【作者】 师超；

【导师】 徐朗莱；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 以小麦栽培品种扬麦158 (Triticum aestivum L., Yangmai 158)为实验材料,研究了小麦种子吸胀及后续幼苗生长过程中胚乳中内肽酶(endopeptidases, EPs)的变化和生化特性,以及胚乳中20S蛋白酶体的生化特性和其对种子萌发过程的响应；并研究了盐胁迫下小麦幼苗根尖的氧化伤害对20S蛋白酶体的影响。主要研究结果如下：1.小麦种子吸胀后胚乳中半胱氨酸内肽酶和20S蛋白酶体的研究采用与明胶共聚4-20%梯度凝胶电泳的方法研究了小麦种子吸胀后胚乳中内肽酶的变化及其生化特性。在小麦种子吸胀0-7 d内,小麦胚乳内有4种半胱氨酸内肽酶(EP1,EP2,EP3和EP4)逐渐出现,且它们的活性都随着实验时间的延长而逐渐增加；在这些EPs中,EP2不仅最先出现,而且它的活性在整个实验过程中也一直是最高的。这4种EPs的最适pH都是4.0,最适温度都是40℃,它们的活性都能被25μME-64完全抑制,因此,这4种EPs都属于半胱氨酸内肽酶。4种EPs对热都不很稳定,特别是EP1的热稳定性最差。此外,这4种EPs对成熟小麦籽粒贮藏蛋白质,清蛋白,球蛋白,醇溶蛋白和麦谷蛋白有不同的底物专一性。在4种EPs中,EP2对球蛋白,醇溶蛋白和麦谷蛋白均有最高的蛋白水解活性。综合以上结果,我们认为EP2可能是与小麦种子萌发及其幼苗生长相关最紧密且最重要的一种内肽酶。首先采用连续3次离心和梯度PAGE(GPAGE)后切胶回收的方法纯化小麦胚乳中的20S蛋白酶体,通过GPAGE及Western印迹检测证明获得了纯的20S蛋白酶体,然后对20S蛋白酶体和其在小麦种子萌发及幼苗生长过程中的生化特性进行了研究。结果表明,提纯的20S蛋白酶体能明显地切割模式合成底物Z-GGL-AMC和Z-GGR-AMC的芳-肽键,表示该20S蛋白酶体具有胰凝乳蛋白酶和胰蛋白酶的活性,这两种蛋白酶活性的最适pH都为8.0,而其胰凝乳蛋白酶和胰蛋白酶活性的最适温度分别为55℃和37℃,但这两种蛋白酶活性都能被MG115和PMSF显著性地抑制。以上结果表明,我们提纯的蛋白酶是一种典型的20S蛋白酶体。结果同时还表明,在小麦种子吸胀0-7 d内,胚乳内多聚泛素化蛋白的含量保持不变。但是与糊粉层相关的20S蛋白酶体的酪蛋白水解活性和蛋白含量在1-2 d内逐渐降低,但在2-4 d期间逐渐增加,在4 d达到最高水平,并保持最高水平一直到7 d。在2-4 d期间,20S蛋白酶体的β5-亚基mRNA水平增加到较高的水平,20S蛋白酶体的酪蛋白水解活性和蛋白含量相应地从3 d开始逐渐增加。此外,小麦胚乳的主要贮藏蛋白不能被20S蛋白酶体降解。以上结果进一步表明,在小麦种子吸胀后的这段时间内,胚乳中20S蛋白酶体的主要作用可能不是用来降解胚乳中大量贮藏蛋白的。2.盐胁迫下小麦根尖的氧化伤害对20S蛋白酶体的影响为研究盐胁迫下小麦根尖的氧化伤害对20S蛋白酶体的影响,用200 mM NaCl胁迫小麦幼苗12、24、48和72 h,之后分别取其根尖。测定了其中H2O2含量、O2-产生的速率、膜脂过氧化程度和质膜完整性；及20S蛋白酶体的羰基化水平、酪蛋白水解活性及蛋白含量变化等生化特性。同时也测定了小麦根尖细胞内羰基化蛋白和泛素化蛋白的含量。结果表明,小麦根尖中H2O2含量、O2-产生的速率、TBARS含量和细胞死亡数都随着胁迫时间的延长而逐渐增加。对根尖进行组织染色发现,膜脂过氧化和质膜完整性破坏的程度和分布范围随着胁迫时间的延长而逐渐加重和扩大。在72 h盐胁迫过程中,小麦根尖中总酪蛋白水解活性和20S蛋白酶体的酪蛋白水解活性都逐渐增加；与此同时,小麦根尖中总蛋白的羰基化水平和20S蛋白酶体的羰基化水平也都逐渐增加。但是,在这个过程中,20S蛋白酶体的含量和泛素化蛋白的含量却反而减少。Fe2+催化的对照小麦根尖可溶性蛋白的氧化实验证明,氧化处理也能使20S蛋白酶体的酪蛋白水解活性逐渐增加,但其蛋白含量却逐渐减少。这些结果表明,在盐胁迫诱导的小麦根尖细胞中,20S蛋白酶体轻度的氧化修饰能使其含量逐渐降低,但反而使其活性逐渐增加。更多还原

【Abstract】 Wheat cultivar Yangmai 158 (Triticum aestivum L., Yangmai 158) is the experimental material of this study, the changes of cysteine endopeptidases and their biochemical characteristics were investigated in wheat endosperm during seed germination and subsequential seedling growth, and enzymatic properties of wheat endosperm 20S proteasome and the response of which to this germination process were also investigated; simultaneity, the effect of salt-induced oxidative damage to the 20S proteasome was also studied in wheat root tips under salt stress. The main contents of this dissertation as followings:1. The investigation of cysteine endopeptidases and 20S proteasome in wheat endosperm after seed imbibitionThe endopeptidases (EPs) in wheat endosperm after seed imbibition were characterized by 4-20% gradient PAGE with gelatin copolymerized into the gel. Four cysteine EPs (EP1, EP2, EP3 and EP4) were detected in wheat endosperm during the 7 d growth after seed imbibition. The results showed that the activities of all of these EPs increased continuously, and EP2 first appeared and had the highest proteolytic activity among the four EPs in this experimental process. The optimum pH and temperature of all four EPs were 4.0 and 40.0℃. All EPs were completely inhibited by 25μM E-64 and had no good thermal stabilities, especially EP1. In addition, these EPs had different substrate specificities to albumins, globulins, gliadins and glutenins; the main storage proteins of mature wheat endosperm. Among them, EP2 had the highest proteolytic activities on globulins, gliadins and glutenins, and might be the most important and specific EP with potential to be tightly correlated with seedling development.The 20S proteasome from wheat endosperm was purified by three sequential centrifugations and gradient PAGE (GPAGE) to apparent homogeneity, as judged by GPAGE and western blotting. The results showed that the purified 20S proteasome could clearly cleave the peptidyl-arylamide bonds in model synthetic substrates Z-GGL-AMC and Z-GGR-AMC, used for reflecting its chymotrypsin-like and trypsin-like activity respectively. Both of optimum pH were 8.0, but the optimum temperatures of chymotrypsin-like and trypsin-like activity were 55℃and 37℃respectively, the activities above were clearly inhibited by MG115 or PMSF. The results also showed that polyubiquitinated proteins remained constant from 0 to 7 d after seed imbibition. But the caseinolytic activity and amount of 20S proteasome associated with aleurone layer decreased from 1 to 2 days after imbibition (DAI), then increased from 2 to 4 DAI, and reached the highest level on 4 DAI and kept the level till 7 DAI. With the mRNA level increase ofβ5-subunit for 20S proteasome from 2 DAI, the caseinolytic activity and amount of 20S proteasome increased accordingly from 3 DAI. In addition, the main storage proteins of wheat endosperm could not be hydrolyzed by the 20S proteasome. The evidences above suggested that the main role of 20S proteasome may not be to degrade massive proteins of wheat endosperm after seed imbibition.2. Effect of salt-induced oxidative damage to the 20S proteasome in wheat root tips under salt stressIn order to study the effect of salt-induced oxidative damage to the 20S proteasome in wheat root tips under salt stress, the root tips from wheat seedlings treated with 200 mM NaCl for 12,24,48 and 72 h were used for studying the content of H2O2, production rate of O2-, the degree of lipid peroxidation and plasma membrane integrity; and the carbonyl level, caseinolytic activity and protein amount of 20S proteasome and other biochemical characteristics. The contents of carbonylated and ubiquitinated proteins (Ub-P) in root tips were also investigated. The results showed that the contents of H2O2 and TBARS, production rate of O2-, and the quantity of cell death in root tips all gradually increased along with the stress time prolonged. Histochemical detection of lipid peroxidation and loss of plasma membrane integrity found that the degree and distribution of the histochemical staining patterns of these two events were aggravated and extended along with the prolonged treatment time. Caseinolytic activities of the total and the 20S proteasome both gradually increased, with a concomitant increase of the carbonyl level of total proteins and the 20S proteasome during the salt treatment. But the amounts of 20S proteasome and Ub-P decreased oppositely during this process. Metal-catalyzed oxidation of soluble proteins from control root tips validated that oxidative treatment could increase the caseinolytic activity of 20S proteasome, but oppositely decrease its protein amount. These results indicated that the amount and activity of 20S proteasome could be contrarily altered by mild oxidative modification in wheat root tips under salt stress.更多还原