【摘要】： 中華白蛉(Phlebotomus chinensis Newstead,1843)隸屬毛蠓科(Psychodidae)、白蛉亞科(Phlebotominae)、白蛉屬(Phlebotomus)。中華白蛉在我國(guó)的分布廣泛,是其分布區(qū)利什曼病的重要傳播媒介。多年的研究證實(shí)中華白蛉在不同地理分布區(qū),其形態(tài)特征、生態(tài)習(xí)性和傳病能力存在一定差異,具有相當(dāng)?shù)膹?fù)雜性,部分地區(qū)其分類地位存疑多年。有鑒于此,深入探討中華白蛉的分類地位和群體遺傳結(jié)構(gòu)具有重要意義。 本課題的研究策略如下：首先,在我國(guó)中華白蛉的分布地,廣泛收集樣本；然后,應(yīng)用形態(tài)特征與分子特征相結(jié)合的方法,對(duì)應(yīng)分類鑒別中華白蛉及其他蛉種,并探討中華白蛉的分類地位；其次,篩選中華白蛉具有多態(tài)性的微衛(wèi)星DNA位點(diǎn),描述其基本特征；再者,應(yīng)用mtDNA-Cytb基因和多態(tài)微衛(wèi)星DNA位點(diǎn)兩個(gè)分子標(biāo)志,闡明我國(guó)中華白蛉不同群體間的基因交流、基因漸滲、遺傳差異程度以及群體分化等問題,并分析影響群體遺傳結(jié)構(gòu)的主要因素。取得如下主要結(jié)果： 1.本研究獲得我國(guó)河南、陜西、四川和甘肅4省6個(gè)地點(diǎn)的中華白蛉樣本,涵蓋不同的地理環(huán)境、生態(tài)習(xí)性、海拔高度和疾病流行區(qū)。采集獲得模式產(chǎn)地(四川九寨溝,海拔2000m以上)的四川白蛉,以及新疆的吳氏白蛉和長(zhǎng)管白蛉。 2.檢視了來自不同采集地中華白蛉132只、四川白蛉26只、吳氏白蛉29只,以及長(zhǎng)管白蛉23只。吳氏白蛉和長(zhǎng)管白蛉具有典型的形態(tài)鑒別特征,而中華白蛉與四川白蛉的差異主要體現(xiàn)在翅長(zhǎng),及其雄蛉與雌蛉的翅長(zhǎng)度比例。 3.對(duì)應(yīng)形態(tài)鑒別確認(rèn)的中華白蛉、四川白蛉和吳氏白蛉,單蛉抽提基因組DNA,擴(kuò)增和測(cè)定rDNA-ITS2和mtDNA-Cytb序列。序列比對(duì)結(jié)果顯示,ITS2序列在中華白蛉、四川白蛉和吳氏白蛉種間缺乏解析度,Cytb序列的種間解析度比ITS2理想。中華白蛉與四川白蛉的分子特征已具有一定明顯差別,且中華白蛉種內(nèi)也出現(xiàn)遺傳分化。提示目前將四川白蛉暫列為獨(dú)立種較為合理,四川白蛉與中華白蛉的親緣關(guān)系非常近,四川白蛉應(yīng)處于獨(dú)立種的分化中(speciation)。 4.在已測(cè)定的中華白蛉微衛(wèi)星DNA的154個(gè)陽性克隆序列中,獲得有效的微衛(wèi)星DNA序列共118條。在此基礎(chǔ)上,選擇了22個(gè)位點(diǎn)進(jìn)行多態(tài)性篩選,共獲得了多態(tài)的微衛(wèi)星DNA位點(diǎn)14個(gè)。同時(shí),對(duì)其中的10個(gè)位點(diǎn)做了基因掃描,描述了各位點(diǎn)的基本特征。 5.應(yīng)用mtDNA-Cytb基因和10個(gè)微衛(wèi)星DNA位點(diǎn)研究了中華白蛉的群體遺傳結(jié)構(gòu),兩種分子標(biāo)志的研究結(jié)果均顯示：來自河南群體的中華白蛉遺傳分化程度最高,陜西的兩個(gè)群體間FST為負(fù)值,其間差異極小,總體趨勢(shì)是基因流水平隨著地理距離的增大而減小,群體遺傳分布格局符合距離隔離模型。但基于Cytb基因序列的結(jié)果顯示基因流水平為0.0594,群體間分化水平高,其間遺傳差異程度與地理距離密切相關(guān)；基于微衛(wèi)星DNA的結(jié)果是群體間分化水平低,基因流為6.599,其間遺傳差異程度與種的分化密切相關(guān),也與地理距離存在一定相關(guān)性。導(dǎo)致上述差異的原因是兩種分子標(biāo)志本身的特點(diǎn)和進(jìn)化速率不同,也與Cytb基因序列的樣本量較少有一定關(guān)系。綜合判斷,微衛(wèi)星位點(diǎn)的結(jié)果應(yīng)較為符合實(shí)際情況。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R384.1

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