基于蛋白组学鉴定猪精子中的性染色体基因表达及其生物学功能  

Identification of Porcine(Sus scrofa)Sperm Sex Chromosome Gene Expression and Its Biological Function Based on Proteomics

作　　者：石咏 刘怡冰 刘华涛 王楚端[1] 邢凯 SHI Yong;LIU Yi-Bing;LIU Hua-Tao;WANG Chu-Duan;XING Kai(College of Animal Science and Technology,China Agricultural University,Beijing 100193,China;College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China)

机构地区：[1]中国农业大学动物科学技术学院,北京100193 [2]北京农学院动物科技学院,北京102206

出　　处：《农业生物技术学报》2022年第9期1747-1762,共16页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2019YFE0106800)。

摘　　要：动物的性别控制在畜牧生产中有着重要意义,性控精液在生产中是控制家畜后代性别比例常用的商品,现有性控精液的商用多集中用于奶牛(Bos taurus),其在奶牛良种扩繁中有重要作用;流式细胞仪分选是主流的性控精液的分选方法,但分选过程中对精子的机械损伤和分选后精子活率降低都不可避免,因此利用免疫学方法分选X/Y精子是理想的方法;随着蛋白质组学技术进步,探索猪精子中的蛋白质组成也成为可能。本研究为了明确猪(Sus scrofa)精子中性染色体上蛋白编码基因并分析其生物学功能,寻找猪精子中可能用于性别控制的抗原,收集10只健康种公猪的精液,利用流式细胞仪去除精浆并把精子分离成X/Y精子,通过Label-free蛋白组学技术对分离的猪精子中的蛋白质表达进行定性、定量分析,将其中分布于性染色体上的表达基因提取出来,并进行生物学功能分析,同时将得到的性染色体上表达的蛋白质通过TMHMM 2.0进行跨膜区预测,利用WoLF PSORT进行亚细胞定位预测,利用SignalP 5.0进行信号肽预测,并对筛选得到的部分抗原进行验证。蛋白质组测序共得到蛋白1925个,肽段13558个,蛋白质对应的基因有2976个,其中有67个性染色体基因表达蛋白质,包括65个X染色体基因和2个Y染色体基因。将性染色体上表达的基因进行GO和KEGG分析发现,其主要参与了精子运动相关的能量产生通路。预测到45个蛋白含跨膜结构,47个蛋白位于细胞质膜上或细胞外,28个蛋白能产生信号肽(secretory signal peptides/signal peptides,Sec/SPI),筛选出脱氧核糖核酸酶1(deoxyribonuclease like 1,DNASE1L1)、ATP酶H+运输辅助蛋白1(ATPase H+transporting accessory protein 1,ATP6AP1)、四跨膜蛋白6(tetraspanin6,TSPAN6)、NADH:泛醌氧化还原酶亚基A1(NADH:ubiquinone oxidoreductase subunit A1,NDUFA1)、磷酸核糖焦磷酸合成酶2(phosphoribosyl pyrophosphate synthetase 2,PRPS2)、NADPH�It is critical to the actual output of animal husbandry that the sex control of animals be properly managed.A frequent commodity in the manufacturing of sex-controlled semen is used to regulate the sex ratio of their progeny.The commercial use of sex-controlled semen is mostly concentrated in dairy cows(Bos taurus),and sex-controlled frozen semen plays an important role in the propagation of improved breeds of dairy cows.Both the mechanical damage and the reduction of sperm motility after sorting are inevitable,so immunological methods to sort X/Y sperm is an ideal method.Semen from 10 healthy male boars was collected to study the gene expression of proteins on the sex chromosomes of porcine(Sus scrofa)sperm and its biological function,as well as to search for possible antigens for sex control in porcine sperm.Flow cytometry was used to remove seminal plasma and separate the sperm into X/Y sperm.The protein expression in the isolated porcine sperm was analyzed qualitatively and quantitatively by Label-free proteomics technology,and the expressed genes distributed on the sex chromosome were extracted and analyzed their biological function.Simultaneously,the protein expressed on the sex chromosome was predicted the transmembrane region by TMHMM 2.0.Subcellular localization was predicted by Wo LF PSORT,signal peptide was predicted by SignalP 5.0;and some selected antigens were validated.A total of 1925 proteins and13558 peptides were obtained by proteinomic sequencing.According to the results of this study,there were 2976 proteins-coding genes,of which 67 were expressed by sex-specific genes,with 65 X and 2 Y chromosomes genes.The GO and KEGG analyses of the genes expressed on allosome revealed that they were mostly engaged in the energy generation pathway associated with sperm motility.45 proteins were expected to contain the transmembrane structure,47 proteins were located on the plasma membrane or extracellular region,and 28 proteins could generate signal peptides(secretory signal peptides/signal peptides,Sec/SPI

关 键 词：猪精子 蛋白质组学 候选基因 

分 类 号：S828.95[农业科学—畜牧学]