【摘要】：鐵蛋白是廣泛存在于生物體細(xì)胞中的一類鐵貯藏蛋白,具有調(diào)節(jié)鐵代謝平衡以及去除亞鐵離子毒性的雙重功能。與動(dòng)物鐵蛋白相比,關(guān)于植物鐵蛋白的結(jié)構(gòu)和功能的研究甚少。目前已發(fā)現(xiàn)豆類植物中90%以上的鐵以鐵蛋白的形式貯藏在淀粉體中。因此,豆類種子鐵蛋白是研究植物鐵蛋白的理想模型。植物鐵蛋白與動(dòng)物鐵蛋白相比,其在結(jié)構(gòu)上具有三個(gè)明顯的特征：1、植物鐵蛋白N端含有EP肽段,而動(dòng)物鐵蛋白沒(méi)有。該EP肽段位于植物鐵蛋白的外殼表面,參與鐵的結(jié)合和氧化。2、動(dòng)物鐵蛋白由H和L型亞基組成,而植物鐵蛋白只含有H型亞基,H-1和H-2。H-1和H-2型亞基具有-80%的同源性,二者在鐵氧化沉淀過(guò)程中起著協(xié)同作用。在不同來(lái)源的植物鐵蛋白中,H-1和H-2亞基的組成比例不同。3、植物鐵蛋白的三重軸通道和四重軸通道均為親水性通道,而動(dòng)物鐵蛋白的三重軸通道為親水性通道,四重軸通道為疏水性通道。鐵蛋白的通道是與外界進(jìn)行物質(zhì)交換的重要途徑。本文以植物鐵蛋白(H-1和H-2亞基)為研究對(duì)象,一方面,從植物鐵蛋白的結(jié)構(gòu)特點(diǎn)出發(fā),研究植物鐵蛋白的三重軸和四重軸通道在鐵氧化沉淀過(guò)程中的作用,揭示為什么植物鐵蛋白的三重軸和四重軸通道為親水性通道?另一方面,從鐵蛋白的應(yīng)用出發(fā),在體外和體內(nèi)研究鐵蛋白的消化穩(wěn)定性,進(jìn)而對(duì)不同來(lái)源的植物鐵蛋白的吸收率進(jìn)行鑒定,闡明其細(xì)胞吸收機(jī)制。研究成果將為開(kāi)發(fā)高效的功能性補(bǔ)鐵制劑奠定理論基礎(chǔ)。具體研究結(jié)果如下： 1.利用分子克隆、點(diǎn)突變、載體構(gòu)建、表達(dá)及重組蛋白的分離純化的方法制備了重組大豆鐵蛋白rH-1、rH-2、及rH-2的突變體H193AH197A, E165IE167AE171A和E165IE167AE171AH193AH197A,采用柱層析方法純化得到了電泳純的rH-2及其突變體。得到了充足的植物鐵蛋白,為后續(xù)蛋白質(zhì)的性質(zhì)研究提供了保障。 2.通過(guò)比較重組大豆鐵蛋白(rH-2)及其三重軸和四重軸突變體(H193AH197A, E165IE167AE171A, E165IE167AE171AH193AH197A)的鐵氧化沉淀活性、聚合特性以及鐵釋放活性,初步闡明rH-2鐵蛋白的親水性(三重軸和四重軸)通道均為鐵離子擴(kuò)散和氧化沉淀的主要途徑。 3.通過(guò)建立體外消化模型比較不同亞基組成的植物鐵蛋白(rH-1, rH-1:rH-2=1:1, rH-1:rH-2=1:2; rH-1:rH-2=2:1和rH-2)的消化穩(wěn)定性以及在體外和體內(nèi)研究加工條件和食品組分對(duì)鐵蛋白消化穩(wěn)定性的影響,闡明大部分植物和動(dòng)物鐵蛋白能夠抵抗住腸胃的消化作用到達(dá)小腸,并且食品組分中脫脂奶粉對(duì)鐵蛋白具有保護(hù)作用。 4.采用同位素59Fe標(biāo)記不同亞基組成的植物鐵蛋白(rH-1,rH-1:rH-2=1:1,rH-1:rH-2=1:2;rH-1:rH-2=2:1和rH-2)和動(dòng)物鐵蛋白,通過(guò)Caco-2細(xì)胞模型鑒定其鐵的吸收率和鐵蛋白與細(xì)胞表面的相互作用。本文研究發(fā)現(xiàn)大豆鐵蛋白;rH-l:rH-2=1:1)的吸收率最高,與細(xì)胞表面的特異性結(jié)合強(qiáng)度最大。因此,大豆鐵蛋白是一種潛在的新型高效補(bǔ)鐵功能因子。
[Abstract]:Ferritin is a kind of ferric storage protein widely found in biological cells. It has the dual functions of regulating the balance of iron metabolism and removing the toxicity of ferrous ions. Compared with animal ferritin, there are few studies on the structure and function of plant ferritin. At present, more than 90% of iron in legume has been stored in amyloplasts in the form of ferritin. Therefore, soybean seed ferritin is an ideal model for the study of plant ferritin. Compared with animal ferritin, plant ferritin has three distinct structural features: 1: 1. Plant ferritin contains EP peptide in N-terminal, but not in animal ferritin. The EP peptide is located on the outer surface of plant ferritin and is involved in the binding and oxidation of iron. The animal ferritin consists of H and L subunits, while the plant ferritin contains only H-type H-1 and H-2.H-1 and H-2 subunits with -80% homology. They play a synergistic role in the process of iron oxidation and precipitation. The composition of H-1 and H-2 subunits in plant ferritin from different sources is different. The triple axis channel and quadruple axis channel of plant ferritin are hydrophilic channel, while the triplex axis channel of animal ferritin is hydrophilic channel. The quadruple axis channel is hydrophobic channel. The ferritin channel is an important way to exchange substances with the outside world. In this paper, plant ferritin (H-1 and H-2 subunits) was studied. On the one hand, based on the structural characteristics of plant ferritin, the role of triplex and quadruplex channels in the process of iron oxidation was studied. Why triplex and quadruplex channels of plant ferritin are hydrophilic? On the other hand, based on the application of ferritin, the digestion stability of ferritin was studied in vitro and in vivo, and then the absorption rate of plant ferritin from different sources was identified, and the mechanism of cell absorption was elucidated. The research results will lay a theoretical foundation for the development of efficient functional iron supplements. The results are as follows: 1. Using molecular cloning, point mutation, vector construction, expression and separation and purification of recombinant protein, the recombinant soybean ferritin rH-1rH-2, and the mutants of rH-2, H193AH197A, E165IE167AE171A and E165IE167AE171AH193AH197A197A were prepared. The purified rH-2 and its mutants were purified by column chromatography. Sufficient plant ferritin was obtained, which provided a guarantee for further study on the properties of plant ferritin. 2. The iron oxidation precipitation activity, polymerization characteristics and iron release activity of recombinant soybean ferritin (rH-2) and its triple axis and quadruplex axons (H193AH197A, E165IE167AE171A, E165IE167AE177AH19AH197A) were compared. It is shown that the hydrophilic channels of rH-2 ferritin (triplex axis and quadruple axis) are the main pathways of iron ion diffusion and oxidation precipitation. The digestive stability of plant ferritin with different subunits (rH-1, rH-1: rH-2H 1: 1: 1: 1: 1: 2; rH-1: rH-2H 2: 1 and rH-2) was studied in vitro and in vivo by establishing an in vitro digestion model, and the effects of processing conditions and food components on the digestion stability of ferritin were studied in vitro and in vivo. It is shown that most plant and animal ferritin can resist the digestion of stomach to reach the small intestine, and skim milk powder has protective effect on ferritin. 4. The plant ferritin (rH-1rH-1: 1: 1: 1rH-1rH-2) and animal ferritin were labeled with the isotope 59Fe. The iron absorption rate and the interaction between ferritin and cell surface were determined by Caco-2 cell model. It was found that the absorption rate of soybean ferritin was the highest and the specific binding intensity to cell surface was the highest. Therefore, soybean ferritin is a potential new and efficient iron supplement functional factor.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R151

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 胡菊;廖夏云;鄧建軍;胡小松;趙廣華;;黃豆鐵蛋白提取新方法及其與豌豆鐵蛋白活性比較[J];高等學(xué)�；瘜W(xué)學(xué)報(bào);2009年10期

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本文編號(hào)：2121134