摘要：我们通过前期工作分离、纯化了一株具有抑菌活性的内生真菌，保藏于江苏师范大学江苏省药食植物生物技术国家重点实验室培育点。我们利用形态鉴定方法结合分子生物学方法对其进行了鉴定。形态学观察显示，将该菌株与25℃下培养8天，可见白色菌落，菌丝绒毛状，菌落直径6.5cm，边缘规则，菌落背面初无色，培养7日后中部开始呈暗红色，并逐渐加深；分生孢子梗不一，细长、简单，分枝不规则，分生孢子无色，不一，有大小型两种，大型分生孢子长棒形，小型分生孢子卵圆形，常聚生。利用氯化苄法提取菌株的基因组DNA，对其ITS区域进行PCR扩增及测序，并在NCBI网站上进行核酸BLSAT分析，结果表明所测序列与一株Fusarium sp.的ITS序列（登录号：KM513579.1）相似度达99%。应用MEGA 6.0软件构建系统发育树，结果表明，该菌株与上述Fusarium sp.聚类于同一末端分支。结合形态学方法和分子生物学方法，将该菌株鉴定为Fusarium sp.。49318

毕业论文关键词： 内生真菌、分离、纯化、鉴定  

Title:Isolation, purification and identification of an endophytic fungus

Abstract : We have isolated and purified an endophytic fungal strain with antimicrobial activity in the previous work, preserved in the Key Laboratory of Biotechnology for Medical Plant of Jiangsu Province, Jiangsu Normal University.We identified the strain using both the morphological method and DNA-based techniques. The strain was cultured at 25 ℃ for 8 days, a visible white colony developed, the  diameter of which was 6.5cm. The edge was rule and the back of the colony was colorless. After it was cultured 7 days, the middle of the culture turned dark red and gradually deepen. The conidiophores was simple, long and thin, branched irregularly. There were two kinds of spores, large and small, both colorless.The large type of spores were long-stick, and the small type of spores were oval and often in groups. we used benzyl chloride method to extract strain’s genomic DNA and conducted PCR amplification for its ITS region. The BLSAT analysis was carried out on the NCBI site. The result showed that the similarity of the measured sequence and the ITS sequence of a strain of Fusarium sp. (GenBank accession number: KM513579.1) was as high as 99%. Phylogenetic tree was conducted using MEGA 6 software, showing that the strain was at the same end of the branch with the above Fusarium sp. The strain was thus identified as Fusarium sp. 

Key words:endophytic fungi; isolation; purification; identification

目录

引言 5

1材料与方法 6

1.1材料 6

1.2仪器 6

1.3试剂 6

1.4实验方法 7

1.4.1 内生真菌的分离、纯化 7

        1.4.2形态学鉴定 7

1.4.3菌株ITS序列分析 7

2实验结果 9

2.1 形态学鉴定 9

2.2 ITS序列分析 10

3讨论 12

致谢 13

文献参考 14

引言

随着内生真菌在医药、工业中的广泛应用，人们对内审真菌的研究越来越深入。内生真菌一词“endophyte”最初是由德国科学家巴里于1886年提出[1，2]，主要指自然界中普遍在植物中生存，某一阶段或全部阶段生活在寄主组织中但不引植物产生明显病害的一类真菌。