影响山羊产羔数lncRNA的筛选及功能分析

doi:10.11843/j.issn.0366-6964.2020.08.009

摘要/Abstract

摘要： 旨在分析高、低繁殖力川中黑山羊卵巢组织中长链非编码RNA（long non-coding RNA，lncRNA）的表达谱，探讨lncRNA与山羊生殖调控的关系，为解释lncRNA在山羊繁殖调控中的分子机制提供理论依据。本研究选择3胎以上的10头健康川中母羊（3.5~4.5岁），将其分为高繁组和低繁组。高繁组为每胎产羔数在2头以上的母羊（n=6），低繁组为每胎产羔数为1头的母羊（n=4）。同期发情后，采集高繁和低繁山羊的卵巢样品，并从中提取总RNA构建测序文库，通过HigSeq X ten平台进行测序，筛选出差异lncRNAs，通过对差异显著的lncRNAs与mRNAs位置关系以及结合能的判定预测顺式作用的靶基因，并对其进行GO和KEGG通路分析。最后，随机抽取6个差异lncRNAs进行实时荧光定量验证。结果表明，本研究共鉴定出168个差异表达lncRNAs，其中，163个lncRNAs在高繁组卵巢显著上调，5个lncRNAs在低繁组卵巢显著上调。筛选出的差异表达lncRNAs可能为调控山羊繁殖力的候选lncRNAs，包括ENSCHIG00000001479、ENSCHIG00000002617和ENSCHIG00000002622等。这些候选lncRNAs的靶基因（MYC、CDH2、FGF9、PPARGC1A等）主要富集于Jak-STAT、细胞黏附分子及AMPK等信号通路，ENSCHIG00000001479的靶基因MYC参与了Jak-STAT、TGF-β和MAPK通路；ENSCHIG00000002617和ENSCHIG00000002622的靶基因CDH2参与了细胞黏附分子通路。通过qRT-PCR验证发现，定量结果与测序结果基本一致。研究结果推断，ENSCHIG00000001479、ENSCHIG00000002617和ENSCHIG00000002622等lncRNAs可能对山羊生殖发育过程中的卵巢发育及排卵等进程具有重要的调控作用，认为鉴定出的差异表达lncRNAs与山羊产羔数有关。本研究利用RNA-Seq技术筛选高、低繁殖力山羊的差异表达lncRNAs，并对其进行GO和KEGG功能分析，为探讨山羊产羔数的繁殖机制提供更完善的转录组数据。

关键词: 川中黑山羊, 产羔数, 卵巢, 高通量测序

Abstract: The objectives of this study were to explore the correlation between lncRNAs and the reproductive regulation for goat by analyzing the expression profile of lncRNAs in ovarian tissue of Chuanzhong Black goats with high and low fecundity, and to provide the base for further interpreting the molecule mechanism of lncRNA in reproduction of goats. A total of 10 healthy Chuanzhong female goats (3.5 to 4.5 years old) with more than 3 litters were divided into high fecundity group and low fecundity group. The litter size of female goats in high fecundity group (n=6) were more than 2 per litter, the litter size of female goats in low fecundity group (n=4) were only 1 per litter. Ovary tissue of high and low fecundity goats were collected to construct the RNA sequencing libraries by extracting total RNA after estrus synchronization. Total RNA sequencing was performed by HigSeq X ten platform and differentially expressed lncRNAs were screened. Then the cis-targeted genes of lncRNAs were predicted by exploring the positional relation and binding energy between lncRNAs and mRNAs, and the GO and KEGG pathway enrichment analysis were also performed. Finally, 6 randomly selected differentially expressed lncRNAs were verified by quantitative real-time PCR. The results showed that 168 differentially expressed lncRNAs were identified. Among them, 163 lncRNAs were significantly up-regulated in goat ovaries in high fecundity group and 5 lncRNAs were significantly up-regulated in goat ovaries in low fecundity group. The screened differentially expressed lncRNAs included ENSCHIG00000001479, ENSCHIG00000002617, and ENSCHIG00000002622, which might be candidate lncRNAs for regulating fecundity of goats. The target genes of candidate lncRNAs (MYC, CDH2, FGF9, PPARGC1A, etc) were mainly involved in Janus kinase/signal transducer and activator of transcription (Jak-STAT), cell adhesion molecules, adenosine monophosphate-activated protein kinase (AMPK) signaling pathways. The target gene MYC of ENSCHIG-00000001479 participated in Jak-STAT, transforming growth factor beta (TGF-β), MAPK pathways, and the target gene CDH2 of ENSCHIG00000002617 and ENSCHIG00000002622 participated in cell adhesion molecules pathway. The results of qRT-PCR were basically consistent with the sequencing results. It was inferred that those lncRNAs(such as ENSCHIG00000001479, ENSCHIG00000002617 and ENSCHIG00000002622) might play an important role in the regulation of ovarian development and ovulation of goats. It was considered that differentially expressed lncRNAs identified were associated with litter size of goats. In this study, RNA-Seq was used to obtain and analyze differentially expressed lncRNAs in high and low fecundity goats, and GO and KEGG functional analysis was performed. This provided more transcriptome data for further exploring the reproductive mechanisms of litter size of goats.

Key words: Chuanzhong Black goats, litter size, ovary, high throughput sequencing

中图分类号:

S827.3

李耀坤, 许香萍, 陆婷婷, 练志全, 邹娴, 赵智锋, 柳广斌, 刘德武, 邓铭. 影响山羊产羔数lncRNA的筛选及功能分析[J]. 畜牧兽医学报, 2020, 51(8): 1853-1865.

LI Yaokun, XU Xiangping, LU Tingting, LIAN Zhiquan, ZOU Xian, ZHAO Zhifeng, LIU Guangbin, LIU Dewu, DENG Ming. Screening and Functional Analysis of lncRNA Affecting Litter Size in Goats[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(8): 1853-1865.

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