【作者】 王丽；

【导师】 乐祥鹏；

【作者基本信息】 兰州大学 ， 畜牧学， 2020， 硕士

【摘要】 Y染色体雄性特异区基因在性别决定和雄性繁殖力方面具有重要的作用。先前的研究表明ZNF280AY、ZNF280BY和PRAMEY基因是牛科动物特有的Y染色体多拷贝基因,在睾丸中高表达或特异性表达,其拷贝数变异与公牛繁殖性状显著相关。由于绵羊Y染色体测序没有完成,故尚未有研究对绵羊Y染色体多拷贝基因进行报道。本研究基于牛ZNF280AY、ZNF280BY和PRAMEY基因的序列以及本课题组前期绵羊睾丸组织RNA-Seq数据设计雄性特异性引物,采用cDNA末端快速扩增技术（Rapid amplification of cDNA ends,RACE）和实时定量PCR（quantitative PCR,qPCR）技术获得绵羊ZNF280BY和PRAMEY基因cDNA全长以及它们在不同组织和睾丸不同发育阶段的表达特征,并利用相关软件对序列进行生物信息学分析,预测基因结构和功能;同时利用qPCR技术检测ZNF280AY、ZNF280BY和PRAMEY基因在8个品种723只绵羊中的拷贝数变异（Copy number variation,CNV）,并分析CNV与湖羊睾丸大小相关指标的相关性。主要研究结果如下:（1）首次扩增得到绵羊ZNF280BY基因的cDNA全长为1993 bp,开放阅读框为1632 bp（NCBI GenBank access No:MN850837）,编码543个氨基酸残基。预测其蛋白分子量为60750.11 Da,等电点为8.14,分子式为C₂₆₆₁H₄₂₀₃N₇₅₁O₈₁₃S₃₂,为不稳定的亲水性蛋白,不存在跨膜区域和信号肽序列,磷酸化位点包括丝氨酸（Ser）、苏氨酸（Thr）和酪氨酸（Tyr）,有1个潜在的糖基化位点,存在Pfam:DUF4195和ZnF_C2H2两种结构域,亚细胞定位预测其主要分布在细胞膜上。绵羊PRAMEY基因的cDNA全长为2099 bp,开放阅读框为1536 bp（NCBI GenBank access No:MN850838）,编码511个氨基酸。PRAMEY蛋白分子量为57950.06 Da,等电点为6.57,分子式为C₂₆₀₄H₄₁₇₈N₆₉₂O₇₃₀S₃₄,为不稳定的疏水性蛋白。不存在跨膜区域和信号肽序列,有31个丝氨酸、10个苏氨酸和2个酪氨酸磷酸化位点,2个潜在的糖基化位点,二级结构由?-螺旋、无规则卷曲和延伸链组成。（2）半定量PCR鉴定出绵羊ZNF280AY基因在各组织中均不表达,推测绵羊ZNF280AY基因可能是一个假基因;qPCR结果表明绵羊ZNF280BY在各组织中均有表达,且在睾丸中极显著高于其他组织（P<0.01）,PRAMEY仅在睾丸和肌肉中表达,且睾丸中的表达量极显著高于肌肉中（P<0.01）。ZNF280BY和PRAMEY都随着发育阶段上升,表达量增加,且6月龄大睾丸组mRNA的表达量显著高于小睾丸组（P<0.05）。同时分析表达量与睾丸大小相关指标的关系发现ZNF280BY和PRAMEY基因mRNA表达量与湖羊睾丸大小相关指标均显著正相关（P<0.05）。（3）利用qPCR分析了8个品种723只绵羊个体Y染色体基因ZNF280AY、ZNF280BY和PRAMEY的拷贝数变异情况。结果表明,ZNF280AY基因的中值拷贝数为42,变化范围为15-122,滩羊群体中的中值拷贝数最低（36,范围25-60）,在杜泊羊群体中的中值拷贝数最高（69,范围42-93）,显著高于湖羊、萨福克羊、南非肉用美利奴羊、滩羊、特克塞尔羊和白萨福克羊（P<0.01）;ZNF280BY基因的中值拷贝数为188,变化范围为17到514,ZNF280BY基因在东弗里生羊群体中的中值拷贝数最低（123,范围103-151）,在特克塞尔羊群体中的中值拷贝数最高（306,范围103-512）;PRAMEY基因拷贝数在2-25之间变化,中值拷贝数为4,滩羊群体中的中值拷贝数最低（3,范围2-7）。基因拷贝数变异与湖羊睾丸大小相关指标的关联分析发现,ZNF280AY基因拷贝数与左侧睾丸长、左侧睾丸宽及右侧睾丸长呈显著负相关,ZNF280BY基因拷贝数与睾丸总重量、左侧睾丸重量、左侧睾丸宽、左侧附睾重、右侧睾丸重量和右侧睾丸长呈显著负相关,说明ZNF280AY和ZNF280BY基因低拷贝数有利于公羊睾丸发育。ZNF280BY和PRAMEY基因拷贝数与其对应基因mRNA表达量均无显著相关。更多还原

【Abstract】 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.更多还原