| In mammals,prior to the determination and development of gonads,female and male embryos have shown differences in proliferation and differentiation.Bovine pre-implantation male embryos develop more rapidly than female embryos,which is associated with transcriptional differences between the two sexes.Trace amounts of RNA in preimplantation embryos cannot meet the basic requirements for conventional transcriptome sequencing library construction,whereas single-cell RNA sequencing(sc RNA-seq)technology allows analysis of gene expression at the level of individual embryos and becomes a useful tool for studying transcriptome differences between the two sexes.In this study,we identified the sex of embryos by nested amplification of bovine tooth enamel gene(Amelogenin,AMEL)sequence fragments with ultramicro DNA templates.Single female and male blastocysts of definite sex were selected as test materials and individual embryos were constructed by Smart-Seq2 amplification technology for transcriptome sequencing libraries.Illumina Hi Seq Xten high-throughput sequencing platform was applied to perform sc RNA-Seq on individual embryos,and the differentially expressed genes(DEGs)were classified by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases,and DEGs related to developmental differences between male and female blastocysts were identified.At the same time,this study also cloned and screened the CDS sequence of the gene(SLC22A14),and conducted bioinformatics analysis to detect its expression in female and male blastocysts in cattle.The main research work and results are as follows:1.Optimizing the technology system of nested amplification of bovine enamel gene(AMEL)DNA sequence fragments to identify embryo sex.Primers were designed according to the sequence of the bovine AMEL gene(AMELX and AMELY)in Gen Bank,and a nested PCR system was established by factorial method.The specificity and sensitivity of the primers were tested with sex-specific(female and male)blood genomic DNA diluted by 10-fold serial dilution method,with the help of a micromanipulator,5 to 8embryonic blastomeres were aspirated with a microscopic glass needle,and were directly cleaved by proteinase K and used as nested PCR templates for gender identification.The sensitivity of the bovine embryo sex identification system established in this study can reach about 10 pg(3-4 cells),and the g DNA present in the sample is used to amplify its corresponding gene instead of using the traditional DNA isolation method.The accuracy rate of identifying embryo sex was 100%(16/16).2.Construct transcriptome sequencing libraries of single bovine female and male blastocysts and perform sc RNA-Seq.using an optimized embryo sex identification system to determine the sex of blastocysts in cattle,constructing single embryo sequencing libraries using Smart-Seq2 amplification technology with sexdetermining blastocysts as test material,applying Illumina Hi Seq Xten high-throughput sequencing platform sc RNA-Seq was performed,and transcriptome data were screened,assembled and compared using HISAT2,String Tie,and DEseq2 packages,and gene differential expression analysis,GO functional analysis and KEGG pathway analysis were performed.A total of 6 160 DEGs were identified between the two groups,including 675 female-specific genes and 305 male-specific genes.GO annotation revealed that femalespecific genes were significantly annotated in the entries of nucleotide binding,signal transduction regulation,and multicellular biogenesis,and male-specific genes were significantly annotated in the entries of oxidative phosphorylation,mitochondria,and ribosomes;KEGG enrichment analysis identified seven entries associated with female KEGG enrichment analysis identified seven pathways associated with differences in female and male blastocyst development,namely metabolic pathway,glycolysis,pentose phosphate pathway,cellular senescence,oxidative phosphorylation,signaling pathway regulating stem cell pluripotency.We also screened nine differentially expressed genes: SLC22A14、DNMT3A、GADD45G、MRPL1、 G6PD、HPRT1、FHL2、 CAPN6、 FBP1 using GO and KEGG results.3.Cloning of the CDS region of the differentially expressed gene SLC22A14 and detection of its expression in bovine female and male blastocysts.The c DNAs of individual embryos were synthesized and preamplified by a one-step method,and the CDS sequences of the bovine SLC22A14 gene were obtained by cloning and sequencing.The results showed that the CDS region of the bovine SLC22A14 gene totaled 1941 bp and encoded 646 amino acids;bioinformatics analysis showed that SLC22A14 protein was an unstable hydrophobic protein with 12 transmembrane structural domains and 56 phosphorylation sites and no signal peptide.Real-time fluorescent quantitative PCR results showed that the expression of SLC22A14 gene in male blastocysts was significantly higher than that in female blastocysts(P<0.05).In summary,in this study,we successfully constructed transcriptome sequencing libraries of single bovine female and male blastocysts and obtained transcriptome data of bovine blastocysts using sc RNA-Seq technology.The significant enrichment of female-specific genes in pathways associated with the metabolic rate of the embryo,especially those related to the control of oxygen radical quantity and glucose consumption,and the significant enrichment of male-specific genes in pathways related to oxidative phosphorylation,mitochondria,and ribosomes,resulted in faster development of male blastocysts compared to female blastocysts.In addition,real-time fluorescent quantitative PCR results showed that the expression level of SLC22A14 gene in male blastocysts was significantly higher than that in female blastocysts,indicating that the up-regulated expression of SLC22A14 gene in male blastocysts may be one of the reasons for the faster development of male blastocysts.This will provide a reference for further research on the mechanism of differential proliferation and differentiation of female and male embryos. |
PDF Full Text Request