Investigating structural impact of a valine to isoleucine substitution on anti-Müllerian hormone in silico and genetic association of the variant and AMH expression with egg production in chickens

Anti-Mullerian hormone(AMH)acts in maintaining orderly cyclic recruitment of early follicles,suggesting that it is a promising can didate for in flue ncing an imal reproductive efficiency.This study aimed to elucidate the effect of a missense mutati on of Val566lle on the structure of AMH protein and the genetic association of Val566lle and AMH expression with egg production in chickens.Structural perturbations of Val566lle were predicted by homology modeling.The association of the variant with the number of eggs was tested using a quantitative trait transmission disequilibrium test model.AMH expression in granulosa cells in Lueyang black-boned chickens was compared with that in Nick chickens.The Vai566 of AMH is a non?conservative amino acid among mammals and birds,but its hydrophobicity is completely conservative.The substitution of Vai566 for lie566 pote ntially disrupted hydroge n bonds and solve nt accessibility of 22 residues and created a short a-helix in the C terminus of AMH.Despite having striking structure-disrupting potential,the variant was not statistically associated with the number of eggs(P>0.05)in the Lueyang black-boned chickens.We did not detect differential expression of AMH betwee n Lueya ng black-b oned chicke ns and Nick chickens(P>0.05).These results con firmed the structural impact of Val566lle,but suggested that Val566lle and AMH expression might not be the major genetic determinants for egg production in Lueyang black-boned chickens.

Northern Blot and Immunohistochemistry Analysis of HMOX1Expression in Blue- and Brown-shelled Chickens

HMOX1 is an important functional candidate gene for chicken blue egg in view of its role in biosynthesis of biliverdin for blue egg coloration. To elucidate molecular mechanism of blue egg formation, this study detected expression of HMOX1 in blue-shelled chickens and brown-shelled chickens. Expression and alternative splicing of HMOX1 were detected by Northern blot, expression traits of HO-1 protein in shell glands of blue- （n=4） and brown-shelled （n=4） chickens were analyzed by immunohistochemistry. 3＇ UTR of HMOX1 was cloned using 3＇RACE. Results showed that the expression of HMOX1 at mRNA level had no significant difference between two groups of chickens, but at protein level HO-1 protein was highly expressed in blue-shelled chickens. Immunohistochemistry analysis showed that HO-1 protein expression was predominately located in villus epithelial cell of shell gland. Length of HMOX1 3＇ UTR were 586 bp. In 3＇ UTR we found a SNP of rs13866562 showing significant association with blue egg phenotype. Further miRNA prediction showed that it might influence interaction of some miRNAs and target sequences. The data suggested that blue egg is relevant to high expression of HO-1 in villus epithelial cell of shell gland. Further experimental validation for biological relevance of miRNAs is dispensable to elucidate reason for differential expressions of HO-1 protein.