三种室内饲养鱼类肠道微生物群落PCR-DGGE指纹分析  

PCR-DGGE FINGERPRINTING ANALYSIS ON INTESTINAL MICROBIAL COMMUNITY OF THREE INDOOR REARING FISHES

作　　者：李学梅[1,2] 余育和[1] 解绶启[3] 颜庆云[1] 陈宇航[3,2] 董小林[3,2] 

机构地区：[1]中国科学院水生生物研究所中国科学院水生生物多样性与保护重点实验室,武汉430072 [2]中国科学院研究生院,北京100049 [3]中国科学院水生生物研究所淡水生态与生物技术国家重点实验室,武汉430072

出　　处：《水生生物学报》2011年第3期423-429,共7页Acta Hydrobiologica Sinica

基　　金：国家重点基础研究发展计划(2007CB109205);国家自然科学基金(30970358)资助

摘　　要：以室内饲养的斑点叉尾(Ictalurus punctatus)、银鲫和异育银鲫(中科三号)(Carassius auratus gibelio)幼鱼为对象,通过PCR-DGGE指纹技术对其肠道微生物群落进行了探索研究。在三种鱼的肠道中检测到不同的PCR-DGGE指纹谱带,其中斑点叉尾的平均谱带数(7.5)相对于银鲫和异育银鲫的谱带数(分别为15和14)要少。基于PCR-DGGE指纹谱带及各谱带相对丰度的UPGMA聚类和MDS排序结果显示银鲫和异育银鲫肠道微生物相似性高,而与斑点叉尾的差异比较大;rank-abundance散点图及回归分析也显示斑点叉尾与银鲫、异育银鲫肠道微生物群落存在显著差异(P<0.05),而银鲫和异育银鲫之间无显著差异(P=0.383)。在斑点叉尾肠道中检测到的菌群主要是变形杆菌,包括γ-变形杆菌和α-变形杆菌;而银鲫和异育银鲫肠道中菌群主要包括梭杆菌属中的类群,还包括变形杆菌门中的气单胞菌属,以及一些未知的类群。以上结果均表明在所研究的三种鱼的幼鱼阶段,其肠道微生物组成在不同种类鱼中存在差异,且该差异受基因型的影响可能更大。PCR-DGGE fingerprinting was explored to study intestinal microbial community of three indoor rearing ju-venile fishes（channel catfish,silver prussian carp and hybridized prussian carp）.Each fishes with three repetitions were reared in a semi-recirculating system in Institute of Hydrobiology.During the experiment,water temperature was 23℃—30℃,and fish were fed to satiation twice daily（9：00 and 15：00）.After 30 days,3 fishes of each treatment were randomly collected to extract microbial DNA.Gastrointestinal samples of all fishes were obtained by aseptically dis-secting the fish and carefully extracting the entire gastrointestinal tract.After dissecting,different segments of each gastrointestinal tracts from 3 fishes of the same treatment were pooled to analyse the bacterial communities.DNA from species samples were obtained according to the traditional Phenol-chloroform method.Bacterial domain-specific prim-ers F357（5＇-CCTACGGGAGGCAGCAG-3＇ with GC clamp in the 5＇ end） and R518（5＇-ATTACCGCGGCTGC TGG-3＇） were used to amplify the variable V3 region of 16S rRNA genes.PCR products were separated on a 9% polyacrylamide gel（ratio of acrylamide to bisacrylamide was 37.5∶1） in a 1×TAE buffer and a denaturing gradient from 35% to 50% of urea and formamide.Unweighted pair-group method using arithmetic averages（UPGMA） and Multidimensional Scaling（MDS） were used to investigate community similarity of bacterial among different fish species.Moreover,rank– abun-dance plots were used to determine differences in bacterial community structure based on taxa relative abundance.For each plot,a linear regression model was fitted,and it was represented by the equation,log10y = a ＋ bx,where a was the intercept and b was the slope of the plot.The slope（b） was subsequently used as a descriptive statistic for changes in community structure.Linear regressions,coefficients of determination（r） and significance（P） were calculated by using SPSS 13.0 software.Special DNA bands on DGG

关 键 词：斑点叉尾 银鲫 异育银鲫 PCR-DGGE 肠道微生物群落 

分 类 号：Q145.2[生物学—生态学] Q781[生物学—普通生物学]