肠道细菌乳糖酶基因多样性分析通用引物  

Universal primer for analysis of the diversity of intestinal bacterial lactase gene

作　　者：龙承星[1,2] 贺璐[1] 刘又嘉[1] 惠华英[1] 谭周进[1] 李丹丹[1] 

机构地区：[1]湖南中医药大学,长沙410208 [2]湖南人文科技学院数学与金融学院,娄底417000

出　　处：《应用与环境生物学报》2017年第4期758-763,共6页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(81573951)资助~~

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.In order to further study the diversity of the intestinal bacterial lactase gene, seven pairs of universal primers were designed and synthesized by using the premier 5.0 software according to the bacterial lactase gene （beta-galactosidase （lacZ）） sequence published in NCBI database, and metagenomic DNA was screened through PCR amplification, sequencing, and bioinformatics analysis. Four pairs of primers of 436R, 375R, 217R, and 406R did not amplify the target fragment in PCR amplification, while primers of 436, 324, or 194 all had the target fragment amplified. For amplification on the content sample, the expected bands of primer 436 were the most bright and clear with high sensitivity of the primer. The band quality between 194 and 324 showed no difference; for amplification on the mucosal sample, primer 324 amplified expected bands, which were clear and bright. Primer 436 amplified one sample only, and its sensitivity was poor. Primer 194 amplified multiple bands and the specificity was poor. In the aspect of sample sequence statistics, the final sequence ratio of primers 194, 324, and 436 was higher than 98% in the content samples. In the mucosal samples, the specificity of the primer 194 was the lowest. Primer 436 was found to be the best, followed by primer 324 after removal of the host sequence. In the aspect of OTU clustering and annotation, in the content samples, primer 324 amplified more species than primer 436. Primer 324 was able to cluster most species in the mucosal samples. For alpha diversity, in the content samples, the Chao1, ACE, Simpson, and Shannon indices of primer 324 were higher than those of primer 436. The Simpson and Shannon indices of primer 436 were higher than those of primer 194. The Chao1, ACE, Simpson, and Shannon indices of primer 324 were all higher than those of primer 436 in the mucosal sample. In the comprehensive analysis, the universal primer 324 was found to be the best to study the diversity of the bacterial lactase gene in the intestinal contents and mucosa.

关 键 词：细菌乳糖酶 通用引物 Miseq宏基因组测序 PCR扩增 功能基因 肠道微生物 

分 类 号：Q933[生物学—微生物学]