双叉犀金龟(Trypoxylus dichotomus)幼虫肠道微生物多样性及纤维素降解菌筛选

Exploration of microbial diversity and identification of cellulose-degrading bacteria in the gut of Trypoxylus dichotomus larvae

摘要:

【目的】揭示双叉犀金龟(Trypoxylus dichotomus)幼虫肠道微生物群落的结构组成和功能特征，挖掘纤维素降解细菌资源。【方法】通过宏基因组测序，对肠道细菌的结构组成和功能进行分析。以羧甲基纤维素(sodium carboxymethyl cellulose,CMC)为唯一碳源从幼虫肠道分离筛选纤维素降解细菌，进一步采用形态学观察和分子生物学方法对菌株进行鉴定。【结果】肠道微生物以细菌为主，占比为81.3%，门水平分布上，厚壁菌门(Firmicutes,45.8%)和拟杆菌门(Bacteroidota,20.3%)为优势菌门，丰度排名前三的菌属为梭菌属(Clostridium,3.90%)、拟杆菌属(Bacteroides,3.52%)、营发酵单胞菌属(Dysgonomonas,2.41%)。宏基因组功能水平分析显示，肠道微生物基因主要与碳水化合物、氨基酸和能量代谢相关，在KEGG数据库注释中，碳水化合物代谢相关基因占据主导地位。CAZy注释结果表明，48 856个(7.43%)基因成功注释到344个碳水化合物代谢酶家族，糖苷水解酶(glycoside hydrolase,GH)(48.67%)是肠道细菌中占最大优势的酶家族，相对丰度前十的功能酶类有6个属于GH家族。此外，从肠道中分离获得3株纤维素降解细菌TRC-3(Bacillus subtilis)、TRC-5(Bacillus subtilis)和TRC-6(Bacillus safensis)，其中TRC-3的滤纸酶、内切葡聚糖酶、外切葡聚糖酶和β-葡萄糖苷酶活性更强。【结论】双叉犀金龟幼虫肠道微生物群落具有较高的多样性和复杂性，拥有大量编码碳水化合物活性酶的基因，存在丰富的纤维素降解细菌资源。

Abstract:

[Objective] To elucidate the structural and functional characteristics of the gut microbiota of Trypoxylus dichotomus larvae and isolate cellulose-degrading bacteria. [Methods] Metagenomic sequencing was employed to analyze the structure and functions of the gut microbiota. Cellulose-degrading bacteria were isolated and screened from the larval gut with carboxymethyl cellulose (CMC) as the sole carbon source. The strains were identified based on morphological characteristics and molecular evidence. [Results] The gut microbiota was dominated by bacteria, which accounted for 81.3%. At the phylum level, Firmicutes (45.8%) and Bacteroidota (20.3%) were the dominant phyla. The top three abundant genera were Clostridium (3.90%), Bacteroidia (3.52%), and Dysgonomonas (2.41%). The functional analysis of metagenome data revealed that the genes of the gut microbiota were mainly associated with carbohydrate, amino acid, and energy metabolism. The annotation in the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the genes related to carbohydrate metabolism were predominant. The annotation in the carbohydrate-active enzyme database (CAZy) indicated that 48 856 (7.43%) genes were successfully annotated to 344 carbohydrate metabolism enzyme families, with glycoside hydrolase (GH, 48.67%) being the most dominant enzyme family in the gut bacteria. Among the top ten functionally abundant enzymes, six belonged to the GH family. Additionally, three strains of cellulose-degrading bacteria, TRC-3 (Bacillus subtilis), TRC-5 (B. subtilis), and TRC-6 (B. safensis), were isolated from the gut. TRC-3 exhibited stronger activities of filter paper enzyme, endoglucanase, exoglucanase, and β-glucosidase. [Conclusion] The gut microbiota of Trypoxylus dichotomus larvae exhibits high diversity and complexity, carrying a large number of genes encoding carbohydrate-active enzymes and harboring rich cellulose-degrading bacteria.