Molecular Cloning,Characterization And Tissue Expression Analysis Of The Ovine Actin Gamma Genes

The sheep actin gamma family gene consists of actin gamma 1(ACTG1)and actin gamma 2(ACTG2).In the present work,the complete cDNA sequence of the Small-tailed Han sheep was cloned using reverse transcription-polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE).The gene structures and characteristics of the predicted amino acids sequences were analyzed using bioinformatics analysis software.Gene expression analyses were performed using quantitative reverse transcription PCR(qRT-PCR).All of the works are to explore the main structure and the expression characteristics of the genes of Small-tailed Han sheep from the molecular level.The main results were showed as follows:(1)The cDNA sequences of the ACTG1 and ACTG2 genes from Small-tailed Han sheep were cloned and sequenced.The cDNA sequence of ACTG1 is 1960 bp.Compared with the predicted sequence of the NCBI database,the cloned gene sequence was similar to the database.ACTG1 had a mutation site,and there were two mutation sites of ACTG2.(2)Bioinformatics analysis showed that the two protein were weakly acidic,stable in structure and had good hydrophilicity and high mobility.The proteins were predicted to be non-secretory proteins with several phosphorylation sites and N-glycosylation sites and without any transmembrane helices.ACTG1 protein contains isoleucine(7%)than ACTG2(8%),so ACTG1 protein more hydrophilic than ACTG2.The mobility of two protein was relatively large,and there was no transmembrane domain,which contained multiple phosphorylation sites and glycosylation sites.The secondary structural analysis predicted that the proteins were mainly composed of random coils,accounting for 63.2% and 64.9%,respectively.(3)Multiple alignments demonstrated that the amino acid sequence was highly conserved between the investigated species.The phylogenetic tree showed that for the ACTG1 protein,Ovis shared the closest evolutionary relationship with the goats,wild boar,human.For ACTG2 protein,the closest evolutionary relationships were observed with Striped Hamster,moles,flying foxes.(4)The expression level of ACTG1 and ACTG2 gene in Small Tail Han sheep and Dorper sheep was detect using the qRT-PCR test method in multiple groups of mRNA.ACTG1 was highly expressed in the pancreas and stomach tissues,which was significantly higher than that in other tissues(p<0.05),followed by the ovary,spleen and lung,and the expression of the medium and Small Tail Han sheep was significantly higher than that of p<0.05.In general,ACTG1 gene was expressed in almost all tissues.The high expression of ACTG2 in the heart and spleen,significantly higher than other tissues(p<0.05),followed by lung,ovary,the expression in heart tissue was significantly higher than that of Small Tail Han sheep and Dorper sheep(p<0.05)in the ovarian tissue expression of Dorper sheep were significantly higher than small tail Han sheep(p<0.05).(5)The expression levels of two proteins differed among the various tissues of the two sheep species It was found that the molecular weight of two actin was 48 KD,which was similar to the results of bioinformatics analysis.ACTG1 protein was expressed in Small Tail Han sheep and Dorper sheep muscles triceps muscle of arm,except in the intercostal muscle,triceps muscle of arm expression but also in lung tissues.All in all the ACTG2 protein expression in longissimus muscle,intercostal muscle,triceps muscle of arm,heart,lung,stomach,brain and liver tissues in low,no significant difference in varieties.In conclusion,the ACTG1 and ACTG2 genes of the Small Tail Han sheep were cloned successfully,and a systematic study was carried out.Exploring the characteristics and functions of these genes at the molecular level would provide basic theoretical foundation for the subsequent study of the gene function and molecular breeding.To explore the characteristics and functions of these genes at the molecular level,and to provide a theoretical basis for the subsequent study of gene function and molecular breeding.