兰州大学机构库 >草地农业科技学院
两种草原毛虫肠道菌多样性的比较分析
Alternative TitleComparative analysis of the diversity of gut bacteria of two Gynaephora species
杨兴卓
Thesis Advisor袁明龙
2019-05-01
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Degree Discipline草学
Keyword草原毛虫 青藏高原 生态适应 高寒草甸 肠道菌 分离培养 16S扩增子 宏基因组 高通量测序
Abstract草原毛虫是我国青藏高原高寒草甸的重大害虫,不仅取食优质牧草与畜争食,而且可改变高寒草甸的植被群落结构,已成为高寒草甸持续利用及健康发展的重要限制因素之一。草原毛虫大面积暴发、持续危害,显示出其对高寒草甸低氧、低温及强紫外辐射等极端生态环境的良好适应性。本论文基于分离培养法及高通量测序技术,比较分析了栖息于高寒草甸不同海拔的门源草原毛虫和黄斑草原毛虫的肠道菌组成、多样性及功能,旨在从肠道菌的视角解析草原毛虫对高海拔环境适应的生态学机制。主要结果如下:1. 基于6种培养基,对两种草原毛虫5龄幼虫的肠道菌进行分离、培养及纯化,并通过菌落形态特征及16S测序进行菌种鉴定。结果显示,LB培养基、牛肉膏蛋白胨培养基、高氏培养基、无机盐培养基,沙门菌培养基,这5种培养基能够培养草原毛虫肠道菌。门源草原毛虫共分离获得5种肠道菌,其中3种(M1、M2、M3)属厚壁菌门的葡萄球菌属,另2种属变形菌门的Lelliottia(M4)和勒克氏菌属(M5)。黄斑草原毛虫共分离得到6种肠道菌,其中3种属厚壁菌门的葡萄球菌(N1和N5)和肠球菌属(N4),其余3种属变形菌门的沙雷氏菌属(N2和N6)和假单胞菌属(N3)。沙雷氏菌已知在协助昆虫降解纤维素、木质素上具有积极作用,提示该菌也有可能协助黄斑草原毛虫降解植物纤维。2. 门源草原毛虫肠道菌16S扩增子共获得了57个OTU,注释到:4个门,其中优势门为变形菌门;7个纲,优势纲为α变形菌纲和γ变形菌纲;12个目,优势目为立克次氏体目和假单胞菌目;15个科,优势科为立克次氏体科(83.8%)和假单胞杆菌科;13个属,优势属为Wolbachia和假单胞菌属;7个种,优势种为酪黄肠球菌、Staphylococcus sciuri和Methylotenera mobilis。门源草原毛虫肠道宏基因组共注释到:5个界,相对丰度最高的是细菌界(71.8%)和真菌界(12.1%);27个门,优势门为变形菌门、厚壁菌门和拟杆菌门;51个纲,优势纲为α变形菌纲、γ变形菌纲和芽孢杆菌纲;107个目,优势目为立克次氏体目、肠球菌目和芽孢杆菌目;178个科,优势科为无形体科、肠杆菌科;236个属,优势属为Wolbachia、埃希氏菌属。3. 黄斑草原毛虫肠道菌16S扩增子测序共获得了91个OTU,注释到:5个门,厚壁菌门相对丰度最高;7个纲,优势纲为杆菌纲和γ变形菌纲;10个目,优势目为乳杆菌目和芽孢杆菌目;11个科,优势科为肉杆菌科和肠球菌科;9个属,优势属为肉食杆菌属和肠球菌属;8个种,优势种为铅黄肠球菌和壁芽孢杆菌。黄斑草原毛虫肠道宏基因组共注释到:5个界,相对丰度最高的为细菌界(57.3%)、真菌界(17.7%)和病毒界(11.6%);27个门,优势门为变形菌门、拟杆菌门和厚壁菌门;56个纲,优势纲为γ变形菌纲、芽孢杆菌纲和纤维黏网菌;119个目,优势目为肠杆菌目、芽孢杆菌目和噬纤维菌目;200个科,优势科为肠杆菌科、动球菌科和Cyclobacteriaceae;262个属,优势属为埃希氏菌属、土壤芽孢杆菌属和Cecembia。4.草原毛虫肠道宏基因组注释到与新陈代谢、碳水化合物运输、氨基酸合成等代谢密切相关的基因,表明肠道菌可能参与了草原毛虫的碳水化物代谢、物质合成、物质降解及吸收等生理过程,在草原毛虫适应高寒草甸极端生态环境中发挥重要作用。16S扩增子测序表明,门源草原毛虫肠道菌相对丰度较高的为Wolbachia和假单胞菌,而黄斑草原毛虫肠道菌相对丰度较高的为肉食杆菌属和肠球菌。这种差异可能与两种草原毛虫栖息的差异性海拔环境相关。门源草原毛虫肠道菌α多样性未显著高于黄斑草原毛虫(P>0.05),但在不考虑肠道菌相对丰度的情况下,不动杆菌属、无色杆菌、芽单胞菌属、噬氢菌属、甲氧菌属、Wolbachia、肠球菌属、假单胞菌属和肉食杆菌属等9个属在两种草原毛虫间差异显著(P<0.01)。据此推测,这些菌可能与两种草原毛虫适应差异性海拔环境相关。
Other AbstractGynaephora spp. (Lepidoptera: Erebidae: Lymantriinae) are one group of major pests of alpine meadow on the Tibet Plateau in China. They not only feed on high quality forage as a tough competitor of livestock, but also change the vegetation community structure of alpine meadow, which means it has become a restrictive factor and barrier for the sustainable utility and healthy development of alpine meadow. Their outbreak in many areas and continuous damage of grassland caterpillars show good adaptation to extreme ecological environments such as low oxygen, low temperature and strong ultraviolet radiation in the alpine meadow. In this thesis, the composition, diversity and function of gut bacteriain Gynaephora menyuanensis and Gynaephora alpherakii inhabiting different altitudes in alpine meadow were compared and analyzed based on the isolation and culture methods and high-throughput sequencing technology, so as to dissect the ecological mechanisms of Gynaephora’s adaptation to high-altitude environments from a perspective of gut bacteria. The main results are as follows: 1. Gut bacteria of fifth-instar larvae of two Gynaephora species were isolated, cultivated, and purified based on six kinds of culture media. Species identification of gut bacteria was conducted by using morphological characteristics of bacterial colony and 16S sequencing techonology. The results showed that five culture media can be used to isolate Gynaephora gut bacteria. A total of five species of gut bacteriawere isolated from G. menyuanensis, of which three species (M1, M2, M3) belonged to Staphylococcus of Firmicutes, and two species belonged to Lelliottia (M4) and Leckerella (M5) of Proteobacteria. A total of 6 gut bacteria were isolated from G.alpherakii, three of which belonged to Staphylococcus (N1 and N5) and Enterococcus (N4) of Firmicutes, and the remaining three belonged to Serratia (N2 and N6) and Pseudomonas (N3) of Proteobacteria. Serratia is known to have a positive role in assisting insects to degrade cellulose and on lignin, suggesting that this bacterium may also assist G.alpherakii to degrade plant fibers. 2. A total of 57 OTUs were obtained from 16S amplicon sequencing of gut bacteria from G. menyuanensis, which were annotated as: 4 phyla, of which the dominant phylum was Proteobacteria; 7 classes, of which the dominant classes were Alphaproteobacteria and Gammaproteobacteria; 12 orders, of which the dominant orders were Rickettsiales and Pseudomonadales; 15 families, of which the dominant families were Rickettsiaceae and Pseudomonadaceae; 13 genera, of which the dominant bacteria were Wolbachia and Pseudomonas; and 7 species, of which the dominant species were Enterococcus tyrococcus, Staphylococcus sciuri, and Methylotenera mobilis. The sequencing results of the gut metagenome of G. menyuanensis were annotated as: 5 kingdoms, of which Bacteria (71.8%) and Eumycetes (12.1%) showed the highest relative abundance; 27 phyla, of which Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla; 51 classes, of which Alphaproteobacteria, Gammaproteobacteria and Bacillus were the dominant classes; 107 orders, of which Rickettsiales, Enterococcales, and Bacillus were the dominant orders; 178 families, of which Anaplasmataceae and Enterobacteriaceae were the dominant families; and 236 genera, of which Wolbachia and Escherichia were the dominant genera. 3. A total of 91 OTUs were obtained by 16S amplicon sequencing of G.alpherakii, which were annotated as: 5 phyla, of which Firmicutes showed the highest relative abundance; 7 classes, of which Bacilli and Gammaproteobacteria were the dominant classes; 10 orders, of which Lactobacillus and Bacillus were the dominant orders;11 families, of which Carnobacteriaceae and Enterococcaceae were the dominant families; 9 genera, of which Carnobacteriales and Enterococcaceae were the dominant genera; 8 species, of which Enterococcus casseliflavus and Bacillus muralis were the dominant species . The sequencing results of the gut metagenome of G.alpherakii were annotated as: 5 kingdoms, of which Bacterial (57.3%), Eumycetes (17.7%) and Virus (11.6%) showed the highest abundance; 27 phyla, of which Proteobacteria, Bacteroidetes, and Firmicutes were the dominant phyla; 56 classes, of which Gammaproteobacteria, Bacillales, and Cellulosimicrobiales were the dominant classes; 119 orders, of which  Enterobacteriales, Bacillales, and Cellulophagales were the dominant orders; 200 families, of which Enterobacteriaceae, Arthroceae, and Cyclobacteriaceae were the dominant families; and 262 genera, of which Escherichia, Agrobacteriales, and Cecembia were the dominant genera. 4. Many annotated genes obtained from metagenomic sequencing of gut bacteria were related with metabolism, carbohydrate transport, amino acid synthesis and so on, indicating that the gut bacteria may be involved in physiological processes such as carbohydrate metabolism, material synthesis, material degradation and absorption of the host, and thus play an important role in ecological adaptation of Gynaephora to extreme environments of alpine meadow. 16S amplicon sequencing showed that Wolbachia and Pseudomonas were the most abundant species in gut bacteria of G.menyuanensis, while Carnivora and Enterococcus in gut bacteria of G.alpherakii. This difference in the most abundant gut bacteria may be associated with adaptation of the two Gynaephoras species to different altitudes.The α diversity of gut bacteria in G.menyuanensis was not significantly higher than that of G.alpherakii (P>0.05), but nine genera including Acinetobacter, Achromobacter, Blastomonas, Hydrogenophaga, Methanobacterium, Wolbachia, Enterococcus, Pseudomonas, and Carnivorawere significantly different between the two Gynaephora species (P<0.01) when the relative abundance of gut bacteria were not considered. Therefore, it was proposed that these nine generas may be associated with the adaptation of the two Gynaephora species to different altitude environments.
Pages82
URL查看原文
Language中文
Document Type学位论文
Identifierhttp://ir.lzu.edu.cn/handle/262010/338102
Collection草地农业科技学院
Affiliation草地农业科技学院
Recommended Citation
GB/T 7714
杨兴卓. 两种草原毛虫肠道菌多样性的比较分析[D]. 兰州. 兰州大学,2019.
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