利用线粒体DNA上的cytB鉴定林蛙及林蛙油
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摘要
目的:以线粒体DNA上cytB为研究对象,采用聚合酶链式反应(PCR)手段,建立林蛙及林蛙油鉴定方法.方法:采用市售成年雌蛙的新鲜组织和林蛙油为实验材料,通过试剂盒提取总DNA;以已报道的东北林蛙、中国林蛙、黑斑蛙和蟾蜍的cytB为模板,设计四对引物; PCR扩增产物回收测序.结果:仅东北林蛙引物的PCR体系获得1 100 bp左右大小的产物,产物序列与东北林蛙cytB序列一致性为99%,说明实验对象林蛙为东北林蛙,林蛙油来源为东北林蛙.讨论:利用mt DNA独特的优势,应用PCR扩增cytB,并测序确定了林蛙品种,建立了林蛙种属以及林蛙油基源的鉴定方法,为林蛙油类药材的鉴定提供科学理论依据.
Objective: The mitochondria DNA was employed to constructing the identified method of Oviductus Ranae by PCR.Methods: The total DNA of the adult female frog oil and the fresh issues of frog was extracted by the kit; the cytB of Rana dybowskii,Rana chensinensis,Pelophylax nigromaculatus Yamaguchi,Bufo raddei was used as the template to design four pairs of primers,and the PCR system contained above DNA and primers and the products was extracted and sequenced. Results: The DNA fragment only gained from the system contained Rana dybowskii primers and the sequence showed the identified in 99% which illustrated the experimental materials were from the Rana dybowskii.Discussion: Taking advantage of mt DNA and applying the PCR,the identified method of Oviductus Rana was established,and this can provide theory basis for the identification of Rana and Oviductus Rana.
Objective: The mitochondria DNA was employed to constructing the identified method of Oviductus Ranae by PCR.Methods: The total DNA of the adult female frog oil and the fresh issues of frog was extracted by the kit; the cytB of Rana dybowskii,Rana chensinensis,Pelophylax nigromaculatus Yamaguchi,Bufo raddei was used as the template to design four pairs of primers,and the PCR system contained above DNA and primers and the products was extracted and sequenced. Results: The DNA fragment only gained from the system contained Rana dybowskii primers and the sequence showed the identified in 99% which illustrated the experimental materials were from the Rana dybowskii.Discussion: Taking advantage of mt DNA and applying the PCR,the identified method of Oviductus Rana was established,and this can provide theory basis for the identification of Rana and Oviductus Rana.
引文
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[2]赵立香,李荣权,肖振晶.东北林蛙开发与利用的研究现状[J].畜牧兽医科技信息,2014(4):4-5.
[3]刘仁杰,孙旸,王刚,等.中国林蛙产品的开发现状与研究进展[J].粮油加工,2014(12):72-75.
[4]沈雪,崔浩,杨秀东,等.林蛙油水溶性蛋白质的制备与相对分子量测定[J].吉林化工学院学报,2016,33(9):11-14.
[5]张丽华,李明成,王冰梅.貂组织线粒体DNA及细胞色素b鉴定及特征[J].吉林大学学报(医学版),2008,34(5):790-793.
[6]邓莹,李明成,张丽华.复合聚合酶链反应鉴别龟甲正品与伪品的特征[J].中国药学杂志,2014,49(12):1022-1026.
[7]曲莉,王淼,邓莹,等.龟甲DNA检测试剂盒的研制与评价[J].天然产物研究与开发,2016(5):668-672.
[8]曲萌,崔继春,董志恒,等.鸡内金的分子鉴定研究[J].中国中药杂志,2009,34(24):3192-3194.
[9]崔浩,胡伟,周鸿立.玉米须总DNA提取方法的建立[J].吉林化工学院学报,2016,33(11):36-39.