蚜虫内共生菌基因组DNA提取方法的比较和优化

Comparison and optimization of genomic DNA extraction from endosymbionts of aphids

为探究蚜虫共生菌基因组DNA的提取方案,以桃蚜为试验材料,比较了目前较为常用的4种蚜虫基因组DNA提取方法,从DNA纯度、完整性、PCR扩增效率及稳定性等方面进行了比较和评价,并通过调整蛋白酶K用量和水浴温度及时间对STE法进行了优化。结果表明,4种方法提取的蚜虫共生菌基因组DNA均可用于共生菌的PCR扩增检测;CTAB法和SDS法提取的DNA纯度较高,条带较完整,稳定性相对较高,不易降解,而STE法和PCR缓冲液法操作简便,适于快速提取单头蚜虫共生菌的基因组DNA,但纯度相对较低;可根据试验条件和要求进行选择。STE法优化条件为:用30μL STE缓冲液将蚜虫匀浆,加入1.5μL 20 mg/m L蛋白酶K,于56℃水浴1.5 h;再加入0.1μL 10 mg/L RNA酶,于37℃培养1 h,95℃下处理5 min,5 000 r/min离心3 min,将提取到的DNA于-20℃保存或直接用于PCR扩增。优化后的STE法可作为提取蚜虫共生菌基因组DNA经济而快捷有效的方法。

In order to explore the optimum protocol of extracting DNA from aphid endosymbionts,four commonly used genomic DNA extraction methods,CTAB,SDS,STE and PCR buffer,were compared with the peach aphid Myzus persicae( Sulzer) as the experimental species. DNA purity,integrity,efficiency of PCR amplification,and stability were compared and evaluated. The results showed that the genomic DNA of aphid endosymbionts extracted by all the four methods could be used in the PCR amplification of the endosymbiont detection. The genomic DNA extracted by CTAB and SDS methods had higher purity and stripe completeness compared with the other two methods. Both of the STE and PCR buffer methods were suitable for rapid extraction of single-aphid endosymbiont genomic DNA, but extracted DNA with these two methods had lower purity compared with the other two methods. It should be chosen according to the specific experimental tests and requirements. STE method is the most economical and time-saving method for the extraction of genomic DNA of aphid endosymbionts. To optimize the STE method,the dosages of the proteinase K,and water bath temperature and incubatingtime were adjusted. The optimum conditions of the STE are: homogenizing aphid body with 30 μL STE buffer in a PCR tube,adding 1. 5 μL 20 mg / m L proteinase K,and mixing well and incubating the mixture in water bath at 56 ℃ for 1. 5 h,then adding 0. 1 μL 10 mg / m L RNase A to the mixture,and incubating inside an incubator at 37 ℃ for 1 h,then at 95 ℃ for 5 min to denature the enzymes,centrifuged at a table-top centrifuge at 5 000 r / min for 3 min,and at last collecting the supernatant containing DNA directly for experiment or stored at- 20 ℃. Optimized STE method is an economical and time-saving method for the extraction of genomic DNA of aphid endosymbionts.