中国农业科学 ›› 2018, Vol. 51 ›› Issue (8): 1577-1589.doi: 10.3864/j.issn.0578-1752.2018.08.015

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

基于RNA-Seq技术的牦牛体外受精胚胎发育转录组分析

字向东1,罗斌1,夏威1,郑玉才1,熊显荣1,李键1,钟金城2,朱江江2,张正帆1

 
  

  1. 1西南民族大学生命科学与技术学院, 成都 610041;2西南民族大学青藏高原研究院, 成都 610041
  • 收稿日期:2017-07-04 出版日期:2018-04-16 发布日期:2018-04-16
  • 作者简介:字向东,Tel:028-85528868;E-mail:zixd2000@sina.com
  • 基金资助:
    国家“973”前期专项(2007CB116204)、中央高校基本科研业务费专项资金(2015NZYTD02)

Transcriptomic Analysis of IVF Embryonic Development in the Yak (Bos grunniens) Via RNA-Seq

ZI XiangDong1, LUO Bin1, XIA Wei1, ZHENG YuCai1, XIONG XianRong1, LI Jian1, ZHONG JinCheng2ZHU JiangJiang2, ZHANG ZhengFan1   

  1. 1College of Life Science and Technology, Southwest Minzu University, Chengdu 610041; 2Institute of Qinghai-Tibetan Plateau, Southwest Minzu University, Chengdu 610041
  • Received:2017-07-04 Online:2018-04-16 Published:2018-04-16

摘要: 【目的】探明不同发育阶段牦牛体外受精(IVF)胚胎的转录组差异,了解差异表达基因(DEG)在功能、分类和代谢通路的差异,为揭示牦牛早期胚胎发育调控机制,促进牦牛胚胎体外生产技术的发展提供理论基础。【方法】以IVF技术生产的牦牛2-细胞、4-细胞、8-细胞、桑椹胚和囊胚5个发育阶段的胚胎为样本分别提取总RNA,采用Smart-Seq2扩增技术构建5个测序文库,应用HiSeqTM 2500高通量测序技术进行转录组测序,获得的有效序列进行功能注释及相关生物信息学分析。【结果】牦牛IVF后的卵裂率和囊胚率分别为69.3%和26.2%。2-细胞、4-细胞、8-细胞、桑椹胚和囊胚5个发育阶段的牦牛胚胎Clean reads为47 355 57050 855 888条,其中有85.65%90.02%reads能比对到牦牛参考基因组序列上;8-细胞的胚胎比对上的转录本最多(14 893),而囊胚比对上的转录本最少(9 827)。牦牛胚胎转录本主要有5种可变剪接类型,转录起始区域可变剪接(TSS)和转录结束区域可变剪接(TTS)所占比例最大牦牛2-细胞、4-细胞、8-细胞、桑椹胚和囊胚基因组分别有116 601、234 131、196 420、70 841和94 840个位点存在单核苷酸多态性(SNP)。在4-细胞、8-细胞、桑椹胚、囊胚期开始表达的基因分别有1 221、1 116、142和564个;随着胚胎发育的进行,BMP15KITGDF9STAT3ZP3ZP4等母源基因的表达量逐渐减少,而SARSIL18、ACO2、TXN2、ATP5BPCGF4UBE3AMAPK13SNURFJUP等胚胎基因的表达量则逐渐增加。|log2ratio| ≥1且Q-value < 0.05为筛选标准,在2-细胞和4-细胞胚胎、4-细胞和8-细胞胚胎、8-细胞胚胎和桑椹胚以及桑椹胚和囊胚比对中分别筛选到6 922、7 601、8 071和10 555个DEGs。GO分析表明,4个发育阶段的DEGs归类注释都涉及生物过程(BP)、细胞组分(CC)和分子功能(MF)3大类62个二级条目。通过KEGG pathway数据库分析,2-细胞和4-细胞期胚胎的DEGs参与到308条通路中,显著富集剪接体、RNA转运和泛素介导的蛋白水解等11条通路;4-细胞和8-细胞胚胎的DEGs参与到310条通路,显著富集嗅觉转导、神经活性配体-受体互作和核苷酸切除修复等9条通路;8-细胞期胚胎桑椹胚的DEGs参与到316条通路,显著富集嗅觉转导、泛素介导的蛋白水解和神经活性配体-受体互作等10条通路;桑椹胚和囊胚的DEGs参与到315条通路,显著富集剪接体和RNA转运2条通路。【结论】利用高通量测序技术对牦牛IVF胚胎不同发育阶段的转录组进行测序和分析,揭示了牦牛胚胎发育不同阶段差异表达基因的数量,获得了差异表达基因的功能、分类和代谢通路。为丰富牦牛胚胎转录组信息,揭示牦牛胚胎发育分子调控机制及完善其胚胎体外培养技术研究奠定基础。

关键词: 牦牛, 体外受精, 胚胎, 转录组, RNA-Seq

Abstract: 【Objective】The objectives of this study were to investigate the transcriptome differences and identify function, classification and metabolic pathways of the differentially expressed genes (DEG) at different developmental stages of yak embryos derived from in vitro fertilization (IVF), which are necessary to better understand the mechanism that regulates embryonic development and provide theoretical basis for improving in vitro embryo production in the yak (Bos grunniens). 【Method】Total RNA was extracted from IVF derived yak embryos at 2-cell, 4-cell, 8-cell, morula and blastocyst stages and amplified via the Smart-seq2 method, and the constructed RNA libraries were sequenced using the HiSeqTM2500 high-throughput sequencing method. 【Result】After IVF, the average cleavage rates and blastocyst rates were 69.3% and 26.2%, respectively. A total of 47 355 570 to 50 855 888 clean reads were obtained from 2-cell, 4-cell, 8-cell, morula and blastocyst stages, respectively, of which, 85.65% to 90.02% were covered in the yak reference genome. In total, the number of transcripts mapped to yak genome was highest for 8-cell (14 893) and lowest for blastocysts (9 827). The transcripts mainly had five patterns of alternative splice, of which, the two largest proportions were transcription start site (TSS) and transcription terminal site (TTS). The SNP numbers of the five stages of yak embryonic transcripts were 116 601, 234 131, 196 420, 70 841 and 94 840, respectively. A total of 1 221, 1 116, 142 and 564 transcripts were first detected at the 4-cell, 8-cell, morula and blastocyst stages, respectively. As embryo development proceeded, maternally derived transcripts such as BMP15, KIT, GDF9, STAT3, ZP3 and ZP4 etc.were decreased, whereas embryonic transcripts such as SARS,IL18, ACO2, TXN2, ATP5B, PCGF4, UBE3A, MAPK13, SNURF and JUP etc. were increased at specific stages. When |log2ratio| ≥1 and Q-value<0.05 were set as thresholds for identifying DEGs, a total of 6 922, 7 601, 8 071 and 10 555 DEGs were identified from 2-cell vs. 4 cell, 4-cell vs. 8-cell, 8-cell vs. morula, and morula vs. blastocyst, respectively. The GO distributions of the DEGs were classified into three categories: biological processes (BP), cellular components (CC), molecular functions (MF) with a total of 62 subcategories of two successive stages. KEGG enrichment analysis of DEGs showed that DEGs of 2-cell vs. 4-cell participated in 308 pathways, and significantly enriched in 11 pathways such as spliceosome, RNA transport and ubiquitin mediated proteolysis etc. DEGs of 4-cell vs. 8-cell participated in 310 pathways, and significantly enriched in 9 pathways such as olfactory transduction, neuroactive ligand-receptor interaction and nucleotide excision repair etc. DEGs of 8-cell vs. morula participated in 316 pathways, and significantly enriched in 10 pathways such as olfactory transduction, ubiquitin mediated proteolysis and neuroactive ligand-receptor interaction etc. DEGs of morula vs. blastocyst participated in 315 pathways, significantly enriched in 2 pathways i.e., spliceosome and RNA transport.ConclusionThis is the first study for analyzing the transcriptomes of IVF derived yak-embryos at different stages using high-throughput sequencing. A number of DEGs and their function, classification and metabolic pathways were discovered, which enriched transcriptome information for yak embryos. In addition, the results provided a foundation and reference to uncover the mechanism that regulates embryonic development and improves in vitro embryo production of the yak species. 

Key words: yak, in vitro fertilization, embryo, transcriptome, RNA-Seq