背景:在卵母细胞和胚胎中表达的基因的适当调节对于获得哺乳动物的发育能力至关重要。这里,我们假设在卵母细胞和植入前胚胎中表达的几个基因仍然未知.我们的目标是使用短读和长读序列重建卵母细胞(生发囊泡和中期II)和植入前牛胚胎(胚泡)的转录组,以鉴定推定的新基因。
结果:我们鉴定出274,342个转录序列和3,033个这些基因座与官方注释中存在的基因不匹配,因此是潜在的新基因。值得注意的是,63.67%(1,931/3,033)的潜在新基因表现出编码潜力。同样值得注意的是,97.92%的推定新基因与转座因子的注释重叠。转录物丰度的比较分析鉴定出1,840个新基因(最近添加到注释中)或潜在的新基因在发育阶段之间差异表达(FDR<0.01)。我们还确定,与卵母细胞相比,八个细胞胚胎中的522个新的或潜在的新基因(分别为448和34个)上调(FDR<0.01)。在八细胞胚胎中,共表达了102个新的或推定的新基因(|r|>0.85,P<1×10-8),并注释了与多能性维持和胚胎发育有关的基因本体论生物学过程。CRISPR-Cas9基因组编辑证实,在八细胞胚胎中高表达的新基因之一的破坏降低了胚泡发育(ENSBTAG00000068261,P=1.55×10-7)。
结论:我们的结果揭示了一些推测的新基因,需要仔细注释。许多推定的新基因在植入前发育过程中具有动态调节,并且是参与多能性和胚泡形成的基因调节网络的重要组成部分。
BACKGROUND: Appropriate regulation of genes expressed in oocytes and embryos is essential for acquisition of developmental competence in mammals. Here, we hypothesized that several genes expressed in oocytes and pre-implantation embryos remain unknown. Our goal was to reconstruct the transcriptome of oocytes (germinal vesicle and metaphase II) and pre-implantation cattle embryos (blastocysts) using short-read and long-read sequences to identify putative new genes.
RESULTS: We identified 274,342 transcript sequences and 3,033 of those loci do not match a gene present in official annotations and thus are potential new genes. Notably, 63.67% (1,931/3,033) of potential novel genes exhibited coding potential. Also noteworthy, 97.92% of the putative novel genes overlapped annotation with transposable elements. Comparative analysis of transcript abundance identified that 1,840 novel genes (recently added to the annotation) or potential new genes were differentially expressed between developmental stages (FDR < 0.01). We also determined that 522 novel or potential new genes (448 and 34, respectively) were upregulated at eight-cell embryos compared to oocytes (FDR < 0.01). In eight-cell embryos, 102 novel or putative new genes were co-expressed (|r|> 0.85, P < 1 × 10-8) with several genes annotated with gene ontology biological processes related to pluripotency maintenance and embryo development. CRISPR-Cas9 genome editing confirmed that the disruption of one of the novel genes highly expressed in eight-cell embryos reduced blastocyst development (ENSBTAG00000068261, P = 1.55 × 10-7).
CONCLUSIONS: Our results revealed several putative new genes that need careful annotation. Many of the putative new genes have dynamic regulation during pre-implantation development and are important components of gene regulatory networks involved in pluripotency and blastocyst formation.