环状RNA ame_circ_000115调节蜜蜂球囊菌胁迫下意大利蜜蜂幼虫肠道基因表达

Circular RNA ame_circ_000115 regulates expression of genes in larval gusts of Apis mellifera ligustica stressed by Ascosphaera apis

摘要:

环状RNA (circular RNA, circRNA)是一类新型非编码RNA，已被证实可通过竞争性内源RNA (competing endogenous RNA, ceRNA)调控网络等多种方式参与调节昆虫基因表达和免疫应答。目前，circRNA在蜜蜂免疫应答中的作用尚不清楚。本研究对意大利蜜蜂(Apis mellifera ligustica, 简称意蜂) ame_circ_000115 (简称ac115)的反向剪切(back splicing, BS)位点进行验证。采用RT-qPCR检测蜜蜂球囊菌(Ascosphaera apis)胁迫下意蜂幼虫肠道内ac115的表达谱，利用双荧光素酶报告基因试验验证ac115与ame-miR-13b的结合关系。通过饲喂特异性siRNA对蜜蜂球囊菌胁迫的意蜂幼虫肠道内ac115进行干扰，进而检测干扰ac115对宿主免疫应答相关的6个基因表达量的影响。结果表明，ac115的BS位点真实存在；相较于未接种组，蜜蜂球囊菌接种组4日龄幼虫肠道内ac115的表达量极显著上调(P＜0.000 1)，5和6日龄幼虫肠道内ac115显著上调表达(P＜0.05)；siRNA-circ_000115饲喂组4、5和6日龄幼虫肠道内ac115的特异性条带亮度逐渐减弱，而siRNA-scramble饲喂组4、5和6日龄幼虫肠道内ac115的特异性条带亮度均较高且无明显变化；与siRNA-scramble饲喂组相比，siRNA-circ_000115饲喂组4日龄幼虫肠道内ac115的表达量显著下调(P＜0.05)，5和6日龄幼虫肠道内ac115的表达量均极显著下调(P＜0.001)；ame-miR-13b在意蜂幼虫肠道内真实存在和表达；ac115和ame-miR-13b之间存在真实的结合关系；相比于siRNA-scramble饲喂组，siRNA-circ_000115饲喂组6日龄幼虫肠道内抗菌肽基因hymenoptaecin和abaecin的表达量均显著上调(P＜0.05)，而蜕皮激素受体(ecdysone receptor, Ecr)的表达量极显著下调(P＜0.01)。研究结果表明，ac115在意蜂幼虫肠道内真实表达，ac115中的BS位点真实存在，饲喂siRNA的方式能实现意蜂幼虫肠道内ac115的有效干扰，ac115通过吸附ame-miR-13b潜在调控Ecr表达，通过非ceRNA的方式调控hymenoptaecin和abaecin表达进而参与宿主的免疫应答。

Abstract:

Circular RNAs (circRNAs) are a new class of non-coding RNAs, which have been confirmed to regulate insect gene expression and immune response through multiple manners such as competing endogenous RNA (ceRNA) regulatory network. Currently, function of circRNA in honey bee immune response remains unclear. In this study, PCR and Sanger sequencing were performed to validate the back splicing (BS) site of ame_circ_000115 (in short ac115). RT-qPCR was used to detect the expression profile of ac115 in larval guts of Apis mellifera ligustica stressed by Ascosphaera apis. Dual-luciferase reporter gene assay was conducted to verify the binding relationship between ac115 and ame-miR-13b. Interference of ac115 in larval guts was carried out by feeding specific siRNA, followed by determination of the effect of ac115 interference on expression of six genes relevant to host immune response. The results confirmed the existence of BS site within ac115. Compared with the un-inoculated group, the expression of ac115 in 4-day-old larval gut of the A. apis-inoculated group was up-regulated with extreme significance (P＜0.000 1), while that in 5- and 6-day-old larval guts were significantly up-regulated (P＜0.05). The brightness of specific band for ac115 in 4-, 5- and 6-day-old larval guts of the siRNA-circ_000115-fed group gradually became weak, whereas that of the siRNA-scrambl-fed group was pretty high without obvious variation. Compared with that of the siRNA-scramble-fed group, the expression of ac115 in 4-day-old larval gut of the siRNA-circ_000115-fed group was significantly down-regulated (P＜0.05), whereas that of the 5- and 6-day-old larval guts were down-regulated with extreme significance (P＜0.001). Ame-miR-13b was truly existed and expressed in A. m. ligustica larval guts, and there was true binding relationship between ac115 and ame-miR-13b. Compared with that of the siRNA-scramble-fed group, the expression of antimicrobial peptide genes hymenoptaecin and abaecin in 6-day-old larval gut of the siRNA-circ_000115-fed group was significantly up-regulated (P＜0.05), while that of ecdysone receptor (Ecr) was down-regulated with extreme significance (P＜0.01). These results indicate that ac115 is truly expressed in A. m. ligustica larval guts, BS site truly exists within ac115, and effective interference of ac115 in A. m. ligustica larval guts can be achieved via feeding siRNA. Moreover, ac115 potentially regulates Ecr expression through adsorption of ame-miR-13b and expression of hymenoptaecin and abaecin using a non-ceRNA manner, further participating in host stress-response.