【作者】 徐芬；

【导师】 王国秀；

【作者基本信息】 华中师范大学 ， 动物学， 2005， 硕士

【摘要】 索科线虫是一类昆虫寄生线虫,其形态多样而且具有较高的宿主特异性。由于这类昆虫病原线虫具有重要的生防价值,所以有必要研究它们的遗传变异和系统演化。本文首次报道了有关中国的索科线虫的分子系统发育关系,对索科线虫中的部分分类混淆问题进行了分析,为更完善地构建索科线虫系统进化树奠定了一定的理论基础。 本文采用RAPD技术构建了索科线虫4属5种的指纹图谱。从47个引物中筛选出12个稳定性好、多态性高的引物,共扩增出161条谱带,其中150条谱带具遗传多态性,占93.17%。所获片段长度大小为200-3200bp,单个引物扩增的条带数在11～16间,平均为13.42条。采用RAPDistance软件及MEGA程序,计算Nei氏相似系数和遗传距离,建立了UPGMA和NJ聚类图。两个聚类图拓扑结构基本相同,将5种索科线虫可分为两大分枝:同属于蚊幼寄生罗索属(Romanomermis)的食蚊罗索线虫(R.culicivorax)与武昌罗索线虫(R.wuchangensis)亲缘关系最近,先聚在一起;再与同翅目(Homoptera)寄生长沙多索线虫(Agamermis changshaensis)聚为一枝;鳞翅目(Lepidoptera)寄生中华卵索线虫(Ovomermis sinensis)和同翅目寄生两索属线虫(Amphimermis sp.)亲缘关系较近,两者聚为一枝。从5种索线虫之间的遗传距离来看,属内种间的距离较小,食蚊罗索线虫与武昌罗索线虫之间遗传距离仅为0.1789;而属间的遗传距离较大,在0.4471～0.5488之间。结果表明:RAPD技术可以应用于索科线虫亲缘关系的分析,能够反映出不同线虫间的遗传差距,从而进行属、种的分类及进化问题研究。 本文比较了13种索科线虫(其中8种索科线虫序列为本文所测定,5种索科线虫序列从互联网GenBank检索得到)的18S rDNA的同源序列。计算核苷酸的使用频率,并采用邻接法(NJ)和最大简约法(MP)构建分子系统发育树。在获得的773bp的序列片段中有200个位点为多态性位点,大部分的碱基改变是转换(transition)。研究结果表明:用两种方法构建的系统树结果基本一致,索科线虫分为两大类群,第一大类群是以罗索属武昌罗索线虫更多还原

【Abstract】 Mermithidae is a group of insect’s parasitic nematode with diversified morphological characters and host specificity. Because of the biological control value of these entomopathogenic nematodes, it is necessary to study the genetic variation and phylogenetic evolution of them. It was the first report to infer the phylogenetic relationships of Mermithidae from China and figure out some confusions of classifying system in Mermithidae.In this study, the genetic relationships among five species belonging to four genera of Mermithidae were detected using RAPD. Twelve primers were screened from 47 random primers and amplified 161 bands (from 200bp to 3200 bp). 150 (93.17%) of these bands were polymorphic bands. The number of obtaining segments of individual primer was between 11-16, the average was 13.42. Nei’s similarity coefficients and genetic distance were calculated using the RAPDistance and MEGA softwares. The phylogenetic trees were constructed with the methods of UPGMA and NJ. The results from two methods of cluster analysis were similar in general, and the relationship indicated that the five species of Mermithidae could be distinguished with RAPD markers and were grouped into two distinct clusters. In the phylogenetic trees, Romanomermis culicivorax and R. wuchangensis (all parasite in larvae of mosquitoes) clustered together, then they clustered with Agamermis changshaensis (parasite in Homoptera) into one group. Ovomermis sinensis (parasite in Lepidopters) and Amphimermis sp. (parasite in Homoptera) clustered into one group. The genetic distances of five mermithid species showed that the relationships of intra-genus were nearer than that of inter-genus (the genetic distance between R. culicivorax and R. wuchangensis was 0.1789, but the genetic distance between inter-genus were 0.4471-0.5488). All these results suggested that RAPD was suitable for analysis of phylogenetic relationships among Mermithidae.Nucleotide sequences of 18S rDNA (773 base pairs) were compared among 13 samples (8 samples were sequenced in this study and 5 samples were from GenBank). A molecular phylogenetic tree was constructed by using maximum parsimony (MP) and neighbor joining (NJ) methodologies. The sequence data revealed considerable variation in 200 nucleotide sites among the sequencesanalyzed, with transitions outnumbering transversions. All two phylogenetic analysis generated similar affinities, with 13 mermithid species of 7 genera grouped into two distinct clusters. The congeners Romanomermis culicivorax and R. wuchangensis clustered with Aranimermis sp. (Araneae parasites), then clustered with Amphimermis sp. and Mermis nigrescens (Orthoptera parasites). A second cluster was formed by Ovomermis sinensis, Agamermis changshaensis and Octomyomermis sp. (parasitic in of mosquito larvae). The genetic distances of these 13 mermithid samples revealed closer intrageneric distances (0.29% nucleotide dissimilarity) relative to intergeneric comparisons (4.33-14.78%). Furthermore, phylogenetic analysis based on the 18S rDNA of 4 unidentified mermithid samples can be used to resolve relationships among Mermithidae.RAPD and 18S rDNA markers were used to detect the genetic distance among several mermithid species. The results showed that these two molecular markers were all suitable for study phylogenetic relationships among Mermithidae. In addition, RAPD marker was superior to 18S rDNA marker when analyse relationships of intra-genus and the latter was suitable for inter-genus.更多还原