Studies On Molecular Cloning,expression And Polymorphism Of Apolipoprotein A5 Gene In Pig

Fat traits are important economic traits of pig,which greatly affect meat quality traits.The results showed that apolipoprotein A5 resides on VLDL,HDL and CM particles.Apo A5 gene plays an important roles in lipid metabolism,such as the transport of lipids,conjugation with lipids,stability of lipoprotein structure,regulation of the key enzyme activity of lipoprotein metabolism and identification of lipoprotein receptors.1.The full-length c DNA of Apo A5 gene was cloned from Mashen pig liver by methods of reverse transcription PCR and rapid amplification of c DNA ends,which was initial code of ATG and a termination code TAA.The obtained full-length c DNA of 1917 bp contained a 1092 bp open reading frame,a 24 bp untranslational region(UTR)at 5? end,an 801 bp UTR at 3? end that includes three signal sequences of AATAAA for adding the poly-A tail.The ORF encodes 364 amino acids and predicted protein size was 41.15 k Da.The sequence data has been submitted to Gen Bank(Accession number: HM991258).The cloning of Apo A5 gene provides the basis for the further studies on functions of Apo A5 in pig.2.The Apo A5 protein was predicted and analyzed on aspects of their basic properties,basic properties,structures and function by bioinformatics methods.The phylogenetic tree based on Apo A5 amino acids sequences of various species was constructed with neighbor-joining(NJ)method.The Apo A5 amino acids sequence of Mashen pig was analyzed the identity with those of other mammals.The results showed that: The Apo A5 was not a trans-membrane protein.The phosphorylation sites were predicted at Ser and Thr,and O-glycosylation site was not found in amino acids encoded Apo A5.There are 98.07% ?-helix and 1.93% random coil in the probable secondary structure of Apo A5.The homology-modeling was constructed with the human Apo A5 model,and three-dimensional structure of pig Apo A5 was predicted.Pig Apo A5 encoded 364 amino acid residues,which shared 81.7%?80.0%?78.3%?77.5%?76.5%?73.6%?67.1% and 66.7% identity with those of Bos taurus,Equus caballus,Homo sapiens,Canis familiaris,Macaca mulatta,Oryctolagus cuniculus,Rattus novegicus and Mus musculus,respectively.Molecular cloning and bioinformatics analysis of Apo A5 of pig will be helpful for the further analysis the functions of Apo A5 protein.3.The Apo A5 m RNA tissue distribution and developmental expression profiles of Mashen pig and Yorkshire pig at the age of 60,90,120,150,and 180 days,were detected by quantitative real-time PCR method.Results showed that Apo A5 gene expressed in liver,subcutaneous adipose,longissimus dorsi,small intestine,kidney,pancreas,heart and stomach,and the expression level of Apo A5 was significantly different in various tissues.It indicated that the expression of Apo A5 m RNA was influenced by tissue.The expression levels of Apo A5 m RNA in liver of Mashen and Yorkshire pig all peaked at the age of 180 days,but the developmental expression patterns at the age of 60,90,120 and 120 days were significantly different between two breeds.The expression of Apo A5 in liver of Yorkshire at the age of 60,90 and 120 days were significantly higher than that of Mashen pig(P<0.01).The expression patterns of Apo A5 m RNA in longissimus dorsi tissue differed between breeds.The Apo A5 expression in longissimus dorsi of Yorkshire increased gradually,peaked at the age of 180 days and the expression levels among different months were significantly different(P<0.01).But Apo A5 expression of Mashen pig in longissimus dorsi increased first and then decreased.The peak of expression occurred earlier in Mashen and peaked at the age of 180 days.The Apo A5 expression in subcutaneous adipose of Yorkshire decreased gradually,and the expression level peaked earlier at the age of 60 days then declined.The expression level of Mashen pig peaked at the age of 60,120 and 180 days and the highest expression abundance were 180 days.The expression of Apo A5 m RNA in subcutaneous adipose of Yorkshire was lower than that of Mashen at whole days of age,respectively(P<0.01).These results indicated that Apo A5 gene have potential association with regulatory mechanisms of lipid metabolism and effects on meat production traits in pigs.4.The Apo A5 CDS was cloned,based on a pair of specific primers,and inserted into the expression vector p ET32a+.After confirmation by the sequencing and restriction enzyme analysis,the recombinant plasmid was transformed into E.coli BL21.SDS-PAGE results showed that the recombinant protein was expressed and then the expression level reached the highest level after induced for four hours.Western blotting analysis indicated that the molecular weight of expressed product was the same as predicted size of approximately 46 k Da,and the rabbit anti-Apo A5 antibody specifically conjugated expression product,testing to higher efficiency of expressed plasmid of p ET32a-Apo A5.These data provided the important information for further studying the physiological functions of pig Apo A5 gene.5.The Apo A5 protein expression profiles were detected in different tissues of Mashen pig and Yorkshire pig by Western blot.Results showed that the expression level of Apo A5 m RNA was higher in liver,subcutaneous adipose and small intestine,medium in longissimus dorsi,heart and pancreas,and lower in other tissues.The expression level of Apo A5 was highest in liver,and the expression of Apo A5 in liver of Yorkshire and Mashen pig was significantly higher than other tissues.Moreover,the expression patterns of Apo A5 m RNA in liver differed between breeds.The expression levels of Apo A5 protein in liver of Mashen pig decreased gradually,and the expression peaked at the age of 60 days in Mashen pig,then declined.However,the expression peaked at the age of 90 days in Yorkshire and then declined.The expression of Apo A5 protein in liver of Yorkshire at 90,120,150 and 180 days were significantly higher than that of Mashen pig(P<0.01).These results indicated that Apo A5 gene have potential association with regulatory mechanisms of lipid metabolism in different economic type pigs.6.The polymorphisms of Apo A5 gene were detected by PCR-SSCP in eight population,including of Shanxi White,Mashen pig,Shanxi Black,Duroc,Large White and Landrace,and the associations between polymorphisms and growth traits of Shanxi White pig were analyzed by simple univariate animal model.The results showed that the polymorphisms at the site of 5'-UTR?Intron 2 and Exon/3'-UTR and eleven haplotypes were found,and the polymorphism site exhibited significant generation effect and sex effect on body weight and size traits and backfat thickness.The current study showed an association between the polymorphism of Apo A5 gene and growth traits in Shanxi White pigs.In conclusion,we systematically studied the Apo A5 gene of pig at different levels.These results provided a theoretical foundation for further investigation on the biological function of Apo A5 and the regulation mechanism Apo A5 gene in lipid metabolism.