NO和冷信号交互作用对鞘脂代谢及神经酰胺酶的调控作用

发布时间：2017-12-27 14:32

本文关键词：NO和冷信号交互作用对鞘脂代谢及神经酰胺酶的调控作用　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：鞘脂(sphingolipid)是细胞膜脂的重要成分,对于维持细胞膜正常的流动性起到重要作用。作为一种信号分子,鞘脂参与调节细胞的生长、分化、衰老和程序性死亡等许多重要的信号转导过程。神经酰胺(ceramide)是鞘脂代谢的中心产物,能传递凋亡和生长阻滞,可以被神经酰胺酶(ceramidase)水解生成鞘氨醇和脂肪酸。一氧化氮(nitric oxide,NO)是重要的信号分子,能调控植物多种生理过程,延缓果实衰老,缓解贮藏冷害。植物体内存在着NO和冷信号的交互作用。目前对于NO调控果实冷害的研究主要集中于NO对冷害的缓解效果和果实贮藏品质影响等方面,对于果实细胞膜脂如何感受冷藏期间的冷信号以及NO和冷信号交互作用对细胞膜脂组分和代谢的影响等方面研究较少。本实验以采后肥城桃为材料,提取果实总蛋白并进行同位素标记相对和绝对定量技术(iTRAQ)检测。结果显示,共检测到4776个蛋白质,涉及135个代谢过程。与外源NO处理相比,储存温度的不同更易引起蛋白质的表达差异。COG和GO分析发现,肥城桃果实采后贮藏期间,其主要的生理活动为蛋白质的代谢和糖分的积累。鞘脂代谢过程共检测到31个蛋白质,其中9个是差异蛋白。这些差异蛋白出现在鞘脂合成初期以及下游代谢过程,主要是β-半乳糖苷酶和丝氨酸棕榈酰转移酶,还包括中性神经酰胺酶、3-脱氢鞘氨醇还原酶等。根据iTRAQ结果,选择肥城桃神经酰胺酶(Prunus persica ceramidase,PPCD_(ase))为后续研究的目标。采用同源克隆的方法和cDNA末端快速扩增技术(RACE)克隆获得了PPCD_(ase)基因全长。该全长为2784 bp,开放阅读框有2337bp,共编码778个氨基酸。NCBI网站blast结果和三维结构模拟表明,该基因即是桃中性神经酰胺酶基因,且与梅、梨的中性神经酰胺酶基因相似度较高。蛋白理化性质分析显示,PPCD_(ase)为亲水性蛋白,稳定性较强;二维结构模拟发现,PPCD_(ase)含有16段α螺旋和25段β折叠结构;亚硝基化预测表明,PPCD_(ase)内部有6个S-亚硝基化作用位点,具备发生亚硝基化的条件。根据PPCD_(ase)全长设计带酶切位点的引物,进行PPCD_(ase)重组蛋白的体外表达,并使用镍柱纯化法进行纯化。电泳结果显示,在112 KDa左右出现目的条带,重组蛋白表达和纯化成功。实时荧光定量PCR结果表明,外源NO处理能延缓PPCD_(ase)基因表达量的下降,冷藏效果更显著。
[Abstract]:Sphingolipids (sphingolipid) is an important component of cell membrane lipid fluidity, to play an important role in maintaining normal cell membrane play. As a signal molecule, sphingolipids are involved in the regulation of cell growth, differentiation, senescence and programmed death of many important signal transduction. Ceramide (ceramide) is the center of product of sphingolipid metabolism, can transfer apoptosis and growth arrest can be ceramidase (ceramidase) hydrolysis of sphingosine and fatty acid. Nitric oxide (NO) is an important signal molecule, which can regulate the physiological processes of plants, delay the senescence of fruit and alleviate the storage of cold damage. There is a interaction between NO and cold signals in plants. The present study for NO regulation of fruit chilling injury mainly focused on NO to alleviate the effects of chilling injury and fruit quality factors, cell membrane lipid for the fruit to feel cold during the cold and cold signal and NO signal interaction effect on cell membrane lipid composition and metabolism research. In this experiment, the total fruit protein of Feicheng peach was extracted from Postharvest Peach, and the relative and absolute quantitative technique (iTRAQ) was used to detect the total protein of the fruit. The results showed that 4776 proteins were detected and involved in 135 metabolic processes. Compared with the exogenous NO treatment, the difference of storage temperature is more likely to cause the difference of protein expression. Analysis of COG and GO showed that Feicheng peach fruit during postharvest storage, the main physiological activity of protein metabolism and sugar accumulation. Sphingolipid metabolism processes were detected 31 proteins, 9 of which are proteins. These proteins appear in sphingolipid synthesis early and downstream metabolic processes, mainly beta galactosidase and serine palmitoyltransferase, including neutral ceramidase, 3- dehydrogenation sphingosine reductase. According to the results of iTRAQ, the target of Feicheng peach neuraminidase (Prunus persica ceramidase, PPCD_ (ASE)) is selected as a follow-up study. The full length of PPCD_ (ASE) gene was obtained by homologous cloning and cDNA terminal rapid amplification (RACE) cloning. The total length was 2784 BP, and the open reading frame was 2337bp, and 778 amino acids were coded in total. The blast results and three-dimensional structure simulation of NCBI website showed that this gene is peach neutral neurosase gene, and has a high similarity to neutral neurosase gene from plum and pear. Protein physicochemical properties analysis showed that PPCD_ (ASE) as a hydrophilic protein, strong stability; two-dimensional structure simulation, PPCD_ (ASE) containing 16 alpha helix and 25 p-sheet; nitrosylation prediction shows that PPCD_ (ASE) has 6 internal S- nitrosylation sites have occurred nitrosylation condition. The expression of PPCD_ (ASE) recombinant protein was expressed in vitro by PPCD_ (ASE) full length design with the primers with the enzyme cut site, and purified by the nickel column purification method. The electrophoretic results showed that the target band appeared at about 112 KDa, and the recombinant protein was expressed and purified successfully. The results of real time fluorescence quantitative PCR showed that exogenous NO treatment could delay the decrease of PPCD_ (ASE) gene expression, and the effect of cold storage was more significant.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q946

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