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68.水牛瘤胃微生物多樣性分析

廖仁寶⁽¹⁾、 黃文瑛⁽¹⁾、 魏良原⁽¹⁾、 林正鏞⁽¹⁾、 吳明哲⁽¹⁾、 李佳音⁽²⁾

⁽¹⁾行政院農業委員會畜產試驗所 ⁽²⁾國立台灣大學農業化學系

瘤胃為一特殊複雜的厭氧環境，內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。本研究所採集之水牛瘤胃液樣品，其pH值約為5.6。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、古細菌、真菌及原蟲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，89個株系中所屬菌門有Bacteroidetes (57/89), Firmicutes(20/89), Proteobacteria (2/89),Lentisphaerae(1/89)及unclassified Bacteria (8/89)。在古細菌多樣性分析中，88個株系中所分屬之菌綱有Thermoplasmata (84/88)與Methaobacteria(1/88)，另外三株則屬於unclassified Euryarchaeota菌門。在真菌多樣性分析中，40個株系中有36個屬於Neocallimastix frontalis.，二株各分屬於Aspergillus penicillioides, Paecilomyces sp.,另二株則屬於未培養的真菌。在原蟲多樣性分析中，42個株系分屬於6種原蟲，由以上得知，瘤胃中細菌之多樣性較其他三種微生物最為豐富，應該可從其中藉由多源基因體學方式找到新穎之水解酵素基因。

關鍵語：瘤胃、微生物、多樣性、水解酵素

ANALYSIS OF MICROBIAL DIVERSITY FROM WATER BUFFALO RUMEN

R. B. Liaw⁽¹⁾, W. Y. Huang⁽¹⁾ , L. Y. Wei⁽¹⁾, J. Y. Lin⁽¹⁾, M. C. Wu⁽¹⁾ and C. Y. Lee⁽²⁾, 

⁽¹⁾Livestock Research Institute, Council of Agriculture, Executive Yuan;

⁽²⁾Deparment of Agricultural chemistry, National Taiwan University

Rumen is a special, complicated, and anaerobic environment consisting of different microorganisms such as bacteria, archaea, 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 water buffalo was about 5.6.  the microbial DNAs of rumen samples were extracted directly using a commercial kit.  further, the small subunit ribosomal RNA genes of bacteria, archaea, fungi and protozoa were amplified with their 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 in bacterial diversity analysis of 89 clones they belonged to bacterial phyla of Bacteroidetes (57/89), Firmicutes (20/89), Proteobacteria (2/89), spirochaetes (1/89), Lentisphaerae (1/89), and unclassified Bacteria (8/89).  According to archaea diversity analysis of 88 clones, there were two Classes of Thermoplasmata (84/88) and Methanobacteria (1/88) and the other three clones belonged to Phylum of unclassified Euryarchaeota.  In fungal diversity analysis, thirty six of 40 clones were identified as Neocallimastix frontalis and two clones as Aspergillus penicillioides and Pacecilomyces sp., repectively.  And the other two clones were recognized as uncultured fungi.  Besiders, six species of protozoa found in protozoan diversity analysis of 42 clones.  based upon the results above, the bacterial diversity of rumen was the most abundant that the other three kinds of microbes.  Therefore, it is possible to discover some novel hydrolase genes from such a sample through metagenomic approach.

Key Words: rumen, Microorganism, Diversity, Hydrolase.