本文關(guān)鍵詞： 雷氏按蚊 分子鑒別 群體遺傳 微衛(wèi)星DNA　出處：《第二軍醫(yī)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：雷氏按蚊Anopheles lesteri Baisas Hu 1936,隸屬按蚊屬(Genus Anopheles)、赫坎按蚊種團(An. hyrcanus group),分布于菲律賓、中國、日本、韓國和關(guān)島等地,就醫(yī)學(xué)重要性而言,僅在我國中部具有較高的傳瘧效能。多年的調(diào)查顯示,雷氏按蚊在我國不同地理分布區(qū),生態(tài)習(xí)性和傳病能力也存在一定差異,故亟待深入研究雷氏按蚊的群體的遺傳差異。本研究應(yīng)用分子特征首先鑒別我國的按蚊樣本;在研究雷氏按蚊多態(tài)微衛(wèi)星DNA位點特征的基礎(chǔ)上,應(yīng)用多態(tài)的微衛(wèi)星DNA位點檢測我國雷氏按蚊群體遺傳結(jié)構(gòu)。依據(jù)分子特征鑒別我國的按蚊樣本,提高了準(zhǔn)確性,在一定程度上補充和更新了各地按蚊的本地資料。雷氏按蚊不同群體的遺傳差異程度,以及群體分化等問題的闡明,可望為制定媒介區(qū)的瘧疾控制策略提供理論依據(jù)。 課題研究策略如下:首先,對我國不同分布地的按蚊樣本依據(jù)rDNA的2個標(biāo)志,即ITS2和D3,進行分子鑒定;之后,對課題組已分離的雷氏按蚊微衛(wèi)星DNA位點應(yīng)用現(xiàn)場樣本進行GENESCAN掃描,研究各位點的基本特征;應(yīng)用獲得的多態(tài)微衛(wèi)星位點檢測我國雷氏按蚊的群體內(nèi)和群體間的遺傳差異。取得如下主要結(jié)果: 1.本研究分子鑒定的樣本來自云南、湖北、貴州、河南、廣東、山東、遼寧、陜西和海南共126個樣本,序列分析結(jié)果顯示包括:中華按蚊(An. sinensis)(n=44)、迷走按蚊(An. vagus)(n=22)、暗灰按蚊(An. pullus) (n=18)、微小按蚊(An. minimus)(n=13)、林氏按蚊(An. lindesayi)(n=10)、克萊按蚊(An. kleini)(n=4)、筠連按蚊(An. junlianensis)(n=4)、腹簇按蚊(An. kochi)(n=4)、比倫按蚊(An. belenrae)(n=3)、貴陽按蚊(An. kweiyangensis)(n=3)和烏頭按蚊(An. aconitus)(n=1)。 2.序列分析發(fā)現(xiàn)存在4類無法定名的種類,即LL1、LL2、LL3和LL4,除LL4外,其他3類均與赫坎按蚊種團成員種最為近似,應(yīng)是該種團的新成員種。另外,在多種按蚊中發(fā)現(xiàn)個體內(nèi)的堿基雙峰現(xiàn)象,提示這些按蚊物種近期分化活躍,處于隱種形成中(speciation)。 3.應(yīng)用分子鑒定確認(rèn)的雷氏按蚊現(xiàn)場57個樣本,研究了雷氏按蚊11個多態(tài)微衛(wèi)星DNA的基本特征,為進一步檢測雷氏按蚊的群體遺傳結(jié)構(gòu)提供了新的分子標(biāo)志。 4.應(yīng)用9個多態(tài)的微衛(wèi)星DNA位點檢測了采自我國17個,以及韓國1個采集地的雷氏按蚊樣本共216個,各行政省份合并后成1個群體,將獲得的9個群體進行后續(xù)分析。 5.雷氏按蚊群體內(nèi)遺傳差異的分析結(jié)果可見,平均期望雜合度和觀察雜合度的范圍為0.4848(江蘇)～0.7134(遼寧)和0.3309(江蘇)～0.4704(韓國);在遼寧群體中自有等位基因最多(9),而云南群體無自有等位基因;近交系數(shù)范圍為0.2101(海南)～0.5037(湖北),平均為0.3508;經(jīng)檢驗,總共30個位點偏離哈代-溫伯格平衡,遼寧群體中的位點最多(7),在韓國、云南和湖北群體無位點偏離;連鎖不平衡的檢驗結(jié)果顯示,存在連鎖關(guān)聯(lián)的共38對位點(P0.01),位點ANL10與ANL11在所有群體中均出現(xiàn)連鎖關(guān)聯(lián),其他位點無規(guī)律。 6.雷氏按蚊群體間遺傳差異分析時去除江蘇群體(實驗室品系),以及位點ANL11。分析結(jié)果可見,成對群體間的FST范圍為-0.0127(遼寧與韓國)～0.2539(云南與海南);Mantel檢驗結(jié)果顯示群體間遺傳差異與地理距離為正相關(guān)關(guān)系,群體遺傳結(jié)構(gòu)符合地理隔離模型;分子變異等級分析顯示雷氏按蚊的變異主要存在于群體內(nèi),占總變異的86.55%,群體間的變異僅占總變異的13.45%;貝葉斯法的分析結(jié)果均顯示所有群體可分為2個支,分支I包括廣東和遼寧群體,分支II包括即韓國、河南、湖北、廣東、海南和四川群體。 7.雷氏按蚊群體的穩(wěn)定性和規(guī)模的分析結(jié)果可見,河南、湖北、海南和遼寧共4個群體經(jīng)歷了近期群體的擴張;基于連鎖不平衡模型,群體規(guī)模的范圍為1.2(湖北)～33.7(廣東),總體的群體規(guī)模為44.6,95%置信區(qū)間為40.6～49.0。 8.綜合分析雷氏按蚊的群體遺傳結(jié)構(gòu),可以推測雷氏按蚊的祖先群體來自云南,其他群體均為擴散而至,向北、東遷移和擴散,在途中定殖,為適應(yīng)當(dāng)?shù)厣鷳B(tài)環(huán)境出現(xiàn)一定比例的變異。雷氏按蚊分布廣泛,地理屏障雖在一定程度上阻礙基因的交流,但群體間差異并不大,群體遺傳結(jié)構(gòu)符合地理隔離模型(Isolation-by-distance Model)。因其在我國中部的醫(yī)學(xué)重要性,故在分布地長期、大量使用殺蟲劑,導(dǎo)致雷氏按蚊的群體數(shù)量較小,存在大量近交,偏離哈代-溫伯格平衡的現(xiàn)象。
[Abstract]:Anopheles LESTERI Baisas Hu 1936 Anopheles LESTERI, membership (Genus Anopheles), Anopheles HYRCANUS group (An. HYRCANUS group), located in Philippines, Chinese, Japan, South Korea and Guam, terms of medical importance, only high in our country in the middle of the malaria transmission efficiency. According to many years of investigation, Anopheles LESTERI in our country, different geographical distribution, ecological habits and disease ability there exist some differences between the population genetic differences need to be in-depth study of Anopheles LESTERI. The research and application of molecular characteristics first identify our sample of Anopheles LESTERI in Anopheles; polymorphic microsatellite DNA loci based on the characteristics and application of polymorphic microsatellite DNA loci the detection of genetic structure of Anopheles LESTERI group in our country. On the basis of the molecular characteristics of identification in China Anopheles samples, improve the accuracy, supplement and update all Anopheles local data to a certain extent. Ray The explanation of the genetic diversity and population differentiation in different groups of Anopheles' Anopheles is expected to provide a theoretical basis for the formulation of malaria control strategies in the media area.
The following research methods: first, the 2 sign of different distribution in China based on rDNA sample of Anopheles, namely ITS2 and D3, were identified; after GENESCAN scanning of the research group has isolated Anopheles LESTERI microsatellite DNA loci using field samples, study on the basic characteristics of each site; the application of polymorphism microsatellite loci detected genetic differences between Chinese Anopheles LESTERI groups within and between groups. The main results are as follows:
1. of the studies on molecular identification of samples from Yunnan, Hubei, Guizhou, Henan, Guangdong, Shandong, Liaoning, Shaanxi and Hainan, a total of 126 samples, the results of sequence analysis showed: Anopheles sinensis (An. sinensis) (n=44), Anopheles vagus (An. vagus) (n=22), dark grey (An. pullus). (n=18) (An. minimus), Anopheles minimus (n=13), Anopheles (An. LINDESAYI) Lin (n=10), Klein (An. KleinI) from Junlian (n=4), Anopheles (An. junlianensis) (n=4), abdominal cluster (An. Kochi) of Anopheles (n=4), (An. belenrae) than Aaron Anopheles (n=3 Guiyang (An.), Anopheles kweiyangensis and Anopheles) (n=3) (An. aconitus) (n=1).
2. sequence analysis shows that there are 4 kinds of species can not be named, namely LL1, LL2, LL3 and LL4, except LL4, the other 3 were with HYRCANUS group members for the most similar, is a new member of this kind should be group. In addition, the phenomenon of Shuangfeng within the individual bases found in a variety of Anopheles. These species of Anopheles cryptic species in recent differentiation of active form (speciation).
3., 57 samples from Anopheles Rex were identified by molecular identification. The basic characteristics of 11 polymorphic microsatellite DNA were studied, providing new molecular markers for further detection of the population genetic structure of Anopheles leani.
4., 9 polymorphic microsatellite DNA loci were used to detect 17 loci collected from China, and 1 samples of Anopheles leani collected from 1 places in South Korea were collected from 216 provinces.
Analysis of 5. groups of Anopheles LESTERI genetic differences within the visible range, the average expected heterozygosity and the observed heterozygosity was 0.4848 (Jiangsu) to 0.7134 (Liaoning) and 0.3309 (Jiangsu) to 0.4704 (South Korea); in the Liaoning group has most alleles (9), and Yunnan population without its own allele inbreeding coefficient; range of 0.2101 (Hainan) to 0.5037 (Hubei), the average was 0.3508; after inspection, a total of 30 loci deviated from Hardy Weinberg equilibrium, the Liaoning population was the largest (7), in South Korea, Yunnan and Hubei groups site deviation; linkage disequilibrium test results showed that a total of 38 para point linkage Association (P0.01), ANL10 and ANL11 loci were associated with a chain in all groups, other sites without the law.
6. groups of genetic differences between Anopheles LESTERI analysis group (Jiangsu removal laboratory strain), and site ANL11. analysis indicated that the paired groups FST range of -0.0127 (Liaoning and Korea) to 0.2539 (Yunnan and Hainan); Mantel test results show that the genetic diversity and geographic distance among populations was positive correlation between population genetic the structure with geographical isolation model; molecular variance analysis showed that the variation of Anopheles LESTERI mainly existed within populations, accounting for 86.55% of the total variation, variation between groups accounted for 13.45% of the total variance; Bayesian analysis method showed that all groups can be divided into 2 branches, including Guangdong and Liaoning branch of I group, II branch including South Korea, Henan, Hubei, Guangdong, Hainan and Sichuan populations.
7. groups of Anopheles LESTERI stability analysis and scale results, Henan, Hubei, Hainan and Liaoning a total of 4 groups experienced a recent population expansion; linkage disequilibrium model based on the range of population size is 1.2 (Hubei) to 33.7 (Guangdong), the overall population size 44.6,95% CI 40.6 ~ 49.0.
A comprehensive analysis of the genetic structure of 8. Anopheles LESTERI groups, presumably Anopheles LESTERI ancestral populations from Yunnan and other groups are spread to North eastward, and the spread of colonization in the way, in order to adapt to the variation of the local ecological environment. A certain percentage of Anopheles LESTERI distributed widely, although the geographical barrier hindering gene exchange to a certain extent, but the difference between groups is not large, population genetic structure with geographical isolation model (Isolation-by-distance Model). Because of its importance in medicine of central China, so in the long-term distribution, extensive use of pesticides, resulting in the Anopheles LESTERI population is small, there are a lot of inbreeding, deviations from Hardy Weinberg equilibrium phenomenon.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R384.1

【相似文獻】

相關(guān)期刊論文 前10條

1 陳至善!200052上海,熊顯華!200052上海;腎癌細胞增殖活性與預(yù)后的關(guān)系[J];臨床腫瘤學(xué)雜志;1998年02期

2 張秀榮,孟如松，

本文編號：1458157