科学研究
舞毒蛾标本DNA提取和COI基因扩增
2020-08-21     阅读量:409

           徐瑶王鸿斌王梅李国宏

【摘要】 [目的]探讨适合舞毒蛾标本的保存和DNA提取方法,分析舞毒蛾标本的保存时间和保存方式对基因序列扩增的影响。[方法]利用SDS法、磁珠法以及E.Z.N.A.TM昆虫DNA提取试剂盒3种方法对舞毒蛾新鲜样本、干燥成虫标本和福尔马幼虫林标本进行DNA提取,并对3种方法提取DNA的浓度和纯度进行分析比较。利用1956年至1996年收集的舞毒蛾干燥成虫标本和福尔马林幼虫标本分析并比较保存时间和保存方式对15对COI基因(cytochrome oxidase subunit I)引物(目的片段长度为216 bp~977 bp)扩增成功率的影响。[结果]表明与SDS法和磁珠法相比,E.Z.N.A.TM昆虫DNA提取试剂盒对舞毒蛾3种标本的DNA提取效果最好,提取过程中未对标本DNA产生额外损伤。标本保存时间越久扩增长序列目的片段的成功率越低,福尔马林幼虫标本总体扩增成功率比干燥成虫标本的略高。[结论]E.Z.N.A.TM昆虫DNA提取试剂盒更适合舞毒蛾新鲜样本、干燥成虫标本和福尔马林幼虫标本的DNA提取。舞毒蛾标本的保存时间和保存方式均会对基因序列扩增产生影响。

【Abstract】 [Objective] The preservation and DNA extraction methods for Lymantria dispar specimens were discussed, and the effects of storage time and preservation methods of L. dispar specimens on amplification of gene sequences were analyzed. [Method] Genomic DNA of L. dispar specimens(dried adult, formalin-fixed larval and fresh larval specimens) was extracted by SDS method, the magnetic bead method, and the E.Z.N.A.TM Insect DNA extraction kit. Moreover, the concentration and purity of extracted genomic DNA obtained by the three methods were analyzed and compared. The L. dispar specimens collected from 1956 to 1996 were used to analyze and compared the effects of storage time and preservation methods on the amplification success rate of 15 pairs of COI(cytochrome oxidase subunit I) gene primers(the length of the target fragment is 216 bp to 977 bp). [Result] The results showed that the concentration and purity of extracted genomic DNA by E.Z.N.A.TMInsect DNA extraction kit was the highest compared with the SDS method and the magnetic bead method. And the specimens’ DNA was not damaged during the extraction process. The amplification success rate of specimens with longer storage time was reduced. Moreover,the overall amplification success rate of formalin-fixed larval specimens was slightly higher than that of dried adult specimens. [Conclusion] The E.Z.N.A. TM Insect DNA extraction kit is more suitable for DNA extraction of L. dispar specimens(dried adult, formalin-fixed larval and fresh larval specimens). The amplification of gene sequences is affected by the storage time and preservation method of L. dispar specimens. 

【关键词】 舞毒蛾标本DNA提取福尔马林COI基因
【Key words】 Lymantria disparspecimensDNA extractionformalinCOI gene

【基金】 中国林业科学研究院中央非营利研究机构基础研究基金(CAFYBB2017ZE002)



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