Study Of The Expression Of PRLR And Other Related Genes In Skin Tissue Of Different Feathering Type Bashang Long-tail Chickens

Phenotype of feathering type can be used for sex discrimination,and there was difference in production performance between early and slow feathering chickens.In order to study the effects of PRLR,SPEF2 and others genes on feather developments of Bashang long-tail chickens,the primary and the secondary flight feathers of the early and slow feathering Bashang Long-Tail chickens of E19,E20 and D0 were measured for the length.Bashang Long-Tail chickens follicle tissues of embryo period12,15 and 19 days and after hatched 0,15,20,35 and 56 days were used to extract DNA and RNA.RFLP was used to detect the feather type and semi-quantitative RT-PCR was used to detect expression of the two PRLR spliceosomes.Amino acid physicochemical properties and structure of two PRLR spliceosomes were predicted with Protparam,PSORT II,SignalP4.1,TMHMM,SOPMA and SWISS-MODEL softwares.And PRLR promoter active region was explored.The expression of aPRLR,SPEF2,FUSION,and FST,BMP2 was measured by RT-qPCR.The results showed that partial repeated sequence of PRLR and SPEF2 gene was the basis of feathering type,which had no relation with the repeated sequence of unoccupied site for ev21.The primary and the secondary flight feathers of fast feathering chickens on E20 both were longer than on E19?P<0.05?,while the two feathers were the same with D0?P>0.05?.In constrast to that the primary flight feathers of slow feathering chickens on E20 were not significantly different with on E19?P>0.05?,but were significantly shorter than on D0?P<0.05?.And the secondary flight feathers of slow feathering chickens were significantly different on the three time points and grew longer with days?P<0.05?.It was concluded that the primary flight feathers of slow feathering had slower growth rate than fast feathering between 19²0 embryoic days,and the secondary flight feathers of slow feathering chickens remained the same growth rate between 19²0 embryoic days with 20²1embryoic days,but that of the fast feathering chickens decreased.RT-PCR showed that there was no PRLR spliceosome type with exon 8 in hair follicles of Bashang Long Tail chicken,but there were 2883?PRLRS1?and 2400?PRLRS2?spliceosome.There was no significant difference in the expression of the two spliceosomes between the hemp and black feather Bashang Long Tail chicken hair follicles?P>0.05?.The expression of PRLR_S1 in slow feathering chickes was significantly lower than fast feathering on D0?P<0.05?,on the contrary,it was higher on D15?P<0.05?.However,the expression of PRLR_S2 in slow feathering chickes was significantly higher than fast feathering on E19?P<0.05?,and significantly lower on D35?P<0.05?.PRLR_S1 was expresed significantly higher than PRLR_S2 in fast feathering chickens on E15,E19 and D56?P<0.05?,while it was significantly higher in slow feathering chickens during all the embryoic days and the afer hatching peroids?P<0.05?except D0 and D15,with D0 PRLR_S1 significantly lower than PRLR_S2 and D15 without any significant difference?P>0.05?.It was concluded that:?1?PRLR_S22 was highley expressed in slow feathering chickens on E19^D0,which might be related to the decrease of growth rate of primary feathers of slow feathering chickens on E19²0.And PRLR_S22 might inhibit the growth of primary feathers of slow feathering chickens.?2?PRLR_S11 was highley expressed in fast feathering chickens on D0,which might be related to the decrease of growth rate of secondary feathers of fast feathering chickens.And PRLR_S11 might inhibit the growth of secondary feathers of slow feathering chickens.?3?PRLR_S11 was highley expressed in two feathering types chickens on most time points,which illustrated again that PRLR_S11 had a role in inhibiting of growth of secondary feathers.And the higher expression of PRLR_S22 in slow feathering chickens on D0 showed again PRLR_S2had a role in inhibiting of growth of primary feathers of slow feathering chickens.The PRLR_S1 protein molecular formula was C₄₁₆₆H₆₄₇₂N₁₁₀₆O₁₂₉₈S₄₀ and relative molecular weight was 94102.25,including 831 amino acid residues.The PRLR_S2protein molecular formula was C₃₄₆₁H₅₃₈₂N₉₂₆O₁₀₇₆S₃₄ and relative molecular weight was 78270.41,including 688 amino acid residues.Both are unstable acidic hydrophilic proteins.There were 5 differences in the two spliceosomes,the PRLR_S1were S,M,L,P,and S and the PRLR_S2 were N,I,F,S,and N,respectively.It is predicted that PRLR_S1 had a signal peptide at amino acid 24,whereas PRLR_S2 had no signal peptide.PRLR_S1 mainly existed in the extracellular,Golgi,endoplasmic reticulum and cell membrane;PRLR_S2 mainly existed in the nucleus,PRLR_S1 and PRLR_S2 had transmembrane regions.Analysis of the secondary structure of two proteins showed thery were conventional protein.Cloning the PRLR gene upstream promoter regions P2 from-37 bp^-1738 bp and P4 from-10252 bp^-8957 bp,and to construct a luciferase reporter gene recombinant plasmid vector,DF-1 cells were used for transient transfection.The results showed that there was no significant difference in the activity ratio between the two segments and the negative control?P>0.05?,indicating that the predicted two sequences did not contain a promoter active region.RT-qPCR analysis showed that FST expression:slow feather expressions were higher than fast feathers on E15,the hemp feather expressions were higher than black feathers;slow feather expressions were lower than fast feather on E19,mainly as slow feather hemp chicken low expression;Slow feather expressions were lower than fast feather on D35,mainly as fast hemp feather high expression.The effect of FST on feather development needs to be further explored.SPEF2 expression:hemp chickens were significantly higher than black chickens on E15,showing the fast hemp feather high expression.From E19 to D0,SPEF2 gene expression increased in both fast and slow feather,but did not reach significant levels,and there was no significant difference between fast and slow feather.It should be further studied whether SPEF2had an effect on feather growth.BMP2 expression:hemp chickens were significantly higher than black chickens on E19 and D0,and the expression of slow feathers was significantly higher than fast feathers,showing slow hemp chickens high expression.It could not be considered that there was a correlation between BMP2 and feather development.aPRLR expression:the expression was significantly higher on E15 and E19 than the other time points?P<0.05?,and was also higher on E19 in slow feathering chickens than fast feathering?P<0.05?.But feathering color showed no effects on the expression.All showed that the higher experssion of PRLR might be related to the development of feathers during embryoic late stages.FUSION expression:the expression of FUSION showed a similar character with PRLR,which was higher expression on E15 and E19 and no effects from feather color on expression.But FUSION was only expressed in slow feathering chickens.All showed that the higher experssion of FUSION might be related to the development of feathers of late feathering chickens during embryoic late stages.Sequencing revealed that PRLR_S1 contained 7 mutated bases,including 2synonymous and 5 missense mutations.Synonymous mutations were 309 bp and1068 bp in PRLR_S1 sequences,and missense mutations were located in 106 and 476,1492,1726,and 1883 bp,where amino acid mutations at positions 106 and 476 are located in the ligand binding region of the ectodomain and may affect the bind of PRLR and PRL;the others are located in the intracellular domain,and the mutation at the 1883 position,resulted in the appearance of an N-glycosylation site.aPRLR regulates the formation of slow feather during embryonic stage;BMP2and FST genes were antagonistic in embryos.FUSION,aPRLR and BMP2 genes had a synergistic effect on the development of the follicles of the slow feather chicken.In summary,the main wind feather differentiation of the Bashang Long-tail chickens early and slow feathering appeared at E19-E20,and the covered main wing feather appeared at E20-D0.There were PRLR_S1 and PRLR_S2 spliceosomes in the hair follicles of the Bashang Long-tail chicken;the high expression of PRLR_S1 in early feather D0 may be related to the growth rate decrease of fast feathering main wing feathers in E20 to D0;the high expression of PRLR_S22 in slow feather E19 may be related to the growth rate decrease of the slow feather main wing feather at E19 to E20.The expression of aPRLR gene in slow feather embryonic was higher than that in fast feather;FUSION gene was only expressed in slow feathered chickens and was highly expressed in E15 and E19,which may be related to the formation of slow feather phenotypes;relationship of expressions of FST,SPEF2 and BMP2 gene and the feathering type needs to be further explored.Regions of PRLR gene up to-37 bp^-1738 bp and-10252 bp^-8957 bp have no promoter activity.There are 7 SNPs in PRLR gene mRNA,including 5 missense mutations and 2 synonymous mutations.