【作者】 师丽敏；

【导师】 嵇保中；

【作者基本信息】 南京林业大学 ， 森林保护学， 2006， 硕士

【摘要】 本文选用了天牛线粒体DNA的特定片段,采用直接测序法对不同种类天牛的线粒体DNA进行了测序,并通过序列比对分析了天牛科不同亚科的进化关系。同时,在研究中针对出现假基因的情况作了初步探讨。研究的主要结论如下:通过对天牛科五个亚科的天牛线粒体DNA的Cytb和16S rRNA基因片段进行了测序,根据序列的差异建立了系统发育树,并对天牛科高级阶元的分类进行了初步探讨。从对Cytb序列进行分析的Bootstrap树和N-J系统树中可以看出,幽天牛亚科的光胸断眼天牛与花天牛亚科的双斑厚花天牛先聚为一枝,随后与沟胫天牛亚科的光肩星天牛的分枝相聚。三者相聚在一个节点后,再与天牛亚科的家茸天牛和锯天牛亚科的中华薄翅天牛相聚在同一节点,且中华薄翅天牛明显离节点较远。显示出幽天牛亚科与花天牛亚科的亲缘关系较近。其次是沟胫天牛亚科,再次是天牛亚科,最后是锯天牛亚科,即五个天牛亚科呈现如下的关系:锯天牛亚科→天牛亚科→沟胫天牛亚科→花天牛亚科→幽天牛亚科。从对16SrRNA序列构建的Bootstrap树和N-J系统树中可以看出,幽天牛亚科的光胸断眼天牛和花天牛亚科的双斑厚花天牛先聚在同一分枝,随后与沟胫天牛亚科的粒肩天牛的分枝相聚。三者相聚在一个节点后,再与天牛亚科的家茸天牛相聚在同一节点,最后,与锯天牛亚科的中华薄翅天牛和吉丁虫科的花曲柳窄吉丁虫相聚。显示出五个天牛亚科也呈现出如下的进化关系:锯天牛亚科→天牛亚科→沟胫天牛亚科→花天牛亚科→幽天牛亚科。通过对天牛科4个亚科13个属的16种共30个天牛样品及吉丁虫科的花曲柳窄吉丁虫和叶甲科的二纹柱萤叶甲的mtDNA COⅠ基因的一段序列进行了分析,对天牛科基因条码的构建进行了初步的研究。结果显示,样品的碱基差异率由种内到种间、属间呈逐渐增大的趋势,天牛不同分类阶元的种间具有明显的碱基差异,且差异分布各不相同,的确就像基因条码一样,因此笔者确信该基因可以作为天牛种类鉴定的依据,该段基因适合于基因条码的构建。对其灵敏度进行了分析,显示了引物的高度灵敏性与稳定性,同时也说明了该反应体系的稳定性和可重复性。但本研究测序天牛种类还较少,有必要选择各分类阶元主要种及重要检疫性天牛一一测序,以建立天牛基因条码系统,为天牛的鉴定和遗传学、系统学研究提供更为科学的基础。在对不同地理来源的光肩星天牛进行测序时发现,线粒体COⅠ-2基因的序列明显分为两组并且差异较大,通过比较用长片段扩增技术和通用引物扩增技术对来自河北霸州的光肩星天牛测得序列后发现,其中有一组有可能是出现了假基因,并对假基因的原因进行了初步探讨,认为提取的总DNA中线粒体DNA含量少且采用通用引物进行扩增是导致假基因出现的直接原因。更多还原

【Abstract】 Some fragment of mtDNA of samples were respectively sequenced,and these samples including individuals from different subfamilies of Cerambycidae.The relation ships of these samples from different subfamilies and the reasons brought numts were studied. The results were an follows:We sequenced Cytb DNA and 16S rRNA of the samples from different subfamilies and builded phylogenetic tree.The tree was builded according to the differences between the sequences of the samples.We studied the classification of these samples.The result of the analysis of Cyt b DNA sequence indicated that the Tetropium castaneum and Pachyta bicuneata were grouped in a same clade of the phylogenetic tree and subsequently integrated the clade of the Anoplophora glabripennis.After they integrated at same joint,the three subfamilies finally meet Trichofreus compestris and Megopis sinica(White) at the other joint.The distance from joint of Megopis sinica(White) was farer than Trichofreus compestris. According to the result we can included that the relationship of the five subfamilie as follows: Prioninae→Cerambycinae→Lamiinae→Aseminae→Lepturinae. The tree which builded according to the sequence of 16S rRNA indicated that the Tetropium castaneum and Pachyta bicuneata were grouped in a same clade of the phylogenetic tree and subsequently integrated the clade of the Apriona germari Hope . After they integrated at same joint,the three subfamilies finally meet Trichofreus compestris and Megopis sinica(White) at the other joint.Also,the five subfamilies relationship as follow: Prioninae→Cerambycinae→Lamiinae→Aseminae→Lepturinae.Result of the two studies jointly prove the same relationship of the five subfamilies.We sequenced 30 samples of Cerambycidae,1 samples of Buprestidae and 1 samples of Chrysomelidae. We studied the feasibility of the building of the gene code .The result indicated that the difference of the sequence was more larger within than between species. So we believed that this fragment of mtDNA COⅠcan be used as a marker to build the gene code of all individual of Cerambycidae. We inspect the delicacy of this PCR system,and the result proved that the system had a high veracity and stability. The number of species in this study was inadequate and important species and quarantine species were should be sequenced in next study.This study will provide scientific base of the inspection, genetics and phylogenesis.We found that the sequences of COⅠ-2 of Anoplophora glabripennis form different area were divided into two group on base of the difference of sequence.After sequenced the Anoplophora glabripennis form Hebei province with the long PCR,we believe that the numts was existed in one group.The reason of appearance of the numts were analyzed then we found the possible reason.We believe that the conserved primers and the low content of mtDNA were the most possible reasons.更多还原