本文選題：中華按蚊 + 赫坎按蚊種團�。� 參考：《第二軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：中華按蚊Anopheles sinensis Wiedemann,1828隸屬按蚊屬（Genus Anopheles）、按蚊亞屬（Subgenus Anopheles）的赫坎按蚊種團（An. hyrcanus group）。中華按蚊在我國分布廣泛，是其分布區(qū)瘧疾和絲蟲病的重要傳播媒介。多年的研究顯示中華按蚊在我國不同地理分布區(qū)，生態(tài)習(xí)性和媒介效能存在一定差異，故亟待深入闡明其遺傳學(xué)基礎(chǔ)。本研究基于mtDNA-COI和免疫相關(guān)基因的多態(tài)性，檢測中華按蚊群體的遺傳變異和分化程度，并分析免疫相關(guān)基因在赫坎按蚊種團成員種的適應(yīng)性進化。 本課題的研究策略如下：①對中華按蚊部分基因組序列進行生物信息學(xué)分析，以期發(fā)現(xiàn)與岡比亞按蚊免疫相關(guān)基因同源性高的contig。②設(shè)計引物，，建立擴增中華按蚊，及其近緣種免疫相關(guān)基因的反應(yīng)體系。③在中華按蚊分子鑒別的基礎(chǔ)上，分析Toll6和SRPN14基因，以及mtDNA-COI的多態(tài)性，檢測我國中華按蚊群體的遺傳差異和結(jié)構(gòu)，探討不同群體的遺傳分化現(xiàn)象。④分析赫坎按蚊種團不同成員種的SRPN14及Toll6基因序列，探討該基因的適應(yīng)性進化和選擇模式。 取得如下主要結(jié)果： 1、在中華按蚊基因組中獲得了19條contig與GenBank數(shù)據(jù)庫的免疫相關(guān)基因同源性高，其中17條為岡比亞按蚊的免疫相關(guān)基因。 2、成功擴增獲得了中華按蚊的2個免疫相關(guān)基因部分片段，分別與岡比亞按蚊的信號通路基因Toll6和編碼絲氨酸蛋白酶抑制蛋白的效應(yīng)基因SRPN14同源。 3、分析了來自我國16個群體459個個體的中華按蚊mtDNA-COI序列，比對顯示共251個單倍型，其中41個為群體間共享，家系網(wǎng)絡(luò)圖顯示各單倍型呈高水平的平行演化；群體內(nèi)序列差異最大的是河南（11.83246±3.59614，6.71265±3.57225），最小的是遼寧（4.30562±1.88608，2.60952±1.65685），各群體內(nèi)單倍型之間的差異性范圍為0.709（云南）-0.998（安徽）。云南群體與其他群體間的遺傳分化程度均較大，最大的是與四川群體之間，F(xiàn)st為0.6220；但9對群體之間的Fst為負(fù)數(shù)，提示其間的遺傳分化極小。AMOVA計算結(jié)果顯示群體內(nèi)遺傳變異大于群體間，群體內(nèi)差異占總差異的83.14%，群體間占16.86%。所有群體的Fst值為0.168，提示中華按蚊群體間的遺傳變異為中等水平。Mantel檢驗結(jié)果顯示，群體間遺傳差異隨地理距離的增大而增大（R2=0.006，P=0.073），群體遺傳分布格局符合距離隔離模型。中性檢驗和錯配分析結(jié)果均顯示我國中華按蚊群體處于近期擴張狀態(tài)。 4、分析中華按蚊16個群體的免疫相關(guān)基因Toll6和SRPN14的多態(tài)性，結(jié)果均顯示其序列類型數(shù)目多，而在群體間共享的非常少；序列的變異在群體間非常小，僅占總變異的5.05%（Toll6）和5.37%（SRPN14），差異主要存在群體內(nèi)個體間；所有群體的Fst值為0.055（Toll6）和0.054（SRPN14），其間的遺傳分化程度很低；分析Toll6和SRPN14基因中華按蚊群體間的適應(yīng)性進化，結(jié)果顯示均受到負(fù)選擇壓力的影響，足以維持其結(jié)構(gòu)和功能的穩(wěn)定。 5、分析赫坎按蚊種團9成員種的免疫相關(guān)基因Toll6和SRPN14的多態(tài)性，結(jié)果均顯示其多態(tài)性程度在種間大于種內(nèi)；聚類關(guān)系顯示，基于Toll6基因的拓?fù)浣Y(jié)構(gòu)與依據(jù)ITS2和形態(tài)特征的種間親緣關(guān)系一致；而基于SRPN14基因的結(jié)果存在部分差異；Toll6和SRPN14基因的進化在成對種間均是受到負(fù)選擇壓力。 媒介按蚊對瘧原蟲的免疫反應(yīng)可決定其在蚊體內(nèi)的生存和發(fā)育，故按蚊免疫相關(guān)基因應(yīng)是新型瘧疾控制方法中最具潛力的目標(biāo)基因，按蚊免疫相關(guān)基因多態(tài)性研究也將為病原體-媒介的共進化關(guān)系提供新的解釋，為制定有效的媒介控制策略提供理論依據(jù)。
[Abstract]:sinensis Wiedemann , 1828 belong to the genus of the genus mosquito , according to An . hyrcanus group . Based on the polymorphism of mtDNA - COI and immune - related genes , this study examines the genetic variation and differentiation degree of the population of the sinensis , and analyses the adaptive evolution of the immune - related genes in the species of anophelikis .

On the basis of molecular identification of sinensis , the genetic diversity and structure of Toll6 and SRPN14 genes , and the polymorphism of mtDNA - COI were analyzed .

The following main results are obtained :

1 . In the genome of the sinensis , 19 primers were obtained which were highly homologous to the immune - related genes of GenBank database , of which 17 were the immune - related genes in the Gambia .

2 . Two fragments of immune - related genes were amplified successfully , which were homologous to the signal pathway gene Toll6 and the gene SRPN14 encoding serine protease inhibitory protein respectively .

3 . The mtDNA - COI sequence from 459 individuals from 16 individuals in China was analyzed , compared with 251 haplotype pairs , among which 41 were inter - population sharing , and the family network graph showed a high level of parallel evolution for each haplotype ;
The difference of intra - population was Henan ( 11.83246 鹵 3.59614 , 6.71265 鹵 3.57225 ) , and the smallest was Liaoning ( 4.30562 鹵 1.88608 , 2.60952 鹵 1.65685 ) . The difference of single fold between groups was 0.709 ( Yunnan ) - 0.998 ( Anhui ) .
The results showed that the genetic variation among populations was greater than that of population . The results showed that the genetic variation among populations was higher than that of population . The results of Mantel test showed that the genetic variation among populations increased with geographic distance ( R2 = 0.006 , P = 0.073 ) .

4 . Analysis of the polymorphism of the immune - related genes Toll6 and SRPN14 in 16 populations of the sinensis showed that the number of sequences was much , and the sharing among groups was very little ;
The variation of sequence was very small among groups , accounting for only 5.05 % ( Toll6 ) and 5.37 % ( SRPN14 ) of the total variation .
The Fst values of all groups were 0.055 ( Toll6 ) and 0.054 ( SRPN14 ) , and the degree of genetic differentiation was very low .
The adaptive evolution between Toll6 and SRPN14 genes was analyzed . The results showed that both of Toll6 and SRPN14 genes were affected by negative selection pressure , which was sufficient to maintain its structure and function .

5 . Analysis of the polymorphism of the immune - related genes Toll6 and SRPN14 of 9 member species in Hekan group showed that the degree of polymorphism was more than species in species .
The clustering relation shows that the topological structure based on Toll6 gene is consistent with the inter - species affinity according to ITS2 and morphological characteristics ;
Based on the results of SRPN14 gene , there were some differences .
The evolution of the Toll6 and SRPN14 genes was negatively selected between paired species .

The immune response of mosquitoes to Plasmodium falciparum can determine its survival and development in mosquitoes . Therefore , the immune - related gene should be the most potential target gene in the new malaria control method , and the research on the polymorphism of the immune - related gene of mosquitoes will provide a new explanation for the co - evolution of pathogen - vector and provide a theoretical basis for the formulation of effective media control strategies .

【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】：2012
【分類號】：R384.1

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