【作者】 单林娜；

【导师】 杨莲芳；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2004， 博士

【摘要】 本研究首次将核酸序列分析方法用于毛翅目幼虫鉴定，测定了毛翅目完须亚目昆虫：具钩竖毛螯石蛾Apsilochorema unculatum、黄褐鳞石蛾Lepidostoma flavum、傅氏鳞石蛾L. fui、弓突鳞石蛾L. arcuatum、莫氏斑胸鳞石蛾Paraphlegopteryx morsel成、幼虫和黄氏竖毛螯石蛾Apsilochorema hwangi成虫的线粒体DNA细胞色素氧化酶Ⅰ和Ⅱ基因(CO Ⅰ和Ⅱ)序列，通过对序列特征的比较分析，完成了前述5种昆虫的成幼虫联系；并首次记述了傅氏鳞石蛾、黄褐鳞石蛾、弓突鳞石蛾、莫氏斑胸鳞石蛾、具钩竖毛螯石蛾和长叉瘤石蛾幼虫的形态。初步探讨了DNA多态性在4种环须亚目(不筑可携带巢)种类的成、幼虫联系中的应用技术。同时，对mtDNA COⅠ序列的不同区段与系统学分析的关系进行了探讨。研究表明利用核酸序列分析方法鉴定毛翅目幼虫是可行的，为加快水生昆虫的幼虫分类研究提供了新途径。 一、线粒体DNA-CO Ⅰ、Ⅱ基因序列分析在毛翅目成幼虫联系中的应用 采用克隆测序方法测定成幼虫克隆片段的序列。利用“BLAST”工具(NCBI站点)进行序列分析、DNA序列检索及同源性比较。采用Dnastar Package中的Editseq软件进行序列编辑、ORF查找；采用Clustal X软件进行序列比对(alignment)；比对结果输入MEGA2.1软件计算各样品间的遗传距离，并基于Kjmura 2-Parameter模型，用邻接法(neighbor-jojning，NJ)构建系统发生树，通过自展(bootstrap1000次)检验获得系统树分支的置信度。 测定了具钩竖毛螯石蛾成虫、蛹、幼虫和黄氏竖毛螯石蛾成虫的mtDNA-COⅠ、COⅡ及tRNA基因序列，在获得的2236bp序列中，A％+T％平均为67.9％，编码COⅠ、COⅡ的密码子第三位点的A％+T％则分别是78.6％、80.9％，具钩竖毛螯石蛾成幼虫之间有7个变异位点，均为转换位点，且均发生在COⅠ区段，成虫与蛹、蛹与幼虫之间分别有19和24个变异位点，而具钩竖毛螯石蛾幼虫与黄氏竖毛螯石蛾成虫之间有267个变异位点。具钧竖毛螯石蛾成虫、蛹与幼虫间的核酸歧异度均为1％，而具钩竖毛螯石蛾成虫、蛹、幼虫与黄氏竖毛螯石蛾成虫间的核酸歧异度为13％。线粒休DNA一COI、U基因序列在毛翅目成幼虫联系中的应用研究 扩增并测定了4种鳞石蛾的成、幼虫的mtDNACOI、COH及tRNA一Leu基因序歹‘J。在获得的220obp左右的序歹，J中，A%+T%平均为70.4%，编码COI和COH的密码子第三位点的A%+T%则分别是82.9%和850%。对鳞石蛾8个COI和CO 11序歹.{的比对分析表明:在COI区的一45一个比对位点中，有1260个保守位点，191个变异位点，其中98个转换位点，93个颠换位点;发生在密码子第三、第一和第二位点的变异分别为78%、18%和4%;种内序列歧异度为O一0.9%，而种间为一4%一24%。在CO Ix区的591个比对位点中，有5 16个保守位点，75个变异位点，其中46个转换位点，29个颠换位点;发生在密码子第三、第一和第二位点的变异分别为71%、25%和6%;种内序列歧异度为O一0.5%，而种间为16%一19%。 序列分析结果表明:(l)供试的具钩竖毛鳌石蛾、黄揭鳞石蛾、傅氏鳞石蛾、弓突鳞石蛾和莫氏斑胸鳞石蛾各成、幼虫之间的序列歧异度即种内序列歧异度《1%，而种间序列歧异度>】3%，由此可确认上述5种昆虫的成幼虫联系;(2)COI区4种鳞石蛾的种间序列歧异度为14%一24%，变异幅度远大于COH区的16%一19%，且COI区比CO 11区有更高的颠换率，似表明COI区比CO 11区更适于种属水平的分类。(3)以COI+CO 11、COI、CO 11核普酸序列和氨基酸序列构建的鳞石蛾成幼虫的NJ树呈现出不同的分支式样，但均显示在4种鳞石蛾中以莫氏斑胸鳞石蛾分化较早。COH核普酸序列的NJ树反映出弓突鳞石蛾与黄褐鳞石蛾有更近的亲缘关系，与根据成虫形态学建立的系统关系相吻合。 根据原双栖纹石蛾成幼虫的PCR反应电泳图谱、序列同源性和酶切图谱的一致性，基本确认了其成幼虫联系。分别扩增得到了裂茎侧枝纹石蛾成虫、瓦尔侧枝纹石峨成虫、多斑短脉纹石蛾成幼虫和侧枝纹石蛾属一种幼虫(编号HyL0002)约2.2kb的mtDNA CO 1 COH基因片段，为其成幼虫联系打下了一定基础。二、线粒体Col序列不同区段在4种鳞石蛾系统树重建中的作用 将COI序列按其碱基位点分成第430一779、第780一1 1 79和第1 1 80一1569位.氛3个区段，以COI全序歹，」、CO 11序歹，}和COI+11序歹，」为参照，利用MEGAZ.l软件，分析各序列区段的碱基构成及变异情况，基于KimuraZ一Parameter模型，计算其平均遗传距离(含所有取代位点)，并用邻接法(neighbor-joining，NJ)、最小进化法(minimum evolution，ME)和最大简约法(maximum parsimony，Mp)重建COI序列及其各区段的系统发生树。 结果表明:①4种鳞石蛾CO 13个区段的分子树呈现出不同的分支式样，变中文摘要异位点在该基因不同区段的分布的不均匀性可能与其分子树的拓扑学结构有密切关系.②第1 180一1569位区段分子树表明黄纹鳞石蛾与弓突鳞石蛾的亲缘关系较近，与根据形态学建立的系统关系相吻合。该区段序歹}}的平均歧异度高达21%，表明鳞石蛾科内物种的遗传差异较大，而更多还原

【Abstract】 Identification of the larval stage of aquatic insects is one of most important factors in biomonitoring water quality. The morphological diagnostic characters for many mature and early instar larvae are still lacking in China. Partial sequences of the mitochondrial cytochrome oxydase I and II and a transfor RNA ( CO I CO II and tRNA ) gene of the adults and larvae from caddisflies were. Lepidostomaflavum, L.fui, L. arcuatum, Paraphlegopteryx morsei, Apsilochorema unculatum and Apsilochorema hwangi, and the larval and adult stages of these species were sequenced and associated. The DNA-based identification of ecologically important hydropsychids larvae was primarily studied. The role of CO I gene and its fragments on phylogenetic reconstruction of four lepidostomatids were discussed. Larvae of all reared species and Goera ramose were described for the first time except for Apsilochorema hwangi.The association of larval and adult stages of ecologically important Caddisfly (Insecta: Trichoptera) using mitochondrial DNA sequencesLarval and adult amplification fragments were cloned and sequenced. DNA sequences were aligned using Clustal X software (Gene Codes Corporation). Analysis of genetic and phylogenetic relationship was performed using MEGA2.1. Genetic relationships among haplogenotypes were estimated based on the pair-wise matrix of sequence divergences by Kimura’s Two-parameter method. Phylogenetic trees were constructed by the Neighbor-Joining (NJ) method. Confidence levels for NJ tree were assessed by bootstrapping from 1000 pseudo-replications.The partial sequences of the mitochondrial CO I , CO II and tRNA gene of theadults and larvae of 4 species from Lepidostomatidae and the adults and larvae ofApsilochorema unculatum and A. hwangi were sequenced and compared in this study. In the sequences obtained (1718~2239bp), A % + T% was about 69.2 %, 267 nucleotide sites were substituted in Apsilochorema and 266 nucleotide sites were substituted in Lepidostomatidae. AT bias in the third codon position site was much higher than the other two sites, reaching about 81.9%.The results of sequence analysis showed that intraspecific divergence is <1% between the adult and the larva and interspecific divergence is 14-24% in Lepidostomatidae, and that intraspecific divergence is <1% between the adult and the larva of Apsilochorema unculatum, and interspecific divergence is 13% between the larva of Apsilochorema unculatum and the adult of A. hwangi, which identified the association of larval and adult stages of 4 species from Lepidostomatidae and larval and adult stages of A. unculatum.The study indicates that mitochondrial DNA sequence analysis is a useful tool to associate the larval and adult stages of Trichoptera, in turn, it will be valuable for health assessment of stream ecosystems.The role of cytochrome oxidase I gene and its fragments on phylogenetic reconstruction of 4 lepidostomadonIt is well known that the phylogenetic trees reconstructed from different genes forthe same set of organisms are often different. The differences between phylogenetic trees reconstructed may be due to insufficient sampling of nucleotides or codons, different assumptions used in the analysis of patterns of nucleotide or amino acid substitutions, different sorting of genes, etc., but in most cases it is difficult to know which of the reconstructed trees is the correct one because the true tree is unknown. This paper reported some initial results of studying about the role of a fragment of the CO I gene on phylogenetic reconstruction of 4 lepidostomatids.The CO I sequence is subdivided into 3 fragments according to its base position: Pos. # 430-779, Pos. #780-1179 and Pos. #1180-1569. Complete CO I sequence, CO II sequence and CO I + II sequence were used for comparison. Nucleotide composition and variation of each sequence fragment were analyzed using MEGA2.1.Geneticrelationships among haplogenotypes were estimated based on the pair-wise matrix of sequence divergences by Kimura’s Two-parameter更多还原