Transformation And Function Analysis Ofbovine α-actin Gene Promoter

Cultivating the high yield and high quality beef 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 beef 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 beef. Skeletal muscle α-actin encoded by the α-actin gene is the principal actin in skeletal muscle. We aimed to study the bovine α-actin gene promoter, to recombine the fragment of the promoter through molecular cloning technology. We hope to screening promoters with higher activity and muscle tissue-specific, in order to provide appropriate muscle specific promoters for relatedtransgenic cattle study in the future.The bovine skeletal muscle α-actin gene promoters were recombined by PCR site-specific mutagenesis of the regulatory elements, ligating introns to the upstream and downstream of the promoter, connecting Myo G gene promoter to construct dual promoter,adding positive cis-acting elements copies. 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. At last, the reformed promoters with higher activity were chosen to exam the its effection to the expression of the bovine skeletal muscle α-actin. The main contents and results are as follows:1. PCR site-directed mutagenesis was used to study the function of specific nucleotide sequence of the α-actin promoter according to the bovine skeletal muscle α-actin gene promoter. The Sp1/KLFs on the 389 bp was mutated and its mutation promoter activity increased, indicating that Sp1/KLFs acted as negative cis-acting element. The ZF5 F on the 430 bp was mutated and its mutation promoter activity decreased, indicating that ZF5 F acted as positive cis-acting element. The Pax3 on the 490 bp was mutated and its mutation promoter activity decreased, indicating that Pax3 acted as positive cis-acting element.2. The first and second intron of bovine α-actin gene was ligated to the upstream and downstream of p GL3-α-actinp262 respectively according to the published sequence of Gen Bank. The results revealed that introns located to the upstream of promoter increased the promoter activity and the introns located to the downstream of promoter decreased the promoter activity. This showed that the intron could affect the activity of α-actin promoter with its position.3. The Myo G gene promoter was ligated to the upstream of α-actin promoter to construct the dual promoter vector p GL3-Myo Gp373-α-actinp262. The results revealed that the activity of dual promoter vector increased compared to the single promoter of Myo G and α-actin gene. The dual promoter kept higher muscle specificity meanwhile.4. The sequence with multiple positive regulatory elements of bovine α-actin gene promoter was cloned by PCR, its length is 170 bp. It was inserted into the upstream of promoter to construct the recombinant plasmid containing two or three copies of cis-acting elements. The artificial promoters of bovine skeletal muscle α-actin gene were constructed through addition of Myo G gene promoter cis-acting elements. The activity of these artificial promoters increased significantly and kept higher muscle specificity meanwhile.5. The bovine α-actin gene from the start codon and stop codon was cloned according to the sequence published on Gen Bank. The recombinant plasmid containing multiple copies of cis-acting elements was linked to its upstream to test the expression of α-actin gene by RT-PCR, Western Blot, immunofluorescence and microfilament analysis. It confirmed that the recombinant promoters could increased the expression of α-actin significantly.