未培养技术在瘤胃产甲烷菌群研究中的应用

摘要/Abstract

摘要： 瘤胃中的产甲烷菌为严格厌氧微生物，很难通过分离培养的方法进行研究。现已经培养并公布的瘤胃产甲烷菌只有4种，成千上万种类的瘤胃产甲烷菌不能培养出来。未培养技术的发展与应用突破了传统分离培养检测方法的限制，使瘤胃产甲烷菌的研究有了较大的进展。综述了实时定量PCR、16S rDNA文库、DGGE和高通量测序技术等6项具有代表性的未培养技术及其在瘤胃产甲烷菌研究中的应用，为以后瘤胃产甲烷菌的研究提供方法上的参考。

关键词: 瘤胃, 产甲烷菌, 未培养技术, 全基因组测序, 高通量测序

Abstract: The ruminal methanogen are strictly anaerobic, which are hard to culture.The ruminal methanogen which have been cultured successfully are only 4 species.while hundreds of species of methanogen could not be cultured.The application and development of uncultured technology promotes the research of ruminal methanogen, which overcome the limitation of the tranditional technology of isolation and culturing. Several dominant culture-independent technologies used to study ruminal methanogens were introduced in this paper to provide references on methods for future ruminal methanogen study.

Key words: Rumen, Methanogen, Uncultured methods, Whole genome sequencing, High throughput, DNA sequencing

王兴文, 王加启, 赵圣国, 李发弟, 卜登攀. 未培养技术在瘤胃产甲烷菌群研究中的应用[J]. 生物技术通报, 2014, 0(6): 67-74.

Wang Xingwen,Wang Jiaqi,Zhao Shengguo,Li Fadi,Bu Dengpan. The Application of Uncultured Methods in the Study of Ruminal Methanogen Population[J]. Biotechnology Bulletin, 2014, 0(6): 67-74.

参考文献

[1] IPCC（Intergovernmental Panel on Climate Change）, Climate Change 2007[M] . The Scientific Basis. Cambridge, UK：Cambridge University Press, 2007.
[2] Lin C, Raskin L, Stahl DA. Microbial community structure in gastrointestinal tracts of domestic animals：comparative analyses using rRNA-targeted oligonucleotide probes[J] . FEMS Microbiol Ecol, 1997, 22：281-294.
[3] Janssen PH, Kirs M. Structure of the archaeal community of the rumen[J] . Appl Environ Microbiol, 2008, 74（12）：3619-3625.
[4] Ellermann J, Hedderich R, B?cher R, et al. The final step in methane formation. Investigations with highly purified methyl-CoM reductase（component C）from Methanobacterium thermoautotrophicum（strain Marburg）[J] . Eur J Biochem, 1988, 172（3）：669-677.
[5] Zhou M, McAllister TA, Guan LL. Molecular identification of rumen methanogens：Technologies, advances and prospects[J] . Anim Feed Sci Tech, 2011, 166-167：76-86.
[6] Yanagita K, Kamagata Y, Kawaharasaki M, et al. Phylogenetic anal- ysis of methanogens in sheep rumen ecosystem and detection of Met-hanomicrobium mobile By fluorescence in situ hybridization[J] . Biosci Biotechnol Biochem, 2000, 64（8）：1737-1742.
[7] Whitford MF, Teather RM, Forster RJ. Phylogenetic analysis of methanogens from the bovine rumen[J] . BMC Microbiol, 2001, 1：5.
[8] Skillman LC, Evans PN, Naylor GE, et al. 16S ribosomal DNA-directed PCR primers for ruminal methanogens and identification of methanogens colonising young lambs[J] . Anaerobe, 2004, 10（5）：277-285.
[9] Tajima K, Nagamine T, Matsui H, et al. Phylogenetic analysis of archaeal 16S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens[J] . FEMS Microbiol Lett, 2001, 200（1）：67-72.
[10] Wright AD, Williams AJ, Winder B, et al. Molecular diversity of rumen methanogens from sheep in Western Australia[J] . Appl Environ Microbiol, 2004, 70（3）：1263-1270.
[11] Shin EC, Choi BR, Lim WJ, et al. Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16S rDNA sequence[J] . Anaerobe, 2004, 10（6）：313-319.
[12] Whitehead TR, Cotta MA. Phylogenetic diversity of methanogenic archaea in swine waste storage pits[J] . FEMS Microbiol Lett, 1999, 179（2）：223-226.
[13] DeLong EF. Archaea in coastal marine environments[J] . Proc Natl Acad Sci USA, 1992, 89：5685-5689.
[14] Tokura M, Chagan I, Ushida K, et al. Phylogenetic study of methanogens associated with rumen ciliates[J] . Curr Microbiol, 1999, 39（3）：123-128.
[15] Marchesi JR, Weightman AJ, Cragg BA, et al. Methanogen and bacterial diversity and distribution in deep gas hydrate sediments from the Cascadia Margin as revealed by 16S rRNA molecular analysis[J] . FEMS Microbiol Ecol, 2001, 34（3）：221-228.
[16] Embley TM, Finlay BJ, Thomas RH, et al. The use of rRNA sequences and fluorescent probes to investigate the phylogenetic positions of the anaerobic ciliate Metopus palaeformis and its archaeobacterial endosymbiont[J] . J Gen Microbiol, 1992, 138（7）：1479-1487.
[17] Barns SM, Fundyga RE, Jeffries MW, et al. Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment[J] . Proc Natl Acad Sci USA, 1994, 91（5）：1609-1613.
[18] Baker GC, Smith JJ, Cowan DA. Review and re-analysis of domain-specific 16S primers[J] . J Microbiol Methods, 2003, 55（3）：541-555.
[19] Yu Z, García-González R, Schanbacher FL, et al. Evaluations of different hypervariable regions of archaeal 16S rRNA genes in profiling of methanogens by Archaea-specific PCR and denaturing gradient gel electrophoresis[J] . Appl Environ Microbiol, 2008, 74（3）：889-893.
[20] Bano N, Ruffin S, Ransom B, et al. Phylogenetic composition of Arctic Ocean archaeal assemblages and comparison with Antarctic assemblages[J] . Appl Environ Microbiol, 2004, 70（2）：781-789.
[21] Pinar G, Saiz-Jimenez C, Schabereiter-Gurtner C, et al. Archaeal communities in two disparate deteriorated ancient wall paintings：detection, identification and temporal monitoring by denaturing gradient gel electrophoresis[J] . FEMS Microbiol Ecol, 2001, 37：45-54.
[22] Kleikemper J, Pombo SA, Schroth MH, et al. Activity and diversity of methanogens in a petroleum hydrocarbon-contaminated aquifer[J] . Appl Environ Microbiol, 2005, 71（1）：149-158.
[23] Wright AD, Pimm C. Improved strategy for presumptive identifica-tion of methanogens using 16S riboprinting[J] . J Microbiol Met-hods, 2003, 55（2）：337-349.
[24] van Hoek AH, van Alen TA, Sprakel VS, et al. Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates[J] . Mol Biol Evol, 2000, 17（2）：251-258.
[25] Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation[J] . Microbiol Rev, 1995, 59（1）：143-169.
[26] Zhou M, Hernandez-Sanabria E, Guan LL. Assessment of the microbial ecology of ruminal methanogens in cattle with different feed efficiencies[J] . Appl Environ Microbiol, 2009, 75（20）：6524-6533.
[27] Hales BA, Edwards C, Ritchie DA, et al. Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis[J] . Appl Environ Microbiol, 1996, 62（2）：668-675.
[28] Luton PE, Wayne JM, Sharp RJ, et al. The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfill[J] . Microbiology, 2002, 148（11）：3521-3530.
[29] Hallam SJ, Girguis PR, Preston CM, et al. Identification of methyl coenzyme M reductase A（mcrA）genes associated with methane-oxidizing archaea[J] . Appl Environ Microbiol, 2003, 69（9）：5483-5491.
[30] Denman SE, Tomkins NW, McSweeney CS. Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane[J] . FEMS Microbiol Ecol, 2007, 62（3）：313-322.
[31] Scanlan PD, Shanahan F, Marchesi JR. Human methanogen diversity and incidence in healthy and diseased colonic groups using mcrA gene analysis[J] . BMC Microbiol, 2008, 8：79.
[32] Steinberg LM, Regan JM. Phylogenetic comparison of the methanogenic communities from an acidic, oligotrophic fen and an anaerobic digester treating municipal wastewater sludge[J] . Appl Environ Microbiol, 2008, 74（21）：6663-6671.
[33] Juottonen H, Galand PE, Yrj?l? K. Detection of methanogenic Archaea in peat：comparison of PCR primers targeting the mcrA gene[J] . Res Microbiol, 2006, 157（10）：914-921.
[34] Popova M, Martin C, Eugène M, et al. Effect of fibre and starch-rich finishing diets on methanogenic Archaea diversity and activity in the rumen of feedlot bull[J] . Anim Feed Sci Tech, 2011, 166-167：113-121.
[35] Sundset MA, Edwards JE, Cheng YF, et al. Rumen microbial diversity in Svalbard reindeer, with particular emphasis on methanogenic archaea[J] . FEMS Microbiol Ecol, 2009, 70（3）：553-562.
[36] Zhou M, Chung YH, Beauchemin KA, et al. Relationship between rumen methanogens and methane production in dairy cows fed diets supplemented with a feed enzyme additive[J] . J Appl Microbiol, 2011, 111（5）：1148-1158.
[37] Cheng YF, Mao SY, Liu JX, et al. Molecular diversity analysis of rumen methanogenic Archaea from goat in eastern China by DGGE methods using different primer pairs[J] . Lett Appl Microbiol, 2009, 48（5）：585-592.
[38] Franzolin R, St-Pierre B, Northwood K, et al. Analysis of rumen methanogen diversity in water buffaloes（Bubalus bubalis）under three different diets[J] . Microb Ecol, 2012, 64（1）：131-139.
[39] Huang XD, Tan HY, Long R, et al. Comparison of methanogen diversity of yak（Bos grunniens）and cattle（Bos taurus）from the Qinghai-Tibetan plateau, China[J] . BMC Microbiol, 2012, 12：237.
[40] 裴彩霞, 毛胜勇, 朱伟云. 晋南牛瘤胃中古菌分子多样性的研究[J] . 微生物学报, 2008, 48（1）：8-14.
[41] Tymensen LD, Beauchemin KA, McAllister TA. Structures of free-living and protozoa-associated methanogen communities in the bovine rumen differ according to comparative analysis of 16S rRNA and mcrA genes[J] . Microbiology, 2012, 158（7）：1808-1817.
[42] Hook SE, Northwood KS, Wright AD, et al. Long-term monensin supplementation does not significantly affect the quantity or diversity of methanogens in the rumen of the lactating dairy cow[J] . Appl Environ Microbiol, 2009, 75（2）：374-380.
[43] Frey JC, Pell AN, Berthiaume R, et al. Comparative studies of microbial populations in the rumen, duodenum, ileum and faeces of lactating dairy cows[J] . J Appl Microbiol, 2010, 108（6）：1982-1993.
[44] Danielsson R, Schnürer A, Arthurson V, et al. Methanogenic population and CH₄ production in swedish dairy cows fed different levels of forage[J] . Appl Environ Microbiol, 2012, 78（17）：6172-6179.
[45] Zened A, Combes S, Cauquil L, et al. Microbial ecology of the rumen evaluated by 454 GS FLX pyrosequencing is affected by starch and oil supplementation of diets[J] . FEMS Microbiol Ecol, 2013, 83（2）：504-514.
[46] Thoetkiattikul H, Mhuantong W, Laothanachareon T, et al. Compar-ative analysis of microbial profiles in cow rumen fed with different dietary fiber by tagged 16S rRNA gene pyrosequencing[J] . Curr Microbiol, 2013, 67（2）：130-137.
[47] Fouts DE, Szpakowski S, Purushe J, et al. Next generation sequencing to define prokaryotic and fungal diversity in the bovine rumen[J] . PLoS One, 2012, 7（11）：48289.
[48] Hristov AN, Callaway TR, Lee C, et al. Rumen bacterial, archaeal, and fungal diversity of dairy cows in response to ingestion of lauric or myristic acid[J] . J Anim Sci, 2012, 90（12）：4449-4457.
[49] Lee HJ, Jung JY, Oh YK, et al. Comparative survey of rumen microbial communities and metabolites across one caprine and three bovine groups, using bar-coded pyrosequencing and ¹H nuclear magnetic resonance spectroscopy[J] . Appl Environ Microbiol, 2012, 78（17）：5983-5993.
[50] Tymensen L, Barkley C, McAllister TA. Relative diversity and com-munity structure analysis of rumen protozoa according to T-RFLP and microscopic methods[J] . J Microbiol Methods, 2012, 88（1）：1-6.
[51] Hook SE, Steele MA, Northwood KS, et al. Impact of high-concent-rate feeding and low ruminal pH on methanogens and protozoa in the rumen of dairy cows[J] . Microb Ecol, 2011, 62（1）：94-105.
[52] Yá?ez-Ruiz DR, Macías B, Pinloche E, et al. The persistence of bac- terial and methanogenic archaeal communities residing in the rum-en of young lambs[J] . FEMS Microbiol Ecol, 2010, 72（2）：272-278.
[53] Leahy SC, Kelly WJ, Altermann E, et al. The genome sequence of the rumen methanogen Methanobrevibacter ruminantium reveals new possibilities for controlling ruminant methane emissions[J] . PLoS One, 2010, 5（1）：8926.
[54] Lee JH, Rhee MS, Kumar S, et al. Genome sequence of Methanobr-evibacter sp. strain jh1, isolated from rumen of Korean native cattle [J] . Genome Announc, 2013, 1（1）：2-13.
[55] Wedlock DN, Janssen PH, Leahy SC, et al. Progress in the develo-pment of vaccines against rumen methanogens[J] . Animal, 2013, 7（2）：244-252.
[56] Morgavi DP, Kelly WJ, Janssen PH, et al. Rumen microbial（meta）genomics and its application to ruminant production[J] . Animal, 2013, 7（1）：184-201.