Transformation And Functional Analysis Of Bovine Desmin Gene Promoter

Cultivating the high yield and high quality livestock is always the goal of animal husbandry production.The disadvantage of traditional hybridization breeding methods is low efficiency and long cycle.Transgenic technology can produce new varieties of livestock with high quality traits in a relatively short period,which has extensive application prospect.The efficient tissue-specific promoter could activate expression of foreign genes in the skeletal muscle satellite cells and promote muscle yield of transgenic livestock.Desmin is an important fibrous protein of medium size bone in muscle tissue,it can paly an important role in forming and maintaining of muscle shape,transmiting information between cells,differentiation and regulation of muscle cells,and so on.Desmin gene promoter can guide the specific expression of foreign genes,but as a promoter of livestock production,its transcriptional activity is not enough.So to recombine the fragment of the desmin promoter through molecular cloning technology.We hope to screening promoters with higher activity and muscle tissue-specific.The sequence with multiple positive regulatory elements of bovine desmin300 gene promoter was cloned by PCR.It was inserted into the upstream of promoter to construct the recombinant plasmid of multiple positive regulatory elements;Let the sequence of partial regulatory elements of the Myo G gene promoter and the ?-actin gene promote to inserte into desmin300 promoter,Constructing recombination plamid of two copies of cis-acting elements;Connect multiple positive regulatory elements with desmin300 promoter consecutively,to Construct recombination plamid of multiple copies of cis-acting elements.Dual luciferase reporter gene plasmids were constructed,and transfected into bovine muscle satellite cells and fetal fibroblast cells to test the muscle specificity and activity of the promoters of the bovine desmin.The main results were as follows:1.Based on bioinformatics analysis revealed that the transcriptional regulatory site bovine desmin gene promoter containing different fragments : the 300 bp containing three CTCF,one CPBP,one Myo D,two EGRF,one AP2,one Pax3,one TFIIB,one Sp1.2.The bovine skeletal muscle desmin gene promoters were recombined by PCR site-specific mutagenesis of the CTCF regulatory elements,to construct recombination plamid p GL3-desmin300-CTCF.Through dual luciferase reporter gene to test the activity of the promoter,we find,the activity of p GL3-desmin300 wild type promoter exceed mutation promoter after site-specific mutagenesis of the CTCF.Indicating that CTCF regulatory elements acted as positive cis-acting element.3.At the same time,we let foreign gene p My-CTCF-GFP to have a transient cotransfection with wild type promoter p GL3-desmin300 and site-specific mutagenesis promoter p GL3-desmin300-CTCF.The results show that,the activity of mutagenesis promoter and it cotransfection with foreign gene is similar.The activity of wild type promoter cotransfection with foreign gene exceed itself.This suggests that,CTCF element combined with regulating-site of transcription factors in regulatory area of desmin gene promoter,and it has a positive control function on the transcription.4.This study,we let the sequence of partial regulatory elements of the Myo G gene promoter and the ?-actin gene promote to inserte into desmin promoter.Constructing recombinational plamid p GL3-MD-?D(1)-desmin300 and p GL3-?D(2)-?D(1)-desmin300.The results showed that the activities of p GL3-MD-?D(1)-desmin300 and p GL3-?D(2)-?D(1)-desmin300 were 2.56 and 2.88 times more than p GL3-desmin300,respectively.This proved that the artificial promoter has a higher activity and better muscle specificity.5.We again to inserte the DOUBLE sequence with multiple positive regulatory elements was cloned by PCR into the before the recombinational promoter.The results showed that,the activity of recombinational promoter will be further strengthened and keep higher muscle specificity meanwhile.The activities of p GL3-MD-?D(1)-desmin300-DOUBLE3,p GL3-?D(2)-?D(1)-desmin300-DOUBLE3,p GL3-MD-?D(1)-desmin300-DOUBLE2 and p GL3-?D(2)-?D(1)-desmin300-DOUBLE2 were 3.49,3.68,4.1 and 4.39 times more than wild type promoter p GL3-desmin300.This study to provide appropriate muscle specific promoters for relatedtransgenic livestock study in the future.