Function Analysis And Application Of Porcine Skeletal Muscle/liver-specific Gene MyoG/α1-AT Promoter

Promoters are gene fragments that can control transcription.Tissue-specific promoter is the promoter of gene expressed in specific tissues and organs.Tissue-specific promoters are extremely valuable in many applications such as genetics breeding,traits improvement,and so on.This paper used the expression of porcine muscle-specific genes MyoG drive the peroxisome proliferator-activated receptor gene ectopic expression of in muscle which has closely related to lipid metabolism to enhance intramuscular fat content.And,our study adopt ectopic expression of Saccharomycopsis fibuligera’s β-glucosidase gene which drive from the pork liver-specific gene promoter α1-AT and α1-AT signal peptide in liver.This study provide some theoretical basis for the construction of transgenic mouse model.The results are as follows:1.Used online software MEME to compare the same area of pigs、mice and human,found many conservative motifs in these sequences.So,we cloned the 5’-flanking sequence 806bp of the MyoG gene.Furthermore,the software of TESS and TFSEARCH analysis showed that the sequence contain the promoter element and many transcription factor binding sites,such as TATA box、Spl、E-box、C/EBP、NF-1、Myogenin and so on.We constructed pGL3-MyoG reported vector,transfer to C2C12 differentiating d 0、2、3、4、6 and 3T3-L1 cells.We found that the promoter activity of MyoG reached maximum on third day in differentiation of C2C12 cells,with the extension of the differentiate time,the active gradually reduce.In 3T3-L1 cells,the activity of MyoG promoter had no significant difference with the negative control.Combined transfections of MyoD and pGL3-MyoG or pGL3-MyoG mutation vectors to C2C12 cells cultures in differentiating medium.We found that MyoD could increase the promoter transcriptional activity,and it is verified by EMSA.2.Construct the recombination vector contained MyoG promoter and porcine PPARy gene.The technology of QPCR and western blot detected the PPARy relative expression.The results show that the expression level of PPARy increase about 2 times compare the negative control.In order to building transgenic mouse,make pGL3-MyoG-PPARy linearize.The linear segment in MyoG promoter for regulatory,with the PPARy CDS and Flag label for the expressing region and retaine the component of the poly(A)coming from the pGL3-Basic.Transgenic animals were generated by micro-injection of the resulting expression cassette into fertilized mouse eggs.Four male mice carried the transgene verified by pPCR.3.Contruct the vector contained MyoG promoter and porcine CKM enhancer,transfer to C2C12 cultures in differentiating medium and 3T3-L1cells.The results show that CKM enhancer can significantly improve the activity of MyoG promoter.In 3T3-L1 cells,the activity of chimeric promoter had no significant difference with the negative control.It caused by tissue specificity of the promoter.The vector could be used to solve the problem in the process of transgene requires high efficiency and specificity.4.Analysis the upstream regulation sequence of α1-AT,online software o analysis showed that the sequence contain the promoter element and many transcription factor binding sites,such as Sp1、Ap1、HNF-3b、NF-Kap and so on.According the result of prediction,we cloned the 5’-flanking sequence 1661bp of al-AT successfully.Construct the pGL3-AT dual luciferase report plasmid,transfer to Hepal-6、3T3-L1 and PK15 cells.The results show that the deletion fragment 2 not only has the highest activity but retain the tissue specificity.So we regard the deletion fragment 2 as the α1-AT promoter with liver-specific and the activity.5.Construct the recombination vector contained α1-AT promoter deletion fragment 2 and BGL1 gene.QPCR and western blot detected the BGL1 relative expression in Hepal-6.The results show that the expression level of BGL1 protein under the driving of α1-AT promoter had significant difference with negative control in Hepal-6.