【作者】 吕宝乾；

【导师】 陈家骅；

【作者基本信息】 福建农林大学 ， 农业昆虫与害虫防治， 2003， 硕士

【摘要】 茧蜂是农林害虫重要天敌，开展生物防治的有益资源。世界各地保存了大量的茧蜂标本，为了充分利用标本这一潜在的遗传资源，必须快捷、高效、低成本地提取基因组DNA，以便在分子水平上研究物种的起源、系统进化，生物多样性及天敌的引进、保护、释放等。 1．本文探讨了高温，氧化，化学修饰等影响茧蜂标本基因组DNA降解的因素，对室内保存寄生蜂标本提出应用含50mmol／L乙二胺四乙酸（EDTA）的无水乙醇固定，并在液面覆盖石蜡油等可行性建议。 2．通过对茧蜂标本DNA几种提取方法的比较发现，十六烷基三乙基溴化铵（Cetyltrimethyl Ammonium Bromide，CTAB）法效果最好。本文在原有CTAB法基础上进行了改进，使用优化的方法可以从保存近20年的乙醇浸泡标本中提取出DNA，该基因组DNA可很好地用于RAPD-PCR扩增。针插干标本DNA提取有待于进一步探讨。 3．建立了茧蜂RAPD分析的技术体系：25μl反应体系中，含10mmol／l tris-HCl（PH8.0），50mmol／l KCl，2.5mmol／l MgCl₂，150μmol／l dNTP，0.2μmol／l随机引物，30～60ng模板DNA，1.5U TaqDNA聚合酶。反应程序为：94℃预变性4min；94℃ 15s,36℃ 45s，72℃ 90s，45个循环；72℃维持4min。 4．在12个供试材料中，亚科之间具有多态性。对蚜茧蜂亚科下的4个属进行RAPD分析，25引物共扩增出159条带，多态条带比率（PPB）为75％。依据RAPD标记数据，对蚜茧蜂亚科4个属供试材料进行了聚类分析，结果表明：全脉茧蜂属Ephedrus Haliday和孔蚜茧蜂属Fovephedrus Chen关系最近，他们首先聚类在一起，然后和蚜茧蜂属Aphidius Nees聚在一起，最后是和少脉茧蜂Diaeretiella Stray聚在一起。与传统分类的结果相比分子系统发育关系基本相同。更多还原

【Abstract】 Parastic Hymenoptera are important enemies of pest and benefit resources for biocontrol. In order to make good use of heritage resource of parastic Hymenoptera specimen, we need high quality of genomic DNA. Then we can study the species origin, system evolution, biology diversity, introduction of insect enemy, protection of insect enemy, release of insect enemy in molecular level.(1) we discuss Factors of genomic DNA degradation in Braconidae specimen. These factors include high temperature, oxidation and chemic modification. We should douse Braconidae specimen in 100% ethanol with 50mmol/L EDTA. At the same time, olefin oil should be used on ethanol level(2) By comparing some methods for extracting DNA, we find CTAB (Cetyltrimethyl Ammonium Bromide) method is the best. By optimizedmethod of CTAB, we can extract DNA in Braconidae specimen which preserved in ethanol for almost 20 years. Extracted genomic DNA can be used for RAPD-PCR analysis. The method for dry specimen needs study more.(3) An optimized RAPD-PCR reaction system for RAPD analysis was established: PCR was performed in a 25ul reaction mixture with 30~60ng DNA, 150umol/l of each dNTP, 0.2umol/l random primer, 10mniol/l Tris-HCl(PH8.0) ,50mmol/l KCl,2.5mmol/l MgCl2,andl.5U Taq polymerase. The temperature profile used for PCR was 94 for 4 min, followed by 45 cycles of94 for 15sec, 36 for45sec, and72 for l.5min, and was terminated with a 4 min DNA extension step at 72 .(4)There are polymorphic DNAs in 12 accessions. 159 DNA bands were amplified with 25 RAPD primers among which 119 (75%) were polymorphic in 4 genues of Aphidiidae. Cluster analysis based RAPD showes that Ephedrus Holiday and Fovephedrus Chen have higher genetic similarity, then withAphidius Nees,lasl with Diaeretiella Stray.更多还原