基于高通量测序分析不同品种水牛生乳中细菌多样性  

Bacterial diversity of fresh milk in different buffalo breeds by high throughput sequencing

作　　者：谢芳[1] 谢华德 唐振华[1] 彭开屏 郭艳霞[1] 杨承剑[1] XIE Fang;XIE Hua-De;TANG Zhen-Hua;PENG Kai-Ping;GUO Yan-Xia;YANG Cheng-Jian(Key Laboratory of Buffalo Genetics,Breeding and Reproduction Technology,Ministry of Agriculture(Guangxi),Guangxi Buffalo Research Institute,Chinese Academy of Agricultural Sciences,Nanning 530001,China)

机构地区：[1]中国农业科学院广西水牛研究所/广西水牛遗传繁育重点实验室,南宁530001

出　　处：《食品安全质量检测学报》2020年第20期7527-7535,共9页Journal of Food Safety and Quality

基　　金：广西自然科学基金项目(2018GXNSFBA281006、2018GXNSFAA281162);国家重点研发计划项目子课题(2016YFD0500507、2018YFD0501600-02);广西水牛研究所人才小高地创新团队项目(2016)。

摘　　要：目的利用16S rRNA高通量测序研究广西水牛研究所3品种水牛生鲜乳中的细菌多样性。方法提取当天采集的水牛生乳样本细菌总DNA、PCR扩增细菌的16SrDNA、纯化PCR扩增目的片断,将PCR产物与pMD18-T载体连接,构建其菌群的16S rDNA文库。用Miseq对其16S rRNA基因的V3-V4可变区进行测序以及基于局部比对算法的搜索工具(basic local alignment search tool,BLAST)比对。结果3品种27个样本共获得1223个光转化单元(optical transform unit,OTUs),三品杂水牛乳、摩拉水牛乳、尼里-拉菲水牛乳独有的OTU分别为295、41和42,共有OTU为511。物种组成与丰度差异显示:3品种优势菌门为:变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)。优势菌属为:金黄杆菌属(Chryseobacterium)、不动杆菌属(Acinetobacter)、乳球菌属(Lactococcus)、假单胞菌属(Pseusomonas)。样本层级聚类显示:3品种样本间菌落组成相似度较高。PCA与PLS-DA分析表明:3品种样本组内菌群多样性差异不显著,但组间菌群丰度差异明显。结论摩拉、尼里-拉菲及三品杂水牛生鲜乳中细菌菌群均呈现较丰富的多样性,其共有的优势菌群为金黄杆菌(Chryseobacterium)、不动杆菌(Acinetobacter)、乳球菌(Lactococcus)、假单胞菌(Pseusomonas)。各品种间菌群组成种类整体相似,但其主要菌落丰富度差异明显。Objective To study the bacterial diversity in fresh milk of three species of buffalo in Guangxi Buffalo research institute by 16S rRNA high-throughput sequencing. Methods Total bacterial DNA of buffalo raw milk samples collected on the same day, 16S rDNA of bacteria amplified by PCR, and target fragment of purified PCR amplification were extracted. The PCR products were connected with PMD18-T vector to construct the 16S rDNA library of its bacterial community. The V3-V4 variable region of its 16S rRNA gene was sequenced by Miseq and compared with the basic local alignment search tool(BLAST). Results A total of 1223 optical transform units(OTUs) were obtained from 27 samples of 3 varieties. The OTU unique to Sanpai hybrid buffalo milk, Mora buffalo milk and Nili-Lafite buffalo milk were 295, 41 and 42 respectively, with a total OTU of 511. The difference in species composition and abundance showed that the dominant bacteria of 3 varieties were Proteobacteria, Firmicutes and Bacteroidetes. Dominant fungi were Chryseobacterium, Acinetobacter, Lactococcus and Pseusomonas. The hierarchical clustering of the samples showed that the composition of bacterial colonies among the samples of 3 varieties had a high similarity. PCA and PLS-DA analysis showed that there was no significant difference in bacterial diversity among 3 sample groups, but significant difference in bacterial abundance between the groups. Conclusion The bacterial diversity of fresh milk in 3 buffalo breeds are very high and the dominant bacteria are Chryseobacterium, Acinetobacter, Lactococcus, and Pseusomonas. The composition of the bacterial is similar among the varieties, but the abundance of the main bacterial is obviously different.

关 键 词：水牛 牛奶 细菌多样性 高通量测序 

分 类 号：TS252.1[轻工技术与工程—农产品加工及贮藏工程]