基于转录组数据挖掘外源褪黑激素影响水貂卵巢发育的分子机制

doi:10.11843/j.issn.0366-6964.2024.02.018

摘要/Abstract

摘要： 旨在探究外源褪黑激素(melatonin,MLT)影响水貂卵巢发育的分子机制。本研究随机选取相同饲养条件下、体重相似的200只3月龄健康白色雌性水貂,均分为试验组和对照组。试验组埋植1粒含MLT 18 mg·粒^-1的埋植剂,对照组埋植1粒不含MLT的空粒,当水貂处于准备配种期时随机采集试验组和对照组各4只水貂的卵巢,然后采用Trizol法进行总RNA提取,构建cDNA文库,并利用Illumina平台测序后进行生物信息学分析,对差异表达基因进行GO功能分类和KEGG通路注释,寻找MLT调控卵巢发育的相关候选基因和代谢通路。为进一步验证测序数据的可靠性,随机选取6个差异表达基因进行qPCR验证。转录组结果显示,试验组和对照组的SNPs位点均在120 000个以上,且均主要出现在基因区,两组的SNP突变类型均为转换型多于颠换型和杂合型;试验组与对照组相比,差异表达基因发生SE事件的数量最多;本试验共获得9 631个新基因,其中有1 323个、1 317个新基因分别注释到 TrEMBL 和NR数据库,注释比例明显高于其他数据。试验组与对照组相比共发现206个差异表达基因(其中有36个未被注释的基因),69个为上调基因,137个为下调基因;GO功能富集显示,共计7个差异表达基因富集于生殖过程;KEGG通路富集结果显示,共计7个差异表达基因富集于5条与繁殖相关的通路。综合GO功能分类和KEGG通路注释结果分析,共筛选得到4个与MLT影响卵巢发育相关的候选基因,均为上调基因,分别是KRT19、GSTT1、ALAS1和NR4A1。本研究通过对MLT埋植和未埋植MLT的准备配种期水貂卵巢进行转录组学分析,挖掘出MLT可能通过调节KRT19、GSTT1、ALAS1、NR4A1等基因促进卵巢发育,研究结果对水貂的育种工作提供了理论基础。

关键词: 水貂, 褪黑激素, 转录组, 卵巢发育, 差异表达基因

Abstract: The study aimed to explore the molecular mechanism of exogenous melatonin (MLT) on mink ovarian development. In this study,200 female white and healthy 3 months old minks with similar body weight were selected. They were divided into experimental groups and control groups. The individuals in experimental group was implanted with an implant containing 18 mg·grain^-1 MLT, while the individuals in control group was implanted with an empty implant without MLT. When the minks were in preparation for breeding, the ovaries of 4 minks in the experimental group and 4 minks in the control group were randomly collected.Then the total RNA was extracted by the Trizol method, and the cDNA library was constructed. The Illumina platform was used for sequencing and bioinformatics analysis and differentially expressed genes screening, and GO functional annotation analysis and KEGG analysis of differentially expressed genes were performed to find out the candidate genes and metabolic pathways related to MLT regulating ovarian development.To further verify the reliability of sequencing data, 6 differentially expressed genes were randomly selected for qPCR verification. The transcriptome results showed that both the experimental and the control groups had more than 120 000 SNP sites, and the SNP sites both were predominantly found in the gene region. The SNP mutation types of conversion were more than transversion and heterozygous in both groups. The number of SE events in the experimental group was the largest. A total of 9 631 new genes was obtained in this study, of which 1 323 and 1 317 new genes were annotated respectively in TrEMBL and NR databases, the annotation ratio was significantly higher than that of other data. The annotation ratio was significantly higher than that of other data. A total of 206 differentially expressed genes were found between the experimental group and the control group, including 36 unannotated genes, 69 up-regulated genes, and 137 down-regulated genes. GO functional enrichment showed that a total of 7 differentially expressed genes were enriched in the reproductive process. KEGG pathway enrichment results showed that a total of 7 differential genes was enriched in 5 pathways related to reproduction. Based on the results of GO functional classification and KEGG pathway annotation analysis, a total of 4 regulatory genes related to MLT affecting ovarian development were screened, all of which were up-regulated genes, including KRT19, GSTT1,ALAS1 and NR4A1. In this study, we performed transcriptome analysis of mink ovaries with or without MLT implantation during the pre-mating stage and found that MLT may promote ovarian development by regulating KRT19, GSTT1, ALAS1, and NR4A1.The results provide a theoretical basis for mink breeding work.

Key words: mink, melatonin, transcriptome, ovarian development, differentially expressed genes

中图分类号:

S814

高娅薇, 彭弟, 孙朝阳, 晏子越, 崔凯, 马泽芳. 基于转录组数据挖掘外源褪黑激素影响水貂卵巢发育的分子机制[J]. 畜牧兽医学报, 2024, 55(2): 607-618.

GAO Yawei, PENG Di, SUN Zhaoyang, YAN Ziyue, CUI Kai, MA Zefang. Mining the Molecular Mechanism of Exogenous Melatonin Affecting the Development of Mink Ovary Based on Transcriptome Data[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 607-618.

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