RNAi沉默王浆主蛋白1(Mrjp1)基因对意大利蜜蜂工蜂学习和记忆的影响

doi:10.16380/j.kcxb.2021.10.003

摘要/Abstract

摘要：

【目的】前期研究发现经吡虫啉处理的意大利蜜蜂Apis mellifera ligustica(简称“意蜂”)工蜂学习能力下降，转录组学分析表明王浆主蛋白1(major royal jelly protein 1， MRJP1)基因在吡虫啉处理的蜜蜂脑中显著下调，MRJP1可能参与调控蜜蜂学习能力。本研究旨在采用RNA干扰(RNAinference, RNAi)技术将Mrjp1特异性沉默，验证MRJP1在意蜂工蜂嗅觉学习中的关键作用。【方法】通过克隆技术获得Mrjp1基因cDNA 序列，经测序验证后，设计引物，合成用于RNAi干扰Mrjp1基因表达的dsRNA。注射dsMrjp1的意蜂工蜂作为处理组(dsMrjp1注射组)，注射dsEGFP的意蜂工蜂作为对照组(dsEGFP注射组)，随后通过伸吻反应(proboscis extension response, PER)实验比较两组的嗅觉学习与记忆能力差异。最后采用实时荧光定量PCR(quantitative real-time PCR, qRT-PCR)检测注射dsMrjp1后意大利蜜蜂工蜂脑中Mrjp1的相对表达量。【结果】dsMrjp1注射组与dsEGFP注射组意蜂工蜂学习能力差异显著，dsMrjp1注射组意蜂工蜂的学习能力显著降低。学习后2 h，两组意蜂工蜂的记忆力无显著差异。qRT-PCR结果显示Mrjp1的表达水平在dsMrjp1注射组意蜂工蜂脑中显著低于dsEGFP注射组，表明学习能力降低的处理组意蜂脑内对应的Mrjp1表达水平也降低。【结论】通过RNAi抑制意蜂工蜂Mrjp1基因的表达后，其嗅觉学习能力受到显著性抑制，但记忆力未受到显著影响，提示Mrjp1可能是调控意蜂学习的重要基因之一。本研究结果有助于后续进一步研究蜜蜂嗅觉学习相关的分子机制。

关键词: 意大利蜜蜂, 王浆主蛋白, RNA干扰, 嗅觉学习, 记忆, 伸吻反应

Abstract:

【Aim】 In the previous studies it was found that the learning ability of Apis mellifera ligustica worker bees treated with imidacloprid is decreased, and transcriptomic analyses showed that the expression of major royal jelly protein 1 gene Mrjp1 is significantly down-regulated in the brains of bees treated with imidacloprid, suggesting that MRJP1 may be involved in olfactory learning in honeybees. This study aims to verify the crucial role of MRJP1 in olfactory learning in A. mellifera ligustica workers through silencing Mrjp1 by RNA interference (RNAi). 【Methods】 The cDNA sequence of Mrjp1 was obtained by cloning technique and validated by sequencing, and then the sequence was used for designing primers for generating dsRNA for knockdown of Mrjp1 by RNAi. The worker bees of A. mellifera ligustica injected with dsMrjp1 were assigned to the treatment group (dsMrjp1-injected group), and those injected with dsEGFP were assigned to the control group (dsEGFP-injected group). Then, the olfactory learning and memory abilities of the two groups were compared based on the proboscis extension response (PER) assay. Finally, the relative expression level of Mrjp1 in the brain of A. mellifera ligustica workers after injection of dsMrjp1 was detected by quantitative realtime PCR (qRT-PCR). 【Results】There was a significant difference in the learning ability of A. mellifera ligustica workers between the dsMrjp1injected group and the dsEGFP-injected group, with the learning ability of the dsMrjp1-injected group significantly decreased. However, there was no significant difference in the faculty of memory of A. mellifera ligustica workers at 2 h after learning between the two groups. The qRT-PCR results showed that the expression level of Mrjp1 in the brain of A. mellifera ligustica workers in the dsMrjp1-injected group was significantly lower than that in the dsEGFP-injected group, indicating that A. mellifera ligustica workers with decreased learning ability correspondingly exhibit lower expression level of Mrjp1. 【Conclusion】 After knockdown of Mrjp1 by RNAi, the olfactory learning performance of A. mellifera ligustica workers is significantly decreased, while their memory performance is not significantly affected, suggesting that Mrjp1 is probably one of the key genes regulating learning in A. mellifera ligustica. The results of this study contribute to further understanding of the molecular mechanism related to olfactory learning in honey bees.

Key words: Apis mellifera ligustica, major royal jelly protein, RNA interference, olfactory learning, memory, proboscis extension response

蔚添添, 邱园妹, 候梦赏, 王天宝, 苏松坤, 李志国. RNAi沉默王浆主蛋白1(Mrjp1)基因对意大利蜜蜂工蜂学习和记忆的影响[J]. 昆虫学报, 2021, 64(10): 1145-1152.

YU Tian-Tian, QIU Yuan-Mei, HOU Meng-Shang, WANG Tian-Bao, SU Song-Kun, LI Zhi-Guo . Effects of silencing of major royal jelly protein 1 (Mrjp1) gene by RNAi on learning and memory in worker bees of Apis mellifera ligustica[J]. Acta Entomologica Sinica, 2021, 64(10): 1145-1152.

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RNAi沉默王浆主蛋白1(Mrjp1)基因对意大利蜜蜂工蜂学习和记忆的影响

Effects of silencing of major royal jelly protein 1 (Mrjp1) gene by RNAi on learning and memory in worker bees of Apis mellifera ligustica

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