Designing specific bacterial 16S primers to sequence and quantitate plant endo-bacteriome
Designing specific bacterial 16S primers to sequence and quantitate plant endo-bacteriome
复制标题
设计特异性细菌 16S 引物对植物内细菌组进行测序和定量
DOI：
10.1007/s11427-021-1953-5
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发表时间：
2021-07-22
影响因子：
9.1
通讯作者：
Zhang, Lili
中科院分区：
文献类型：
--
作者：
Chen, Liying;Zhang, Mengting;Zhang, Lili
植物内生细菌定殖于植物内部组织，并与植物密切相互作用。在过去二十年中，新一代16S rRNA基因测序技术在细菌群落研究中的应用日益增多。然而，由于大量植物细胞器DNA会与细菌16S共同扩增，通过这种方法解析植物内生细菌群落颇具难度。在此，我们设计了聚合酶链反应（PCR）引物组，包括799F/1107R、322F/796R以及322F - Dr/796Rs（即322F倒数第二个碱基发生替换的322F/796R引物对），这些引物组能够从植物总DNA中特异性扩增细菌16S。我们通过计算机模拟和实验评估了新设计引物组的特异性、覆盖度和准确性。对于细菌群落丰度极低的植物材料，799F/1107R和322F - Dr/796Rs这两个引物组均能产生不含植物DNA的16S扩增子文库，或将植物DNA污染降低至5%以下。引物组322F - A/796R通过绝对定量PCR用于对水稻叶片或根部内生细菌群落的种群数量进行定量分析，结果显示每克鲜重中分别含有10⁶ - 10⁷和10⁹ - 10¹⁰个细菌。这些16S引物组和扩增方法使得对植物内生细菌群落进行简便且低成本的新一代测序和定量分析成为可能，这将极大地推动与植物相关微生物组的研究。
Plant endophytic bacteria colonize the internal tissues of plants and interact with plants closely. The past two decades have witnessed the increasing application of next-generation 16S rRNA gene sequencing in the investigation of bacterial communities. However, deciphering plant endo-bacterial communities by this method is difficult because of the co-amplification of massive plant organellar DNAs with bacterial 16S. Here, we designed polymerase chain reaction (PCR) primer sets, including 799F/1107R, 322F/796R, and 322F-Dr/796Rs (primer pair 322F/796R with a penultimate-base substitution in 322F), that can specifically amplify bacterial 16S from plant total DNAs. We computationally and experimentally evaluated the specificity, coverage, and accuracy of the newly designed primer sets. Both 799F/1107R and 322F-Dr/796Rs produced plant DNA-free 16S amplicon libraries or reduced plant DNA contamination to lower than 5% for the plant materials with extremely-low-abundance bacterial communities. The primer set 322F-A/796R was used through absolute quantitative PCR to quantitate the population size of rice leaf or root endo-bacteriome, which revealed 106–107and 109–1010bacteria per gram fresh weight, respectively. These 16S primer sets and amplification methods enable the simple and inexpensive next-generation sequencing and quantification of plant endo-bacteriome, which will significantly advance studies on the plant-related microbiome.