基于COI与SRY序列建立梅花鹿马鹿及其杂交鹿茸分子鉴别方法.docVIP

基于COI与SRY序列建立梅花鹿马鹿及其杂交鹿茸分子鉴别方法 　　[摘要] 为了建立梅花鹿、马鹿及其杂交鹿茸的特异性PCR鉴别方法。采集不同来源的梅花鹿、马鹿鹿及其杂交鹿的鹿角或鹿茸样品共48份，其中24份为市场流通的待测样品。分别利用COI与SRY基因序列作为其父母本的鉴别标记，对已知样品的COI与SRY基因进行扩增、测序，进行同源比对后根据其变异位点设计特异鉴别引物，分别基于COI与SRY基因建立对梅花鹿、马鹿及其杂交鹿的鹿茸的特异PCR鉴定方法，并随机对市场流通的24份待测鹿茸样品进行鉴别分析。该实验所构建双位点特异PCR体系，基于COI的鉴别位点，可通过PCR扩增获得232 bp的梅花鹿特异片段，而产生518 bp的马鹿特异片段；而基于SRY的鉴别位点，通过PCR扩增获得803 bp的梅花鹿特异片段，而产生425 bp的马鹿特异片段。随机抽取市场流通中的24个待测鹿茸样品，经鉴定有3个样品?儆诼怼せㄔ咏坏穆谷籽?品。该实验建立对梅花鹿、马鹿及其杂交鹿的鹿茸的PCR鉴别方法简便、可靠。 　　[关键词] 梅花鹿； 马鹿； 杂交； 双位点特异PCR体系； COI； SRY 　　[Abstract] For rapid identification of Cervus nippon， C. elaphus and their hybridize samples， the specific PCR for mutual authentication of them was established based on the SNPs in COI and SRY sequence. C. nippon， C. elaphus and their hybridize samples were collected from different origins， total DNA of 24 identified samples were extracted， and the COI and SRY gene was seqenced. SNPs in the COI and SRY sequences of the samples were found by Clustul X 2.1 program. Primers for identifying C. nippon and C. elaphus were designed according to the SNP site， two multi-PCR reaction system were established to identify them. In addition， 24 samples which were randomly collected in different herbal medicine market were identified. The band special for C. nippon （232 bp）and band special for C. elaphus （518 bp） based on COI sequence，and the band special for C. nippon （803 bp）and band special for C. elaphus （425 bp） based on SRY sequence， were found using multi-PCR reaction， and three of the twenty-four samples were identified as the hybridize samples. The multi-PCR reaction system could be used to identify C. nippon， C. elaphus and their hybridize samples . 　　[Key words] Cervus nippon； Cervus elaphus； hybridize； multi-PCR reaction system； COI； SRY 　　鹿茸是我国传统名药之一，始载于《神农本草经》，具有促进生长发育、强壮身体、抗炎、免疫调节、抗衰老、促进创伤愈合等功能[1-4]。《中国药典》规定鹿茸基原为鹿科动物梅花鹿Cervus nippon Temminck 或马鹿C.elaphus Linnaeus的雄鹿未骨化密生茸毛的幼角，作为名贵中药材品种，鹿茸在市场上供不应求且价格昂贵。 　　杂交有利于提高畜牧产量，我国的茸鹿种间和亚种间的杂种优势利用一直处于国际领先水平，早在1958年吉林省吉林市龙潭山鹿场即用本交的方法开