Preliminaryly Functional Study Of Porcine ACTN3 Gene

Recently, our lab has identified lots of differentially expressed genes in pig by means of suppression subtractive hybridization, mRNA differential expression, gene chip, and proteomic techniques; however, needs further confirmation. ACTN3 gene is identified as the differentially expressed genes in the Longissimus Muscle from Meishan pigs of different ages. This research aimed at the further function of ACTN3 and got the following results:1. Whole cDNA sequence of ACTN3 was obtained, with the length of 2875bp. ACTN3 contains 21 exons, coding for 902 amino acids. Also, part of the genomic sequence of ACTN3 was obtained in our experiment. All exons and introns agree the GT-AG rule.2. Bioinformatics methods was used to analyze the structures of ACTN3 gene and ACTN3 protein, based on which, phyligenetic tree was constructed.3. Semi-quantitative RT-PCR method was used to analyze the tissue expression profile of ACTN3. The result showed that ACTN3 gene was comparatively highly expressed in fat, longissimus muscle, biceps femoris, semitendinosus tendon, masseter muscle; lowly expressed in lung and liver. No expression was detected in other tissues (heart,spleen,kidney,stomach and didymus)4. Real-time PCR methods was applied to analyze the spatio-temporal expression profiles of ACTN3 gene in Longissimus Muscle from Large white pig and Meishan pig, the results showed that ACTN3 gene was expressed in the Longissimus Muscle of day 3,21,60,120 and 180 post-born of either Large white and Meishan pigs. From day 3 to day 60 post-born, the expression of ACTN3 gene was increasing steadily until reached to a stable level in the longissimus muscle of Large white, then maintained this stable level till day 180 post-born. The expression styles of ACTN3 gene were significantly different between3 and21 days in tissues of Meishan pigs while less significantly different in that of Large white pigs with different ages.5. Gene frequencies and genotype frequencies of two SNPs from ACTN3 gene were analyzed, and association analysis was done in Large white×Meishan F2 resources family. The results showed that, in regard of meat quality, exon11 G418A (analyzed by PCR-TspEⅠ-RFLP) was significantly associated with water holding capacity, marbling of biceps femoris and water moisture(m. longissimus Dorsi,LD) (P<0.05), intron19 A512G (analyzed by PCR-SacⅡ-RFLP) was significantly associated with Meat pH (m. Biceps Femoris, BF) and Meat pH (m.Semispinalis Capitis, SC) (P<0.05). As in carcass quality, G418A was highly significantly associated with Fat percentage, Dressing percentage,6-7Rib Fat Thickness, Thorax-Waist Fat Thickness (P<0.01), and was significantly associated with Average Back Fat Thickness and Ratio of Lean Meat versus Fat Meat (P<0.05). A512G was highly significantly associated with Dressing percentage,6-7 Rib Fat Thickness and Average Back Fat Thickness (P<0.01), and was significantly associated with Thorax-Waist Fat Thickness, Ratio of Lean Meat versus Fat Meat, Loin Eye Height (P<0.05).6. Amplified a 1724 bp 5'UTR sequence of ACTN3 gene. Predictions of core promoter region, translation start point, distribution of CpG islands, and TF binding sites were achieved by bioinformatics methods.7. Based on the bioinformatics analysis of promoter,8 recombinant expression vectors containing the predictive promoter regions of ACTN3 gene were constructed successfully by using differentially deleted 5'UTR. Transfection and detection was done in PK15 cell line and by dual-luciferase reporting system. The results showed that the difference between the activity of all the 8 vectors and that of control was significant (P<0.05), indicating that all the sequences contained in these vectors have some promoter activity. The promoter activity decreases from pGL-ACTN120 to pGL-ACTN212, from pGL-ACTN395 to pGL-ACTN696, and from pGL-ACTN1077 to pGL-ACTN1410, indicating that negative regulation sites exist in these regions. The promoter activity increases from pGL-ACTN696 to pGL-ACTN1077 and reaches the highest level, indicating that positive regulation sites exist in this region.