采用不同限制性内切核酸酶研究森林土壤真菌多样性和群落结构

摘要

以秦岭南坡火地塘林区5种主要森林类型(油松纯林、锐齿栎纯林、青扦纯林、华山松纯林和油松-锐齿栎混交林)土壤真菌为研究对象,通过T-RFLP技术探讨了不同限制性内切酶对土壤真菌多样性和群落结构产生的影响.用4种常用内切酶(AluI、HhaI、MspI和RsaI)分别对5种森林类型土壤真菌ITS序列PCR产物进行消化,获得了100个土壤真菌TRF图谱.综合比较不同森林类型土壤真菌多样性指数(Richness S、Shannon H、Simpson D和Evenness E)后发现,限制性内切核酸酶的选择可以明显影响到运用T-RFLP技术对土壤真菌多样性和群落结构的评价.相比MspI和RsaI,用AluI和HhaI消化5种森林类型土壤真菌ITS序列PCR产物能够普遍获得较高的TRF丰富度和多样性,尤以HhaI的各指数表现更为突出,展现了其更加优越的DNA消化能力和更高的酶切位点特异性.根据TRF的有无和数量对5种森林类型土壤真菌群落进行聚类分析和非度量多维尺度分析(NMDS).结果表明,HhaI、MspI和RsaI所产生的TRF的数量和种类较为接近,进而获得了一致的土壤真菌群落相似性结果,而AluI的聚类分析和非度量多维尺度分析结果与其他3种内切核酸酶差异较大.综合多样性和群落结构的分析,具有较强消化能力的HhaI既能较好地揭示5种森林类型土壤真菌多样性,又有利于真菌群落结构的辨识,为适宜限制性内切酶.结果对限制性内切核酸酶选择的探讨可以为更好地运用T-RFLP来量化环境样品中的微生物多样性和群落提供指导.%Taking the soil fungi of five main forest types (pure forests of Pinus tabulaeformis,Quercus aliena var.acuteserrata,P icea wilsonii,and P inus armand i,and mixed forest of P.tabulae f ormis-Q.aliena var.acuteserrata) at Huoditang forest region on the south-facing slope of the Qinling Mountains as objects,this paper discussed the impacts of different restriction endonucleases on soil fungal diversity and community structure through T-RFLP.Four commonly used endonucleases (AluI,HhaI,MspI and RsaI) were applied to digest PCR products of soil fungal ITS sequence from the forest types,which resulted in 100 of soil fungal TRF profiles.Followed comprehensive comparisons of diversity indices (Richness S,Shannon H,Simpson,D,and Evenness E) of soil fungi from different forest types,it was found that the selection of endonucleases exerted significant affection on the application of T-RFLP to evaluating soil fungal diversity and community structure.Compared with MspI and RsaI,generally higher richness and diversity occurred across the five forest types when using AluI and HhaI to digest the fungal ITS PCRproducts,especially HhaI,more outstanding performance in all indices was observed,indicating the superior DNA digestion and higher enzyme site specificity.Based on the absence and quantity of TRF,soil fungal communities of the forest types were subjected to clustering analysis and multidimensional scaling analysis (NMDS),the results showed that HhaI,MspI and RsaI might generated approximate quantity and variety of TRF,further leading to the production of a consistent result in similarity of soil fungal communities,while AluI was significantly different from other 3 endonucleases in the analyses.Combined diversity with community structure,HhaI was in favor of revealing the soil fungal diversity and for identifying fungal community structure of the 5 forest types,and was the best restriction endonuclease in this study.Here,the discussion on the selection of restriction endonucleases may provide guidance and advice for the better use of T-RFLP to quantify microbial communities in environmental samples.