摘要
采用16S rDNA分析方法,分别提取中华鳖体表和体内细菌总DNA,以之为模板进行PCR扩增获得细菌16S rDNA基因片段,并构建16S rDNA克隆文库。从2个文库中分别随机选取100个重组克隆子,抽提重组质粒,用HaeⅢ和MspⅠ限制性内切酶进行酶切,获得酶切指纹图谱,从每种图谱随机挑选克隆子测序,获得其16S rDNA部分序列,构建了系统进化树。在中华鳖体表和体内细菌克隆文库中分别包含13种和10种RFLP谱型,分别挑选了70个和60个克隆子进行测序。结果表明,中华鳖体表和体内文库克隆子分别归为15类和11类OTU,体表和体内菌群多样性较强,优势细菌类群分别为拟杆菌和变形菌,并在文库中检测到了几株益生菌和条件致病菌。
Diversity of external and internal bacteria associated with soft-shelled turtle（Trionyx sinensis）was determined by 16S rDNA gene sequences.With culture-independent approach,total bacterial DNA was extracted from external and internal bacteria respectively.16S rDNA genes were amplified by PCR,and clone library were constructed.The phylogenetic diversity of the bacteria was determined by RFLP-16S rDNA sequencing of the cloned DNA fragments.A total of 100 cloned fragments from each library were digested with restriction endonuclease Hae Ⅲ and MspⅠ.Thirteen and 10 different RFLP types were obtained from external and internal bacterium 16S rDNA clone library respectively.Seventy and 60 cloned partial sequences of external and internal bacterium clone library were acquired;they belonged to 15 and 11 OUT,respectively.The phylogenetic trees showed that they were mainly related to Bacteroidetes and Proteobacteria respectively.Flavobacterium sp.were detected in both cloned librarys,and they were dominate species.Also,several beneficial bacteria（Lactococcus lactis and Pseudomonas sp.）and conditional pathogens（Edwardsiella tarda,and Citrobacter freundii）were detected.Results demonstrated that the prolific diversity of external and internal bacteria associated with the soft-shelled turtle.
出处
《水产科学》
CAS
北大核心
2010年第7期393-398,共6页
Fisheries Science
基金
国家自然科学基金资助项目(30840061)
宁波大学学科项目(XK0715049)
宁波大学人才项目(XR0713017)
浙江省自然科学基金人才培养项目(R305333)
关键词
中华鳖
16S
RDNA
RFLP
细菌多样性
soft-shelled turtle（Trionyx sinensis）
16S rDNA
RFLP
bacterial diversity
