基于高通量测序的犏牛囊胚玻璃化冷冻损伤机制研究

doi:10.11843/j.issn.0366-6964.2017.10.010

摘要/Abstract

摘要：

本研究旨在利用RNA-seq技术从转录组学角度探讨犏牛囊胚玻璃化冷冻的损伤机制。采用体外受精（IVF）技术生产犏牛胚胎（奶牛精子×牦牛卵子），以新鲜犏牛囊胚和经玻璃化冷冻复苏后的冻融囊胚为研究对象，提取总RNA，使用Smart-seq2方法进行扩增并构建文库进行高通量测序。结果表明：新鲜囊胚和冻融囊胚样本经深度测序后，分别得到51 099 116和54 192 358条Clean Reads，其中80%以上Clean Reads被比对上参考基因组。冻融囊胚相对于新鲜囊胚共筛选出11 196个差异表达基因（DEGs），其中上调表达基因有7 570个，下调表达基因有3 626个。新鲜囊胚和冻融囊胚可变剪切数分别有49 016和64 352个；SNP位点数量分别为116 681和224 750个。差异基因GO功能分析主要富集于生物过程、细胞组成和分子功能3大类；KEGG注释结果表明，冻融囊胚与新鲜囊胚间共涉及318条通路，其中14条通路显著富集。推测犏牛囊胚冷冻损伤机制可能是通过剪接体、泛素介导蛋白水解和内质网蛋白加工等通路的相互协调以及Prp19、Prp4、CaM、PKA、Hsp70、CCL2等基因的差异表达来发挥生物学作用。综上表明，本研究利用RNA-seq技术首次从转录组学角度探讨了犏牛囊胚玻璃化冷冻的损伤机制，为完善胚胎的玻璃化冷冻提供新思路，同时也为进一步完善犏牛基因结构信息和胚胎玻璃化冷冻相关的新基因提供理论基础。

Abstract:

This study was conducted to explore the molecular mechanism for vitrified-thawed damage of the cross-bred blastocysts of the yak by RNA-seq. The cross-bred blastocysts were derived from yak oocytes in vitro fertilized with cattle spermatozoa. Total RNA were extracted from these fresh blastocysts (FRB) and vitrified-thawed blastocysts (VTB) and cDNA was amplified via the Smart-seq2 method, and finally RNA libraries were constructed and sequenced. We obtained 51 099 116 and 54 192 358 clean reads from FRB and VTB, respectively, of which more than 80% were mapped to referenced genome. A total of 11 196 differentially expressed genes (DEGs) were detected, in which 7 570 were upregulated, and 3 626 were downregulated in VTB vs. FRB. The FRB and VTB had 49 016 and 64 352 alternative splicing, 16 681 and 224 750 putative SNPs, respectively. GO enrichment analysis of DEGs showed that they were enriched in molecular function, cellular component, and biological process. The KEGG analysis of DEGs showed that there were 318 pathways in VTB and FRB, of which 14 pathways were significantly enriched. We speculated that the coordination between Spliceosome, Ubiquitin mediated proteolysis and protein processing in endoplasmic reticulum pathway's etc. and the differential expression of Prp19, Prp4, CaM, PKA, Hsp70 and CCL2 might play an important role in the mechanism for vitrified-thawed damage. This is the first report to explore the mechanism for damage of VTB using high-throughput sequencing, which might serve as a key resource for improving embryo vitrification technology. The study also provided valuable information to study gene structure of cross-bred yak, and discover new genes related to vitrification damage of embryos.

中图分类号:

S823.8⁺5.2

郑杰, 蒲思颖, 杨远潇, 王琴, 杨绕芬, 字向东. 基于高通量测序的犏牛囊胚玻璃化冷冻损伤机制研究[J]. 畜牧兽医学报, 2017, 48(10): 1871-1881.

ZHENG Jie, PU Si-ying, YANG Yuan-xiao, WANG Qin, YANG Rao-fen, ZI Xiang-dong. Exploring Mechanism for Vitrification Damage of the Cross-bred Blastocysts of the Yak via High-throughput Sequencing[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(10): 1871-1881.

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https://www.xmsyxb.com/CN/Y2017/V48/I10/1871

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