| 引用本文: |
蔡媛,刘浩,王勇庆,谢景,黄建华,劳嘉,贺炜,张水寒.基于16S rRNA基因高通量测序方法分析多花黄精内生细菌群落结构及多样性[J].湖南中医药大学学报,2020,40(7):846-852[点击复制] |
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| 基于16S rRNA基因高通量测序方法分析多花黄精内生细菌群落结构及多样性 |
| 蔡媛,刘浩,王勇庆,谢景,黄建华,劳嘉,贺炜,张水寒 |
| (湖南省中医药研究院中药研究所, 湖南 长沙 410006;绿之韵生物工程集团有限公司, 湖南 长沙 410329) |
| 摘要: |
| 目的 采用高通量测序技术分析多花黄精内生细菌的多样性及菌群结构。方法 通过蒽酮-硫酸法测定多糖含量，采用通用引物对细菌16S rRNA高可变区（V3-V4区）进行PCR扩增，运用Illumina Miseq PE250高通量测序技术对扩增子进行测序，并用QIIME等软件对测序序列进行生物信息学分析。通过Spearman相关性分析菌群与多糖含量的相关性。结果 多花黄精内生菌测序共获得90 628条有效序列和5 287个OTU，稀释曲线和Coverage指数反映测序结果比较全面地覆盖了多花黄精内生细菌群落。Alpha多样性分析表明，其内生细菌多样性程度高。在门水平，其优势菌门为变形菌门（Proteobacteria）、放线菌门（Actinobacteria）、芽单胞菌门（Gemmatimonadetes）、厚壁菌门（Firmicutes）、酸杆菌门（Acidobacteria）。属水平，核心菌群由鞘氨醇单胞菌属（Sphingomonas）、芽单胞菌属（Gemmatimonas）、链霉菌属（Streptomyces）组成。PICRUSt基因预测表明，多花黄精内生细菌以代谢功能为主，主要包括能量代谢、碳水化合物代谢、氨基酸代谢等。通过Spearman相关性分析，在属水平共得到19个菌群与多糖含量相关，其中3个属呈正相关，16个属菌群呈负相关。结论 多花黄精内生细菌的多样性程度高，存在多种不同种类的细菌，且菌群与多糖含量相关。该研究解析了多花黄精内生细菌的多样性、丰度及主要菌属，对深入研究多花黄精内环境有一定指导意义。 |
| 关键词: 多花黄精 16S rRNA 内生细菌 多样性 群落结构 |
| DOI：10.3969/j.issn.1674-070X.2020.07.013 |
| 投稿时间：2020-01-14 |
| 基金项目:国家自然科学青年基金项目（81503197）；湖南省自然科学青年基金项目（2018JJ3310）；湖南省中医药管理局项目（201810）；湖南省中医药研究院科研计划项目（201705）；湖南省科技特派员创新创业（2019NK4143）。 |
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| Analysis of Structure and Diversity of Endophytic Bacteria Community in Polygonatum cyrtonema and Diversity Based on 16S rRNA Gene High-Throughput Sequencing |
| CAI Yuan,LIU Hao,WANG Yongqing,XIE Jing,HUANG Jianhua,LAO Jia,HE Wei,ZHANG Shuihan |
| (Institute of Chinese Materia Medica, Hunan Academy of Traditional Chinese Medicine, Changsha, Hunan 410006, China;Resgreen Group, Changsha, Hunan 410329, China) |
| Abstract: |
| Objective To analyze the bacterial diversity and their community structure from Polygonatum cyrtonema using high-throughput sequencing technology. Methods The polysaccharide content was determined by anthrone-sulfuric acid method. PCR amplification of 16S rRNA high variable region (V3/V4 region) of bacteria was performed by using universal primers. The sequencing of amplicons was conducted by using Illumina Miseq PE250 high-throughput sequencing technology, and bioinformatics analysis of sequencing sequences was performed using software such as QIIME. The correlation between endophytic bacteria and polysaccharide was analyzed by spearman correlation. Results A total of 90 628 effective sequences and 5 287 OTUs were obtained from the sequencing of endophytic bacteria in Polygonatum cyrtonema, and the dilution curve and coverage index reflected that the sequencing results comprehensively covered the endophytic bacterial community of Polygonatum cyrtonema. Alpha diversity analysis showed that the endophytic bacteria were highly diverse. At the level of the phylum, the dominant bacteria were Proteobacteria, Actinobacteria, Gemmatimonadetes, Firmicutes, and Acidobacteria. At the genus level, the core flora consists of Sphingomonas, Gemmatimonas, and Streptomyces. The PICRUSt gene prediction indicated that endophytic bacteria of Polygonatum cyrtonema were mainly metabolized, including energy metabolism, carbohydrate metabolism, and amino acid metabolism. Through spearman correlation analysis, the polysaccharide content associated with endophytic bacteria were obtained 19 bacterias at genus level. Among them, three genera were positively correlated and 16 genera were negatively correlated. Conclusion The diversity of endophytic bacteria in Polygonatum cyrtonema is high. There are many different kinds of bacteria, and the flora is related to polysaccharide content. The study analyzed the diversity, abundance and main genus of endophytic bacteria of Polygonatum cyrtonema, which has certain guiding significance for deep study on the internal environment of Polygonatum cyrtonema to quality. |
| Key words: Polygonatum cyrtonema 16S rRNA endophytic bacteria diversity community structure |
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