Molecular cloning and expression pattern of duck Six1 and its preliminary functional analysis in myoblasts transfected with eukaryotic expression vector

摘要

Skeletal muscle development is regulated by i style="mso-bidi-font-style:normal"Six1/i, an important myogenic transcription factor. However, the functional analysis of duck i style="mso-bidi-font-style:normal"Six1/i has not been reported. Here, we cloned the coding domain sequence (CDS) region of the duck i style="mso-bidi-font-style:normal"Six1/i gene using RT-PCR and RACE methods. Bioinformatics analysis revealed that duck i style="mso-bidi-font-style:normal"Six1/i CDS region comprised of 849 bp and encoded 282 amino acids and had a high degree of homology with other species, suggesting that the functions of duck i style="mso-bidi-font-style: normal"Six1/i gene are conserved among other animals. Real-time PCR used to determine the mRNA expression profiles of duck i style="mso-bidi-font-style: normal"Six1/i in different tissues and different developmental stages showed that i style="mso-bidi-font-style:normal"Six1/i was highly expressed in skeletal muscle and the embryonic stage. Furthermore, the eukaryotic expression vector pEGFP-duSix1 was constructed and transfected into the duck myoblasts; the MTT assay revealed an obvious increase of cell proliferation after transfection. The expression profiles of i style="mso-bidi-font-style:normal"Six1/i, i style="mso-bidi-font-style:normal"Myf5/i and i style="mso-bidi-font-style: normal"MyoD/i showed that their expression levels were significantly increased. These results together suggested that pEGFP-duSix1 vector was constructed successfully and overexpression of duck i style="mso-bidi-font-style:normal"Six1/i in the myoblasts could promote cell proliferation activity and significant up-regulate expression of i style="mso-bidi-font-style:normal"Myf5/i and i style="mso-bidi-font-style: normal"MyoD/ii style="mso-bidi-font-style: normal"./i" xml:lang="en_US