基于DGGE技术的茯砖茶发花过程细菌群变化分析

Analysis of bacterial flora during the fahua-fermentation process of fuzhuan brick tea production based on DGGE technology

变性梯度胶电泳是当前微生物生态学研究重要技术之一。为研究茯砖茶发花过程中细菌群落结构和种类,对发花过程中不同时段细菌16S rDNA的V3可变区扩增,经变性梯度胶电泳(DGGE)后、对细菌DGGE条带进行克隆、测序和比对。结果表明,在发花过程的第0—4天、6—8天、10—14天茯砖茶发花存在3个差异较大的细菌优势种群结构的演变;从16SrDNA的V3可变区比对结果证明黑毛茶发花过程中有短波单胞菌属、诺卡氏菌属、新鞘脂菌属、突那梭菌属、韦龙氏假单胞菌属、乳杆菌属、克雷伯氏菌属以及不可培养ε-变形菌、腐败螺旋菌属、粘球菌属、根瘤菌属和6种未知分类地位的不可培养细菌,说明采用DGGE指纹图谱能更系统、更真实地反映茯砖茶发花发酵过程中细菌群落结构和多样性变化。

Denaturing gradient gel electrophoresis （DGGE） is one of the most important and commonly used techniques in studies of microbial molecular ecology. DGGE has been widely used in the analysis of community structures and biodiversity of bacteria, cyanobacteria, archaea, miniature eukaryotes, eukaryotes, and viruses. This technique can be used for the reliable analysis of multiple samples at the same time. It is suitable for the surveillance of microbial population changes over both space and time, and is used to probe microbial community composition by DNA sequence analysis. This technology can overcome the limitations associated with traditional analysis techniques, such as cultivation, purification, and microscopy. In this research, DGGE spectra analysis was used to examine the microbial diversity during the Fahua-fermentation process of Fuzhuan Brick Tea production. To explore the bacterial community structure during this process, we collected Fuzhuan Brick Tea samples at days 0, 2, 4, 6, 8, 10, 12, and 14 of the Fahua-fermentation process. Total genomic DNA was extracted from each of the samples, and the V3 variable region of the 16S rDNA gene was amplified. The amplified products were separated by DGGE. The separated DGGE bands were extracted and gel-purified then ligated into the pEASY-T vector. Resultant plasmids were transformed into electrocompetent Escherichia coli DH5α cells, and positive clones were identified by blue white screening experiment. Plasmids from the positive clones were then extracted and sequenced, and the resulting sequences were submitted to the GenBank database. Using the BLASTN tool to carry out homology comparisons, we identified sequences in the database that were most similar to the 16S rDNA sequences amplified from the Fahua-fermentation community samples. The results showed three distinctive kinds of bacterial community structure, correlating to fermentation times of 0-4 days, 6-8 days, and 10-14 days. Alignments of the V3 variable regions of the 16S rDNA genes showed that samples contained Brevundimonas aurantiaca, Millisia brevis, Novosphingobium sp, Clostridium ultunense, Pseudomonas veronii, Lactobacillus plantarum, Klebsiella pneumoniae,uncultured Epsilonproteobacteria, uncultured Saprospiraceae bacterium, uncultured Myxococcales bacterium, uncultured Rhizobiales bacterium,and six kinds of unculturable bacteria. This analysis technique provided a detailed spectrum of bacteria at the different time periods during the Fahua-fermentation process, and showed that bacterial species present during this stage of Fuzhuan Brick Tea production are abundant. Amongst the bacterial species, nine strains belonged to the Proteobacteria, three strains were identified as Firmicutes, one strain belonged to the Actinobacteria, and one strain belonged to the phylum Bacteroidetes. We also identified six strains with the greatest similarity to unknown unculturable bacteria, which will require further analysis. This study also identified K. pneumoniae, B. aurantiaca, P. veronii, and a Myxococcales bacterium in the Fahua-fermentation process samples, all of which are generally considered to be conditionally pathogenic bacteria. The roles of these species during the fermentation process remain unknown and will require further investigation. Overall, the results of the current study indicate that DGGE fingerprinting is a useful technique to obtain a comprehensive, detailed bacterial community structure and estimate of diversity variation from samples collected during Fahua-fermentation in the production of Fuzhuan Brick Tea.