利用MiSeq高通量测序技术分析风干处理及青贮发酵对玉米秸秆菌群构成及丰度的影响

doi:10.3864/j.issn.0578-1752.2015.S.011

摘要/Abstract

摘要： 【目的】玉米秸秆等粗饲料由于饲喂量大，其所携带的微生物可能会对家畜营养物质的吸收利用及家畜健康存在潜在影响。通过分析自然风干玉米秸秆以及玉米秸秆青贮饲料中所携带细菌的群落构成及丰度，尝试从微生物角度为玉米秸秆的利用提供参考。【方法】运用MiSeq高通量测序技术测定玉米秸秆原料、自然风干玉米秸秆及玉米秸秆青贮饲料中门、纲、目、科和属5个水平细菌群落构成及丰度，分析风干处理及青贮发酵对菌群的影响及二者差异性。【结果】在序列相似性大于97%时，共检测到相同的操作分类单元（operational taxonomic units，OTU）数为10 887，各样品的稀释曲线均已趋于平坦，测序深度基本可以反映样品中绝大多数的微生物信息。玉米秸秆原料、风干玉米秸秆及青贮后玉米秸秆中菌群群落构成存在差异性，其中，风干玉米秸秆菌群构成最为复杂，其次为玉米秸秆原料，青贮45 d后的玉米秸秆中细菌种类最少。同时，风干玉米秸秆与原料中微生物种类存在相似性，而青贮后玉米秸秆与原料相比其菌落构成差异较大。风干处理后玉米秸秆中变形菌门（Proteobacteria）、γ-变形菌纲（Gammaproteobacteria）、肠杆菌目（Enterobacteriales）和肠杆菌科（Enterobacteriaceae）菌群丰度分别为96.53%、90.67%、81.42%和81.42%，与玉米秸秆原料相比，显著增加（P＜0.05）；青贮发酵后玉米秸秆中变形菌门、γ-变形菌纲、肠杆菌目和肠杆菌科菌群丰度分别为55.83%、37.51%、25.03%和25.03%，与玉米秸秆原料及风干玉米秸秆相比，均显著降低（P＜0.05）。青贮发酵后玉米秸秆中厚壁菌门（Firmicutes）、芽孢杆菌纲（Bacilli）、乳杆菌目（Lactobacillales）、乳杆菌科（Lactobacillaceae）、片球菌属（Pediococcus）菌群丰度分别为39.10%、38.91%、38.81%、32.08%和28.14%，显著高于风干玉米秸秆和玉米秸秆原料（P＜0.05）；而风干处理后玉米秸秆中厚壁菌门、芽孢杆菌纲、乳杆菌目、乳杆菌科和片球菌属菌群丰度分别仅为2.47%、2.45%、2.39%、1.55%和1.32%。【结论】风干处理和发酵处理后，玉米秸秆中菌群构成及丰度差异显著。与风干处理相比，通过青贮发酵方式对利用玉米秸秆效果更佳。通过MiSeq高通量测序技术，能够提供不同处理方式玉米秸秆中整个菌落构成及丰度变化的信息。

关键词: MiSeq, 玉米秸秆, 风干, 青贮, 细菌群落

Abstract: 【Objective】The microbes carried by corn stalk maybe had the potential impact on the nutrients absorption and utilization of nutrients and animal health, due to the large amount of feeding. The objective of the present study was to investigate the effect of ensiling and natural drying of corn stalk silage on its bacterium composition and abundance, and try to provide a theoretical basis for the utilization of corn stalk from the microbiological point of view. 【Method】 The effect of ensiling and natural drying on composition and abundance of bacterium of corn stalk was analyzed using MiSeq sequencing technology. 【Result】The results showed that, the overall number of OTUs detected by the analysis reached 10887 based on ≥ 97% nucleotide sequence identity between reads, and the rarefaction curve of each sample was flattening. These implied that the sequencing depth was sufficient to reflect the vast majority of bacterium in the samples. There were differences and similarities on the composition and abundance of bacterium during stuff, dried and silage samples of corn stalk. Flora constitute of dry corn stalks was the most complex, followed by corn stalk stuff and the silage. The numbers of Proteobacteria plylum, Gammaproteobacteria class, Enterobacteriales order, Enterobacteriaceae family in dry corn stalk were 96.53%, 90.67%, 81.42% and 81.42%, which were significantly increased compared with stuff of corn stalk (P＜0.05); while the numbers of Proteobacteria plylum, Gammaproteobacteria class, Enterobacteriales order, Enterobacteriaceae family in corn stalk silage were 55.93%, 37.51%, 25.03% and 25.03%, which were significantly decreased compared with stuff and dry corn stalk (P＜0.05). 4) The numbers of Firmicutes phylum, Bacilli class, Lactobacillales order, Lactobacillaceae family, and Pediococcus genus in corn stalk silage were 39.10%, 38.91%, 38.81%, 32.08% and 28.14%, which were significantly increased compared with stuff and dry corn stalk (P＜0.05); while the numbers of Firmicutes phylum, Bacilli class, Lactobacillales order, Lactobacillaceae family, and Pediococcus genus in corn stalk silage were only 2.47%, 2.45%, 2.39%, 1.55% and 1.32%. 【Conclusion】 There was significant difference on bacterium composition and abundance between dried and silage samples of corn stalk; the using method of ensiling was better than natural dried corn stalk. MiSeq sequencing technology could provide the information of composition and abundance of flora.

Key words: MiSeq, corn stalk, ensiling, natural drying, bacterium

陶莲，刁其玉. 利用MiSeq高通量测序技术分析风干处理及青贮发酵对玉米秸秆菌群构成及丰度的影响[J]. 中国农业科学, 2015, 48(S): 94-103.

TAO Lian, DIAO Qi-yu. Effects of Ensiling and Natural Drying on Composition and Abundance of Bacterium of Corn Stalk Analyzed by MiSeq Sequencing Technology[J]. Scientia Agricultura Sinica, 2015, 48(S): 94-103.

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