【作者】 刘瑞志；

【导师】 管华诗； 江晓路；

【作者基本信息】 中国海洋大学 ， 药物化学， 2009， 博士

【摘要】 本论文从植物生长与抵抗逆境的特性出发,利用外源性寡糖对其进行作用,通过检测植物生长指标和抗逆指标变化,来研究寡糖在植物促生长与诱导抗逆过程中的作用机制。一方面对褐藻寡糖在植物促生长与抗逆领域的作用机理进行表征,另一方面,也希望能够通过此研究获得具有优良生物功能活性的寡糖制剂,为农业生产抗灾减灾做出贡献。本论文首先选择三种来源(褐藻胶、果胶、壳聚糖)的不同分子量大小的九种寡糖片段进行研究。利用植物的促生长和抗逆指标进行筛选,获得了一种既具有明显的促生长作用,又具有抗逆作用的寡糖片段,为褐藻寡糖HZ3,经过质谱分析,其组分是以二、三糖为主的含有少量四糖和五糖的寡糖混合物。本研究第二部分是利用植物的植株、愈伤组织和悬浮细胞为材料,建立起寡糖促生长的综合性的验证方法。对植株分别选择了双子叶植物豌豆和单子叶植物玉米,利用寡糖浸种,分别考察在种子萌发与幼苗生长初期其体内蛋白酶、淀粉酶和激素含量的变化,结合种子与植株的生长指标,探讨寡糖的促生长作用机理。研究发现,寡糖对豌豆和玉米的促生长作用不同。对双子叶植物豌豆,以0.15%褐藻寡糖效果最好,第7d根和芽干重的增长率分别为79.2%和53.5%,是通过促进激素含量、蛋白酶和脂肪酶的活力来促进种子萌发和幼苗的生长;对单子叶植物玉米,以0.20%褐藻寡糖效果最好,第7d根和芽干重的增长率分别为140%和143.8%,是通过促进激素含量、脂肪酶、淀粉酶和蛋白酶共同作用来促进种子萌发和幼苗生长。通过对愈伤组织的诱导和继代培养的研究,发现,此褐藻寡糖具有激素的作用,在极低的浓度下能够诱导愈伤组织的产生,并能够在有2,4-D的培养基中促进愈伤组织的诱导和生长。对悬浮细胞的研究发现,0.03%寡糖能够明显增强细胞内的激素含量,从而对细胞的生长进行调控。本论文第三部分以植物在各种逆境下的生理指标的变化为检测目标,通过寡糖刺激作用于植物,探讨褐藻寡糖对植物抗逆的影响与作用机制。在低温逆境时,寡糖处理能迅速增强其抗逆酶类,对引起细胞损伤的物质进行清除,从而提高植物的抗逆能力。对植物的干旱逆境,通过寡糖作用,能够使其体内的抗旱指标明显增强,如ABA的含量升高显著,对干旱下植株的生长状态进行调控,降低干旱胁迫对植株的损伤,从而达到诱导抗旱的目的。对植物的抗病性能的提高,通过检测烟草花叶病毒和白粉病对植株的致病作用。对烟草花叶病毒,通过将寡糖作用于病毒与植株的侵染过程、体内复制阶段进行诱导抗病毒研究,发现褐藻寡糖可以明显提高植株的抗病毒能力,同时还能直接作用于病毒,在体外对其进行钝化,降低传染几率,达到抗病毒的效果。对抗白粉病研究,通过体外和体内两条途径对白粉病菌进行抗病能力的检测,发现褐藻寡糖不能抑制白粉病菌的生长,而是通过提高植株的体内抗逆酶类,达到抗病的效果,其对烟草白粉病的最佳防效为4d,比三唑酮的最佳防效减少了2d,缩短了白粉病的治疗时间,降低了烟草的损伤。对植物果实的抗病保鲜的研究,发现褐藻寡糖能够改变草莓的呼吸过程,减少水分散失,降低外界对其造成的损伤。0.20%处理组储藏9d后仍能保留一定的鲜度品质。因此褐藻寡糖对植物的抗逆机制是通过诱导作用实现的。诱导植物体内产生各种抗性物质来缓解逆境因素对植物造成的损伤,从而达到抗逆的目的。为了研究褐藻寡糖与细胞的结合机制与作用规律,利用激光共聚焦技术对标记的寡糖与烟草细胞进行结合,观察其动态结合过程,研究发现,褐藻寡糖能够与植物的细胞壁进行结合,又可以穿过细胞壁进入细胞内部与细胞膜进行结合。通过将蛋白酶以及蛋白变性剂SDS对膜蛋白进行处理后研究发现能够对寡糖的结合产生影响。表明褐藻寡糖的结合是与膜上的蛋白有关。通过封闭细胞膜上的钙离子通道,对其进行阻断后结合寡糖,研究发现,褐藻寡糖与细胞膜的结合与钙离子通道无关。更多还原

【Abstract】 A fragment of alginate-derived oligosaccharide (ADO) HZ3 was screened out from 9 oligosaccharides of different origins by comparing the parameters of growth promotion and stress resistance. This active oligosaccharide fragment was identified by ESI-MS method, with the polymerization degree between 2 and 5. The most content of the oligosaccharides mixture were trisaccharide and disaccharide.The growth promotion effects were determined from 3 aspects: plant (including the seed), callus and suspension cells. By soaking the seed with different concentration of HZ3 and then germination, the growth promotion of plants was measured by detecting the shoot and root length, dry weight, protease, lipase, amylases and growth hormones in germination period of pea and maize. The results showed that 0.15% HZ3 showed the best growth promotion effect of pea by enhancing the activity of protease and lipase and the growth hormones of IAA and GA. The 0.20% HZ3 could enhance the growth of maize by promoting the activity of amylase, lipase, protease and hormones of IAA and GA. By measuring the callus inducing rate, the HZ3 exhibited hormone effect and could induce tobacco callus formation. In the procedure of subculture of tobacco callus, the 0.03 % HZ3 showed the best growth promotion effect. In suspension cells subculture, the presence of HZ3 could promote the tobacco cells growth by increasing the content of IAA and GA in the suspension cells.The inducing resistance of the plants by HZ3 towards different adversities was investigated by detecting the damage parameters (MDA and electrolyte leakage), activities of defense enzymes, osmotic substances (including proline, soluble sugar) and some other parameters. The inducing low temperature resistance was testified by spraying the HZ3 solution on the surface of tobacco leaves and stored at 4℃. The presence of HZ3 could induce the changes of various parameters. The defense enzymes activities were enhanced in a short time which could reduce the content of MDA and electrolyte leakage of tobacco leaves. The most effective HZ3 concentration was determined as 0.20%. However, the presence of HZ3 can neither promote the activity of defense enzymes nor the damage of the leaves, suggesting that this concentration of HZ3 exhibited the activity of protecting tobacco leaves from chilling damage. The effects of HZ3 on tomato seedlings under drought stress were detected with the parameters of leaves damage, defense enzymes, stress inducing ABA, and the osmotic substance. The results indicated that 0.20% HZ3 showed the best drought resistance inducing effect which could reduce plant damage by increasing the defense enzymes and osmotic substances. With the increasing of ABA, the defense reactions of the tomato plant were increased. The disease resistance was measured in two aspects: anti-TMV and anti-powdery mildew. 0.20% HZ3 exhibited the best anti-TMV and anti-powdery mildew and could reduce the cure time of powdery mildew 2 days ahead of triazolone. The HZ3 also exhibited fresh keeping ability of strawberry.In the final area, the combination mechanism of HZ3 with tobacco cells was investigated by Confocal Laser Seanning Mieroseopy. The results showed that the HZ3 could combine with cell wall and membrance and the combination had the relationship with protein in membrance and no effect with Ca2+-CaM.This study showed that this alginate derived oligosaccharide HZ3 has potential for applications in plant growth promotion and adversities stress resistance in modern agricultures.更多还原