【作者】 费晨；

【导师】 钱永华； 鲁兴萌；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2006， 硕士

【摘要】 从家蚕消化道分离得到5株肠球菌(C1、GC、FD、A20、F1-4),其中F1-4对家蚕微孢子虫(Nosema bombycis,简称N.b)体外发芽有抑制作用。本文研究了这5株分离菌株的生理生化特征和16S rDNA序列,并与肠球菌种特异性探针序列进行了比对,以期为进一步研究家蚕肠球菌的多样性及家蚕微粒子病的生物防治奠定基础。CTAB法抽提基因组DNA,采用肠球菌通用引物扩增16S rDNA,对产物进行测序,并用BLAST软件与GenBank中相关属种的16S rDNA序列比较。从GenBank中调取肠球菌属(Enterococcus)32个菌株的16S rDNA序列用于系统发育学分析。用CLUSTAL X (1.8)软件包对16S rDNA全序列进行排序,选择大肠杆菌(Escherichia coli)为外群,用MEGAversion 2软件包中的Kuimura2-Parameter Distance模型计算进化距离,用Neighber-Joining法构建系统发育树,1 000次随机抽样,计算自引导值(Bootstrap)以评估系统发育树的置信度。由系统发育分析结果可知,分离菌株与蒙氏肠球菌(Enterococcus mundtii AJ301836)有高同源性,在系统发育树中聚为一群。比较分离菌株16S rDNA序列和肠球菌种特异性探针序列,C1、F1-4、FD、GC、A20含有与蒙氏肠球菌种特异性探针完全相同的序列,与其它肠球菌探针均不完全相同。生理生化特征测定结果表明,除A20外,分离菌株均与蒙氏肠球菌种的特征基本一致。因此将5株分离菌株鉴定为蒙氏肠球菌。首次发现该菌种存在于家蚕消化道内。更多还原

【Abstract】 asdfgFive Enterococcus strains, namely C1、FD、GC、F1-4 and A20, were isolated from the midgut of the silkworm. Among them, F1-4 could inhibit the spore germination of Nosema bombycis. The biochemical characteristics and 16S rDNA sequences analyses of all isolates were studied in this paper, and the sequence similarity was compared between the 16S rDNA and the species-specific probes of Enterococci. The aim of this study was to obtain some basic information for further studies on the diversity of Enterococcus and utilize the characteristics of Enterococcus effectively for biological control of pebrine.qwerThe genomic DNA of isolates was extracted by CTAB method, and universal primers of Enterococci were used to amplify the sequences of 16S rDNA, and then the results of PCR were sequenced. The 16S rDNA sequences of isolates were compared with the related genus or species in GenBank by BLAST software. 32 sequences of 16S rDNA of Enterococcus were taken from GenBank for phylogenetic analysis. Alignment of the 16S rDNA sequences was done by CLUSTAL X (1.8) package. Escherichia coli as an extra group, evolutionary distances were calculated by using Kuimura2-Parameter Distance model of MEGA software package version 2 and phylogenetic tree was constructed by Neighber-Joining Method. 1 000 samples were chosen randomly to calculate the bootstrap value for evaluating the confidence level of phylogenetic tree. The results of phylogenetic analysis of isolates indicated that the 16S rDNA were highly homologous to the sequence of Enterococcus mundtii AJ301836. The investigation of sequences of the species-specific probes for Enterococcus spp. revealed that the 16S rDNA sequences of isolates included the same sequence as the species-specific probe of Enterococcus mundtii Biochemical characteristics of all isolates except for A20 coincided with that of Enterococcus mundtii. It was concluded that Enterococcus mundtii existed in the midgut of the silkworm. It was the firt time that Enterococcus mundtii was detected in the silkworm midgut.更多还原