2种杀虫剂对舞蹈蜜蜂大脑转录组中可变剪切的影响

The Effects of Two Insecticides in the Transcriptome of the Brains of Dancing Bees on the Alternative Splicing

【目的】蜜蜂是一种重要的经济性社会昆虫,也是一种重要的传粉昆虫,对农业生态系统平衡与植物多样性具有重要贡献。为防治农作物的病虫害,生产中杀虫剂的大量使用,引起蜜蜂中毒事件逐年增加,给养蜂业带来重大的经济损失,同时农作物因为得不到充分授粉,导致产量下降和品质降低,进而影响农业的收益。可变剪切(alternative splicing,AS)是调控基因表达和产生蛋白质多样性的重要机制,也是造成物种之间、器官之间、生理与病理之间差异的重要因素。为了揭示可变剪切基因在蜜蜂应对杀虫剂过程中的作用,对2种亚致死剂量杀虫剂诱导舞蹈蜜蜂基因的可变剪切进行深入分析。【方法】基于前期蜜蜂舞蹈行为学试验和已获得杀虫剂诱导的舞蹈蜜蜂大脑转录组数据,应用tophat分别比对吡虫啉处理组与对照组,溴氰菊酯处理组与对照组包含的所有大脑转录组中剪切位点信息。【结果】在吡虫啉处理组间和溴氰菊酯处理组间中,外显子跳跃(skipped exon,SE)是主要的可变剪切类型,检测到各组之间事件数目占总事件数达60%以上,而SE类型的差异事件数占差异AS事件总数的50%以上。在不同距离(300,500,1000 m)的试验地点,2种杀虫剂处理的舞蹈蜜蜂脑部转录组中发生5种可变剪接的事件总数之间存在显著差异(P<0.001),且发生5种可变剪接的基因数目之间存在显著差异(P<0.001)。根据这些发生可变剪切的基因做了KEGG(kyoto encyclopedia of genes and genomes)富集分析,主要涉及新陈代谢、细胞修复、氨基酸代谢、脂肪酸代谢和信号传导等生物学活动,与重要的信号通路和免疫应答通路密切相关。【结论】2种亚致死剂量杀虫剂能够使蜜蜂体内的基因发生可变剪切,可能改变配体受体结合、转录因子、生理节律、内吞作用以及神经信号传导。可变剪切基因参与蜜蜂响应杀虫剂胁迫过程中,会对细胞功能产生影响,包括免疫应答、学习记忆和飞行定位等信号通路,从而影响蜜蜂的舞蹈行为学。研究2种亚致死剂量杀虫剂诱导的舞蹈蜜蜂内在基因表达和调控情况,不但对吡虫啉和溴氰菊酯的环境毒性风险评价有参考价值,并进一步制定相应的防护措施,对于保护这种农业重要的授粉昆虫具有重要的价值。

[Objective]Honeybee is an important economic and social insect,as well as an important pol⁃linator,which contributes to the balance of agricultural ecosystem and plant diversity.In order to prevent the diseases and pests of the crops,the usage of pesticides is increasing yearly,which inevitably threaten the safe⁃ty of beneficial insects such as honeybees.As a result of the extensive use of pesticides,bee poisoning cases in⁃crease every year,which leads up to substantial economic losses to the apiculture industry.Due to insufficient pollination,the yield and quality of the crops are reduced,which in turn affects the agricultural income.Alter⁃native splicing(AS)is an important mechanism to regulate gene expression and protein diversity,and it is also an important factor to differentiate species,organs,physiology and pathology.In order to reveal the role of alter⁃natively splicing genes in response to sub-lethal doses of insecticides in honeybees,the responses of alterna⁃tively splicing towards two insecticides were analyzed.[Method]Based on previous studies of dance behavior in honeybees and data from the transcriptome of the brain of the insecticide-induced dance honeybees,tophat was used to compare the splice sites between imidacloprid treated group and the control group,as well as be⁃tween deltamethrin treated group and the control group.[Result]Between the imidacloprid treated group and deltamethrin treated group,skipped exon(SE)was the main alternatively spliced type,and the number of events detected between each group accounted for more than 60%of the total number of events,while the num⁃ber of different events of SE type accounted for more than 50%of the total number of different AS events.There were significant differences in the total number of five alternatively splicing events in the brain transcrip⁃tome of two insecticide-treated dancing bees at different trial distances at 300,500,1000 m from hive(P<0.001).And the number of genes with five alternatively splicing events were significant different between the two insecticide-treated groups at different distances(P<0.001).Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis was carried out according to these variable splicing genes,which mainly involved in metabolism,cell repair,amino acid metabolism,fatty acid metabolism,signal transduction and other biologi⁃cal activities,closely related to important signal pathways and immune response pathways.[Conclusion]Both sub-lethal doses of insecticides cause genes in honeybees to undergo alternatively splicing,potentially alter⁃ing ligand receptor binding,transcription factors,circadian rhythms,endocytosis,and nerve signal transduc⁃tion.In response to insecticide stress,alternatively splicing events affect cellular functions,including immune responses,learning,memory and navigation ect,thus influencing honeybee dance behavior.The study on the gene expression and regulation of dance bees induced by the two sub-lethal doses of insecticides not only has reference value for the environmental toxicity risk assessment of imidacloprid and deltamethrin,but also makes corresponding protective measures,it is of great value for the conservation of the agriculture-important pollinating insect.