Diaphorina citri sialoprotein DcMucin antibody as well as preparation method and application thereof

Abstract

The invention discloses an antibody of diaphorina citri sialoprotein DcMucin as well as a preparation method and application of the antibody. Through the preparation of the DcMucin-like polyclonal antibody, it is verified for the first time that DcMucin-like is salivary protein of diaphorina citri, and DcMucin-like is released into plant tissues along with feeding of diaphorina citri to participate in salivary sheath formation. The diaphorina citri salivary protein polyclonal antibody and the preparation method thereof make up the blank of diaphorina citri salivary protein antibody preparation and research, the prepared diaphorina citri salivary protein polyclonal antibody has good specificity on DcMucin recombinant protein, and diaphorina citri salivary sheath is observed based on antibody immunofluorescence labeling; a foundation is laid for further research on biological functions of DcMucin in interaction of the Candidatus Liberibacter asiaticum, the phylloxera and the citrus, and a foundation is laid for further obtaining a specific target for green prevention and control of the phylloxera.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology and plant disease and insect pest control, and in particular to an antibody against a citrus psyllid salivary protein DcMucin and a preparation method and application thereof. Background Art

[0002] The Asian citrus psyllid (Diaphorina citri) belongs to the Psyllidae family of the order Hemiptera. It is a typical piercing-sucking insect. It has a serious impact on citrus production by sucking citrus phloem sap and spreading the citrus Huanglongbing disease (Candidatus Liberibacter). Similar to other Hemiptera insects, the citrus psyllid secretes two types of saliva during feeding, gelatinous saliva and aqueous saliva. Gelatinous saliva is mainly involved in the formation of salivary sheaths to wrap the stylet, which is convenient for piercing plant tissues. Aqueous saliva contains a variety of enzymes that have the functions of degrading plant cells, detoxifying plant allelochemicals, and inhibiting or stimulating plant defense responses. Given that saliva plays an important role in the feeding and interaction of citrus psyllids with citrus, the study of psyllid salivary proteins will help to explore new targets for pest control, thereby providing support for biological control of psyllids.

[0003] The saliva of Hemiptera insects acts as a mediator bridge in the interaction between insects, plants and pathogens. When citrus psyllids suck the phloem sap of citrus plants with bacteria, Huanglongbing bacteria enter the stylet with the sap, pass through the filtration chamber and foregut to reach the midgut system, then break through the midgut release barrier to spread to the hemolymph, infect the salivary glands through the hemolymph system, and finally break through the salivary gland release barrier to complete the infection cycle in the vector citrus psyllid. When the psyllids feed on phloem sap, they are excreted with saliva into new host plants (Chen Q et al., 2022; Ammar E-D et al., 2011; Weintraub PG et al., 2006). The salivary sheath formed by gelatinous saliva is indispensable for insect feeding. It is secreted during the process of the insect stylet probing plant tissues, providing mechanical stability and lubrication for the movement of the stylet (Huang HJ et al., 2019). The salivary sheath can seal the penetration site of the stylet, thereby inhibiting the plant immune response triggered by the leakage of cellular components (Miles PW et al., 1999). In aphids and leafhoppers, interfering with the formation of the salivary sheath will hinder the insects from feeding from the plant sieve tubes. The salivary sheath is composed of a variety of salivary sheath proteins, which may be recognized as relevant molecular patterns of herbivorous insects, thereby activating the immune response of the host plant (Shangguan XX et al., 2018). At present, the understanding of insect salivary sheath proteins is mainly limited to their mechanical functions, and there are few reports on their other functions in the interaction between herbivorous insects and plants.

[0004] Mucin-like proteins play an important role in the formation of insect salivary sheaths. Huang HJ et al. (2016, 2017) found that mucin-like proteins are specifically expressed in the salivary glands of brown planthoppers, playing a role in the virulence of brown planthoppers and adapting to host plant resistance. Brown planthopper (Laodelphax striatellus) salivary sheath protein LsSP1 can be secreted into host plants and bind to the salivary sheath by interacting with mucin-like protein (LsMLP). Overexpression of LsSP1 in plants can significantly reduce LsMLP-induced H2O2 accumulation and defense gene expression, and can rescue insect feeding defects caused by insufficient secretion of LsSP1, indicating the importance of salivary sheath proteins in the interaction between herbivorous insects and host plants (2023). Shangguan XX et al. (2018) also found that the mucus-like protein of brown planthopper can cause plant cell death, induce the expression of defense-related genes and the deposition of callose, indicating that mucus-like protein is also involved in the interaction between insect salivary sheath proteins and host plants.

[0005] The above studies on the functions of salivary sheaths of plant-feeding insects are all based on the preparation of corresponding specific antibodies. At present, there is a lack of antibodies to salivary proteins of citrus psyllids, especially antibodies to salivary sheath proteins of psyllids. Therefore, the preparation of relevant antibodies will help to clarify their biological roles in psyllid feeding and interaction with citrus hosts in the future, laying the foundation for obtaining specific targets for green control of citrus psyllids. Summary of the invention

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

[0007] In order to achieve its purpose, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides an antigenic polypeptide for inducing polyclonal antibodies against citrus psyllid salivary protein, the amino acid sequence of which is the amino acid sequence shown in SEQ ID NO.11.

[0009] The second aspect of the present invention provides a biological material of the above-mentioned antigen polypeptide encoding gene, wherein the biological material is an expression cassette, a plasmid, an expression vector or a host bacterium.

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

[0011] (1) Application in the preparation of antigens for inducing polyclonal antibodies against citrus psyllid salivary proteins;

[0012] (2) Use in the preparation of reagents or kits for detecting salivary proteins of citrus psyllids or their expression levels.

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

[0014] The fifth aspect of the present invention provides a method for preparing the above-mentioned citrus psyllid salivary protein polyclonal antibody, comprising the following steps:

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

[0016] (2) Immunizing animals with the obtained recombinant protein antigen, collecting blood, separating and purifying the antiserum.

[0017] In the above-mentioned preparation method, 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(+);

[0018] The recombinant expression vector is transformed into competent Escherichia coli cells and inoculated into liquid culture medium for culture expansion, and then IPTG inducer is added to induce expression to obtain DcMucin recombinant protein; the preferred induction expression conditions are IPTG final concentration of 0.4-0.6mM, 36-38°C for induction for 7-9h;

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

[0020] DcMucin-F:

[0021] 5'-CAGCAAATGGGTCGCGGATCCATGCAGAGCCAGACCCCTGCACCTGCTGTAAA-3';

[0022] DcMucin-R:

[0023] 5'-GTGGTGGTGGTGGTGCTCGAGTTTCAAAGGTGTCTCAACCAGGTTGTTGGA-3'.

[0024] In the above preparation method, in step (2), the recombinant protein antigen is first mixed with complete Freund's adjuvant and then 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 citrus psyllid salivary protein polyclonal antibodies are obtained by serum separation and purification.

[0025] The sixth aspect of the present invention provides a kit containing the above-mentioned polyclonal antibody to citrus psyllid salivary protein.

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

[0027] In the above-mentioned application technology scheme, the method for detecting citrus psyllid salivary protein is:

[0028] 1) incubating the sample to be tested with the above-mentioned polyclonal antibody to salivary protein of citrus psyllid, wherein the polyclonal antibody specifically binds to the salivary protein in the sample to be tested, thereby forming an immune complex;

[0029] 2) Detecting the presence of immune complexes; the preferred detection method is Western blot.

[0030] The beneficial effects of the present invention are:

[0031] Through the preparation of DcMucin-like polyclonal antibodies, it was verified for the first time that DcMucin-like is a salivary protein of citrus psyllid, and is released into plant tissues as the psyllid feeds and participates in the formation of salivary sheaths.

[0032] The present invention fills the gap in the preparation and research of psyllid salivary protein antibodies. The prepared citrus psyllid salivary protein polyclonal antibody has good specificity for DcMucin recombinant protein. The psyllid salivary sheath is observed based on immunofluorescence labeling with the antibody, laying a foundation for further studying the biological function of DcMucin in the interaction between Huanglongbing bacteria, psyllids and citrus, and laying a foundation for further obtaining specific targets for green control of citrus psyllids. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The differential expression of DcMucin-like in different tissues of citrus psyllid is shown; different letters indicate significant differences, p<0.05.

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

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

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

[0037] Figure 5 The results of real-time fluorescence quantitative PCR (A) and Western blot (B) analysis of the differential expression of DcMucin-like genes in citrus psyllids infected with Huanglongbing fungus are shown. In the figure, ** indicates significant difference, p<0.01; M: medium molecular weight standard protein; CLas-: healthy citrus psyllid; CLas+: citrus psyllid infected with CLas; GAPDH is the internal reference protein.

[0038] Figure 6 The image shows that DcMucin-like is secreted into the plant as the psyllid feeds and participates in the formation of salivary sheaths observed under a microscope. In the image, FTIC (488 nm): FTIC fluorescein channel; Bright: white light channel; Megaed: overlap; Anti-DcMucin (FITC): slices labeled with Anti-DcMucin (FITC) fluorescent antibody; Unlabelled: slices without antibody labeling. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

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

[0041] Example 1

[0042] 1 Materials and methods

[0043] The experiment was completed in 2024 at the National Key Laboratory of Ecological Control of Crop Pests in Fujian and Taiwan, and the Laboratory of Mechanism and Ecological Control of Insect-Transmitted Crop Diseases, Fujian Agriculture and Forestry University.

[0044] 1.1 Test materials

[0045] Test insects: Infected citrus psyllids were collected from citrus orchards in Gutian County, Ningde City, Fujian Province, and healthy citrus psyllids were collected from Murraya exotica plants on both sides of the road in the campus of Fujian Agriculture and Forestry University. After PCR testing, the non-infected and non-infected citrus psyllid populations were separated in an artificial climate culture room. The non-infected psyllids were propagated on healthy Murraya exotica plants for more than 3 generations and confirmed to be non-infected by PCR testing. The infected psyllids were continued to be raised on citrus plants infected with Huanglongbing bacteria, and continued to propagate to establish the infected citrus psyllid and healthy citrus psyllid populations. The temperature in the artificial climate culture room was 27°C, the humidity was 55-65%, and the photoperiod was 16h light and 8h dark.

[0046] Main reagents: pET-28a(+) plasmid, Escherichia coli DH5а and Roessta competent strains were maintained by our laboratory; restriction endonucleases BamH I and Xho I (Baoriyi Biotechnology (Beijing) Co., Ltd.); 2×SuperNova ultra-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 reagents, protein marker (Kangrun Biotechnology Co., Ltd.); 1stStrand cDNA Synthesis SuperMix (gDNA Purge) reverse transcription (Jinan Protein Technology Co., Ltd.); gel recovery kit, plasmid extraction kit, IPTG (Solabo Technology Co., Ltd.); Ni-NTA column (Promega (Beijing) Biotechnology Co., Ltd.); Anti-6×His rabbit polyclonal antibody, HRP-conjugatedAffinipure Goat Anti-Rabbit IgG (H+L) (Wuhan Sanying Biotechnology Co., Ltd.); Freund's complete adjuvant and Freund's incomplete adjuvant (Sigma Merck Co., Ltd.); serum purification Pierce TM Protein A chromatography column (Thermo Fisher Scientific); 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 salivary glands and salivary proteins of citrus psyllids reported in previous studies, the sequence information of mucus-like protein DcMucin-like (DcitrP023480.1.1) was obtained. The signal peptide information of the protein was predicted using the 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 based on the DcMucin-like sequence without signal peptide (SEQ ID NO.1), and a 21bp sequence homologous to the two ends of pET-28a linearized by BamH I and Xho I was introduced at the 5' end of the primer. The primer sequence (DcMucin-F / R) is shown in Table 1. Total RNA of citrus psyllid adults was extracted using TRIGene Plus total RNA extraction reagent, and 1st Strand cDNA Synthesis SuperMix (gDNA Purge) was used as a template for reverse transcription to obtain citrus psyllid cDNA. The above-mentioned DcMucin-F / R primers and 2×SuperNova ultra-fidelity PCR premix were used to perform PCR amplification with the psyllid cDNA as a template. The amplified product was recovered by gel and was 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 Diaphorina citri

[0051] The salivary glands, midgut, ovary and testis tissues of citrus psyllid adults were dissected, and total RNA from each tissue was extracted and reverse transcribed to obtain cDNA as described in 1.2. The real-time fluorescence quantitative PCR specific amplification primers DcMucin-qF / R (Table 1) were designed based on the DcMucin-like sequence. The cDNA of each tissue was used as a template and amplified in a Bio-Rad CFX real-time fluorescence quantitative PCR instrument using 2×RealStar Fast dye method qPCR premix and fluorescence was collected. The citrus psyllid DcActin was used as an internal reference gene and the relative quantification method (2 -△△Ct )The transcriptional level differences of DcMucin-like in various tissues of psyllids were calculated, and the data were statistically analyzed and graphed using GraphPadPrism 8.0 software.

[0052] Table 1. Primers

[0053]

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

[0055] The antigen gene fragments recovered in 1.2 and pET-28a linearized by double restriction digestion with BamH I and Xho I were connected by homologous recombination with EZ-Flex seamless cloning enzyme, and the ligation products were transformed into DH5a competent cells, spread on LB solid medium (containing 100 μg / mL Km), and cultured at 37°C overnight. Pick a single colony in LB liquid medium containing 100 μg / mL Km, shake culture at 37°C until the logarithmic growth phase, use DcMucin-F / R for bacterial liquid PCR, send part of the positive clone bacterial liquid to the sequencing company for sequencing, and add glycerol to the other part and store it at -80°C.

[0056] 1.5DcMucin-like recombinant protein induced expression

[0057] The sequences returned by sequencing were aligned using MegAlign software and online Blast, and the recombinant plasmid pET-28a-DcMucin-like with correct sequencing was extracted and transformed into competent cells of Rosseta E. coli expression strain. 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 was 0.6-0.8, IPTG inducer (final concentration 0.5mM) was added and induced with shaking at 37℃ for 8h. 200mL bacterial solution induced by IPTG was centrifuged at 8000rpm for 10min and then the bacteria were collected. The bacteria were resuspended in 8mL PBS buffer and protease inhibitors were added. The bacteria were ultrasonically broken on ice for 15min, centrifuged at 8000rpm for 10min at 4℃, and the supernatant and precipitate were taken. The expression of recombinant protein was detected by 12% SDS-PAGE gel electrophoresis and Western blot (primary antibody: Anti-6×His rabbit polyclonal antibody; secondary antibody: HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L)).

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

[0059] Add 700 μL Ni 2+ -NTA Agarose, mix well and shake slowly on a horizontal shaker at room temperature for 1.5h, load the mixed solution into a Ni-column and let it flow down slowly, collect the flowing liquid as the penetration liquid, and after the liquid flows out, wash the column with 10mL 20mM imidazole for 3 times, 3mL each time, collect the flowing liquid as the washing liquid, and then elute the target protein with 100mM, 200mM, 300mM and 500mM imidazole in sequence, and collect the eluate. The purification of the target protein in each eluate was detected by 12% SDS-PAGE gel electrophoresis and Western blot (primary antibody: Anti-6×His rabbit polyclonal antibody; secondary antibody: HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L). 300 μL of the above-purified recombinant DcMucin-like protein was emulsified with an equal amount of complete Freund's adjuvant, and a healthy rabbit was selected for the first immunization. Starting from the second immunization, the purified antigen protein was emulsified with incomplete Freund's adjuvant, and a total of 5 immunizations were performed. Blood was collected one week after the last immunization. The collected blood was placed at room temperature for 1 hour, placed at 4°C overnight, and centrifuged at 8000 rpm for 10 minutes. The serum was collected and stored at -80°C as anti-DcMucin-like serum. Pierce TM The antiserum was purified by protein A column 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 antibodies

[0061] First, anti-DcMucin-like serum and purified DcMucin-like IgG were used as primary antibodies (1:1000) to perform Western blot detection on the prokaryotic expressed proteins in 1.4 to confirm the specificity of the antibodies to the recombinant proteins. Total DNA of the citrus psyllid population raised in 1.1 was extracted, and PCR detection was performed according to the CLas-specific detection primers of Jagoueix et al. (1994) (CLas_OI1-F: GCGCGTATGCAATACGAGCGGCA, SEQ ID NO.8; CLas_OI2c-R: GCCTCGCGACTTCGCAACCCAT, SEQ ID NO.9) to obtain the populations of citrus psyllids with and without bacteria. Ten psyllids carrying bacteria and healthy psyllids were taken, and after starvation for 5 hours, RNA of single psyllids was extracted and reverse transcribed to obtain cDNA according to the methods in 1.2 and 1.3. The relative expression of DcMucin-like gene of psyllids was determined by real-time fluorescence quantitative PCR with DcActin gene of psyllids as internal reference (primers are shown in Table 1). At the same time, ten psyllids carrying bacteria and healthy psyllids were taken, total protein of psyllids was extracted, GADPH protein was used as internal reference, and the expression of DcMucin-like in citrus psyllids was detected by Western blot using the prepared polyclonal antibody. After verifying the specificity of the antibody, the antibody was cross-linked with fluorescein isothiocyanate (FITC), and the specific steps were as follows: 100 μL of DcMucin-like polyclonal antibody was transferred into a cross-linked antibody dialysis bag and dialyzed with 0.01 M NaHCO3; after the dialysis, IgG was collected into a 1.5 mL centrifuge tube, and an appropriate amount of FITC was slowly added thereto, mixed evenly, and incubated at 4°C in the dark for 1-2 h; the incubated IgG was transferred to a dialysis bag again and dialyzed in 0.01 M PBS buffer; after the 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 citrus psyllids were collected and starved for 3 hours. The psyllids were then forced to feed on healthy sweet orange plant leaves using a leaf cage method. After 24 hours of feeding, the psyllids were removed and the main veins of the fed leaves were collected. The main veins of the leaves were cut into sections and placed in a freezing mold with an appropriate amount of embedding agent. After freezing and solidification, the sections were set to a thickness of 20 μM for cryosectioning. The sections were collected and fixed in 4% paraformaldehyde solution at room temperature for 6 h; after rinsing 3 times with 0.01 M PBS buffer, they were permeabilized in PBS buffer containing 2% Triton X-100 at room temperature for 3-4 h; after rinsing 3 times with 0.01 M PBS buffer, the sections were placed in a solution containing fluorescent antibody DcMucin-like (FITC) (the antibody was diluted at 1:50-1:100 with PBS buffer containing 3% BSA), and incubated at 37°C for 2 h; after rinsing 3 times with 0.01 M PBS buffer, the petiole tissue sections were transferred to a slide with anti-fluorescence attenuation solution added and covered with a coverslip; the prepared samples were observed on the Leica LAS X confocal microscope platform.

[0064] 2 Results

[0065] 2.1 Differential expression of DcMucin-like in different tissues of citrus psyllid

[0066] In order to clarify the expression level of salivary protein DcMcuin-like in the salivary glands of citrus psyllid, the salivary glands, midgut, ovaries and testes of psyllid were dissected, and the differences in the transcription level of DcMcuin-like in each tissue were analyzed by real-time fluorescence quantitative PCR. Figure 1 As shown, DcMcuin-like was abundantly expressed in the salivary glands of psyllids, and the expression level was significantly higher than that in the midgut, ovary and testis tissues.

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

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

[0069] The amplified DcMucin-like antigen gene nucleotide sequence (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 the purified DcMucin-like recombinant protein collected by the 500mM imidazole eluate, blood was collected to obtain anti-DcMucin-like serum. The antiserum was used as the primary antibody to perform Western blot detection on the prokaryotic expressed protein. The results showed that there was a target band of about 36KDa in the sample containing the DcMucin-like recombinant protein, but there were also non-specific bands ( Figure 4 A). Then, the anti-DcMucin-like serum was purified to obtain anti-DcMucin-like IgG, and the above samples were tested by Western blot again. The results showed that the purified DcMucin-like polyclonal antibody had good specificity for DcMucin-like recombinant protein ( Figure 4 B). The relative expression of DcMucin-like genes in infected psyllids and healthy psyllids showed that ( Figure 5 A), DcMucin-like gene was up-regulated in the infected psyllid. Then the purified DcMucin-like polyclonal antibody was used to detect the DcMucin-like protein level in healthy and infected citrus psyllids. ( Figure 5 B), DcMucin-like polyclonal antibody can specifically recognize DcMucin-like in psyllids, and CLas infection can cause up-regulated expression of DcMucin-like protein in psyllids.

[0075] 2.4 Observation of psyllids feeding on salivary sheaths of citrus leaves

[0076] Most piercing-sucking insects secrete saliva into plant tissues during feeding to help them feed. It has been previously reported that DcMucin-like protein can be detected in the saliva of citrus psyllids collected by membrane feeding. This protein is involved in the formation of salivary sheaths when feeding on plants in other Hemiptera insects. In this study, ultrathin sections were made on the main veins of citrus leaves after citrus psyllids had fed. The tissue sections were labeled with a fluorescein (FITC)-crosslinked DcMucin-like polyclonal antibody. The feeding holes of the psyllids were observed under a confocal microscope, and specific FITC green fluorescence signals were detected around the feeding holes ( Figure 6 A); however, no FITC fluorescence was observed in tissue sections without psyllid feeding and unlabeled antibodies ( Figure 6 B, C). These results confirm that DcMucin-like is a salivary protein of citrus psyllids and is released into plant tissues as the psyllids feed and participates in the formation of salivary sheaths.

[0077] 3 Analysis

[0078] The salivary sheath formed by the gelatinous saliva is indispensable for insects to feed. It is secreted into plant tissues during the probing process of the stylet, and the salivary sheath remains in the plant when the stylet is retracted. In brown planthoppers and white-backed planthoppers, Mucin protein is the main component of their gelatinous saliva and is involved in the formation of the salivary sheath. Silencing Mucin will make it difficult for insects to feed. The salivary sheath proteins of piercing-sucking insects not only have mechanical functions, but also can activate or inhibit plant immunity and 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)). Citrus psyllid is the main vector of citrus Huanglongbing, the "number one killer" of citrus. Research on the function of psyllid salivary sheath proteins can provide new ideas for the prevention and control of citrus Huanglongbing.

[0079] 3.1 Optimization of preparation conditions for DcMucin-like antibodies against citrus psyllids

[0080] Previous studies have shown that DcMucin-like has a high abundance in the salivary proteins of citrus psyllid, but its biological function is still unclear. In this study, the relative expression levels of DcMucin-like in the salivary glands, midgut, ovaries and testes of citrus psyllid were analyzed by qPCR, and the gene was verified to be highly expressed in the salivary glands of psyllids. The preparation of DcMucin-like antibodies can lay the foundation for further clarifying the role of this protein in the feeding and transmission of Huanglongbing bacteria by citrus psyllids. Immunizing animals with antigens obtained by prokaryotic expression of recombinant proteins is a rapid way to obtain polyclonal antibodies. In this study, Escherichia coli Roseate carrying the pET-28a-DcMucin-like recombinant plasmid was induced under the conditions of IPTG final concentration of 0.5mM and induction at 37℃ for 8h, and the DcMucin-like recombinant protein that can be expressed in large quantities in the supernatant of the bacterial lysate was obtained. The recombinant protein was expressed by Ni 2+ -NTAAgarose column and eluted with 500mM imidazole solution to obtain the purified antigen. Rabbits were then immunized with the antigen and the DcMucin-like polyclonal antibody with good specificity was obtained through antiserum IgG purification.

[0081] 3.2 Upregulation of DcMucin-like expression induced by citrus Huanglongbing pathogen

[0082] Mucin protein is a highly glycosylated mucin protein widely found in the salivary glands and intestines of insects. This type of protein has multiple biological functions, including being secreted into plants with saliva to play multiple roles to assist insects in feeding, as well as participating in immune responses and regulating cell signal transduction in epithelial tissues in the body. Mucin is involved in the formation of salivary sheaths of rice white-backed planthoppers and is a key protein for the successful feeding of white-backed planthoppers. Silencing Mucin can reduce the feeding amount of white-backed planthoppers, making it impossible for them to successfully feed on rice phloem sap. At the same time, silencing mucin-like in rice brown planthoppers (Nilaparvata lugens) can cause the configuration of insect salivary sheaths to become shorter and more single-branched, affecting insect growth and development. 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 the infection of Huanglongbing bacteria CLas induces differential expression of DcMucin-like in citrus psyllids, this study used qPCR and Western blot analysis based on specific antibodies and found that CLas infection can stimulate the up-regulation of DcMucin-like in psyllids, both at the mRNA level and the protein level, indicating that DcMucin-like plays an important role in the interaction between CLas and the vector insect psyllids. The transmission of CLas by citrus psyllids is the result of the interaction between citrus psyllids, CLas and host plants. Previous studies have shown that between diseased plants and healthy plants, healthy citrus psyllids will preferentially feed on diseased plants, but fungus-carrying citrus psyllids tend to feed on healthy plants, which may be caused by the different volatile components of the tender shoots of healthy and susceptible citrus plants and the combined results of the interaction between CLas and the vector psyllids.

[0084] The present study used a cross-linked fluorescent DcMucin-like antibody to label the main veins of citrus leaves after psyllid feeding. Confocal microscopy can observe specific fluorescence signals similar to salivary sheaths around the psyllid feeding holes, which verified for the first time that DcMucin-like is a salivary protein of citrus psyllids, and is released into plant tissues as psyllids feed to participate in the formation of salivary sheaths. The present study showed that DcMucin may play an important role in the interaction between CLas, psyllids and citrus plants, providing a new target for citrus disease and pest resistance breeding and citrus psyllid control.

[0085] In summary, the research of the present invention shows that DcMucin is upregulated in citrus psyllid in response to infection with Huanglongbing disease, and can be secreted into citrus plant tissues as the psyllid feeds; soluble DcMucin fusion protein was successfully expressed, and specific DcMucin rabbit polyclonal antibody was prepared, which can be used for Western blot analysis and immunofluorescence labeling.

Claims

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

11.

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

3. Use of the antigen polypeptide according to claim 1 or the biomaterial according to claim 2 in any of the following: (1) Application in the preparation of antigens for inducing polyclonal antibodies against citrus psyllid salivary proteins; (2) Use in the preparation of reagents or kits for detecting salivary proteins of citrus psyllids or their expression levels.

4. A polyclonal antibody against citrus psyllid salivary protein, characterized in that: The antigen of the polyclonal antibody is induced by a polypeptide consisting of the amino acid sequence shown in SEQ ID NO.

11.

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 competent Escherichia coli cells to obtain a recombinant protein antigen; (2) Immunizing animals with the obtained recombinant protein antigen, collecting blood, separating and purifying 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 competent Escherichia coli cells and inoculated into liquid culture medium for culture expansion, and then IPTG inducer is added to induce expression to obtain DcMucin recombinant protein; the preferred induction expression conditions are IPTG final concentration of 0.4-0.6mM, 36-38°C for induction for 7-9h; The antigen gene fragment of the nucleotide sequence shown in SEQ ID NO.10 is obtained by PCR amplification using the DcMucin-like sequence without a signal peptide shown in SEQ ID NO.1 to design specific amplification primers; preferably, the specific amplification primers are as follows: DcMucin-F: 5'-CAGCAAATGGGTCGCGGATCCATGCAGAGCCAGACCCCTGCACCTGCTGTAAA-3'; DcMucin-R: 5'-GTGGTGGTGGTGGTGCTCGAGTTTCAAAGGTGTCTCAACCAGGTTGTTGGA-3'.

7. The preparation method according to claim 5, characterized in that: In step (2), First, the recombinant protein antigen was mixed with complete Freund's adjuvant and then used for the first immunization of animals. Then, the recombinant protein antigen was mixed with incomplete Freund's adjuvant and then used for the second to fourth immunization. The citrus psyllid salivary protein polyclonal antibodies were obtained through serum separation and purification.

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

9. Use of the polyclonal antibody to citrus psyllid salivary protein according to claim 4 or the kit according to claim 8 in preparing a kit for detecting citrus psyllid salivary protein.

10. The use according to claim 9, characterized in that: The method for detecting citrus psyllid salivary proteins is: 1) incubating the sample to be tested with the polyclonal antibody to salivary protein of citrus psyllid as claimed in claim 4, wherein the polyclonal antibody specifically binds to the salivary protein in the sample to be tested, thereby forming an immune complex; 2) Detecting the presence of immune complexes; the preferred detection method is Western blot.

Images