肠道细菌乳糖酶基因多样性分析通用引物

发布时间：2019-04-26 18:58

【摘要】：为深入研究肠道细菌乳糖酶基因多样性,根据NCBI公布的细菌乳糖酶基因[Beta-galactosidase(lac Z)]序列,利用软件Primer Premier 5.0前后设计合成7对通用引物,经过PCR扩增、宏基因组测序和生物信息学分析进行引物筛选.PCR扩增电泳中,436R、375R、217R和406R四对引物没有扩增出目的片段,436、324和194三对引物均有目的条带扩出.内容物样品扩增,436引物条带最清晰,灵敏性高,324和194引物条带质量相差不大;粘膜样品扩增,324引物均扩增出目的条带,条带清晰明亮.436引物只扩增出一个样品的目的条带,灵敏度较差;194引物扩增杂带多,特异性差.样品序列统计方面,删除宿主序列信息后,内容物样品中,194、324和436三对引物最终序列比例均达98%以上;粘膜样品中,194引物特异性最差,436引物最好,324引物居中.OTU聚类和注释方面,内容物样品中,324引物扩增物种也较436引物多;粘膜样品中,324引物能聚类最多的物种.Alpha多样性方面,内容物样品中,324引物的Chao1、ACE、Simpson、Shannon指数较436引物大,436引物的Simpson、Shannon指数较194引物大;粘膜样品中,324引物的Chao1、ACE、Simpson、Shannon指数均较436引物大.综合分析,对肠道内容物和肠道粘膜细菌乳糖酶基因多样性研究选用324通用引物最佳.
[Abstract]:In order to study the diversity of intestinal bacterial lactase gene, seven pairs of universal primers were designed and synthesized by using software Primer Premier 5.0.Based on the sequence of bacterial lactase gene [Beta-galactosidase (lac Z)] published by NCBI, 7 pairs of primers were designed and synthesized before and after PCR amplification. Four pairs of primers, 436R, 375R, 217R and 406R, did not amplify the target fragment, but three pairs of 436324 and 194 pairs of primers all had the target band in the PCR amplification of 436R, 375R, 217R and 406R. The bands of 436 primers were the clearest, the sensitivity was high, and the band quality of 324 and 194 primers had no significant difference. The target bands were amplified with 324 primers and only one sample was amplified with 436 primers with poor sensitivity and 194 primers with many heterozygotes and poor specificity. In terms of sample sequence statistics, after deleting host sequence information, the final sequence proportion of 194324 and 436 pairs of primers was more than 98% in the entrapment samples. In mucosa samples, 194 primers had the worst specificity, 436 primers were the best, 324 primers were in the middle. In OTU cluster and annotation, 324 primers amplified more species than 436 primers in the content sample. In mucosal samples, 324 primers could cluster the most species. Alpha diversity, the Chao1,ACE,Simpson,Shannon index of 324 primer was higher than that of 436 primer, and the Simpson,Shannon index of 436 primer was higher than that of 194 primer. In mucosa samples, the Chao1,ACE,Simpson,Shannon index of 324 primer was higher than that of 436 primer. According to the comprehensive analysis, 324 universal primers were the best for the study of intestinal contents and intestinal mucosal bacterial lactase gene diversity.
【作者单位】：湖南中医药大学;湖南人文科技学院数学与金融学院;
【基金】：国家自然科学基金项目(81573951)资助~~
【分类号】：Q933

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