海门山羊MSTN基因克隆、生物信息学分析及单碱基编辑的研究  

Cloning,bioinformatics analysis and single base editing of MSTN gene in Haimen goat

作　　者：夏潇潇 王佳琳 李玲[1,2] 佰伍加 宋志贤 岳恒 曹少先 袁玉国[1,2] XIA Xiaoxiao;WANG Jialin;LI Ling;KYAW Paing Oo;SONG Zhixian;YUE Heng;CAO Shaoxian;YUAN Yuguo(College of Veterinary Medicine/Key Laboratory of Animal Genetic Engineering,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center of Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China;College of Medicine,Yangzhou University,Yangzhou 225009,China;Institute of Animal Science,Jiangsu Academy of Agricultural Science/Jiangsu Province Engineering Research Center for Precision Animal Breeding/Key Laboratory of Crop and Animal Integrated Farming,Ministry of Agriculture and Rural Affairs,Nanjing 210014,China)

机构地区：[1]扬州大学兽医学院/扬州大学动物基因工程重点实验室,江苏扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [3]扬州大学医学院,江苏扬州225009 [4]江苏省农业科学院畜牧研究所/江苏省畜禽精准育种工程研究中心/农业农村部种养结合重点实验室,江苏南京210014

出　　处：《畜牧与兽医》2025年第5期24-30,共7页Animal Husbandry & Veterinary Medicine

基　　金：江苏省种业振兴“揭榜挂帅”项目(JBGS[2021]025);江苏高校优势学科建设工程项目(PAPD);高等学校学科创新引智计划项目(D18007);扬州市市校合作专项(YZ2023205)。

摘　　要：为探明海门山羊不同组织中肌肉生长抑制素(MSTN)基因表达规律及其编码蛋白结构特点,通过克隆海门山羊MSTN基因CDS序列并分析其生物特点,运用实时荧光定量PCR技术分析MSTN基因在不同组织中的表达水平,在MSTN基因第2、3外显子上设计3个单碱基编辑靶位点,将构建的sgRNA质粒分别与胞嘧啶碱基编辑器YE1-BE3-FNLS电转入培养的海门山羊胎儿成纤维细胞中,筛选48 h后提取细胞基因组DNA,对PCR扩增靶位点序列进行Sanger测序和TA克隆分析。结果:海门山羊MSTN基因的CDS序列编码375个氨基酸,具有高度保守性。海门山羊不同组织中MSTN基因表达水平存在差异,其中骨骼肌的表达量最高;3条sgRNA均可在MSTN基因外显子上进行单碱基编辑,编辑效率分别为25%、37%和29%。结论:研究结果为进一步利用单碱基基因编辑技术培育MSTN基因突变海门山羊新品系提供了新的依据。In order to investigate the expression of the myostatin(MSTN) gene and the structural characteristics of its encoded protein in different tissues of Haimen goat,the CDS sequence of MSTN gene of Haimen goat was cloned and the biological characteristics were analyzed.The expression level of the MSTN gene in different organs and muscle tissues of Haimen goat was checked by RT-qPCR.Three sgRNA plasmids targeting the second and third exons of the MSTN gene were designed and constructed,respectively,and co-transfected with YE1-BE3-FNLS into the fetal fibroblasts cell in Haimen goat.After 48 h of screening,cell DNA was extracted,and target site sequences were amplified by PCR for Sanger sequencing and TA cloning analysis.The data indicated that the CDS sequence for the MSTN gene of Haimengoat encoding 375 amino acids,which was highly conserved.The MSTN gene was expressed in different tissues and organs of Haimen goats,and the expression level of MSTN was the highest in the triceps brachii.Three sgRNA successfully introduced stop codons with editing effi-ciencies of 25%,37%,and 29%,respectively.These data laid a foundation for the use of single base gene editing technology to breed newstrains of Haimen goats with MSTN gene mutation.

关 键 词：海门山羊 肌肉生长抑制素 单碱基编辑 

分 类 号：S826[农业科学—畜牧学]