奶牛精子变形期间Pld6基因表达及蛋白序列分析  

The Transcription of Pld6 during Spermiogenesis and the Analysis of Its Protein Sequence in Dairy Cows

作　　者：李小俊[1] 李欣 陈晓丽[1] 赵毅强 路永强 王栋[1] LI Xiao-jun;LI Xin;CHEN Xiao-li;ZHAO Yi-qiang;LU Yong-qiang;WANG Dong(Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193,China;College of Biological Sciences, China Agricultural University, Beijing 100193,China;Animal Husbandry Station of Beijing,Beijing 100107,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]中国农业大学生物学院,北京100193 [3]北京市畜牧总站,北京100107

出　　处：《畜牧兽医学报》2018年第12期2632-2640,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31372296)

摘　　要：本研究旨在揭示精子线粒体鞘形成机制,提高种公牛繁殖潜力。采集3头健康状况良好、15～17月龄荷斯坦公牛睾丸和附睾组织,通过冷冻切片和激光捕获显微切割(laser capture microdissection,LCM)技术获取圆形和长形精子细胞,浮游法获取附睾尾精子,并采用TRizol法提取各样本RNA,对奶牛精子形成过程中的转录组进行差异分析和q PCR检测,利用生物信息学技术对其中Pld6蛋白结构和功能进行预测分析。结果表明,在精子形成过程中,Pld6基因在圆形～长形精子细胞发育过程中表达升高,而在成熟过程中转录降低甚至关闭。Pld6基因富集在线粒体融合(mitochondrial fusion)条目上,与精子尾部中段线粒体分布和形态结构相关,其蛋白为跨膜蛋白,第10～32位氨基酸是跨膜区域,三级结构呈喇叭状,在喇叭内部具有保守且呈β折叠的H(X) K(X4) D (HKD)酶活性结构域,与小鼠Pld6结构相似,且两者具有最接近的进化趋势。综上表明,根据小鼠研究推断牛精子变形过程中,Pld6基因表达增多可诱导线粒体向精子细胞尾部中段聚集,促进了线粒体鞘的正常形成,对确保牛精子活力及受精能力具有重要作用。In order to reveal the mechanism of sperm mitochondrial sheath formation and improve the fertility of bulls,testis and cauda epididymidis of3health Holstein bulls aged15-17months were collected.The round and elongated sperm cells were collected throung frozen section and(laser capture microdissection,LCM)technique.Epididymal spermatozoa were obtained by floating method.TRizol was used to extract sample RNA.The differences between transcriptomes of germ cells during the spermiogenesis of dairy cows has been analysed and verified by qPCR.To understand the function of the Pld6gene differentially expressed during spermiogenesis,the structure and function of the Pld6protein were predicted by bioinformatics analysis.The results showed that,during spermiogenesis,the transcription of Pld6gene increased during the development from round to elongated spermatids,and then decreased or even shut off during sperm maturation.The gene was enriched in the mitochondrial fusion term and associated with distribution and morphological structure of mitochondria on the midpiece of sperm tail.The10-32th amino acids of Pld6was a transmembrane region in cattle.The tertiary structure of Pld6was horn-like with a conservedβ-sheet active site H(X)K(X4)D(HKD)inside,which was similar to the mouse Pld6structure.And there was a closest evolutionary trend of Pld6between mouse and cattle.Therefore,according to the studies about mouse,it is concluded that,during the process of bovine spermaiogenesis,increasing expression of Pld6gene induces the cluster of mitochondria in the midpiece of sperm tail,which promotes the formation of normal mitochondrial sheaths and plays an important role in ensuring bovine sperm viability and fertilizing ability.

关 键 词：精子形成 Pld6基因 蛋白结构 线粒体鞘 

分 类 号：S823.91.2[农业科学—畜牧学]