摘要
BackgroundJUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for sperm-oocyte fusion; their interaction is indispensable for fertilization.MethodsPCR was used to clone the full-length DNA sequence of the Juno gene in sheep. The single nucleotide polymorphism (SNP) loci of Juno were genotyped by Sequenom MassARRAY®. PCR combined with rapid amplification of cDNA Ends were used to clone the full-length cDNA sequence of Juno and Izumo1. Reverse transcriptase-PCR (RT-PCR) and real time-quantitative-PCR (RT-qPCR) were used to analyze the genes’ expression in tissues of sheep, and single cell RNA-seq was used to analyze the genes’ expression in oocytes, granulosa cells and follicular theca of polytocous and monotocous Small Tail Han ewes. Bioinformatics was used to analyze advanced structure and phylogeny of JUNO and IZUMO1 proteins.ResultsThe full-length DNA sequence of the Juno gene in sheep was cloned and nine SNPs were screened. We found a significant association between the g.848253 C > A locus of Juno and litter size of Small Tail Han sheep (P < 0.05). The full-length cDNA sequence of Juno and Izumo1 genes from Small Tail Han sheep were obtained. We found a new segment of the Izumo1 CDS consisting of 35 bp, and we confirmed the Izumo1 gene has 9 exons, not 8. RT-qPCR showed that Juno and Izumo1 genes were highly expressed in ovarian and testicular tissues, respectively (P < 0.01). Single cell RNA-seq showed Juno was specifically expressed in oocytes, but not in granulosa cells or follicular theca, while Izumo1 displayed little to no expression in all three cell types. There was no difference in expression of the Juno gene in oocyte and ovarian tissue in sheep with different litter sizes, indicating expression of Juno is not related to litter size traits. Bioinformatic analysis revealed the g.848253 C > A locus of Juno results in a nonconservative missense point mutation leading to a change from Phe to Leu at position 219 in the amino acid sequence.ConclusionsFor the first time, this study systematically analyzed the expression, structure and function of Juno and Izumo1 genes and their encoded proteins in Small Tail Han sheep, providing the basis for future studies of the regulatory mechanisms of Juno and Izumo1 genes.
摘要译文
BackgroundJuno和Izumo1是发现对精子卵母细胞融合至关重要的第一个受体 - 配体蛋白对;它们的相互作用对于施肥是必不可少的。方法用于克隆绵羊中juno基因的全长DNA序列。 juno的单个核苷酸多态性（SNP）基因座由亮片Massarray®进行基因分型。 PCR结合CDNA末端的快速扩增克隆juno和Izumo1的全长cDNA序列。逆转录酶-PCR（RT-PCR）和实时定量-PCR（RT-QPCR）用于分析绵羊组织中的基因，而单细胞RNA-SEQ用于分析卵母细胞中的基因表达，Granulosa细胞和多层和单位耳小尾韩母羊的卵泡Theca。生物信息学用于分析Juno和Izumo1蛋白的先进结构和系统发育。克隆绵羊中juno基因的全长DNA序列，筛选九个SNP。我们在G.848253 C> juno和小尾羊羊的垃圾座位之间找到了一个重要的关联（P <0.05）。得到了来自小尾汉羊的juno和Izumo1基因的全长cDNA序列。我们发现了一个由35 bp组成的Izumo1 CD的新段，我们确认了Izumo1基因有9个外显子，而不是8. Rt-qpcr显示juno和Izumo1基因分别在卵巢和睾丸组织中高度表达（P <0.01 ）。单细胞RNA-SEQ显示juno在卵母细胞中特异性表达，但不在颗粒细胞或卵泡Theca中表达，而Izumo1在所有三种细胞类型中没有表达。在具有不同垃圾尺寸的绵羊中的卵母细胞和卵巢组织中的Juno基因表达没有差异，表明Juno的表达与垃圾尺寸特征无关。生物信息分析显示，juno的轨迹导致非可选的畸形点突变导致氨基酸序列中Phe对Leu的影响。第一次进行结论，该研究系统地分析了表达，结构朱诺和Izumo1基因的功能及其在小尾羊群中的编码蛋白，为未来研究朱诺和Izumo1基因的监管机制的研究提供了依据。
Wenping Hu;Xinlong Dong;Mingxing Chu;. Expression, structure and function analysis of the sperm-oocyte fusion genes Juno and Izumo1 in sheep ( Ovis aries )[J]. Journal of Animal Science and Biotechnology, 2021,12(1)