环状RNA通过吸附miR-14调控蜂王产卵的机制研究

Oviposition of honey bee queens is the base of other economic traits in apicultural industry. Study of oviposition genetic mechanism is important for genetic improvement of oviposition in honey bees. To investigate the genetic and regulatory mechanism of oviposition, we analyzed the RNA expression in ovary of honey bee queens by RNA sequencing. The results showed that the key circular RNA circ_16640 and ecdysone receptor (EcR) were significantly up-regulated in ovary activation and oviposition starting processes of queens. Through ceRNA network constructing, we found that circ_16640 may promote the expression of EcR, ovary activation and oviposition by sponging microRNA miR-14. In this study, we will analyze the sponging mechanism between circ_16640 and miR-14 and the regulatory mechanism between miR-14 and EcR through suppressed-expression and over-expression in vitro. And the effect of circ-16640 on ecdysteroids synthesis will be analyzed at the molecular level. Further, the effect of circ_16640, miR-14 and EcR on oviposition will be investigated in vivo via circ_16640, miR-14 and EcR knockout queen model. We expect to elucidate the mechanism of circular RNA affecting oviposition by sponging miR-14. The work helps to further reveal the genetic and regulatory mechanism of oviposition. It will benefit the genetic improvement of oviposition in honey bees.

蜜蜂蜂王产卵性状是蜂群产生经济效益的基础性状，产卵的分子机制研究是蜜蜂产卵性状遗传改良的重要基础。为了解和掌握产卵的遗传调控机制，我们利用全转录组测序技术对蜂王卵巢组织RNA进行分析发现，关键环状RNA(circ_16640)和蜕皮激素受体(EcR)在蜂王卵巢激活和产卵启动时显著上调；通过构建ceRNA网络发现，该环状RNA可能通过吸附小RNA miR-14，上调EcR的表达，进而促进蜂王卵巢激活和产卵启动。本项目在此研究基础上，拟通过抑制表达和超表达等方法，确定环状RNA对miR-14的吸附机制和miR-14对EcR的调控机制，并在分子水平研究环状RNA对蜕皮激素合成的作用；通过环状RNA、miR-14、EcR抑制和超表达蜂王模型，从活体水平分析环状RNA通过吸附miR-14影响蜂王产卵的作用机制。研究有利于进一步阐明蜂王产卵的遗传调控机制，对蜜蜂产卵性状的遗传改良具有理论和实践意义。

为阐明环状RNA通过吸附小RNA miR-14，调控蜕皮激素受体的表达，进而调控蜂王产卵的分子机制，项目以西方蜜蜂为研究对象，通过生物信息学分析，确定了环状RNA circ_16640与miR-14、miR-14与EcR的结合位点，通过双荧光素酶实验确定了circ_16640与miR-14、miR-14与EcR之间的相互作用。通过qRT-PCR和western blot实验，明确了miR-14对EcR mRNA和蛋白表达的影响作用，确定miR-14对EcR为转录抑制。通过qRT-PCR实验，确定了miR-14与circ_16640之间存在表达负相关。通过ELSA实验，确定了circ_16640、EcR的高表达可以促进蜕皮激素的分泌，miR-14的表达水平与蜕皮激素浓度呈负相关。通过体外实验验证了环状RNA circ_16640、EcR和miR-14对蜂王产卵数的作用，结果表明：环状RNA circ_16640和EcR的高表达能能够促进蜂王产卵，抑制miR-14表达能够促进蜂王产卵。研究为进一步阐明蜂王产卵的遗传机制和提高蜂王产卵数提供了理论依据。