内蒙古8种啮齿类动物细胞色素C氧化酶亚基Ⅰ基因引物的优化研究

Optimization of cytochrome C oxidase subunit Ⅰ gene amplification primers for eight kinds of rodents in Inner Mongolia

目的 确定内蒙古鼠疫自然疫源地8种啮齿类动物细胞色素C氧化酶亚基Ⅰ （CO Ⅰ）基因扩增的最佳引物.方法 在内蒙古锡林郭勒盟和乌兰察布市,采集8个鼠种的动物,提取基因组DNA,选择6对已报道的扩增鼠CO Ⅰ基因的引物（F1/R1、F2/R2、F3/R3、F4/R4、VF/VR、F6/R6）,PCR扩增8个鼠种的CO Ⅰ基因,对扩增的产物进行测序,并对测序结果进行生物信息学分析和比较.结果 达乌尔黄鼠扩增CO Ⅰ基因的最佳引物为F3/R3,达乌尔鼠兔和蒙古兔尾鼠均为鸡尾酒引物VF/VR,大沙鼠、蒙古毛足鼠和布氏田鼠均为F6/R6,五趾跳鼠为F2/R2,子午沙鼠为F1/R1.8种啮齿动物的CO Ⅰ序列进行比对后,发现所有DNA条码都是物种所独有,每个物种都有各自的鉴别位点.结论 不同鼠种在扩增CO Ⅰ基因时需要相应种属特异性引物;扩增多种鼠种时,优先选择F6/R6引物或鸡尾酒引物,针对特殊鼠种可选择该鼠种特异引物进行扩增.

Objective To determine the optimal primers of cytochrome C oxidase subunit Ⅰ （CO Ⅰ） gene for 8 kinds of rodents in natural epidemic focus of plague in Inner Mongolia.Methods In Xilingol League and Wulanchabu City of Inner Mongolia,eight kinds of rodents were collected and DNA was extracted;six pairs of targeted primers （F1/R1,F2/R2,F3/R3,F4/R4,VFNR,F6/R6） were used to amplify the CO Ⅰ gene of the 8 species by PCR;PCR products were send to biotechnology company for sequencing,and bioinformatics analysis of the sequencing results was conducted.Results F3/R3 was the optimal CO Ⅰ gene primer for Spermophilus dauricus;cocktail primer （VFNR） was the optimal CO Ⅰ gene primer for Ochotona daurica and Lagurus przewalskii;F6/R6 was the optimal CO Ⅰ gene primer for Rhombomys opimus,Phodopus campbelli and Microtus brandti;F2/R2 was the optimal CO Ⅰ gene primer for Allactaga sibirica,and F1/R1 was the optimal CO Ⅰ gene primer for Meriones meridianus.CO Ⅰ gene sequences of the 8 kinds of rodents were compared;DNA barcoding was unique in each rodent and every rodent had differential point.Conclusions Different rodent needs its own species-specific primers for CO Ⅰ gene amplification.Upon amplification of different rodent,cocktail or F6/R6 primers are the first choices,and choose the species-specific primers for particular species to amplify.