双齿围沙蚕消化道共栖微生物菌群多样性的PCR-DGGE分析

将采自青岛市沿海潮间带的双齿围沙蚕Perinereis aibuhitensis(体质量为4～5 g)置于无菌海水中暂养24 h后,分别从5条双齿围沙蚕消化道中提取微生物基因组总DNA,应用细菌16S rDNA通用引物341f/534r进行细菌16S rDNA基因V3高变异区的PCR扩增,再将PCR产物进行变性梯度凝胶电泳(DGGE),从而获得样品消化道共栖微生物群落特征的DNA指纹图谱。通过对指纹图谱半定量分析发现,采集的双齿围沙蚕消化道共栖微生物菌群多样性丰富,优势条带明显,不同个体间既存在共同的微生物种属,也有各自特异的种属。其中存在一条共同的优势条带,但优势条带含量存在个体间差异。分别对DGGE指纹图谱中公共条带序列进行测序比对,结果表明,产丙酸菌属Propionigenium分别为5个样品中的优势菌群,假交替单胞菌属Pseudoalteromonas广泛分布于双齿围沙蚕消化道中。研究表明,基于16S rDNA的PCRDGGE图谱技术是分析双齿围沙蚕及其他海洋沉积食性无脊椎动物消化道微生物菌群结构较为有效的手段。

PCR-DGGE analysis of immobilized microbial diversity in digestive tract of sand worm Perinereis aibuhitensis

The total microbial genomic DNA was isolated from intestines of 5 healthy sand worm Perinereis aibuhit- ens/s with biomass of approximately 4-5 g collected from shoreline of Qingdao （Shandong Province, China） and held in sterile seawater for 24 hours by Fecal DNA Extraction Kit. The V3 region of the 16S rRNA genes （approxi- mately 230 bp） in the intestinal microorganisms was amplified with specific primers set of GC-341f/534r, and then DGGE （Denaturing gradient gel electrophoresis） of amplified products generated with GC-341f/534r primer set was performed with the Bio-Rad Dcode TM mutation detection system. The DGGE band profiles of immobilized bacteria in the digestive tract of the sand worm were finally generated, which provided a snapshot of composition and diversity of the immobilized bacterial population in the digestive tract of the sand worm. PCR-DGGE DNA pro- files of the V3 region gene of 16S rDNA showed similar profiles of 5 selected individual worms and the maximal di- versity levels were observed. Some common and special bands were identified among 5 samples, and three common predominant bands on the DGGE gel were found, with different relative contents in each predominant band. The se- quencing and blasts of the DGGE common bands indicate that the predominant intestinal mieroflora was Propionige- rtium in the 5 samples and Pseudoalteromonas was widely distributed in the digestive tract of the sand worm. The findings indicate that PCR-DGGE analysis is an effective technique for analysis of immobilized intestinal microbial diversity in the sand worm.