绵羊粪便中植物基因组DNA的提取及其PCR检测  

Plant Genomic DNA Extraction and Amplication from Faeces Samples of Sheep

作　　者：郭艳萍[1] 张英俊[1] 陈文青[2] 张浩[3] 

机构地区：[1]中国农业大学动物科技学院,北京100193 [2]西北农林科技大学动物科技学院,陕西杨凌712100 [3]甘肃农业大学草业学院,甘肃兰州730070

出　　处：《中国畜牧杂志》2016年第23期78-83,共6页Chinese Journal of Animal Science

基　　金："973计划"项目(2014CB138805);牧草产业技术体系项目(CARS-35)

摘　　要：本试验采集了16只圈养绵羊的新鲜粪便,利用试剂盒法(酚-氯仿法)对粪便基因组DNA进行了提取。电泳检测结果表明:从绵羊粪便中获得的DNA纯度较高,完整性好。以提取的基因组DNA为模板,利用文献报道的2对通用引物(r D5-ITS2/rb1-ITS2和rbc LZ1/rbc L19b)以及自行设计的羊草ITS特异性引物(ITS-LC)、碱茅ITS特异性引物(ITS-PD)和芦苇rbc L特异性引物(rbc L-PA)进行PCR扩增。琼脂糖凝胶电泳分析结果表明,5对引物均能获得成功扩增,并得到目的条带。进一步对羊草、碱茅和芦苇进行序列测定,并将测序结果通过Gen Bank数据库进行Blast比对。结果表明:DNA相似性分别为95%、94%和98%,可以证明扩增产物分别属于羊草、碱茅和芦苇的某个片段。This experiment extracted total genomic DNA from faeces of sixteen captive sheeps through Tiangen kit method （phenol： chloroform）. The results showed that the DNA extracted by kit method had high purity and good integrity. Plant genomic DNA from sheep feaces were extracted and amplified by PCR using the 5 pairs of primers, including 2 pairs of general primers （rDS- ITS2/rbl-ITS2 and rbcLZ1/rbcL19b） obtained from related reference, as well as 3 pairs of specific primers （ITS-LC, ITS-PD and rbcL-PA） designed by using ITS or rbcL gene sequences of Leymus chinensis, Puccinellia distans, Phragmites australisas the target. All the five primers could be amplified prospective fragments successfully. The ITS and rbcL fragments were sequenced and aligned by Blast with the corresponding ones published in GenBank database. Their sequences showed 95%, 94% and 98%similarity respectively. Based on these facts, the three of amplified DNA fragments were confirmed to be Leymus chinensis, PuccineUia distans , Phragmites australis .

关 键 词：绵羊 粪便DNA 植物基因 PCR扩增 特异性引物 

分 类 号：S826.4[农业科学—畜牧学]