蝽类昆虫干制标本基因组提取及相关基因扩增  

Extract DNA from Dry Specimens of Pentatomidae and Amplify the Related Genes

作　　者：赵清[1] 张佳庆[2] 张虎芳[1] 

机构地区：[1]山西农业大学农学院,山西太谷030801 [2]南开大学生命科学学院,天津300071

出　　处：《山西农业大学学报（自然科学版）》2015年第5期478-483,共6页Journal of Shanxi Agricultural University(Natural Science Edition)

基　　金：国家自然基金(31440078);山西省教育厅高等学校科技创新基金(2014131);山西农业大学科技创新基金(2014017)

摘　　要：本文以辉蝽(Carbula humerigera)(半翅目:蝽科)为实验材料,研究蝽科昆虫干制标本基因组的提取并扩增相关基因。通过CTAB法、SA裂解法及吸附柱法,对50头辉蝽干制标本进行了基因组提取试验,并针对mtDNA的COI基因部分片段和nrDNA的ITS2基因部分片段进行PCR扩增,共获得COI序列6条,ITS2序列33条。结果表明,核基因中的ITS2基因扩增效果相对较好,而线粒体中的COI基因扩增效果并不理想。通过对PCR产物电泳,结果显示,1994-2008年的干制标本,3种基因组提取方法均可行,且已获得基因序列无明显差别;1974-1989年的干制标本,SA裂解法所造成的核酸损失大,提取效果不理想,而CTAB法、吸附柱法效果明显。由于CTAB法耗时较长、对实验材料需求量大、破坏较强且涉及如β-巯基乙醇等有毒试剂,SA法对基因组损失太大且杂质保留较多,所以提取蝽科干制标本及扩增相关基因的最优方法为二氧化硅膜吸附柱法。This paper took Carbula humerigera （Hemiptera ：Pentatomidae） as main material to study DNA extraction from dried specimens and amplify the related genes .Fifty dried specimens collected from 1974 to 2008 were analyzed . CTAB ,SA lysis and Silical coating column methods were all used to extract genes from one side legs of each specimen . The mitochondrial DNA （COI gene fragment） and the nuclear rDNA （ITS2 gene fragment） were successfully ampli‐fied from six specimens and thirty‐three specimens ,respectively .The comparison of results demonstrates that all the three methods are feasible for the specimens from 1994 to 2008 and no obvious difference was found among the se‐quences from different methods .However ,for the specimens as far back as 1974 to 1989 ,the SA lysis method lost DNA a lot ,while the other methods not .Since the CTAB method costs much time ,destroys specimens a lot by peeling chest muscle and uses toxic organic solvents such as 2‐mercaptoethanol ,in SA method ,the genome lost too much and the impurity contained a lot ,so the Silical coating column method is turned out to be the best method of extracting DNA from dried specimens and amplifying the related genes .

关 键 词：干制标本 基因提取 基因扩增 

分 类 号：S433.3[农业科学—农业昆虫与害虫防治]