基于地鳖线粒体基因组差异鉴定土鳖虫的性别OA

Objective:To establish a rapid and accurate molecular gender identification method for the mixed use of female and male Eudolyphaca sinensis individuals in the medicinal materials of Eupolyphaga Steleophaga,providing a technical support for the qual-ity control of the medicinal materials.Methods:The complete mitochondrial genome sequences of adult female and male Eupolyphaga sinensis were determined respectively by using Illumina high-throughput sequencing technology.The mitochondrial genomic difference loci between female and male individuals were compared and analyzed,and sequencing verification and enzyme digestion site analysis were conducted.Based on specific enzyme digestion sites,a PCR-RFLP molecular identification method was established.Results:Three mitochondrial genome sequences of female and male Eupolyphaga sinensis were successfully obtained,with a total length of 15 551 bp.There were three stable base difference sites(sites 4 113,4 413,8 262)between female and male individuals.Two pairs of universal primers designed based on the fragment where the difference site was located could effectively amplify the target sequence.Further,the PCR-RFLP identification system was successfully established by using the EcoT14 I enzyme digestion site.This method could complete the gender identification of Eupolyphaga sinensis samples within 2.5 hours.Conclusion:The PCR-RFLP gender identification method established in this study is highly accurate,convenient and fast,providing feasible technical solution for the quality control and standard formulation of the gender of Eupolyphaga Steleophaga medicinal materials.