粉尘螨体内微生物群落多样性及功能基因的宏基因组分析

doi:10.12140/j.issn.1000-7423.2025.03.014

中国寄生虫学与寄生虫病杂志 ›› 2025, Vol. 43 ›› Issue (3): 395-402.doi: 10.12140/j.issn.1000-7423.2025.03.014

粉尘螨体内微生物群落多样性及功能基因的宏基因组分析

杨亚雯¹()(), 洪钰婕¹, 钱格格², 凌敏¹, 王子璇³, 俞慧¹, 孙怀宇¹, 陶香林³, 李敏洁⁴, 孙恩涛³^,^*()()

1 皖南医学院公共卫生学院，安徽芜湖 241000
2 皖南医学院病理教研室，安徽芜湖 241000
3 皖南医学院检验学院，安徽芜湖 241000
4 营养健康与食品安全北京市重点实验室，中粮营养健康研究院有限公司，北京 102209

收稿日期:2025-01-08 修回日期:2025-03-04 出版日期:2025-06-30 发布日期:2025-06-16
通讯作者: *孙恩涛（ORCID: 0000-0003-4771-0301），男，博士，教授，从事医学螨类分子生态学研究。E-mail：asdentao@126.com
作者简介:杨亚雯（ORCID: 0009-0007-4950-1227），女，硕士研究生，从事卫生检验研究。E-mail：2736481900@qq.com
基金资助:
国家自然科学基金(31870352);2024安徽省学科（专业）带头人培育项目(DTR2024032)

Macrogenomic next-generation sequencing of microbial community diversity and functional genes in Dermatophagoides farinae

YANG Yawen¹()(), HONG Yujie¹, QIAN Gege², LING Min¹, WANG Zixuan³, YU Hui¹, SUN Huaiyu¹, TAO Xianglin³, LI Minjie⁴, SUN Entao³^,^*()()

1 School of Public Health, Wannan Medical College, Wuhu 241000, Anhui, China
2 Department of Pathology, Wannan Medical College, Wuhu 241000, Anhui, China
3 School of Laboratory Medicine, Wannan Medical College, Wuhu 241000, Anhui, China
4 Beijing Key Laboratory of Nutrition & Health and Food Safety, COFCO Nutrition and Health Research Institute Co., Ltd., Beijing 102209, China

Received:2025-01-08 Revised:2025-03-04 Online:2025-06-30 Published:2025-06-16
Contact: *E-mail: asdentao@126.com
Supported by:
National Natural Science Foundation of China(31870352);2024 Anhui Provincial Discipline Leader Cultivation Project(DTR2024032)

摘要/Abstract

摘要：

目的研究粉尘螨体内微生物群落的组成、结构和功能，并分析其功能基因特征。方法采集3份粉尘瞒样品（每份150只），提取3份粉尘螨总DNA，构建双末端测序文库。桥式PCR扩增后进行宏基因组测序。将获得的基因序列与非冗余蛋白（NR）、直系同源蛋白分组比对（EggNOG）、京都基因与基因组百科全书（KEGG）、碳水化合物活性酶（CAZY）、全面抗生素抗性（CARD）等数据库进行BLAST比对。采用α多样性指数实际观测物种数指数、Ace指数、Chao1指数、香农多样性指数、辛普森多样性指数、覆盖度指数分析粉尘螨体内微生物的基因多样性。结果 3份粉尘螨样品宏基因组测序获得1.18 × 10⁷~1.29 × 10⁷个碱基。经拼接后，得到的重叠群数目为34 527个，N50为25 005个，重叠群的平均长度为11 509个。对粉尘螨体内微生物预测基因进行物种注释，选取水平上相对丰度前20位的物种进行分析，共注释到粉尘螨体内微生物73门430属689种，其中细菌最为丰富，占80.9%，其次为病毒（5.5%），再次为真菌（1.2%）。在细菌群落中，假单胞菌门（97.8%）和厚壁菌门（0.8%）是优势菌门，巴尔通体属（59.0%）和未分类的α变形菌属（11.0%）为优势菌属。在真菌群落中，微孢子门（66.6%）和子囊菌门（19.6%）占主导地位，Pancytospora（41.4%）和Saccharomyces（12.2%）为优势菌属。在病毒群落中，Cressdnaviricota（96.2%）和Uroviricota（2.6%）为优势菌门，环状病毒属（96.2%）为优势菌属。α多样性结果显示，3份粉尘螨样品重复性好。EggNOG数据库蛋白序列注释结果显示，细菌和真菌分别注释到23和22种功能类别，其中翻译/核糖体结构和生物发生类别的基因数在细菌（15.3%）和真菌（27.4%）中均占比最高。KEGG数据库分析基因功能结果显示，细菌中辅因子和维生素的代谢相关基因占主导地位（10.5%），在真菌中，折叠、分类和降解相关的基因更为突出（12.0%）。CAZY数据库对碳水化合物代谢酶家族的注释结果表明，在细菌中，高达98.3%的基因能够成功匹配到65个不同的碳水化合物代谢酶家族，其中以糖基转移酶家族占主导地位（57.6%）。真菌中仅有0.4%的基因能够注释到12个碳水化合物代谢酶家族，糖基转移酶家族也是主要酶家族（87.7%）。CARD数据库的抗性基因注释结果显示，在细菌中，99.1%的基因能够注释到17种不同的抗生素抗性基因，其中多重耐药性基因占比最高（39.5%）。在真菌中，0.3%的基因能够注释到5种抗生素抗性基因，以四环素抗性基因和多重耐药性基因为主，分别占41.6%和39.7%。结论粉尘螨体内微生物群落存在复杂性和多样性，细菌和真菌在基因功能和代谢途径存在差异。

关键词: 粉尘螨, 宏基因组测序, 微生物群落, 功能基因

Abstract:

Objective To investigate the composition, structure and function of the microbial community in Dermatophagoides farinae and to analyze characteristics of their functional genes. Methods Total DNA was extracted from D. farinae and paired-end library was constructed, and metagenomic next-generation sequencing (mNGS) was performed following bridging PCR assay. The obtained gene sequences aligned with BLAST program in the non-redundant protein sequence (NR), Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (EggNOG), the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Carbohydrate Activating Enzymes (CAZY) and the Comprehensive Antibiotic Resistance (CARD) databases. The diversity of microbial genes was analyzed in D. farinae with observed species index, Ace index, Chao1 index, Shannon diversity index, Simpson’s diversity index and coverage index. Results mNGS results of D. farinae yielded (1.18 to 1.29) × 10⁷ bases. Following sequence assembly, there were 34 527 overlapping groups, with an N50 value of 25 005, and the mean length of overlapping groups was 11 509. The top 20 most abundant species were annotated to 73 phyla, 430 genera, and 689 species, and bacteria was the predominant microorganism in D. farina (80.9%), followed viruses (5.5%) and fungi (1.2%) in D. farinae. Within the bacterial community, Proteobacteria (97.8%) and Firmicutes (0.8%) were dominant phyla, with Bartonella (59.0%) and unclassified Alphaproteobacteria (11.0%) as dominant genera. Within the fungal community, Microsporidia (66.6%) and Ascomycota (19.6%) were dominant phyla, with Pancytospora (41.4%) and Saccharomyces (12.2%) as dominant genera. In the viral community, Cressdnaviricota (96.2%) and Uroviricota (2.6%) were dominant phyla, with Orbivirus (96.2%) as the dominant genus. α diversity analysis showed a good repeatability of D. farinae samples. Protein sequence annotation results in the EggNOG database showed that bacteria and fungi were annotated to 23 and 22 functional categories, respectively, with the percentage of genes associated with translation/ribosome structure and biogenesis (bacteria, 15.3%; fungi, 27.4%). KEGG annotation results showed that cofactor- and vitamin metabolism-related genes were dominant in bacteria (10.5), and genes associated with folding, sorting, and degradation were more pronounced in fungi (12.0%). CAZY annotations showed that 98.3% of bacterial genes were matched to 65 distinct CAZY families, with glycosyltransferases as the dominant CAZY family member (57.6%), and only 0.4% of fungal genes were annotated to 12 CAZY families, with glycosyltransferases as the primary CAZY family member (87.7%). CARD annotations showed that 99.1% of bacterial genes were annotated to 17 diverse antibiotic resistance genes, with multidrug resistance genes exhibiting the highest prevalence (39.5%), and 0.3% of fungal genes were annotated to 5 antibiotic resistance genes, with tetracycline resistance genes (41.6%) and multidrug resistance genes (39.7%) as the two most common genes. Conclusion The microbial community within D. farinae exhibits a significant complexity and diversity, and the gene functions and metabolic pathways vary between bacteria and fungi.

Key words: Dermatophagoides farinae, Metagenomic next-generation sequencing, Microbial community, Functional gene

中图分类号:

R384.429

杨亚雯, 洪钰婕, 钱格格, 凌敏, 王子璇, 俞慧, 孙怀宇, 陶香林, 李敏洁, 孙恩涛. 粉尘螨体内微生物群落多样性及功能基因的宏基因组分析[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(3): 395-402.

YANG Yawen, HONG Yujie, QIAN Gege, LING Min, WANG Zixuan, YU Hui, SUN Huaiyu, TAO Xianglin, LI Minjie, SUN Entao. Macrogenomic next-generation sequencing of microbial community diversity and functional genes in Dermatophagoides farinae[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2025, 43(3): 395-402.

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粉尘螨体内微生物群落多样性及功能基因的宏基因组分析

Macrogenomic next-generation sequencing of microbial community diversity and functional genes in Dermatophagoides farinae

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