意大利蜜蜂工蜂幼虫nkd基因的生物信息学分析及功能研究

【目的】意大利蜜蜂(Apis mellifera ligustica，简称意蜂)是西方蜜蜂(Apis mellifera)的亚种之一。蜜蜂球囊菌(Ascosphaera apis)侵染意蜂幼虫导致白垩病。本研究对西方蜜蜂裸表皮蛋白(naked cuticle, Nkd)进行保守基序预测和系统进化分析，并通过RNAi明确nkd基因对意蜂工蜂幼虫体重及宿主响应蜜蜂球囊菌胁迫的免疫应答的影响，以期丰富西方蜜蜂基因nkd的信息，并揭示意蜂幼虫nkd的功能。【方法】通过MEME软件预测西方蜜蜂和其他9个物种Nkd蛋白的保守基序。采用MEGA X软件对西方蜜蜂及其他9个物种的Nkd蛋白进行系统进化分析。通过饲喂dsRNA对意蜂幼虫肠道内的nkd进行RNAi。使用电子天平对幼虫进行称重。利用RT-qPCR检测nkd基因的干扰效率及免疫基因的相对表达量。【结果】西方蜜蜂与东方蜜蜂、柑橘凤蝶、家蚕和金凤蝶的Nkd蛋白均含有3个保守基序(motif 1、motif 2 和motif 3)，说明上述5个昆虫物种的Nkd具有较高的保守性。西方蜜蜂与东方蜜蜂的Nkd蛋白聚为一支，说明二者的亲缘关系近。与dsRNA-egfp组相比，dsRNA-nkd组5日龄和6日龄幼虫肠道内nkd的表达量均极显著下调(P<0.001)，干扰效率分别为49.60%和56.40%。另外，dsRNA-nkd组幼虫体重较dsRNA-egfp组显著下降，说明nkd显著影响幼虫体重。RT-qPCR结果显示，4日龄幼虫肠道内abaecin、apidaecin、birc5、defensin-1和PGRP-S2均被激活表达；5日龄幼虫肠道内abaecin、apidaecin、birc5和defensin-1均被激活表达，PGRP-S2的表达受到抑制；6日龄幼虫肠道内abaecin被激活表达，而apidaecin、birc5、defensin-1和PGRP-S2的表达均受到抑制，说明上述5个免疫基因在宿主响应胁迫的过程中呈不同的表达趋势，均参与宿主的免疫应答，nkd与abaecin和apidaecin的表达存在负向调控关系。【结论】西方蜜蜂的Nkd蛋白含有3个保守基序(motif 1、motif 2和motif 3)，西方蜜蜂与东方蜜蜂的Nkd蛋白亲缘关系最近，通过饲喂dsRNA能有效干扰意蜂工蜂幼虫肠道内nkd表达，nkd影响意蜂工蜂幼虫体重及宿主对蜜蜂球囊菌胁迫的免疫应答。

Bioinformatic analysis and functional study of nkd gene in larvae of Apis mellifera ligustica workers

Objective] Apis mellifera ligustica is one of the subspecies of Apis mellifera. Ascosphaera apis exclusively infects bee larvae and leads to chalkbrood. In this study, we predicted the conserved motifs and analyzed the phylogenetic relationship of naked cuticle (Nkd) protein in A. mellifera. Further, we employed RNA interference (RNAi) to study the effect of nkd gene on the larval body weight of A. m. ligustica and the immune response to A. apis stress, aiming to enrich the information of A. mellifera nkd gene and reveal the role of nkd in A. m. ligustica larvae. Methods] The conserved motifs of Nkd proteins from A. mellifera and nine other species were predicted by MEME. MEGA X was used for the phylogenetic analysis of these Nkd proteins. RNAi of the nkd in the guts of the worker larvae of A. m. ligustica was carried out by feeding dsRNA-nkd. The larvae were weighed via an electronic balance. RT-qPCR was employed to determine the relative expression of nkd and immune response genes. Results] The nkd proteins from A. mellifera, Apis cerana, Papilio xuthus, Bombyx mori, and Papilio machaon contained three conserved motifs (motif 1, motif 2, and motif 3), which indicated that the Nkd proteins from the above-mentioned five insect species were highly conserved. The Nkd proteins in A. mellifera and A. cerana shared the same clade, showing the closest genetic relationship. Compared with the dsRNA-egfp group, the dsRNA-nkd group showed down-regulated expression of nkd in the guts of 5- and 6-day-old larvae (P<0.001), with the interference efficiencies of 49.60% and 56.40%, respectively. Additionally, the body weights of all the larvae in the dsRNA-nkd group were significantly lower than those in the dsRNA-egfp group. The expression of abaecin, apidaecin, birc5, defensin-1, and PGRP-S2 was activated in the guts of 4-day-old larvae. In the guts of 5-day-old larvae, the expression of abaecin, apidaecin, birc5, and defensin-1 was activated, while that of PGRP-S2 was inhibited. In the guts of 6-day-old larvae, the expression of abaecin was activated, while that of apidaecin, birc5, defensin-1, and PGRP-S2 was suppressed. The results suggested that the expression of above-mentioned five genes varied in response to stress and were involved in the host immune response. Moreover, there was a negative regulatory relationship between the expression of nkd and that of abaecin and apidaecin. Conclusion] A. mellifera Nkd protein contains three conserved motifs (motif 1, motif 2, and motif 3). The Nkd proteins from A. mellifera and A. cerana shared the closest genetic relationship. The interference on nkd in the larval guts of A. m. ligustica workers could be achieved via feeding dsRNA, and nkd affected A. m. ligustica workers in terms of larval body weight and immune response to A. apis stress.