MSTN基因编辑牛的鉴定及基因分型新方法  

A New Method for Identifying and Genotyping MSTN Gene-edited Cattle

作　　者：苏秋菊 周翔[1] 李光鹏 白春玲 许文涛[3] 刘榜[1] SU Qiu-ju;ZHOU Xiang;LI Guang-peng;BAI Chun-ling;XU Wen-tao;LIU Bang(Key Laboratory of Agricultural Animal Genetics,Breeding and Reproduction of the Ministry of education,Huazhong Agricultural University,Wuhan 430070;State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock,Inner Mongolia University,Hohhot 010070;College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083)

机构地区：[1]华中农业大学农业动物遗传育种与繁殖教育部重点实验室,武汉430070 [2]内蒙古大学省部共建草原家畜生殖调控与繁育国家重点实验室,呼和浩特010070 [3]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《生物技术通报》2019年第4期208-212,共5页Biotechnology Bulletin

基　　金：转基因重大专项(2018ZX08012-001)

摘　　要：基因编辑是目前进行遗传修饰的重要手段,但因编辑时缺失的片段小而给基因编辑的鉴定和分型带来困难。该研究以肌肉生成抑制素基因(Myostation,MSTN)编辑牛为材料,建立功能核酸PCR(Functional nucleic acid PCR,FNA-PCR)方法对其进行鉴定及基因分型。针对编辑位点的两种等位基因设计两条不同的正向引物和一条共用的反向引物,其中一条正向引物是用于检测野生型等位基因,3'端终止于编辑位点,同时对它的5'端进行功能核酸接头设计;另一条正向引物用于检测编辑型等位基因,3'端跨越编辑位点向后延伸几个碱基。这种设计可使野生型和编辑型等位基因扩增产物大小不同。通过对PCR反应体系和条件的优化,建立了一种通过一次PCR反应准确对三种不同基因型进行分型的FNA-PCR新方法。检测方法灵敏度高,检测限为0.1ng。该研究中所建立的FNA-PCR方法可用于基因编辑检测及其它小片段缺失的基因分型,具有简便、准确、灵敏、成本低等优点。Gene editing is a critical technique to generate genetically modified animals;however,it is hard to identify and genotype gene-edited animal because deleted fragment during editing is quite small.In this study,functional nucleic acid PCR(FNA-PCR)technique was established for identifying and genotyping MSTN gene-edited cattle.Based on 2 alleles for edited sites,2 different forward primers and one common reverse primer were designed.One forward primer was used to identify the allele of wild type samples,its 3'end was terminated at the edited site,and functional nucleic acid joint was designed to its 5'end.The other forward primer was used to detect MSTN gene-edited cattle in which the 3'end extended several nucleotides beyond the gene-edited site.This unique design allowed the amplified allele sizes of gene-edited and wild-type varied.By optimizing PCR reaction system and conditions,a novel FNA-PCR method,3 different genes were accurately genotyped in one PCR reaction,was established.FNA-PCR was high in sensitivity with detection limit 0.1 ng of MSTN gene-edited cattle genome DNA.In conclusion,FNA-PCR is a simple,accurate and rapid for identifying gene-edited animal and genotyping those smallfragment-deletion genes.

关 键 词：基因编辑牛 功能核酸PCR 基因分型 MSTN 

分 类 号：Q78[生物学—分子生物学]