Northern Blot and Immunohistochemistry Analysis of HMOX1 Expression 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.