Antibodies to citrus psylla saliva protein dc mucin and methods of making and using the same

Abstract

The application discloses an antibody of citrus psylla saliva protein DcMucin and a preparation method and application thereof. Through preparation of the DcMucin-like polyclonal antibody, it is verified for the first time that DcMucin-like is saliva protein of the citrus psylla, and is released into plant tissues to participate in saliva sheath formation with the citrus psylla feeding. The application fills the blank of the preparation and research of saliva protein antibodies of the citrus psylla, the prepared saliva protein polyclonal antibody of the citrus psylla has good specificity to the DcMucin recombinant protein, the antibody immunofluorescence labeling observation of the saliva sheath lays a foundation for further research on the biological function of DcMucin in the interaction of Huanglongbing bacteria-citrus psylla-citrus, and lays a foundation for further obtaining a specific target of green prevention and control of the citrus psylla.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology and plant pest control, and particularly relates to an antibody of citrus psylla saliva protein DcMucin and a preparation method and application thereof. BACKGROUND

[0002] Diaphorina citri belongs to Hemiptera and Psyllidae, and is a typical piercing-sucking insect. It causes serious impact on citrus production by sucking citrus phloem sap and transmitting Candidatus Liberibacter. Similar to other Hemiptera, D. citri secretes two types of saliva, namely, gummy saliva and watery saliva. The gummy saliva is mainly involved in forming saliva sheath to wrap the stylet for facilitating penetration into plant tissue, and the watery saliva contains various enzymes and has the functions of degrading plant cells, detoxifying plant allelochemicals, and inhibiting or stimulating plant defense response. Given that saliva plays an important role in the feeding and interaction of D. citri with citrus, the research on psylla saliva protein is helpful to the excavation of new targets for pest control, thereby providing support for the biological control of D. citri.

[0003] The saliva of Hemiptera insects acts as a bridge in the interaction among insects, plants and pathogens. When D. citri sucks the phloem sap of Candidatus Liberibacter-infected citrus plants, the bacteria enter the stylet along with the sap, pass through the filter chamber and foregut to reach the midgut system, then break through the midgut release barrier to diffuse into the hemolymph, infect the salivary glands through the hemolymph system, and finally complete the infection cycle in the vector D. citri and are discharged along with the saliva into the new host plant when D. citri feeds on the phloem sap (Chen Q et al., 2022; Ammar E-D et al., 2011; Weintraub PG et al., 2006). The saliva sheath formed by the gummy saliva is indispensable for insect feeding, and it is secreted during the penetration of the stylet into plant tissue to provide mechanical stability and lubrication for the movement of the stylet (Huang HJ et al., 2019). The saliva sheath can seal the penetration site of the stylet, thereby inhibiting the plant immune response triggered by the leakage of cell components (Miles PW et al., 1999). In aphids and leafhoppers, interference with the formation of saliva sheath will hinder the insects from feeding on the plant sieve tubes. The saliva sheath is composed of various saliva sheath proteins, which can be recognized as a related molecular pattern of phytophagous insects, thereby activating the immune response of the host plant (Shangguan XX et al., 2018). At present, the understanding of insect saliva sheath proteins is mainly limited to their mechanical functions, and there are few reports on their other functions in the interaction between phytophagous insects and plants.

[0004] Mucin-like proteins play an important role in the formation of insect salivary sheath, Huang HJ, et al. (2016, 2017) found that mucin-like proteins were specifically expressed in the salivary glands of the brown planthopper, and played a role in the damage of the brown planthopper and the adaptation to host plant resistance. The salivary sheath protein LsSP1 of the brown planthopper (Laodelphax striatellus) can be secreted into the host plant and combined on the salivary sheath by interacting with the mucin-like protein (LsMLP), overexpression of LsSP1 in plants can significantly reduce the accumulation of H2O2 and the expression of defense genes induced by LsMLP, and can rescue the feeding defects caused by insufficient secretion of LsSP1, which indicates the importance of salivary sheath proteins in the interaction between phytophagous insects and host plants (2023). Shangguan XX et al. (2018) also found that the mucin-like protein of the brown planthopper can cause plant cell death, induce the expression of defense-related genes and the deposition of callose, indicating that the mucin-like protein is also involved in the interaction between insect salivary sheath protein and host plant.

[0005] The above research on the function of the salivary sheath of phytophagous insects is based on the preparation of specific antibodies, and there is currently a lack of antibodies for citrus psylla salivary proteins, especially psylla salivary sheath protein antibodies. Therefore, the preparation of related antibodies will help to clarify their biological role in psylla feeding and interaction with citrus hosts in the future, and lay the foundation for obtaining specific target for green control of citrus psylla. SUMMARY

[0006] The purpose of the present application is to provide an antibody for citrus psylla salivary protein DcMucin and a preparation method and application thereof in view of the above problems.

[0007] In order to achieve the purpose of the present application, the technical scheme adopted by the present application is:

[0008] The first aspect of the present application provides an antigen polypeptide for inducing citrus psylla salivary protein polyclonal antibody, and the amino acid sequence thereof is the amino acid sequence shown in SEQ ID NO. 11.

[0009] The second aspect of the present application provides a biological material of the coding gene of the above-mentioned antigen polypeptide, and the biological material is an expression cassette, a plasmid, an expression vector or a host bacteria.

[0010] The third aspect of the present application provides the application of the above-mentioned antigen polypeptide or the above-mentioned biological material in any of the following:

[0011] (1) the application in the preparation of an antigen for inducing citrus psylla salivary protein polyclonal antibody;

[0012] (2) the application in the preparation of a reagent or kit for detecting citrus psylla salivary protein or the expression amount thereof.

[0013] The fourth aspect of the present application provides a polyclonal antibody of the citrus psylla salivary protein, wherein the antigen of the polyclonal antibody is a polypeptide induced by the amino acid sequence shown in SEQ ID NO. 11.

[0014] The fifth aspect of the present application provides a preparation method of the polyclonal antibody of the citrus psylla salivary protein, comprising the following steps:

[0015] (1) constructing a recombinant expression vector containing the nucleotide sequence shown in SEQ ID NO. 10, transforming the recombinant expression vector into an E. coli competent cell to obtain a recombinant protein antigen;

[0016] (2) immunizing an animal with the obtained recombinant protein antigen, taking blood, separating antisera and purifying to obtain.

[0017] The preparation method, in step (1), the recombinant expression vector is obtained by connecting the antigen gene fragment of the nucleotide sequence shown in SEQ ID NO. 10 to the prokaryotic expression vector pET-28a(+);

[0018] After the recombinant expression vector is transformed into the E. coli competent cell, the cell is inoculated into a liquid culture medium for culture and expansion, and then IPTG inducer is added for induction of expression to obtain the DcMucin recombinant protein; preferably, the conditions for induction of expression are that the final concentration of IPTG is 0.4-0.6 mM, the induction is carried out at 36-38℃ for 7-9 h;

[0019] The antigen gene fragment of the nucleotide sequence shown in SEQ ID NO. 10 is obtained by PCR amplification using the specific amplification primers designed based on the DcMucin-like sequence shown in SEQ ID NO. 1 without signal peptide; preferably, the specific amplification primers are as follows:

[0020] DcMucin-F:

[0021] 5'-CAGCAAATGGGTCGCGGATCCATGCAGAGCCAGACCCCTGCACCTGCTGTAAA-3';

[0022] DcMucin-R:

[0023] 5'-GTGGTGGTGGTGGTGCTCGAGTTTCAAAGGTGTCTCAACCAGGTTGTTGGA-3'.

[0024] The preparation method, in step (2), first mixes the recombinant protein antigen with complete Freund's adjuvant to immunize the animal for the first time, and then mixes the recombinant protein antigen with incomplete Freund's adjuvant to immunize for the second to fourth times, and serum separation and purification obtain the citrus psylla saliva protein polyclonal antibody.

[0025] The sixth aspect of the present application provides a kit containing the above-mentioned citrus psylla saliva protein polyclonal antibody.

[0026] The seventh aspect of the present application provides the use of the above-mentioned citrus psylla saliva protein polyclonal antibody or the above-mentioned kit in the preparation of a kit for detecting citrus psylla saliva protein.

[0027] The application technical solution, the method for detecting citrus psylla saliva protein is:

[0028] 1) Incubate the sample to be tested with the above-mentioned citrus psylla saliva protein polyclonal antibody, and the polyclonal antibody specifically binds to the saliva protein in the sample to be tested, thereby forming an immune complex;

[0029] 2) Detect whether the immune complex exists; preferably, the detection method is Western blot.

[0030] The present application has the following beneficial effects:

[0031] Through the preparation of DcMucin-like polyclonal antibody, it is first verified that DcMucin-like is a saliva protein of citrus psylla, and is released into the plant tissue to participate in the formation of saliva sheath when the psylla feeds.

[0032] The present application fills the blank of psylla saliva protein antibody preparation and research, and the prepared citrus psylla saliva protein polyclonal antibody has good specificity to DcMucin recombinant protein, and the antibody immunofluorescence labeling observation of psylla saliva sheath lays a foundation for further studying the biological function of DcMucin in the interaction of huanglongbing bacteria-psylla-citrus, and lays a foundation for further obtaining a specific target of green prevention and control of citrus psylla. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The figure shows the differential expression of DcMucin-like in different tissues of citrus psylla; different letters represent significant differences, p<0.05.

[0034] Figure 2SDS-PAGE (A) and Western blot (B) detection of recombinant plasmid expression in E. coli Rosetta: In the figure, M: medium molecular weight standard protein; 1: pET28a empty vector 0.5 mM IPTG induced bacterial lysate; 2: pET28a-DcMucin uninduced bacterial lysate; 3: pET28a-DcMucin 0.5 mM IPTG induced bacterial lysate; 4: pET28a-DcMucin 0.5 mM IPTG induced bacterial lysate supernatant; 5: pET28a-DcMucin 0.5 mM IPTG induced bacterial lysate precipitate.

[0035] Figure 3 SDS-PAGE (A) and Western blot (B) analysis of the results of the purified fusion protein after purification, in the figure, M: medium molecular weight standard protein; 1: pET28a empty vector 0.5 mM IPTG induced; 2: pET28a-DcMucin uninduced protein supernatant; 3: pET28a-DcMucin 0.5 mM IPTG induced protein supernatant; 4: pET28a-DcMucin 0.5 mM IPTG induced protein supernatant Ni-NTA column purification through the penetration liquid; 5-9: pET28a-DcMucin 0.5 mM IPTG induced protein supernatant Ni-NTA column eluent with 20 mM, 100 mM, 200 mM, 300 mM and 500 mM imidazole.

[0036] Figure 4 Western blot detection of anti-DcMucin-like serum (A) and IgG (B) specificity results, in the figure, M: medium molecular weight standard protein; 1: pET28a empty vector IPTG induced; 2: pET28a-DcMucin IPTG induced whole protein; 3: pET28a-DcMucin IPTG induced protein supernatant; 4: pET28a-DcMucin 0.5 mM IPTG induced protein supernatant Ni-NTA column penetration liquid; 5-9: pET28a-DcMucin 0.5 mM IPTG induced protein supernatant Ni-NTA column eluent with 100 mM, 200 mM, 300 mM and 500 mM imidazole.

[0037] Figure 5 Real-time fluorescent quantitative PCR (A) and Western blot (B) analysis of the differential expression of DcMucin-like gene after infection of citrus psylla with Huanglongbing bacteria, in the figure, ** indicates significant difference, p<0.01; M: medium molecular weight standard protein; CLas-: healthy citrus psylla; CLas+: citrus psylla infected with CLas; GAPDH is the internal reference protein.

[0038] Figure 6 Microscopy observation of DcMucin-like secretion into the plant involved in salivation sheath formation with the feeding of the psyllid. In the figure, FTIC (488 nm): FTIC fluorescent channel; Bright: white light channel; Meged: overlay; Anti-DcMucin (FITC): Anti-DcMucin (FITC) fluorescent antibody labeled section; Unlabelled: section without antibody labeling. DETAILED DESCRIPTION

[0039] The application will be further described in conjunction with the following examples, but the application is not limited by the examples.

[0040] The experimental methods in the following examples are all routine methods unless otherwise specified.

[0041] Example 1

[0042] 1. Materials and methods

[0043] The experiment was completed in 2024 in the State Key Laboratory of Ecological Control of Crop Pests in Fujian Agriculture and Forestry University, the laboratory of mechanism and ecological control of crop diseases transmitted by insect vectors.

[0044] 1.1 Test materials

[0045] Test insects: The bacteria-carrying citrus psyllid was collected from citrus orchards in Gutian County, Ningde City, Fujian Province, and the healthy citrus psyllid was collected from the roadside Murraya exotica plants in the campus of Fujian Agriculture and Forestry University. After PCR detection, the bacteria-carrying and bacteria-free citrus psyllid populations were separately reared in an artificial climate chamber. The bacteria-free psyllid was propagated on healthy Murraya exotica plants for more than 3 generations and confirmed to be bacteria-free by PCR detection. The bacteria-carrying psyllid was continuously reared on citrus plants infected with HLB bacteria to establish a bacteria-carrying citrus psyllid and a healthy citrus psyllid population. The temperature in the artificial climate chamber was 27°C, the humidity was 55-65%, and the light cycle was 16h light and 8h dark.

[0046] Main reagents: pET-28a(+) plasmid, E. coli DH5a and Roessta strain competent preserved by the laboratory; restriction endonuclease BamH I and Xho I (Bodipy Biotech (Beijing) Co., Ltd.); 2×SuperNova super fidelity PCR premix, 2×RealStar Fast dye method qPCR premix (Low ROX), EZ-Flex seamless cloning kit, TRIGene Plus total RNA extraction reagent and auxiliary reagent, protein Marker (Kangrun Biotechnology Co., Ltd.); 1stStrand cDNA Synthesis SuperMix (gDNA Purge) reverse transcription (Coast Protein Technology Co., Ltd.); gel recovery kit, plasmid extraction kit, IPTG (Solebiotech Co., Ltd.); Ni-NTA column (Promega (Beijing) Biotechnology Co., Ltd.); Anti-6×His rabbit polyclonal antibody, HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Wuhan Synergy Biotech Co., Ltd.); Freund's complete adjuvant and Freund's incomplete adjuvant (Sigma Merck Co., Ltd.); serum purification Pierce TM Protein A chromatographic column (Thermo Fisher Scientific Co., Ltd.); cross-linked antibody dialysis bag MD10 (Viskase).

[0047] 1.2DcMucin-like antigen gene sequence amplification

[0048] According to the proteomics and mass spectrometry analysis results of the salivary glands and salivary proteins of H. aurantii reported in the prior art, the sequence information of a mucus-like protein DcMucin-like (DcitrP023480.1.1) was obtained. The signal peptide information of the protein was predicted by using a signal peptide online analysis software (https: / / services.healthtech.dtu.dk / services / SignalP-4.1 / ). After removing the signal peptide sequence, specific amplification primers were designed according to the sequence of the signal peptide-free DcMucin-like (SEQ ID NO. 1), and 21 bp sequences homologous to the sequences at both ends of pET-28a linearized by double digestion with BamH I and Xho I were introduced at the 5' end of the primers. The primer sequences (DcMucin-F / R) are shown in Table 1. The total RNA of H. aurantii adults was extracted by using TRIGene Plus total RNA extraction reagent, and the cDNA of H. aurantii was obtained by reverse transcription using 1st Strand cDNA Synthesis SuperMix (gDNA Purge) with the total RNA as a template. The above DcMucin-F / R primers and 2x SuperNova super-fidelity PCR premix were used to perform PCR amplification with the cDNA of H. aurantii as a template, and the amplification product was recovered by gel to obtain the DcMucin-like antigen gene fragment.

[0049] The nucleotide sequence of DcMucin-like without signal peptide (SEQ ID NO. 1) is as follows: ATGGATAAGCAAAGTTCTTCTCTGCTGCTTCTGGCCATTTTGGCTGCCTTCAACCTGAACCTATGCACTTCCCAGAGCCAGACCCCTGCACCTGCTGTAAACGGGACCACACCTGCTGTAAACGGGACCACACCTGCGGTAAATGGGACGACACCTGCTGCAAACGGGACAACACCCGCGGGAAATGGAATCACATCTAGTGAGAAACCAAAGGGTAAATTCGAGTACACCAACCTGACCAAGGCTGCTATCTGTATTCCAAATAACAACGAGTCTATCAGCTACTGTGAGACTAAATTGAAGGCTCAAGTGGAGACCCACTTCCATCCTGAACTCAAGCTGGCCAATCTTACCGAAGGGAAATGTGTCTATTTATCTGATGGACCCGAAGTAACTGGCTCAACTCCAGCAGGTAACGGAACCACCCCACCCGGCAACGGAACCACCCCAGCCGGCAACGGAACTACCCCAGCCGGTACCACTCCAGCAGGCACCAGCCCGGCAGGCAACGGTACCACCCCAGCCCCTGGAGGGGCTCCTACAGGACCACTCCGTTGTACCATAACCGGACTCTACACCACCCCGGGGGGAGTTCAAGAGAGAGTGCGTCTCAACTTCTTCAACAATGTCAATGACCCAAAAGGCAAGGACAATGTGATCATGGTGATGGGTTACTATGGTAACGCCAGTCAGTCCAACAACCTGGTTGAGACACCTTTGAAATAG.

[0050] 1.3 Differential expression of DcMucin-like in different tissues of citrus psylla

[0051] The salivary glands, midgut, ovaries and testes tissues of adult H. aurantii were dissected, respectively, and total RNA was extracted from each tissue and reverse transcribed to obtain cDNA according to the method described in 1.2. The specific amplification primers DcMucin-qF / R (Table 1) were designed according to the DcMucin-like sequence, and the cDNA of each tissue was used as a template to amplify and collect fluorescence in the Bio-Rad CFX real-time fluorescence quantitative PCR instrument by 2x RealStar Fast Dye Method qPCR premix. The relative quantification method (2 -△△Ct ) was used to calculate the transcription level difference of DcMucin-like in each tissue of H. aurantii, and the software GraphPad Prism 8.0 was used for data statistical analysis and drawing of charts.

[0052] Table 1. Primers

[0053]

[0054] 1.4 Construction of DcMucin-like prokaryotic expression vector

[0055] The antigen gene fragment recovered in 1.2 and the pET-28a linearized by double enzyme digestion of BamH I and Xho I were connected by EZ-Flex seamless cloning enzyme homologous recombination, and the connection product was transformed into DH5a competent cells, coated on LB solid medium (containing 100 μg / mL Km), and cultured at 37°C overnight. A single colony was picked up in LB liquid medium containing 100 μg / mL Km, and cultured at 37°C with shaking until the logarithmic growth phase. Bacterial liquid PCR was performed using DcMucin-F / R, and a part of the positive clone bacterial liquid was sent to a sequencing company for sequencing, and the other part was preserved at -80°C with glycerol.

[0056] 1.5 Induction expression of DcMucin-like recombinant protein

[0057] The sequence returned by sequencing was aligned using MegAlign software and online Blast, and the correctly sequenced recombinant plasmid pET-28a-DcMucin-like was extracted to transform Rosseta E. coli expression strain competent cells. The expression bacteria containing the recombinant expression plasmid were inoculated into LB liquid medium containing 100 μg / mL Km, and cultured at 37°C with shaking until OD 600When the value is 0.6-0.8, IPTG inducer (final concentration 0.5 mM) is added, and the culture is induced at 37°C for 8 hours. The 200 mL bacterial solution induced by IPTG is centrifuged at 8000 rpm for 10 minutes to collect the bacterial cells. The bacterial cells are resuspended in 8 mL PBS buffer and then added with protease inhibitor. The bacterial cells are ultrasonically broken on ice for 15 minutes. The supernatant and precipitate are obtained by centrifugation at 8000 rpm for 10 minutes at 4°C. The expression of the recombinant protein is detected by 12% SDS-PAGE gel electrophoresis and Western blot (primary antibody: Anti-6xHis rabbit polyclonal antibody; secondary antibody: HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L)).

[0058] 1.6 Purification of the recombinant fusion protein and preparation of DcMucin-like polyclonal antibody

[0059] After the supernatant of the broken bacterial cells is induced, 700 μL Ni 2+ -NTA Agarose is added, and the mixture is slowly shaken at room temperature on a horizontal shaker for 1.5 hours. The mixture is loaded into a Ni-column, and the eluted liquid is collected as the breakthrough liquid. After the liquid is completely eluted, the column is washed with 10 mL of 20 mM imidazole for 3 times, 3 mL each time, and the eluted liquid is collected as the washing liquid. Then, the target protein is eluted with 100 mM, 200 mM, 300 mM and 500 mM imidazole in sequence, and the eluted liquid is collected. The purification of the target protein in each eluted liquid is detected by 12% SDS-PAGE gel electrophoresis and Western blot (primary antibody: Anti-6xHis rabbit polyclonal antibody; secondary antibody: HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L)). 300 μL of the purified recombinant DcMucin-like protein is emulsified with an equal amount of complete Freund's adjuvant, and one healthy rabbit is selected for initial immunization. From the second immunization, the purified antigen protein is emulsified with incomplete Freund's adjuvant, and a total of 5 immunizations are performed. Blood is collected one week after the last immunization, and the blood is placed at room temperature for 1 hour, then placed at 4°C overnight, centrifuged at 8000 rpm for 10 minutes, and the collected serum is stored at -80°C. The Pierce TM Protein A chromatography column is used to purify the antiserum to obtain anti-DcMucin-like IgG, which is the DcMucin-like polyclonal antibody.

[0060] 1.7 Western blot detection of antibody specificity and preparation of fluorescent antibody

[0061] First, Western blot analysis was performed against each prokaryotic protein expressed in 1.4 using anti-DcMucin-like serum and purified DcMucin-like IgG as primary antibodies (1:1000) to confirm the specificity of the antibodies for the recombinant proteins. Total DNA was extracted from the citrus psyllid population reared in 1.1, and PCR was performed using the CLas-specific primers described by Jagoueix et al. (1994) (CLas_OI1-F: GCGCGTATGCAATACGAGCGGCA, SEQ ID NO. 8; CLas_OI2c-R: GCCTCGCGACTTCGCAACCCAT, SEQ ID NO. 9) to identify infected and healthy citrus psyllid populations. Ten infected and healthy psyllids were starved for 5 hours and then RNA was extracted from each individual psyllid. cDNA was obtained by reverse transcription according to the methods described in 1.2 and 1.3. The relative expression of the DcMucin-like gene in the psyllids was determined by real-time quantitative PCR using the DcActin gene as an internal reference (primers are shown in Table 1). Total protein was extracted from ten infected and healthy psyllids, and the expression of DcMucin-like in citrus psyllids was detected by Western blot using the prepared polyclonal antibody, using GADPH protein as an internal reference. After verifying the specificity of the antibody, the antibody was cross-linked with fluorescein isothiocyanate (FITC). The specific steps are as follows: 100 μL of DcMucin-like polyclonal antibody was transferred into a cross-linked antibody dialysis bag and dialyzed against 0.01 M NaHCO3; after dialysis, IgG was collected into a 1.5 mL centrifuge tube, and an appropriate amount of FITC was slowly added thereto. After uniform mixing, the mixture was incubated at 4°C in the dark for 1-2 h; the incubated IgG was transferred to a dialysis bag again and dialyzed against 0.01 M PBS buffer; after dialysis, the cross-linked fluorescent antibody DcMucin-like (FITC) was collected and stored at -20°C in the dark.

[0062] 1.8 Observation of citrus psyllids feeding on salivary sheaths of citrus leaves

[0063] 50 adult D. citri were collected and starved for 3 h. Then, the D. citri were allowed to feed on healthy sweet orange leaves using a clip-cage method. After 24 h of feeding, the main veins of the leaves were collected and cut into small pieces. The small pieces were placed in a frozen mold with a suitable amount of embedding agent. After freezing and solidification, the pieces were cut into 20 μM-thick slices. The slices were fixed in 4% paraformaldehyde solution at room temperature for 6 h. After three rinses in 0.01 M PBS buffer, the slices were permeabilized in PBS buffer containing 2% Triton X-100 at room temperature for 3-4 h. After three rinses in 0.01 M PBS buffer, the slices were incubated in a solution containing fluorescent antibody DcMucin-like (FITC) diluted in PBS buffer containing 3% BSA at a ratio of 1:50-1:100 for 2 h at 37°C. After three rinses in 0.01 M PBS buffer, the leaf-vein tissue slices were transferred to a glass slide to which anti-fluorescence quenching solution was added, and a cover glass was placed on top. The prepared sample was observed on a Leica LAS X confocal microscope platform.

[0064] 2 Results

[0065] 2.1 Differential expression of DcMucin-like in different tissues of D. citri

[0066] To determine the expression level of the salivary protein DcMucin-like in the salivary glands of D. citri, the salivary glands, midgut, ovary, and testis tissues of D. citri were dissected, respectively, and the differences in the transcriptional level of DcMucin-like in each tissue were detected and analyzed using real-time fluorescent quantitative PCR. As shown in Table 1, DcMucin-like was highly expressed in the salivary glands of D. citri, and the expression level was significantly higher than that in the midgut, ovary, and testis tissues. Figure 1

[0067] 2.2 Prokaryotic expression and purification of DcMucin-like protein

[0068] The specific amplification of the DcMucin-like antigen sequence (SEQ ID NO. 10) was inserted into the expression vector pET28a by homologous recombination to construct the recombinant prokaryotic expression plasmid pET28a-DcMucin. The correct recombinant plasmid was verified by sequencing and transformed into E. coli Rosetta. After induction and expression, the bacterial cells were broken. SDS-PAGE electrophoresis analysis showed that, compared with the empty vector and uninduced control samples, the E. coli containing the recombinant plasmid had an obvious band at about 36 KDa after IPTG induction (A), which was consistent with the expected size of the recombinant fusion protein. Western blot analysis using an anti-His tag antibody showed that the recombinant fusion protein mainly existed in the supernatant of the bacterial cell lysate (B). Figure 2 Figure 2 ​​B), indicating that the recombinant DcMucin-like protein expressed in E. coli is mainly in the form of soluble protein. The supernatant was purified by Ni 2+ -NTA column, and SDS-PAGE electrophoresis found that the Ni 2+ -NTA column, and SDS-PAGE electrophoresis found that the Ni Figure 3 A), and the results of Western blot detection using anti-His tag antibody further confirmed that 500 mM imidazole is the best eluent for the recombinant DcMucin-like protein Figure 3 B), indicating that the recombinant DcMucin-like protein is successfully purified.

[0069] The nucleotide sequence of the amplified DcMucin-like antigen gene (SEQ ID NO. 10) is as follows :

[0070] CGCGGATCCATGCAGAGCCAGACCCCTGCACCTGCTGTAAACGGGACCACACCTGCTGTAAACGGGACCACACCTGCGGTAAATGGGACGACACCTGCTGCAAACGGGACAACACCCGCGGGAAATGGAATCACATCTAGTGAGAAACCAAAGGGTAAATTCGAGTACACCAACCTGACCAAGGCTGCTATCTGTATTCCAAATAACAACGAGTCTATCAGCTACTGTGAGACTAAATTGAAGGCTCAAGTGGAGACCCACTTCCATCCTGAACTCAAGCTGGCCAATCTTACCGAAGGGAAATGTGTCTATTTATCTGATGGACCCGAAGTAACTGGCTCAACTCCAGCAGGTAACGGAACCACCCCACCCGGCAACGGAACCACCCCAGCCGGCAACGGAACTACCCCAGCCGGTACCACTCCAGCAGGCACCAGCCCGGCAGGCAACGGTACCACCCCAGCCCCTGGAGGGGCTCCTACAGGACCACTCCGTTGTACCATAACCGGACTCTACACCACCCCGGGGGGAGTTCAAGAGAGAGTGCGTCTCAACTTCTTCAACAATGTCAATGACCCAAAAGGCAAGGACAATGTGATCATGGTGATGGGTTACTATGGTAACGCCAGTCAGTCCAACAACCTGGTTGAGACACCTTTGAAACTCGAGCAC.

[0071] The amino acid sequence of the recombinant DcMucin-like protein (SEQ ID NO. 11) is as follows :

[0072] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMQSQTPAPAVNGTTPAVNGTTPAVNGTTPAANGTTPAGNGITSSEKPKGKFEYTNLTKAAICIPNNNESISYCETKLKAQVETHFHPELKLANLTEGKCVYLSDGPEVTGSTPAGNGTTPPGNGTTPAGNGTTPAGTTPAGTSPAGNGTTPAPGGAPTGPLRCTITGLYTTPGGVQERVRLNFFNNVNDPKGKDNVIMVMGYYGNASQSNNLVETPLKLEHHHHHH.

[0073] 2.3DcMucin-like polyclonal antibody specificity detection

[0074] After immunizing rabbits 5 times with 500 mM imidazole elution collected purified DcMucin-like recombinant protein, we obtained anti-DcMucin-like serum by taking blood. Western blot detection of prokaryotic expression protein with anti-serum as the first antibody showed that there was a 36 KDa band in the sample containing DcMucin-like recombinant protein, but there were also non-specific bands Figure 4 A). Subsequently, Western blot detection of the above samples was performed again after IgG purification of anti-DcMucin-like serum to obtain anti-DcMucin-like IgG, and the results showed that the purified DcMucin-like polyclonal antibody had good specificity for DcMucin-like recombinant protein Figure 4 B). Figure 5 A), DcMucin-like gene was up-regulated in CLas-infected D. citri. Subsequently, we found that Figure 5 B), DcMucin-like polyclonal antibody can specifically recognize DcMucin-like in D. citri, and CLas infection can cause up-regulation of DcMucin-like protein in D. citri.

[0075] 2.4Observation of D. citri salivary sheath after feeding on citrus leaves

[0076] Most piercing-sucking insects will secrete saliva into plant tissues to help them feed during the feeding process. It has been reported that DcMucin-like protein can be detected in citrus psylla saliva collected by membrane feeding, which is involved in the formation of salivary sheath when other hemipteran insects feed on plants. In this study, we observed the main vein of citrus leaves after D. citri feeding by ultrathin sectioning, and observed the feeding holes of D. citri and specific FITC green fluorescent signals around the feeding holes by labeling tissue sections with FITC cross-linked DcMucin-like polyclonal antibody under confocal microscope Figure 6 A); but no FITC fluorescence was observed in tissue sections without D. citri feeding and without labeled antibody Figure 6 B, C). These results verify that DcMucin-like is a salivary protein of D. citri and is released into plant tissues to participate in the formation of salivary sheath with D. citri feeding.

[0077] 3 Analysis

[0078] Saliva sheath formed by the gelatinous saliva is indispensable for insect feeding, which is secreted into plant tissues during the process of stylet probing and remains in the plant when the stylet is retracted. In Nilaparvata lugens and Sogatella furcifera, Mucin protein is the main component of their gelatinous saliva, which is involved in saliva sheath formation, and silencing Mucin will cause feeding difficulties in insects. Saliva sheath proteins of piercing-sucking insects not only have mechanical functions, but also activate or inhibit plant immunity to participate in the game between insects and host plants (Reference 1: Huang HJ, Zhang CX, Hong XY. How does saliva function in planthopper-host interactions? Archives of Insect Biochemistry and Physiology. 2019, 100(4); Reference 2: Huang HJ, Wang YZ, Li LL, Lu HB, Lu JB, Wang X, Ye ZX, Zhang ZL, He YJ, Lu G, Zhuo JC, Mao QZ, Sun ZT, Chen JP, Li JM, Zhang CX. Planthopper salivary sheath protein LsSP1 contributes to manipulation of rice plant defenses. Nature Communications. 2023, 14(1)). Diaphorina citri is the main transmission vector of citrus Huanglongbing, the “number one killer” of citrus. Studying the function of saliva sheath protein of woodlice can provide new ideas for the prevention and control of citrus Huanglongbing.

[0079] 3.1 Optimization of the preparation conditions of DcMucin-like antibody of Diaphorina citri

[0080] Previous studies have shown that DcMucin-like is highly abundant in citrus psylla saliva proteins, but its biological function is not clear. In this study, we analyzed the relative expression level of DcMucin-like in the salivary glands, midgut, ovary and testis of citrus psylla by qPCR, and verified that the gene is highly expressed in the saliva glands of psylla. The preparation of DcMucin-like antibody can lay the foundation for further elucidating the role of this protein in citrus psylla feeding and transmitting Candidatus Liberibacter spp. The antigen obtained by prokaryotic expression of recombinant protein is a way to quickly obtain polyclonal antibodies. In this study, E. coli Roseate carrying pET-28a-DcMucin-like recombinant plasmid was induced at an IPTG final concentration of 0.5 mM at 37°C for 8 h, and DcMucin-like recombinant protein was expressed in the supernatant of bacterial lysate. The recombinant protein was purified by Ni 2+ -NTAAgarose column and eluted with 500 mM imidazole solution to obtain purified antigen. Then the antigen was used to immunize rabbits, and the specific DcMucin-like polyclonal antibody was obtained by purifying the antiserum IgG.

[0081] 3.2 Candidatus Liberibacter spp. induced up-regulation of DcMucin-like expression in citrus psylla

[0082] Mucin protein is a highly glycosylated mucin protein that exists widely in the salivary glands and intestines of insects. This type of protein has various biological functions, including being secreted into plants with saliva to play multiple roles to assist insect feeding, and participating in immune response and regulating cell signaling in epithelial tissues in vivo. Mucin is involved in the formation of the saliva sheath of the white-backed planthopper, which is a key protein for successful feeding of the white-backed planthopper, and silencing Mucin can reduce the feeding amount of the white-backed planthopper, making it unable to successfully feed on the phloem sap of rice. Meanwhile, silencing the mucin-like of the brown planthopper (Nilaparvata lugens) can cause the configuration of the insect saliva sheath to be shorter and single-branched, affecting the growth and development of the insect. In addition, Mucin protein can help pathogens adhere to the surface of insect epithelial cells, which helps pathogens enter cells and replicate, thereby exacerbating infection.

[0083] To explore whether DcMucin-like is differentially expressed in response to CLas infection, qPCR and Western blot analysis were performed. The results showed that the expression of DcMucin-like was up-regulated at both mRNA and protein levels in response to CLas infection, indicating that DcMucin-like plays an important role in the interaction between CLas and the vector citrus psyllid. The transmission of CLas by citrus psyllid is the result of the interaction among citrus psyllid, CLas and host plant. Previous studies have shown that healthy citrus psyllids prefer to feed on diseased plants, while CLas-infected citrus psyllids prefer to feed on healthy plants. This may be due to the different volatile components of healthy and diseased citrus plant shoots, as well as the interaction between CLas and the vector citrus psyllid.

[0084] In the present application, DcMucin-like antibody labeled with cross-linked fluorescein was used to mark the main veins of citrus leaves after feeding by citrus psyllid. Confocal microscopy showed specific fluorescent signals around the feeding holes of citrus psyllid, which were similar to saliva sheath. This is the first time that DcMucin-like has been identified as a saliva protein of citrus psyllid, and it is released into plant tissues after feeding by citrus psyllid to participate in saliva sheath formation. The present application shows that DcMucin may play an important role in the interaction among CLas, citrus psyllid and citrus plant, and provides a new target for citrus disease and pest resistance breeding and citrus psyllid control.

[0085] In summary, the present application shows that DcMucin is up-regulated in response to CLas infection, and can be secreted into citrus plant tissues after feeding by citrus psyllid. Soluble DcMucin fusion protein was successfully expressed, and specific DcMucin rabbit polyclonal antibody was prepared. This antibody can be used for Western blot analysis and immunofluorescence labeling.

Claims

1. An antigenic polypeptide for inducing polyclonal antibodies against citrus psyllid salivary proteins, characterized in that: Its amino acid sequence is the amino acid sequence shown in SEQ ID NO.

11.

2. A biological material containing the gene encoding the antigen polypeptide according to claim 1, wherein the biological material is an expression cassette, plasmid, expression vector or host bacteria.

3. Use of the antigenic polypeptide according to claim 1 or the biological material according to claim 2 in preparing an antigen for inducing polyclonal antibodies against citrus psyllid salivary proteins.

4. A polyclonal antibody against citrus psyllid salivary protein, characterized in that: The polyclonal antibody is induced by the antigen polypeptide according to claim 1.

5. The method for preparing the polyclonal antibody against citrus psyllid salivary protein according to claim 4, characterized in that: The steps include: (1) constructing a recombinant expression vector containing the nucleotide sequence shown in SEQ ID NO. 10, and transforming the recombinant expression vector into Escherichia coli competent cells to obtain a recombinant protein antigen; (2) Immunize animals with the obtained recombinant protein antigen, collect blood, separate and purify the antiserum.

6. The preparation method according to claim 5, characterized in that: The recombinant expression vector in step (1) is obtained by connecting the antigen gene fragment with the nucleotide sequence shown in SEQ ID NO.10 with the prokaryotic expression vector pET-28a(+); The recombinant expression vector is transformed into Escherichia coli competent cells and then inoculated into liquid culture medium for culturing and expansion, and then IPTG inducer is added to induce expression to obtain DcMucin recombinant protein; The antigen gene fragment of the nucleotide sequence shown in SEQ ID NO.10 is a fragment without a signal peptide as shown in SEQ ID NO.

1. DcMucin-like The sequence was designed and amplified using specific primers by PCR.

7. The preparation method according to claim 6, characterized in that: The induction expression conditions are as follows: IPTG final concentration 0.4-0.6 mM, 36-38°C induction for 7-9 h; The specific amplification primers are as follows: DcMucin-F: 5'-CAGCAAATGGGTCGCGGATCCATGCAGAGCCAGACCCCTGCACCTGCTGTAAA-3'; DcMucin-R: 5'-GTGGTGGTGGTGGTGCTCGAGTTTCAAAGGTGTCTCAACCAGGTTGTTGGA-3'.

8. The preparation method according to claim 5, characterized in that: In step (2), the recombinant protein antigen is first mixed with complete Freund's adjuvant and then the animal is immunized for the first time, and then the recombinant protein antigen is mixed with incomplete Freund's adjuvant and then immunized for the second to fourth times, and the serum is separated and purified to obtain the citrus psyllid salivary protein polyclonal antibody.

9. A kit comprising the polyclonal antibody against citrus psyllid salivary protein according to claim 4.

10. Use of the polyclonal antibody to citrus psyllid salivary protein according to claim 4 or the kit according to claim 9 in preparing a kit for detecting citrus psyllid salivary protein.

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