我国中华白蛉的鉴别和分子群体遗传结构研究

发布时间：2018-09-06 19:22

【摘要】： 中华白蛉(Phlebotomus chinensis Newstead,1843)隶属毛蠓科(Psychodidae)、白蛉亚科(Phlebotominae)、白蛉属(Phlebotomus)。中华白蛉在我国的分布广泛,是其分布区利什曼病的重要传播媒介。多年的研究证实中华白蛉在不同地理分布区,其形态特征、生态习性和传病能力存在一定差异,具有相当的复杂性,部分地区其分类地位存疑多年。有鉴于此,深入探讨中华白蛉的分类地位和群体遗传结构具有重要意义。本课题的研究策略如下：首先,在我国中华白蛉的分布地,广泛收集样本；然后,应用形态特征与分子特征相结合的方法,对应分类鉴别中华白蛉及其他蛉种,并探讨中华白蛉的分类地位；其次,筛选中华白蛉具有多态性的微卫星DNA位点,描述其基本特征；再者,应用mtDNA-Cytb基因和多态微卫星DNA位点两个分子标志,阐明我国中华白蛉不同群体间的基因交流、基因渐渗、遗传差异程度以及群体分化等问题,并分析影响群体遗传结构的主要因素。取得如下主要结果： 1.本研究获得我国河南、陕西、四川和甘肃4省6个地点的中华白蛉样本,涵盖不同的地理环境、生态习性、海拔高度和疾病流行区。采集获得模式产地(四川九寨沟,海拔2000m以上)的四川白蛉,以及新疆的吴氏白蛉和长管白蛉。 2.检视了来自不同采集地中华白蛉132只、四川白蛉26只、吴氏白蛉29只,以及长管白蛉23只。吴氏白蛉和长管白蛉具有典型的形态鉴别特征,而中华白蛉与四川白蛉的差异主要体现在翅长,及其雄蛉与雌蛉的翅长度比例。 3.对应形态鉴别确认的中华白蛉、四川白蛉和吴氏白蛉,单蛉抽提基因组DNA,扩增和测定rDNA-ITS2和mtDNA-Cytb序列。序列比对结果显示,ITS2序列在中华白蛉、四川白蛉和吴氏白蛉种间缺乏解析度,Cytb序列的种间解析度比ITS2理想。中华白蛉与四川白蛉的分子特征已具有一定明显差别,且中华白蛉种内也出现遗传分化。提示目前将四川白蛉暂列为独立种较为合理,四川白蛉与中华白蛉的亲缘关系非常近,四川白蛉应处于独立种的分化中(speciation)。 4.在已测定的中华白蛉微卫星DNA的154个阳性克隆序列中,获得有效的微卫星DNA序列共118条。在此基础上,选择了22个位点进行多态性筛选,共获得了多态的微卫星DNA位点14个。同时,对其中的10个位点做了基因扫描,描述了各位点的基本特征。 5.应用mtDNA-Cytb基因和10个微卫星DNA位点研究了中华白蛉的群体遗传结构,两种分子标志的研究结果均显示：来自河南群体的中华白蛉遗传分化程度最高,陕西的两个群体间FST为负值,其间差异极小,总体趋势是基因流水平随着地理距离的增大而减小,群体遗传分布格局符合距离隔离模型。但基于Cytb基因序列的结果显示基因流水平为0.0594,群体间分化水平高,其间遗传差异程度与地理距离密切相关；基于微卫星DNA的结果是群体间分化水平低,基因流为6.599,其间遗传差异程度与种的分化密切相关,也与地理距离存在一定相关性。导致上述差异的原因是两种分子标志本身的特点和进化速率不同,也与Cytb基因序列的样本量较少有一定关系。综合判断,微卫星位点的结果应较为符合实际情况。
[Abstract]:Phlebotomus chinensis Newstead (1843) belongs to Psychodidae, Phlebotominae, and Phlebotomus. Phlebotomus chinensis is widely distributed in China, and is an important vector of Leishmaniasis in its distribution areas. Many years of research has confirmed that Phlebotomae chinensis belongs to different geographical distribution areas, its morphological characteristics and ecological habits. There are some differences between sex and disease transmission ability, and the taxonomic status of some areas is doubtful for many years. Therefore, it is of great significance to study the taxonomic status and population genetic structure of Chinese white fly.
The research strategy of this subject is as follows: firstly, the samples are collected extensively in the distribution area of Chinese white maggot; then, the morphological and molecular characteristics are combined to identify the Chinese white maggot and other maggot species, and the taxonomic status of Chinese white maggot is discussed; secondly, the polymorphic microsatellite DNA loci of Chinese white maggot are screened. Furthermore, two molecular markers, mtDNA-Cytb gene and polymorphic microsatellite DNA loci, were used to elucidate the problems of gene exchange, gene infiltration, genetic diversity and population differentiation among different populations of Chinese white grub, and to analyze the main factors affecting population genetic structure.
1. Samples from 6 locations in Henan, Shaanxi, Sichuan and Gansu provinces were collected, covering different geographical environments, ecological habits, altitude and epidemic areas.
2. A total of 132 Chinese white maggots, 26 Sichuan white maggots, 29 Wu's white maggots and 23 long-tube white maggots from different collections were examined. The typical morphological characteristics of Wu's white maggots and long-tube white maggots were observed. The differences between Chinese white maggots and Sichuan white maggots were mainly reflected in the wing length and the ratio of the wings of male maggots to female maggots.
3. Genomic DNA was extracted from the three species, and rDNA-ITS2 and mtDNA-Cytb sequences were amplified and determined. Sequence alignment results showed that ITS2 sequence was lack of resolution among the three species, and Cytb sequence was better than ITS2. The results showed that it is reasonable to classify Sichuan white grub as an independent species at present, and the relationship between Sichuan white grub and Chinese white grub is very close. Sichuan white grub should be in the differentiation of independent species.
4. Out of 154 positive microsatellite DNA clones, 118 microsatellite DNA sequences were obtained. On this basis, 22 sites were selected for polymorphism screening, and 14 microsatellite DNA loci were obtained. At the same time, 10 of them were gene scanned, and the basic characteristics of each site were described. Sign.
5. Using mtDNA-Cytb gene and 10 microsatellite DNA loci to study the population genetic structure of Chinese white grub, the results of two molecular markers showed that the genetic differentiation degree of Chinese white grub from Henan population was the highest, and the FST between the two populations in Shaanxi was negative, the difference was very small. The general trend was that the level of gene flow along with geographical distance. The genetic distribution pattern of the population conformed to the distance isolation model with the increase of the distance, but the results based on Cytb gene sequence showed that the level of gene flow was 0.0594 and the level of inter-population differentiation was high, and the degree of genetic difference was closely related to geographical distance. The results based on microsatellite DNA showed that the level of inter-population differentiation was low, and the gene flow was 6.599. The difference of transmission is closely related to species differentiation and geographic distance. The reason for the difference is that the characteristics and evolutionary rate of the two molecular markers are different, and the number of Cytb gene sequences is less.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R384.1

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