Cloning And Activity Analysis Of Pig Microphthalmia-associtated Transcription Factor (MITF) Gene Promoter

Using gene cloning, sequencing, dual luciferase report system and RNA interference, this experiment studied the promoter regions of pig MITF-M gene. And the results are as follows:1, We got the scarffords containing the MITF gene from the Tibetan pig genome. And we designed primers using scarffords as reference sequence. Then we used three pairs of overlapping primers to clone about 7.8 kb transcription regulatory regions of pigs MITF-M with using PCR method. This is the first time that we obtain the transcription regulatory regions of pigs MITF-M.2, According to the sequence of MITF-M regulatory regions, we designed primers to clone the promoter fragment using the "nested" PCR. With enzyme digestion, connection, transformation, validation, we successfully built ten recombined vectors:PGL3-1397, PGL3-1140, PGL3-2470, PGL3-3487, PGL3-3487, PGL3-5201, PGL3-5429, PGL3-6036, PGL3-6274, PGL3-6274.3, We transfected ten recombined vectors into B16 cells. After 48h, Using double fluorescein report system, we detected and compared the luciferase activity of the different transcription regulatory regions in B16 cells. Two important core regions of MITF-M regulatory region was found, located in negative regulatory region:-2470～-3487 bp and positive regulatory region:-5429～-6036 bp.4, According to the transcription factor prediction software JASPAR, interference RNA of KLF4, the results were found that when the expression of KLF4 was reduced, PGL3-2470 luciferase activity fall down significantly, while pGL-3487 rose up significantly. And KLF4 influenced MITF-M transcription activity.This research laid the foundation to further study the pig MITF gene expression mechanism, and was of great significance to explore the transcription activity of pig MITF-M and the mechanism of expression regulation.