| With the improvement of living standard and the change of consumption concept,people have higher demands on the quality of pork.In the process of production,oxidative stress is an important reason for producing inferior meat such as PSE.There was significant difference in oxidation resistance between individual muscle tissues of different pigs,and the native pig breeds and the imported pigs also had different anti-stress abilities.Nrf2(nuclear factor erythroid 2-related factor)is an important transcription regulatory factor that regulate the balance of oxidative and antioxidant levels,and plays an important role to maintain the body's normal physiological activities.Nrf2 has become the important regulatory factors in antioxidant stress.In order to understand the differences in the antioxidant capacity of skeletal muscle between different pig breeds,further understand the structure and function of Nrf2 gene,and to reveal the regulation mechanism of Nrf2 gene transcription,we made this research:1.SOD activity,MDA level and T-AOC in muscle of six pig breeds were measured.The difference of SOD activity and total antioxidant capacity(T-AOC)between Dapulian pig and Yantai black pig is not obvious(P > 0.05).In Dapulian pig,Licha black pig,Yimeng black pig and DLY(Duroc * Landrace * Yorkshire),the significant difference of content of malondialdehyde(MDA)was found.2.The result showed that the full sequence of Nrf2 cDNA is 2358 bp,containing 87 bp sequence of 5'UTR and 495 bp sequence of 3'UTR,and coding region(CDS,1776 bp)encodes 591 amino acids.The prediction of structure and function was analyzed by bioinformatics technology.The isoelectric point(pI)of the protein is 4.66,and the molecular weight is 66.4027 kDa.This sequence didn't have a transmembrane region.The secondary structure was mainly consisted of alpha helix,and Nrf2 was not secreted protein.Compared with other sequences,nucleotide sequence and amino acid sequence of Nrf2 in Dapulain pigdetected the highest homologous between swine and human.3.Nrf2 mRNA widely exists in the tissues and cells,which was detected in muscle,heart,liver,spleen,lung and kidney,and significant difference expression was found between kidney and heart(P<0.05).The high expression levels of Nrf2 mRNA were detected in kidney,muscle and lung,but the lowest expression levels were observed in heart.In muscle,Dapulian pig Nrf2 mRNA expression level was significantly higher than that of Landrace(P<0.05).This result indicated that the kidney plays an important role in the antioxidant stress of pigs,and revealed the muscle,kidney and lung perform important functions in the antioxidant stress of Dapulian pigs.4.2091 bp 5 'flanking fragment of Nrf2 gene was cloned by Genome Walking technique.Nrf2 gene 5'flanking sequence that was predicted contains typical TATA-box,excluding the CpG island.A series of different lengths of promoter fragments were built by nested PCR and inserted into pGL3-Basic vectors.The recombinant vectors and pGL4.74 were transfected into 293 T cells,and the promoter activities were measured by the dual-luciferase assay system after 48 hours.The results demonstrated that the activity of the region from-785 bp to-710 bp in Nrf2 gene promoter was higher than other regions,and there was no significant difference between the region from-785 bp to-710 bp and the region from-903 bp to-785 bp.This result indicated that there was a positive regulation zone or enhancer in the region from-903 bp to-710 bp.In addition,potential transcription factor binding sites which may be involved in the transcriptional regulation of Nrf2 gene were found in this region. |
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