CN110357956B

CN110357956B - Preparation method and application of oryza sativa auxin efflux protein OsPIN1b antibody

Info

Publication number: CN110357956B
Application number: CN201910652459.2A
Authority: CN
Inventors: 杜云龙; 万媛媛; 韩丽; 赵宜婷
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-07-19
Filing date: 2019-07-19
Publication date: 2023-03-24
Anticipated expiration: 2039-07-19
Also published as: CN110357956A

Abstract

A preparation method of a rice auxin efflux protein OsPIN1b antibody comprises the following steps: analyzing a rice auxin efflux protein OsPIN1b by a bioinformatics method, selecting a polypeptide sequence with a QSSRNPTPRGSFNC sequence from an amino acid sequence of the OsPIN1b, synthesizing a polypeptide according to the polypeptide sequence, coupling the polypeptide with a carrier to obtain a polypeptide antigen, injecting the polypeptide antigen into a New Zealand white rabbit, performing 3 times of boosting immunity after primary immunity, and performing serum separation and purification to obtain the rice auxin efflux protein OsPIN1b polyclonal antibody. The polyclonal antibody prepared by the method can specifically recognize auxin efflux protein OsPIN1b in rice through an immunoblotting technology, provides a theoretical basis for disclosing a polar transport and plant development mechanism of auxin regulated auxin of an auxin efflux carrier protein PIN family, and has wide application in detection and functional identification of the auxin efflux protein PIN 1.

Description

Preparation method and application of oryza sativa auxin efflux protein OsPIN1b antibody

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method and application of a rice auxin efflux protein OsPIN1b antibody.

Background

Auxin is the first discovered phytohormone, and the characteristic of polar transport is its unique feature that distinguishes it from several phytohormones. At present, it is widely believed that the uneven distribution of auxin influx and efflux carriers on the cytoplasmic membrane realizes the polar transport of auxin and establishes the gradient distribution of auxin in plants. Polar transport of auxin is involved in many important growth and development processes of plants, including branching, tropism, growth and development of root systems of plants. Auxin and its polar transport not only affect the morphological characteristics of plants, but also have a close relationship with the absorption, transport and distribution of mineral elements.

The PIN protein family is the most studied auxin export carrier at present, and the PIN protein is a transmembrane protein, and the polar positioning of the PIN protein on a cell membrane determines the spatial distribution of auxin. In arabidopsis thaliana, 8 genes are cloned from a PIN gene family; 12 homologous genes for PIN were predicted in rice. OsPIN1 is expressed in root primordium and vascular tissues, and the generation and development of adventitious roots in transgenic plants with OsPIN1 silenced are inhibited, which shows that OsPIN1 plays an important role in the process of the generation of the adventitious roots depending on auxin. The invention researches and prepares a polyclonal antibody of the rice auxin efflux protein OsPIN1b for detecting the location, distribution and cell endocytosis of the rice auxin efflux protein OsPIN1b in a cell membrane, and lays a foundation for further developing and analyzing the functions of the rice auxin efflux protein OsPIN1b.

At present, a rice auxin output carrier gene OsPIN1b is obtained by cloning and various rice varieties are successfully transformed, and the high expression of the gene OsPIN1b in rice root tips is realized; the transportation of auxin and the elongation of root primordium are influenced by regulating the activity of the root tip tissue of the rice. It is presumed that the OsPIN1b gene may be involved in the geotropism of rice roots.

The preparation of the rice auxin output carrier OsPIN1b antibody is beneficial to further observing the cell membrane positioning of auxin output protein OsPIN1b in rice and other plants by immunohistochemistry, immunoblotting and other methods, revealing the action mechanism of auxin polarity transport regulation and control of plant growth and further clarifying the internal relation between the interaction of phytohormones and plant growth, thereby laying the experimental foundation for regulating the plant growth and development by utilizing auxin.

The invention discloses a preparation method of an antibody of a rice auxin efflux protein OsPIN1b and immunohistochemical application of the antibody for the first time.

Disclosure of Invention

The invention aims to fill the blank in the field of detection of the rice auxin efflux protein OsPIN1b, and provides a preparation method of a polyclonal antibody aiming at the rice auxin efflux protein OsPIN1b.

The invention aims to solve the problem of providing a preparation method of a polyclonal antibody of a rice auxin efflux protein OsPIN1b, and the invention adopts the following technical scheme:

a preparation method of a polyclonal antibody of a rice auxin efflux protein OsPIN1b comprises the following steps:

1) OsPIN1b, a protein expressed by OsPIN1b (LOC _ Os02g50960 or Os02g 0743400) of a rice Oryza sativa subsp.

2) Selecting a section of polypeptide sequence QSSRNPTPRGSFNC from the antibody coding protein OsPIN1b, wherein the specific method comprises the following steps: according to the structural complexity, the easy oxidation degree, the synthesis difficulty, the amino acid category and the distribution of a peptide chain and the like, the 15 th amino acids at the 244 th to 258 th positions of the protein coded by the antibody are finally determined as the amino acid sequence of the synthesized polypeptide, wherein the 258 th amino acid is H → C, and the sequence is QSSRNPTPRGSSSFNC.

3) Synthesizing polypeptide according to the polypeptide sequence in the step 2;

4) Coupling polypeptide and a carrier to obtain a polypeptide antigen;

5) Injecting the polypeptide antigen into a New Zealand white rabbit, performing 1 boosting immunization every 2 weeks after 4 weeks of primary immunization, treating for three times in total, and performing serum separation and purification to obtain a rice auxin efflux protein OsPIN1b polyclonal antibody, wherein the IgG concentration of the antibody is 0.51mg/mL;

6) The indirect ELISA method was used to evaluate the titer of polyclonal antibodies.

The test shows that the antibody is qualified rabbit polyclonal antibody, and the detection value is 0.718 when the antibody is diluted by 1.

The invention also applies the rice auxin efflux protein OsPIN1b antibody to an immunoblotting test.

The rice auxin efflux protein OsPIN1b antibody is also applied to detecting the cell positioning and distribution rule of the auxin efflux protein OsPIN1b in the rice, corn, rape, potato, wheat and soybean root tip epidermal cells.

The invention also applies the rice auxin efflux protein OsPIN1b antibody to the detection of the cell endocytosis of the rice auxin efflux protein OsPIN1b.

The invention analyzes and contrasts the rice antibody coding protein OsPIN1b to select a specific polypeptide sequence QSSRNPTPRGSFNC, synthesizes the polypeptide according to the polypeptide sequence to obtain the polypeptide, couples the polypeptide with BSA to obtain a polypeptide antigen, and immunizes New Zealand white rabbits for a plurality of times by using the polypeptide antigen to obtain the polyclonal antibody of the specific auxin efflux protein OsPIN1b for the first time. The OsPIN1b polyclonal antibody prepared by the method can specifically identify the rice auxin efflux protein OsPIN1b, can realize the cell membrane positioning of the auxin efflux protein OsPIN1b in rice root tip epidermal cells and embryos, can identify the distribution and the cell endocytosis of the rice auxin efflux protein OsPIN1b, and has wide application in the detection, functional identification and research of the auxin efflux protein PIN 1. In addition, the antibody can also be used for positioning cell membranes of auxin export protein PIN1 in roots and embryos of rape, corn, potato, wheat and soybean.

Drawings

FIG. 1 is an immunoblot image (Western blot);

FIG. 2 is an immunohistochemical map: detecting the cell membrane localization of PIN1 protein in the root tips of six grain crops of rice, corn, rape, potato, wheat and soybean by using the prepared Anti-rabbitit OsPIN1b antibody, wherein Bar =10 mu m;

FIG. 3 is an immunohistochemical map: detecting the cell membrane location of PIN1 protein in rice, corn, wheat and soybean embryo cells of grain crops by using the prepared Anti-rabbitit OsPIN1b antibody, wherein Bar =10 mu m;

fig. 4 is an immunohistochemical map: the prepared Anti-rabbitit OsPIN1b antibody is used for detecting the endocytosis of OsPIN1b protein in root tip epidermal cells of two rice varieties LTH and Nipponbare, and Bar =10 mu m.

Detailed Description

Example 1:

the preparation method of the polyclonal antibody of the oryza sativa auxin efflux protein OsPIN1b provided by the embodiment comprises the following steps:

1) A rice Oryza sativa subsp.japonicum (Nipponbare) gene OsPIN1b (LOC _ Os02g50960 or Os02g 0743400) expression protein OsPIN1b was analyzed by bioinformatics.

2) Selecting a section of polypeptide sequence QSSRNPTPRGSFNC from the antibody coding protein OsPIN1b, wherein the specific method comprises the following steps: according to the complexity of the polypeptide structure, the easy oxidation degree, the synthesis difficulty, the amino acid type and distribution and the like, the 15 th amino acid of the 244 th-258 th protein of the antibody coding protein is finally determined as the amino acid sequence of the synthetic polypeptide, wherein the 258 th amino acid is H → C, and the sequence is QSSRNPTPRGSFNC.

3) Synthesizing the polypeptide by using a polypeptide synthesizer according to the polypeptide sequence in the step 2, wherein the specific procedure is as follows: 1. the Fmoc-protected resin was removed to expose the amino groups. Deprotection reagent was 20% pip prepared with DMF; 2. dissolving amino acid with DMF; 3. the program automatically extracts 4mL of amino acid solution in turn; 4. each 4mL aliquot of the amino acid solution was loaded and the procedure extracted the carboxyl activating agent 0.4M HBTU (in DMF) and the aminocarboxyl condensing agent 0.8M DIEA (in DMF) to allow condensation of the next amino acid exposed to the carboxyl group upon activation with the previous amino acid exposed to the amino group upon deprotection, thus forming the dipeptide. Cycling in this manner until the last amino acid is condensed to the end of the peptide chain. Each step in the synthesis process requires washing the deprotected group with DMF, DCM and eluting the unreacted amino acid residue from the reaction system.

4) Coupling of the synthesized polypeptide to BSA using SPDP ligation: 4.6mg SPDP was dissolved in 740uL DMSO, at a final concentration of 20mM.0.1008g BSA was dissolved in 2mL PBS-EDTA solution and allowed to stand at room temperature for 1h. Excess SPDP is eluted using a desalting column. 4mg of polypeptide was added to the coupled BSA-SPDP system overnight at room temperature. Coupling the polypeptide and the BSA polypeptide coupled carrier through carboxyl or amino terminal residues to obtain a polypeptide antigen;

5) Injecting the polypeptide antigen into a healthy New Zealand white rabbit, performing 1 boosting immunization every 2 weeks after 4 weeks of primary immunization, treating for three times in total, wherein the injection way is the same as that of the primary immunization, performing otogenic venous blood collection, separating serum, and separating and purifying IgG antibody by a Protein G column: the Protein G affinity column was equilibrated with PBS (pH 7.4), eluted with 0.1M Gly-HCl (pH 3.0), and dialyzed against PBS (pH 7.4). Obtaining a rice auxin efflux protein OsPIN1b polyclonal antibody, wherein the IgG concentration of the antibody is 0.51mg/mL;

6) The indirect ELISA method is used for evaluating the titer of the polyclonal antibody, and comprises the following specific steps:

a. coating: adding 200 mu L of antigen OsPIN1b to each hole of an enzyme label plate by using a pipette gun at the concentration of 1 mu g/mL, and standing overnight at 4 ℃;

b. and (3) sealing: adding 200 μ L of 1% BSA solution, and incubating at 37 deg.C for 1 hour;

c. diluting: the rabbit polyclonal antibodies were raised to 1:200,1:1000,1:5000,1:10000,1:20000,1:60000,1:240000 diluting;

d. sample adding: adding diluted rabbit polyclonal antibody 50 μ L/well, placing in a thermostat, and incubating at 37 deg.C for 1 hr;

e. washing: wash well 2 times with detergent TBST at 200. Mu.L/well;

f. adding an enzyme-labeled secondary antibody: add to each well a mixture of 1:5000 of diluted goat anti-rabbit HRP solution was used as an enzyme-labeled secondary antibody, incubated at 37 ℃ for 45min and washed 3 times with TBST at 200. Mu.L/well;

g. color development: adding 100 mu L of TMB color development liquid into each hole, and developing for 10 minutes at 37 ℃;

i. and (4) terminating: adding 2 mol/l sulfuric acid 50 microliter/well, and reading the light absorption value of each well by using an enzyme-labeling instrument.

And (3) detection results: when the antibody was diluted 1.

Example 2:

application of OsPIN1b antibody in immunoblotting:

and (3) detection results: as shown in figure 1, western blot detects the expression of OsPIN1b protein in rice root tissue, and the expression ratio of rice root tissue sample 1: at 500-fold dilution, the protein showed the target band at 65 kD.

Example 3:

the OsPIN1b antibody is positioned in the root tip epidermal cells of six different grain crops.

1. And (3) test crops: rice, corn, canola, potato, wheat and soybean;

2. experimental materials: microcloth, 4% paraformaldehyde, PBS buffer (pH 7.4), triton X-100, draselase, DMSO, nonidet-P40, BSA, sterile water, and a tablet. A first antibody: anti-OsPIN1b antibody;

secondary antibody: donkey Anti-rabbitIgG (H + L) -Alexafluor 488coupled.

3. Experimental procedure

a. Obtaining of materials: the six grain crops are hydroponically planted for 7 days, and after the crops grow roots, the root tips are taken for about 1cm and are cleaned by sterile water. Placed in a 24-well plate packed with Microcloth and flushed with PBS three times.

b. Adding 1mL of 4% paraformaldehyde fixing solution into the holes filled with the tissue, and fixing for 1h at room temperature in a vacuum state; purge 5 times with 1mL of PBS +0.5% Triton X-100, then purge 2 times with double distilled water, each for 10min; finally, the tissue is soaked in double distilled water and placed at 37 ℃ for 12min.

c. Treatment with 1mL of 2% crashase (Driselase) for 1h at 37 ℃ and flushing with 1mL of PBS +0.5% Triton X-100 5 times for 12min each; 1mL of permeate (10% DMSO and 2% Nonidet-P40) was added, treated at room temperature for 1.5h, and flushed with PBS for 6 times 10min each.

d. Add 1mL 2% BSA solution, block for 1h at 37 ℃; add 1mL of primary antibody (1-fold dilution) and stand overnight at 4 ℃; purge 5 times with 1mL PBS +0.5% Triton X-100, 12min each time.

e. 1mL of secondary antibody (1-fold diluted) was added and left at 37 ℃ for 4h; purge 5 times with 1mL PBS +0.5% Triton X-100, 12min each time.

f. A drop of the encapsulated tablet was placed on a glass slide, the treated material was placed therein, and the tablet was observed with a confocal laser microscope.

4. The experimental results are as follows: as can be seen from figure 2, the OsPIN1b antibody can be well combined with rice OsPIN1b protein, the antibody has different combining ability with PIN1 protein in six major grain crops, the combining ability of corn and potato is stronger, wheat and soybean have inferior effect, and can hardly generate immune reaction with rape, thus proving that the antibody is suitable for rice, corn, potato, wheat, soybean and the like. In addition, the cell membrane localization in the crop root system can be carried out on the PIN1 export protein through the combination position of the OsPIN1b antibody and the PIN1 protein.

Example 4

The experimental materials were taken from four grain crop embryos of rice, corn, wheat and soybean: seeds of four crops were soaked in water overnight, the next day, the seed shells were removed with a scalpel, and the embryos in the seeds were removed, and the other experimental procedures were the same as in example 3.

The experimental results are as follows: as can be seen from FIG. 3, the OsPIN1b antibody can also be combined with PIN1 protein on the cell membranes of rice and wheat embryos, has better combination capability with PIN1 protein on the cell membranes of wheat embryos, and can clearly see green fluorescence on the cell membranes. The prepared OsPIN1b antibody is not only suitable for detecting the positioning of the PIN1 protein of roots, but also can be used for detecting the cell positioning of the PIN1 protein in different plant embryos, thereby laying a theoretical foundation for disclosing the polar transportation and plant development mechanism of auxin regulated auxin of an auxin export carrier protein PIN family.

Example 5:

the OsPIN1b antibody is used for detecting the cell endocytosis of OsPIN1b protein in the root tips of LTH and Nipponbare of different rice varieties: before an immunohistochemical experiment, rice root tips are treated by a protein transport inhibitor brefeldin a (BFA), and BFA bodies formed by OsPIN1b protein aggregation in epidermal cells of different rice root tips are observed after the treatment.

1. Materials and reagents:

rice LTH, rice Nipponbare, 0.25. Mu.M BFA solution, microcloth, 4% paraformaldehyde, PBS buffer (pH 7.4), triton X-100, the enzyme crashing Driselase, DMSO, nonidet-P40, BSA, sterile water, and a sealed tablet;

a first antibody: anti-rabbitOsPIN 1b antibody;

secondary antibody: donkey Anti-rabbitIgG (H + L) -Alexafluor 488coupled.

2. The experimental method comprises the following steps:

a. before an immunohistochemical experiment, rice root tips are treated by a protein transport inhibitor brefeldin a (BFA), and BFA bodies formed by aggregation of OsPIN1b protein in different rice epidermal cells are observed after the treatment.

b. Cutting the radicle tip of the rice seedling with a razor blade to 1cm, carefully placing the root tip into a container containing 1mL ddH ₂ O wells of a twenty-four well plate, carefully purged with a 1mL pipette, taking care not to injure the roots; the liquid was aspirated and 1mL ddH was added ₂ Soaking the root tip for about 15min to ensure the culture of the surface of the root tipThe base is cleaned, and the medicament is not influenced to enter the apical cells;

c. sucking out the liquid, adding 1mL of 0.25 μ M BFA solution, and treating for 90min in darkness (wrapping twenty-four-hole plate with tinfoil); after the treatment, the rice root tips were removed and placed in a 24-well plate packed with Microcloth and flushed with PBS three times.

d. Adding 1mL of 4% paraformaldehyde fixing solution, and fixing at room temperature for 1h in a vacuum state; purging with 1mL of PBS +0.5% Triton X-100 solution 5 times, and then purging with double distilled water 2 times, each for 10min; finally, the tissue is soaked in double distilled water and placed at 37 ℃ for 12min.

e. Treating with 1mL of 2% solution of crashase (Driselase) at 37 deg.C for 1h, and purging with 1mL of PBS +0.5% Triton X-100 solution 5 times for 12min each time; 1mL of permeate (10% DMSO and 2% Nonidet-P40) was added, treated at room temperature for 1.5h, and flushed with PBS for 6 times 10min each.

f. Adding 1mL of 2% BSA, blocking at 37 ℃ for 1h; add 1mL primary antibody (1-fold dilution) and stand overnight at 4 ℃; purge 5 times for 12min with 1mL of PBS +0.5% Triton X-100 solution.

g. 1mL of secondary antibody (1-fold diluted) was added and left at 37 ℃ for 4h; purge 5 times for 12min with 1mL of PBS +0.5% Triton X-100 solution.

h. Dropping a drop of the encapsulated tablet on a glass slide, putting the treated rice root tip tissue into the encapsulated tablet, and observing the encapsulated tablet by using a laser confocal microscope after the encapsulated tablet is pressed.

3. The experimental results are as follows:

as can be seen in FIG. 4, the OsPIN1b antibody in rice root cells is combined with the OsPIN1 protein in rice to form BFA corpuscles under the action of a protein transport inhibitor BFA on the rice roots of both Lijiang New group black rice LTH and Nipponbare. The experimental result successfully verifies that the OsPIN1b antibody can be well used for detecting the cell endocytosis experiment of the auxin efflux protein OsPIN1b in different rice, thereby being beneficial to researching the polar transport mechanism of the auxin and providing a theoretical basis for the growth and development relationship of an auxin efflux carrier and plants.

Claims

1. A preparation method of a rice auxin efflux protein OsPIN1b antibody is characterized by comprising the following steps:

1) Analysis of rice by bioinformaticsOryza sativa subsp.japonica.NipponbareA protein OsPIN1b expressed by LOC _ Os02g50960 or Os02g0743400 of the gene OsPIN1b;

2) Selecting 15 amino acids from 244 th to 258 th positions from the protein OsPIN1b as an amino acid sequence of a synthetic polypeptide, and on the basis, changing the 258 th position amino acid from H → C, wherein the mutated amino acid sequence is QSSRNPTPRGSFNC;

3) Synthesizing polypeptide according to the polypeptide sequence in the step 2);

4) Coupling polypeptide and a carrier to obtain a polypeptide antigen;

5) Injecting the polypeptide antigen into a New Zealand white rabbit, performing 3 times of boosting immunity after primary immunity, and obtaining a rice auxin efflux protein OsPIN1b polyclonal antibody through serum separation and purification, wherein the IgG concentration of the antibody is 0.51mg/mL;

2. The method for preparing an antibody against OsPIN1b, which is the auxin efflux protein of rice as described in claim 1, wherein the antibody against OsPIN1b is applied to immunoblotting.

3. The rice auxin efflux protein OsPIN1b antibody prepared by the preparation method of the rice auxin efflux protein OsPIN1b antibody as described in claim 1 is applied to detecting the cell positioning and distribution rule of the auxin efflux protein PIN1 in the root tip, embryo, leaf epidermal cells and cell membranes of rice, corn, potato, wheat and soybean.

4. The method for preparing an antibody against the rice auxin efflux protein OsPIN1b as described in claim 1, wherein the antibody against the rice auxin efflux protein OsPIN1b is used for detecting the endocytosis of the auxin efflux protein PIN1 in rice, corn, potato, wheat, soybean.