Cloning and expression differential analysis of different subtypes of ACOX1 gene in Luchuan pigs

【Objective】 This study was aimed to clone different subtypes of acyl-coenzyme A oxidase 1 （Acyl-CoA oxidase 1， ACOX1）gene in Luchuan pig and conduct bioinformatics analysis， analyze the expression of ACOX1 gene and its subtypes in longissimus dorsi muscle of different breeds of pigs. So as to provide reference for further exploring the functional roles of ACOX1 gene in Luchuan pigs.【Method】 Based on pig ACOX1 gene sequence published in GenBank（NM_ 001101028.1）and the coding region（CDS）sequence of the Luchuan pig ACOX1 gene， the specific primers and validation primers were designed. Total RNA was extracted from the liver of Luchuan pig aged 0 month and the longissimus dorsi muscle of Luchuan pig， Duroc pig， Dulu pig and Ludu pig aged 8 months by TRIzol method，reverse transcription cDNA was synthesized，using 0-month-old Luchuan pig liver cDNA as template for cloning and validation，to obtain different subtypes of ACOX1 gene sequences of Luchuan pig，and bioinformatic analysis was performed using various online tools，and real-time fluorescent quantitative PCR was used to detect the expression of different subtypes of ACOX1 gene in longissimus dorsi muscle tissue.【Result】There were two subtypes of ACOX1 gene in Luchuan pigs（ACOX1-Ⅰ and ACOX1-Ⅱ）， the GenBank accession numbers were OQ701687 and OQ701688，the full length sequence of ACOX1 gene CDS in Luchuan pigs were 1986 bp， encoding 661 amino acids residues， the two isomers were mainly selective cleavage between sequences between positions 270 to 429. The molecular weight of Luchuan pig ACOX1-Ⅰ encoded protein was 74786.50 Da， and its theoretical isoelectric point（pI）was 7.36，the molecular weight of ACOX1-Ⅱ encoded protein was 74670.49 Da， and pI was 8.17. The secondary structure of ACOX1-Ⅰ protein In Luchuan pig was composed of α-helix （47.96%）， β-turn（10.14%）， extended chain（14.52%） and random coil（27.38%）. The secondary structure of ACOX1-Ⅱ protein in Luchuan pig was composed of α-helix（47.66%）， β-turn（10.89%），extended chain（14.37%）and random coil （27.08%） . The ACOX1 protein of Luchuan pig had no transmembrane domain. The expression of ACOX1-Ⅰ gene in the longissimus dorsi muscle of Luchuan pigs was the highest， that of Duroc pig was the lowest，but the difference was not significant among the four breeds（P>0.05） . The expression of ACOX1-Ⅱ gene in the longissimus dorsi muscle of Luchuan pig and Duroc pig were significantly higher than Dulu pig and Ludu pig（P<0.05） .【Conclusion】 There are two subtypes of ACOX1 gene（ACOX1-Ⅰ and ACOX1-Ⅱ）in Luchuan pigs，and the expression of ACOX1 gene and its subtypes is greatly different in different breeds of pigs. The results can provide reference for elucidating the regulation mechanism of ACOX1 gene in Luchuan pig fat deposition and metabolism.