The Isolation, Genetic Effects Analysis Of Pig Prdx2 And Prdx6 Gene

PRDX2 and PRDX6 gene belong to the same superfamily of antioxidant protein.And they play an important role in oxidative stress and free radical scavenging by the elimination of peroxide or superoxide during metabolism. This laboratory filtered out two differential proteins PRDX2 and PRDX6 by differential proteomics when studying the molecular mechanism of the formation of skeletal muscle between the typical lean pigs(Large White)and Chinese indigenous pigs(Meishan). In view of this,the present research chose PRDX2 and PRDX6 as a candidate gene for meat quality of pigs and carred out these research of genetic structure,temporal expression profiling,polymorphisism trait association analysis and so on.We got the following results:1,We obtained 765bp cDNA sequence of porcine PRDX6 gene and predicted the ORF using GENESCAN.The 675bp sequence of cDNA was coding area and included 224 amino acids.In the research of pig DNA database, the full-length cDNA of PRDX6 gene exactly matched with a pig chromosome 9 genomic clones (pig clone CH242-230A16). The full-length DNA of PRDX6 gene was 165kb and included 5 exons.The analysis of PRDX6 gene structure by Spidey procedure and the conservation of cleavage site indifferent species showed that the splicing way of 4 introns of pig PRDX6 gene was in line with"gt-ag"rule.We obtained 611bp cDNA sequence of porcine PRDX2 gene and predicted the ORF using GENESCAN.The 597bp sequence of cDNA was coding area and included 198 amino acids.In the research of pig DNA database, the full-length cDNA of PRDX2 gene exactly matched with a pig chromosome 2 genomic clones(pig clone CH242-275M24). The full-length DNA of PRDX2 gene was 181kb and included 6 exons.The analysis of PRDX2 gene structure by Spidey procedure and the conservation of cleavage site indifferent species showed that the splicing way of 5 introns of pig PRDX2 gene was in line with"gt-ag"rule.2,Quantitative RT-PCR technique of porcine PRDX6 gene in RNA level was carried out to analyze whether there were differences between the developmental stages and cultivars.The results showed that PRDX6 gene of Large White pigs had the highest expression of four months after birth in RNA level, consistent with the results abtained from proteomics; while PRDX2 gene of Large White pigs had the highest expression of 60 days after birth in RNA level, inconsistent with the results abtained from proteomics (PRDX2 protein highly expressed in embryonic 65 days). 3,Quantitative RT-PCR technique of porcine PRDX2 and PRDX6 gene in RNA level were carried out to analyze whether there were differences towards tissue expression. The results showed that PRDX6 gene of Large White pigs had the highest expression in liver and biceps femoris,moderately expression in stomach,relatively low expression in other tissues; while PRDX2 gene of Large White pigs had the highest expression in stomach,moderately expression in liver and kidney,low expression in spleen,relatively low expression in other tissues.4,Two potenial SNPs were found in the fourth exon by comparison of the abtained sequence from different pig breeds Large White and Meishan, one at 417bp (C/T) and the other at 423bp (A/G), both of which did not change the amino acid. Furthermore,genetic variation and trait association were performed in six purebred pig population and two hundred and forty seven F2"Large White×Meishan"resource population by pyrosequencing method,respectively.For C417T, C allele was predominant in Western pig breeds,while T allele was predominant in Chinese indigenous breeds,and this SNP was significantly associated with the Intramuscular fat,Water moisture(P<0.05);for A423G, A allele was dominant in Western pig breeds,while G allele was dominant in Chinese indigenous breeds,and this SNP was significantly associated with Drip water rate,Water holding capacity,Intramuscular fat,Water moisture (P<0.05).5,2000bp of porcine PRDX6 gene 5'flanking sequence was amplified by PCR and predicted by bioinformatics.6,The correlation analysis between the spatio-temporal expression profile of PRDX6 gene and the promoter methylation:(1)The first results showed that PRDX6 gene had the same tendency between protein and mRNA expression in Longissimus Muscle from Large White pig and the opposite tendency between their expression and the promoter methylation.lt was worth to say that PRDX6 gene of Large White pigs had the highest expression of four months after birth, while the lowest methylation of four months after birth.What is more, the negative correlation between the expression of PRDX6 gene of Meishan pigs and the promoter methylation was inconsistent. (2)The second results showed that tissue expression was consistent with promoter methylation besides the brain and kidney.