PCR-DGGE方法分析玉米及苜蓿青贮动态发酵体系中菌群多样性

Analysis on the Bacterial Diversity in the Dynamic Fermentation System of Corn and Alfalfa Silage by PCR-DGGE

[目的]探讨玉米青贮和苜蓿青贮动态发酵过程。[方法]以玉米和苜蓿为青贮原料，利用氯化苄方法对青贮中微生物基因组DNA进行提取，以P2f和P3r为引物扩增目的片段并利用PCR—DGGE方法对玉米青贮及苜蓿青贮进行动态发酵多样性研究。[结果]玉米青贮的pH值在发酵过程中维持在4．0～4．5，而苜蓿青贮则维持在5．5—6．0。PCR-DGGE分析显示2种物料青贮在0和1d、3和7d、20和60d相似性均很高，并且它们具有基本相似的谱带类型，其中条带1、3、13、14、17、21、22为所有样品共有，只是亮度上有差异。DGGE研究结果表明大多数序列代表乳酸菌（LAB）类细菌，并以Lactobacillus类最多；其次为Lactococcus和Weissella。[结论]该研究为青贮添加剂的制作奠定基础。

[ Objective ] The aim was to discuss the dynamic fermentation process of corn silage and alfalfa silage. [ Method ] With corn and alfalfa as silage materials, the genome DNA from silage microbe was extracted by benzyl chloride method. The target fragment was amplified by using primers of P2f and P3r. The dynamic fermentation diversity of corn silage and alfalfa silage were studied by PCR-DGGE. [ Result] The pH value of corn silage maintained in 4.0 - 4.5 and alfalfa silage maintained in 5.5 - 6.0 during fermentation process. PCR-DGGE analysis showed that the similarity of the 2 silages were all high between 0 and 1 d, 3 and 7 d, 20 and 60 d. The 2 silages had basically similar band types. Among them, the band 1, 3, 13, 14, 17, 21 and 22 belonged to all samples and the difference was only in brightness. DGGE result showed that most sequence represented lactic-acid bacteria, and the Lactobacillus was most, followed by Lactococcus and WeisseUa. [ Conclusion] The research laid the foundation for preparation of silage additive.