NbREM1.1 Carboxyl-Terminal Region Protein Crystal, Preparation Method and Application

Abstract

The present invention discloses a crystal of the carboxyl-terminal region protein of NbREM1.1, a preparation method and applications thereof, belonging to the field of biotechnology. The crystal includes the amino acid sequence of the carboxyl-terminal region of the NbREM1.1 protein, has a P21 space group, and the unit cell parameters are: #imgabs0##imgabs1# α = γ = 90° and β = 95.8°. The present invention genetically engineers the remorin protein NbREM1.1 derived from the natural host rice of rice stripe virus, obtains the carboxyl-terminal of the remorin protein NbREM1.1, highly expresses, purifies and crystallizes it in Escherichia coli, and analyzes the three-dimensional crystal structure of the protein of the 97th to 207th amino acids at the carboxyl-terminal of the remorin protein NbREM1.1, providing a theoretical guidance for the later prevention and control of rice stripe disease.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a protein crystal of the carboxyl-terminal region of NbREM1.1, a preparation method and an application thereof. Background Art

[0002] Rice stripe disease caused by Rice stripe virus (RSV) seriously threatens the yield and quality of rice in East Asia. RSV is transmitted by the small brown planthopper (Laodelphax striatellus) in a persistent manner. Once the small brown planthopper is infected with the virus, it can transmit the virus through eggs throughout its life and multiply in the insect body. Once the disease breaks out, the virus-carrying rate of its transmission vector will remain for several years, seriously threatening rice production in China and should be given sufficient attention. At present, no effective chemical drug control method has been developed for rice stripe virus disease, and the main way of prevention and control is to control the occurrence and migration of the transmission vector. Searching for host factors that regulate RSV infection of plants and studying the protein crystal structure of host factors are conducive to exploring the interaction mechanism between RSV and plant hosts and providing reference strategies for the prevention and control of rice stripe disease.

[0003] Remorin proteins are a family of plant-specific oligomeric filamentous proteins that are associated with the plasma membrane and located in membrane microdomains. They were first discovered in tomato (Solanum lycopersicum) and potato (Solanum tuberosum). They are hydrophilic proteins that are tightly bound to the plasma membrane. Remorin proteins are thought to be involved in hormone signal transduction, and can enhance the drought and salt tolerance of plants, and can increase seedling biomass, germination rate and survival rate. The functions of many remorin proteins are still unclear. Previous studies have shown that remorin proteins are also involved in plant-pathogen interactions.

[0004] Genomic studies of remorin proteins have shown that they have a highly conserved carboxyl-terminal coiled-coil domain and an amino-terminal domain with relatively high sequence variability. The carboxyl-terminal domain of Remorin is called REM-CA (REMORIN C-terminal Anchor). Equivalent spectra obtained in a hydrophobic environment indicate that REM-CA can fold into an α-helical conformation. REM-CA is a necessary region for oligomerization reactions and is directly involved in the interaction with other proteins. The oligomerization of Remorin proteins is of great significance for their targeting and function. At present, there is no experimental data on the oligomeric state of REM-CA in plants.

[0005] Previous studies have shown that NbREM1.1 is a negative regulator of RSV infection in Nicotiana benthamiana, and at the same time, the RSV movement protein NSvc4 can interact with NbREM1.1. NbREM1.1 can regulate the callose accumulation and plasmodesmata permeability in Nicotiana benthamiana cells, thereby affecting the intercellular movement of RSV and interfering with the ability of the RSV movement protein NSvc4 to complement the intercellular movement of PVX.

[0006] Therefore, analyzing the three-dimensional crystal structure of the carboxyl terminus of the plant remorin protein NbREM1.1 not only has important scientific significance for revealing the mechanism of plant-microbe interaction, but also can provide important reference strategies and theoretical guiding significance for the prevention and control of rice stripe disease. Summary of the Invention

[0007] The object of the present invention is to provide a method for preparing and applying a protein crystal of the carboxyl-terminal region of NbREM1.1 to solve the problems existing in the above-mentioned prior art. By analyzing the structure of the protein crystal of the carboxyl-terminal region of NbREM1.1, important reference strategies and theoretical guiding significance for the prevention and control of rice stripe disease are provided.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a protein crystal of the carboxyl-terminal region of NbREM1.1, and the crystal comprises an amino acid sequence of the carboxyl-terminal region of the NbREM1.1 protein; the crystal has a P21 space group, and the unit cell parameters are: α = γ = 90° and β = 95.8°.

[0010] Preferably, the carboxyl-terminal region of NbREM1.1 is the 97th to 207th amino acids at the carboxyl terminus of the NbREM1.1 protein, with a total of 111 amino acids, a molecular weight of 12.66 kDa, and the amino acid sequence is as shown in SEQ ID NO: 1.

[0011] Preferably, there are two protein molecules in one crystallographic asymmetric unit of the three-dimensional structure of the crystal.

[0012] Preferably, two carboxyl-terminal regions of NbREM1.1 form a long and straight single helix, which together form an antiparallel helix bundle.

[0013] The present invention also provides a method for preparing the protein crystal of the carboxyl-terminal region of NbREM1.1, comprising the following steps:

[0014] Concentrate the purified protein solution of the carboxyl-terminal region of NbREM1.1 to 8-15 mg / mL, and form the protein crystal of the carboxyl-terminal region of NbREM1.1 by vapor diffusion method at a temperature of 4-20 °C;

[0015] Among them, the NbREM1.1 carboxyl-terminal region protein crystal grows in a solution containing the protein solution and buffer with a volume ratio of 1:1. The buffer is HEPES at pH 7.5, 0.2 M ammonium acetate, 25% PEG3350; 0.5% n-octyl-β-D-glucopyranoside (n-Octyl-β-D-glucoside).

[0016] When purifying the carboxyl-terminal region of the NbREM1.1 protein above, it is preferred to use prokaryotic cells of Escherichia coli for expression (but other expression systems are not excluded, such as expression in other eukaryotic cells). It is preferred to use His-tag to express the above protein in the form of a fusion protein to obtain soluble expression (but other tags are not excluded, such as GST or MBP tags may also have soluble expression). The method of recognizing the specific tag His is through an affinity chromatography column nickel column, and the method of removing the tag is through enzymatic digestion with protease TEV. Further, the protein is separated and purified by methods such as gel filtration chromatography to obtain the purified protein of the carboxyl-terminal region of the NbREM1.1 protein. The purity of the protein is determined by gel electrophoresis. In this way, a high-purity carboxyl-terminal region of the NbREM1.1 protein is obtained.

[0017] Preferably, the protein solution of the carboxyl-terminal region of NbREM1.1 is concentrated to 11 mg / mL; the temperature is 16 °C.

[0018] The present invention also provides an application of the NbREM1.1 carboxyl-terminal region protein crystal in the prevention and control of rice stripe disease.

[0019] The present invention discloses the following technical effects:

[0020] Based on gene modification technology, the present invention obtains the carboxyl-terminal region of the plant remorin protein NbREM1.1, expresses the carboxyl-terminal region of NbREM1.1 through a prokaryotic expression system, and then obtains a high-purity protein solution of the carboxyl-terminal region of NbREM1.1 through separation and purification. The obtained protein solution is used to screen crystal growth conditions by the vapor diffusion method to obtain crystals. Through structural analysis of the crystals, it is found that: there are two protein molecules in one crystallographic asymmetric unit of the three-dimensional structure of the carboxyl-terminal domain of the NbREM1.1 protein. Two carboxyl-terminal domains of the NbREM1.1 protein form a long and straight single helix, jointly forming an antiparallel helix bundle. Further experimental verification shows that: the carboxyl-terminal region of the NbREM1.1 protein is related to the intercellular movement ability of rice stripe virus. Therefore, the present invention analyzes the three-dimensional structure of the carboxyl-terminal region of the plant remorin protein NbREM1.1, which has important scientific significance for revealing the interaction mechanism between rice and rice stripe virus, and provides important structural guidance for the later prevention and control of rice stripe disease. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 15% SDS–PAGE electrophoresis analysis of the purification process of the carboxyl terminus (97-207AA) of the plant remorin protein NbREM1.1; S: supernatant after disruption; P: precipitate after disruption; FL: supernatant after passing through the nickel column (flow-through); 20: 20 mM imidazole eluent; 40: 40 mM imidazole eluent; 300: 300 mM imidazole eluent. 2FL: flow-through after the target protein, Trx-tag, and 6His-tag are cleaved by TEV enzyme and then purified through the nickel column for the second time; 2-20, 20 mM imidazole eluent for the second nickel column purification; M: standard protein Maker

[0023] Figure 2 Molecular sieve diagram of NbREM1.1-97-207AA

[0024] Figure 3 Selenomethionine crystal of the carboxyl terminus region (97-207AA) of the plant remorin protein NbREM1.1

[0025] Figure 4 Dimer structure of the carboxyl terminus region (97-207AA) of the plant remorin protein NbREM1.1

[0026] Figure 5 Effect of point mutations at the dimer interaction interface site of the protein crystal on the negative regulation of the intercellular movement of RSV by the Nicotiana benthamiana remorin protein NbREM1; (A) Wild-type remorin protein NbREM1 infects Nicotiana benthamiana; (B) Remorin protein NbREM1 (K54A Mutant) after site-directed mutation infects Nicotiana benthamiana Detailed Embodiments

[0027] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0028] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are merely exemplary.

[0031] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0032] Example 1 Expression and Purification of the Carboxyl-Terminal (97 - 207 AA) of the Plant Remorin Protein NbREM1.1

[0033] The carboxyl-terminal of the remorin protein NbREM1.1 from the natural host rice of RSV has a total of 111 amino acids. The amino acid sequence is shown as follows, and the molecular weight is 12.66 kDa.

[0034] KRLSLIKAWEESEKSKAENKAQKNVSAIAAWENSKKANLEAELKKMEEQVEKKKAEYIEK MKNKIALLHKEAEEKRAMIEAKRGEDLLKAEELAAKYRATGTAPKKLLGCF.

[0035] The remorin protein NbREM1.1 from the natural host rice of RSV was genetically engineered. Its full length was truncated, and its carboxyl-terminal was cloned onto the vector pET32a(+). Among them, the primers for amplifying the amino acid fragment from the 97th to the 207th position of NbREM1.1 were:

[0036] NbREM1.1 97F - BamHI: CGCGGATCCAAGAGACTGTCACTAATC;

[0037] NbREM1.1R - Not I: ATAAGAATGCGGCCGCTCAAAAACATCCAAGGAG.

[0038] The amplification reaction system is: 50 μL PCR reaction system [2x PrimeSTAR GC buffer, 25 μL; dNTPs (2.5 mM each), 4 μL; 100 μM primer - F, 0.15 μL; 100 μM primer - R, 0.15 μL; genomic DNA, 0.2 μL;

[0039] PrimeSTAR HS DNA polymerase, 0.5 μL; dd H2O, 20 μL].

[0040] The amplification reaction program is: pre - denaturation at 94°C for 5 min; [denaturation at 98°C for 10 sec; annealing at 55 - 60°C for 5 sec; extension at 72°C for 1 min 10 sec;], 33 cycles; extension at 72°C for 10 min.

[0041] The carboxyl - terminal domain of NbREM1.1 obtained by amplification was cloned into the vector pET32a(+) to construct a recombinant vector for expressing a fusion protein with 6 His and Trx tags fused to the amino - terminus. The recombinant vector was transformed into Escherichia coli BL21(DE3), and cultured with shaking at 37°C until the OD 600 reached 0.6 - 1.0 (preferably 0.8), then IPTG with a final concentration of 0.1 - 1.0 mM (preferably 0.3 mM) was added for induction expression. The expression conditions were: shaking culture at 16°C, 200 rpm for 20 hours, and then the bacteria were collected by centrifugation (4500 rpm, 15 min) for purification.

[0042] The centrifugally collected expressed bacteria were resuspended in an appropriate amount of buffer [50 mM Tris (pH 7.5), 500 mM NaCl, 5% glycerol, 10 mM imidazole], and the bacterial cells were lysed using a high-pressure cell disruptor. The precipitate and other particulate impurities were removed by centrifugation at 18,000 rpm for 20 min. Then, chromatography was performed using an affinity chromatography column, a nickel column. Specifically, after the supernatant after centrifugation was combined with Ni-NTA affinity medium, the medium was rinsed with buffer [50 mM Tris (pH 7.5), 500 mM NaCl, 5% glycerol, 20 mM imidazole] to remove miscellaneous proteins; then, the medium was further rinsed with buffer [50 mM Tris (pH 7.5), 500 mM NaCl, 5% glycerol, 40 mM imidazole] to further remove miscellaneous proteins. Finally, the target protein was eluted from the affinity medium with eluent [50 mM Tris (pH 7.5), 500 mM NaCl, 5% glycerol, 300 mM imidazole]. There is a TEV enzyme cleavage site on pET32a(+). Therefore, after digestion with TEV enzyme and dialysis overnight, the second nickel column purification was carried out to obtain a carboxyl-terminal domain protein of NbREM1.1 without a tag with higher purity. The eluent was concentrated using a 10 kDa cut-off concentrator tube. The concentrated protein solution was further purified by gel filtration chromatography, and the target protein eluted was concentrated to a concentration of 11 mg / mL using buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM DTT] and stored at -80 °C for crystallization experiments.

[0043] The results are as Figure 1 shown. After purification and separation, the protein at the carboxyl terminus of the remorin protein NbREM1.1 was obtained. The size of the carboxyl terminus (97 - 207 AA) of the tagged plant remorin protein NbREM1.1 is approximately 32 kDa; after excision of the tag with the protease TEV, it is approximately 12 kDa.

[0044] Example 2 Crystallization of the carboxyl terminus (97 - 207 AA) protein of the plant remorin protein NbREM1.1

[0045] The carboxyl terminus of the plant remorin protein NbREM1.1 (NbREM1.1-97-207AA) purified by the above method was concentrated to a concentration of about 11 mg / mL. A crystallization kit (CrystalScreen Kit I / II from Hampton Research; PEGs from Qiagen, JCSG and Classics Suites, etc.) was used as the primary screening condition for crystal growth. According to the kit instructions, the sitting-drop vapor diffusion method was used to screen the crystal growth conditions at 4-20 °C. The present invention obtained initial crystals under multiple different crystallization reagent conditions, as shown in Figure 2 . Through subsequent optimization and adjustment, the protein was mixed with the crystallization reservoir solution at a volume ratio of 0.7:0.7, preferably a buffer solution of HEPES (pH 7.5); 0.2 M ammonium acetate; 25% PEG3350; 0.5% w / v n-Octyl-β-D-glucoside, at 16 °C, as the crystal growth condition. A set of X-ray diffraction data was collected: the resolution was . The crystal structure space group was P21. The unit cell parameters were: α = γ = 90°, β = 95.8°. The present invention achieved the synthesis of selenomethionine by adding selenomethionine (final concentration: 50 mg / L) to the bacterial culture medium. Seleno-crystals grew under the same conditions as the native protein crystals (see Figure 3 ).

[0046] Example 3 Data collection and structure analysis of the carboxyl terminus (97-207AA) of the plant remorin protein NbREM1.1

[0047] The X-ray diffraction data of the crystal was collected at the BioMacromolecule Crystallography Beamline Station (BL18U1) of the Shanghai Synchrotron Radiation Facility (SSRF). The data was processed using XDS, and further data and model operations were performed using CCP4 software. The SeSAD phase was processed using the CRANK2 software, and refinement was performed using REFMAC5. In addition, the model was further corrected in coot. Finally, the crystal structure of the protein was obtained. Specifically, the three-dimensional structure of the carboxyl terminus domain of the remorin protein NbREM1.1 has two protein molecules within one crystallographic asymmetric unit. The two carboxyl terminus domains of the remorin protein NbREM1.1 form a long and straight single helix, jointly forming an antiparallel helix bundle (see Figure 4 ). Such an arrangement buries the hydrophobic side chains, making the remorin protein soluble, and the remorin protein dimer can dissociate in the hydrophobic plasma membrane environment.

[0048] The atomic coordinate group of the carboxyl terminus protein crystal of the plant remorin protein NbREM1.1 is shown in Table 1 below.

[0049] Table 1. The atomic coordinate groups of the NbREM1.1-97-207 protein crystal are as follows:

[0050] Crystal space group: P21

[0051] The unit cell parameters are: α = γ = 90°, β = 95.8°.

[0052] / <σ(I)>a: 13.9.

[0053] R work / R free are respectively: 0.250 and 0.292.

[0054] Resolution range 78.39–2.50.

[0055] Data integrity (%): 99.8.

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[0142] Example 4 Functional verification of the carboxyl-terminal region of the plant remorin protein NbREM1.1 based on protein crystal structure analysis According to the results of the three-dimensional protein crystal structure of the carboxyl-terminal region of the plant remorin protein NbREM1.1 analyzed above, site-directed mutation K54A (Mutant) was designed at the dimer interaction interface, as follows:

[0143] The primers were:

[0144] REMJ-K54A-F: GCAGGTGGAGAAAAAGGCGGCAGAATATATTGAGA;

[0145] REMJ-K54A-R: TCTCAATATATTCTGCCGCCTTTTTCTCCACCTGC.

[0146] The amplification reaction system is as follows: 50 μL PCR reaction system [2x PrimeSTAR GC buffer, 25 μL; dNTPs (2.5 mM each), 4 μL; 100 μM primer-F, 0.15 μL; 100 μM primer-R, 0.15 μL; template (wild type),

[0147] 0.2 μL; PrimeSTAR HS DNA polymerase, 0.5 μL; dd H2O, 20 μL].

[0148] The amplification reaction program is as follows: pre-denaturation at 94 °C for 5 min; [denaturation at 98 °C for 10 sec; annealing at 55 - 60 °C for 5 sec; extension at 72 °C for 6 min;], 33 cycles; extension at 72 °C for 10 min.

[0149] The amplified gene sequence is based on gene technology and infects Nicotiana benthamiana by conventional transformation methods, and then observes the changes in the intercellular movement ability of the virus on the leaves of wild type and point mutants (Reference: Fu S, Xu Y, Li C, Li Y, Wu J, Zhou X. Rice Stripe Virus Interferes with S-acylation of Remorin and Induces Its Autophagic Degradation to Facilitate Virus Infection. Mol Plant. 2018 Feb 5;11(2):269 - 287. doi: 10.1016 / j.molp.2017.11.011. Epub 2017 Dec 9. PMID: 29229567.).

[0150] The results showed that: the expression of wild type (WT) NbREM1 protein inhibits the intercellular movement of the virus, showing smaller fluorescent dots ( Figure 5 in A); after point mutation, the area of the fluorescent dots on the leaves is larger than that of WT ( Figure 5 in B), indicating that the ability of the NbREM1 protein after point mutation to inhibit the intercellular movement of the virus is weakened. Therefore, the analysis of the three-dimensional crystal structure of the plant remorin protein NbREM1.1 from amino acid positions 97 to 207 at the carboxyl terminus has important scientific significance for revealing the interaction mechanism between rice and RSV, and provides important structural guidance for the prevention and control of rice stripe disease in the later stage.

[0151] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. NbREM1.1 carboxyl-terminal region protein crystal, characterized in that, The crystal comprises the amino acid sequence of the carboxyl-terminal region of the NbREM1.1 protein; the crystal has a space group of P21, and the unit cell parameters are: a = 26.6 Å, b = 91.1 Å, c = 128.7 Å, α = γ = 90°, and β = 95.8°; The carboxyl-terminal region of NbREM1.1 is the amino acids at positions 97 to 207 at the carboxyl terminus of the NbREM1.1 protein, and the amino acid sequence is as shown in SEQ ID NO: 1, and a K54A mutation is made at position 54 of the amino acid sequence; There are two protein molecules in one crystallographic asymmetric unit of the three-dimensional structure of the crystal; The two carboxyl-terminal regions of NbREM1.1 form a long and straight single helix, which together form an antiparallel helix bundle; The method for preparing the protein crystal of the carboxyl-terminal region of NbREM1.1 comprises the following steps: Concentrate the purified protein solution of the carboxyl-terminal region of NbREM1.1 to 8 - 15 mg / mL, and form the protein crystal of the carboxyl-terminal region of NbREM1.1 by the vapor diffusion method at a temperature of 4 - 20 °C; Among them, the protein crystal of the carboxyl-terminal region of NbREM1.1 grows in a solution containing the protein solution and the buffer in a volume ratio of 1:1, and the buffer is HEPES at pH 7.5, 0.2 M ammonium acetate, 25% PEG3350, and 0.5% n-octyl-β-D-glucopyranoside.

2. The preparation method according to claim 1, characterized in that, The protein solution of the carboxyl-terminal region of NbREM1.1 is concentrated to 11 mg / mL; the temperature is 16 °C.

3. The application of the protein crystal of the carboxyl-terminal region of NbREM1.1 as described in claim 1 in the prevention and control of rice stripe disease.

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