凡纳滨对虾肠道红杆菌科细菌富集碳源筛选及其定向分离

Screening of carbon sources for enrichment and directional isolation ofRhodobacteraceae from the gut of Litopenaeus vannamei

摘要:

【目的】红杆菌科(Rhodobacteraceae)细菌为凡纳滨对虾肠道微生物的优势类群，在健康对虾肠道中具有较高的相对丰度，是指示对虾健康的关键类群，探究对虾肠道红杆菌科细菌定向富集和分离方法，可为对虾养殖益生菌菌剂的研发提供基础。【方法】利用16S rRNA基因高通量测序技术研究不同碳源添加对凡纳滨对虾肠道中红杆菌科细菌的富集作用，筛选对红杆菌科细菌有显著富集作用的碳源；利用纯培养技术从红杆菌科细菌富集的样品中定向分离红杆菌科细菌，并对其进行鉴定和遗传多样性分析。【结果】添加短链脂肪酸(乙酸、丙酸、丁酸、戊酸)和碳酸氢钠对红杆菌科细菌有显著富集作用，主要富集到Cribrihabitans、Tritonibacter、Rhodovulum、Ruegeria、Sagittula和Thalassobius属相关菌株；对红杆菌科细菌相对丰度最高的样品进行稀释涂布培养，共分离纯化出303株细菌，分属于2门12科，其中红杆菌科细菌为主导类群共119株，主要包括Tritonibacter (90株)、Phaeobacter (25株)、Sulfitobacter (1株)、Ruegeria (1株)、Roseovarius (1株)和Aliiroseovarius (1株)等6个属；分离的各属红杆菌科细菌占总分离菌株比例与高通量测序结果中红杆菌科各属细菌占比相似。【结论】本研究基于高通量测序技术探究了添加不同碳源连续传代培养对红杆菌科细菌富集的影响，筛选出了5种富集红杆菌科细菌的碳源，并定向分离获得119株红杆菌科细菌，构建了一种有效定向富集分离红杆菌科细菌的方法。

关键词:凡纳滨对虾;红杆菌科;碳源;16S rRNA 基因高通量测序;定向分离

Abstract:

[Objective]Rhodobacteraceae, the dominant group of gut microbiota in Litopenaeus vannamei, usually has higher relative abundance in the gut of healthy shrimps, and some members of this family have been identified as the indicators for shrimp health. Therefore, clarifying the method for the directional enrichment and isolation of Rhodobacteraceae from shrimp gut can provide a basis for the development of probiotics for shrimp farming. [Methods]The 16S rRNA gene high-throughput sequencing was applied in the screening of the suitable carbon sources for the enrichment of Rhodobacteraceae. Then, the bacteria were directionally isolated from the enriched samples via the pure culture method. Finally, the taxonomic status and genetic diversity of the isolates were determined.[Results] The addition of short-chain fatty acids (acetic acid, propionic acid, butyric acid, and valeric acid) and sodium bicarbonate facilitated the enrichment of Rhodobacteraceae, which mainly included Cribrihabitans, Tritonibacter, Rhodovulum, Ruegeria, Sagittula, andThalassobius. A total of 303 bacterial strains belonging to 12 families of 2 phyla were isolated from the samples with high relative abundance of Rhodobacteraceae, and 119 out of the 303 strains were identified as members of Rhodobacteraceae, including 90 strains of Tritonibacter, 25 strains ofPhaeobacter, 1 strain of Sulfitobacter, 1 strain of Ruegeria, 1 strain ofRoseovarius, and 1 strain ofAliiroseovarius. In addition, all the isolates of Rhodobacteraceae had the relative abundance similar to the results of high-throughput sequencing. [Conclusion] We explored the effect of different carbon sources on the enrichment and directional isolation of Rhodobacteraceae by using high-throughput sequencing and screened out five carbon sources for isolating 119 strains of Rhodobacteraceae.

Key words:Litopenaeus vannamei;Rhodobacteraceae;carbon source;16S rRNA gene high-throughput sequencing;directional isolation