本文關(guān)鍵詞：NO和冷信號(hào)交互作用對(duì)鞘脂代謝及神經(jīng)酰胺酶的調(diào)控作用　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鞘脂 神經(jīng)酰胺酶 同位素標(biāo)記相對(duì)與絕對(duì)定量技術(shù)(iTRAQ) 一氧化氮 基因克隆

【摘要】：鞘脂(sphingolipid)是細(xì)胞膜脂的重要成分,對(duì)于維持細(xì)胞膜正常的流動(dòng)性起到重要作用。作為一種信號(hào)分子,鞘脂參與調(diào)節(jié)細(xì)胞的生長(zhǎng)、分化、衰老和程序性死亡等許多重要的信號(hào)轉(zhuǎn)導(dǎo)過(guò)程。神經(jīng)酰胺(ceramide)是鞘脂代謝的中心產(chǎn)物,能傳遞凋亡和生長(zhǎng)阻滯,可以被神經(jīng)酰胺酶(ceramidase)水解生成鞘氨醇和脂肪酸。一氧化氮(nitric oxide,NO)是重要的信號(hào)分子,能調(diào)控植物多種生理過(guò)程,延緩果實(shí)衰老,緩解貯藏冷害。植物體內(nèi)存在著NO和冷信號(hào)的交互作用。目前對(duì)于NO調(diào)控果實(shí)冷害的研究主要集中于NO對(duì)冷害的緩解效果和果實(shí)貯藏品質(zhì)影響等方面,對(duì)于果實(shí)細(xì)胞膜脂如何感受冷藏期間的冷信號(hào)以及NO和冷信號(hào)交互作用對(duì)細(xì)胞膜脂組分和代謝的影響等方面研究較少。本實(shí)驗(yàn)以采后肥城桃為材料,提取果實(shí)總蛋白并進(jìn)行同位素標(biāo)記相對(duì)和絕對(duì)定量技術(shù)(iTRAQ)檢測(cè)。結(jié)果顯示,共檢測(cè)到4776個(gè)蛋白質(zhì),涉及135個(gè)代謝過(guò)程。與外源NO處理相比,儲(chǔ)存溫度的不同更易引起蛋白質(zhì)的表達(dá)差異。COG和GO分析發(fā)現(xiàn),肥城桃果實(shí)采后貯藏期間,其主要的生理活動(dòng)為蛋白質(zhì)的代謝和糖分的積累。鞘脂代謝過(guò)程共檢測(cè)到31個(gè)蛋白質(zhì),其中9個(gè)是差異蛋白。這些差異蛋白出現(xiàn)在鞘脂合成初期以及下游代謝過(guò)程,主要是β-半乳糖苷酶和絲氨酸棕櫚酰轉(zhuǎn)移酶,還包括中性神經(jīng)酰胺酶、3-脫氫鞘氨醇還原酶等。根據(jù)iTRAQ結(jié)果,選擇肥城桃神經(jīng)酰胺酶(Prunus persica ceramidase,PPCD_(ase))為后續(xù)研究的目標(biāo)。采用同源克隆的方法和cDNA末端快速擴(kuò)增技術(shù)(RACE)克隆獲得了PPCD_(ase)基因全長(zhǎng)。該全長(zhǎng)為2784 bp,開放閱讀框有2337bp,共編碼778個(gè)氨基酸。NCBI網(wǎng)站blast結(jié)果和三維結(jié)構(gòu)模擬表明,該基因即是桃中性神經(jīng)酰胺酶基因,且與梅、梨的中性神經(jīng)酰胺酶基因相似度較高。蛋白理化性質(zhì)分析顯示,PPCD_(ase)為親水性蛋白,穩(wěn)定性較強(qiáng);二維結(jié)構(gòu)模擬發(fā)現(xiàn),PPCD_(ase)含有16段α螺旋和25段β折疊結(jié)構(gòu);亞硝基化預(yù)測(cè)表明,PPCD_(ase)內(nèi)部有6個(gè)S-亞硝基化作用位點(diǎn),具備發(fā)生亞硝基化的條件。根據(jù)PPCD_(ase)全長(zhǎng)設(shè)計(jì)帶酶切位點(diǎn)的引物,進(jìn)行PPCD_(ase)重組蛋白的體外表達(dá),并使用鎳柱純化法進(jìn)行純化。電泳結(jié)果顯示,在112 KDa左右出現(xiàn)目的條帶,重組蛋白表達(dá)和純化成功。實(shí)時(shí)熒光定量PCR結(jié)果表明,外源NO處理能延緩PPCD_(ase)基因表達(dá)量的下降,冷藏效果更顯著。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q946

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