| Myostatin (MSTN) gene was a potent negative regulator of muscle and was related to the production traits in livestock, and it has been considered as an inhibitor of myogenic differentiation; ankyrin-repeat domain 2 (Ankrd.2), a novel skeletal muscle gene was involved in skeletal muscle cell proliferation and apoptosis, which plays an important role in skeletal muscle hypertrophy. It plays an important role in skeletal muscle growth and development. In this study, the rabbit skeletal muscle satellite cells (SCs) were obtained from 1 day old of New Zealand rabbit to detect the effect of siRNA-mediated silencing of MSTN, Ankrd2 on cell proliferation and differentiation on rabbit SCs.Phosphoglycerate mutase (PGAM2) was an important enzyme during glycolysis and gluconeogenesis. PGAM2 plays key roles in the glycolysis and controlling growth rate in livestock. Kell blood group complex subunit-related family, member 4 (XKR4) gene was involved in many biological processes, which was considered as an important candidate gene for feed intake, rump fat thickness and growth rate. In the current study, a total of four breeds of animals were used including Tianfu, Ira, Champagne and New Zealand rabbits, and then genotyped by PCR-RFLP or HRM method to investigate the association between the polymorphisms in the CDS regions of PGAM2, XKR4 and growth traits in rabbits. All the results in the current study were showed as follows:(1) For isolation and purification rabbit SCs, a 1 day old New Zealand rabbit was used. The muscle tissues were digested by collagenase and cells were purified with differential adherent. We observed the cellular morphology under the general microscope. In different stages, the cellular morphology was in keeping with the characters of SCs; The Desmin was expressed in cytoplasm in the second generation by immunohistochemical. These results indicated the cells we obtained were SCs.(2) For selecting the most valuable siRNA, three pairs of siRNA were designed for MSTN and Anrkd2, respectively. si-MSTN-01, si-MSTN-02, si-MSTN-03 and si-Ankrd2-01, si-Ankrd2-02, si-Ankrd2-03 were used, each of them was transfected to the SCs by LipofectamineTM 2000. The relative expression levels of MSTN and Anrkd2 were tested by Real Time quantitation PCR after 24h,48h and 72h. The data demonstrated 48h was the better time than 24h or 72h. And si-MSTN-02, si-Ankrd2-01 at 48h were the highest efficient to MSTN and Ankrd2 respectively.(3) For evaluating the proliferation ability of the tests, Cell Counting Kit-8 (CCK-8) was used. The results were reflected by the microplate reader after transfected with si-MSTN-02 or Anrkd2 after 48h. The proliferation ability was significantly promoted when MSTN was inhibited (p< 0.05) and highly significant promoted when Anrkd2 was inhibited (p< 0.01) in SCs. The results indicated MSTN and Anrkd2 are negative regulators in the SCs proliferation process.(4) MyoD and Caveolin-3, the specific markers of SCs were detected by Real Time quantitation PCR, when the relative expression level of MSTN was down-regulated, MyoD and Caveolin-3 were expressed in higher levels, and it was occurred when Ankrd2 was down-regulated, too. These results indicated that MSTN and Anrkd2 are negative regulators in the SCs differentiation process.(5) A total of three single nucleotide polymorphisms (SNPs) were identified in the CDS of PGAM2, including c.-10C>T, c.195C>T and c.414+17C<T. The c.195C>T didn’t cause amino acid change, was a synonymous mutation, and it was genotyped by PCR-RFLP. Association analysis indicated CT genotype of c.195C>T was associated significantly (P< 0.05) with greater body weight at 84 days old (W84) and average daily weight gain (ADG).(6) A total of three SNPs were identified in the CDS of XKR4, including c.804-8C>T, c.1668C>T and c.1698C>T. The SNP of c.1668C>T and c.1698C>T, located in the coding region, didn’t case amino acid change, were synonymous mutations. They were complete linkage by direct sequencing. They were genotyped by HRM method and association analysis revealed the individuals of CTCT haplotype were significantly with a higher W70 than that of CCCC (P<0.05), in addition, individuals of CTCT haplotype had a highly significant performance of ADG from 35 to 70 day old than the homozygote individuals (P<0.01).In this study, we found the ability of SCs proliferation and differentiation were obviously enhanced when MSTN and Ankrd2 was down-regulated, it provided molecular breeding markers for improving meat production and quality. The mutations in the CDS of PGAM2 or XKR4 were related to growth rate in meat rabbits, both of them could act as the candidate genes affecting growth traits and provide molecular breeding markers. |
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