利用小亚基核糖体RNA技术分析温室黄瓜近根土壤古菌和真菌多样性

Diversity analysis of archaeal and fungal communities in adjacent cucumber root soil samples in greenhouse by small-subunit rRNA gene cloning

土壤古菌和真菌在温室生态系统是仅次于细菌的微生物,具有类似于细菌的重要生态功能。通过构建古菌16S rRNA和真菌18S rRNA基因克隆文库,分析温室黄瓜近根土壤古菌和真菌群落结构组成,为开发利用温室这一特殊的生态环境中丰富的微生物资源以及理解微生物与植物间的互作提供参考依据。采用研磨-冻融-溶菌酶-蛋白酶K-SDS热处理以及CTAB处理等理化方法,提取和纯化微生物总DNA,构建古菌16S rRNA和真菌18S rRNA基因克隆文库。利用DOTUR软件将古菌和真菌序列按照相似性97%的标准分成若干个可操作分类单元(OTUs)。土壤古菌克隆文库主要包括泉古菌门和未分类的古菌两大类,并有少部分广域古菌类群,所有泉古菌均属于热变形菌纲,共45个OTUs;真菌克隆文库包括真菌门的大多数亚门真菌,共24个OTUs,未发现担子菌亚门真菌。古菌多样性比较丰富,且发现少量的广域古菌(甲烷菌),这一情况可能与温室长期高温高湿,高有机质含量,土壤处于缺氧环境有关;土壤真菌的优势种群为子囊菌,占到土壤真菌的80%以上,这可能与绝大多数植物真菌性病害属于土传病害,通过菌丝体、菌核或子囊壳在土壤病残体中越冬有一定的关系。

Soil archaea and fungi play important roles in the greenhouse soil ecosystem.To develop and apply rich microbial resources in greenhouse ecological environment,and to understand the interaction between microbes and plants,we constructed archaeal 16S rRNA and fungal 18S rRNA gene libraries to analyze the compositions of archaeal and fungal communityies.Total greenhouse soil DNA was directly extracted and purified by skiving-thawing-lysozyme-proteinase K-SDS hot treatment and treatment of cetyltriethylammnonium bromide（CTAB）.After PCR amplification,retrieving,ligating,transforming,screening of white clones,archaeal 16S rRNA and fungal 18S rRNA gene libraries were constructed.The sequences of archaea and fungi were defined into operational taxonomic units（OTUs） when 97% similarity threshold for OTU assignment was performed by using the software DOTUR.Phylogenetic analysis showed that crenarchaeota and unidentified-archaea were the two major sub-groups and only a few of euryarchaeota existed in the archaeal clone library,total 45 OTUs.All the crenarchaeota belonged to thermoprotei;except for Basidiomycotina,the other four sub-group fungi were discovered in the fungal library,total 24 OTUs.The diversities of archaea were very abundant and a few euryarchaeota（methanebacteria） existed in the archaeal clone library,it might be directly related to the long-term high temperature,high humidity,and high content of organic matter.The limitation of oxygen was the other reason for causing this phenomenon;Ascomycotina（over 80%） was the dominant sub-groups in fungal library.It was because most of the plant fungal diseases belonged to soil-borne diseases which gone through the winter by the ways of scierotium or perithecium and became the sources of primary infection.