敲除热敏瞬时受体电位锚蛋白1(BmTRPA1)基因后家蚕蛹脑-咽下神经节复合体的转录组学分析

doi:10.16380/j.kcxb.2025.05.005

昆虫学报 ›› 2025, Vol. 68 ›› Issue (5): 585-595.doi: 10.16380/j.kcxb.2025.05.005

敲除热敏瞬时受体电位锚蛋白1(BmTRPA1)基因后家蚕蛹脑-咽下神经节复合体的转录组学分析

白新宇1, 冯嘉伟1, 张明蔚1, 包玫1, 邢东旭2, 杨婉莹1,*

(1. 华南农业大学动物科学学院, 广东省农业动物基因组学与分子育种重点实验室, 广州 510642; 2. 广东省农业科学院蚕业与农产品加工研究所, 广州 510610)

出版日期:2025-05-20 发布日期:2025-06-06

Transcriptomic analysis of the pupal brain-suboesophageal ganglion complex after knocking out the thermal sensitive transient receptor potential ankyrin 1 (BmTRPA1) in Bombyx mori

BAI Xin-Yu¹, FENG Jia-Wei¹, ZHANG Ming-Wei¹, BAO Mei¹, XING Dong-Xu², YANG Wan-Ying^1,*

(1. Agricultural Animal Genomics and Molecular Breeding Laboratory of Guangdong Province, College of Animal Science, South China Agricultural University, Guangzhou 510642, China; 2. Institute of Sericulture and Agricultural Products Processing, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, China)

Online:2025-05-20 Published:2025-06-06

摘要/Abstract

摘要： 【目的】二化性家蚕Bombyx mori卵在25 ℃下催青时，热敏瞬时受体电位锚蛋白1(TRPA1)被激活，随后可能激活一条与滞育激素(diapause hormone, DH)释放相关的未知信号通路。本研究旨在研究家蚕BmTRPA1调控DH分泌的分子机制，以及BmTRPA1与GABAergic信号通路的关系。【方法】利用CRISPR/Cas9敲除25 ℃下催青的家蚕BmTRPA1基因，通过转录组测序筛选野生型(WT)和BmTRPA1^-/-突变型家蚕在蛹期第3天脑-咽下神经节(brain-suboesophagealganglion, Br-SG)复合体中的差异表达基因；对差异表达基因和特异表达基因分别进行GO分类和KEGG功能富集分析； qRT-PCR检测6个差异表达基因、调控GABAergic信号通路10个关键基因和神经肽基因K5的相对表达量。【结果】WT的Br-SG复合体中有13 039个基因表达， BmTRPA1^-/-Br-SG复合体中有12 937个基因表达，其中12 484个基因在WT和BmTRPA1^-/-Br-SG复合体中共表达，有555个基因在WT中Br-SG复合体特异表达，453个基因在BmTRPA1^-/-Br-SG复合体中特异表达；相比于WT Br-SG复合体，BmTRPA1^-/-Br-SG复合体有32个基因表达上调，13个基因表达下调。这些差异表达基因主要富集在正反馈调节过程、代谢、应激和细胞调控等关键生命活动过程，其中富集于蛋白质转运过程与内分泌的差异表达蛋白GAT属于质膜GABA转运蛋白，是调控家蚕DH释放的关键蛋白。qRT-PCR检测结果显示， GAT和K5在BmTRPA1^-/-Br-SG复合体中的相对表达量与WT Br-SG复合体中的相比显著下调。【结论】本研究在组学水平上证明，敲除TRPA1将降低GAT的表达，从而调控GABAergic信号通路，减少DH释放。研究结果为后续探索家蚕响应环境温度诱导滞育发生的分子调控机制提供了靶标蛋白和数据参考。

关键词: 家蚕, 滞育, 瞬时受体电位锚蛋白, 转录组, 脑-咽下神经节复合体

Abstract: 【Aim】 The thermal sensitive transient receptor potential ankyrin 1 (TRPA1) is activated when the eggs of biovoltine Bombyx mori are stimulated under 25 ℃, which may then activate an unknown signaling pathway related to the release of diapause hormone (DH). The aim of this study is to investigate the molecular mechanism of BmTRPA1 regulating DH secretion and the relationship between BmTRPA1 and GABAergic signaling pathway in B. mori. 【Methods】 CRISPR/Cas9 was used to knockout BmTRPA1 in B. mori stimulated under 25 ℃. Transcriptome sequencing was used to screen the differentially expressed genes in the brain-suboesophageal ganglion (Br-SG) complex between B. mori wild type (WT) and BmTRPA1 mutant type BmTRPA1^-/-on the day-3 of the pupal stage. GO classification and KEGG functional enrichment analysis were performed for the differentially expressed genes and specifically expressed genes. The relative expression levels of six differentialy expressed genes, 10 key genes in GABAergic signaling pathway and neuropeptide gene K5 were detected by qRT-PCR. 【Results】 A total of 13 039 genes were expressed in the Br-SG complex of WT and 12 937 genes were expressed in the Br-SG complex of BmTRPA1^-/-, among them 12 484 genes were co-expressed in the Br-SG complex of WT and BmTRPA1^-/-, 555 genes were specifically expressed in the Br-SG complex of WT and 453 genes were specifically expressed in the Br-SG complex of BmTRPA1^-/-. Compared with Br-SG complex of WT, 32 genes in the Br-SG complex of BmTRPA1^-/- were up-regulated and 13 genes in the Br-SG complex of BmTRPA1^-/- were down-regulated. These differentially expressed genes were mainly enriched in key life processes such as positive feedback regulation, metabolism, response to stimulus, and cell regulation. The differentially expressed protein GAT, which is enriched in the process of protein transport and endocrine, belongs to the plasma membrane GABA transporter and is a key protein in regulating the release of DH from B. mori. qRT-PCR result showed that the relative expression levels of GAT and K5 in the Br-SG complex of BmTRPA1^-/-were significantly down-regulated as compared with those in the Br-SG complex of WT. 【Conclusion】 This omics level study showed that the knockout of TRPA1 can reduce the expression of GAT, thereby regulating GABAergic signaling pathway and reducing DH release. The results provide target proteins and data references for further exploration of molecular regulatory mechanisms of diapause induced by B. mori in response to ambient temperature.

Key words: Bombyx mori, diapause, transient receptor potential ankyrin, transcriptome, brain-suboesophageal ganglion complex

白新宇, 冯嘉伟, 张明蔚, 包玫, 邢东旭, 杨婉莹. 敲除热敏瞬时受体电位锚蛋白1(BmTRPA1)基因后家蚕蛹脑-咽下神经节复合体的转录组学分析[J]. 昆虫学报, 2025, 68(5): 585-595.

BAI Xin-Yu, FENG Jia-Wei, ZHANG Ming-Wei, BAO Mei, XING Dong-Xu, YANG Wan-Ying. Transcriptomic analysis of the pupal brain-suboesophageal ganglion complex after knocking out the thermal sensitive transient receptor potential ankyrin 1 (BmTRPA1) in Bombyx mori[J]. Acta Entomologica Sinica, 2025, 68(5): 585-595.

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敲除热敏瞬时受体电位锚蛋白1(BmTRPA1)基因后家蚕蛹脑-咽下神经节复合体的转录组学分析

Transcriptomic analysis of the pupal brain-suboesophageal ganglion complex after knocking out the thermal sensitive transient receptor potential ankyrin 1 (BmTRPA1) in Bombyx mori

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