM多胺浸种处理后,与对照相比,蛋白质、粗脂肪和淀粉含量下降更显著,但各种多胺处理间差异不大,仅0.5 mM Spd对可溶性糖的增加和淀粉含量的下降作用效果更好。多胺生物合成抑制CHA和MGBG处理,对种子内的蛋白质和淀粉含量的下降均有不同程度的延缓作用,但粗脂肪含量的下降受MGBG的促进。用SNP浸种可明显降低淀粉和增加可溶性糖含量,0.05 mM SNP就有显著的作用效果。时间进程曲线研究表明,种子内的蛋白质、粗脂肪和淀粉含量逐渐下降,而可溶性糖含量逐渐增加,经0.1 mM SNP处理后,蛋白质、粗脂肪含量进一步降低,但与对照相比差异不大;与此不同的是,淀粉含量的下降和可溶性糖含量的增加更显著,处理24 h后,与对照相比差异就达显著性水平。用NO生物合成抑制1.0 mM钨酸钠和0.2 mML-NAME浸种,对种子内含物质含量变化呈现相似的影响。对种子蛋白质和粗脂肪含量的下降有一定的延缓作用,但量值与对照相比差异不大,而对淀粉降解的抑制作用非常明显,36 h后便观察到可溶性糖含量的显著下降。测定种子萌发时水解酶活性变化结果表明,α-淀粉酶、脂肪水解酶和硝酸还原酶活性随种子的萌发而逐渐增加,而β-淀粉酶活性在36 h时活性达最高峰后逐渐下降。多胺可显著促进α-淀粉酶和β-淀粉酶活性,最适浓度为0.5 mM,Spd作用最好;对脂肪水解酶活性也有不同的促进作用,但作用浓度需大于0.5 mM时才有效果;而对硝酸还原酶活性则呈现出一定的抑制作用。此外,还发现不同种类的外源多胺对水解酶活性的作用有差异,Spd和Put对α-淀粉酶、β-淀粉酶和脂肪水解酶活性的促进作用大于Spm。多胺生物合成抑制剂CHA和MGBG处理结果表明,两者都可明显抑制α-淀粉酶和β-淀粉酶活性,CHA对β-淀粉酶活性的抑制作用更为显著,CHA的抑制效果好于MGBG。硝酸还原酶活性也受不同浓度的CHA和MGBG的强烈抑制。与CHA显著抑制脂肪水解酶活性不同,MGBG处理脂肪水解酶活性有受促进的趋势,但与对照相比差异不显著。SNP处理结果表明,种子的α-淀粉酶和β-淀粉酶活性受低浓度SNP的促进,而受高浓度SNP的抑制;硝酸还原酶活性也受到促进,0.1 mM SNP效果佳;大于0.1 mM SNP对脂肪水解酶活性才有显著的促进作用。时间进程曲线研究表明,α-淀粉酶、硝酸还原酶和脂肪水解酶活性明显受0.1 mM SNP处理的促进,且随种子的萌发而增加;β-淀粉酶活性在36 h时达最大值后逐渐下降,也明显受SNP处理的促进。经1.0 mM T-Na和0.2 mM L-NAME处理后,种子的硝酸还原酶和脂肪水解酶活性受到强烈抑制,处理12 h就有明显的作用效果;对α-淀粉酶活性抑制的作用时间有差异,T-Na在萌发前12 h就起作用,L-NAME在处理36 h后才表现强烈的抑制效果。对β-淀粉酶活性,L-NAME在处理24 h后就表现出强的抑制作用。上述结果暗示,多胺和NO是通过调节α-淀粉酶、β-淀粉酶活性、硝酸还原酶和脂肪水解酶活性,影响种子体内贮藏物质的降解,而影响莴苣种子萌发。其中,对淀粉的降解和可溶性糖的增加作用更为明显。更多还原

【Abstract】 Spermine (Spm), spermidine (Spd) and their diamine obligate precursor putrescine (Put) are micro molecular aliphatic polycations that are ubiquitous in all plant cells, participate in a wide range of physiological and biochemical processes. Nitric oxide (NO) is a physiologically reactive substance, as a plant cell signaling molecule, it play an important role in plant root growth and other physiological processes. Our previous results suggest that both polyamines and NO promote the seed germination and the development of lateral roots in lettuce, and that polyamines can induce NO production in plant tissues. However, there is no report about what changes the seed storage substances and corresponding hydrolases are in seed germination after treatments with exogenous polyamines and NO ? In this study we choose a normal breed of lettuce seed (Lactuca sativa L. cv. Guasihong) as an experimental material to investigate the role of polyamine and NO in the process of lettuce seed germination. The results are as follows:Treatments with exogenous polyamines at low concentrations promoted early germination of lettuce seed. Spm was more effective than Put, but less than Spd. The detection of time course of seed germination showed that the promoting effect of polyamines disappeared after 48 h. In addition, treatment with 0-0.1 mM SNP markedly promoted seed germination with optimal concentration at 0.1 mM, whereas 0.2 mM SNP failed to increase germination. The detection of time course of seed germination showed that the promoting effect of 0.1 mM SNP on the germination of lettuce seed was obvious in 36 h after treatment, and this effect disappeared in 48 h.The detection of reserve changes showed that the protein, crude fat and starch contents decreased and soluble sugar increased gradually with seed germination. In contrast with the control, the protein, crude fat and starch contents significantly declined after treatments with 0.5 mM polyamines. The differences between polyamine treatments were not observed and only 0.5 mM Spd had strong effects on the decline of starch content and the increment of soluble sugar. Treatments with polyamine synthetic inhibitors (MGBG and CHA) could retard the decreases of the protein and starch contents in various degrees, whereas the crude fat content was inhibited by MGBG.The imbibition with NO donor SNP could obviously reduce starch content and increase soluble content, and 0.05 mM SNP had significant effects. The detection of time course showed that the contents of the protein, crude fat and starch declined and the soluble sugar increased gradually. After treatments with 0.1 mM SNP, the protein and crude fat contents further decreased, but no differences occurred in contrast with the control. Conversely, only starch content decreased and the soluble sugar increased rapidly, the differences after treatments for 24 h were obvious as contrast with the control. The similar effects of the biosynthetic inhibitor of nitric oxide (1.0 mM tunsgate and 0.2 mM L-NAME) on seed reserves were observed. They both had a certain retarding effect on the decline of the protein and the crude fat contents though with no differences as contrast to the control, while the starch degradation was strongly inhibited and the soluble sugar content reduced only after treatment for 36 h.The determination of the activities of various hydrolases during seed germination showed that the activities ofα-amylase, nitrate reductase and lipase increased gradually with lettuce seed germination, andβ-amylase activity declined after its activity reached the peak at 36 h. The imbibition with exogenous polyamines at different concentrations could significantly enhancedα-amylase andβ-amylase activities with optimal concentration at 0.5 mM, and Spd was most effective. Lipase activity was also increased but the polyamine concentrations over 0.5 mM were needed, while nitrate reductase activity reduced. In addition, different kinds of polyamines showed different effects, the promoting effects of Spd and Put onα-amylase,β-amylase and lipase activities were more effective than Spm.In agreement with these results, treatments with the polyamine biosynthetic inhibitors, CHA and MGBG, could significantly inhibit the activities ofα-amylase andβ-amylase activities. The inhibitory effect of CHA onβ-amylase activities was more obvious and better than MGBG. Similarly nitrate reductase activity was also strongly retarded by CHA and MGBG at different concentrations. Unlike with CHA, MGBG imbibition could slightly promote lipase activity though with no difference as contrast with the control.The results caused by NO donor SNP treatment showed that the activities ofα-amylase andβ-amylase in lettuce seeds were promoted by low SNP, but inhibited by high SNP concentration. The nitrate reductase activity was also improved with optimal concentration at 0.1 mM SNP. The concentration exceeding 0.1 mM SNP exhibited a promotive effect on lipase activity. The detection of time course showed that the activities ofα-amylase, nitrate reductase and lipase increased with lettuce seed germination, and significantly enhanced by 0.1 mM SNP treatment.β-amylase activity declined after it reached the peak at 36 h and also improved by SNP.After treatments with NO biosynthetic inhibitor tunsgate and L-NAME, the nitrate reductase and lipase activities strongly reduced, the effects occurred only at 12 h after inhibitor treatments. There was difference of inhibitory effect ofα-amylase on acting time between tunsgate and L-NAME,. Tunsgate showed its inhibitory effect at 12 h after seed germination, while L-NAME behaved at 36 h.β-amylase activity decreased only at 24h after L-NAME treatment.Altogether, Polyamines and nitric oxide may regulate the germination of lettuce seeds through manipulating the activities ofα-amylase,β-amylase, nitrate reductase and lipase and affecting reserve degradation. Treatment with polyamine and nitric oxide mainly caused starch degradation and soluble sugar increment.更多还原