【作者】 杜波；

【导师】 迟德富；

【作者基本信息】 东北林业大学 ， 森林保护学， 2007， 硕士

【摘要】 异色瓢虫(Harmonia axyridis Pallas)是一种多型性昆虫，其鞘翅上的色斑变化丰富，许多分类学者常以此对其归纳分类。本论文运用PCR-SSCP方法对13种不同色斑型异色瓢虫的COⅠ基因进行分析，以此来研究该基因在不同色斑型之间的关系。针对扩增出的COⅠ基因，优化了SSCP技术的实验条件，摸索出一套稳定而合适的SSCP方法，并结合DNA测序对实验结果进行检验和分析，得到的研究结果如下：首先，利用改良的CTAB法，成功得提取了单只异色瓢虫的基因组DNA，扩增出的目的条带能很好的满足PCR-SSCP实验的要求。与传统的剪碎虫体，匀浆提取的方法相比：该方法程序简单，所用时间短，而且费用低廉。其次，在运用PCR-SSCP方法对COⅠ基因的分析中，优化了PAGE的电泳条件，适合于本实验的电泳条件是：凝胶浓度：5％、交联度：29：1、电压：150V(15h)、甘油：无、电泳缓冲液：1×TBE、样品和变性剂比例：3：3。在该条件下，DNA单链能明显地区分开，但各色斑型之间单链的构象变化不明显。通过DNA测序进一步分析，所扩增的目的基因片段在685bp左右。各色斑型之间，有约600bp的区域高度保守，在其中有碱基变化的有：二斑变型、拟月斑变型、16斑变型和眼斑变型。各斑型之间的遗传相似度比较高：94％-100％，其中16斑变型和眼斑变型、19斑变型和暗黄变型之间的遗传相似度为100％。说明在这13种色斑型中，他们之间的亲缘关系最近。更多还原

【Abstract】 Harmonia axyridis Pallas has pleomorphism. There are many mottlings in their proalacrustacea,and some taxonomists classed them according to the mottlings.This thesis has analysed the COⅠof 13 phenotypes of Harmonia axyridis Pallas havingdifferent mottlings,using the method of PCR-SSCP, to study the kinship of them. According tothe COⅠ,optimize the experiment condition to obtain a stable and suitable condition for theexperiment,then check and analyse the results with DNA sequencing.The results:First,extract the genomic DNA of one Harmonia axyridis successfully with the method ofCTAB and it is suit for the experiment. Compared with the traditionary method for extractingthe DNA of insect using homogenate approach, this method is simpler and save more time andmoney.Second, optimize the electrophoresis condition of PAGE, and the suitable condition is: gelstrength: 5%, degree of cross linking:29:1, voltage:150V(15h), glycerol: none, electrophoresisbuffer: 1×TBE, the ratio between the sample and denaturing agent:3:3. The ssDNA can beseparated clearly under this condition. But the conformation of ssDNA change little betweenthe 13 Harmonia axyridis. The sequence of COⅠgene has about 685bp,and about 600bp ofthem are very conservative. During the 13 phenotypes of Harmonia axyridis, Harmoniaaxyridis ab. 1347-octosigna ta Yuan, ab.falca ta Hem、ab. 12345678-sedecimsi-gna ta Mls andab. circumscrip ta Hem have base mutation. The homology between the 13 Harmonia axyridisare 94%-100%. The genetic similarity between Harmonia axyridis ab. 12345678-sedecimsi-gna taMls and ab. circumscrip ta Hem is 100%, and that between Harmonia axyrisdisab.1/2123456789-undevigintisigna ta Fald and ab. succinea Hope is 100% too. This implys thattheir kinship are nearest in the 13 phenotypes of Harmonia axyridis.更多还原