Monoclonal antibody for detecting cotton drought tolerance related protein KAT2 and application thereof

Abstract

The invention belongs to the technical field of cotton detection, and particularly relates to a monoclonal antibody for detecting cotton drought tolerance related protein KAT2 and application of the monoclonal antibody. The amino acid sequence of the protein KAT2 is as shown in SEQ ID NO. 1. A mouse is immunized by using the protein KAT2 as an immunogen to obtain lymphocytes, fusion cells are prepared by using a hybridoma cell fusion technology, a cell strain capable of producing a monoclonal antibody is obtained, and the monoclonal antibody secreted by the cell strain is used for detecting the protein KAT2 related to the drought tolerance of cotton. The monoclonal antibody of the KAT2 provided by the invention has high affinity and high specificity to the protein KAT2, can be widely used for detecting the protein KAT2, and particularly can be used for detecting the expression condition of the protein KAT2 of various varieties of drought-enduring cotton which are widely planted in agriculture.

Description

Technical Field

[0001] The invention belongs to the technical field of cotton drought tolerance protein detection, and particularly relates to a monoclonal antibody for detecting protein KAT2 related to cotton drought tolerance and an application thereof. Background Art

[0002] Universal stress protein (KAT2) is an enzyme that catalyzes fatty acid β-oxidation. It participates in ABA signaling during plant seedling germination and growth, and in the biosynthesis of jasmonic acid during senescence. KAT2 positively regulates ABA signaling in all major ABA responses, including ABA-induced inhibition of Arabidopsis seed germination and postgermination growth, as well as ABA-induced stomatal closure and inhibition of stomatal opening. KAT2 plays an important role in the production of reactive oxygen species (ROS) by ABA, indicating that KAT2 regulates ABA signaling at least in part by regulating ROS homeostasis in plant cells. At the same time, KAT2 may act downstream of an important WRKY transcriptional repressor, WRKY40, which may link KAT2 to signaling mediated by the ABA receptor ABAR / CHLH.

[0003] Previous studies have found that when cotton is in drought conditions, there is a clear regulatory relationship between the expression of the protein KAT2 and the drought tolerance of cotton, that is, the expression of the protein KAT2 determines the drought tolerance of cotton. Currently, there are a large number of different drought-resistant cotton varieties being planted in agricultural cultivation, most of which are naturally selected and bred natural drought-resistant varieties. However, there are currently no antibodies specifically targeting the cotton protein KAT2 on the market. Therefore, at the protein detection level, there is a lack of effective antibody tools for this research field, and it cannot be used for basic research on the broad-spectrum drought tolerance of cotton, as well as protein-level detection of widely planted drought-resistant cotton varieties. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a monoclonal antibody for detecting protein KAT2 related to cotton drought tolerance and its application. The protein KAT2 is a full-length recombinant protein expressing KAT2, and the amino acid sequence of protein KAT2 is: MEKAINRQQVLLQHLNPSASSFSSHQNNSALSASACLAGDSAAYQRTNVFGDDVVIVAAYRTALCKSKRGGFKDTYPDDLLAPVLRAVIEKTNLNPSEVGDIVVGTVLAPGSQRASECRMAAFYAGFPETVPVRTVNRQCSSGLQAVADVAAAIKAGFYEIGIGAGLESMTTNPMAWEGSVNPRVKQMEQAQNCLLPMGITSENVAHRFGVTRQE QDQAAVESHRKAAAATASGKFKDEIVPVATKIVDPKTGDEKPVTISVDDGIRANTSVSDLGKLKPVFKKNGTTTAGNSSQVSDGAGAVLLMKRSVAMRKGLPILGVFRTFAAVGVDPAIMGVGPAVA IPAAVKSAGLELDDVDLFEINEAFASQFVYCQKKLELDPQKINVNGGAMAIGHPLGVTGARCVATLLHEMKRRGKDCRFGVVSMCIGTGMGAAAVFERGDCVDELCNARKVETNSLLSKDAR as SEQ Shown as ID NO.1.

[0005] A monoclonal antibody for detecting protein KAT2 related to cotton drought tolerance, wherein the amino acid sequence of protein KAT2 is shown in SEQ ID NO.1; using protein KAT2 as an immunogen, mice are immunized to obtain a monoclonal antibody for detecting protein KAT2 related to cotton drought tolerance. The monoclonal antibody provided by the present invention targets protein KAT2 for detection, and determines whether a cotton variety is drought-tolerant by the expression level of protein KAT2, so that the expression changes of protein KAT2 in different drought-tolerant cotton varieties widely planted in agriculture and the protein network map can be studied in depth.

[0006] The present invention provides a monoclonal antibody for detecting a protein KAT2 related to cotton drought tolerance, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region sequence comprises the amino acid sequence GLNIKDYY of CDR1-1 shown in SEQ ID NO.2, the amino acid sequence IDPENGNT of CDR2-1 shown in SEQ ID NO.3, and the amino acid sequence NVRWE of CDR3-1 shown in SEQ ID NO.4; the light chain variable region sequence comprises the amino acid sequence KSVSTSGYSY of CDR1-2 shown in SEQ ID NO.5, the amino acid sequence LVS of CDR2-2, and the amino acid sequence QHIRELT of CDR3-2 shown in SEQ ID NO.6.

[0007] The invention discloses a method for preparing a monoclonal antibody for detecting a protein KAT2 related to drought tolerance of cotton. The method comprises the following steps: selecting healthy Balb / c mice, preparing a protein KAT2 antigen solution, immunizing the Balb / c mice with the protein KAT2 antigen solution, obtaining lymphocytes after immunization, and using hybridoma cell fusion technology to obtain fused cells, thereby obtaining a cell line capable of producing monoclonal antibodies. The monoclonal antibody secreted by the cell line is a monoclonal antibody for detecting a protein KAT2 related to drought tolerance of cotton.

[0008] Preferably, in the method for preparing a monoclonal antibody for detecting the protein KAT2 associated with drought tolerance of cotton, the mice are 8- to 12-week-old Balb / c mice.

[0009] Preferably, in the method for preparing a monoclonal antibody for detecting protein KAT2 associated with drought tolerance of cotton, the protein KAT2 antigen solution is obtained by dissolving protein KAT2 in physiological saline.

[0010] Preferably, the method for preparing a monoclonal antibody for detecting the protein KAT2 related to cotton drought tolerance is to obtain the whole spleen and lymph nodes of mice, fuse them with the myeloma SP2 / 0 cell line, culture the fused cells, collect the supernatant, and select at least 60 parent clones with the highest immunogen affinity to enter subclone; subclone and screening are continued by limiting dilution method and ELISA method to obtain positive fusion cells.

[0011] Preferably, in the method for preparing a monoclonal antibody for detecting the protein KAT2 related to drought tolerance of cotton, a cell line capable of producing the monoclonal antibody is injected into the abdomen of Balb / c mice, and the produced ascites antibody is purified with ProteinA / G to obtain a purified monoclonal antibody.

[0012] The present invention provides an application of the monoclonal antibody, wherein the monoclonal antibody is used for distinguishing drought-tolerant cotton varieties from drought-intolerant cotton varieties.

[0013] Preferably, the monoclonal antibody is used to test the expression level of protein KAT2, and the drought resistance of cotton varieties is identified by the expression level of protein KAT2, thereby identifying and distinguishing drought-tolerant cotton varieties from drought-intolerant cotton varieties.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides a monoclonal antibody for detecting protein KAT2 related to cotton drought tolerance, which has high affinity and high specificity to protein KAT2 and can be widely used for detecting protein KAT2, and is particularly useful for detecting the expression of protein KAT2 in various varieties of drought-resistant cotton widely planted in agriculture.

[0016] The monoclonal antibody of the present invention provides a tool for studying the targeted protein KAT2, so that the regulatory relationship between the expression of the protein KAT2 in different drought-resistant cotton varieties and whether the cotton can eventually develop effective drought resistance can be effectively studied. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the ELISA test curve result of ascites antibody.

[0018] Figure 2 The results of using ascites antibody to detect the expression level of protein KAT2 in drought-tolerant cotton varieties (Yumian 508) and drought-intolerant cotton varieties (Liaomian 31) by Western Blot method.

[0019] Figure 3 To purify the antibody Anti-KAT2, the Western Blot method was used to detect the expression levels of KAT2 in drought-tolerant cotton varieties (Yumian 508) and drought-intolerant cotton varieties (Liaomian 31) at different stress periods.

[0020] Figure 4 To purify the antibody Anti-KAT2, the Western Blot method was used to detect the quantitative analysis of the expression level of protein KAT2 in different stress periods in the drought-resistant cotton variety (Yumian 508).

[0021] Figure 5 yes Figure 4 Quantitative detection results of protein KAT2 in samples. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0023] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0024] Example 1

[0025] 1.1 Preparation of Antigens

[0026] By cloning the full-length gene of KAT2, the full-length protein of KAT2 was recombinantly expressed. The amino acid sequence of protein KAT2 is as follows:

[0027] MEKAINRQQVLLQHLNPSASSFSSHQNNSALSASACLAGDSAAYQRTNV

[0028] FGDDVVIVAAYRTALCKSKRGGFKDTYPDDLLAPVLRAVIEKTNLNPSEVGDI

[0029] VVGTVLAPGSQRASECRMAAFYAGFPETVPVRTVNRQCSSGLQAVADVAAAI

[0030] KAGFYEIGIGAGLESMTTNPMAWEGSVNPRVKQMEQAQNCLLPMGITSENV

[0031] AHRFGVTRQEQDQAAVESHRKAAAATASGKFKDEIVPVATKIVDPKTGDEKP

[0032] VTISVDDGIRANTSVSSDLGKLKPVFKKNGTTTAGNSSQVSDGAGAVLLMKRS

[0033] VAMRKGLPILGVFRTFAAVGVDPAIMGVGPAVAIPAAVKSAGLELDDVDLFEIN

[0034] EAFASQFVYCQKKLELDPQKINVNGGAMAIGHPLGVTGARCVATLLHEMKR

[0035] RGKDCRFGVVSMCIGTGMGAAAVFERGDCVDELCNARKVETNSLLSKDAR, and use it as an immunogen (antigen) to immunize mice to prepare monoclonal antibodies.

[0036] 1.2 Immunization of mice

[0037] Select healthy 8-week-old Balb / c mice, dissolve the protein KAT2 prepared in 1.1 as an antigen in physiological saline to prepare a 1 mg / mL protein KAT2 antigen solution, and use the protein KAT2 antigen solution to immunize Balb / c mice. The specific scheme is as follows:

[0038] 1) First immunization: On day 1, mice were injected with a 1 mg / mL antigen solution and an equal volume of the first mixed emulsion of Freund's complete adjuvant as an injection, with each mouse being injected with 100 μL of the emulsion.

[0039] 2) Second immunization: On the 14th day, each mouse was injected with 100 μL of the second mixed emulsion, and the formula of the second mixed emulsion was the same as that of the first mixed emulsion for the first immunization.

[0040] 3) 3rd immunization: On day 21, each mouse was injected with 100 μL of the third mixed emulsion, the formula of the third mixed emulsion was the same as that of the first mixed emulsion for the first immunization.

[0041] 4) 4th immunization: On day 28, each mouse was injected with 100 μL of the fourth mixed emulsion, the formula of the fourth mixed emulsion was the same as that of the first mixed emulsion for the first immunization.

[0042] 5) 5th immunization: On the 35th day, each mouse was injected with 100 μL of the fifth mixed emulsion, the formula of the fifth mixed emulsion was the same as the formula of the first mixed emulsion for the first immunization.

[0043] Starting from the first immunization, the serum titer and titer of mice are monitored every 7 days to determine the optimal number of immunizations. The above immunization method can produce high-affinity IgG subtype antibodies against most antigens. After the first immunization, there will be 3 to 4 boosters.

[0044] Among them, the serum detection and screening methods are as follows:

[0045] Blood was collected from the orbits of the immunized mice, and the serum titer was tested by ELISA (protein KAT2 was used as an immunogen coating). The serum titer must be greater than 10K, otherwise, the immunization was continued.

[0046] The titer was the highest after the 5th immunization (after the booster immunization on the 35th day), and mice with qualified titers were prepared for fusion.

[0047] 1.3 Fusion

[0048] On the third day after the fifth immunization, the whole spleen and 1 / 2 of the lymph nodes of the mice were taken and fused with myeloma SP2 / 0 cells. The fused cells were spread on four 384-well plates, with 10 cells inoculated in each well. 4The supernatants of all wells were collected and screened by ELISA. The positive wells that showed successful hybridization under microscopic examination were transferred to 96-well plates for further culture. After a few days of growth, the supernatants of all wells were collected and tested for reaction with the immunogen (protein KAT2) by ELISA. The 60 parent clones with the highest affinity for the immunogen were selected for subcloning.

[0049] 1.4 Subcloning and screening

[0050] Subcloning and screening were continued by limiting dilution method and ELISA method to obtain monoclonal hybridoma cells. The specific method was:

[0051] 1) The parent clone cells of 1.3 were plated in a 96-well plate and cultured until they covered about 1 / 6 of the bottom of the well. The supernatant of each well was tested for the response to the immunogen by ELISA, and two wells with high OD values ​​and good cell status were selected for the next round of subcloning.

[0052] Repeat the above step 1) until the positive rate of the cell line in the well is 100%. At this time, we obtain the monoclonal cell line 105. After the last round of subcloning, all positive cells 105 are immediately expanded and cultured, a part of which is frozen for later use, and the other part is used for supernatant or ascites preparation.

[0053] 1.5 Antibody supernatant preparation and purification

[0054] The monoclonal cell line 1O5 finally obtained in 1.4 was taken as a fusion cell, which is a cell line capable of producing monoclonal antibodies. The monoclonal cell line 1O5 was injected into Balb / c mice through the abdomen to produce monoclonal antibodies for detecting the protein KAT2 related to cotton drought tolerance. The produced ascites antibody was purified with ProteinA / G to obtain a purified monoclonal antibody, which was recorded as purified antibody anti-KAT2 and used for subsequent detection.

[0055] The monoclonal cell line 105 was cultured and total RNA was extracted, and the mRNA was reverse transcribed into the first-strand cDNA; the heavy chain and light chain genes were amplified by PCR and cloned into a sequencing vector, and multiple positive clones were sequenced to obtain the final sequence results.

[0056] RNA extraction was performed using an RNA kit (Thermo Fisher Scientific) according to the kit instructions.

[0057] The results showed that the monoclonal antibody included a heavy chain variable region and a light chain variable region; the heavy chain variable region sequence contained the amino acid sequence GLNIKDYY of CDR1-1 shown in SEQ ID NO.2, the amino acid sequence IDPENGNT of CDR2-1 shown in SEQ ID NO.3, and the amino acid sequence NVRWE of CDR3-1 shown in SEQ ID NO.4; the light chain variable region sequence contained the amino acid sequence KSVSTSGYSY of CDR1-2 shown in SEQID NO.5, the amino acid sequence LVS of CDR2-2, and the amino acid sequence QHIRELT of CDR3-2 shown in SEQ ID NO.6.

[0058] In order to prove the effect of the present invention, the present invention performs ELISA detection, protein imprinting, immunoprecipitation plus mass spectrometry, antibody chip and other verifications on the obtained ascites antibody and purified antibody anti-KAT2 to determine the most effective antibody. The following experimental methods and results are included.

[0059] (1) ELISA verification of monoclonal antibody and protein KAT2 antigen solution

[0060] Experiment 1: 100 μL of the ascites antibody of 1.5 of Example 1 was coated on a 96-well ELISA plate, 100 μL of protein KAT2 antigen solution was added, incubated at 37°C for 1 h, washed and blocked with skim milk overnight, washed with PBS (0.01 mol / L, pH 7.0), and stored at 4°C for use.

[0061] Experiment 2 (PC): Incubation with protein KAT2 antigen solution and washing with PBS (0.01 mol / L, pH 7.0).

[0062] Experiment 3 (NC): A blank control of PBS (0.01 mol / L, pH 7.0) was set up at the same time.

[0063] HRP-labeled detection antibodies were added to the ELISA plates incubated with the above-mentioned experiments 1 to 3. TMB color reaction was performed and the readings were obtained using an ELISA reader. The antibody titers are shown in Table 1 and Figure 1 The results in Table 1 show that even if the protein KAT2 antigen solution is diluted 100,000 times, the antigen can still be detected, with a low detection limit and high sensitivity. Figure 1 The results correspond to those in Table 1.

[0064] Table 1 Antibody ELISA test data

[0065]

[0066] Note: K means that the protein KAT2 antigen solution is diluted 1000 times based on the previous value, such as 3.125K means that the protein KAT2 antigen solution is diluted 3125 times; PC, positive control, NC, negative control (sterile ddH 2 O).

[0067] (2) Endogenous protein blotting (WB) verification

[0068] Using the root protein lysate of drought-tolerant cotton varieties (Yumian 508) and drought-intolerant cotton varieties (Liaomian 31), the purified antibody anti-KAT2 was diluted to a concentration of 1:1000 for WB verification. The experimental results showed that the purified antibody anti-KAT2 prepared in 1.5 of Example 1 could specifically identify the KAT2 band in the WB verification, which was consistent with the expected size. At the same time, the protein KAT2 was significantly expressed in drought-tolerant cotton varieties, and the band brightness was significantly higher than that of drought-intolerant cotton varieties, such as Figure 2 shown.

[0069] In order to prove the effect of the present invention, the present invention also carried out the following experiments.

[0070] Experiment 4: Application of a monoclonal antibody against KAT2 in the detection of drought stress in cotton

[0071] Cotton drought stress treatment method:

[0072] The cotton strains were planted in an environmental incubator. The formula of the nutrient soil for planting was a mixture of garden soil and vermiculite = 3:1. When the seedlings grew to three leaves and one heart, the seedlings with the same growth were selected for the experiment. The experiment adopted the drought treatment method without watering.

[0073] Cotton strains include drought-intolerant and drought-tolerant varieties.

[0074] Samples were taken at 0h, 12h, 24h, 72h, and 120h of treatment to prepare root whole protein lysate of the samples. The purified antibody Anti-KAT2 was diluted to a concentration of 1:1000. WB was used to verify the expression of the test protein KAT2. The results are shown in Figure 3 and Figure 4 The results showed that except for 0 hour, the protein KAT2 was expressed at other time points, but the brightness was different. This is the result of qualitative analysis.

[0075] At the same time, quantitative tracking and detection of protein KAT2 expression at different stages can be used to predict the quantitative basis for cotton drought tolerance. Figure 5 As shown in Figure 2, the expression level of KAT2 in drought-tolerant varieties changes with the different drought stress treatment times, which is consistent with Figure 4The band brightness of the drought-tolerant varieties showed a consistent trend. For example, the expression level of KAT2 was the highest at 12h and 120h after drought stress treatment. Figure 4 The corresponding sample's band brightness is also the most obvious.

[0076] It should be noted that drought tolerance is the drought tolerance of cotton plants themselves, so compared with drought-intolerant cotton varieties, the expression of protein KAT2 is increased. Drought stress treatment explores the effect of external drought factors on the expression of protein KAT2, so Figure 2 The expression levels of KAT2 protein in drought-tolerant cotton and drought-intolerant cotton are different. Figure 3 to Figure 5 The expression results of protein KAT2 under different drought stress treatments were different.

[0077] It should be noted that, since the monoclonal antibody of the present invention has unique specificity for protein KAT2, it can be used as a detection antibody specifically for detecting the drought tolerance of cotton and different drought stress treatment conditions, or it can be further developed into a detection kit according to conventional technical means in the art.

[0078] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0079] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A monoclonal antibody for detecting the protein KAT2 related to cotton drought tolerance, characterized in that: The amino acid sequence of protein KAT2 is shown in SEQ ID NO.

1. Protein KAT2 is used as an immunogen to immunize mice, thereby obtaining a monoclonal antibody of protein KAT2 for detecting drought tolerance of cotton.

2. The monoclonal antibody for detecting the protein KAT2 related to cotton drought tolerance according to claim 1, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region sequence comprises the amino acid sequence GLNIKDYY of CDR1-1, the amino acid sequence IDPENGNT of CDR2-1 and the amino acid sequence NVRWE of CDR3-1; the light chain variable region sequence comprises the amino acid sequence KSVSTSGYSY of CDR1-2, the amino acid sequence LVS of CDR2-2 and the amino acid sequence QHIRELT of CDR3-2.

3. The method for preparing a monoclonal antibody for detecting a protein KAT2 related to drought tolerance of cotton according to claim 1, characterized in that: A protein KAT2 antigen solution was prepared, and healthy Balb / c mice were immunized with the protein KAT2 antigen solution. After the immunization, lymphocytes were obtained, and fusion cells were obtained by hybridoma cell fusion technology to obtain a cell line capable of producing monoclonal antibodies. The monoclonal antibody secreted by the cell line is a monoclonal antibody for detecting the protein KAT2 related to the drought tolerance of cotton.

4. The method for preparing a monoclonal antibody for detecting a protein KAT2 related to drought tolerance of cotton according to claim 3, characterized in that: The mice are 8- to 12-week-old Balb / c mice.

5. The method for preparing a monoclonal antibody for detecting the protein KAT2 related to cotton drought tolerance according to claim 3, characterized in that: The protein KAT2 antigen solution is obtained by dissolving the protein KAT2 in physiological saline.

6. The method for preparing a monoclonal antibody for detecting the protein KAT2 related to cotton drought tolerance according to claim 3, characterized in that: The concentration of the protein KAT2 antigen solution is 1 mg / mL to 5 mg / mL.

7. The method for preparing a monoclonal antibody for detecting a protein KAT2 related to drought tolerance of cotton according to claim 3, characterized in that: The cell line was injected into the abdomen of Balb / c mice, and the ascites antibodies produced were purified with ProteinA / G to obtain purified monoclonal antibodies.

8. The use of the monoclonal antibody according to claim 1, characterized in that: The monoclonal antibody is used to identify drought-tolerant cotton varieties and drought-intolerant cotton varieties.

9. The use of the monoclonal antibody according to claim 8, characterized in that: The monoclonal antibody is used to test the expression level of protein KAT2, and the drought resistance of cotton varieties is identified by the expression level of protein KAT2.

10. A detection kit, characterized in that: The invention comprises the monoclonal antibody for detecting the protein KAT2 related to drought tolerance of cotton as described in claim 1.

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