Research Of The Relationship Between PRLR Expression And Feather Types In Taihang Chickens

The PRLR and SPEF2 tandem repeat genes long for 176kb and located at the short arm of Z chromosome are the molecular basis of late feather type,but their specific sequence structure is not clear.The purpose of this study was to clarify the embryonic age at which the length difference of the primaries(Ps)and the primary coverts(PCs)between the two types of chicken was formed,to reveal the structural characteristics of PRLR and SPEF2 repetitive genes and the relationship between its expression and how to regulates the growth of the Ps and the PCs,and to provide a theoretical basis for the molecular regulation mechanism of the late feathering(LF)phenotype.The primaries and the primary coverts of fast and LF Taihang chickens from 18 to 21 embryonic ages were collected,and their length was measured.RNA and DNA were extracted from the feather pulp of the Ps and the PCs and the livers,respectively.The molecular identification of feather type,sex and ev21 integration was carried out by PCR,and the relationship of embryonic age,feather type,sex and ev21 with feather length was analyzed.Semi-quantitative PCR was used to detect the expression level of the two spliceosomes of PRLR,and the copy number of the common region of PRLR and SPEF2 gene to reveal the structural characteristics of the 176 kb repeat gene.RT-qPCR was used to detect the expression level of PRLR,SPEF2,BMP2 and FST gene.And the relationship between gene expression and length changes of Ps and PCs of chicken embryo with different feather types and genders along with embryonic age was analyzed.The 3' and 5' terminal sequences of PRLR gene mRNA were obtained by RACE,and their structural characteristics were analyzed.The results showed that the embryonic age,feather type and sex effected on the length of the Ps and the PCs.The length of the wing feathers of Taihang chickens increased significantly with the embryonic ages(P<0.05).The Ps of early feathering(EF)chickens were significantly longer than its PCs and the Ps and the PCs of LF chickens(P<0.05).There was no significant difference between the length of the Ps and the PCs of LF chickens at E18-E21(P>0.05).The primaries of EF females at E18-E21 were significantly longer than those of EF males(P<0.05),the PCs of EF females embryonic-aged 21 were significantly longer than those of EF males(P<0.05),and the Ps and the PCs of LF chickens showed no gender difference(P>0.05).No effects of ev21 integration on the length of the wing feathers were found(P>0.05).In addition,it was found that the growth rate of the primaries of EF chickens at E18?E21 was faster than that of the PCs,while the growth rate of the PCs of LF chickens was faster than that of the Ps.At E21,the primaries of EF chickens was more than 2 mm longer than the PCs,while the PCs of LF chickens was slightly longer than the Ps.RT-qPCR analysis showed that the expression of PRLR in EF chickens was significantly lower than that in LF chickens(P<0.05),and the expression at E21 was significantly lower than at E19 and E20(P<0.05),and the expression level of EF hens was significantly lower than that in other chickens(P<0.05).The FST gene expression in EF chickens was significantly lower than LF chickens(P<0.05).There was no significant difference among E18,E19 and E20(P>0.05),but the expression of E21 was significantly lower than that of E18,E19 and E20(P<0.05).The expression of fast feathering females was the lowest and that of slow feathering females was the highest.The expression of SPEF2 gene in EF chickens was significantly lower than that of LF feathering chickens(P<0.05),and the expression level of EF females was the lowest.The expression of BMP2 gene was only closely related to the feather,in which the expression of the primary covert was significantly higher than that of the primaries,and that of the primary covert was significantly higher in EF chickens(P<0.05).There were PRLRS1(2883 bp)and PRLRS2(2400 bp)two spliceosomes in Taihang chicken follicles.Embryonic age and gender had no significant effects on the expression of PRLRS1 and PRLRS2(P>0.05),but the expression of PRLRS2 in LF chicken was significantly higher than that in EF chicken at E19(P<0.05).Semi-quantitative PCR was used to detect the structure of the head-to-head junction of PRLR and SPEF2.The gray values indicated the copies of a common region of this region was consistent with the expected ratio of 4:3:2:2:1(homozygous LF males:heterozygous LF males:EF males:LF females:EF females),so it was inferred the length of tandem repeat region of PRLR and SPEF2 gene to be 188.7 kb.In conclusion,the differences in the length of Ps and PCs,as well as the differences in the length of the two kinds of feathers between LF and EF chickens have been highlighted when Taihang chickens were 18 embryonic age,and the EF females had the fastest growth rate in the primaries.The high or low expression of PRLR,SPEF2 and FST genes in LF chicken and the specific high or low expression of PRLR,SPEF2 and FST gene at E19 were related to the growth inhibition of the PCs and the Ps of LF chickens and the growth speed changes of the PCs and the primaries after E20.The high expression of BMP2 in the PCs might be related to the growth and development of the PCs,but not the feather types.PRLRS2 participated in the feather types forming of PRLR gene regulation.The tandem repeat region of PRLR and SPEF2 in LF chickens would be long for 188.7 kb.