Bovine MYOZ1 And MYOZ2 Gene Promoter Activity Analysis By Dual-luciferase Reporter

MYOZ1 gene and MYOZ2 gene encode sarcomere including Z disk proteins. Z-disc widely regulate sarcomere assembly and proportion of different muscle fiber. MYOZ1 gene was specific expressed in fast-twitch muscles, while MYOZ2 gene was specific expressed in slow-twitch muscles. The study of MYOZ1 gene promoter focused on pigs and mice, and the study of MYOZ2 gene promoter focused on mice, there is still no report about the transcription regulatory mechanism of bovine MYOZ1 or bovine MYOZ2 gene. Our research was conducted to detect the core region activities of bovine MYOZ1 and MYOZ2 promoter, and to lay a foundation for further research on transcription regulation of bovine MYOZ1 and MYOZ2 gene.5’ Rapid amplification of cDNA ends(5’RACE) was used to determine the transcription initiation site of the gene, and the 5’ flanking region of target gene was obtained by PCR. 7 subclones were obtained by the designed primers, and inserted into pGL3-Basic vectors. The recombinant plasmids were transfected into the C2C12 cells, and the relative transcriptional activities were measured by Dual-Luciferase Reporter Assay System.The main results were as follows:1. According the results of amino acid sequence alignment, the similarity of amino acids for bovine MYOZ1 is as high as 96% that with goat and ovis, the similarity of amino acids for bovine MYOZ2 is as high as 98.9% that with goat and ovis, these results revealed that they are very conservative in ruminants;2. 5’ Rapid amplification of cDNA ends(5’RACE) was used to determine the transcription initiation site of two gene. The transcription initiation site of bovine MYOZ1 gene is C, and the transcription initiation site of bovine MYOZ2 gene is A;3. The 7 subclones of bovine MYOZ1 gene promoter were successfully insert into dual-luciferase report gene vector, and the 7 subclones of bovine MYOZ2 gene promoter were also successfully insert into dual-luciferase report gene vector;4. The results of dual-luciferase reporter assays showed that, the core region of bovine MYOZ1 promoter were confirmed to locate in-116/+61, and the core region of bovine MYOZ2 promoter were confirmed to locate in-84/+125;5. The result of bioinformatics analysis revealed that, there are several possible transcription regulatory elements in the core promoter sequence of bovine MYOZ1 gene, such as CAAT,SRF,and MyoD, and several possible transcription regulatory elements in the core promoter sequence of bovine MYOZ2 gene, such as SRF, MyoD, and MEF2.