谷胱甘肽促进牛体外受精胚胎发育的转录组初探

Transcriptome of Bovine IVF Embryos Treated with Glutathione

旨在探究牛体外受精胚胎的培养液中添加3mmol·L^(-1)谷胱甘肽(Glutathione,GSH)后,8~16-细胞期和桑椹期的牛体外胚胎在转录组水平上的变化。收集8~16-细胞期和桑椹期的体外受精胚胎,构建文库,通过Illumina平台进行测序;筛选差异基因,并进行功能注释和代谢途径分析;采用定量PCR对10个基因的表达水平进行检测,以验证测序结果的准确性;在获得的新转录本中挑选3组进行RNA和蛋白结构的预测。通过OOSP1、THAP9等10个基因的定量检测、测序结果的质量评价(碱基错误率、Q20、Q30、GC含量)、reads与牛基因组的比对及其在基因组的分布统计分析,均说明该测序结果准确、可靠。测序获得差异表达基因4 100个(其中,处理组8~16-细胞期胚胎和桑椹胚中3 952个,对照组中884个),已知基因2 225个,未知基因1 875个。这些差异基因主要富集到与ATP合成、呼吸链、DNA合成、翻译过程和物质代谢相关的84条GO terms上,参与细胞黏附、柠檬酸循环、谷胱甘肽代谢、溶酶体以及氨基酸代谢等27条通路。另外,与已知的转录本相比,5个新转录本中均发现不同长度的新外显子;预测的蛋白结构中除包含各自的保守结构域(丝氨酸/苏氨酸蛋白激酶的催化结构域、MAD同源结构域)外,还发现2个新的蛋白结构域(低复杂区、dwarfins蛋白家族B结构域)。综上表明,在培养液中添加GSH能导致胚胎转录谱的变化,显著提高胚胎的体外发育能力。

To elucidate the effects of exogenous glutathione（GSH）on embryos transcriptome,bovine IVF embryos at 8-16-cell and morula stage were collected for high-throughput RNA sequencing（RNA-seq）.Quantitative determination of genes expression was used to verify the RNA-seq data.Gene ontology and KEGG pathway enrichment analysis of differential expression genes were performed.The accuracy and validity of RNA-seq data were confirmed with combination of error rate of base,mapping of reads to bovine genome,distribution region analysis and consistency between qPCR and RNA-seq results of ten genes.A total of 4 100 genes were differentially expressed between embryos at two stages and 3 952 genes were identified as expressed in embryos treated with GSH.A high enrichment of genes involved in 84 GO terms,such as ATP synthesis,respiratory chain,DNA synthesis and translation.Pathway analysis revealed 27 pathways involved in cell adhesion,TCA cycle,GSH metabolism and amino acid metabolism.Additionally,2known conserved domains（S-TKc,MH1）and 2new domains（LCR,DWB）were found in 5new tran-scripts by prediction,compared with known transcripts.To sum up,GSH treatment can result into the comprehensive change at transcriptional level of bovine IVF embryos and improve their development consequently.