|
|
|
| Molecular Cloning and Tissue Expression Analysis of TNNI1 Gene in Gaoyou Duck (Anas platyrhynchos) |
|
|
|
|
Abstract Abstract Slow skeletal muscle troponin I 1 (TNNI1) located in slow-twitch myofibers that provides a calcium-sensitive switch for the contraction of striated muscle and plays an important role in muscle growth. The aim of this study was to clone the cDNA full length of TNNI1 gene of Gaoyou duck (Anas platyrhynchos domestica), analyze its sequence characteristics, mRNA expression profile in different tissues/organs, which might provide basis to further study the function of TNNI1 gene in muscle development in duck. Gaoyou duck was selected as materials, the full length of TNNI1 cDNA was cloned from breast muscle by 5' and 3' rapid-amplification of cDNA ends (RACE) and its homologies with TNNI1 gene of other animals was analyzed. The TNNI1 mRNA levels in different tissues/organs at sexual age (70 d), as well as in the breast and leg muscles during the developmental stages were measured using quantitative real-time PCR (qRT-PCR). The results showed that the full length of TNNI1 cDNA sequence was 965 bp, containing a 56 bp 5′UTR, a 345 bp 3′UTR and a 564 bp open reading frame (ORF), encoding a protein of 187 amino acids (GenBank accession number: KY926794). Sequence analysis revealed that the TNNI1 amino acid sequence of the Gaoyou duck is similar with those of Beijing duck (Anas platyrhynchos, 97.9%), quail (Coturnix japonica, 97.9%), chicken (Gallus gallus, 97.3%) and mammals (87.7%). Phylogenetic tree analysis showed that Gaoyou duck has the closest phylogenetic relationship to the Peking duck, quail and chicken, but were far away with mammals TNNI1 in amino acid sequences. The qRT-PCR results showed that TNNI1 were expressed in different organizations. And the mRNA level of TNNI1 in leg muscle were significantly higher than that of other tissues (P＜0.01), and the heart were the second. There was no significant difference between the mRNA level of breast muscle and glandular stomach (P＞0.05), while the lowest level was in brain, liver, spleen, and hypothalamus. During embryogenesis and post-hatching development, the expression pattern of TNNI1 in breast muscle and leg muscle showed an extreme significant difference as time went on. The mRNA level peak of TNNI1 in breast muscle was at embryonic age of 21 days, and in leg muscle was at embryonic age of 27 days. The mRNA level of TNNI1 gene in breast muscle was significantly lower than that of leg muscle at the embryonic days 27 and the neonatal age of 5 days, but was significantly higher than that of leg muscle at the embryonic age of 21 days. The results of these experiments showed that the amino acid sequence of TNNI1 belonged to the same branch as that of poultry, and was far away from mammals. qRT-PCR results showed that TNNI1 could be expressed in different tissue of Gaoyou duck, and existed some difference. The present study also found that there were significant differences in mRNA level of TNNI1 gene between the breast muscle and the leg muscle at different developmental stages. Therefore, structural characterization and tissue expression of TNNI1 gene in Gaoyou duck lay the foundation for further research on the function of TNNI1 gene in duck muscle development.
|
|
Received: 02 November 2017
Published: 21 May 2018
|
|
|
|
|
|
|
