Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (20): 4466-4477.doi: 10.3864/j.issn.0578-1752.2021.20.019

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

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

CHEN Yuan(),CAI He,LI Li,WANG LinJie,ZHONG Tao,ZHANG HongPing()   

  1. Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130
  • Received:2020-09-09 Accepted:2021-05-13 Online:2021-10-16 Published:2021-10-25
  • Contact: HongPing ZHANG E-mail:Chyuan201301@163.com;zhp@sicau.edu.cn

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

Fig. 1

Molecular structure and interaction proteins of Troponin T (B) and alternative spliced exons of three TnT isoforms (A), (Adapted from literature[20-22]) A: Alternative spliced exons of three TnT isoforms: Red box indicated alternatively spliced exons; Blue box indicated embryonic specific exons; Green box indicated avian exons; B: Proteins interact with TnT"

Table 1

Information of primer sequence for the TNNT3 genes"

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

Fig. 2

PCR amplification, cloning results of TNNT3 gene in goats and comparison results of different alternative transcripts sequences with the goat genome database A: PCR products of TNNT3 full-length CDS region (835bp); B: PCR products of TNNT3 isoforms from monoclonal bacteria solution; C: qPCR products with TNNT3 transcripts at alternative fragment (Exon4-8: 193bp-253bp ; M1: DNA Marker DL 2000 bp; M2: DNA Marker DL 5000 bp; D: Comparison results of different alternative transcripts sequences with the Goat genome database; The red box is an exon site that can be alternative splicing; E: Amino acid sequences of different alternative transcripts of TNNT3 in goats; Green box: The first degraded product in skeletal muscle[18]; Red box: the Exon 9-11 of TNNT3_3 is repeated with 46 amino acid; Blue box: Exon 16 and 17 coding amino acid are alternatively spliced in a mutually exclusive manner[25]; F: TNNT3 alternative transcripts domain prediction"

Table 2

Amino acid sequence information predicted of goat TNNT3 alternative transcripts"

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

Fig. 3

Phylogenetic tree and evolutionary analysis of the TNNT3 amino acid sequence"

Fig. 4

The different alternative transcripts of goat TNNT3 distribution and expression levels at different stages A: The mRNA level of TNNT3 in different tissues; B: The semi-quantitative result of goat TNNT3 alternative transcripts in different tissues; C: The mRNA level of TNNT3 in different development stages of LD and SM; D: The semi-quantitative result of TNNT3 alternative transcripts in different development stages of LD and SM; The different capital letter/ lowercase letter indicated extremely significant difference (P<0.01);During the same period, the significant of expression between the two tissues was expressed as * (P<0.05) ** (P<0.01)"

Fig. 5

Transcription in vitro and overexpression of TNNT3_3 and its effect on the differentiation in goat MuSCs A: Construction of TNNT3_3 transcriptional vector in vitro; B: Western blot results of TNNT3_3 transcriptional protein in vitro; C: mRNA levels of marker genes in the proliferative/ differentiation stage of MuSCs treated with TNNT3_3 vector; D: MyHC immunofluorescence results after differentiation for 7 days; M1:DNA marker DL 2000 bp;M2:DNA marker DL 5000 bp;M3:Protein marker"

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