特定乙酰度壳寡糖诱导小麦抗盐作用及其机理研究

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	特定乙酰度壳寡糖诱导小麦抗盐作用及其机理研究
	邹平
学位类型	博士
导师	李鹏程
	2015-05-22
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋药物学
关键词	特定乙酰度壳寡糖小麦抗盐活性抗盐机理基因表达
摘要	中国盐渍土面积广阔，类型繁多，据统计，我国现代盐渍土面积约为3693万公顷，残余盐渍土面积约为4487万公顷，潜在盐渍土面积约为1733万公顷，各类盐渍土面积总计约为1亿公顷，占全国可利用土地的13.3%，严重制约了我国农业的可持续发展。且我国人口众多，粮食短缺，提高盐渍土中种植作物的产量是合理开发利用盐渍土的研究重点和热点，对我国粮食安全以及社会、经济发展都具有重大的意义。目前提高盐渍土作物产量的方法包括选育耐盐作物品种、通过基因工程改造作物以及外源物质的应用，其中外源物质的使用无疑是提高盐渍土作物产量最为有效而简单的方式。壳聚糖及其衍生物能够诱导植物产生广谱抗性，提高植物抗病性，缓解高盐、低温和干旱等逆境胁迫对植物的伤害，因此壳聚糖及其衍生物作为一种外源物质在农业生长中具有十分广阔的应用前景。分子量和乙酰度（DA）是影响壳聚糖及其衍生物诱抗活性的重要因素，然而目前对壳聚糖及其衍生物诱抗活性的研究均未考虑这两者对其活性的影响，造成了研究结果的不一致，大大阻碍了壳聚糖及其衍生物在农业生产中的应用。基于此，本论文首先研究了不同分子量及不同乙酰度壳聚糖诱导小麦抗盐作用，确定了诱抗活性最强的壳聚糖的分子量及乙酰度；然后进一步探讨了其诱导小麦抗盐作用的机理。本论文主要取得了以下研究成果： 1. 壳聚糖分子量对其诱导小麦抗盐作用具有重要影响，随着壳聚糖分子量的降低，其诱导小麦抗盐活性增强，其中1300 Da壳寡糖诱导小麦抗盐活性最强。结果显示壳寡糖能够降低100 mmol/LNaCl胁迫下小麦叶片丙二醛含量，减少细胞膜遭受的氧化损伤；提高小麦体内可溶性糖及可溶性蛋白含量，增强其在盐胁迫下的渗透调节能力；减缓盐胁迫下小麦叶片叶绿素的降解，保证光合作用的进行；提高小麦体内超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）的活性，清除小麦体内过量的活性氧，减少盐胁迫对细胞器及细胞膜的氧化损伤，从而提高小麦的抗盐性。 2. 在确定了诱导小麦抗盐作用最佳壳寡糖分子量之后，以此分子量为基础，制备了不同乙酰度壳寡糖，并研究了其诱导小麦抗盐作用。结果发现壳寡糖乙酰度对其诱抗活性具有重要影响，不同乙酰度壳寡糖诱导小麦抗盐活性表现为DA50%＞DA68%＞DA29%＞DA2%。实验结果表明，壳寡糖能降低小麦体内丙二醛含量，增加可溶性糖、可溶性蛋白以及脯氨酸等渗透调剂物质的含量，保护细胞膜结构并提高细胞渗透调节能力；增强小麦体内SOD、POD以及CAT活性，减少盐胁迫导致的氧化损伤对小麦幼苗的伤害。盐胁迫下，壳寡糖处理能增加小麦叶片叶绿素的含量；加大气孔导度（gs）来增加CO2摄取从而提高光合速率（Pn）；并且能够提高小麦叶绿素荧光参数qP值，降低NPQ值来增加小麦吸收的光能用于电子传递的份额，减少热耗散，从而保证实际光化学效率。 3. 确定了诱导小麦抗盐作用最佳的壳寡糖分子量及乙酰度后，本文研究了特定分子量及特定乙酰度的壳寡糖（1300 Da，DA50%）诱导小麦抗盐作用的机理。结果发现：（1）1300 Da，DA50%壳寡糖通过调节小麦细胞内离子转运将过量Na+转运至细胞外或者液泡内，来维持植物细胞内较高的K+含量和较低的Na+/K+，从而保证光化学反应中心的电子传递，缓解气孔关闭，促进光合作用。（2）壳寡糖通过增强植物体内抗氧化酶活性及提高抗氧化剂含量，来清除植物体内过量活性氧，减轻其对植物膜系统包括叶绿体类囊体膜的氧化损伤，保证植物光合器的完整，从而促进光合作用，提高植物抗盐性。 4. 小麦盐胁迫相关抗性基因的表达结果表明，1300Da，DA50%壳寡糖处理能够诱导小麦SOS和NHX基因的高效表达，协调盐胁迫下小麦幼苗体内离子转运，减轻高浓度盐离子对植物的伤害；壳寡糖还能诱导小麦抗氧化酶基因SOD、POD、CAT、APX、DHAR和GR表达，各种抗氧化酶基因协调作用，增强植物清除活性氧的能力，减少盐胁迫对植物的氧化损伤，提高植物抗盐性。在本论文中，壳寡糖诱导小麦抗盐活性与其分子量和乙酰度密切相关，壳寡糖乙酰基可能在其与植物细胞膜的识别与结合过程中发挥重要作用。其中，分子量为1300 Da，DA50%壳寡糖具有最强的诱导小麦抗盐活性，本研究为新型抗盐制剂的研发奠定基础。
其他摘要	China has a vast area and various types of saline soil. According to statistics, there are about 36.93 million hectares modern saline soil, 44.87 million hectares residual saline soil and 17.33 million hectares potential saline soil in China. All kinds of saline soils have a total of about 100 million hectares, accounting for 13.3% of the available land which severely impedes the agricultural development of our country. With the rising population and food shortage, improving the output of crops in saline soil is the emphases and hotspot of the study on the reasonable development and utilization of saline soil and of importance in promoting food security, social and economic development. The current methods to improve crop yield in saline soil include breeding salt-tolerant crops, genetically engineered crops and the application of exogenous substances. The application of exogenous substances is undoubtedly the most effective and simple way to improve crop yield in saline soil. Chitosan and its derivatives can induce broad-spectrum resistance, enhance plant resistance, inhibit plant pathogenic microbial growth, and alleviate the damage of salt stress, low temperature stress and drought stress. The chitosan and its derivatives have a broad application prospects in agricultural. The molecular weight (Mw) and degrees of acetylation (DA) are the most important parameter influencing the chitosans’ various properties. The effectiveness of chitosan in various applications appears to be dependent on the Mw and DA. However, the current researches are not considering the influence of the Mw and DA on its activity, causing the inconsistent results which greatly hinder the application of chitosan and its derivatives in agricultural production. Hence, this paper studied the effect of chitosan with different Mw and DA on wheat under salt stress and studied the mechanism of induced resistance of wheat to salt stress. In this paper, the main results are as follows: 1. Chitosan with different Mw could reduce the content of malondialdehyde (MDA) to protect the cell membranes from oxidative damage, improve the contents of soluble sugar and soluble protein in wheat to enhance the osmotic adjustment ability of plants, alleviate the degradation of chlorophyll to ensure the photosynthesis of plant.What’s more, chitosan with different Mw could enhance the antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities to scaveng the excess reactive oxygen species, reduce salt stress oxidative damage of cell membrane and organelles, thereby improve the salt resistance of wheat to protect wheat from oxidative damage and to improve the salt tolerance of wheat. And with the reduction of the Mw, the resistance activity of chitosan to salt stress increases. The oligochitosan with 1300 Da has the strongest activity of inducing resistance to salt stress. 2. In determining the molecules, oligochitosans with different DAs were preparated. The results showed that treatment with exogenous oligochitosans with different DAs could decrease the concentration of MDA, increase the contents of soluble sugar, soluble protein and proline which protect cell membrane structure and improve the osmotic regulation ability. And the oligochitosans with different DAs could enhance the activities of SOD, POD and CAT to reduce the oxidative damage caused by salt stress on wheat seedlings. Under salt stress, application of oligochitosans with different DAs could increase chlorophyll content of wheat leaves, increase stomatal conductance (gs) to increase intake of CO2 in order to improve photosynthetic rate (Pn), improve the qP value and reduce the NPQ value to increase the absorption of light energy to the electronic transfer and reduce the heat dissipation, thus ensure the actual photochemical efficiency ФPSⅡ. The resistance to salt stress of oligochitosans with different DAs perform as DA50 % > DA68 % > DA29 % > DA2 % which suggest that DA has important influence on its resistance activity. 3. The physiological mechanisms of exogenous oligochitosan with certain DA on wheat tolerance to salt stress were processed. And the results showed that: (1) Oligochitosan with DA 50% could adjust the ion transport in wheat cells and maintain a higher K+ content and lower Na+/K+ in plant cells to ensure the electron transfer, photosynthesis enzyme activity and alleviate the stomatal closure, promote the photosynthesis. (2) Oligochitosan with DA 50% could increase the antioxidant enzyme activity and the antioxidant content to alleviate the oxidative damage to membrane system including plant chloroplast thylakoid membrane and to ensure the integrity of plant photosynthetic apparatus, so as promote the photosynthesis and improve salt resistance of plant. 4. The mRNA expression level examination of several salt stress response genes suggested that oligochitosan with DA 50% can effectively regulate the expression of SOS and NHX genes in wheat seedlings under salt stress to reduce the damage of salt ions with high concentration. Furthermore, oligochitosan with DA 50% can regulate the expression of SOD, POD, CAT, APX, DHAR and GR genes to enhance the ability of scavenging active oxygen, reduce oxidative damage to plants and improve salt resistance of plant. In this paper, the observed elicitor activity of oligochitosan with DA 50% and the other DAs may be explained by a mechanism dependent on specific receptors. The interaction between oligochitosan and plant is dependent on specific recognition of the N-acetyl moieties, which suggest that N-acetyl plays an important role in inducing its activation. Oligochitosans with different DAs may form various structures to interact with elicitor receptor on plant cell membrane because of their strict structural requirement. However, the mechanisms of recognition of elicitors such as COS and signal transduction of plant needed to be further studied.
学科领域	海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/23273
专题	海洋生物技术研发中心
作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	邹平. 特定乙酰度壳寡糖诱导小麦抗盐作用及其机理研究[D]. 北京. 中国科学院大学,2015.

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