三疣梭子蟹肠道菌群结构及代谢表型分析

Gut Microbiota and Metabolic Phenotype of Portunus trituberculatus

建立16S rRNA基因的高通量测序和基于核磁共振（NMR）的代谢组学技术分析三疣梭子蟹肠道菌群结构及代谢物组成的方法。经对梭子蟹肠道样品提取基因组DNA和16 S rRNA基因的V3-V4可变区片段的扩增,产物用于MiSeq测序。经对测序结果进行聚类和注释分析,发现梭子蟹肠道的细菌群落结构以变形杆菌门、拟杆菌门、梭杆菌门、软壁菌门和酸杆菌门为主,其中以发光杆菌属、Paludibacter和产丙酸菌属的细菌丰度最高。梭子蟹肠道样品经组织破碎、甲醇-水（2∶1,V/V）提取、高速离心和冷冻干燥后,重悬在钠钾磷酸盐缓冲液中,用于NMR分析。采用标准的Noesypr1D脉冲序列采集^1H NMR谱,设置90°脉冲宽度约为10μs,等待时间为2 s,混合时间为100 ms,谱宽为20 ppm,采样点数为32 K,自由感应衰减累加次数为64。再利用2D NMR谱对肠道代谢物进行归属,共获得包括氨基酸、有机羧酸和有机胺类等30种代谢产物。本方法可用于系统分析梭子蟹肠道菌群及其代谢物组成。

16 S rRNA gene high-throughput sequencing and nuclear magnetic resonance （ NMR ）-based metabolomics techniques were established to understand the composition of gut microbiota and metabolites of Portunus trituberculatus. Swimming crab gut samples were used for the extraction of bacterial genomic DNA, followed by the amplification of hypervariable domain V3-V4 of 16 S rRNA gene. The obtained PCR products were used for analysis on an Illumina MiSeq platform. The bacterial phylotypes were then clustered and assigned. The results showed that swimming crab gut was dominated by proteobacteria, bacteroidetes, fusobacteria, tenericutes, and acidobacteria at the phylum level. At the genus level, Photobacterium, Paludibacter, and Propionigenium were enriched in it. Crab intestinal samples were extracted with CH3 OH/H2 O （ 2：1, V/V ） solution by shaking with a tissuelyser. After the removal of methanol, the resultant supernatants were lyophilized. Each of lyophilized extract was dissolved into Na＋/K＋phosphate buffer and then centrifuged at high speed for NMR analysis. A standard noesypr1D was used to acquire 1 H NMR spectra. The 90o pulse length was adjusted to approximately 10 μs. The recycle delay and mixing time were set to 2 s and 100 ms in sequence. The spectral width was set to 20 ppm. Sixty-four transients were collected into 32 k data points for each spectrum. A range of two-dimensional NMR spectra were acquired for the resonance assignment. The results showed that swimming crab gut metabolome comprised 30 metabolites including some amino acids, organic acids, and amines. In summary, this study provided a method for the systematic analysis of the compositions of gut bacterial community and metabolites of swimming crab.