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| Cloning and Expression Analysis of Capra hircus FGF10 Gene |
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Abstract Fibroblast growth factor 10 (FGF10) is a critical growth factor, which can promote the differentiation of Mus musculus and Homo sapiens preadipocytes. In order to clarify the expression profile of FGF10 gene of Capra hircus, elucidate its expression changes during intramuscular preadipocyte differentiation, and analyze the correlation between gene expression and intramuscular fat deposition, Jianzhou Big-Eared goat (C. hircus) was used as laboratory animal in this study and the C. hircus intramuscular preadipocytes were obtained usingⅡcollagenase. Reverse transcription PCR (RT-PCR) was used to clone FGF10 gene and Real-time quantitative PCR (qPCR) was used to detect the expression level of genes. The expression level of FGF10 was detected both in different tissues and in various stages during C. hircus muscle development, including stages of kid, youth and adult. Besides, the temporal expression profile of FGF10 during intramuscular preadipocyte differentiation was also constructed. Following the determination of intramuscular fat (IMF) content which was performed by soxhlet extraction method, a correlation between FGF10 mRNA expression and IMF content was built in longissimus dorsi. The full-length sequence of the Jianzhou Big-Eared goat FGF10 gene was 1 252 bp including a complete 642 bp ORF flanked by 166 bp 5' untranslated region (UTR) and 444 bp 3' UTR, encoding 213 amino acids (Accession no. KT899958). The amino acid identity of FGF10 protein between the C. hircus and Ovis aries, Bos Taurus, Rattus norvegicus and Homo sapiens was 100%, 100%, 97% and 94%. The secondary structure prediction of C. hircus FGF10 protein showed that 12.68% α-helix, 27.70% extended strand and 59.62% random coil might be formed. The FGF10 protein of C. hircus possessed a transmembrane region and a FGF region, as well as a signal peptide structure whose cleavage site was between Ala38 and Leu39. Phylogenetic tree analysis showed that FGF10 was conserved among different species. FGF10 widely expressed in all examined tissues of Jianzhou Big-Eared goat, including heart, liver, spleen, lung, kidney, adipose tissue, longissimus dorsi, biceps femoris and triceps brachii. The highest expression level of FGF10 was found in lung which was significantly higher than that in other tissues (P＜0.05). And it also highly expressed in spleen and adipose tissue while less expressed in heart, longissimus dorsi and triceps brachii. The qPCR results also showed that FGF10 continuously expressed during the growth of longissimus dorsi. And it's expression level was increased with the muscle development and reached the highest in adult goat, significantly higher than in kid and youth (P＜0.05). Correlation analysis result showed that the expression level of FGF10 in longissimus dorsi of C. hircus was significantly positively correlated with IMF content (P＜0.05). The mRNA expression of FGF10 in the intramuscular preadipocytes was the lowest and reached the highest at 2th day after the induction of differentiation. Then the mRNA level decreased significantly in 4 d (P＜0.05), but gradually increased again in the following days. These results provide theoretical basis for further elucidating the molecular mechanism of FGF10 gene in regulating C. hircus IMF deposition.
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Received: 15 May 2016
Published: 01 October 2016
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