中华按蚊免疫相关基因多态性研究

发布时间：2018-05-07 00:22

本文选题：中华按蚊 + 赫坎按蚊种团　；参考：《第二军医大学》2012年硕士论文

【摘要】：中华按蚊Anopheles sinensis Wiedemann,1828隶属按蚊属（Genus Anopheles）、按蚊亚属（Subgenus Anopheles）的赫坎按蚊种团（An. hyrcanus group）。中华按蚊在我国分布广泛，是其分布区疟疾和丝虫病的重要传播媒介。多年的研究显示中华按蚊在我国不同地理分布区，生态习性和媒介效能存在一定差异，故亟待深入阐明其遗传学基础。本研究基于mtDNA-COI和免疫相关基因的多态性，检测中华按蚊群体的遗传变异和分化程度，并分析免疫相关基因在赫坎按蚊种团成员种的适应性进化。本课题的研究策略如下：①对中华按蚊部分基因组序列进行生物信息学分析，以期发现与冈比亚按蚊免疫相关基因同源性高的contig。②设计引物，，建立扩增中华按蚊，及其近缘种免疫相关基因的反应体系。③在中华按蚊分子鉴别的基础上，分析Toll6和SRPN14基因，以及mtDNA-COI的多态性，检测我国中华按蚊群体的遗传差异和结构，探讨不同群体的遗传分化现象。④分析赫坎按蚊种团不同成员种的SRPN14及Toll6基因序列，探讨该基因的适应性进化和选择模式。取得如下主要结果： 1、在中华按蚊基因组中获得了19条contig与GenBank数据库的免疫相关基因同源性高，其中17条为冈比亚按蚊的免疫相关基因。 2、成功扩增获得了中华按蚊的2个免疫相关基因部分片段，分别与冈比亚按蚊的信号通路基因Toll6和编码丝氨酸蛋白酶抑制蛋白的效应基因SRPN14同源。 3、分析了来自我国16个群体459个个体的中华按蚊mtDNA-COI序列，比对显示共251个单倍型，其中41个为群体间共享，家系网络图显示各单倍型呈高水平的平行演化；群体内序列差异最大的是河南（11.83246±3.59614，6.71265±3.57225），最小的是辽宁（4.30562±1.88608，2.60952±1.65685），各群体内单倍型之间的差异性范围为0.709（云南）-0.998（安徽）。云南群体与其他群体间的遗传分化程度均较大，最大的是与四川群体之间，Fst为0.6220；但9对群体之间的Fst为负数，提示其间的遗传分化极小。AMOVA计算结果显示群体内遗传变异大于群体间，群体内差异占总差异的83.14%，群体间占16.86%。所有群体的Fst值为0.168，提示中华按蚊群体间的遗传变异为中等水平。Mantel检验结果显示，群体间遗传差异随地理距离的增大而增大（R2=0.006，P=0.073），群体遗传分布格局符合距离隔离模型。中性检验和错配分析结果均显示我国中华按蚊群体处于近期扩张状态。 4、分析中华按蚊16个群体的免疫相关基因Toll6和SRPN14的多态性，结果均显示其序列类型数目多，而在群体间共享的非常少；序列的变异在群体间非常小，仅占总变异的5.05%（Toll6）和5.37%（SRPN14），差异主要存在群体内个体间；所有群体的Fst值为0.055（Toll6）和0.054（SRPN14），其间的遗传分化程度很低；分析Toll6和SRPN14基因中华按蚊群体间的适应性进化，结果显示均受到负选择压力的影响，足以维持其结构和功能的稳定。 5、分析赫坎按蚊种团9成员种的免疫相关基因Toll6和SRPN14的多态性，结果均显示其多态性程度在种间大于种内；聚类关系显示，基于Toll6基因的拓扑结构与依据ITS2和形态特征的种间亲缘关系一致；而基于SRPN14基因的结果存在部分差异；Toll6和SRPN14基因的进化在成对种间均是受到负选择压力。媒介按蚊对疟原虫的免疫反应可决定其在蚊体内的生存和发育，故按蚊免疫相关基因应是新型疟疾控制方法中最具潜力的目标基因，按蚊免疫相关基因多态性研究也将为病原体-媒介的共进化关系提供新的解释，为制定有效的媒介控制策略提供理论依据。
[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 卤 3.59614 , 6.71265 卤 3.57225 ) , and the smallest was Liaoning ( 4.30562 卤 1.88608 , 2.60952 卤 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 .

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R384.1

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