【作者】 王书珍；

【导师】 丁毅；

【作者基本信息】 武汉大学 ， 遗传学， 2013， 博士

【摘要】 植物核糖体失活蛋白(ribosome-inactivating proteins, RIPs)是一类可以将真核细胞28SrRNA的SRL区域的A4324脱去,并破坏延伸因子与核糖体的结合,从而将蛋白质的生物合成抑制在延伸阶段的蛋白质家族。研究表明RIPs具有免疫调节、骨髓净化、抗病毒、抗细菌、抗真菌、抗突变、抗肿瘤等多种活性,并能够增强宿主植物对病原菌的抵抗作用。RIPs在植物界中分布的比较广泛,尤以葫芦科家族(如苦瓜、丝瓜、南瓜)中含量最多。从苦瓜种子中提取的α-苦瓜素因能在体外抑制兔网织红细胞裂解物和小麦胚乳系统的蛋白质翻译过程而被认为是RIPs家族的成员之一。目前对α-苦瓜素的活性研究并不深入,并且该蛋白也尚未得到充分的应用。因此,本研究将大肠杆菌异源表达系统和亲和纯化技术相结合得到了高纯度的α-苦瓜素蛋白,并对其酶活性进行了研究,结果如下：1.将含有α-苦瓜素基因的重组表达载体pET-MC转化到大肠杆菌Rosetta (DE3) pLysS菌株内,得到了富含a-苦瓜素基因的工程菌株。此外,我们还分析了培养条件(培养时间：6-18h; IPTG诱导剂的浓度：0.5-1.5mM;诱导温度：22-37℃；诱导的菌体浓度OD600:0.6-0.8)对重组蛋白质得率的影响,最终优化出能够使大部分重组α-苦瓜素蛋白在细胞质内积累的培养条件。诱导后的菌液经SDS-PAGE检测后发现重组蛋白的表达量是115mg/L。经Ni-NTA亲和层析后得到的蛋白质产量为85mg/L,即回收率为73.9%, SDS-PAGE检测结果表明重组蛋白的纯度为94.7%。2.首先用水稻的rRNAs验证重组α-苦瓜素的RNA N-糖苷酶活性,结果发现该酶活性是浓度依赖性的。低浓度蛋白仅仅影响了28S rRNA,并产生了典型的"Endo’s fragment";而高浓度蛋白则将RNAs完全降解成弥散条带。我们还发现重组α-苦瓜素对尖镰孢(Fusarium oxysporum L.)和腐皮镰孢(Fusarium solani L.)的生长有较强的抑制作用。此外,α-苦瓜素蛋白也能抑制铜绿脓杆菌(Pseudomonas aeruginosa L.)的生长和繁殖。3.在DNase-like活性的研究过程中,我们发现该酶活性是浓度依赖性、二价金属离子依赖性、温度敏感型、pH稳定型的。圆二色谱(CD)检测结果表明α-苦瓜素仅仅影响了质粒DNA的碱基堆积作用,但是对DNA的右手螺旋却没有影响。通过点突变技术将对多聚核苷酸：腺嘌呤糖苷酶活性关键的五个氨基酸残基Tyr93、Tyr134、Glu183、Arg186、Trp215突变成Ala,然后通过大肠杆菌异源表达得到纯化的突变体蛋白,并进一步分析DNase-like活性。研究发现Y93A突变体完全丧失了1DNase-like酶活性,而其余4个突变体的酶活性略受影响,充分说明多聚核苷酸：腺嘌呤糖苷酶活性DNase-like酶活性是由不同但是却紧密相关的结构域行使的。4.为了开发与α-苦瓜素基因紧密连锁的SSR分子标记,我们采用FIASCO (Fast Isolation by AFLP of Sequence COntaining Repeats)扩增和磁珠富集技术从苦瓜基因组DNA内分离出16对多态性的SSR分子标记,并在5个群体(广西、湖南、四川、江西、湖北)共36份材料内进行了多态性检测。这16对引物扩增的等位基因数为3-8,Ho和He的范围分别为0.000-1.000和0.325-0.867,并且这些标记之间不存在严重的连锁不平衡现象。这些SSR标记在苦瓜的近缘属植物西葫芦(Cucurbita pepo L.)、丝瓜(Luffa cylindrical L.)、葫芦(Lagenaria siceraria L.)、黄瓜(Cucumis sativus L.)内具有比较强的通用性。这些标记将为α-苦瓜素基因的定位分析奠定有力的基础,同时也将对苦瓜及近缘种属的分类、亲缘关系的研究,以及育种工作有重要的指导意义。更多还原

【Abstract】 Ribosome-inactivating proteins (RIPs) are a family of enzyme, which could remove a specific adenine A4324from the highly conserved, surface-exposed, α-sarcin/ricin loop in the28S rRNA of eukaryotic ribosomes. The depurination destroyed the interaction between elongation factors and ribosome, thereby resulting in inactivation of ribosomes and inhibition of protein synthesis. Studies show that RIPs not only have immunomodulatory,"clean-up" of bone marrow, antivirus, antimicrobial, antifungal, antimutagenic, antitumor, but also could effectively enhance the resistance of host plant to pathogens. RIPs are widely distributed in the plant kingdom, especially the Cucurbitaceae family including bitter gourd, sponge gourd and pumpkin. Alpha-Momordicin (α-MC), a RIP isolated from Momordica charantia L., could inhibit protein synthesis in rabbit reticulocyte lysate and wheat endosperm system. Studies on the enzamital activity were not complete, while its applications have not yet been fully developed. In this study, the highly purified α-MC was obtained from E.coli heterologous expression system coupled with Ni-NTA affinity chromatography. Researches on the enzymatic activities of α-MC were as followes:1. We have constructed the recombinant expression vector pET-MC and transformed it into E. coli Rosetta (DE3) pLysS strain. After optimizing the cultural condition by varying induction time (6-18h), IPTG concentration (0.5-1.5mM), culture temperature (22-37℃), and the cell concentration (0.6-1.0for OD600), the recombinant α-MC protein was mostly present in the soluble fraction. Subsequently, α-MC protein was recovered from the bacterial cells using Ni-NTA affinity chromatography. The protein concentration was estimated to be85mg/L with Bradford method. SDS-PAGE electrophoresis showed the94.7%purity and the recovery of73.9%. 2. Results showed that the RNA N-glycosidase activity was concentration-dependent. Only the28S rRNA was affected when being incubated with low concentrations of a-MC, releasing the typical "Endo’s fragment". With the increase of protein concentration, total rRNAs were completely degraded, as smear bands appearing on the agarose gel. The antifungal activity towards F. oxysporum and F. solan were tested from both molecular and cell levels. In addition, a-MC could also effectively inhibit the growth of P. aeruginosa.3. The DNase-like activity of a-MC towards plasmid was concentration-dependent, metal-dependent, temperature-sensitive, and pH-stable. Circular dichroism was used to detect the conformational change in plamid DNA. To explore the relationship between the DNase-like activity and polynucleotide:adenosine glycosidase activity, five amino acids critical for the polynucleotide:adenosine glycosidase activity were mutated to alanine, respectively. After prokaryotic expression and affinity purification, five homogenous mutant proteins were obtained. The DNase-like activity was completely lost in the Y93A mutant. However, the DNase-like activity was more or less affected in the other four mutants. We speculated that the polynucleotide: adenosine glycosidase activity and DNase-like activity might be implemented by distinct but related domains.4. In order to develop SSR markers linked with a-MC gene, the FIASCO technology was utilized to isolate microsatellites from bitter melon genomic DNA. In total,16loci showed polymorphic in36accessions collected from five populations (Guangxi, Hunan, Sichuan, Jiangxi, and Hubei). These loci contained three to eight alleles, with He and Ho ranging from0.325to0.867and from0.000to1.000, respectively. No significant linkage disequilibrium was found between these polymorphic loci. These markers could also amplify corresponding fragments in Cucurbita pepo L., Luffa cylindrical L., Lagenaria siceraria L., and Cucumis sativus L. These marks will facilitate the mapping of a-MC gene, and are also useful for genus classification, relation studies, as well as the molecular marker assisting breeding of bitter melon and other closely related species.更多还原