Functional Study of Goat CSRP3 Gene on Myoblast Differentiation

Skeletal muscle growth and development is one of the important factors affecting meat quality of goat. This study aimed to clone the coding sequence (CDS) of the goat CSRP3 gene (Cysteine and Glycine Rich Protein 3), conduct bioinformatics analysis using the online software, investigate the expression patterns of the goat CSRP3 gene in different tissues and at different differentiation stages of goat myoblasts, and clarify the regulatory role of this gene in myoblast differentiation.

Tissues including the heart, liver, spleen, lung, kidney, and longissimus dorsi muscle were collected from goats. Total RNA was extracted from these tissues using the Trizol method and was reverse-transcribed to cDNA. Using the cDNA from the longissimus dorsi muscle as the template, RT-PCR was performed to amplify the CDS region of the goat CSRP3 gene, and online software was used for bioinformatics analysis. cDNA from different goat tissues and myoblasts at different differentiation stages was used as templates to detect the CSRP3 gene expression level by quantitative Real-Time PCR (qPCR). Through the construction of an overexpression vector, siRNA synthesis, and cell transfection experiments, qPCR and immunofluorescence staining were employed to determine the relative mRNA expression level of myoblast differentiation marker genes and the formation of myotubes after overexpression or interference of the goat CSRP3 gene.

The full-length of the goat CSRP3 gene is 1103 bp, with a CDS sequence of 585 bp. Phylogenetic tree analysis showed that the goat CSRP3 gene has the closest genetic relationship with sheep and cattle, indicating high conservation of this gene among closely related species. The goat CSRP3 gene encodes 194 amino acids, and the encoded protein is a hydrophilic and stable basic protein. Protein-protein interaction analysis revealed that the goat CSRP3 protein may be involved in muscle growth and development through interactions with muscle function-related proteins. The relative mRNA expression level of the CSRP3 gene in goat longissimus dorsi muscle and heart was significantly higher than that in other tissues (P < 0.05). During myoblast differentiation, the goat CSRP3 gene expression was low at the initial stage, increased gradually with differentiation time, peaked on day 3 (P < 0.01), and then decreased, showing a dynamic change of first increasing and then decreasing. This suggested that the goat CSRP3 gene may play an important regulatory role in the transition of myoblasts from proliferation to differentiation. Overexpression of the goat CSRP3 gene significantly increased the relative mRNA expression levels of myoblast differentiation marker genes and promoted myotube formation, indicating myoblast differentiation. Conversely, interference with the CSRP3 gene significantly reduced the relative mRNA expression levels of myoblast differentiation marker genes and inhibited myotube formation, impairing the differentiation ability of myoblasts.

The CDS of CSRP3 was successfully cloned (the cloned sequence is 603 bp in length, including a 585 bp CDS region), and its sequence characteristics and physicochemical properties were clarified. The CSRP3 tissue and temporal expression profiles in goats were established. It was confirmed that the CSRP3 gene exerts a positive regulatory effect on goat myoblast differentiation. These results preliminarily indicate that the CSRP3 gene plays an important biological function in the muscle growth and development of meat goats.