CN110498847A - Preparation and crystallization method of rice receptor protein RGA5A_S - Google Patents

Abstract

The invention discloses a preparation and crystallization method of rice receptor protein RGA5A_S. The preparation method of the protein RGA5A_S is as follows: the rice receptor protein RGA5A_S is expressed using a prokaryotic expression system, and after cell lysis, the lysed supernatant is sequentially passed through an affinity chromatography column and a molecular sieve chromatography column to finally obtain a purified target protein; for the protein RGA5A_S It contains the characteristics of metal ion domain, and when the recombinant bacteria are induced to shake culture, 100μM Zn ²⁺ is added to the medium to improve the protein stability. The invention also provides a method for crystallizing the protein RGA5A_S. Shotgun method and Jiugongge method were used to screen and optimize protein crystallization conditions. As a result, protein RGA5A_S was obtained under the conditions of 0.18-0.22MNH ₄ NO ₃ and 18%-22% PEG3350. it is good.

Description

Preparation and crystallization method of rice receptor protein RGA5A_S

technical field

The invention belongs to the field of protein crystallization, and in particular relates to the preparation and crystallization method of rice receptor protein RGA5A_S.

Background technique

Rice is one of the three major economic crops in the world and is the staple food of more than 50% of the world's population (Liu and Wang, 2016). Food security has become the top priority in today’s global economic production, and the continuous growth of population is the original driving force for the development of the world’s rice industry (Zhou Xiyue et al., 2010). By 2030, the world’s rice must grow by at least 38% to meet human needs (Zhang Jing et al., 2014) . Therefore, it is of great strategic significance to ensure the stable yield and quality of rice, which is directly related to issues such as food security supply and people's food and clothing. However, the safe production of rice is endangered by pests and diseases all the year round, and rice blast caused by Magnaportheoryzae is the most common and harmful fungal disease among rice pests and diseases. The occurrence of Magnaporthe grisea has the characteristics of "many spots and wide areas", large disease area, concentrated occurrence area, serious damage degree, and large difference in disease characteristics between regions. Not only can it infect rice, but it can also infect a variety of grass crops and weeds such as barley, wheat, and teff, resulting in a decline in crop quality. On average, rice blast fungus causes 10% to 30% yield loss of rice every year, which is enough to solve the problem of food and clothing for 212 million to 742 million people (Fisher et al., 2012). Therefore, Magnaporthe grisea is rated as the top ten pathogenic fungi (Dean et al., 2012). At present, disease-resistant varieties and chemical fungicides are mainly used in agricultural production to prevent and control blast fungus. With the improvement of people's living standards and environmental protection awareness, people pay more and more attention to food safety and quality, and their green life, the use of chemical pesticides is more and more restricted. Therefore, the selection and breeding of disease-resistant varieties has become very important in the work of controlling rice blast; however, the selection and breeding of disease-resistant varieties has defects such as long cycle and poor resistance stability. In order to meet people's increasing food demand, it is urgent to deeply understand the molecular mechanism of co-evolution of rice receptors and blast fungus, so as to provide a scientific and reasonable theoretical basis for disease-resistant variety breeding and variety distribution.

Protein is the basic organic matter that constitutes cells, and is also the main bearer and executor of life activities. Analyzing its molecular structure is of great significance for understanding its function. Structural biology mainly focuses on biological functions. With the help of molecular biophysics and biochemistry, it analyzes the three-dimensional structure of biological macromolecules and their complexes, and explains the molecular mechanisms of proteins, nucleic acids, etc. that exert biological functions from the perspective of atoms. Therefore, an in-depth understanding of the three-dimensional structure of proteins is not only conducive to people's in-depth understanding of protein biological functions, but also provides a theoretical basis for genetic improvement and rational distribution of rice disease-resistant varieties, drug design, vaccine development and other application fields. X-ray crystallography is recognized as the most reliable method for analyzing the three-dimensional structure of proteins. However, on the one hand, this technology has strict requirements on protein purity (95%-99%), stability, biological activity, etc.; on the other hand, the bottleneck is the crystallization method. Therefore, obtaining high-quality protein samples and stable and reproducible high-quality crystals is of great significance for advancing structural biology to analyze protein-protein, protein-nucleic acid and other biomacromolecular interaction mechanisms.

Rice and Magnaporthe grisea are one of the main model systems for the study of plant-pathogen interactions. So far, 25 rice R genes and 11 rice blast fungus avirulence genes have been cloned, and the interaction between the two conforms to the typical "gene-to-gene hypothesis" (Flor, 1971). Studies have shown that a single R gene in rice is not enough to resist the invasion of pathogenic bacteria, and it needs to rely on paired R genes to participate in the defense response. The rice disease resistance gene Pia consists of two R genes, RGA4 and RGA5, which translate to form the CC-NBS-LRR protein; homologous and heterologous complexes can be formed in the host (Cesari et al., 2014; Cesari et al., 2013 ; de Guillen et al., 2015). When RGA4 exists alone, it can spontaneously induce the host to produce allergic necrosis, and this activation activity is inhibited by RGA5. Further analysis found that there is a non-conserved non-LRR domain (RGA5A_S domain, including the metal ion binding domain RATX1) at the C-terminus of RGA5 protein, which is directly involved in the recognition of two sequence-discordant effectors AVR1-CO39 and AVR-Pia. RGA5RATX1 is the core binding domain involved in the recognition of pathogenic bacteria. It releases the inhibition of RGA4 by directly binding to AVR1-CO39 and AVR-Pia, thereby triggering the disease resistance induced by effectors (Cesari et al., 2014; Cesari et al. , 2013). There are other domains in RGA5 that are directly involved in the recognition of the effector AVR-Pia, which regulates the host immune response (Ortiz et al., 2017). AVR1-CO39 stimulated the ROS burst innate immune response produced by Pia rice plants, which not only controlled the invasion of rice blast fungus (M. oryzae), but also inhibited the Gram-positive bacteria Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzaepv. rice bacterial blight and rice bacterial streak caused by oryzicola (Xoc) (Hutin et al., 2016). At present, the interaction mechanism of RGA5A_S recognizing the rice blast fungus effector AVR1-CO39 is not clear and needs further study. Therefore, it is necessary to provide a crystal preparation and growth technique for the rice receptor protein Pia RGA5A_S to provide a structural basis for in-depth understanding of the plant receptor R protein's recognition of fungal effectors.

Previous studies have found that the rice receptor protein RGA5A_S protein has problems such as temperature sensitivity and easy precipitation. Using the existing protein crystallization method, it is easy to cause problems such as blockage of the crystal growth manipulator pipeline, serious protein precipitation, and low crystal quality.

Contents of the invention

Aiming at the problems that the RGA5A_S protein is sensitive to temperature, easy to precipitate when concentrated, poor crystallization efficiency and low crystal diffraction quality, the invention provides a preparation and crystallization method of the rice receptor protein RGA5A_S.

In order to realize the object of the present invention, the present invention provides a kind of preparation method of rice receptor protein RGA5A_S, and it is to utilize prokaryotic expression system to express rice receptor protein RGA5A_S, through thalline lysing, cleavage supernatant passes through affinity chromatography column successively, Molecular sieve chromatography column to finally obtain the purified target protein. Wherein, 100 μM Zn ²⁺ (ZnCl ₂ ) was added to the culture medium when the recombinant bacteria were induced and shaken.

In the aforementioned method, the recombinant bacterium is constructed by transforming the expression vector carrying the gene encoding the rice receptor protein RGA5A_S into Escherichia coli.

The amino acid sequence of the protein RGA5A_S of the present invention is shown in SEQ ID NO:2. Preferably, the sequence of the gene encoding the rice receptor protein RGA5A_S is shown in SEQ ID NO:1.

Preferably, the expression vector is pETM13. The Escherichia coli is E.coli BL21(DE3).

Specifically, the preparation method of the rice receptor protein RGA5A_S of the present invention comprises the following steps:

(1) Induced expression of recombinant bacteria: Shaking culture of recombinant bacteria at 37°C and 220 rpm until the OD ₆₀₀ value of the bacterial solution is 0.6-0.8, adding a final concentration of 100 μM ZnCl ₂ and 0.1 mM IPTG, and inducing at 18°C and 180 rpm for 16-18 hours;

(2) Phytolysis: collect the bacterium liquid induced by IPTG, centrifuge at 6000rpm at room temperature for 8min, discard the supernatant, and collect the bacterium; resuspend the bacterium with lysis buffer (20mM PBS, pH 7.0, 500mM NaCl); ultrasonically lyse, Turn on for 2s, turn off for 4s, power 400-600W, 10min; 2 cycles; the lysate is centrifuged at 12 000-13 000rpm, 4°C for 25-30min to obtain the cell lysate supernatant;

(3) Affinity chromatography: 100mL cell lysate supernatant was divided into 3 parts, respectively added to 3 affinity chromatography columns pretreated with 200 mM ZnCl ₂ (chelating fast flow agarose column); the penetrating solution was re-hanged on the column once; then gradient elution was performed with elution buffers containing different concentrations of imidazole 0, 20, 60, and 500 mM;

(4) Molecular sieve chromatography: collect 60 and 500 mM imidazole eluents respectively, add DTT (dithiothreitol) with a final concentration of 4 mM, and concentrate to 1 mL respectively; then add samples to 75 10/300GL gel column, eluted with chromatography buffer, and combined the collected flow-through, which contained the purified target protein.

Preferably, the affinity chromatography column used in the present invention is chelating fast flow agarose column.

The base material of the elution buffer used in step (3) is: 20 mM PBS, pH 7.0, 500 mM NaCl.

Wherein, the elution volumes corresponding to the elution buffers with imidazole concentrations of 0, 20, 60, and 500 mM are 40, 10, 10, and 10 mL.

The formula of the chromatography buffer used in step (4) is: 20mM Tris-HCl, pH 8.0, 150mM NaCl, 5mM DTT and 4mM EDTA.

The above steps (2)-(4) should be carried out on ice or at 4°C as much as possible.

The present invention also provides the crystallization method of rice receptor protein RGA5A_S, the target protein prepared by the above method is prepared into a protein solution with a concentration of 6.4 mg/mL with a buffer, and then mixed in 0.2M NH ₄ NO ₃ and 20% PEG3350 (v/v ) in the presence of ₁₆ °C in a constant temperature incubator for _3-5 days to obtain a small amount of crystals; In the presence of /v), the single crystal of the rice receptor protein RGA5A_S is obtained by statically culturing in a constant temperature incubator at 16° C. for 3-5 days.

Preferably, the seed crystals are statically cultured in the presence of 0.18M NH ₄ NO ₃ and 20% PEG3350 to obtain single crystals.

Wherein, the formula of the buffer solution is: 20mM PBS, pH 7.0, 150mM NaCl, 5mM DTT and 4mM EDTA.

By virtue of the above technical solutions, the present invention has at least the following advantages and beneficial effects:

(1) In view of the characteristic that the target protein RGA5A_S contains a metal ion domain (with MxCxxS motif, x represents any amino acid), Zn ²⁺ was added to the culture medium during shaking culture of the recombinant bacteria, which played an important role in stabilizing the state of the protein .

(2) Shotgun method and Jiugongge method were used to screen and optimize protein crystallization conditions. It was found that protein RGA5A_S can obtain a large number of single crystals under the conditions of 0.18M NH ₄ NO ₃ and 20% PEG3350. The rate is high, and the method has good repeatability.

(3) Since the protein RGA5A_S (does not contain 6×His tag), relies on the non-conserved metal ion binding motif MxCxxS to bind to the Zn ²⁺ column, and its binding force is worse than that of 6×His-tag. Therefore, the present invention uses The elution buffer of imidazole performs gradient elution on the target protein on the column to ensure that the impurity protein is removed at low concentration, and the high-purity target protein is completely eluted at high concentration.

Description of drawings

Fig. 1 is a polarizing microscope photo of the tiny crystals of the rice receptor protein RGA5A_S obtained in Example 2 of the present invention.

Fig. 2 is a polarized light microscope photo of a single crystal of the rice receptor protein RGA5A_S obtained in Example 2 of the present invention.

Fig. 3 is the X-ray diffraction data of the rice receptor protein RGA5A_S crystal in Example 2 of the present invention.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

The preparation of embodiment 1 rice receptor protein RGA5A_S

1. Construction of recombinant expression vector

(1) Primer design

The target sequence of RGA5_RATX1 (GenBank: BAK39930.1) was obtained from NCBI, and the gene sequence was synthesized after codon optimization. Primers were designed using the Sticky-end method, and restriction enzymes (NcoI/XhoI) were selected according to the restriction enzyme sites of the selected vector and the restriction enzyme analysis of the target sequence, and two sets of primers were designed using DNAMAN 8.0.

F1-NcoI: catg CTGTCTAACA TGGAATCTGT

R1-XhoI:g CATAGTGCTG CACGGGTT

F2-NcoI: CTGTCTAACA TGGAATCTGT AGTAG

R2-XhoI: tcgag CATAGTGCTG CACGGGT

(2) PCR amplification target fragment and product recovery

Using the Sticky-end method (Pham et al., 1998), the PCR amplification product was annealed to generate a sticky end complementary to the end of the vector.

(3) Denaturation-annealing produces sticky ends

Use a NaNo Drop K550 micro-spectrophotometer to detect the concentration of PCR purified products, and mix them according to the molar ratio of 1:1. Denaturation and renaturation were performed using a PCR instrument to obtain double-stranded DNA with overhanging ends.

Denaturation and renaturation conditions: Denaturation at 94°C for 5 minutes, denaturation at 65°C for 15 minutes, cooling at 16°C.

(4) Enzyme cutting expression vector pETM13

Carry out enzyme digestion to the target fragment, double enzyme digestion to the vector, incubate at 37°C for 4-5h,

(5) The vector is connected to the target fragment

Under the action of T4 DNA ligase, the PCR product with cohesive ends after renaturation was ligated to the carrier with the same ends after digestion, and incubated overnight at 16°C.

(6) Heat shock transformation and resistance screening

Take out the competent cells E.coli JM109/DH5α from the refrigerator at -80°C, and dissolve them on ice;

Add 50 μL of competent cells to the ligation product, and let stand on ice for 30 minutes;

Shock in a water bath at 42°C for 90 seconds, and immediately place it on ice for 2 minutes;

Add 500 μL of SOC medium into the ultra-clean bench, shake and incubate at 37°C, 220rpm for 40min;

Apply the culture medium evenly to the LB plate containing the same resistance as the carrier, and air-dry (operate in the ultra-clean bench);

Inverted in a 37°C constant temperature incubator to grow overnight.

(7) Recombinant verification

Randomly pick a single clone and place it in 1 mL of the corresponding resistant LB liquid medium;

37°C, 220rpm shaking culture for 5-10h;

Bacterial liquid PCR to verify whether the inserted fragment is correct;

After the amplified product was detected by agarose gel, select the bacterial solution with the same size band as the target fragment (SEQ ID NO: 1) and send it to the company for sequencing. Use DNAMAN8.0 or NCBI to compare the sequencing results with the target sequence, and confirm that the restriction site carried by the target sequence in the recombinant plasmid is consistent, and whether there is a frameshift or point mutation between the bases, it means that the recombinant expression vector is successfully constructed. can be used in subsequent experiments.

2. Induced expression of recombinant bacteria and separation and purification of target protein

(1) Transform the above recombinant expression vector into Escherichia coli E.coli BL21(DE3), shake culture at 37°C and 220 rpm until the OD ₆₀₀ value of the bacterial solution is 0.8, add the final concentration of 100μM ZnCl ₂ and 0.1mM IPTG, 18 ℃, 180rpm induction 18h.

(2) Phytolysis: collect the bacterium induced by IPTG, centrifuge at 6000rpm at room temperature for 8min, discard the supernatant, and collect the bacterium; resuspend the bacterium with lysis buffer (20mM PBS, pH 7.0, 500mM NaCl); ultrasonically lyse, Turn on for 2s, turn off for 4s, power 400-600W, 10min; 2 cycles; lysate is centrifuged at 12 000-13 000rpm, 4°C for 25-30min to obtain cell lysate supernatant.

(3) Affinity chromatography: 100mL cell lysate supernatant was divided into 3 parts, respectively added to 3 affinity chromatography columns pretreated with 200 mM ZnCl ₂ ; Gradient elution was performed with elution buffers containing different concentrations of imidazole 0, 20, 60, and 500 mM.

Among them, the affinity chromatography column used is chelating fast flow agarose column.

The base of the elution buffer used was: 20 mM PBS, pH 7.0, 500 mM NaCl.

The elution volumes corresponding to the elution buffers with imidazole concentrations of 0, 20, 60, and 500 mM are 40, 10, 10, and 10 mL.

(4) Molecular sieve chromatography: Collect 60 and 500 mM imidazole eluents respectively, add DTT with a final concentration of 4 mM, and concentrate to 1 mL respectively; then add samples to 75 10/300GL gel column, eluted with chromatography buffer, and the purified target protein was contained in the flow-through solution.

Wherein, the formula of the chromatographic buffer used is: 20mM Tris-HCl, pH 8.0, 150mM NaCl, 5mM DTT and 4mM EDTA.

The crystallization method of embodiment 2 rice receptor protein RGA5A_S

1. Screening conditions for protein crystallization by shotgun method

The protein RGA5A_S prepared in Example 1 was dissolved in a buffer solution (20 mM PBS, pH 7.0, 150 mM NaCl, 5 mM MDTT, 4 mM EDTA) to obtain a protein solution with a concentration of 6.4 mg/mL. Shotgun crystal kit screening was then performed. Static culture in a constant temperature incubator at 16°C for 5 days, under the C4 condition of JCSG+ kit (Hampton research) [NH ₄ NO ₃ and PEG3350 were added to the protein solution, and the final concentrations were 0.2M and 20 (v/v)% respectively] Tiny crystals can be observed (Figure 1). While under other crystal growth reagent conditions, almost no crystals can be obtained.

2. Jiugong grid and inoculation strategy to optimize crystal growth conditions

In order to obtain high-quality diffractive crystals, the NH ₄ NO ₃ and PEG3350 concentration gradients were optimized using the Jiugongge method, and the crystal growth conditions were optimized using the microcrystals obtained in step 1 as seeds. It was found that RGA5A_S can obtain a large number of single crystals in the presence of 0.18M NH ₄ NO ₃ and 20% PEG3350 (v/v) (Fig. 2). Diffraction under X-rays can obtain higher resolution diffraction data (generally left and right resolution) (Figure 3). Finally, the crystal structure of the protein can be obtained by using relevant analysis software to perform indexing (Index), intensity integration (Integration) and normalization (Scaling) processing on the collected diffraction data.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

references

Cesari,S.,et al.(2014).″A novel conserved mechanism for plant NLRprotein pairs: the“integrated decoy”hypothesis.” Frontiers in plant science 5.

Cesari, S., et al.(2013). "The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding." The Plant Cell 25(4):1463-1481.

de Guillen, K., et al.(2015). "Structure analysis uncovers a highly diverse but structurally conserved effector family in phytopathogenic fungi." PLoS pathogens 11(10):e1005228.

Dean, R., et al. (2012). "The Top 10 fungal pathogens in molecular plant pathology." Molecular plant pathology 13(4): 414-430.

Fisher, MC, et al. (2012). "Emerging fungal threats to animal, plant and ecosystem health." Nature 484(7393):186-194.

Flor, HH (1971). "Current status of the gene-for-gene concept." Annual review of phytopathology 9(1):275-296.

Hutin, M., et al.(2016). "Ectopic activation of the rice NLR heteropair RGA4/RGA5 confers resistance to bacterial blight and bacterial leaf streak diseases." The Plant Journal 88(1):43-55.

Liu, W. and G.-L. Wang(2016). "Plant innate immunity in rice: a defense against pathogen infection." National Science Review 3(3):295-308.

Ortiz, D., et al.(2017). "Recognition of the Magnaporthe oryzae effector AVR-Pia by the decoy domain of the rice NLR immune receptor RGA5." The Plant Cell Online 29(1):156-168.

Zhang Jing, et al.(2014). "World food development in 2030 and its impact on the national strategic choices of China and the United States." World Agriculture (3): I0001-I0001.

Zhou Xiyue, et al.(2010). "The Development Status, Trend and Enlightenment of China's Rice Industry in the World." Agricultural Modernization Research 31(5):525-528.

sequence listing

<110> China Agricultural University

<120> Preparation and Crystallization of Rice Receptor Protein RGA5A_S

<130> KHP181112033.0

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<170> SIPOSequenceListing 1.0

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<213> Rice (Oryza sativa)

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ctgtctaaca tggaatctgt agtagagtct gcgctgaccg gccagcgtac taagatcgtg 60

gttaaggtac acatgccgtg tggcaaaagc cgcgctaaag ctatggcgct ggcagcaagc 120

gtgaacggtg tggactctgt agaaattacc ggcgaagaca aagatcgcct ggtagttgta 180

ggccgtggca tcgatccggt tcgtctggtg gcgctgctgc gtgaaaaatg cggcctggcg 240

gaactgctga tggtagaact ggttgaaaaa gaaaagaccc agctggctgg cggcaaaaag 300

ggcgcgtaca agaaacaccc gacctacaac ctgagcccgt tcgattacgt tgaatacccg 360

ccgtccgctc cgattatgca ggacattaac ccgtgcagca ctatg 405

<210> 2

<211> 135

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<213> Rice (Oryza sativa)

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Leu Ser Asn Met Glu Ser Val Val Glu Ser Ala Leu Thr Gly Gln Arg

1 5 10 15

Thr Lys Ile Val Val Lys Val His Met Pro Cys Gly Lys Ser Arg Ala

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Lys Ala Met Ala Leu Ala Ala Ser Val Asn Gly Val Asp Ser Val Glu

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Ile Thr Gly Glu Asp Lys Asp Arg Leu Val Val Val Gly Arg Gly Ile

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Asp Pro Val Arg Leu Val Ala Leu Leu Arg Glu Lys Cys Gly Leu Ala

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Gly Gly Lys Lys Gly Ala Tyr Lys Lys His Pro Thr Tyr Asn Leu Ser

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Pro Phe Asp Tyr Val Glu Tyr Pro Pro Ser Ala Pro Ile Met Gln Asp

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Ile Asn Pro Cys Ser Thr Met

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Claims (10)

1. The preparation method of rice receptor protein RGA5A_S is characterized in that, utilize prokaryotic expression system to express rice receptor protein RGA5A_S, through thalline lysing, cracking supernatant passes through affinity chromatography column, molecular sieve chromatography column successively, finally obtains Purified protein of interest; Wherein, 100 μM Zn ²⁺ is added to the culture medium when the shake culture is induced by the recombinant bacteria. 2 . The method according to claim 1 , wherein the recombinant bacterium is constructed by transforming an expression vector carrying a gene encoding the rice receptor protein RGA5A_S into Escherichia coli. 3. the method according to claim 2, is characterized in that, the sequence of described rice receptor protein RGA5A_S coding gene is as shown in SEQ ID NO:1, and described expression vector is pETM13, and described escherichia coli is Escherichia.coli BL21(DE3). 4. The method according to claim 3, characterized in that, comprising the steps of: (1) Induced expression of recombinant bacteria: Shaking culture of recombinant bacteria at 37°C and 220 rpm until the OD ₆₀₀ value of the bacterial solution is 0.6-0.8, adding a final concentration of 100 μM ZnCl ₂ and 0.1 mM IPTG, and inducing at 18°C and 180 rpm for 16-18 hours; (2) Cell lysis: collect the bacterial liquid induced by IPTG, centrifuge at room temperature 6000rpm for 8min, discard the supernatant, and collect the bacterial cells; resuspend the bacterial cells with lysis buffer; ultrasonic lysis, turn on for 2s, turn off for 4s, power 400-600W , 10min; 2 cycles; the lysate was centrifuged at 12000-13 000rpm and 4°C for 25-30min to obtain the cell lysate supernatant; (3) Affinity chromatography: 100mL cell lysate supernatant was divided into 3 parts, respectively added to ₃ affinity chromatography columns pretreated with 200mM ZnCl2; Gradient elution with different concentrations of imidazole 0, 20, 60, 500mM elution buffer; (4) Molecular sieve chromatography: Collect 60 and 500 mM imidazole eluents respectively, add DTT with a final concentration of 4 mM, and concentrate to 1 mL respectively; then add samples to 75 10/300GL gel column, eluted with chromatography buffer, combined the collected flow-through, which contained the purified target protein; Wherein, the lysis buffer used in step (2) is: 20mM PBS, pH 7.0, 500mM NaCl. 5. The method according to claim 4, wherein the base material of the elution buffer in step (3) is: 20mMPBS, pH 7.0, 500mM NaCl; The elution volume corresponding to the debuffer is 40, 10, 10, 10mL. 6. The method according to claim 4, wherein the formulation of the chromatography buffer used in step (4) is: 20mM Tris-HCl, pH 8.0, 150mM NaCl, 5mM DTT and 4mM EDTA. 7. The method according to any one of claims 4-6, characterized in that steps (2)-(4) are carried out on ice or at 4°C. 8. The crystallization method of rice receptor protein RGA5A_S is characterized in that, the target protein prepared by the method according to any one of claims 1-7 is mixed with a protein solution of concentration 6.4mg/mL with a buffer, and then in 0.2M NH In the presence of ₄ NO ₃ and 20% _PEG3350 , culture it statically in a constant temperature incubator at 16°C for 3-5 days to obtain trace crystals _; In the presence of -22% PEG3350, the single crystal of the rice receptor protein RGA5A_S is obtained by statically culturing in a constant temperature incubator at 16° C. for 3-5 days. 9 . The method according to claim 8 , wherein the seed crystals are statically cultured in the presence of 0.18M NH ₄ NO ₃ and 20% PEG3350 to obtain single crystals. 10. The method according to claim 8 or 9, wherein the formulation of the buffer is: 20mM PBS, pH7.0, 150mM NaCl, 5mM DTT and 4mM EDTA.