LncRNA63及其共表达基因Hr3在家蚕中的表达和功能分析

doi:10.16380/j.kcxb.2022.05.002

摘要/Abstract

摘要： 【目的】本研究旨在通过对一个新的长链非编码RNA (long non-coding RNA, lncRNA)及其共表达基因在家蚕Bombyx mori中的表达和功能进行分析，进一步阐明lncRNA在调控家蚕变态发育中的分子机制。【方法】基于前期家蚕脂肪体转录组测序数据，我们发现在蜕皮激素20E(2 μg/μL)处理5龄幼虫早期的脂肪体中lncRNA63表达显著上调，家蚕激素受体基因Hr3与之共表达。利用qRT-PCR对lncRNA63和Hr3在家蚕不同发育时期(4-5龄幼虫、蛹和成虫)、5龄幼虫不同组织(头、体壁、血淋巴、中肠、马氏管、脂肪体、丝腺、精巢和卵巢)和2 μg/μL 20E处理2, 6, 12和24 h后5龄幼虫脂肪体中的表达谱进行分析；采用核质分离检测和原位杂交技术检测lncRNA63在家蚕BmN细胞中的定位；利用dsRNA干涉家蚕5龄幼虫个体中lncRNA63的表达，并利用qRT-PCR检测RNA干涉lncRNA63对头、丝腺、脂肪体和体壁中Hr3表达的影响。【结果】qRT-PCR结果表明，lncRNA63和Hr3在家蚕羽化前的蛹末期有一个表达高峰；二者在家蚕5龄幼虫头、体壁和丝腺中都有较高表达；lncRNA63和Hr3在2 μg/μL 20E处理2, 6和12 h的家蚕5龄幼虫脂肪体中较对照组(无水乙醇处理组)都显著上调，24 h下降到与对照组相当水平，二者的表达趋势完全一致。LncRNA63主要定位于BmN细胞核，但20E可诱导lncRNA63的核质迁移。利用dsRNA干涉lncRNA63表达后，Hr3的表达显著下调。【结论】20E不仅可调控lncRNA63的表达，还可诱导lncRNA63的核质迁移；lncRNA63可通过调控共表达基因Hr3的表达参与家蚕的变态发育过程。

关键词: 家蚕, 长链非编码RNA, 脂肪体；表达分析, 变态发育

Abstract: 【Aim】 This study aims to analyze the expression and function of a new long non-coding RNA (lncRNA) and its co-expression gene in the domesticated silkworm, Bombyx mori, so as to further clarify the molecular mechanism of lncRNA in regulating the metamorphosis and development of B. mori. 【Methods】 Based on the transcriptome sequencing data of the fat body of B. mori, we found that the expression of lncRNA63 was significantly up-regulated in the fat body of the early 5th instar larvae treated with 20-hydroxyecdysone (20E)(2 μg/μL) and the hormone receptor gene Hr3 of B. mori was co-expressed with lncRNA63. qRT-PCR was used to analyze the expression profiles of lncRNA63 and Hr3 in different developmental stages (4th-5th instar larva, pupa and adult), different tissues (head, integument, hemolymph, midgut, Malpighian tubules, fat body, silk gland, testis and ovary) of the 5th instar larvae and the fat body of the 5th instar larvae of B. mori treated with 20E (2 μg/μL) for 2, 6, 12 and 24 h. The localization of lncRNA63 in BmN cells of B. mori was detected by nucleocytoplasmic separation and in situ hybridization. The expression of lncRNA63 in the 5th instar larvae of B. mori was interfered by dsRNA, and the effects of RNA interference of lncRNA63 on the expression of Hr3 in the head, silk gland, fat body and integument were detected using qRT-PCR. 【Results】 The qRT-PCR results showed that there was an expression peak of lncRNA63 and Hr3 at the end of pupal stage before eclosion, and both are highly expressed in the head, integument and silk gland of the 5th instar larvae of B. mori. The expression of lncRNA63 and Hr3 in the fat body of the 5th instar larvae of B. mori treated with 2 μg/μL 20E for 2, 6 and 12 h was significantly up-regulated as compared to the control group (ethanol treatment group) and decreased to the level of the control group at 24 h after treatment, with their expression trend being completely consistent. LncRNA63 was mainly localized in the nucleus of BmN cells, but 20E induced the nucleocytoplasmic translocation of lncRNA63. After interference of the expression of lncRNA63 with dsRNA, the expression of Hr3 was significantly down-regulated. 【Conclusion】 20E can not only regulate the expression of lncRNA63, but also induce the nucleocytoplasmic translocation of lncRNA63. LncRNA63 may participate in the metamorphosis and development of B. mori by regulating the expression of its co-expressed gene Hr3.

Key words: Bombyx mori, long non-coding RNA, fat body, expression analysis, metamorphosis and development

王博, 李苗苗, 王景娅, 胡晓玥, 阚云超, 乔惠丽. LncRNA63及其共表达基因Hr3在家蚕中的表达和功能分析[J]. 昆虫学报, 2022, 65(5): 548-557.

WANG Bo, LI Miao-Miao, WANG Jing-Ya, HU Xiao-Yue, KAN Yun-Chao, QIAO Hui-Li. Expression and functional analysis of lncRNA63 and its co-expression gene Hr3 in Bombyx mori[J]. Acta Entomologica Sinica, 2022, 65(5): 548-557.

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LncRNA63及其共表达基因Hr3在家蚕中的表达和功能分析

Expression and functional analysis of lncRNA63 and its co-expression gene Hr3 in Bombyx mori

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