山羊TNNT3基因可变剪切及其对骨骼肌细胞分化的作用

doi:10.3864/j.issn.0578-1752.2021.20.019

摘要/Abstract

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,其中在骨骼肌中高度富集(P < 0.01),心脏及肺次之,其余组织中较低;TNNT3 mRNA在背最长肌和半膜肌中的表达始终处于一个动态变化中,胚胎期TNNT3在半膜肌的表达量高于背最长肌（P<0.05）;出生后则背最长肌中高于半膜肌（P<0.05）。④山羊TNNT3基因转录本TNNT3_3重复出现保守的外显子9—11（138bp）,体外翻译实验显示其可编码蛋白且蛋白大小与预期基本相符（37 kD）;相较于对照组,在山羊MuSCs中过表达该转录本使肌分化标志基因Myomaker、MyoG和MyH4 mRNA极显著升高(P < 0.01)。【结论】获得了山羊TNNT3基因具有完整CDS区5个新可变剪切体,TNNT3主要在肌肉组织（背最长肌和半膜肌）中高表达,在哺乳动物中高度保守且促进成肌分化。初步表明TNNT3基因在动物肌肉生长发育中具有重要的生物学功能。

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

Abstract:

【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】① A 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. ② Nucleotide 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. ③ The 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). ④ The conserved exon 9-11 (138 bp) of goat TNNT3 was repeated in the transcript TNNT3_3. TNNT3_3 was amplified, and it was found out that which could encode protein, and the protein size was basically the same as expected (37 kDa) in TnT transcriptional translation system in vitro. Moreover, the transfection of TNNT3_3 into goat MuSCs induced mRNA levels of myogenic differentiation marker genes, including Myomaker, MyoG, and MyH4, comparing with the control (P < 0.01). 【Conclusion】Five isoforms with complete CDS region of the TNNT3 gene were obtained in goat muscles. The sequence and expression of TNNT3 were highly conserved in mammals and enriched in muscles, indicating that potentially TNNT3 gene functions critically in muscle growth and development.

Key words: goat, TNNT3, alternative splicing, quantitative PCR, TnT transcriptional translation in vitro, Western blotting, myogenic differentiation

陈媛,蔡禾,李利,王林杰,仲涛,张红平. 山羊TNNT3基因可变剪切及其对骨骼肌细胞分化的作用[J]. 中国农业科学, 2021, 54(20): 4466-4477.

CHEN Yuan,CAI He,LI Li,WANG LinJie,ZHONG Tao,ZHANG HongPing. Alternative Splicing of TNNT3 and Its Effect on the Differentiation of MuSCs in Goat[J]. Scientia Agricultura Sinica, 2021, 54(20): 4466-4477.

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	引物名称 Primer name	引物序列（5′— 3′） Primer sequence （5′— 3′）	产物长度 Product length (bp)	退火温度 Annealing temperature (℃)	用途 Purpose
P1	TNNT3	F: ACCATGTCGGACGAGGAAGT R: CACTCTACTTCCAGCACCC	835	58.0	克隆CDS区全长 Cloning the full length of CDS
P2	q-TNNT3	F：AGGAGGGCTGAGGACGAT R：CGGTCTCCAGCTTGTAC	253	52.7	定量PCR Quantitative PCR
P3	TNNT3_1-5	F: ACCATGTCGGACGAGGAAGT R：CTCCTTGAGGGCGACCAGC	253	60.0	半定量PCR Semi-quantitative PCR
P4	TNT- TNNT3_3	F：CCCAAGCTTATGTCGGACGAGGAAGTCGA R：CCGCTCGAGCTTCCAGCGCCCGCCAACTT	957	58.9	TNT体外转录载体构建 Construction of TNT transcriptional vector in vitro
P5	pTNNT3_3	F：CCGCTCGAGATGTCGGACGAGGAAGTCGA R：CCCAAGCTTCTTCCAGCGCCCGCCAACTT	957	59.2	过表达载体构建 Construction of overexpression vectors
P6	β-actin	F：CCTGCGGCATTCACGAAACTAC R：ACAGCACCGTGTTGGCGTAGAG	87	59.7	定量PCR Quantitative PCR

序列名称 Sequence name	CDS区长度 Length of CDS (nt)	外显子数 Exon count	氨基酸长度 Amino acid length (AA)	蛋白质分子量 Protein molecular weight (kD)	等电点 Isoelectric point
TNNT3_1	783	15	260	30.64	8.61
TNNT3_2	753	15	250	29.73	8.30
TNNT3_3	954	20	317	37.40	6.77
TNNT3_4	801	16	266	31.39	6.72
TNNT3_5	741	14	246	29.17	9.15