山羊TNNT3基因可变剪切及其对骨骼肌细胞分化的作用  

Alternative Splicing of TNNT3 and Its Effect on the Differentiation of MuSCs in Goat

作　　者：陈媛 蔡禾 李利 王林杰 仲涛 张红平 CHEN Yuan;CAI He;LI Li;WANG LinJie;ZHONG Tao;ZHANG HongPing(Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province,Sichuan Agricultural University,Chengdu 611130)

机构地区：[1]四川农业大学畜禽遗传资源发掘与创新利用四川省重点实验室,成都611130

出　　处：《中国农业科学》2021年第20期4466-4477,共12页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31672402,31772578)。

摘　　要：【目的】快速骨骼肌肌钙蛋白T(fast skeletal troponin T3,TNNT3)作为肌钙蛋白(troponin,Tn)家族成员,调节横纹肌收缩、参与骨骼肌的生长发育并影响家畜肉质性状。通过获得山羊TNNT3基因的可变剪切体,分析山羊TNNT3基因可变剪切的表达模式及其在肌细胞分化中的作用,深入解析TNNT3基因在山羊骨骼肌生长发育过程中的作用机制。【方法】基于NCBI已公布山羊TNNT3基因(NM_001314210.1)和牛TNNT3基因(XM_010821200)mRNA序列,使用软件Primer Premier 6.0设计引物,以简州大耳羊胚胎期和出生后7个阶段骨骼肌为试验材料,克隆测序获得山羊TNNT3基因的CDS区可变剪切体,利用软件ORF Finder、EditSeq、DNAMAN、ClustalW和MEGA_X_10.1.8等对序列进行生物信息学分析;进一步设计实时荧光定量(real-time PCR,RT-qPCR)及半定量引物,研究TNNT3基因剪切体在7个不同组织(背最长肌(longissimus dorsi muscle,LD)、半膜肌(semimembranosus muscle,SM)、心、肝、脾、肺、肾)和7个发育阶段(胚胎期E75、E90、E105和出生后B3、B45、B150、B300)肌肉组织(背最长肌和半膜肌)中表达模式;此外,对转录本TNNT3_3进行体外编码能力检测确定其具有编码蛋白的能力,并在山羊骨骼肌卫星细胞(skeletal muscle satellite cells,MuSCs)中过表达,观察细胞形态变化以及检测标志基因的表达变化,研究其对山羊MuSCs分化的作用。【结果】①TNNT3(NM_001314210.1)CDS区全序列主要含有18个外显子,其中外显子16/17相互排斥,转录后单一表达。克隆发现山羊TNNT3基因5个新转录本(TNNT3_1—5),其外显子数分别是15、15、20、16、14。②生物信息学分析结果显示山羊TNNT3基因核苷酸序列和氨基酸序列与绵羊、牛、猪等哺乳动物具有很高的一致性,而与鱼类和爬行类动物的一致性较低,说明TNNT3基因序列在哺乳动物高度保守。③TNNT3 mRNA在背最长肌、半膜肌、心、肝、脾、肺、肾7个组织中都有表达,其�【Objective】As a member of the troponin(Tn)family,TNNT3(Fast Skeletal Troponin T3)involves skeletal muscle contraction,growth,development,and even meat characteristics of domestic animals.This study initially aimed to identify the alternative splicing of goat(Capra hirus)TNNT3.【Method】Based on goat TNNT3(NM_001314210.1)and cattle TNNT3(XM_010821200)mRNA sequence from NCBI(National Center for Biotechnology Information),the primers were designed by using the Primer Premier 6.0 software,subsequently,TNNT3 was amplified from skeletal muscles of embryo Jianyang Bigear Goat.The obtained TNNT3 sequences were then bioinformatically analyzed by using ORF Finder,EditSeq,DNAMAN,ClustalW,and MEGA_X_10.1.8.Furthermore,the levels of TNNT3 isoforms were quantified by using real-time fluorescence quantitative PCR(RT-qPCR)and semi-quantitative PCR in longissimus dorsi(LD)muscle,semimembranosus(SM)muscle,heart,liver,spleen,lung,and kidney,at seven stages(E75,E90,E105,B3,B45,B150,and B300),respectively.Additionally,the coding ability of transcript TNNT3_3 in vitro and its effect on the differentiation of goat skeletal muscle satellite cells(MuSCs)were explored.【Result】①A total of five isoforms(named TNNT3_1-5)of the TNNT3 gene were identified in pooled RNA extracted from goat muscles,and the complete coding sequence(CDS)sequence mainly contained 18 exons.②Nucleotide sequence and amino acid sequence of the goat TNNT3 gene were highly consistent with sheep,cattle,pig,and other mammals,but low with that of fish and reptiles,indicating the high evolutionary conservation of the TNNT3 gene in mammals.③The TNNT3 mRNA was presented in all seven detected tissues but highly enriched in LD and SM muscles(P<0.01),followed by cardiac muscle and lung.Furthermore,the levels of TNNT3 mRNA in SM muscles were higher than that in LD muscles in prenatal goats(P<0.05),while the verse results were presented postnatal(P<0.05).④The conserved exon 9-11(138 bp)of goat TNNT3 was repeated in the transcript TNNT3_3.TNNT3_3 was amplified,and

关 键 词：山羊 TNNT3 可变剪切体 荧光定量PCR 体外转录翻译 蛋白免疫印迹杂交 肌原分化 

分 类 号：S827.2[农业科学—畜牧学]