基于单细胞转录组测序技术筛选牛体内囊胚性别特异性基因

doi:10.16431/j.cnki.1671-7236.2022.12.007

摘要/Abstract

摘要： 【目的】探究牛体内雌性和雄性囊胚在mRNA水平上的特异性差异，挖掘雌性和雄性囊胚发育差异相关候选基因。【方法】参考GenBank中牛牙釉质基因（AMEL）序列（AMELX基因，登录号：NM_001014984.1；AMELY基因，登录号：NM_174240.2）设计引物，以胚胎内细胞DNA为模板，采用巢式PCR扩增技术鉴定单个牛体内囊胚的性别。选用确定性别的单个雌性和雄性囊胚为试验材料，采用Smart-Seq2扩增技术构建单个胚胎测序文库，应用Illumina HiSeq Xten高通量测序平台对单个胚胎进行单细胞转录组测序（scRNA-Seq），并进行了基因差异表达、GO功能和KEGG通路分析。【结果】通过巢式PCR扩增胚胎内细胞中的AMEL基因，确定了胚胎性别；基因表达分析筛选出两组之间差异表达基因（DEGs）6 160个，其中雌性特异性基因675个，雄性特异性基因305个；GO功能注释发现雌性特异性基因显著注释在核苷酸结合、信号转导调控、多细胞生物发育、细胞骨架等条目，雄性特异性基因显著注释在氧化磷酸化、线粒体、线粒体内膜、核糖体等条目；KEGG通路富集分析发现7条与雌性和雄性囊胚发育差异相关的通路，分别为代谢途径、糖酵解、磷酸戊糖途径、细胞衰老、氧化磷酸化、调节干细胞多能性的信号通路和Wnt信号通路；并利用GO和KEGG结果筛选出5个在牛体内囊胚期胚胎发育过程中具有直接或间接作用的基因：FBP1、GADD45G、FHL2、FOSB和WNT2B。【结论】牛体内雌性和雄性囊胚之间存在广泛的转录差异，性别特异性基因FBP1、GADD45G、FHL2、FOSB和WNT2B可能是直接或间接影响胚胎发育的关键基因。

关键词: 牛体内囊胚; 单细胞转录组测序(scRNA-Seq); 性别特异性基因; 富集分析

Abstract: 【Objective】 The purpose of this study was to explore the specific differences in mRNA levels between female and male blastocysts in cattle and to explore candidate genes related to the development of female and male blastocysts.【Method】 The primers were designed concerning the GenBank sequence of the bovine enamel gene (AMEL) (AMELX gene, accession No.:NM_001014984.1;AMELY gene, accession No.:NM_174240.2), and the intra-embryonic cellular DNA was used as the template to identify the sex of individual bovine blastocysts by nested PCR amplification.Single female and male blastocysts of definite sex were selected as test materials, and a single embryo sequencing library was constructed using Smart-Seq2 amplification technology.Illumina HiSeq Xten high-throughput sequencing technology was applied to single-cell transcriptome sequencing (scRNA-Seq) of single embryos, and differential gene expression analysis, GO functional analysis and KEGG pathway analysis were performed.【Result】 Determination of embryo sex by nested PCR amplification of the AMEL gene present in intra-embryonic cellular. Gene expression analysis screened 6 160 differentially expressed genes (DEGs) between the two groups, including 675 female-specific genes and 305 male-specific genes.GO function annotation revealed that female-specific genes were significantly annotated in nucleotide binding, signal transduction regulation, multicellular biogenesis, cytoskeleton and other entries, and male-specific genes were significantly annotated in oxidative phosphorylation, mitochondria, mitochondrial inner membrane, ribosome and other items.KEGG pathway enrichment analysis identified seven pathways associated with differences in the development of female and male blastocyst, including metabolic pathway, glycolysis, pentose phosphate pathway, cellular senescence, oxidative phosphorylation, signaling pathway regulating stem cell pluripotency and Wnt signaling pathway.Five genes with direct or indirect roles in the development of bovine embryos were screened by GO and KEGG:FBP1, GADD45G, FHL2, FOSB and WNT2B.【Conclusion】 Extensive transcriptional differences existed between female and male blastocysts in cattle, and sex-specific genes FBP1, GADD45G, FHL2, FOSB and WNT2B might be key genes that directly or indirectly influence embryo development.

Key words: in vivo-produced bovine blastocyst; single-cell transcriptome sequencing (scRNA-Seq); sex-specific genes; enrichment analysis

中图分类号:

S814.8

崔宝山, 黄飞, 王杰, 李楠, 高庆华. 基于单细胞转录组测序技术筛选牛体内囊胚性别特异性基因[J]. 中国畜牧兽医, 2022, 49(12): 4573-4581.

CUI Baoshan, HUANG Fei, WANG Jie, LI Nan, GAO Qinghua. Screening of Sex-specific Genes in vivo-Produced Bovine Blastocysts Based on Single-cell RNA Sequencing Technology[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4573-4581.

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