CN101368214B - 用于古细菌多样性分析的针对16S rRNA基因的引物 - Google Patents

用于古细菌多样性分析的针对16S rRNA基因的引物 Download PDF

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CN101368214B
CN101368214B CN2008102002805A CN200810200280A CN101368214B CN 101368214 B CN101368214 B CN 101368214B CN 2008102002805 A CN2008102002805 A CN 2008102002805A CN 200810200280 A CN200810200280 A CN 200810200280A CN 101368214 B CN101368214 B CN 101368214B
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archeobacteria
rrna gene
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CN101368214A (zh
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宋任涛
濮佳
胡启雯
贺其其
李莉
马中良
许政暟
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University of Shanghai for Science and Technology
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Abstract

本发明涉及一对用于古细菌多样性分析的针对16S rRNA基因的引物。该引物的碱基序列为:f,(5’-ACGGCTCAGTAACACG-3’);r,(5’-CCGCCAATTCCTTTAAGT-3’)。本发明可用于聚合酶链式反应(PCR),通过实验证明能很好反映实验对象中古细菌的多样性。

Description

用于古细菌多样性分析的针对16S rRNA基因的引物
技术领域
本发明涉及一对用于PCR的引物。特别是一对用于古细菌多样性分析的针对16S rRNA基因的引物。
背景技术
在过去的二十年中,微生物多样性研究的方法有了飞速的发展。这些手段主要依赖于聚合酶链式反应(PCR)对目的基因的扩增。16S rRNA基因是这类研究中使用最有效最普遍的分子标记。这些分子生物学方法的进展使我们能对未能培养的微生物进行一定的研究,有助于我们对来自各种环境中的未知微生物群落进行分类学和生态学层面上的研究。古细菌在形态学和生理学上都是多种多样的。它们可以是球形、杆形、螺旋形、不规则形和多态形等。有些是单细胞的,而有些是形成菌丝体或团聚体。直径范围一般在0.1μm至15μm,一些菌丝体能长到200μm。古细菌通常在极端生境中被发现,例如产甲烷古细菌大量生长在含丰富有机物的厌氧环境中:沼气池、动物的瘤胃和肠道系统、水域中的沉积物、沼泽湿地、温泉,甚至是厌氧原生动物体内。目前,古细菌研究正在世界范围内升温,这不仅因为古菌中蕴藏着远多于另两类生物的、未知的生物学过程和功能,以及有助于阐明生物进化规律的线索,而且因为古细菌有着不可估量的生物技术开发前景。目前,微生物多样性的研究主要依赖于对16S rRNA基因这类研究中使用最有效最普遍的分子标记的分析。而这种分析通常需要使用PCR技术。引物是PCR的关键,直接影响到能否全面反映微生物多样性。尽管有多种古细菌的16S rRNA引物在研究中被用到,但缺乏适合于单方向测序可读取长度的的此类引物。
发明内容
本发明的目的在于提供一对用于古细菌多样性分析的针对16S rRNA基因的引物。
为达到上述目的,本发明搜集了Sulfolobus acidocaldarius,Sulfolobus solfataricus,Sulfolobus tokodaii,Archaeoglobus fulgidus,Haloarcula marismortui,Halobacterium sp.,Haloquadratum walsbyi,Natronomonas pharaonis,Methanosphaera stadtmanae,Methanothermobacter thermautotrophicus,Methanococcus maripaludis,Methanospirillumhungatei,Methanopyrus kandleri,Methanococcoides burtonii,Methanosarcinaacetivorans,Methanosarcina barkeri,Methanosarcina mazei,Pyrococcus abyssi,Pyrococcus furiosus,Pyrococcus horikoshii,Thermococcus kodakarensis,Picrophilustorridus,Thermoplasma acidophilum,Thermoplasma volcanium,Nanoarchaeum equitans的全长16S rRNA基因,运用MEGA4.0软件进行序列排列,挖掘其保守区段的序列。随后运用引物设计软件Primer Premier5.0进行引物设计。考虑到后续测序能力,每条序列单方向测序的有效读取长度一般小于1000个碱基,所以设计出扩增片段长度约800个碱基对的引物,该引物的碱基序列为:
f,(5’-ACGGCTCAGTAACACG-3’);r,(5’-CCGCCAATTCCTTTAAGT-3’)。
与现有技术相比,本发明的用于古细菌多样性分析的针对16S rRNA基因的引物具有很好的通用性和实用性,能良好反映实验对象中古细菌的多样性,可以适用于来源于土壤,沉积物,人畜胃肠道等环境样品的分析。
附图说明
图1是实施例1中的PCR电泳图,S为PCR产物
图2是实施例2中,根据所构建的16S rDNA克隆文库的测序结果所建立的系统发育进化树的一个小分支。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体实验条件的实验方法,通常按照常规条件,如分子克隆(Molecular Cloning:A Laboratory Manual,3rd ed.)中所述条件,或按照制造厂商所建议的条件。
实施例一:使用本发明引物进行对环境微生物基因组DNA样品的扩增
取来源于沼气池浆中微生物基因组DNA样品,进行PCR扩增。PCR的反应体系为50μl:10×buffer:5μl,10mM dNTP:1μl,10mM primerf:2.5μl,10mM primerr:2.5μl,Taq酶(2U/μl):1μl,DNA:0.5μl,ddH2O:37.5μl。PCR参数设计如下:起始变性5分钟,95℃;30个循环(变性30秒,95℃;退火30秒,55℃;延伸50秒,72℃);最后延伸7分钟,72℃。琼脂糖凝胶电泳所用琼脂糖浓度为1%的凝胶,含0.5μg/mL溴化乙锭,所用DNA分子量标记(Marker)为北京天为时代科技有限公司的100bp ladder。电泳结果见图1。
实施例二:由本发明扩增得到的PCR产物构建16S rDNA克隆文库,并建立系统发育进化树。具体方法为:使用实施例1中的方法得到PCR产物。将PCR产物经
Figure G2008102002805D0002152143QIETU
PCR Purification Kit Columns纯化,并与T载体连接,经电击转化构建16S rDNA克隆文库。从每个样品中随机挑选出克隆进行96孔板扩增培养,使用碱法板抽转化子质粒。获得的质粒使用DYEnamicTM ET Dynamic Terminator Sequencing (AppliedAmersham)和2pm的测序引物M13R(5’-CAGGAAACAGCTATGAC-3’)做测序反应,然后使用MegaBACE4500DNA测序仪(Applied Amersham)进行单向测序。对获得的序列剔除属于载体的序列和质量值低于20的序列,保留可读长度约700个碱基的序列做进一步的系统分类分析。
序列经MEGA4.0排列。依据测序图谱,对每条序列进行人工编辑,只保留测序碱基位点明确的序列做后续分析,引物的序列被去除。对所获得序列使用MEGA4.0建系统发育进化树。图2表示了进化树中的一个小分支,上部的两条所得序列是和已知古细菌菌种Metnanosaeta concilii和Methanosarcina sp.一致,说明通过使用这对引物,能检测已知的古细菌;而下部13条序列与已知菌种的序列存在差异,根据系统发育树显示的进化关系说明它们属于古细菌,这些序列在NCBI(http://www.ncbi.nlm.nih.gov/)的登录号为:012F06,EU838472;012B04,EU838440;012D11,EU838458;011E08,EU838404;014C11,EU838558;012F02,EU838469;011D06,EU838394;005H04,EU838491;011H10,EU838431;012C08,EU838450;012B06,EU838368;013D05,EU838526;012A06,EU838435说明通过使用这对引物,能检测出样品中所含古细菌的多样性。
实施例三:对由本发明扩增得到的PCR产物构建16S rDNA克隆文库进行多样性评价:对由实施例2得到的PCR-16S rDNA克隆文库进行多样性评价。使用软件DOTUR(Distance-based OTUs and Richness)计算产生评价多样性的Shannon指数。所有文库中的序列经MEGA4.0整合的ClustalW程序进行了排列,然后使用程序Philip计算出距离矩阵。将距离矩阵作为输入文件导入DOTUR程序进行运算,得到Shannon指数。
如下表所示:
Figure G2008102002805D00031
序列差异度的取值直接影响到Shannon指数的值,由表所示当序列差异度取值为0.05时,Shannon指数是1.73,说明此文库内所含古细菌具有可观的遗传多样性。因此表明本发明引物能很好地来探测古细菌的多样性。
序列表
<110>上海大学
<120>用于古细菌多样性分析的针对16S rRNA基因的引物
<160>2
<210>1
<211>16
<212>DNA
<213>人工序列
<400>1
Figure G2008102002805D00051
<210>2
<211>21
<212>DNA
<213>人工序列
<400>2
Figure G2008102002805D00052

Claims (1)

1.一种用于古细菌多样性分析的针对16S rRNA基因的引物,其特征在于该引物的碱基序列为:
f:5’-ACGGCTCAGTAACACG-3’;
r:5’-CCGCCAATTCCTTTAAGT-3’。
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