Study On Three Bovide-specific Y Chromosome Mutlicopy Genes In Sheep

The genes in the male specific region of Y chromosome（MSY）play important roles in sex determination and male reproduction.Previous studies have shown that the ZNF280AY,ZNF280BY and PRAMEY genes are bovide-specific Y chromosome mutlicopy genes,which are mainly or solely expressed in the testis,and copy number variations are significantly related to the reproductive efficiency of bulls.Since sheep Y chromosomes has not been sequenced,no experiment has been conducted to investigate multi-copy genes of sheep Y chromosomes.In this study,male-specific primers were designed based on the sequence of bull ZNF280AY,ZNF280BY and PRAMEY genes and our previous RNA-Seq data.Rapid amplification of cDNA ends（RACE）and quantitative polymerase chain reaction（qPCR）were used to obtain the full-length cDNA of sheep ZNF280BY and PRAMEY genes and their expression profiles in different tissues and testicular development stages,and related softwares were used to predict gene structure and function.We analyzed the copy number variations（CNVs）of ZNF280AY,ZNF280BY and PRAMEY genes using qPCR in723 individuals from 8 sheep breeds.Meanwhile,the correlation between CNV and Hu sheep testis size related indexes was analyzed.The major findings are as follow:（1）The full-length cDNA of ovine ZNF280BY gene was 1993 bp,and open reading frame（ORF）was 1632 bp（GenBank accession No:MN850837）,encoding543 amino acids.Bionformatic analysis showed predicted molecular weight,isoeletric point value,predicted molecular formula of ZNF280BY protein were 60750.11 Da,8.14 and C₂₆₆₁H₄₂₀₃N₇₅₁O₈₁₃S₃₂,respectively.It is an unstable hydrophilic protein,and did not have transmembrane region and signal peptide sequence.There were serine（Ser）,threonine（Thr）and tyrosine（Tyr）phosphorylation sites,and a potential glycosylation site.Conserved domain search revealed that ZNF280BY was composed of two functional domains（Pfam:DUF4195 and ZnF_C2H2）.The secondary structure consists of random coils,α-helix,and extended chains.Subcellular localization predicts that it is mainly distributed on the cell membrane.The full-length cDNA of ovine PRAMEY gene is 2099 bp,and open reading frame（ORF）was 1536 bp（GenBank accession No:MN850838）,encoding 511 amino acids.Bionformatic analysis showed predicted molecular weight,isoeletric point value,predicted molecular formula of PRAMEY protein were 57950.06 Da,6.57 and C₂₆₀₄H₄₁₇₈N₆₉₂O₇₃₀S₃₄,respectively.It is an unstable hydrophilic protein,and did not have transmembrane region and signal peptide sequence.There are thirty-one Ser,ten Thr,and two Tyr phosphorylation sites,and two potential glycosylation sites.The secondary structure consists ofα-helix,random curls and extended chains.（2）The mRNA of ovine ZNF280AY was not expressed in all tissues analyzed,indicating it may be a pseudogene.The qPCR results showed that ovine ZNF280BY was expressed in all tissues,and the expression level in testis was significantly higher than that in other tissues（P<0.01）.PRAMEY is only expressed in the testis and muscle,and the expression in the testis is significantly higher than that in the muscle（P<0.01）.The expression level of ZNF280BY and PRAMEY gene increased with the testicular development,and the mRNA expression in the large testis group was significantly higher than that in the small testis group（P<0.05）.Meanwhile,the expression level of ZNF280BY and PRAMEY was significantly correlated with testicular size related indicators.（3）The quantitative PCR（qPCR）method was applied to measure the copy number（CN）of ZNF280AY,ZNF280BY and PRAMEY gene in 723 individuals of 8sheep breeds.The results showed that the median copy number（MCN）of the ZNF280AY gene was 42,ranging from 15 to 122.Tan sheep had the lowest MCN（36,range 25-60）,whereas Dorper sheep possessed the highest MCN（69,range 42-93）,and significantly higher than Hu sheep,Suffolk sheep,South African Mutton sheep,Tan sheep,Texel sheep and White Suffolk sheep（P<0.01）.The MCN of the ZNF280BY gene is 188 with a range of 17 to 514.The ZNF280BY gene had the lowest MCN in the East Friesian sheep population（123,range 103-151）and in the Texel sheep population had highest MCN（306,range 103-512）;The PRAMEY gene CN between 2-25,and the MCN was 4,with the lowest MCN in the Tan sheep（3,range 2-7）.Association analysis revealed that the ZNF280AY CN was correlated negatively with left testicular length,left testicular width,and right testicular length.Association analysis revealed that the CNV of ZNF280BY was correlated negatively with total testicular weight,left testicular weight,left testicular width,left epididymal weight,right testicular weight and right testicular length.The low CN of the ZNF280AY and ZNF280BY genes were conducive to testicular development.There were no significant correlation between CN and mRNA expression levels of ZNF280BY and PRAMEY.