MSTN基因突变纯合子兔的繁育和表型分析  

Propagation and phenotypic analysis of mutant rabbits with MSTN homozygous mutation

作　　者：尚利青 宋绍征 张婷[1] 严坤宁 蔡何清 袁玉国[1] 成勇[1] SHANG Liqing;SONG Shaozheng;ZHANG Ting;YAN Kunning;CAI Heqing;YUAN Yuguo;CHENG Yong(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,Jiangsu,China;Department of Basic Medicine,Wuxi Taihu University,Wuxi 214064,Jiangsu,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]无锡太湖学院基础医学系,江苏无锡214064

出　　处：《生物工程学报》2022年第5期1847-1858,共12页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(政府间合作项目)(2016YFE0126000)。

摘　　要：肌肉生长抑制素(myostatin,MSTN)基因是动物骨骼肌生长的负调控基因。本研究在获得MSTN基因双等位突变原代兔的基础上,培育具有“双肌”表型性状和稳定遗传的MSTN^(–/–)纯合突变兔,建立原代MSTN双等位突变兔繁育纯合子后代的方法。应用CRISPR/Cas9基因编辑技术制备MSTN–/–突变原代兔,原代双等位突变兔与野生型兔配种,产生F1代兔。同一突变系的F1代兔半同胞交配或回交繁殖F2代纯合子兔;后代兔利用基因组DNA PCR扩增产物序列分析和T载体克隆测序鉴定用于筛选纯合子兔;14–19周龄MSTN基因突变兔称量体重。MSTN基因突变兔臀大肌组织切片,统计分析肌纤维横截面积。获得5只MSTN基因突变原代兔,其中3只用于纯合子培育,共获得15只(5种突变子)纯合突变兔(M2-a:3只;M2-b:2只;M3-a:2只;M7-a:6只;M7-b:2只)。在14–19周龄,MSTN^(–/–)纯合突变兔体重极显著高于同龄MSTN+/+野生型兔(平均(2718±120)g和(1969±53)g,P<0.01,同龄体重提高38.0%);纯合突变兔的臀大肌纤维平均截面积极显著高于野生型兔((3512.2±439.2)μm^(2)和(1274.8±327.3)μm^(2),P<0.01),也显著高于MSTN^(+/–)半合子兔((3512.2±439.2)μm^(2)和(2610.4±604.4)μm^(2),P<0.05)。本研究成功培育了5种MSTN^(–/–)基因纯合突变兔,且有明显瘦肉表型。研究表明,应用CRISPR/Cas9基因编辑获得的原代双等位突变兔为非嵌合体突变兔,突变性状遗传后代;原代双等位突变兔后代分离为两个不同突变子,并都呈现“双肌”表型;F1代单等位突变兔通过近交或回交可获得MSTN^(–/–)纯合突变兔。该研究在利用基因编辑技术培育优良家畜品种方面取得了实验进展。Myostatin gene(MSTN)encodes a negative regulator for controlling skeletal muscle growth in animals.In this study,MSTN^(–/–)homozygous mutants with“double muscle”phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits,with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits.MSTN^(–/–)primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology.The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits,whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain.Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits.The MSTN mutant rabbits with 14–19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed.Five primary rabbits with MSTN gene mutation were obtained,among which three were used for homozygous breeding.A total of 15 homozygous rabbits(5 types of mutants)were obtained(M2-a:3;M2-b:2;M3-a:2;M7-a:6;M7-b:2).The body weight of MSTN^(–/–)homozygous mutant rabbits aged 14–19 weeks were significantly higher than that of MSTN^(+/+)wild-type rabbits of the same age((2718±120)g vs.(1969±53)g,P<0.01,a 38.0%increase).The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits((3512.2±439.2)μm^(2) vs.(1274.8±327.3)μm^(2),P<0.01),but also significantly higher than that of MSTN^(+/–)hemizygous rabbits((3512.2±439.2)μm^(2) vs.(2610.4±604.4)μm2,P<0.05).In summary,five homozygous mutants rabbits of MSTN^(–/–)gene were successfully bred,which showed a clear lean phenotype.The results showed that the primary breeds were non-chimeric mutant rabbits,and the mutant traits could be inherited from the offspring.MSTN^(–/–)homozygous mutant rabbits of F2 generatio

关 键 词：肌肉生长抑制素 繁育 突变 CRISPR/Cas9 新西兰兔 

分 类 号：S829.1[农业科学—畜牧学]