昆虫学报 ›› 2021, Vol. 64 ›› Issue (11): 1235-1243.doi: 10.16380/j.kcxb.2021.11.001

• 研究论文 •    下一篇

家蚕5龄幼虫精巢和卵巢microRNA芯片及转录组比较分析

张冰1, 李娜1, 阚云超1,2,*   

  1. (1. 南阳师范学院, 河南省伏牛山昆虫生物学重点实验室, 昆虫生物反应器河南省工程实验室, 河南南阳 473061; 2. 河南大学生命科学学院, 河南开封 475004)
  • 出版日期:2021-11-20 发布日期:2021-11-03

Comparative microRNA microarray and transcriptome analyses of the testis and ovary of the 5th instar larvae of the silkworm, Bombyx mori

 ZHANG Bing1, LI Na1, KAN Yun-Chao1,2,*   

  1.  (1. Henan Key Laboratory of Insect Biology in Funiu Mountain, Henan Provincial Engineering Laboratory of Insects Bio-reactor, Nanyang Normal University, Nanyang, Henan 473061, China; 2. School of Life Sciences, Henan University, Kaifeng, Henan 475004, China)
  • Online:2021-11-20 Published:2021-11-03

摘要: 【目的】本研究旨在通过对家蚕Bombyx mori 5龄幼虫精巢和卵巢组织微小RNA (microRNA, miRNA)基因芯片及转录组进行分析,找到参与家蚕性腺发育相关的miRNA分子及可能的靶基因。【方法】采用新一代高通量测序平台对家蚕5龄幼虫精巢和卵巢(分别定义为Test和Control)进行miRNA基因芯片检测及转录组测序分析,根据P<0.05且log2(fold change, FC)≥2的标准,通过比较筛选出Test vs Control的差异表达miRNA;根据q≤0.05且|log2(fold change)|≥1的标准,通过比较筛选出Test vs Control的差异表达基因 (differentially expressed genes, DEGs);随机选取8个上调和12个下调差异表达miRNA,对其表达及其预测的5个靶基因进行qRT-PCR验证;对DEGs以及差异表达miRNA的靶基因进行KEGG通路富集分析。【结果】从精巢和卵巢样本中(Test vs Control)分别鉴定出68个差异表达miRNA和3 991个DEGs,其中上调和下调miRNA分别为36和32个,上调和下调DEGs分别为2 033和1 958个。差异表达miRNA的qRTPCR验证结果均与芯片数据一致。KEGG通路富集分析结果显示DEGs在新陈代谢及核糖体的信号通路显著富集。对差异表达miRNA在DEGs中的可能靶基因进行预测,结果找到了4组表达趋势相反的miRNA与靶基因:分别是bmo-miR-2774a与LOC101745556;bmo-miR-92b与LOC101735954以及bmo-miR-3266与LOC733130和LOC778467;1组表达趋势一致的miRNA与靶基因:bmo-miR-3321与LOC101744895。5个靶基因的qRT-PCR验证结果与转录组测序结果一致。【结论】本研究获得了家蚕5龄幼虫精巢和卵巢转录组及miRNA芯片数据,筛选并验证了4组差异表达和1组一致表达miRNA及潜在靶基因,为探究家蚕精巢和卵巢发育差异奠定了基础。

关键词: 家蚕, 精巢, 卵巢, miRNA, miRNA芯片, 转录组

Abstract: 【Aim】 This study aims to identify microRNA (miRNA) and possible target genes involved in the gonadal development of Bombyx mori through miRNA microarray and transcriptome analyses of the testis and ovary of the 5th instar larvae of B. mori. 【Methods】 A new generation of highthroughput sequencing platform was used to perform miRNA microarray analysis and transcriptome sequencing of the testis and ovary (defined as test and control, respectively) of the 5th instar larvae of B. mori. According to the criteria of P<0.05 and log2(fold change, FC)≥2, the differentially expressed miRNAs of test vs control were screened. According to the criteria of q≤0.05 and [log2 (fold change)]≥1, the differentially expressed genes (DEGs) of test vs control were screened by comparative analysis. Eight up-regulated and 12 down-regulated differentially expressed miRNAs were randomly selected,and their expression and their predicted five target genes were verified by qRT-PCR. The DEGs and target genes of the differentially expressed miRNAs were enriched by KEGG pathway analysis. 【Results】 Sixty-eight differentially expressed miRNAs and 3 991 DEGs were identified from the testis and ovary (test vs control). Among them, 36 and 32 miRNAs were up-regulated and down-regulated, respectively, and 2 033 and 1 958 DEGs were up-regulated and down-regulated, respectively. qRT-PCR verification results of differentially expressed miRNAs were consistent with the microarray data. KEGG pathway enrichment analysis showed that the DEGs were significantly enriched in the signal pathways involved in metabolism and ribosome. The possible target genes of differentially expressed miRNAs in the DEGs were predicted. Four groups of miRNAs and their target genes showing the opposite expression trend were found, including bmo-miR-2774a and LOC101745556, bmo-miR-92b and LOC101735954, bmo-miR-3266 and LOC733130 and LOC778467, respectively. One group of miRNA and its target gene showing the consistent expression trend was bmo-miR-3321 and LOC101744895. The results of qRT-PCR of five target genes were consistent with the results of transcriptome sequencing. 【Conclusion】 In this study, the transcriptome and miRNA microarray data of the testis and ovary of the 5th instar larvae of B. mori have been obtained, and four groups of miRNAs and their target genes showing the opposite expression trend and one group of miRNA and its target gene showing the consistent expression trend screened and verified, laying a foundation for exploring the development differences between the testis and ovary of B. mori.

Key words: Bombyx mori, testis, ovary, miRNA, miRNA microarray, transcriptome