我国常见白蛉DNA条形码和中华白蛉转录组研究

发布时间：2018-04-30 21:02

本文选题：白蛉 + 生态习性　；参考：《第二军医大学》2017年硕士论文

【摘要】：白蛉是一类体积较小的吸血昆虫,隶属双翅目(Diptera)、毛蠓科(Psychodidae),在我国分布广泛,基于形态特征分类已记录白蛉40余种,部分蛉种可传播利什曼病、巴尔通体病和白蛉热等疾病,具有非常重要的医学意义。对白蛉的准确鉴别,了解白蛉的生态习性,深入阐明传病蛉种的生物学特点尤为重要。本课题的研究策略:首先,在我国河南、四川和海南等地灯诱和人工采集白蛉,调查当地白蛉的生态习性,分子鉴定白蛉的血源动物,依据海南海口全年的灯诱采集标本,初步分析阐明其季节消长规律。其次,检视白蛉的形态特征,鉴定种类;扩增对应样本的mt DNA-COI、Cytb和rDNA-18S基因片段,测定和分析序列,结合形态鉴定的结果,分析分子特征在种内与种间的差异,筛选适合的DNA条形码用于白蛉种类鉴别。再者,应用高通量测序技术,对现场采集的中华白蛉成虫分为雌、雄的头部、胸腹部4组样本,分别进行转录组测序、功能注释与差异表达基因分析。获得以下主要结果:1、我国四川和河南两地的中华白蛉的生态习性存在一定差异。采自四川的白蛉孳生地为山洞和土洞,近家栖,河南的白蛉孳生地为人居的土窑洞,家栖。分子鉴定的血源动物包括:猪、兔、鸡、狗、牛和人等,四川的白蛉主要吸血对象是猪,而河南是鸡,两地均检测到人和狗,其中人的比例分别为4.54%(四川)和41.67%(河南)。2、2015年8月-2016年9月在海南海口全年共捕获377只白蛉,种群密度总体不高,高峰呈双峰,为6月和11月。3、本研究共检视和记录了2属7种白蛉的形态鉴别特征,包括:白蛉属的中华白蛉和施氏白蛉,司蛉属的贝氏司蛉、应氏司蛉、歌乐山司蛉、鳞喙司蛉和鳞胸司蛉。获得对应蛉种的mt DNA-COI和Cytb序列,以及rDNA-18S序列(除歌乐山司蛉)。综合分析结果显示,mtDNA-COI、Cytb和rDNA-18S均可作为DNA条形码鉴别上述白蛉种类。4、贝氏白蛉复合体存在多个隐种,我国的贝氏司蛉与泰国的贝氏司蛉关系密切,与贝氏司蛉A和B种的mtDNA-COI序列差别大,暂定为贝氏司蛉C种。5、高通量测序获得中华白蛉的转录组序列,共133,835,342条原始reads,大于Q20的reads数128,090,788条,占原始数据95.71%,共31.7kM。组装获得32,628条unigene,分别注释到Nr、Swiss-Prot、KEGG、COG和GO数据库。筛选获得4374个具有显著性表达差异的基因,经qRT-PCR验证10个基因片段表达量和转录组测序结果保存一致。中华白蛉的头部和胸腹部的差异表达基因与其功能关系密切,不同性别间的差别基因也是与其摄食和生殖功能相关。
[Abstract]:Lacewing is a small blood-sucking insect belonging to Diptera Diptera. It is widely distributed in China. More than 40 species have been recorded on the basis of morphological classification. Some of them can spread leishmaniasis, Bartonella disease and phlegm fever. Has very important medical significance. It is very important to identify the laceflies accurately, understand their ecological habits and elucidate the biological characteristics of disease transmission species. The research strategies of this subject are as follows: first of all, in Henan, Sichuan and Hainan provinces in China, the ecological habits of the local sandflies were investigated, and the blood source animals were identified by molecular analysis, and the samples were collected according to the annual lamp trap in Haikou, Hainan Province. A preliminary analysis was made to illustrate the seasonal variation and variation. Secondly, the morphological characteristics of the sandfly were examined to identify the species, the mt DNA-COII Cytb and rDNA-18S gene fragments of the corresponding samples were amplified, the sequences were determined and analyzed, and the differences of molecular characteristics between species and species were analyzed by combining the results of morphological identification. The suitable DNA bar code was selected for the species identification of sandflies. Furthermore, high-throughput sequencing technique was used to analyze four groups of female, male head, chest and abdomen samples of adult sandfly collected in the field, respectively, for transcriptional sequencing, functional annotation and differentially expressed gene analysis. The main results are as follows: 1. There are some differences in ecological habits between Sichuan and Henan provinces in China. The breeding ground of sandfly collected from Sichuan is cave and soil cave, near home habitat, and Henan lacewing breeding place is soil cave of human settlement. Molecular identification of the origin of blood animals include: pigs, rabbits, chickens, dogs, cattle and people, Sichuan sandfly is the main blood sucking pigs, while Henan is chicken, both human and dog were detected. The proportion of people was 4.54 and 41.67, respectively. From August 2015 to September 2016, 377 sandflies were captured in Haikou, Hainan Province. The population density was not high, and the peak was double peak. For June and Nov. 3, this study examined and recorded the morphological characteristics of 2 genera and 7 species of sandflies, including: the Chinese and Scheffler sandflies of the genus Phlewing, the Bessler, the Spinelian, and the Geles of the genus, respectively. The squame-beak and the squamothorax. The mt DNA-COI and Cytb sequences and rDNA-18S sequences of the corresponding species were obtained. The results of comprehensive analysis showed that both mtDNA-COICytb and rDNA-18S could be used as DNA bar codes to identify the species of the above mentioned species. There were many codon species in the complex, and there was a close relationship between China and Thailand. The mtDNA-COI sequences of the species A and B were different from those of species A and B, and were tentatively classified as C. 5. The transcriptome sequences were obtained by high-throughput sequencing. A total of 133835342 primordial READSs and 128090788 reads sequences larger than Q20 were obtained, accounting for 95.71 and 31.7kMof the original data. 32628 Unigenees were assembled and annotated into the NrSwiss-ProtKEGG COG and go databases, respectively. 4374 genes with significant difference in expression were screened and confirmed by qRT-PCR. The expression levels of 10 gene fragments were consistent with the results of transcriptome sequencing. The differentially expressed genes in head, chest and abdomen were closely related to their functions, and the difference genes between genders were also related to their feeding and reproductive functions.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R384.1

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