【作者】 荆贝贝；

【导师】 郭蔼光；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 抗菌肽(Antimicrobial peptides,AMPs)也称为肽类抗生素(Peptideantibiotics)或天然抗生素(Natural antibiotics),是一类对细菌、真菌等微生物及某些昆虫和动植物细胞具有抑制作用的小分子多肽。研究抗菌肽为开发新的抗菌素、抗病毒和肿瘤抑制剂开辟了广阔的前景。本实验的目的旨在从银杏果仁中分离纯化出一种抗菌肽、对其性质进行研究,并建立从银杏种仁中反转录克隆cDNA的方法体系,为克隆基因和研究其抗菌机理奠定基础。本实验以银杏种仁为研究材料,以常压柱层析法、离心式离子交换层析加电洗法脱、AKTA柱层析法等三种方法从银杏种仁中提取出一种抗菌肽,并对其纯度、分子量、抗菌活性、热稳定性、对极端pH值的抗性、对高盐浓度的抗性等性质进行了分析。通过异硫氰酸胍法、TRIzoL法,CTAB法以及SDS法分别提取银杏种仁的总RNA,建立反转录体系。主要获得以下结果:1、三种方法纯化出银杏种仁抗菌肽,此蛋白在SDS-PAGE上显示单条带,表观分子量为13000Da。AKTA柱层析法所得蛋白在双向电泳中达到单点纯。2、该蛋白在离子交换层析中以高盐浓度被洗脱,是一种碱性蛋白。对照其双向电泳图谱,其等电点在9.8左右。3、纯化的蛋白对黄瓜镰刀孢菌(Fusarium oxysporum)辣、椒早疫病菌(Alternariasolani)、小麦赤霉病菌(Fusarium graminearum)、葡萄灰霉病菌(Botrytis cinereaPers)、黄瓜炭疽病菌(Colletotrichum lagenarium)等14种真菌有抑制作用。4、该蛋白浓度达到2.5mg/ml以上即表现出明显的抑菌活性。具有较高热稳定性,在90℃处理30min对黄瓜镰刀孢菌抑菌活性无明显变化。在酸性条件下,对供试的黄瓜镰刀孢菌有很强的抑菌活性。对革兰氏阳性菌代表菌金黄色葡萄球菌和革兰氏阴性菌代表菌大肠杆菌均有一定的抑制作用。5、CTAB法提取得总RNA纯度高,完整性好,可用于反转录试验。更多还原

【Abstract】 The Antimicrobial peptides are acturally small peptides which can restain bacterium,fungi, insect cells, plant cells and animal cells. We can also call them Peptide antibiotics orNatural antibiotics. The research on Antimicrobial peptides can provide candidates forfiltering anti anticancer and antivirus medicament. The targets of the experiment are topurifying an Antimicrobial peptide from Ginkgo biloba seeds , to analyzing it’s qualities andestablish an RTPCRsysterm for Ginkgo biloba seeds. The whole work is the foundation ofit,s gene cloning and antimicrobil mechanism in molecularthe level.Using the seeds of ginkgo biloba as experiment material, an antimicrobial peptide isobtained by three methods. We also analyse it’s qualities of molecular weight, antimicrobialactivity, hot stability, extreme pH value stability and high salt concentration stability. We alsoobtain the total RNA of the Ginkgo biloba seed by four methods and establish an RTPCRsysterm.The following results was obntained:1 An antimicrobial peptide is obtained by three methods. In SDSPAGEthe peptideappears as a single band with a molecular weight is 13000 Da. The peptide obtained fromAKTA systerm appears as a single dot in 2Dspectrum.2 The peptide can be eluted from the CM ion exchange chromatography by high saltconcentration elution, so it is a basic peptide. It’s isoelectric point is 9.8 according to it’s 2Dspectrum.3 The peptide exerted potent antifugal activity against 14 fungi, including Fusariumoxysporum, Fusariumgraminearum,Botrytis cinerea, Cloeosporium orbiculare andFusarium oxysporium.4 The peptide appears atrong antifugal activity when its concentration is 2.5 mg/ml. Thepeptide has high hot stability, and it shows strong antifugal activity against Fusariumoxysporum when heating at 90℃for 30 minutes. it also shows strong antifugal activityaagainst Fusarium oxysporum in acidic circumstance. And the peptide exhibite a moderateantibacterial action against staphylococcus aureus and Escherichia Coli.5 The total RNA isolated by CTAB methode is with good quality, high purity andquantity. It is quite fit for the RTPCR.更多还原