【作者】 何平；

【导师】 郑哲民；

【作者基本信息】 陕西师范大学 ， 动物学， 2003， 硕士

【摘要】 金龟子为重要的农林害虫，也是重要的经济昆虫，在国民生产生活中占有重要的地位。鳃角类昆虫其幼虫称作“蛴螬”，其中很多种类是农作物、果树林木、药用植物的重要害虫，有些种类具有极大观赏价值，部分种类尚可入药。鉴于其在国民生产生活中的重要意义，国内外许多科学工作者都对其有研究。但目前对金龟子的系统学研究还多停留在形态学的层次上，并对其分类系统意见不一。本论文从分子生物学的角度入手，以PCR和测序的实验方法，分析金龟子线粒体DNA的Cyt b基因序列特征，探讨该保守区段的序列信息，并应用分子生物学软件对测序结果进行分析处理，建立分子系统进化树，以期得到研究类群间的系统关系，并试图确定其分类定位。以弥补形态学的不足。为进一步研究金龟子的生物防治和合理利用该生物资源提供基础的资料。 研究过程可分为以下几部分：基因组DNA的提取、mtDNA Cyt b基因目的片段的扩增、测序，及对序列结果的分析和研究。 1．基因组DNA的提取及测序：所用标本大部分为无水乙醇浸泡标本和部分保存多年的干标本，基因组提取方法采用SDS蛋白酶K消化法，提取总DNA，以此扩增线粒体基因Cyt b基因的特异性片段，将扩增效果良好的样品进行纯化、测序(委托上海生工生物工程有限公司)。 2．序列校正：对所得测序片段进行校对，使用ContigExpress软件进行正反链的拼接，在EditSeq软件中补齐相应序列的上游引物部分，经与序列图仔细核对、校正后，共得到485bp(含引物部分共52bp)的片段，将序列保存为fasta格式文件。 3．同源性比较：使用ClustalX1.8软件进行多重比对，采用精确慢比对，并将比对的序列保存为Clustal和Phylip格式。 4．构建系统树：使用MEGA2.1统计核苷酸四种碱基的使用频率，计算遗传距离和转换值、颠换值及多态性位点。基于Kimura双参数模型及其它模型，用NJ法(Neighbor-Joining)、ME法和最大简约法(Maximum parsimony method，MP)构建分子系统树，同时使用Phylip 3.6a软件包中的距离法、 简约法和极似然法构建分子系统树。通过对结果的分析讨论，可得到如下结论： 1．通过对所得的Cyt b基因485hp（含引物部分）序列片段的分析，知该片 段序列中富含A、T碱基，这与原有的结论相符合。序列变异位点集中在 第三位点和第二位点，序列的两两比对所得转换和颠换值随分歧时间的 先后而逐渐减小。同一科内的物种间转换值比颠换值要高，不同科间的 转换值比颠换值低。所测基因共编码144个氨基酸，其中有75个发生变 ．异，占52．08％。2应用Cyt b基因标记金龟子部分种类的系统关系，由结果得知同一属内的 种间以很高的置信度优先相聚为一枝，同科内的不同属间表现为单系性。 且枝间分歧大于枝内分歧。花金龟科同属内种问亲缘关系近，白星花金龟 和多纹星花金龟分歧最小，优先相聚为一枝，并与铜绿花金龟聚为姐妹群。 丽金龟科粗绿彩丽金龟和墨绿彩丽金龟同属于彩丽金龟属，优先相聚，然 后再与弧丽金龟属的棉弧丽金龟以45％的置信度汇合。3．科间关系为：腹部气门处于同一位置的上气门系丽金龟科、花金龟科、斑 金龟科。犀金龟科和鳃金龟科相聚为一个聚类簇，然后依次与侧气I”二系 的粪金龟科和金龟科相聚，且在不同的建树方法中都支持这一结果。但 其它科间的关系还有待进一步探讨。4．本论文应用单一基因遗传分子标记，进行系统进化分析，结果表明Cytb基 因对科下物种的系统分析更为适合。所得系统关系与传统的分类学基本一 致。科间的系统关系，还有待进一步的研究。更多还原

【Abstract】 Chafers are important pests and economical insects , which play a significant role in agriculture and forest .The larvae of chafers named grub, which harm many kinds of plants and fruit trees .So, lots of researchers had studied them at home and abroad. The present systematics study of chafers still stays on the level of morphology, and the system of chafers is not identical. In this study the phylogeny of some chafers was based on partial nucleotide sequences (about 433bp) of the mitochondrial cytochrome b gene of 12 taxa .The sequences character were analyzed by bio-software package to construct molecular phylogenetic trees. This study will provide basic data for utilizing and preventing them deeply .The experiment progress included three steps, extraction of genome; amplyfication and sequence of Cyt b gene; analysis and discussion about data .1. Extracting and seguencing gene: The method of extraction was SDS-protein enzyme K digestion from samples kept in pure alcohol or storaged samples , then amplifying target DNA . The better results were purified and sequenced . (entrusting Sangon company to sequence )2. Editing sequence: the sequence was modified by ContigExpress, then edited by Editseq software . The total sequence 485 bp (including primer) of them were exported in fasta format.3. Aligning sequence : the target sequence were complete multiple alignment, and were exported in Clustal and phylip format.4. Constructing phylogentic tree: The sequence data were analyzed by Mega 2.1 and Phylip 3.6a software Package, nucleotide composition and genetic distance computed . Molecular phylogenetic tree constructed by Neighbor-Joining based on Kimura-2 Parameter model , parsimonymethods and Maximum likelihood method . From the analysis of results, conclusions were as follow:1. A and T base are rich in the obtained sequence of cyt b gene. Thedivergent sites concentrated on the third and second site , the ratio of transition and transversion became lower in company with the divergence time of them .The obtained sequence coded total 144 AA, in which 52.08% was variation.2. The phylogenetic relationships Sarabaeidae inferred from Cytb gene datashowed that the same genus assembled first one branch the clade of Protaetial (liocolaj Brevitarsis: (lewis,) and Protaetia (Potosiaj Famelica (Janson) was clearly separated from P.(poios\a)metallica Herbst.. M.holosericea (Fabricius,) and Mimelasplendens (Gyllenhal,) belonged to Popillia Silville genus .The amount of sequence divergence between these clade was significantly greater than whin the clade .3. Relationships of family: Phylogenetic tree showed that Trichiidae, Cetoniidae, Rutelidae, Dynastidae and Melolonthidae all belonged to pleurostict, they concentrated on one group in all molecular phylogentic trees. Scarabaeidae and Geotrupidae all belong to laparostict, which is far from the pleurostict.4. In this paper the phylogenetic trees inferring from single gene was not completely same. Cyt b gene was fit for distinction in intragenus and family. The relationship of molecular trees was almost parity the traditional Systematics. But, the more research about chafers was essential in future.更多还原