基于高通量测序分析西藏沙棘根瘤内生菌的多样性

Abstract

根瘤是微生物侵染植物根部并与之形成的共生结构，这些微生物都可被称为植物内生菌。豆科植物根瘤中的内生菌常常又被称为根瘤菌，而侵染非豆科植物形成根瘤的主要是放线菌弗兰克氏菌，这些非豆科植物又被称为放线菌结瘤植物。西藏沙棘是一种典型的放线菌结瘤植物，由于其分布生境的特殊性，对其根瘤内生菌的研究具有重要的生态意义。对于西藏沙棘根瘤内生菌的研究，培养方法因难以模拟自然条件而不易获得纯培养，高通量测序技术对其多样性的研究提供了便利。因此，本研究以生长在甘肃省天祝县金强河河滩地的西藏沙棘根瘤为材料，采用16S rRNA基因扩增子高通量测序方法，结合OTU分析，对西藏沙棘根瘤内生菌的多样性进行探讨。实验结果表明，西藏沙棘根瘤内生菌具有丰富的多样性，根瘤内的优势属为共生固氮的弗兰克氏菌属（Frankia），其相对丰度为47.63%，共检测到7个弗兰克氏菌属的OTUs；根瘤内除弗兰克氏菌外，还存在大量的非弗兰克氏菌，共检测到1523个OTUs，隶属于22个门、33个纲、69个目、113个科和202个属，相对丰度排名前9的属中有25个非弗兰克氏菌属的OTUs。该研究也表明，西藏沙棘根瘤内生菌具有丰富的多样性，西藏沙棘根瘤中不仅存在着可共生固氮的弗兰克氏菌，并且还分布着非弗兰克氏菌；在同一根瘤样品中，弗兰克氏菌属还具有不同的物种。本研究不仅拓展了西藏沙棘根瘤内生菌多样性的研究方法，还为同一寄主植物中弗兰克氏菌多样性的研究提供了分析思路。;Root nodules are the symbiotic structure formed by microorganisms invading the roots of host plants. These microorganisms can be collectively referred to endophytes. The endophytic bacteria in the nodules of leguminous plants are often called rhizobia, while the actinomycetes are the main bacteria that infect non-leguminous plants to form nodules. The main endophytic actinomycetes in non-leguminous plants is Frankia spp.. These non-leguminous plants are also known as actinorhizal plants. Hippophae tibetana is a typical actinorhizal plant. It is very important to study the endophytes of H. tibetana nodules because of the particularity of H. tibetana distribution habitat. For the study of endophytes from H. tibetana root nodules, the culture method is difficult to simulate the natural conditions and it is difficult to obtain pure culture, and the high-throughput sequencing technology provides convenience for the study of their diversity. In this study, the root nodules of H. tibetana, which grew in Jinqiang River beach in Tianzhu County, Gansu Province, were used as the material. The 16S rRNA gene amplification high-throughput sequencing method was adopted to analyze the diversity of Frankia and non-Frankia endophytic bacteria associated with H. tibetana root nodules combined with Operational Taxonomic Units (OTUs). The results showed that the endophytes of H. tibetana root nodules had a rich diversity. Frankia was single dominant genus comprising of 47.63% of the relative abundance of total endophytes, and a total of 7 OTUs of Frankia were detected in H. tibetana root nodules. In addition to Frankia, there were also a large number of non-Frankia endophytic bacteria in H. tibetana root nodules. A total of 1523 OTUs of non-Frankia endophytes were detected, belonging to 22 phyla, 33 classes, 69 orders, 113 families, and 202 genera. There are 25 non-Frankia OTUs among the top 9 genera in relative abundance. This research also revealed that there was rich diversity of endophytes associated with root nodules of H. tibetana. There was not only Frankia but also non-Frankia in H. tibetana root nodules. There were different species of Frankia in the same nodule sample. This study can provide reference for the analysis of Frankia spp. diversity based on high-throughput sequencing and enrich the database of host plants diversity and Frankia species diversity. This study not only expanded the methods of studying the diversity of endophytic bacteria in H. tibetana root nodules, but also provided an analytical approach for the diversity of Frankia spp. in the same host plant.