摘要
为了解醉马草内生细菌的组成和多样性,采用液氮研磨法提取醉马草总DNA,利用内生细菌16S rRNA基因特异性引物对醉马草总DNA进行16S rRNA基因扩增,构建醉马草内生细菌16S rRNA基因文库。对筛选到249个克隆进行HaeШ酶切分析,得到57个操作分类单元(OTUs)。系统发育分析将其归为4个门:变形杆菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)以及1个未知类群。其中74%的克隆与可培养细菌中的37个属具有高的16SrRNA基因序列相似性(97%—100%),其中芽孢杆菌属(Bacillus spp.)、红球菌属(Rhodococcus spp.)、黄杆菌属(Flavobacterium spp.)、黄单胞杆菌属(Xanthomonas spp.)最为丰富。另外26%的克隆序列与GenBank中已存细菌16S rRNA基因序列相似性小于96%。研究结果表明,醉马草中可培养内生细菌丰富,多样并且可能存在一些潜在新物种或新类群。
Achnatherum inebrians（Hance） Keng,which is belonged to genus Achnatherum Beauv,Gramineae family,also known as Oxytropis glabra,is one of poisonous perennial herbs on grassland and is wildly distributed in Inner Mongolia,Ningxia,Gansu,Qinghai and Xinjiang province of northwest China.It is toxic to hoofed mammals of the genus Equus,however,they are hardy and easy to grow in arid and semi-arid areas and played active role in improving ecological environment.The toxic ingredients extracted from the plant are usually used as anti-cancer drugs,immune modulators;such as Swainsonine,one of well studied anti-cancer drugs that often used to stimulate bone marrow cell proliferates.Therefore,the plant is worth of fullly usage and utilizing it rationally.In this study,a culture-independent of analysis were chosen to investigate the endophytic bacterial composition and diversity associated in Achnatherum inebrians.Total community DNA were extracted by modified CTAB-SDS DNA extraction protocol from plant stem,leaf and root which were grinded up using liquid nitrogen grinding method after strictly surface-sterilization.To construct 16S rDNA clone libraries,endophytic bacterial specific primer pair 799F and 1492R were selected to amplify bacterial 16S rDNA partial gene,and then PCR products were ligated into a PMD18-T Vector and transformed into Escherichia coli DH 5α.Transformants were selected by blue and white selection.Positive clones were analyzed by Restriction Fragment length polymorphism（RFLP）,and clones with unique RFLP patterns were selected for sequencing,alignment and constructing 16S rRNA gene phylogenetic tree.In total 249 clones were screened,which could be grouped into 57 operational taxonomic units（OTUs）,based on Hae III digestion.These were divided into four phyla（Proteobacteria,Firmicutes,Actinobacteria,Bacteroides） and one unknown group by phylogenetic analysis.Of them,74% of clones classified into 37 genera of bacteria phylum were highly related to cultivable species with high 16S rRNA gene s
出处
《生态学报》
CAS
CSCD
北大核心
2011年第8期2178-2187,共10页
Acta Ecologica Sinica
基金
973计划前期研究专项(2008CB417214)
新疆特殊环境微生物实验室开放课题(XJYS0203-2009-02)
关键词
醉马草
内生细菌
免培养法
生物多样性
Achnatherum inebrians
endophytic bacterial
culture-independent
biodiversity
