Guanling Cattle MyoD1 Promoter Functional Characterization And Transcription Factor Binding Sites Screening

This experiment used Guanling cattle,the local bovine species in Guizhou as experimental animals to research the promoter region of MyoD1 gene.Main content includes:According to the sequence of Bovine MyoD1 gene published in GenBank,the primers were designed to amplify the promoter region of Guanling cattle MyoD1 gene by PCR.Then used the transcription factor screening kit to screen out the transcription factor binding sites on the promoter region of Guanling cattle MyoD1 gene.According to the screening result,this experiment cloned the CDS regions of Guangling Cattle MyoD family and constructed recombinant cloning vector pcDNA3.1(+)-MyoD,pcDNA3.1(+)-MyoG,pcDNA3.1(+)-Myf5,pcDNA3.1(+)-Myf6,pGL3-P1,pGL3-P2,pGL3-P1-MD,pGL3-P2-MD,pGL3-P1-MEF1 and pGL3-P2-MEF1.This experiment transfected mice C2C12 cells by recombinant plasmid and tested dual luciferase activity after 30 hours.The experiment analyzed the effect of MyoD1 gene promoter activity by MyoD family and some transcriptional regulatory elements.The results of the study are as follows:(1)The MyoD1 promoter of Guanling cattle has MyoD,TFIID,Pax3,MEF1,VDRand MEF2 transcription factor binding sites.(2)This experiment cloned the CDS areas of Guanling cattle MyoD gene family and constructed recombinant plasmides pcDNA3.1(+)-MyoD,pcDNA3.1(+)-MyoG,pcDNA3.1(+)-Myf5,pcDNA3.1(+)-Myf6,pGL3-P1,pGL3-P2,pGL3-P1-MD,pGL3-P2-MD,pGL3-P1-MEF1 and pGL3-P2-MEF1 successfully.(3)The ransfection results of recombinant plasmid showed that exogenous overexpression transcription factors MyoD,Myf5 or Myf6 could significantly improve the transcription activities of Guanling cattle MyoD1 gene promoter P1(P<0.05).But exogenous overexpression transcription MyoD family could not significantly improve the transcription activities of Guanling cattle MyoD1 gene core promoter P2.(4)Point mutation experiments showed that the mutation of MyoD transcription factor binding site and MEF1 element of Guanling cattle MyoD1 gene core promoter could not significantly effect the transcription activity of Guanling cattle MyoD1 gene promoter P1.But the luciferase activity of restructuring mutation plasmid pGL3-P2-MD and pGL3-P2-MEF1 in mice C2C12 cells were much higher than wild type pGL3-P2.The mutations of MyoD transcription factor binding sites and MEF1 element on pGL3-P2 could make its promoter activity raised significantly(P<0.01).The two point played negative regulatory roles to Guanling cattle MyoD1 gene core promoter region.