miRNAs调控意大利蜜蜂哺育蜂与采集蜂行为转变的机制

MicroRNAs, can cause mRNAs degradation or inhibtion of translation by specifically base pairing with targets, have been implicated in many biological function. While roles for miRNAs in development and phathological states are well established,how miRNAs influence animal behavioral plasticity is still obscure. We applied Solexa sequencing technology to detect small RNAs in nurses and foragers, and discovered nine differertially expressed miRNAs between them after differential expression analysis.In this study,we detected the expression pattern of miRNAs in different development stages of honey bees by using qRT-PCR and in situ hybridization, then predicted their targets through miranda and Cytoscape softwares;miRNAs was input into honeybees through feeding or injecting. The differences of profiles of mRNAs in honeybees heads between nurses and foragers before and after the treatment were detected by mciroarray, and the significantly differentially expressed mRNAs were verified further by qRT-PCR.The function of targets were analyzed through bioinformatics method to find the mechnism of miRNAs in regulating honeybees behavioral transition.Our results provide clue for a better undertanding of the function of miRNAs in animal behavioral plasticity,and help people to catch the rule of development of a colony to guide the production of bee-keeping as well.

miRNAs通过与靶基因mRNA特异性的碱基互补配对，引起靶基因降解或者抑制其翻译。MiRNAs发挥多种生物调节功能，其在发育和病理方面的研究较深入,但在动物行为可塑性中的作用尚未明确。前期我们结合Solexa测序和生物信息分析，在哺育蜂和采集蜂中发现9个表达差异显著的miRNAs。本课题利用实时荧光定量PCR和原位杂交检测miRNAs在哺育蜂和采集蜂头部的表达模式，利用miranda 和Cytoscape软件预测并初步筛选靶基因。通过饲喂或注射方式，将miRNAs导入蜜蜂体内，利用芯片检测处理前后蜜蜂头部mRNAs表达谱差异，实时荧光定量PCR进一步验证。最后结合生物信息技术，分析miRNAs对靶基因的表达调控，寻找miRNAs调控蜜蜂行为可塑性的机制。本课题研究结果为认识miRNAs在动物行为可塑性调控中作用提供思路，同时有助于更好的掌握蜂群发展规律，对养蜂生产有重要的指导意义。

miRNAs通过与靶基因mRNA特异性的碱基互补配对，引起靶基因降解或者抑制其翻译。MiRNAs发挥多种生物调节功能，其在发育和病理方面的研究较深入,但在动物行为可塑性中的作用尚未明确。蜜蜂(Apis mellifera)，典型的社会性昆虫，一直是人类研究动物复杂行为的最佳模式昆虫。蜜蜂有一个的典型劳动分工行为，即成年工蜂刚出房一周，从事巢内哺育任务（哺育蜂），随后一至两周则从事巢内清理或者食物贮存等工作，蜜蜂出房3周以后则开始外出从事采粉采蜜等工作（采集蜂）。前期我们结合Solexa测序和生物信息分析，在哺育蜂和采集蜂头部中发现9个表达差异显著的miRNAs，其中ame-miR-31a、ame-let-7、ame-miR-279a、ame-miR-275在哺育蜂中上调，ame-miR-13b、ame-miR-133、 ame-miR-210、 ame-miR-278、ame-miR-92a在采集蜂中上调。本课题利用实时荧光定量PCR和northern blot检测发现，ame-let-7, ame-miR-13b和ame-miR-279a在工蜂中的表达具有明显的时间特异性；不管在正常蜂群还是同日龄蜂群中，ame-let-7, ame-miR-13b 在采集蜂脑部的表达量总是显著高于哺育蜂脑部的表达，而ame-miR-279a在哺育蜂脑部的表达总是显著高于在采集蜂脑部的表达。通过饲喂方式，将人工合成的ame-miR-279a mimic、ame-miR-279a inhibitor导入蜜蜂体内，使蜜蜂脑部ame-miR-279a过表达或表达受到抑制。利用PER对ame-miR-279a过表达或表达受到抑制的蜜蜂进行糖反应能力检测，发现采集蜂脑部ame-miR-279a过表达时的糖反应能力下降，而ame-miR-279a表达受到抑制时，采集蜂的糖反应能力则变强。实时荧光定量PCR进一步验证发现，蜜蜂脑部ame-miR-279a过表达或表达受到抑制时，其脑部Mblk-1基因及蛋白表达都相应的下降及上升。最后，我们构建荧光载体质粒pAc5.1-fluc-UTR、pAc5.1-fluc-UTR及pAc5.1-pri-miRNA，将质粒分别共转染至果蝇S2细胞，检测细胞的荧光信号值，结果证明ame-miR-279a直接作用于Mblk-1的3’UTR。以上研究表明ame-miR