Cloning Of Alternative Splicing Variants Of MEF2A And Reseaching About Their Spatio-temporal Expression In Pig

Myocyte Enhancer Factor 2(MEF2)belong to the MADS-Box family of transcription factors.There are four vertebrate MEF2 genes,MEF2A,MEF2B,MEF2C and MEF2D,which are expressed in distinct,but overlapping patterns during embryogenesis and adult tissues.Different types of MEF2 protein will be formed because of the different alternative splicing,MEF2A mainly promotes the formation of slow-twitch(type 1)muscle fibers in adlut pig.The aim of this study was to clone the alternative splicing variants of MEF2A in pigs,and to analyze the tissue distribution and developmental expression patterns of different isoforms.In this study,the Large white and Mashen pigs as experimental animal,according to research group RNA-seq sequencing results,using RT-PCR technology combined with the method of TA cloning and sequencing,identificating the alternative splicing of MEF2A in pigs muscle tissue of different period.We analysed the biological characteristics of splicing variants of the MEF2A by bioinformatic methods,qRT-PCR was used to detect the relative expression of the MEF2A gene in 13 tissues of the 90 days pigs,and detected their expression pattern in the psoas major tissue,biceps femoral tissue and longissimus dorsi tissue of pigs(1,90,and 180 days).The main results are follows:1.We successfully obtained 4 splicing variants of MEF2A for the first time,named as MEF2A1,MEF2A2,MEF2A3 and MEF2A4(GenBank accession number:KY313741,KY305402,KY313742,KY313743).The 5th to 8th exon of the MEF2A1 spliced normally.MEF2A2 lacked the 5th exon,and the 5 ' end of the 6th exon was 138nt longer,the 3' end of the 7th exon was 102nt longer.MEF2A3 lacked the 5 th exon,and the 5' end of the 6th exon was 138nt longer.The 3' end of the 7th exon of MEF2A4 was 102nt longer.The nucleotide sequence homology was 99.6%of MEF2A1 and MEF2A1 of pig submitted in GenBank(GenBank accession number:EF576922.1).The nucleotide sequence homology was 99.4%of MEF2A2 and MEF2A2 of pig submitted in GenBank(GenBank accession number:EF576923.1).The results showed that the MEF2A3 and MEF2A4 obtained in this experiment were the newly discovered MEF2A variants.2.The results of bioinformatics analysis showed that the potential phosphorylation sites were predicted in MEF2A1,MEF2A2,MEF2A3 and MEF2A4,indicating that the activity of MEF2A gene was regulated by the phosphorylation of multiple sites.But no signal peptide sequence were detected,indicating that MEF2A is a secreted protein.Subcellular localization results showed that the 4 splicing variants of MEF2A were mainly localized in the nucleus and also found in the cytoplasm,consistently with previous research results.Amino acid domain detection results show that MEF2A1 and MEF2A4 contain a conserved domain HJURP_C,MEF2A2 and MEF2A3 contain two conserved domains,HJURP_C and Med13_N.Amino acid homology and phylogenetic tree resulted showed that the 4 splicing variants of MEF2A belong to two subgroups,MEF2A1,MEF2A4 and MEF2A1 of pig submitted in GenBank belonged to a subgroup,amino acid homology up to 98.9%.The MEF2A2,MEF2A3 and MEF2A2 of pig submitted in GenBank belonged to a subgroup,the amino acid homology was 98.2%and 98.9%respectively.The 4 splicing variants of MEF2A were not in the same group,but the genetic distance was farthest between them and cattle and sheep.3.The results of qRT-PCR showed that the 4 alternative splicing variants of MEF2A were expressed in 13 tissues of Large white pig and Mashen pig.MEF2A1 is mainly expressed in the the biceps femoris of Large white pig and the small intestine of Mashen pig;MEF2A2 is mainly expressed in the myocardium of Large white pig and the small intestine of Mashen pig;MEF2A3 is mainly expressed in the psoas major of Large white pig and the longissimus dorsi tissues of Mashen pig;MEF2A4 is mainly expressed in the liver of Large white pig and the pancreas of Mashen pig.4.To detect the developmental expression of 4 alternative splicing variants of MEF2A in the psoas major,biceps femoris and the longissimus dorsi tissues,the results of this study found that the MEF2A1,MEF2A2 and MEF2A3 had the highest expression in the psoas major at 180 days of Large white pigs,MEF2A1 and MEF2A2 had the highest expression in biceps femoris and longissimus dorsi tissues at the 90 days of Large white pigs,it may be that the longissimus dorsi and biceps femoris tissues are mainly composed of glycolytic fibers,and the proportion of oxidized muscle fibers decreased with the increasing of day,caused the age of the highest expression of MEF2A gene in advance.Comparison of two breed,the expression levels of MEF2A4 in the psoas major,biceps femoris and the longissimus dorsi tissues at one-day in Mashen pig,was significantly lower than that of the large white pig(P<0.05),because the growth of speed is slower than large white pig.The expression of MEF2A2 in biceps femoris and longissimus dorsi tissues of Mashen pig in 180 day old,was most significantly higher than that of large white pig(P<0.01).The expression of MEF2A3 in bicps femoris and longissimus dorsi tissues of Mashen pig in 90 day old,as compared with the Large white pig,the muscle fiber diameter of Mashen pig was thinner,unit area density was biger,and the content of type I muscle fibers are higher.In conclusion,this study has obtained the 4 alternative splicing variants of MEF2A gene for the first time and reseaching about their spatio-temporal expression in pig,laid the foundation for further study of MEF2A gene function.