16SrDNA克隆文库法分析拜城油鸡盲肠细菌多样性研究
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摘要
为了研究拜城油鸡盲肠细菌物种多样性,本研究采用基于16SrDNA基因序列系统发育分析的免培养方法进行细菌物种多样性分析,以盲肠内容物样品总DNA为模板,利用细菌通用引物扩增16SrDNA基因,构建16SrDNA基因克隆文库,并对文库中的插入序列进行RFLP分析。结果表明:随机挑选的252个克隆为阳性克隆,阳性率为65.7%,经过RFLP筛选出95个不同图谱的重组克隆并测序,比对分析后发现:拜城油鸡盲肠细菌分属于厚壁菌门(Firmicutes)(65.26%)、拟杆菌门(Bacteroidetes)27.37%)和变形菌门(Proteobacteria)(4.21%)。在纲水平有梭菌纲(Clostridia)、拟杆菌纲(Bacteroidia)和ε-变形菌纲(Epsilonproteobacteria)相对含量最多,分别占总菌的64.21%、26.32%和4.21。在科水平上以瘤胃菌科(Ruminococcaceae)(21.05%)、毛螺菌科(Lachnospiraceae)(20%)和理研菌科(Rikenellaceae)(12.63%)相对含量最多,为优势科。在属水平上仅有31.58%的序列可以鉴定到属,Lachnoclostridium的相对含量最多,占总菌的12.63%,其次为另枝菌属(Alistipes)和瘤胃梭菌属(Ruminiclostridium)。拜城油鸡盲肠内容物中的细菌组成具有丰富的多样性,并有新细菌分类单位的潜在资源,值得进一步进行开发研究。
In order to study Baicheng chicken cecal bacterial species diversity,16 SrDNA gene sequence for phylogenetic analysis with free culture method for analysis of bacterial species diversity was conducted in the present research.With total DNA of cecum sample as template and universal primer of 16 SrDNA gene for amplification,a 16 srDNA clone library was established,and the insertion sequence were analyzed with RFLP.The results showed that 252 randomly selected clones were positive,with a rate of 65.7%.RFLP screened 95 different profiles of recombinant clone and sequenced to find that,three phyla,4classes,9families and 8genera were identified via taxonomic summary.The dominant phyla among them were Firmicutes(65.26%),Bacteroidetes(27.37%)and Proteobacteria(4.21%).The most abundant classes were Clostridia,Bacteroidia and Epsilonproteobacteria,accounting for 64.21%,26.32%and 4.21,respectively.Ruminococcaceae(21.05%),Lachnospiraceae(20%)and Rikenellaceae(12.63%)were the predominant families.Only 31.58% of the sequences at the genus level can be identified,the most abuadant genera were Lachnoclostridium(12.63%),followed by Alistipes and Ruminiclostridium.Baicheng Soy Sauce Chicken had a rich diversity ef bacterial composition in cecal ccntents and new potential bacterial faxon,ready for further research and development.
In order to study Baicheng chicken cecal bacterial species diversity,16 SrDNA gene sequence for phylogenetic analysis with free culture method for analysis of bacterial species diversity was conducted in the present research.With total DNA of cecum sample as template and universal primer of 16 SrDNA gene for amplification,a 16 srDNA clone library was established,and the insertion sequence were analyzed with RFLP.The results showed that 252 randomly selected clones were positive,with a rate of 65.7%.RFLP screened 95 different profiles of recombinant clone and sequenced to find that,three phyla,4classes,9families and 8genera were identified via taxonomic summary.The dominant phyla among them were Firmicutes(65.26%),Bacteroidetes(27.37%)and Proteobacteria(4.21%).The most abundant classes were Clostridia,Bacteroidia and Epsilonproteobacteria,accounting for 64.21%,26.32%and 4.21,respectively.Ruminococcaceae(21.05%),Lachnospiraceae(20%)and Rikenellaceae(12.63%)were the predominant families.Only 31.58% of the sequences at the genus level can be identified,the most abuadant genera were Lachnoclostridium(12.63%),followed by Alistipes and Ruminiclostridium.Baicheng Soy Sauce Chicken had a rich diversity ef bacterial composition in cecal ccntents and new potential bacterial faxon,ready for further research and development.
引文
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[10]沙尔山别克·阿不地力大,李海英,努尔江·买地亚尔,等.不同饲养方式对拜城油鸡生长、屠宰性能及肉品质的影响[J].新疆农业科学,2011(11):2 121-2 128.
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[12]黄进强,刘哲,王建福,等.盐碱池塘养殖鲤肠道菌群的分子分析[J].农业生物技术学报,2013(6):707-715.
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[14]韩晓雪,吕玲玲,罗晓霞,等.罗布泊糖霉菌属放线菌系统进化分析[J].塔里木大学学报,2014,26(4):8-15.
[15]熊婧,张思璐,魏霜,等.属特异性T-RFLP技术在双歧杆菌群落分析中的应用[J].微生物学通报,2014,41(12):2 538-2 546.
[16]贾晓宇,贺江舟,关统伟,等.新疆红井子盐碱土壤非培养放线菌多样性[J].微生物学通报,2012,39(5):606-613.
[17]李萍,曾军,祖丽皮亚·玉努斯,等.新疆乌鲁木齐10号冷泉古菌群落结构多样性[J].微生物学报,2013,53(3):230-240.
[18]Lumpkins BS,Batal AB,Lee MD.Evaluation of the bacterial community and intestinal development of different genetic lines of chickens[J].Poult Sci,2010,89(8):1 614-1 621.
[19]林奕岑,徐帅,倪学勤,等.利用Illumina MiSeq测序平台分析肉鸡盲肠微生物多样性[J].中国农业大学学报,2016,21(12):65-73.
[20]李荣才,邵路,刘兴义.博尔塔拉蒙古自治州赛里木湖区域放养拜城油鸡治蝗试验效果初报[J].新疆畜牧业,2012(3):28-29.
[2]Savage DC.Microbial ecology of the gastrointestinal tract[J].Annu Rev Microbiol,1977,31:107-33.
[3]雷蕾,张日俊.鸡肠道正常菌群的研究进展[J].中国微生态学杂志,2008,20(3):298-299,303.
[4]何维明,于致茂,严宝英.鸡空肠和盲肠微生物区系的研究[J].西北农林科技大学学报(自然科学版),1998,26(1):49-53.
[5]刘晨黎,高巍.鸡盲肠微生物微生态调控研究进展[J].中国家禽,2000,22(10):30-32.
[6]徐爱蕾,黄毅,王为.16SrRNA基因序列分析技术在肠道菌群检测中的应用进展[J].临床消化病杂志,2015,27(3):186-189.
[7]王小霞,鲁建国,张敏,等.新疆拜城油鸡[J].新疆畜牧业,1995(4):42-43.
[8]格明古丽,许宗运,吾买尔江.艾孜木等.新疆拜城油鸡[J].中国家禽,2003,25(1):38-39.
[9]格明古丽·木哈台,王永,刘利林等.拜城油鸡种质特性测定[J].中国家禽,2013,25(22):57-60.
[10]沙尔山别克·阿不地力大,李海英,努尔江·买地亚尔,等.不同饲养方式对拜城油鸡生长、屠宰性能及肉品质的影响[J].新疆农业科学,2011(11):2 121-2 128.
[11]杨飞,冯隆祖,韩春梅,等.新疆拜城油鸡3种生长模型的比较研究[J].江苏农业科学,2011(5):292-293.
[12]黄进强,刘哲,王建福,等.盐碱池塘养殖鲤肠道菌群的分子分析[J].农业生物技术学报,2013(6):707-715.
[13]Jiang H,Dong H,Zhang G,et al.Fields MW.Microbial diversity in water and sediment of Lake Chaka,an athalassohaline lake in northwestern China[J].Appl Environ Microbiol,2006,72(6):3 832-3 845.
[14]韩晓雪,吕玲玲,罗晓霞,等.罗布泊糖霉菌属放线菌系统进化分析[J].塔里木大学学报,2014,26(4):8-15.
[15]熊婧,张思璐,魏霜,等.属特异性T-RFLP技术在双歧杆菌群落分析中的应用[J].微生物学通报,2014,41(12):2 538-2 546.
[16]贾晓宇,贺江舟,关统伟,等.新疆红井子盐碱土壤非培养放线菌多样性[J].微生物学通报,2012,39(5):606-613.
[17]李萍,曾军,祖丽皮亚·玉努斯,等.新疆乌鲁木齐10号冷泉古菌群落结构多样性[J].微生物学报,2013,53(3):230-240.
[18]Lumpkins BS,Batal AB,Lee MD.Evaluation of the bacterial community and intestinal development of different genetic lines of chickens[J].Poult Sci,2010,89(8):1 614-1 621.
[19]林奕岑,徐帅,倪学勤,等.利用Illumina MiSeq测序平台分析肉鸡盲肠微生物多样性[J].中国农业大学学报,2016,21(12):65-73.
[20]李荣才,邵路,刘兴义.博尔塔拉蒙古自治州赛里木湖区域放养拜城油鸡治蝗试验效果初报[J].新疆畜牧业,2012(3):28-29.