伊犁鹅产蛋前后各期卵巢转录谱的构建及卵泡发育相关基因的分析

doi:10.16431/j.cnki.1671-7236.2022.12.017

摘要/Abstract

摘要： 【目的】构建产蛋前后各期伊犁鹅卵巢转录组文库，结合生物信息学分析揭示不同产蛋期伊犁鹅卵巢组织差异表达基因，并鉴定出影响鹅卵巢发育的关键基因，为伊犁鹅的繁殖调控提供理论参考。【方法】选择处于开产期（KL组）、产蛋期（CL组）及休产期（XL组）的伊犁鹅各4只，屠宰后取其卵巢组织，用于构建卵巢转录组文库。通过新基因挖掘、基因差异表达、基因注释以及蛋白互作网络分析筛选出与鹅卵泡发育相关的候选基因；随机挑选8个差异表达基因，应用实时荧光定量PCR验证其表达情况。【结果】伊犁鹅卵巢组织学结果表明，在开产期时，伊犁鹅卵巢表面有大量初级卵泡，而产蛋期卵巢则显示出卵泡的层级性，在休产期卵巢中可观察到卵泡出现向内凹陷、闭锁的现象。通过转录组测序（RNA-Seq），从构建的12个伊犁鹅卵巢cDNA文库中获得有效读数57 811 186~85 328 377条，Q30值均>93.38%，每个样品所产的测序读数于鹅参考基因组上的比对率在82.79%~89.24%。在卵巢中注释的新基因共有1 112个，单核苷酸多态性（SNP）位点总数为1 642 273~2 425 069个；SNP和插入缺失（InDel）均主要注释于内含子区域；各时期的可变剪切类型主要集中为最后1个外显子可变剪切（TSS）及第1个外显子可变剪切（TTS）。在KL vs CL、XL vs CL及XL vs KL组中分别有337、1 136和525个差异表达基因，共有差异表达基因为α2A肾上腺素能受体（ADRA2A）、表皮蛋白（CP）、非转移性黑色素瘤糖蛋白B （GPNMB）及α-1-抗胰蛋白酶样（LOC106033756）。GO功能富集分析发现，差异表达基因主要富集在肽基酪氨酸磷酸化的正调控、细胞黏附及质膜外侧等过程。KEGG通路富集分析发现，差异表达基因主要显著富集于神经活性配体-受体相互作用、ECM-受体相互作用及类固醇生物合成等。结合蛋白互作网络分析，筛选到与鹅卵巢发育相关的潜在调控因子Bruton’s酪氨酸激酶（BTK）、血小板源性生长因子受体α（PDGFRA）、整合素β3（ITGB3）等。实时荧光定量PCR结果显示，RNA-Seq结果准确可靠。【结论】本研究揭示了产蛋前后各期伊犁鹅卵巢组织中的基因表达差异，筛选到影响伊犁鹅卵巢发育的神经活性配体-受体相互作用、ECM-受体相互作用、类固醇生物合成等重要通路与BTK、PDGFRA和ITGB3等关键候选基因，为了解鹅卵巢组织调控产蛋性能的分子机制提供了理论支撑。

关键词: 伊犁鹅; 产蛋周期; 卵巢发育; 转录组

Abstract: 【Objective】 The experiment was aimed to construct a transcriptome library of Yili goose ovaries before and after egg laying, combined with bioinformatics analysis, reveal differentially expressed genes in ovarian tissues of Yili geese at different egg laying stages, identify key genes affecting goose ovarian development, and provide theoretical references for reproductive regulation of Yili geese.【Method】 Four Yili geese at the prelaying period(KL), laying period(CL), and ceased period(XL) were selected, and the ovarian tissues were obtained after slaughter for the construction of ovaries transcriptome libraries.Candidate genes associated with follicle development were screened by novel gene mining, differential gene expression, gene annotation, and protein interaction network analysis.Eight differentially expressed genes were randomly selected and Real-time quantitative PCR was used to verify their expression.【Result】 Histological results of the ovaries of Yili geese showed that a large number of primary follicles on the surface of the ovaries at the prelaying period, while the ovaries at the laying period showed a hierarchy of follicles, and follicles at the ceased period could be observed to appear sunken and atretic inwards.By RNA sequencing (RNA-Seq), 57 811 186 to 85 328 377 clean reads were obtained from the 12 constructed Yili goose ovary cDNA libraries, all with Q30 values were greater than 93.38%, and the sequencing reads produced by each sample were compared to the goose reference genome at a rate of 82.79% to 89.24%.A total of 1 112 novel genes were annotated in ovaries, and the total number of single nucleotide polymorphism loci (SNP) was ranged from 1 642 273 to 2 425 069.SNP and InDel were mainly annotated in the intron region.The alternative splicing in each period were mainly concentrated in TSS and TTS.There were 337, 1 136 and 525 differentially expressed genes in KL vs CL, XL vs CL and XL vs KL groups, respectively, and the total differentially expressed genes were adrenoceptor alpha 2A (ADRA2A), cuticular protein (CP), glycoprotein non-metastatic melanoma protein B (GPNMB) and alpha-1-antitrypsin-like (LOC106033756).GO function enrichment analysis revealed that the differential mRNAs were mainly enriched in positive regulation of peptidyl-tyrosine phosphorylation, cell adhesion and external side of plasma membrane.KEGG pathway enrichment analysis revealed that differentially expressed genes were significantly enriched mainly in neuroactive ligand-receptor interaction, ECM-receptor interaction and steroid biosynthesis.Combined with protein interaction network analysis, potential regulatory factors Bruton’s tyrosine kinase (BTK), platelet derived growth factor receptor alpha (PDGFRA), integrin subunit beta 3 (ITGB3) associated with goose ovarian development were screened.The results of Real-time quantitative PCR showed that RNA-Seq results were accurate and reliable.【Conclusion】 This study revealed the gene expression differences in ovarian tissues of Yili geese before and after egg laying, and screened out important pathways (neuroactive ligand-receptor interaction, ECM-receptor interaction, steroid biosynthesis) and key candidate genes (BTK, PDGFRA and ITGB3), which could provide theoretical support for understanding the molecular mechanism of ovarian tissues in regulating egg laying performance of Yili geese.

Key words: Yili geese; egg laying cycle; ovary development; transcriptome

中图分类号:

S835

赵晓钰, 吴盈萍, 李海英, 姚莹莹, 李家辉, 姚洋, 卢庆庆. 伊犁鹅产蛋前后各期卵巢转录谱的构建及卵泡发育相关基因的分析[J]. 中国畜牧兽医, 2022, 49(12): 4674-4687.

ZHAO Xiaoyu, WU Yingping, LI Haiying, YAO Yingying, LI Jiahui, YAO Yang, LU Qingqing. Construction of Ovarian Transcriptional Profiles and Analysis of Follicle Development-Related Genes in Yili Geese at Various Stages Before and After Egg Laying[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4674-4687.

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