中华按蚊肠道细菌分子多态性研究
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摘要
中华按蚊(Anopheles sinensis)是我国传播疟疾的主要媒介,在我国分布广,种群数量大。通过研究中华按蚊肠道细菌多样性,可以为探讨其肠道细菌对中华按蚊的生理功能和生态学意义奠定基础,为进一步了解中华按蚊与其共生菌的协同进化、物种进化途径提供新的认知,最终为防治中华按蚊和控制疟疾提供新思路。
本研究通过利用16S rRNA基因序列分析和PCR—DGGE分析技术研究了中华按蚊幼虫、成虫和不同地理种群成虫肠道细菌多样性。
在中华按蚊幼虫肠道中分离出28种细菌,系统发育地位上分属5大菌纲:丙型变形菌纲(Gammaproteobacteria)、黄杆菌纲(Flavobacteria)、放线菌纲(Actinobacteria)、乙型变形菌纲(Betaproteobacteria)和厚壁菌纲(Firmicutes),其中丙型变形菌纲和放线菌纲的细菌在肠道细菌中约占80%。对于中肠,丙型变性菌纲的细菌所占比例为57.6%,为中肠的主要细菌群落;放线菌纲在后肠的比例为37.6%,是后肠的主要细菌群落。而对于整个肠道,放线菌纲Leucobacter属和黄杆菌纲金黄杆菌属细菌占整个肠道细菌19%和10.6%,为中华按蚊幼虫肠道优势菌。
采用16S rRNA克隆文库技术对中华按蚊成虫肠道细菌多样性进行研究时共检测到12条16S rRNA基因序列。在系统发育地位上主要归X3个细菌纲:丙型变形菌纲、黄杆菌纲和放线菌纲,其中有59.8%的细菌属于丙型变形菌纲。丙型变性菌纲在中肠和后肠分别占54%和65.6%,占绝对优势,成为中后肠主要细菌群落。与Enterobacter sp.(EF599680)高度同源(同源性99%)的细菌和杆菌纲金黄杆菌属的Chryseobacterium meningosepticum在成虫肠道中分布比例分别为16.4%和13.8%,成为中华按蚊成虫肠道优势菌。
在研究中华按蚊5个不同地理种群成虫肠道细菌多样性时发现:中华按蚊不同地理种群肠道中都存在肠杆菌属和金黄杆菌属的细菌,这些细菌的存在与地理位置和生态环境无对应关系。通过聚类分析得知,中华按蚊不同地理种群肠道细菌可分为2大类:第一类包括实验种、湖北武汉种、湖南娄底种和四川邛崃种,第二类包括浙江衢州种和湖北黄梅种,同一类的中华按蚊肠道细菌种类相似系数大,存在一定的相似性。这些地区的地理分布、气候等外界环境对肠道菌群的结构有一定影响,取食不同食物在内的各种因素也会对肠道微生物的菌群结构及含量产生影响。
Anopheles sinensis is the most important vector transmitted Malaria in China, they are numerous and widespread distributing.Diversity research of the intestinal bacterial community of An.sinensis,explore intestinal bacteria on the impact of the physiological function and ecological significance of An.sinensis,understand coevolution between An.sinensis and its symbiotic bacteria,provide new knowledge for species evolution ways.Finally,these bacteria could be a potential way of reseach for new mosquito control methods and controlling malaria.
In this study,the bacterial communities in larvae gut,adults gut and different geographic populations of An.sinensist were analysed using Denaturing Gradient Gel Electrophoresis(DGGE)of amplified 16S rRNA sequences.
Twenty eight different DGGE profiles of bacteria clones were obtained from the larvae gut.With sequence similarities and phylogenetic tree,five major phyla of bacteria were observed:Gammaproteobacteria,Flavobacteria,Actinobacteria, Betaproteobacteria and Firmicutes.Gammaproteobacteria and Actinobacteria represented 80%of the total clones.Gammaproteobacteria has 57.6%,mainly in the midgut bacterial community;Actinobacteria has 37.6%,they are the main bacterial community in the hindgut.Leucobacter and Chryseobacterium genus have 19%and 10.6%in the whole intestinal bacteria,they are preponderant bacterium in lavae gut.
The results showed that the intestinal microbial flora of An.sinensist belongs to three major phyla of bacteria:Gammaproteobacteria,Flavobacteriaa,Actinobacteria. Gammaproteobacteria represented 59.8%of the total clones.Gammaproteobacteria are mainly bacterial community respectively in the midgut and hindgut,respectively has 54%and 65.6%.Enterobacter sp.(alignment similarity 99%) and Chryseobacterium meningosepticum represented 16.4%and 13.8%,predominant in adults gut.
Research diversity of the intestinal bacterial community in different population of An.sinensis,the results showde:Enterobacter sp.and Chryseobacterium meningosepticum are all existting in different population,the presence of these bacteria with no geographical and ecological environment relationship.Through cluster analysis that intestinal bacterial of different geographic population of Anopheles sinensis can be divided into two types:the first type including laboratory populations,Wuhan in Hubei,Loudi in Hunan and Qionglai in Sichuan Loudi species, the other type including Quzhou in Zhejiang and Huangguang in Hubei species,the same type of the intestinal bacterial species have big similarity coefficients,there are some similarities.In addition to geographical distribution,climate external environment,eating different foodand other the various factors have an impact on the structure and content of the intestinal microbial flora.
本研究通过利用16S rRNA基因序列分析和PCR—DGGE分析技术研究了中华按蚊幼虫、成虫和不同地理种群成虫肠道细菌多样性。
在中华按蚊幼虫肠道中分离出28种细菌,系统发育地位上分属5大菌纲:丙型变形菌纲(Gammaproteobacteria)、黄杆菌纲(Flavobacteria)、放线菌纲(Actinobacteria)、乙型变形菌纲(Betaproteobacteria)和厚壁菌纲(Firmicutes),其中丙型变形菌纲和放线菌纲的细菌在肠道细菌中约占80%。对于中肠,丙型变性菌纲的细菌所占比例为57.6%,为中肠的主要细菌群落;放线菌纲在后肠的比例为37.6%,是后肠的主要细菌群落。而对于整个肠道,放线菌纲Leucobacter属和黄杆菌纲金黄杆菌属细菌占整个肠道细菌19%和10.6%,为中华按蚊幼虫肠道优势菌。
采用16S rRNA克隆文库技术对中华按蚊成虫肠道细菌多样性进行研究时共检测到12条16S rRNA基因序列。在系统发育地位上主要归X3个细菌纲:丙型变形菌纲、黄杆菌纲和放线菌纲,其中有59.8%的细菌属于丙型变形菌纲。丙型变性菌纲在中肠和后肠分别占54%和65.6%,占绝对优势,成为中后肠主要细菌群落。与Enterobacter sp.(EF599680)高度同源(同源性99%)的细菌和杆菌纲金黄杆菌属的Chryseobacterium meningosepticum在成虫肠道中分布比例分别为16.4%和13.8%,成为中华按蚊成虫肠道优势菌。
在研究中华按蚊5个不同地理种群成虫肠道细菌多样性时发现:中华按蚊不同地理种群肠道中都存在肠杆菌属和金黄杆菌属的细菌,这些细菌的存在与地理位置和生态环境无对应关系。通过聚类分析得知,中华按蚊不同地理种群肠道细菌可分为2大类:第一类包括实验种、湖北武汉种、湖南娄底种和四川邛崃种,第二类包括浙江衢州种和湖北黄梅种,同一类的中华按蚊肠道细菌种类相似系数大,存在一定的相似性。这些地区的地理分布、气候等外界环境对肠道菌群的结构有一定影响,取食不同食物在内的各种因素也会对肠道微生物的菌群结构及含量产生影响。
Anopheles sinensis is the most important vector transmitted Malaria in China, they are numerous and widespread distributing.Diversity research of the intestinal bacterial community of An.sinensis,explore intestinal bacteria on the impact of the physiological function and ecological significance of An.sinensis,understand coevolution between An.sinensis and its symbiotic bacteria,provide new knowledge for species evolution ways.Finally,these bacteria could be a potential way of reseach for new mosquito control methods and controlling malaria.
In this study,the bacterial communities in larvae gut,adults gut and different geographic populations of An.sinensist were analysed using Denaturing Gradient Gel Electrophoresis(DGGE)of amplified 16S rRNA sequences.
Twenty eight different DGGE profiles of bacteria clones were obtained from the larvae gut.With sequence similarities and phylogenetic tree,five major phyla of bacteria were observed:Gammaproteobacteria,Flavobacteria,Actinobacteria, Betaproteobacteria and Firmicutes.Gammaproteobacteria and Actinobacteria represented 80%of the total clones.Gammaproteobacteria has 57.6%,mainly in the midgut bacterial community;Actinobacteria has 37.6%,they are the main bacterial community in the hindgut.Leucobacter and Chryseobacterium genus have 19%and 10.6%in the whole intestinal bacteria,they are preponderant bacterium in lavae gut.
The results showed that the intestinal microbial flora of An.sinensist belongs to three major phyla of bacteria:Gammaproteobacteria,Flavobacteriaa,Actinobacteria. Gammaproteobacteria represented 59.8%of the total clones.Gammaproteobacteria are mainly bacterial community respectively in the midgut and hindgut,respectively has 54%and 65.6%.Enterobacter sp.(alignment similarity 99%) and Chryseobacterium meningosepticum represented 16.4%and 13.8%,predominant in adults gut.
Research diversity of the intestinal bacterial community in different population of An.sinensis,the results showde:Enterobacter sp.and Chryseobacterium meningosepticum are all existting in different population,the presence of these bacteria with no geographical and ecological environment relationship.Through cluster analysis that intestinal bacterial of different geographic population of Anopheles sinensis can be divided into two types:the first type including laboratory populations,Wuhan in Hubei,Loudi in Hunan and Qionglai in Sichuan Loudi species, the other type including Quzhou in Zhejiang and Huangguang in Hubei species,the same type of the intestinal bacterial species have big similarity coefficients,there are some similarities.In addition to geographical distribution,climate external environment,eating different foodand other the various factors have an impact on the structure and content of the intestinal microbial flora.
引文
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