Hu Min, Li Fangbai, Qiao Jiangtao, Yuan Chaolei, Yu Huanyun, Zhuang Li
Guangdong Key Laboratory of Integrated Agro-environmental Pollution Control and Management, Institute of Eco-environmental and Soil Sciences, Guangdong Academy of Sciences, Guangzhou, China.
National-Regional Joint Engineering Research Center for Soil Pollution Control and Remediation in South China, Guangzhou, China.
Front Microbiol. 2021 Oct 6;12:691913. doi: 10.3389/fmicb.2021.691913. eCollection 2021.
Gene encoding the large subunit of As(III) oxidase (AioA), an important component of the microbial As(III) oxidation system, is a widely used biomarker to characterize As(III)-oxidizing communities in the environment. However, many studies were restricted to a few sequences generated by clone libraries and Sanger sequencing, which may have underestimated the diversity of As(III)-oxidizers in natural environments. In this study, we designed a primer pair, 1109F (5'-ATC TGG GGB AAY RAC AAY TA-3') and 1548R (5'-TTC ATB GAS GTS AGR TTC AT-3'), targeting gene sequence encoding for the conserved molybdopterin center of the AioA protein, yielding amplicons approximately 450 bp in size that are feasible for highly parallel amplicon sequencing. By utilizing analyses and the experimental construction of clone libraries using Sanger sequencing, the specificity and resolution of 1109F/1548R are approximated with two other previously published and commonly used primers, i.e., M1-2F/M3-2R and deg1F/deg1R. With the use of the 1109F/1548R primer pair, the taxonomic composition of the genes was similar both according to the Sanger and next-generation sequencing (NGS) platforms. Furthermore, high-throughput amplicon sequencing using the primer pair, 1109F/1548R, successfully identified the well-known As(III)-oxidizers in paddy soils and sediments, and they also revealed the differences in the community structure and composition of As(III)-oxidizers in above two biotopes. The random forest analysis showed that the dissolved As(III) had the highest relative influence on the Chao1 index of the genes. These observations demonstrate that the newly designed PCR primers enhanced the ability to detect the diversity of aioA-encoding microorganisms in environments using highly parallel short amplicon sequencing.
编码微生物砷（III）氧化系统重要组成部分——砷（III）氧化酶大亚基（AioA）的基因，是用于表征环境中砷（III）氧化菌群的一种广泛使用的生物标志物。然而，许多研究仅限于克隆文库和桑格测序产生的少数序列，这可能低估了自然环境中砷（III）氧化菌的多样性。在本研究中，我们设计了一对引物，1109F（5'-ATC TGG GGB AAY RAC AAY TA-3'）和1548R（5'-TTC ATB GAS GTS AGR TTC AT-3'），靶向编码AioA蛋白保守钼蝶呤中心的基因序列，产生大小约为450 bp的扩增子，适用于高度平行的扩增子测序。通过利用分析以及使用桑格测序构建克隆文库的实验，1109F/1548R的特异性和分辨率与另外两个先前发表并常用的引物，即M1-2F/M3-2R和deg1F/deg1R相近。使用1109F/1548R引物对，根据桑格测序和下一代测序（NGS）平台，aioA基因的分类组成相似。此外，使用1109F/1548R引物对进行的高通量扩增子测序成功鉴定了稻田土壤和沉积物中著名的砷（III）氧化菌，并且还揭示了上述两种生物群落中砷（III）氧化菌的群落结构和组成差异。随机森林分析表明，溶解态砷（III）对aioA基因的Chao1指数具有最高的相对影响。这些观察结果表明，新设计的PCR引物增强了使用高度平行的短扩增子测序检测环境中编码aioA微生物多样性的能力。
