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