Use of gss1 protein as internal reference protein of oral secreted protein of plutella xylostella

Abstract

The application discloses application of GSS1 protein as an internal reference protein of oral cavity secreted protein of Plutella xylostella. The application has the advantages that GSS1 can be detected in different tissues of Plutella xylostella, especially in oral cavity secretions, and the detection is stable and not induced by substrate thioglycoside, so the GSS1 can be used as an internal reference protein for quantification of total oral cavity secreted protein and specific secreted protein of Plutella xylostella, and can well serve the quantification of oral cavity secreted protein of the insect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to the application of GSS1 protein as a reference protein of oral secretion protein of Plutella xylostella. BACKGROUND

[0002] Insects can release a large amount of oral secretion proteins (OS) that are perceived by plants during feeding. These oral secretion proteins interact with proteins encoded by important genes in plants, change the plant hormone signaling pathway, and thus regulate the plant's resistance to insects. The composition of insect oral secretion proteins is diverse and complex, and can be mainly divided into two categories, i.e. feeding-related molecular pattern proteins and effector proteins. The composition of oral secretion proteins of insects with different mouthpart types differs greatly. Although mass spectrometry and other detection methods can be used to analyze the components and contents of collected insect oral secretion proteins, this technology relies on large equipment and professional operation, and is suitable for extensive investigation. How to further quantify and confirm specific target proteins requires the use of routine Western Blotting (WB) detection methods in the laboratory.

[0003] The WB detection method for protein quantification has the characteristics of relatively low cost and easy operation. However, how to ensure the uniformity of the loading amount between different samples of extracted total proteins, a stable expression of a reference protein is an important link. For the quantification of specific proteins in insect nuclei or cytoplasm, there are many reference antibodies based on cytoskeletal structures or constituent proteins that can be selected, and the commonly used protein references are GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and cytoskeletal proteins actin or tubulin. However, for secreted proteins in the oral cavity, since common reference proteins cannot be secreted, it is necessary to explore effective and stable expression of proteins in oral secretion proteins as reference proteins. By preparing antibodies corresponding to candidate reference proteins, the stability of the expression of the candidate reference proteins in oral secretion proteins is verified by WB technology, thereby serving the quantification of other secreted proteins, which is an important prerequisite and basis for understanding the secretion degree of specific proteins, and is also an important support for the interaction between insects and host plants.

[0004] The diamondback moth (DBM) is a global pest that exclusively feeds on cruciferous crops, causing significant damage to human agricultural activities. Cruciferous plants have a classic glucosinolate and myrosinase dual defense system. When plants are damaged by insects, the substrate glucosinolate and myrosinase meet to produce a series of toxic substances represented by cyanides to harm insects. However, the DBM has a high and stable expression of glucosinolate sulfatase (GSS) in its body, which gives the DBM the ability to widely damage cruciferous plants. Among them, GSS1 and GSS2 are repeat genes with the same length and more than 96% protein homology (thus the corresponding antibody cannot distinguish GSS1 and GSS2). However, whether GSS1 and GSS2 are contained in the DBM oral secretion protein and whether they are suitable for the quantification of other oral secretion proteins have not been reported. SUMMARY

[0005] To solve the problem of lack of internal reference protein of DBM oral secretion protein in the prior art, the application proposes the application of GSS1 protein as the internal reference protein of DBM oral secretion protein.

[0006] To achieve the above-mentioned purpose, first of all, the prepared GSS1 and GSS2 universal antibodies are used to detect the expression characteristics of endogenous GSS1 and GSS2 proteins in different material samples. WB and immunohistochemical techniques are used in combination. The results of WB show that GSS1 and GSS2 proteins are highly expressed in the larval stage, especially in the 3rd and 4th instar larvae (L3 and L4). In larvae, in addition to high expression in tissues such as salivary glands (Sag), foregut (Fg), midgut (Mg) and hindgut (Hg), they are also obviously present in secretions, including hemolymph (Hl) and gut contents (Gc) Figure 1 ). Secondly, the results of immunohistochemistry also confirm that GSS1 and GSS2 proteins are widely present in hemolymph (Hl) and gut contents (Gc) and related tissues Figure 2 ). In addition, in order to verify whether GSS1 and GSS2 exist in the DBM oral secretion protein and can be released in a natural state, we carried out an in situ detection experiment of GSS1 and GSS2 proteins on the leaves of Arabidopsis thaliana after feeding. The results show that compared with the half leaf wrapped with tin foil paper (CK), the wound site (Attack) and the leaf vein of the other half leaf can obviously detect the signal of GSS1 / 2 Figure 3 ).

[0007] Due to the high sequence homology between GSS1 and GSS2, the antibody cannot distinguish the proportion of the two in the oral secretion. Therefore, this study uses the conventional gene editing technology-CRISPR / Cas9 to construct GSS1 - / - , GSS2 - / -Plutella xylostella single mutants of GSS1 and Plutella xylostella double mutants of DM (GSS1 and GSS2). After Plutella xylostella mutants were allowed to feed on Arabidopsis leaves for 12 h, total proteins of the corresponding leaves were extracted for WB experiments. The corresponding results showed that obvious GSS1 content could be detected after WT (wild type Plutella xylostella) and GSS2 - / - (GSS2 mutant Plutella xylostella) feeding. However, no signal could be detected in the GSS1 - / - group, which was consistent with the treatment group of DM double mutants feeding Figure 4 ), indicating that the protein secreted by the oral cavity of Plutella xylostella was GSS1.

[0008] The present application further verified whether GSS1 protein would be induced to express by substrates. Because the selection of a reference antibody, the corresponding protein expression should be stable expression in most cases. Therefore, different types of glucosinolates were used to smear artificial feed to feed Plutella xylostella, including commercially purchased GSS1 specific substrate glucosinolate sinigrin (20 μM) and total glucosinolate extracted from Arabidopsis (850 μM). The GSS1 content of both treatments did not change significantly (p>0.05) from the beginning of 3rd instar larvae (0 h) to the end of 4th instar (before pupation, i.e. 12 h and 24 h). Figure 5 These experiments showed that GSS1 protein was stably expressed in the body of the larvae and did not affect the normal oral secretion amount.

[0009] In order to verify whether GSS1 protein could be applied to the quantification of specific oral cavity secreted proteins of Plutella xylostella, WB technology was used to quantify the oral cavity secreted protein glucose oxidase (GOX) reported in lepidopteran insects. The results of WB showed that GOX existed in the collected Plutella xylostella oral cavity secreted protein samples (OS#1-3) in three replicates. By scanning the pictures in gray scale and converting them into numerical values, it was revealed that the protein expression amount of GOX relative to GSS1 was about 0.4, indicating that GSS1 could be effectively applied to the quantification and analysis of oral cavity proteins.

[0010] The present application has the advantage that GSS1 is widely distributed in different tissues, especially in oral cavity secretions, and can be stably detected and not induced by substrate glucosinolate, which can be used as a reference protein for the quantification of total oral cavity secreted proteins and specific secreted proteins of Plutella xylostella, and can well serve the quantification of oral cavity secreted proteins of the insect. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1To detect the expression characteristics of insect endogenous GSS1 and GSS2 proteins based on WB technology; E: egg; L1-4: 1-4 instar Plutella xylostella larvae; P: pupa; A: adult; Sag: salivary gland; Sg: silk gland; Fg: foregut; Mg: midgut; Hg: hindgut; Mt: Malpighian tubule; Rb: remaining residue; Hl: hemolymph; Gc: gut content;

[0012] Figure 2 To detect the expression characteristics of insect endogenous GSS1 and GSS2 proteins based on immunofluorescence technology; Hd: head; Gc: gut content; Hl: hemolymph; Ec: intestinal epithelial cell;

[0013] Figure 3 To detect the presence of GSS1 and GSS2 in oral secretory proteins based on protein in situ detection technology; CK: mechanical damage (artificially created wounds); attack: Plutella xylostella larvae feeding;

[0014] Figure 4 To detect the release of oral GSS1 based on different mutant Plutella xylostella; WT: wild type Plutella xylostella; GSS1 - / - : GSS1 mutant Plutella xylostella based on CRISPR / Cas9 technology; GSS2 - / - : GSS2 mutant Plutella xylostella based on CRISPR / Cas9 technology; DM: GSS1 and GSS2 double mutant Plutella xylostella based on CRISPR / Cas9 technology;

[0015] Figure 5 To detect that GSS1 is not induced to express by substrate glucosinolate based on WB technology; CK-0h: 3 instar Plutella xylostella larvae fed with artificial diet containing no glucosinolate; CK-12h and CK-24h: larval samples after 3 instar Plutella xylostella larvae were fed with artificial diet containing no glucosinolate for 12h and 24h; Sin-12h and Sin-24h, larval samples after 3 instar Plutella xylostella larvae were fed with artificial diet added with 20μM sinigrin (A) glucosinolate for 12h and 24h; Total: larval samples after 3 instar Plutella xylostella larvae were fed with artificial diet added with 850μM total glucosinolate (B) extracted from Arabidopsis thaliana for 12h and 24h;

[0016] Figure 6 To quantify GOX protein content with GSS1 as the internal reference protein; A is the oral secretory protein collected by finger pressure method from 4 instar larvae, and the contents of GOX and GSS1 in the sample are detected simultaneously by WB technology, with 3 biological replicates; B is quantified for GOX after gray scale conversion to corresponding numerical values by image J software. DETAILED DESCRIPTION

[0017] The application of GSS1 protein as an internal reference protein of oral secretion protein of Plutella xylostella is further described in detail below in combination with specific examples.

[0018] 1. Detection of expression characteristics of endogenous GSS1 and GSS2 proteins of insects based on WB technology

[0019] The relative protein expression amount is quantitatively analyzed by image J software on the bands on a polyvinylidene fluoride (a commonly used solid support in Western blotting), wherein, the endogenous total protein of Plutella xylostella at different developmental stages and tissues uses alpha-Tubulin as an internal reference protein.

[0020] 1.1 Sample collection

[0021] Insect sample collection Figure 1 A) : Plutella xylostella at different developmental stages fed with artificial feed were taken, including eggs, four instar larvae, pupae and adults (three biological replicates were included in each stage) ; tissue sample collection Figure 1 B and C) : four instar larvae were taken as the dissected sample and placed in PBS, a dissecting forceps was used to first open the foot of the insect body, and then 30 larvae were placed in a 500 μL volume of a bottom-opened centrifuge tube, and placed in a 2 mL centrifuge tube for 500xg centrifugation to obtain hemolymph. For the larval sample, the corresponding tissues were selected by opening the tail end of the larva, the Malpighian tube and the intestinal tract were separated, and the intestinal tract and its contents were distinguished, and the intestinal tract was further divided into three parts of the front, middle and back. The head and chest of the insect were opened to obtain the salivary gland and silk gland, and the remaining material was uniformly collected as the remaining residue.

[0022] 1.2 Protein extraction

[0023] Protein extraction of different developmental stages and tissues: the sample prepared in step 1.1 was ground with liquid nitrogen, 500 μL DNA extraction phenol reagent (Solabio, Beijing) and 500 μL Dense Buffer (formula: 0.1 mol·L -1 pH 8.0 Tris-HCl, 2% SDS, 5% β-mercaptoethanol, 30% sucrose) were added and mixed well; after standing at room temperature for 3 min, 10000xg centrifugation was performed for 5 min, and the supernatant was transferred to a new centrifuge tube, 80% acetone was added in an amount of 5 times the volume of the supernatant, and mixed well, and then the mixture was placed at -20°C for more than 2 h; after standing, 10000xg centrifugation was performed for 5 min, and 1 mL of methanol was used for washing; after air drying, 50 μL of a dissolving solution (formula: 5 mL of 0.1 mol·L -1 pH 8.0 Tris-HCl, 2.4 g of urea, 1% SDS) was added, and the mixture was placed in a 50°C metal bath for 1 h to dissolve the soluble protein, and then the mixture was stored at -20°C after the dissolution was completed.

[0024] 1.3 Protein concentration determination

[0025] Protein concentration determination was performed according to the instructions of the BCA Protein Concentration Determination Kit of Solarbio: BSA standard system was prepared (0, 0.4, 0.8, 1.2, 1.6, 2 mg / mL, respectively). The standard or diluted 10 times protein sample was added to each well of the enzyme-labeled plate at 20 μL, and 100 μL of color developing reaction solution was added. After incubation at 37°C for 30 min, the OD value was read at single wavelength 562 nm. According to the standard curve prepared, the sample concentration was calculated.

[0026] 1.4 Western blot

[0027] 10 μg of protein sample was taken, and an appropriate amount of protein loading buffer was added, and denatured at 99°C for 7-10 min. The total protein was separated by 10% SDS-PAGE gel electrophoresis. After electrophoresis, the PVDF membrane (Bio-Rad, 1703959) was transferred to the PVDF membrane by semi-dry method at a constant voltage of 15V for 30 min. The blocked PVDF membrane was blocked with 5% skim milk powder blocking buffer dissolved in 1xTBST (Solarbio, T1081, Beijing) for 1 h on a shaker. The membrane was washed with 1xTBST three times for 10 min each time. The blocked PVDF membrane was incubated with GSS1 and GSS2 common first antibody (ABclonal) (1:2000) at room temperature for 2 h, and washed with 1xTBST three times for 10 min each time. Goat anti-rabbit IgG HRP-labeled secondary antibody was added and incubated for 1 h (1:10000, Affinity, S0001), and washed with 1xTBST three times for 10 min each time. Color development was performed with special hypersensitive ECL chemiluminescent substrate (Bi Yun Tian, P0018AS). After color development, the results were photographed, edited and saved by AMERSHAM IMAGER 600 ultra-sensitive multifunctional imager. The monoclonal antibody α-Tubulin was used as an internal reference antibody (1:5000, Sigma, T6074).

[0028] 1.5 Protein quantification based on WB gray value

[0029] To quantify the protein content of GSS1 or GSS2, the picture was quantified by ImageJ (version 1.51). The procedure is referred to the software instructions.

[0030] 2. Detection of insect endogenous GSS1 and GSS2 protein expression characteristics based on immunofluorescence technology

[0031] 2.1 Paraffin-embedded 4th instar larvae of P. xylostella were deparaffmized to water by placing the paraffin sections in xylene I 15 min-xylene II 15 min-xylene III 15 min-absolute ethanol I 5 min-absolute ethanol II 5 min-85% ethanol 5 min-75% ethanol 5 min-distilled water.

[0032] 2.2 Antigen retrieval: The tissue sections were placed in a microwave oven in a box filled with citrate antigen retrieval buffer (pH = 6.0) for antigen retrieval, and the temperature was raised to boiling for 8 min, then the power was turned off for 8 min for incubation, and then the temperature was raised to medium-low for 7 min. During this process, the buffer should not be allowed to evaporate too much, and the sections should not be allowed to dry. After natural cooling, the slides were placed in PBS (pH = 7.4) and shaken on a decolorizing shaker for 3 times, 5 min each time.

[0033] 2.3 Blocking endogenous peroxidase: The sections were placed in a 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 min. The slides were then placed in PBS (pH = 7.4) and shaken on a decolorizing shaker for 3 times, 5 min each time.

[0034] 2.4 Serum blocking: After the sections were shaken dry, a circle was drawn around the tissue with a histological pen to prevent the antibody from flowing away. 3% BSA was added to the circle to cover the tissue evenly, and the sections were incubated at room temperature for 30 min. (The primary antibody was goat-derived, and rabbit serum was used for blocking, while other sources were blocked with BSA)

[0035] 2.5 Addition of primary antibody: The blocking solution was shaken off, and PBS was added to the sections to dilute the GSS1 and GSS2 primary antibodies to a certain ratio. The sections were then placed in a wet box and incubated at 4°C overnight. (A small amount of water was added to the wet box to prevent the antibody from evaporating)

[0036] 2.6 Addition of secondary antibody: The sections were placed in PBS (pH = 7.4) and shaken on a decolorizing shaker for 3 times, 5 min each time. After the sections were shaken dry, the corresponding secondary antibody (HRP-labeled) was added to the circle to cover the tissue, and the sections were incubated at room temperature for 50 min.

[0037] 2.7 DAB color development: The sections were placed in PBS (pH = 7.4) and shaken on a decolorizing shaker for 3 times, 5 min each time. After the sections were shaken dry, fresh DAB color development solution was added to the circle, and the color development time was controlled under a microscope. The positive result was a brownish yellow color, and the color development was stopped by rinsing the sections with tap water.

[0038] 2.8 Nuclear counterstaining: Hematoxylin counterstaining was performed for about 3 min, followed by rinsing with tap water, differentiation with hematoxylin differentiation solution for a few seconds, rinsing with tap water, and returning to blue with hematoxylin returning solution, and finally rinsing with running water.

[0039] 2.9 Dehydration and mounting: Put the sections into 75% alcohol for 5 min, 85% alcohol for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, xylene I for 5 min, and then take the sections out and dry for a while, and then mount with neutral resin.

[0040] 3. Detection of GSS1 in oral secretion proteins based on different mutants of P. xylostella

[0041] 3.1 Material collection

[0042] The artificial diet strain larvae were starved for 4 h in advance. The larvae were transferred to detached Arabidopsis leaves. One half of the leaves was artificially wounded and covered with tin foil to prevent the larvae from feeding as the un-fed control group (CK), and the other half was placed with starved larvae for feeding.

[0043] 3.2 Imaging

[0044] After 6 h of feeding, the larvae were removed, and the Arabidopsis leaves were first photographed under bright field, and then semi-dried and transferred to the membrane. The treatment of the membrane, the transfer conditions, and the color development process were the same as step 1.4. The colored photograph was superimposed on the bright field photograph to obtain the protein in situ hybridization results of GSS1 or GSS2.

[0045] 3.3 Obtaining different mutants of P. xylostella

[0046] Obtained by using the conventional CRISPR / Cas9 technology. The process is referred to the paper published by the author: Functions of duplicated glucosinolate sulfatases in the development and host adaptation of Plutella xylostella, doi.org / 10.1016 / j.ibmb.2020.103316.

[0047] 3.4 Detection of total proteins of Arabidopsis fed by different mutants based on WB technology

[0048] The artificial diet strain larvae (WT), P. xylostella single mutants of GSS1 - / - , GSS2 - / - gene, and P. xylostella double mutants of DM (GSS1 and GSS2) were starved for 4 h in advance and then transferred to Arabidopsis leaves. The Arabidopsis leaves fed for 12 h were subjected to protein extraction and WB color development. The process was the same as step 1.4.

[0049] 4. Detection of GSS1 not induced by substrate based on WB technology

[0050] 4.1 Sample collection

[0051] The artificial feed was cut to 2x1.5x0.2cm size, 200μL of total glucosinolate extract of Arabidopsis and single glucosinolate Sinigrin (Sigma-Aldrich, 85440) were evenly smeared on the surface of the artificial feed respectively, and the three-day-old larvae after 4h starvation were placed on the feed for 12h and 24h, and samples were collected at two time points; the collected samples were rapidly cooled in liquid nitrogen and stored in a-80℃ refrigerator.

[0052] 4.2 Protein extraction, concentration, WB and expression analysis of synchronous steps 1.2-1.5.

[0053] 5. Quantification of GOX protein content based on GSS1 as the internal reference protein of oral secretion

[0054] 5.1 Sample collection

[0055] A sterile culture dish was placed on ice, and 10μL of 1xPBS containing 1mM protease inhibitor PMSF was added to the center, and the abdomen of 4th instar Plutella xylostella larvae (about 100) was squeezed by finger, and the visible oral secretion of each larva was transferred to PBS, and the collected sample was spotted on a PVDF membrane with 2μL, and after air drying at room temperature for 5min, the dot blotting experiment was performed.

[0056] 5.2 WB experiment

[0057] Blocking of the spotted PVDF, incubation of GSS1 and GSS2 common polyclonal antibodies, washing of the membrane and membrane color development process of synchronous steps 1.4.

[0058] 5.3 Identification of the content of glucose oxidase (GOX)

[0059] It is known that the oral secretion of Plutella xylostella contains glucose oxidase (GOX), so the GOX polyclonal antibody (laboratory self-made) was also incubated with the insect oral sample, and the sample collection process and dot blotting operation synchronous steps 5.1 and 5.2; membrane development synchronous step 1.4; protein quantification based on WB gray value synchronous step 1.5, the gray value of GOX is compared with the gray value of GSS1, and the protein content of GOX is obtained Figure 6 B).

[0060] The above only describes the preferred embodiments of the present application and does not limit the present application, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be limited by the protection scope defined by the claims.

Claims

1. Use of GSS1 protein as an internal reference protein for oral secreted proteins of Plutella xylostella, characterized in that, The applicability of GSS1 was verified by the following steps: (1) Construction of GSS1 - / - , GSS2 - / - single mutant and GSS1 / GSS2 diamondback moth double mutant DM of diamondback moth by CRISPR / Cas9 technology, and Western Blot detection of total proteins of Arabidopsis leaves after wild type diamondback moth WT, GSS1 - / - , GSS2 - / - and DM fed on Arabidopsis leaves, confirming that only GSS1 exists in oral secretion proteins; (2) By feeding experiment of GSS1 specific substrate glucosinolate, it was proved that the expression of GSS1 protein was not induced by substrate, and the expression amount was stable, wherein the glucosinolate was sinigrin and total glucosinolate extracted from Arabidopsis thaliana; (3) Taking GSS1 as the internal reference protein, the glucose oxidase in the oral secretion protein of Plutella xylostella was quantified to verify the feasibility of GSS1 as the internal reference.

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