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新型壳聚糖衍生物的制备及对小麦幼苗抗旱作用研究
尹秀晶
Subtype硕士
Thesis Advisor刘松
2021-05-19
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name工程硕士
Degree Discipline生物工程
Keyword壳聚糖 氨基丁酸 衍生物制备 诱导抗旱 代谢组学
Abstract

      干旱已成为影响作物生长和产量的重要非生物胁迫。小麦是中国乃至全世界最重要的粮食作物之一,约70% 的小麦被种植在干旱、半干旱地区,如何提高小麦的抗旱能力从而提高其产量和品质已经成为国内外学者研究的热点之一。通过施用外源物质来增强作物抗旱能力是目前提高作物抗旱性的最为有效而简单的方式,因而研制安全、高效的抗旱剂显得尤为迫切。壳聚糖能够缓解干旱、盐等非生物逆境对植物的伤害,并能诱导植物产生广谱抗性,且具有无毒、可降解等优点,壳聚糖作为一种外源物质在农业生产中具有广阔的应用前景。
      壳聚糖通过功能化修饰往往会赋予不同的功能特性或具有新的生物活性。氨基丁酸是一种小分子非蛋白类氨基酸,具有提高植物抗逆性作用,在植物的逆境胁迫响应中起着重要作用。基于活性结构拼接的思路,本论文利用β和γ两种构型的氨基丁酸对壳聚糖进行修饰,制备了新型壳聚糖氨基丁酸衍生物,并研究了其对干旱胁迫下小麦幼苗的抗旱作用。采用代谢组学技术,研究了小麦幼苗受衍生物影响的代谢调控情况,初步探讨衍生物对小麦幼苗的诱导抗旱机理。本论文主要取得了以下研究成果:
      1.利用壳聚糖氨基与氨基丁酸羧基发生酰胺化反应,合成了两类共6种新型壳聚糖氨基丁酸衍生物,并通过核磁碳谱、核磁氢谱以及傅里叶变换红外光谱表征了衍生物的结构,取代度达15%以上。
      2.土培盆栽干旱和水培模拟干旱两种干旱模型均使小麦幼苗的生长发育受到明显抑制,苗高、苗鲜重、苗干重均显著下降。衍生物处理与干旱模型组相比,显著降低丙二醛含量和电解质渗出率,缓解了干旱对叶片细胞膜的损伤;提高渗透调节物质含量,增加了叶片相对含水量;提高了光合性能,改善幼苗的生长发育状况,有利于茎的伸长和生物量的积累,提高小麦幼苗的抗旱性。水培模拟干旱实验结果更稳定,且实验过程操作更加简便、可控性好。
      3.采用含20%聚乙二醇6000的营养液模拟干旱环境进行6种衍生物的诱导抗旱活性筛选研究。结果发现6种衍生物对小麦幼苗均具有诱导抗旱作用,抗旱活性表现为3K-B>9K-G>1K-B>9K-B>3K-G>1K-G,衍生物中氨基丁酸的构型及壳聚糖的分子量均能影响诱导抗旱活性,其中氨基丁酸构型的影响更为显著,含有β氨基丁酸的衍生物抗旱活性更好。3K-B具有最强的诱导抗旱活性,较干旱组生物量增加70%以上,与壳聚糖、氨基丁酸和二者复配相比,生物量增加40%以上。
     4.根据抗旱活性筛选结果,选取活性最好的衍生物3K-B,采用基于UHPLC-Q-TOF-MS技术的代谢组学方法进一步研究了小麦幼苗代谢受壳聚糖氨基丁酸衍生物影响的情况。代谢组学结果表明,与干旱对照组相比,正常培养、水杨酸、β-氨基丁酸、壳聚糖、衍生物以及复配处理组分别得到了20、18、15、26、24、28个差异代谢物。结合对差异代谢物的KEGG代谢通路进行富集分析发现,与干旱组相比,壳聚糖衍生物3K-B处理可能通过调控小麦糖代谢、氨基酸和核苷酸代谢等途径来缓解干旱的影响,诱导多种氨基酸代谢下调,嘌呤和嘧啶代谢的上调,有利于蛋白质积累;诱导葡萄糖、果糖、甘露糖等含量上调,降低了细胞渗透势,提高了叶片的保水能力;诱导愈伤酸和吲哚乳酸等植物激素含量的上调,促进小麦幼苗的生长。3K-B处理与其他外源物质处理对比,主要调控了愈伤酸、糖类、核酸以及脂肪酸等物质的代谢,明显上调核苷酸代谢,并且对氨基酸代谢的调节更加明显,进一步揭示了衍生物能够调控小麦幼苗的碳氮代谢并提高其抗旱能力,还可能激活了特异的信号通路调控幼苗抗旱。 
       本论文制备了两种构型的6种新型壳聚糖氨基丁酸衍生物,衍生物的诱导抗旱活性与壳聚糖的分子量和氨基丁酸的构型相关,其中用β-氨基丁酸修饰3000 Da壳聚糖得到的衍生物3K-B具有最强的诱导抗旱活性。3K-B可能通过激活特异的信号通路调控某些蛋白质的合成来调控小麦抗旱。本研究为新型植物抗旱剂研发奠定了基础。

Other Abstract

      Drought has become an important abiotic stress affecting the growth and yield of crops. Wheat is one of the most important food crops in China and the world, and about 70% of wheat is grown in arid and semi-arid areas. The most effective and simple way to improve the drought resistance of crops is to enhance the drought resistance through the application of exogenous substances, so it is urgent to develop safe and efficient drought inhibitors. Chitosan can alleviate the damage caused by abiotic adversities such as drought and salt, and induce broad-spectrum resistance in plants, and has the advantages of being non-toxic and degradable. As an exogenous substance, chitosan has a promising application in agricultural production.
       Chitosan is often modified by functionalization to confer different functional properties or to have new biological activities. Aminobutyric acid is a small-molecule non-protein amino acid that has a role in enhancing plant stress resistance and plays an important role in plant response to adversity stress. Based on the idea of active structure splicing, this thesis prepared novel chitosan-aminobutyric acid derivatives by modifying chitosan with two configurations of β and γ aminobutyric acid. The drought tolerance of wheat seedlings under drought stress was also investigated. Metabolomics technology was used to study the metabolic regulation of wheat seedlings affected by derivatives and to preliminarily explore the mechanism of drought tolerance induced by derivatives on wheat seedlings. The main research results of this thesis were as follows:
     1.A total of six new chitosan-aminobutyric acid derivatives of two types were synthesized by amidation reaction of chitosan amino group with carboxyl group of aminobutyric acid, and the structures of the derivatives were characterized by carbon-13 nuclear magnetic resonance spectroscopy(13C-NMR), proton-1 nuclear magnetic resonance spectroscopy(1H-NMR)and Fourier transform infrared spectroscopy(FT-IR) with substitution degree of more than 15%.
    2.Both drought models, soil-potted drought and artificial drought, significantly inhibited the growth and development of wheat seedlings, significantly decreased seedling height, seedling fresh weight and seedling dry weight. Compared with the drought treatment alone, the derivative treatment significantly reduced malondialdehyde content and electrolyte leakage rate, alleviated drought damage to membranes; increased soluble sugar and soluble protein content, increased relative water content of leaves; improved photosynthetic performance, promoted the growth and development of seedlings under drought stress, facilitated stem elongation and biomass accumulation, and improved the drought resistance of wheat seedlings. The results of the artificial simulation experiment were more stable, and the experimental process was easier and more controllable.
    3.A nutrient solution containing 20% polyethylene glycol-6000 was used to simulate the drought environment to screen the induced drought resistance activities of six derivatives. The results showed that all the six derivatives could induce drought resistance in wheat seedlings, and the drought resistance activity was 3K-B > 9K-G > 1K-B > 9K-B > 3K-G> 1K-G. The configuration of aminobutyric acid and the molecular weight of chitosan in the derivatives could affect the induced drought resistance activity, especially the configuration of aminobutyric acid, and the derivatives containing β -aminobutyric acid had better drought resistance activity. 3K-B has the strongest drought-resistance inducing activity, and the biomass increased by more than 70% compared with the drought group and more than 40% compared with 3K, B and 3KB group.
     4.Based on the results of drought activity screening, the best active derivative 3K-B was selected and the metabolic response of wheat seedlings to chitosan-aminobutyric acid derivatives was further investigated using a metabolomic approach based on UHPLC-Q-TOF-MS technique. The metabolomic results showed that 20, 18, 15, 26, 24, and 28 differential metabolites were obtained in the normal culture, salicylic acid, β-aminobutyric acid, chitosan, derivative, and mixture treatment groups, respectively, compared with the drought control group. Combination of different metabolites KEGG metabolic pathway enrichment analysis found that compared to the drought group, the derivative treatment may mitigate the effects of drought by regulating sugar metabolism, amino acid and nucleotide metabolism of wheat seedlings, induced down-regulation of many amino acid metabolism and up-regulation of purine and pyrimidine metabolism, and promoted protein accumulation; induced up-regulation of glucose, fructose and mannose contents, reduced cellular osmotic potential and improved water retention capacity of leaves; induced the up-regulation of plant hormones such as traumatic acid and indole lactic acid to promote the growth of wheat seedlings. The 3K-B treatment mainly regulated the metabolism of traumatic acid, sugar, nucleic acid and fatty acid metabolism, significantly up-regulated nucleotide metabolism, and more significantly regulated amino acid metabolism in wheat compared with other exogenous substances, further revealing that the derivatives could regulate carbon and nitrogen metabolism and improve the drought tolerance of wheat seedlings.
      A total of six novel chitosan-aminobutyric acid derivatives with two configurations were prepared in this thesis, and the drought resisitance-inducing activity of the derivatives was correlated with the molecular weight of chitosan and the configuration of aminobutyric acid. The drought-inducing activity of the derivative 3K-B obtained by modifying 3000 Da chitosan with β-aminobutyric acid was the strongest. 3K-B may activate specific signaling pathway to adjust the synthesis of certain proteins to regulate drought resistance in wheat. The present study lays the foundation for the development of novel plant drought-resistant agents.

Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/170646
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
尹秀晶. 新型壳聚糖衍生物的制备及对小麦幼苗抗旱作用研究[D]. 中国科学院海洋研究所. 中国科学院大学,2021.
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