转录组测序筛选山羊妊娠早期胚胎附植相关基因

doi:10.11843/j.issn.0366-6964.2022.05.014

摘要/Abstract

摘要： 旨在通过胚胎附植前、后的山羊子宫角组织高通量测序筛选妊娠早期胚胎附植的关键基因。本研究选取2～4岁体重相近((44.76±3.49)kg)的经产努比亚母山羊16只，在同期发情-人工输精配种后的第15(D15)和第30天(D30)分别随机选取3只羊颈动脉放血处死。采集子宫角组织利用Illumina HiSeq进行高通量转录组测序(RNA-Seq)，筛选差异表达基因(differentially expressed genes, DEGs)并对DEGs进行功能注释，基因功能查询进一步筛选与胚胎附植直接相关的调控基因，荧光定量PCR(qRT-PCR)对筛选的基因进行表达量验证分析。D15和D30子宫角组织转录本比较分析发现了 2 000个DEGs，其中上调基因620个，下调基因1 380个。GO功能聚类分析共分为3大类52组，其中细胞组分17组，生物过程23组，分子功能12组，获得细胞连接与增殖，生物粘附与调节，分子活性与转导等重要生物途径。以D15表达量高低为排序标准，从表达量最高的10个基因中筛选出了与胚胎附植有关的基因MGP和TAGLN；从上调和下调幅度最大的前10个差异表达基因中，获得参与妊娠相关糖蛋白合成与分泌的基因PAG-3、PAG-8、PAG-12，参与生长因子结合与生长激素释放激素受体活性相关基因GHRHR和胰岛素样生长因子结合蛋白基因IGFBP1。qRT-PCR结果显示，随机选取的6个基因在D15和D30子宫角中表达变化趋势与RNA-Seq结果一致。获得的基因MGP、TAGLN、PAG-3、PAG-8、PAG-12、GHRHR、IGFBP1在努比亚山羊胚胎附植中具有重要作用。

关键词: 努比亚山羊, 妊娠早期, 子宫角, 胚胎附植, 转录组测序

Abstract: The study focused on screening key genes of embryo implantation in early pregnancy through high throughput sequencing of goat uterine horn tissue before and after embryo implantation. Sixteen multiparous female Nubian goats (Capra hircus) with similar weights((44.76±3.49) kg) and age(2-4 years old) were selected for simultaneous estrus and artificial insemination. On the 15th day (D15) and 30th day (D30) after mating, 3 goats were randomly selected and sacrificed by carotid bloodletting, respectively. The uterine horn tissues were collected after sacrifice for Illumina HiSeq high fluxtranscriptome sequencing (RNA-Seq). Differentially expressed genes (DEGs) were screened from the sequencing results, and their functions were annotated. Then, the regulatory genes related to embryo attachment were further screened through literature search. Finally, fluorescent quantitative PCR (qRT-PCR) was used to perform expression verification analysis of the selected genes. Comparative analysis of D15 and D30 uterine horn tissue transcripts obtained 2 000 DEGs, including 620 up-regulated genes and 1 380 down-regulated genes. GO functional cluster analysis was divided into 3 major categories and 52 groups, including 17 groups of cellular components, 23 groups of biological processes, and 12 groups of molecular functions, which obtained some important biological pathways such as cell connection and proliferation, biological adhesion and regulation, molecular activity and transduction, etc. Taking the expression level of genes on D15 as the standard, the gene MGP and TAGLN related to embryo attachment were screened from the 10 genes with the highest expression. From the top 10 differentially expressed genes with the largest up-regulation and down-regulation, the gene PAG-3, PAG-8 and PAG-12 related to pregnancy-associated glycoprotein synthesis and secretion, GHRHR related to growth factor binding and growth hormone releasing hormone receptor activity and IGFBP1 related to insulin-like growth factor binding protein were obtained. The results of qRT-PCR showed that the change trend of the expression of 6 randomly selected genes was consistent with the results of RNA-Seq. The obtained gene MGP, TAGLN, PAG-3, PAG-8, PAG-12, GHRHR, IGFBP1 may play an important role in the implantation and development of embryos in Nubian goat.

Key words: Nubian goat, early pregnancy, uterine horn, embryo attachment, transcriptome sequencing

中图分类号:

S827.3

骆金红, 陈祥, 尚以顺, 敖叶, 李鹏程. 转录组测序筛选山羊妊娠早期胚胎附植相关基因[J]. 畜牧兽医学报, 2022, 53(5): 1465-1474.

LUO Jinhong, CHEN Xiang, SHANG Yishun, AO Ye, LI Pengcheng. Transcriptome Sequencing Screening the Genes Related to Goat Embryo Attachment in Early Pregnancy[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1465-1474.

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