| This experiment was conducted to clone and analyze the complete coding sequences of juvenile Percocypris pingi Defensin,Hepcidin,TNF-a and IL-10 genes, besides the expression patterns of these genes in tissues of Percocypris pingi fed with dietary in various protien levels were studied. In addition, the study investigated the stability of EF-1a,B2M,ODC,β-actin and GAPDH gens under the same condition. This research would enrich and develop immunonutrition and provide theoretical basis for healthful aquaculture of Percocypris pingi.The sequences of immuno-related genes were obtained with method of gene cloning and sequencing. With the random single factor guoup divison method, a total of 480 healthy Percocypris pingi (weigh,15.2± 1.1 g) were randomly assigned to four groups (3 replicates per group,40 per replicate), and fed with dietary protien at various percent (37.41 %(Ⅰ),40.10%(Ⅱ),44.34%(Ⅲ),49.20%(Ⅳ). After 80d feeding, six fish in each were selected randomly to collect brain, gill, head kidney, spleen, liver, hindgut, skin and heart, and the stability of reference genes was analyzed with RefFinder and geNorm. Results showed as follows:1 The coding sequences of Hepcidin, TNF-a, Defensin and IL-10 genes with 282, 711,204 and 540 bp, respectively, encode 93,236,67,179 amino acids, and the cleavage site of signal peptide is between 24th and 25th,50th and 51th,24th and 25th,22th and 23th amino acid, besides phylogenetic trees of amino acid match the morphological status. Furthermore, isoelectric point of predicted protein with molecular weight of 10.6kDa,26.4kDa,7.2kDa and 20.9kDa, respectively, is 6.39, 5.25,8.22 and 8.42. Moreover, protein encoded by defensin is hydrophobic, while that encoded by the others is hydrophilic.2 There is no single stable reference gene appropriate for the expression analysis in different tissues with various protien levels. The most stable expression gene among tissues with various protein levels was EF-1α, the most suitable to be single reference gene. In addition, the most stable gene was not same in different tissues with various protien levels, and the most stable expression gene in liver and skin was EF-1α, while it was B2M the most stable in hindgut, spleen, gill, heart and head kidney, and that in the brain was ODC.3 The four immune genes were detected in all tissues with tissue specificity. The expression of defensin in skin was significantly higher than that in other tissues(P<0.05); Hepcidin expressed highest in liver, and the expression in spleen and hindgut was followed (P<0.05); Expression of IL-10 in gill was significantly higher than that in other groups (P<0.05), and that in spleen, hindgut and head kidney was high relatively; There were no significant differences of the TNF-α expression among tissues (P>0.05). IL-10 and TNF-α expressed lower than defensin and hepcdin in the overall level.4 With the increasing dietary protein, the expression of defensin in brain and IL-10 in gills and hindgut was increased first and then decreased, and the highest expression was in group Ⅱ, significantly higher than that in other groups(P<0.05), while there were no big significant differences in the expression of these genes in other tissues in all groups(P>0.05); The expression of Hepcdin in brain, liver and spleen was increased first and then decreased, and the highest value was in group Ⅲ, while that in other tissues did not change significantly (P> 0.05); Except that expression of TNF-α in heart was increased first and then decreased in the heart, there were no significant differences in expression of TNF-α in other tissues (P<0.05).The results demonstrate that, EF-1α could be selected as a single reference gene among tissues with dietary of various protein levels, while appropriate reference gene should be selected in each tissue with various protein levels, EF-1α could be selected in liver and skin, B2M could be selected in hindgut, spleen, gill, heart and head kidney, and ODC could be selected in brain as single reference. Dietary with appropriate protein level can promote the expression of immuno-related genes, and with dietary of 40.10% protien, the expression of defensin and IL-10 was the highest with the best growth performance, in addition,with dietary of 44.34% protien, Hepcdin expressed the highest.The optimal protein level might be 40.10%-44.34% for the balance of immune regulation. |
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