第三十五卷(2006)
59.乳牛瘤胃微生物多樣性分析
廖仁寶(1)、江家豐(1)、黃文瑛(1)、謝昌賢(1)、蕭宗法(1)、吳明哲(1)、李佳音(2)
行政院農業委員會畜產試驗所(1) 國立台灣大學農業化學系(2)
瘤胃為一特殊複雜的厭氧環境含之微生物包括細菌、古細菌、真菌、原蟲，且85-95%尚未能以人工分離培養。本研究所採集之乳牛瘤胃液樣品，其pH值約為6.8，於顯微鏡下觀察，可發現原蟲與格蘭氏陰性和陽性的細菌。使用商業核酸萃取套組直接萃取瘤胃液中之微生物核酸。其後分別以細菌與真菌小單位核醣體核酸基因專一性之引子進行PCR增幅放大，並將增幅片段選殖於TA選殖套組，進行DNA定序與瘤胃微生物多樣性分析。其結果顯示在細菌多樣性分析中，52個株系中有521個是屬於未被分離培養或未被鑑定者，僅一株屬於 Fibrobacter succinogenes (相同度為96%)，而所屬菌門則有bacteroidetes(32/52), Fibrobacteres (1/52), Firmicutes (18/52)及unclassified Bacteria(1/52)。在真菌多樣性分析中，75個株系中有74個屬於Neocallimastix sp.，另僅一株屬於Trichosporon jirovecii。由以上得知，瘤胃中細菌之多樣性較為豐富，應該可從其中藉由多源基因體學方式找到新穎之水解酶基因。
關鍵語：瘤胃、微生物、多樣性
Rumen is a special, complicated, and anaerobic environment consisting of different microorganisms such as bacteria, archeae, fungi, and protozoa, and 85% to 95% of these microbes are very hard to cultivate individually and artificially. In this study, the pH value of the rumen fluid from dairy cow was about 6.8. Protozoa and Gram positive and negative bacteria were found under microscopic investigation. The microbial DNAs of rumen samples were extracted directly using a commercial kit. Further, the small subunit ribosomal RNA genes of bacteria and fungi were amplified with bacterial and fungal specific sets of primers by PCR, respectively. Then the amplicons were ligated into TA cloning vector for DNA sequencing and microbial diversity analyses. The results indicated that 51 of 52 clones were classified as uncultured or unidentified bacteria in bacterial diversity analysis and only one was Fibrobacter succinogenes (identities: 96%). And they belonged to bacterial phyla of Bacteroidetes (32/52), Fibrobacteres (1/52), Firmicutes, and unclassified Bacteria (1/52). In fungal diversity analysis, 74 of 75 clones were identified as Neocallimastix sp. and only one as Trichosporon jirovecii. Based upon the results above, the bacterial diversity of rumen was more abundant than that of fungi. Therefore, it is possible to discover some novel hydrolase genes from such a sample through metagenomic approach.
Key Words: Rumen, Microorganism, Diversity
  
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