植物内生菌16S rRNA基因扩增子高通量测序中降低宿主污染的方法

Methods to Reduce Host Pollution in High Throughput Sequencing of 16S rRNA Gene of Plant Endophytic Bacteria

植物内生菌能够帮助植物生长,增强其抗逆性与抗病性,研究植物内生菌对了解植物入侵机制、保护生态环境与农业的健康发展具有重要意义。通过阅读相关文献,总结归纳了降低植物内生菌16S rRNA基因扩增子高通量测序(16S-seq)中宿主基因污染的4种方法,即:①寻找特异性错配引物用来扩增细菌16S rRNA基因;②添加特异性阻断物用来抑制宿主16S rRNA基因扩增,如PNA-PCR和LNA-RCR钳位技术;③在文库构建过程中剪切宿主细胞器的16S rRNA基因,如Cas-16S-seq方法;④改变PCR扩增流程,如巢式PCR技术。了解上述各种方法的特点,有助于在植物内生菌16S-seq中选择合适的方法,避免或者减少宿主基因的污染,更准确地进行植物内生细菌群落的研究。

Endophytes can help plants to grow,and enhance their stress and disease resistance.The study of endophytes is of great significance for understanding the mechanism of plant invasion and protecting the healthy development of ecological environment and agriculture.In this paper,we summarized from previous studies about four methods currently used to reduce host DNA contamination in endophyte 16S rRNA gene amplification(16S-seq):①using bacterial specific primers for 16S-deq;②designing specific blockers to inhibit the amplification of host genes,such as peptide nucleic acids(PNA)and locked nucleic acid(LNA)oligonucleotide-PCR clamping technique;③cutting the 16S rRNA gene from host organelles during gene library construction,such as Cas-16S-seq;③changing the PCR protocols in 16S-seq,such as nested PCR.These methods are helpful to avoid or reduce host gene contamination,and get a more accurate determination of endophytic bacterial community.