中国农业科学 ›› 2025, Vol. 58 ›› Issue (2): 281-290.doi: 10.3864/j.issn.0578-1752.2025.02.006

• 植物保护 • 上一篇    下一篇

柑橘木虱黏液样蛋白DcMucin-like抗体制备及应用

蒋丽琴(), 苏巧灵, 李猷, 魏太云, 宾羽()   

  1. 福建农林大学植物保护学院/闽台作物有害生物生态防控国家重点实验室,福州 350002
  • 收稿日期:2024-08-10 接受日期:2024-09-19 出版日期:2025-01-21 发布日期:2025-01-21
  • 通信作者:
    宾羽,E-mail:
  • 联系方式: 蒋丽琴,E-mail:17873374973@163.com。
  • 基金资助:
    国家自然科学基金青年科学基金项目(32402347); 国家自然科学基金青年科学基金项目(32202395); 福建省自然科学基金面上项目(2022J01577)

Preparation and Application of DcMucin-like Antibodies in Diaphorina citri

JIANG LiQin(), SU QiaoLing, LI You, WEI TaiYun, BIN Yu()   

  1. College of Plant Protection, Fujian Agriculture and Forestry University/State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fuzhou 350002
  • Received:2024-08-10 Accepted:2024-09-19 Published:2025-01-21 Online:2025-01-21

摘要:

【目的】黏液样蛋白(mucin-like protein)参与半翅目昆虫取食过程中唾液鞘的形成。本研究旨在制备柑橘木虱(Diaphorina citri)黏液样蛋白DcMucin-like特异性抗体并对木虱取食孔进行免疫荧光标记,为DcMucin-like生物学功能研究提供依据。【方法】通过解剖柑橘木虱唾液腺、中肠、卵巢和精巢组织,根据木虱DcMcuin-like序列设计特异性引物,利用实时荧光定量PCR检测分析各组织中DcMcuin-like在转录水平的差异;扩增无信号肽的DcMucin-like序列并插入pET-28a中构建重组质粒,测序验证后转化Rosetta表达菌株,以终浓度0.5 mmol·L-1 IPTG在37 ℃下振荡培养8 h诱导重组蛋白表达,SDS-PAGE凝胶电泳检测重组蛋白表达情况;扩大培养后裂解菌体,裂解液上清经Ni-NTA纯化作为抗原免疫家兔5次,纯化血清IgG获得DcMucin-like多克隆抗体,Western blot检测抗体特异性;利用实时荧光定量PCR和Western blot比较健康木虱和感染黄龙病菌(Candidatus Liberibacter asiaticus,CLas)木虱中DcMucin-like在转录水平和蛋白水平的差异;利用交联荧光素(FITC)的DcMucin-like多克隆抗体标记木虱取食后的柑橘叶片,在共聚焦显微镜下观察柑橘木虱取食孔和唾液鞘。【结果】实时荧光定量PCR检测显示DcMcuin-like在木虱唾液腺中大量表达,且表达量显著高于中肠、卵巢和精巢组织。携带有pET28a-DcMucin的Rosetta表达菌株经IPTG诱导后在菌体裂解液的上清中获得大量表达的重组蛋白。重组蛋白免疫家兔后取抗血清纯化IgG,Western blot结果表明获得特异性好的DcMucin-like多克隆抗体。实时荧光定量PCR和Western blot分析结果显示DcMucin-like在木虱响应黄龙病菌侵染时上调表达。DcMucin-like(FITC)荧光抗体标记木虱取食后的柑橘叶片组织切片,共聚焦显微镜下观察到木虱的取食孔,并在取食孔周围检测到特异的FITC荧光信号,表明DcMucin-like随木虱取食释放到植株组织中参与唾液鞘形成。【结论】DcMucin-like在柑橘木虱唾液腺中高表达并响应黄龙病菌侵染上调表达;成功制备了特异性好的柑橘木虱DcMucin-like多克隆抗体;验证了DcMucin-like随木虱取食分泌到柑橘植株组织中。研究结果可为深入探究DcMucin-like在黄龙病菌-木虱-柑橘三者互作中的作用打下基础。

关键词: 柑橘木虱, 唾液鞘蛋白, DcMucin-like, 抗体制备, 免疫荧光

Abstract:

【Objective】Mucin-like proteins are integral to the formation of salivary sheaths in Hemiptera insects. This research seeks to prepare a specific antibody targeting the Diaphorina citri mucin-like protein (DcMucin-like) and to employ immunofluorescent labeling to identify the feeding sites of D. citri, so as to provide a basis for the study of the biological functions of DcMucin-like.【Method】The salivary glands, midgut, ovaries, and testes of D. citri were dissected for analysis. Specific primers were designed based on the DcMucin-like sequence of the psyllid, and real-time fluorescent quantitative PCR was employed to assess the transcriptional level differences of DcMucin-like across various tissues. The DcMucin-like sequence, excluding the signal peptide, was amplified and subsequently inserted into the pET-28a vector to construct a recombinant plasmid. Following sequence verification, the plasmid was transformed into Rosetta expression strains. The expression of recombinant protein was induced using 0.5 mmol·L-1 IPTG at 37 ℃ with agitation for 8 h. The presence of the recombinant protein was confirmed via SDS-PAGE gel electrophoresis. Following large-scale bacterial culture, the cells were lysed, and the supernatant was subjected to purify using Ni-NTA affinity chromatography to obtain the antigen. This antigen was subsequently used to immunize rabbits five times. The resulting purified serum IgG yielded the DcMucin-like polyclonal antibody, whose specificity was assessed through Western blot analysis. Real-time fluorescent quantitative PCR and Western blot analyses were employed to compare the transcriptional and protein expression levels of DcMucin-like between healthy and Candidatus Liberibacter asiaticus (CLas) infected D. citri. The feeding sites of D. citri on citrus leaves post-ingestion were labeled using DcMucin-like polyclonal antibodies conjugated with fluorescein isothiocyanate (FITC). These feeding sites, along with the salivary sheaths of D. citri, were subsequently examined under a confocal microscope.【Result】Real-time fluorescent quantitative PCR analysis revealed that the DcMucin-like exhibited significantly elevated expression level in the salivary gland of D. citri compared to the midgut, ovary, and testis. Rosetta expression strains harboring the pET28a-DcMucin were induced with IPTG, resulting in the production of substantial quantities of recombinant protein in the supernatant of the bacterial lysate. The recombinant protein was utilized to immunize rabbits for the production of antiserum, from which purified IgG was subsequently employed to generate DcMucin-like polyclonal antibodies. Western blot analysis confirmed the successful acquisition of specific DcMucin-like polyclonal antibodies. Furthermore, DcMucin-like expression was found to be upregulated in D. citri response to CLas infection. The DcMucin-like (FITC) fluorescent antibody-labeled tissue sections of citrus leaves, following D. citri feeding, were examined using a confocal microscope. Specific FITC fluorescence signals were detected in proximity to the feeding sites, suggesting that DcMucin-like was released into plant tissues during D. citri feeding to participate in the formation of salivary sheaths.【Conclusion】DcMucin-like is highly expressed in the salivary glands of D. citri and exhibits upregulation in response to CLas infection. Specific polyclonal antibodies targeting the DcMucin-like salivary protein of D. citri were successfully generated, demonstrating high specificity. Additionally, it was confirmed that DcMucin-like was secreted into citrus plant tissues during D. citri feeding. These findings provide a foundational basis for further investigation into the role of DcMucin-like in the interactions among CLas, D. citri, and citrus plants.

Key words: Diaphorina citri, salivary sheath protein, DcMucin-like, antibody preparation, immunofluorescence