China Animal Husbandry and Veterinary Medicine ›› 2022, Vol. 49 ›› Issue (12): 4573-4581.doi: 10.16431/j.cnki.1671-7236.2022.12.007

• Biotechnology • Previous Articles     Next Articles

Screening of Sex-specific Genes in vivo-Produced Bovine Blastocysts Based on Single-cell RNA Sequencing Technology

CUI Baoshan1, HUANG Fei1, WANG Jie2, LI Nan1, GAO Qinghua1,3   

  1. 1. College of Animal Science and Technology, Tarim University, Aral 843300, China;
    2. College of Life Sciences and Technology, Tarim University, Aral 843300, China;
    3. Key Laboratory of Tarim Animal Husbandry Science and Technology of Xinjiang Production & Construction Corps, Aral 843300, China
  • Received:2022-05-05 Online:2022-12-05 Published:2022-12-01

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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

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