Transcription Regulation And Biological Function Analysis Of Porcine 14-3-3 Theta Gene

The 14-3-3 proteins are small (-30 kD) acidic proteins, which highly conserved in vary different species. There are seven different isoforms which were found in mammalian in present.These proteins function in form of both homo-and heterodimers in organisms. Reseach showed 200 proteins have been proved to couple with 14-3-3 proteins to take part in biological functions among which most proteins were phosphorylation protein.14-3-3 theta plays a critical antiapoptotic function in cardiomyocytes through antagonizing ASK1 signaling.While other evidences indicate that 14-3-3 theta effects survival of cardiomyocytes involved in several signal pathways. There are many researches on 14-3-3 theta gene. However, the transcriptional regulation and fuction of 14-3-3 theta on myogensis are still unclear. Several evidences have indicated that 14-3-3 theta gene plays an important role in muscle biological function. The results were as follows:1. The expression of 14-3-3 theta gene in Meishan and Yorkshire breeds at four periods (fetal 65d, and after birth 3d,60d,120d)during longissmus dorsi (abbreviated to LD) were analyzed. The expression of 14-3-3 theta gene was up regulated from the stage fetal 65d to after birth 60d in Yorkshire breeds. In the mean while, it reaches expression peak at after birth 60d in Meishan breeds.14-3-3 theta gene were widely expressed in vary tissue, with higher expression level in skeletal muscle and smooth muscle, but with lower expression level in cardiac muscle.2.1814bp promoter sequence of the 14-3-3 theta gene were cloned and sequenced, then subcloned in to PGL3-basic report gene vector. A series of 5'-deleted constructs were made and luciferase reporter assays were carried out in C2C12 and PK-15 cell lines. The transcription regulation region among-918-669bp which is near the first putative transcription initiation site have the ability to increase promoter activity, while the second putative transcription initiation site concludes to be a real transcription initiation site. And the effect of TSA to promoter activity is not only limited to region which is putatively combined by transcription factor p300, but the whole region near transcription initiation site. 3.14-3-3 theta protein can be rapidly induced during the period range from 0 to 1h in PK-15 cells and porcine satellite cells which were treated with 33nM TSA. And the effect is not coordinate with the result mentioned above.4., C2C12 cell line was applied as a model to illustrate the 14-3-3 theta gene function during myogensis. Accompanied with differentiation procedure of C2C12 cells, the expression of 14-3-3 theta proteinwas decrease dramatically in both TSA treated or matched group with no significant difference.5.675bp cording sequence was obtain, which encoding a protein of 224 amino acids, and subcloned to a FLAG-tag vector prk-flag 3.1, fusion protein were expressed and detected by western blotting in different cell lines...