【作者】 白承之；

【导师】 王转花；

【作者基本信息】 山西大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 植物源抗真菌蛋白或抗真菌肽(AFPs)种类丰富,来源广泛,因其广谱,无毒的特征一直受到学者的关注。抗真菌蛋白的研究与农业,食品工业密切相关。本文选取了三种我国的特色作物种子为研究材料,从中纯化抗真菌蛋白或抗真菌肽,并分析其生物化学特性及抗真菌活性。首先以苦荞种子为实验材料,经提取,热处理,Resource S阳离子交换层析及Superdex Peptide分子筛层析,纯化得到一种抗真菌肽,Tricine-SDS-PAGE显示其表观分子质量约为8.0 kDa,表观纯度很高。表面增强激光解吸电离飞行时间质谱(SELDI-TOF)显示其实际分子质量为3.909 kDa。该抗菌肽对白腐菌(Panus conchatus),绿色木霉(Trichoderma reesei)和链格孢霉(Alternaria alternata)等均表现出显著的生长抑制活性。绿色木霉的形态学分析显示受到影响的菌丝生长停滞,分支加剧,基内菌丝端部膨大,原生质凝缩。以莜麦(裸燕麦)种子为实验材料经过Resource S阳离子交换层析和superdex 75凝胶排阻层析,纯化得到两种抗真菌蛋白,分别命名为α-avechin和β-avechin,其中,β-avechin的含量约为α-avechin的三倍。SDS-PAGE显示两种蛋白的分子量均为35 kDa左右,推测是两种相似蛋白或是同一种蛋白的两种修饰形式应用固相pH梯度胶条测定β-avechin的等电点为8.9。两种蛋白均对白腐菌(Panus conchatus),绿色木霉(Trichoderma reesei)显示出显著的生长抑制作用并呈现剂量依赖性,其中,对于绿色木霉的最低抑制量为0.21μg。胶内酶解和串联质谱实验的匹配结果显示,该蛋白与莜麦种子中的几丁质酶具有显著的相似性。以胶体几丁质为底物测定了该蛋白的几丁质酶活性,结果显示该蛋白是一种几丁质酶,其比活力为3.59U／mg,同时测定了酶活力的最适pH和最适温度。以薏苡种子为实验材料,经过Resource S阳离子交换层析和superdex 75凝胶排阻层析等步骤纯化得到一种28 kDa的薏苡抗真菌蛋白,该蛋白此前未见报道,对链格孢霉(Alternaria alternate),绿色木霉(Trichoderma reesei)和白腐菌(Panus conchatus)三株丝状真菌具有显著生长抑制活性,并呈现出剂量依赖的特征。更多还原

【Abstract】 Antifungal peptides(AFPs), serve to protect organisms against fungal invasion, have been isolated from a variety of plants species.The experimental materials of present investigation was seeds of tartary buckwheat (Fagopyrum tataricum).The purification procedure involved extraction with aqueous buffer, heat treatment, cation exchange chromatography on Resource S column, and size exclusion chromatography (SEC) on Superdex Peptide.The apparent molecular weight of purified peptide was approximately 8.0 kDa by Tricine-SDS-PAGE,and actual molecular weight was 3.909 kDa by surface-enhanced laser desorption ionization-time of flight (SELDI-TOF). This peptide showed strong antifungal activity against Panus conchatus, Trichoderma reesei and Alternaria alternata. Light microscopic examination disclosed antifungal peptide induced swelling of hyphal tips, hyphal distortions and fragmentation in the Trichoderma reesei.A chitinase with antifungal activity was purified from naked oat (Avena chinensis) seeds by extraction with an aqueous buffer, cation exchange and size exclusion chromatography. The molecular weight and isoelectric point of the purified chitinase were estimated at 35 kDa and 8.9, respectively. Peptide mass fingerprint analysis indicated the primary structure of the purified naked oat (Avena chinensis) chitinase is highly homologic to oat (Avena sativa) class I chitinase.When colloidal chitin is used as a substrate, with a specific activity 3.596 U/mg, which is consistent with reported chitinase activity, the purified chitinase is highly active.The chitinase activity is dependent on both pH and temperature, with an optimum pH of 7.0 and optimum temperature of 40℃.Measurement of in vitro antifungal activity demonstrated that the purified chitinase has potent, dose-dependent inhibitory activity against fungi Panus conchatus and Trichoderma reesei. All these results provided evidence that the chitinase purified from naked oat seeds is a class I chitinase with antifungal activity..To isolate and purify an antifungal proteins from seeds of adlay(Coix chinensis Tod.)and analyze its antifungal activity. Methods The procedure involved extraction with aqueous buffer, ion exchange chromatography on Resource S,and size exclusion chromatography (SEC) on Superdex 75.Results The molecule weight of antifungal protein is approximately 28kDa, which was determined by SDS-PAGE.It shows strong fungal growth inhibitory activity against Alternaria alternate, Trichoderma reesei and Panus conchatus, in a dose-dependent manner. Conclusion The method is simple,and the purify of protein is over 95%,it can be used for purification of some novel antifungal protein from plant seeds.更多还原