基于DGGE和T-RFLP分析采食不同粗饲料梅花鹿瘤胃细菌区系  

Analysis of Bacterial Diversity in Rumen of Sika Deer(Cervus nippon) fed Different Forages Using DGGE and T-RLFP

作　　者：李志鹏[1] 姜娜 刘晗璐[1] 崔学哲[1] 荆祎[1] 杨福合[1] 李光玉[1] 

机构地区：[1]中国农业科学院特产研究所吉林省特种经济动物分子重点实验室,长春130112 [2]农业部环境保护科研监测所生态农业研究室,天津300191

出　　处：《中国农业科学》2014年第4期759-768,共10页Scientia Agricultura Sinica

基　　金：国家科技支撑计划(2011BAI03B02);中国农业科学院特产研究所创新工程专项经费

摘　　要：【目的】细菌区系在梅花鹿(Cervus nippon)瘤胃发酵中发挥着重要作用,然而有关梅花鹿瘤胃细菌区系的研究鲜有报道。研究梅花鹿瘤胃细菌区系,为梅花鹿瘤胃发酵调控提供分子生物学依据。【方法】选取4头2岁龄的装有永久性瘤胃瘘管的成年雄性梅花鹿为研究对象,分别饲喂以柞树叶(OL组,梅花鹿A和B)和玉米秸秆(CS组,梅花鹿C和D)为主要粗饲料的日粮,持续饲喂30 d。通过瘤胃瘘管取瘤胃内固液混合物,提取瘤胃微生物基因组DNA。扩增瘤胃细菌16S rRNA基因V3区以及16S rRNA基因,分别用于DGGE和T-RFLP分析。DGGE图谱进行聚类分析,并切取优势条带进行克隆测序,鉴定瘤胃内细菌组成。根据T-RFLP图谱结果计算细菌群落的多样性、优势度、均匀度和丰富度,通过Microbial Community AnalysisⅢ(MiCAⅢ)数据库推测T-RFs可能代表的微生物种类,并进行T-RFLP图谱的聚类分析。【结果】DGGE图谱聚类表明,CS组和OL组瘤胃细菌区系相似性低于65%,表明粗饲料种类影响梅花鹿瘤胃细菌区系。OL组梅花鹿A和B的DGGE图谱相似度大于70%,CS组梅花鹿C和D的DGGE图谱相似性大于75%,而且同组不同个体之间瘤胃细菌区系存在差异。OL组和CS组分别获得20和24个DGGE特异性条带。序列分析表明,CS组条带可归类为拟杆菌门、厚壁菌门和变形菌门,而OL组条带可归类为拟杆菌门、厚壁菌门、变形菌门和互养菌门。OL组与CS组中存在大量Prevotella spp.,但不同组中Prevotella spp.在种水平组成不同,主要纤维降解菌为Clostridium spp.与Eubacterium spp.。T-RFLP结果显示,梅花鹿D(CS组)具有最高的丰富度、多样性、均匀度和最低的优势度,梅花鹿A和B的各项指数相近但低于梅花鹿D,说明OL组中的粗饲料(柞树叶)影响瘤胃中微生物的相对生物量。梅花鹿C和D的各项指数相差较大而且梅花鹿C的指数低于梅花鹿A和B,表明同组不同个体之间存在差异。81�[ Objective ] Bacterial communities play critical roles in the rumen fermentation of Sika deer （Cervus nippon）, while the bacterial composition in the rumen of Sika deer is rarely reported. The objective of present study is to investigate the bacterial diversity in the rumen of sika deer, which can provide a molecular basis for manipulation of rumen fermentation. [ Method ] Four two year old male rumen-cannulated Sika deers fed oak leaf （OL group, Sika deer A and B） and corn stover （CS group, Sika deer C and D） based diets were used in the present study. After 30 days of feeding, rumen contents including solid and liquid fractions were sampled, and the microbial genomic DNA was extracted. V3 region of ruminal bacterial 16S rRNA gene and 16S rRNA gene was amplified, which was used in the DGGE and T-RFLP analysis, respectively. The clustering analysis was applied to DGGE results. The dominant bands in DGGE profiles were obtained, and then used to clone sequencing in order to indentify the bacterial communities. The results of T-RFLP were also applied to clustering analysis, and the possible bacterial structure was speculated by Microbial Community AnalysisIII （MiCAIII） dataset. [ Result] The clustering patterns of DGGE revealed that the similarity of bacterial diversity between CS group and OL group was lower than 65%, indicating that the bacterial diversity was affected by forage source. The similarity between Sika deer A and B in the OL group, and between Sika deer C and D in the CS group was greater than 70% and 75%, respectively. In addition, the differences were found between animlas in the same group. A total of 20 and 24 unique DGGE bands were obtained from the OL and CS groups, respectively. Sequences analysis of DGGE showed that the bacteria in the OL group were composed of Bacteroides, Firmicutes and Proteobacteria phyla, while they were composed of Bacteroides, Firmicutes, Proteobacteria and Synergistetes phyla in the CS group. Prevotella spp. were the dominant bacteria in the OL and CS g

关 键 词：梅花鹿 细菌区系 普雷沃氏菌 单宁 

分 类 号：S825[农业科学—畜牧学]