GSS高通量测序在川芎内生细菌群落多样性分析中的应用

Application of GSS in Analysis of Endophytic Bacterial Diversity in Ligusticum chuanxiong

目的:宿主线粒体DNA(mtDNA)及叶绿体DNA(cpDNA)污染严重影响植物内生细菌高通量测序分析,该研究旨在探索和评价药用植物内生细菌高通量测序中宿主基因扩增抑制的新策略。方法:以川芎为研究材料,在其内生细菌的16S rDNA聚合酶联式反应(PCR)过程中引入绿盾测序(GSS)技术以屏蔽宿主基因的非靶扩增,对比分析GSS-PCR及常规PCR在内生细菌及根际土壤细菌高通量测试分析中的性能差异。结果:与常规扩增相比,GSS-PCR显著降低了川芎高通量测序中mtDNA及cpDNA扩增,非靶基因占比降幅超过60%;且内生细菌群落多样性指数显著提高,物种注释信息成倍增加,但不影响对优势菌群物种组成信息的提取。对川芎16S rDNA V4区的GSS扩增比对V3-V4区的扩增显示出更低的宿主污染率和更丰富的内生细菌多样性。结论:GSS扩增策略可以显著降低川芎内生细菌的高通量分析中宿主基因污染,在同等测度深度下提高内生细菌多样性分析的准确性,有效改善内生细菌的高通量分析质量,这使得内生细菌的16S rDNA V3-V4区、V4区的高通量测序变得可行,结果更加可靠。该研究所采用的GSS技术为其他药用植物内生细菌的分析提供了方法参考。

Objective:Host mitochondrial DNA(mtDNA)and chloroplast DNA(cpDNA)contamination severely affects high-throughput sequencing of endophytic bacteria in plants.This study aims to explore and evaluate a novel strategy of inhibiting host gene amplification in high-throughput sequencing of endophytic bacteria in medicinal plants.Method:Green Shield Sequencing(GSS)was introduced in the 16S rDNA polymerase chain reaction(PCR)of endophytic bacteria to shield the non-target amplification of genes in the host(Ligusticum chuanxiong).The performance was compared between GSS-PCR and conventional PCR in the high-throughput sequencing of endophytic bacteria and rhizosphere soil bacteria.Result:Compared with conventional PCR,GSS-PCR significantly reduced the amplification of mtDNA and cpDNA in L.chuanxiong in high-throughput sequencing,decreasing the non-target genes by more than 60%.Moreover,this strategy significantly increased the diversity of endophytic bacteria and multiplied the species without compromising the extraction of the information about the dominant bacteria.The GSS amplification of 16S rDNA V4 region of L.chuanxiong showed lower host contamination rate and higher endophytic bacterial diversity than that of V3-V4 regions.Conclusion:GSS can significantly reduce host gene contamination in the high-throughput sequencing of L.chuanxiong endophytic bacteria and improve the accuracy of endophytic bacterial diversity analysis at the same sequencing depth,thus improving the high-throughput analysis quality of endophytic bacteria in plants.Accordingly,this strategy improves the feasibility and reliability of high-throughput sequencing for the 16S rDNA V3-V4 and V4 regions of endophytic bacteria.GSS used in this study provides a method reference for studying the endophytic bacteria in other medicinal plants.