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
作者:
基金项目:

国家自然科学基金(31702190);国家现代农业产业技术体系(CARS-44-KXJ7);福建农林大学硕士生导师团队项目(郭睿);江西省蜜蜂生物学与饲养重点实验室开放基金(JXKLHBB-2020-04);福建省大学生创新创业训练计划(202210389127,202210389131)


Bioinformatic analysis and functional study of nkd gene in larvae of Apis mellifera ligustica workers
Author:
  • 摘要
  • | |
  • 访问统计
  • |
  • 参考文献 [32]
  • |
  • 相似文献 [20]
  • | | |
  • 文章评论
    摘要:

    【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abaecinapidaecinbirc5defensin-1PGRP-S2均被激活表达;5日龄幼虫肠道内abaecinapidaecinbirc5defensin-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apidaecinbirc5defensin-1PGRP-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,nkdabaecinapidaecin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。

    Abstract:

    [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.

    参考文献
    [1] Morfin N, Anguiano-Baez R, Guzman-Novoa E. Honey bee (Apis mellifera) immunity. The Veterinary Clinics of North America: Food Animal Practice, 2021, 37(3):521-533.
    [2] 梁勤, 陈大福. 蜜蜂保护学. 2版. 北京:中国农业出版社, 2009.
    [3] Zeng W, Wharton KA, Mack JA, Wang K, Gadbaw M, Suyama K, Klein PS, Scott MP. Naked cuticle encodes an inducible antagonist of Wnt signalling. Nature, 2000, 403(6771):789-795.
    [4] Rousset R, Mack JA, Wharton KA, Axelrod JD Jr, Cadigan KM, Fish MP, Nusse R, Scott MP. Naked cuticle targets disheveled to antagonize Wnt signal transduction. Genes Development, 2001, 15(6):658-671.
    [5] Chan CC, Zhang S, Rousset R, Wharton KA. Drosophila naked cuticle (Nkd) engages the nuclear import adaptor importin-alpha3 to antagonize Wnt/beta-catenin signaling. Developmental Biology, 2008, 318(1):17-28.
    [6] Wang R, Xie H, Yang L, Wang P, Chen MM, Wu HY, Liao YL, Wang MY, Wang Q, Gong XX, Cheng Q, Cheng L, Xie FY, Bi CL, Fang M. Naked cuticle inhibits wingless signaling in Drosophila wing development. Biochemical and Biophysical Research Communications, 2021, 576:1-6.
    [7] Lei H, Juan AH, Kim MS, Ruddle FH. Mouse naked cuticle 2(mNkd2) as a direct transcriptional target of Hoxc8 in vivo. Journal of Experimental Zoology Part A, Ecological Genetics and Physiology, 2007, 307(1):1-6.
    [8] Wharton KA, Zimmermann G, Rousset R, Scott MP. Vertebrate proteins related to Drosophila naked cuticle bind dishevelled and antagonize Wnt signaling. Developmental Biology, 2001, 234(1):93-106.
    [9] Zhao S, Kurenbekova L, Gao Y, Roos A, Creighton CJ, Rao P, Hicks J, Man TK, Lau C, Brown AM, Jones SN, Lazar AJ, Ingram D, Lev D, Donehower LA, Yustein JT. NKD2, a negative regulator of Wnt signaling, suppresses tumor growth and metastasis in osteosarcoma. Oncogene, 2015, 34(39):5069-5079.
    [10] Li W, Evans JD, Huang Q, Rodríguez-García C, Liu J, Hamilton M, Grozinger CM, Webster TC, Su S, Chen YP. Silencing the honey bee (Apis mellifera) naked cuticle gene (nkd) improves host immune function and reduces Nosema ceranae infections. Applied and Environmental Microbiology, 2016, 82(22):6779-6787.
    [11] 陈华枝, 蒋海宾, 祝智威, 范元婵, 许雅静, 孙明会, 刘佳美, 熊翠玲, 郑燕珍, 付中民, 徐国钧, 陈大福, 郭睿. 蜜蜂球囊菌菌丝和孢子中环状RNA的鉴定及比较分析. 微生物学报, 2021, 61(5):1299-1314. Chen HZ, Jiang HB, Zhu ZW, Fan YC, Xu YJ, Sun MH, Liu JM, Xiong CL, Zheng YZ, Fu ZM, Xu GJ, Chen DF, Guo R. Identification and comparison of circular RNAs in Ascosphaera apis mycelium and spore. Acta Microbiologica Sinica, 2021, 61(5):1299-1314. (in Chinese)
    [12] 孙明会, 刘佳美, 王思懿, 朱乐冉, 王紫馨, 叶亚萍, 钱加珺, 顾小雨, 徐细建, 陈大福, 郭睿, 徐国钧. 结合三代测序与二代测序技术揭示蜜蜂球囊菌孢子转录组的复杂性. 微生物学报, 2022, 03(22):1-16. Sun MH, Liu JM, Wang SY, Zhu LR, Wang ZX, Ye YP, Qian JJ, Gu XY, Xu XJ, Chen DF, Guo R, Xu GJ. Unraveling the complexity of transcriptome in Ascosphaera apis spore:based on third-generation and next-generation sequencing. Acta Microbiologica Sinica, 2022, 03(22):1-16. (in Chinese)
    [13] Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS. MEME SUITE:tools for motif discovery and searching. Nucleic Acids Research, 2009, 37:W202-W208.
    [14] Xu H, Hao Z, Wang L, Li S, Guo Y, Dang X. Suppression of transferrin expression enhances the susceptibility of Plutella xylostell a to Isaria cicadae. Insects, 2020, 11(5):281.
    [15] 王倩, 孙亮先, 肖培新, 刘锋, 康明江, 胥保华. 室内人工培育中华蜜蜂幼虫技术研究. 山东农业科学, 2009(11):113-116. Wang Q, Sun LX, Xiao PX, Liu F, Kang MJ, Xu BH. Study on technology for indoor artifical feeding of Apis cerana cerana larvae. Shandong Agricultural Sciences, 2009(11):113-116. (in Chinese)
    [16] Yang X, Cox-Foster DL. Impact of an ectoparasite on the immunity and pathology of an invertebrate:evidence for host immunosuppression and viral amplification. PNAS, 2005, 102(21):7470-7475.
    [17] Borsuk G, Ptaszyńska AA, Olszewski K, Domaciuk M, Krutmuang P, Paleolog J. A new method for quick and easy hemolymph collection from Apidae adults. PLoS One, 2017, 12(1):e0170487.
    [18] 陈华枝, 付中民, 王杰, 祝智威, 范小雪, 蒋海宾, 范元婵, 周丁丁, 李汶东, 熊翠玲, 郑燕珍, 徐国钧, 陈大福, 郭睿. 中华蜜蜂6日龄幼虫响应蜜蜂球囊菌胁迫的环状RNA应答. 微生物学报, 2020, 60(10):2292-2310. Chen HZ, Fu ZM, Wang J, Zhu ZW, Fan XX, Jiang HB, Fan YC, Zhou DD, Li WD, Xiong CL, Zheng YZ, Xu GJ, Chen DF, Guo R. Circular RNA response of Apis cerana cerana 6-day-old larvae to Ascosphaera apis stress. Acta Microbiologica Sinica, 2020, 60(10):2292-2310. (in Chinese)
    [19] Rodríguez-García C, Heerman MC, Cook SC, Evans JD, Chen YP. Transferrin-mediated iron sequestration suggests a novel therapeutic strategy for controlling Nosema disease in the honey bee, Apis mellifera. PLoS Pathogens, 2021, 17(2):e1009270.
    [20] 汤贤春, 钱倩, 罗才林, 王蕾, 钱刚. 千里光脂肪醛脱羰基酶(CER1蛋白)的保守基序(CSM)与功能结构域分析. 生命科学研究, 2016, 20(5):395-400. Tang XC, Qian Q, Luo CL, Wang L, Qian G. Analysis of conserved sequence motif (CSM) and functional/structural domains of fatty-aldehyde decarbonylase (CER1 protein) in Senecio scandens buch.-ham.ex D.Don. Life Science Research, 2016, 20(5):395-400. (in Chinese)
    [21] Guo XJ, Wang Y, Sinakevitch I, Lei H, Smith BH. Comparison of RNAi knockdown effect of tyramine receptor 1 induced by dsRNA and siRNA in brains of the honey bee, Apis mellifera. Journal of Insect Physiology, 2018, 111:47-52.
    [22] Jarosch A, Moritz RF. Systemic RNA-interference in the honeybee Apis mellifera:tissue dependent uptake of fluorescent siRNA after intra-abdominal application observed by laser-scanning microscopy. Journal of Insect Physiology, 2011, 57(7):851-857.
    [23] Xiao G, Liu ZH, Zhao M, Wang HL, Zhou B. Transferrin 1 functions in iron trafficking and genetically interacts with ferritin in Drosophila melanogaster. Cell Reports, 2019, 26(3):748-758.
    [24] Gordon MD, Dionne MS, Schneider DS, Nusse R. WntD is a feedback inhibitor of Dorsal/NF-kappaB in Drosophila development and immunity. Nature, 2005, 437(7059):746-749.
    [25] Jenny FH, Basler K. Powerful Drosophila screens that paved the wingless pathway. Fly, 2014, 8(4):218-225.
    [26] Luiz DP, Almeida JF, Goulart LR, Nicolau-Junior N, Ueira-Vieira C. Heterologous expression of abaecin peptide from Apis mellifera in Pichia pastoris. Microbial Cell Factories, 2017, 16(1):76.
    [27] Zhou XX, Wang YB, Pan YJ, Li WF. Nisin-controlled extracellular production of apidaecin in Lactococcus lactis. Applied Microbiology and Biotechnology, 2008, 78(6):947-953.
    [28] Bucekova M, Sojka M, Valachova I, Martinotti S, Ranzato E, Szep Z, Majtan V, Klaudiny J, Majtan J. Bee-derived antibacterial peptide, defensin-1, promotes wound re-epithelialisation in vitro and in vivo. Scientific Reports, 2017, 7(1):7340.
    [29] Wang Q, Ren M, Liu X, Xia H, Chen K. Peptidoglycan recognition proteins in insect immunity. Molecular Immunology, 2019, 106:69-76.
    [30] Kordaczuk J, Sułek M, Wojda I. General overview on the role of peptidoglycan recognition proteins in insect immunity. Acta Biochimica Polonica, 2020, 67(3):319-326.
    [31] Zhu Y, Hu M, Ngowo J, Gao X, Chen X, Yan H, Yu W. Deacetylation of BmAda3 is required for cell apoptosis caused by Bombyx mori nucleopolyhedrovirus infection. Archives of Insect Biochemistry and Physiology, 2021, 108(2):e21838.
    [32] Obeng E. Apoptosis (programmed cell death) and its signals-a review. Brazilian Journal of Biology, 2021, 81(4):1133-1143.
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

郭意龙,余岢骏,赵萧,钱加珺,赵浩东,张婕,张扬,赵红霞,徐细建,骆群,陈大福,郭睿. 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[J]. 微生物学报, 2022, 62(12): 5005-5017

复制
分享
文章指标
  • 点击次数:333
  • 下载次数: 948
  • HTML阅读次数: 716
  • 引用次数: 0
历史
  • 收稿日期:2022-04-01
  • 最后修改日期:2022-05-31
  • 在线发布日期: 2022-12-08
文章二维码