Nunes Francis M F, Aleixo Aline C, Barchuk Angel R, Bomtorin Ana D, Grozinger Christina M, Simões Zilá L P
Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, 14049-900, Brazil.
Departamento de Biologia Celular, Tecidual e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade Federal de Alfenas, Alfenas, Minas Gerais, 37130-000, Brazil.
Insects. 2013 Jan 4;4(1):90-103. doi: 10.3390/insects4010090.
RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.
RNA干扰已被频繁应用于在产生和维持突变体具有挑战性的生物体中调节基因功能，如在我们的研究模型蜜蜂（Apis mellifera）中。绿色荧光蛋白（GFP）衍生的双链RNA（dsRNA-GFP）目前常用于蜜蜂RNA干扰实验中的对照，因为其基因不存在于西方蜜蜂基因组中。尽管预计dsRNA-GFP不会在处理过的蜜蜂中引发RNA干扰反应，但经常观察到对基因表达、色素沉着或发育时间的不良影响。在这里，我们使用微阵列进行了三项独立实验，以研究dsRNA-GFP处理（通过喂食引入）对发育中的工蜂整体基因表达模式的影响。我们的数据显示，近1400个基因的表达因dsRNA-GFP而发生改变，约占已知蜜蜂基因的10%。表达变化似乎是直接脱靶效应和间接下游二级效应共同作用的结果；事实上，从dsRNA-GFP构建体产生的假定小干扰RNA与表达水平发生改变的基因之间存在多个序列相似的实例。一般来说，受影响的基因参与与RNA加工和转运、激素代谢、免疫、对外部刺激和应激反应相关的重要发育和代谢过程。这些结果表明，在蜜蜂的RNA干扰研究中应采用多种dsRNA对照。此外，在我们的研究中，任何涉及受dsRNA-GFP影响的这些基因的RNA干扰研究都应使用不同的dsRNA对照。
