Distribution Of Chicken Selenoprotein N In Vivo And Selenoprotein N Gene Regulation By Selenium In Muscle Tissues

Selenium(Se) is an essential trace element that is critical to the normal physiology of a wide range of species, including humans. Se deficiency can to be correlated with the incidence of Keshan desease and Kaschin-Beck disease in human and white muscle disease of livertock.Selenoprotein is the main form of Se. Se exerts its biological effects mainly through its incorporation into selenoprotein. Levels of Se intake are associated with the change of selenoproteins and involved in the regulation of selenoprotein. SelN is the first selenoprotein shown to be responsible for a genetic myopathy. Mutations in the SelN gene cause a group of neuromuscular disorders now referred to as SEPN1-related myopathy. SelN is present at a high level in several human fetal tissues and at a lower level in adult ones. Its high expression in cultured myoblasts is also down-regulated in differentiating myotubes, which suggest SelN play an important role in early development and in cell proliferation or regenesis.At present, the studies about SelN are mainly carried out in mammalian, especially genetic myopathy of human. But researches about tissue distribution, gene expression and regulation of SelN are seldom carried out in poultry. In this study, Coding sequence of SelN gene was cloned by the methods of molecular biology. SelN was expressed in prokaryotic system by genetic engineering technology. The polyclonal antibody was prepared using expressed fusion protein. The distribution of SelN in vivo was detected by the applications of RT-PCR and Western blot.The expression levels of SelN, SecS, SPS1 mRNA and Myf5, Pax7, MEF2A mRNA were determined by real-time PCR, when chicken, chick embryo and myoblasts were supplied with Se. The main results were showed as follows:1. Fusion proteins of SelN were expressed in prokaryotic system. The polyclonal antibody were prepared by using expressed fusion proteins.2. SelN mRNA was expressed in all 27 tissues detected, including tongue, esophagus, craw, glandular stomach, muscular stomach, duodenum, small intestine, cecum, rectum, cerebrum, interbrain, chorda spinalis, pituitary gland, thymus, bursa of Fabricius, spleen, lung, liver, kidney, testis, ovary, pancreas, breast muscle, flight muscle, shank muscle, thigh muscle, heart. SelN were detected in 16 tissues except pancreas in all the detected 17 tissues including cerebrum, interbrain, chorda spinalis, skeletal muscle, heart, muscular stomach, craw and cecum, spleen, thymus, bursa of Fabricius, lung, liver, kidney, pancreas, testis and ovary. All these data suggest the expression of SelN is ubiquitous in chicken.3. when 1,2,3,5 mg/kg Na2SeO3 was added in animal feeds, we confirmed that Se could influence Se content and SelN, SecS, SPS1 mRNA expression in muscular tissue of adult cock. SelN mRNA expression in chicken muscles were increased in groups supplymented with a low concentration of Se and decreased in high concentration groups, when compared with control group. SecS and SPS1 mRNA expression were gradually increased with the increasing Se supplyment in feeds. All these results indicate that Se could regulate SelN, SecS and SPS1 mRNA expression on the transcriptional level, but the decrease of SelN expression at high concentration group could due to the Se toxicity.4. Se supplement and Se dificiency affected Se content and SelN, SecS, SPS1 mRNA expression of chicken muscle tissues within different day of age. In Se over-supply groups, regulation of Se on SelN mRNA expression differed in different tissues and ages of chicken. SelN mRNA level were lower in cardiac muscle in Se dificiency groups than that of control group, while a higher SelN mRNA level was observed in skeletal. It was concluded that the importance of SelN in skeletal muscle make the expression priority. Se could affect SecS and SPS1 mRNA expression level, however, the regulation mechanism of the latter two gene on SelN expression need further research.5. Chick embryo were injected with different concentration of Na2SeO3. The results indicated that Se altered SelN, SecS, SPS1 mRNA expression in muscles of chick embryo. SelN,SecS,SPS1 mRNA expression level in breast muscle and leg muscle were significantly decreased in 21 day of age chick embryo in control group and Se-supply groups. The effect of Se on gene expression was negligible. It was concluded that gene transcription could be regulated by Se, but the mechanism need a further study.6. Chick embryo myoblasts were cultured for different time in vitro and the addition of Se in the culture system could change all gene mRNA expression detected. SelN mRNA was increased significantly in the proliferating cells and decreased in the differentiation process of myoblasts into myotubes. Results revealed that the expression of Myf5, Pax7, MEF2A genes have some correlations with cell proliferation, differentiation. Taken togetherm, Se supplement could influence the SelN, SecS, SPS1, Myf5, Pax7 and MEF2A mRNA expression levels, in addition, A correlations were observed between SelN mRNA expression level and Myf5, Pax7, MEF2A mRNA expression level during the whole cultivation process. In short, genetic transcription could changed during the proliferation and differentiation of myoblasts, no matter Se supplied or not, but the mechanism need further study.7. During the development of embryo from E12 to 35 day of age, SelN mRNA expression level exhibited an irregular changes in cardiac muscle. SelN mRNA expression in skeletal mucsle sustained a high level in embryo stage and decreased postnatal stage. It could conclude that SelN play an important role in skeletal muscle of chick embryo. The changes of SecS and SPS1 mRNA expression were in accorded with that of SelN mRNA expression level. It concluded that SelN, SecS and SPS1 could exert diverse roles in different tissues and development stages.Taken together, the expression of chicken SelN was ubiquitous from transcriptional and protein level. Se could regulate SelN, SecS and SPS1 mRNA expression on transcriptional level. A correlation was observed between SelN mRNA and Myf5, Pax7, MEF2A mRNA in myoblasts. SelN mRNA expression level decreased dramatically in skeletal muscle during development suggesting a role for SelN in early development of embryo muscle.