Identification of rice NAL1 protein conformation and use thereof

Abstract

Provided are the identification of a rice NAL1 protein conformation and the use thereof. On the basis of the structural characteristics of a protein, by means of modifying the polarity of the amino acid at position 240 in indica NAL1, not only is the conformational opening of indica NAL1 realized, but indica NAL1 is also provided with new functions of positively regulating plant height and degrading a substrate protein. Further provided is a method for altering the indica rice NAL1 protein conformation, which method comprises disrupting the salt bridge between the amino acid at position 233 and the amino acid at position 240 by means of amino acid replacement, thereby altering the indica rice NAL1 protein conformation, wherein the indica rice NAL1 protein has a nucleotide sequence as shown in SEQ ID NO: 4.

Description

Rice NAL1 protein conformation identification and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to rice NAL1 protein conformation identification and application thereof, in particular to the precise directional modification of the molecular conformation of NAL1 by taking advantage of the difference in the molecular conformation of NAL1 between japonica and indica, so as to improve the plant type and yield of rice. BACKGROUND

[0002] The three elements of rice yield, panicle number, grain number per panicle and 1000-grain weight, regulate the final yield of rice from different dimensions. Leaf is the main place for photosynthesis, and the leaf width, spatial distribution of leaf and other factors directly affect the light receiving area of the whole plant, and the leaf thickness and other factors have an important influence on the photosynthetic efficiency per unit area.

[0003] The currently cloned leaf width regulating genes include NAL1, NAL2, NAL7 and NAL9, among which NARROW LEAF 1 (NAL1) is closely related to the nitrogen and chlorophyll content of leaf, the photosynthetic efficiency per unit leaf area and the like (Takai et al., 2013; Zhang et al., 2014; Wang et al., 2015). Gly-257-Asp single amino acid mutation or deletion of 10 amino acids from position 375 to 384 of NAL1 will cause the leaf of rice to become narrow, the plant to become dwarf, and the grain number per panicle to decrease (Qi et al., 2008; Huang et al., 2018). The NAL1 protein sequence has a trypsin-like domain, and it has been found that it can reduce the accumulation of FZP (rice panicle development gene) protein, thereby promoting the development of secondary branch of rice panicle, and plays an important role in rice domestication (Huang et al., 2018). It has also been found that NAL1 can promote the protein degradation of TPR2, regulate the histone acetylation level of genes related to hormone signal pathway, realize the expression regulation of these genes, and ultimately affect the growth and development of rice plants and rice yield (Li et al., 2023). There are four SNPs in the exon of NAL1, and three amino acid mutations (Xu et al., 2015).

[0004] However, the existing research only verifies the protease activity of NAL1 and its influence on the downstream target protein, and does not start from the protein structure to explore why the natural variation of NAL1 can cause great difference in its function, nor does it imagine that the precise regulation of rice development can be realized by regulating the conformation of NAL1 protein, so as to achieve the purpose of increasing yield.

[0005] SUMMARY

[0006] Japonica and indica are two subspecies of rice, each evolved independently. In breeding, it is found that there is heterosis in the genomes of indica and japonica, and there are different natural variations of the same gene between the two subspecies. Some variations show obvious advantages for special environment and planting mode in terms of nitrogen utilization rate, photosynthetic efficiency, plant type, yield, and even rice quality. Studies have shown that introducing japonica NAL1 into indica varieties can increase the rice yield of the latter by 18-30%, and at the same time improve the quality of rice. Phenotypic analysis of rice shows that the introduction of the above system makes the rice have wider flag leaves, fewer ineffective tillers, thicker panicle neck, and more grains per panicle, ultimately increasing the yield, in addition to the increase in overall milled rice rate and the decrease in chalkiness rate.

[0007] Haplotype analysis shows that the highly concentrated NAL1 at the 233rd amino acid is histidine in japonica varieties (referred to as japonica NAL1 in the present application, denoted by capital letter NAL1), which has a significant effect on flag leaf width and panicle morphology, while indica usually uses arginine at this position (referred to as indica NAL1 in the present application, denoted by small letter nal1) (Wang et al., 2015).

[0008] The present application first found that the 233rd amino acid of japonica NAL1 is histidine, which weakens the interaction between the two rings, making it have a unique open conformation, that is, the difference in the 233rd amino acid of NAL1 ultimately leads to the difference in the conformation of japonica / indica NAL1 protein. Further mutation of the 240th aspartic acid, which is the amino acid interacting with the ring at the 233rd amino acid, shows that indica NAL1 also has an open conformation through cryo-EM structure analysis, which determines the improvement direction for high-yield breeding based on NAL1 molecular modification.

[0009] The present application found that indica NAL1 only needs to change the 240th aspartic acid to alanine to obtain a similar protein conformation as japonica NAL1.

[0010] Specifically, the present application provides the following technical solutions:

[0011] In one aspect, the present application provides a method for identifying the conformation of rice NAL1 protein, which comprises the following steps:

[0012] a. expressing rice NAL1 protein;

[0013] b. purifying rice NAL1 protein, which comprises the following steps:

[0014] First, use a Ni-NTA affinity chromatography column;

[0015] Second, use an anion exchange column;

[0016] Third step, using phenyl sepharose hydrophobic interaction column;

[0017] Fourth step, using molecular sieve;

[0018] c. Using liquid ethane to complete the glassy quick-freezing of the sample;

[0019] d. Collecting electron microscope data of NAL1 protein on cryo-EM;

[0020] e. Collecting electron microscope data and processing to obtain the conformation of NAL1 protein.

[0021] In another aspect, the present application provides a method for changing the conformation of indica rice NAL1 protein, the method comprising disrupting the salt bridge between the amino acid at position 233 and the amino acid at position 240 by amino acid substitution, thereby changing the conformation of indica rice NAL1 protein, the nucleotide sequence of which is shown as SEQ ID NO: 4.

[0022] In the present application, examples of the group of amino acids with side chains having similar chemical properties include aliphatic amino acids, aromatic amino acids, and heterocyclic amino acids. Aliphatic amino acids include 1) neutral amino acids containing one amino-one carboxyl group: glycine, alanine, valine, leucine, and isoleucine; 2) hydroxyl-containing amino acids: serine and threonine; 3) sulfur-containing amino acids: cysteine and methionine; 4) amide-containing amino acids: asparagine and glutamine; 5) acidic amino acids containing one amino dicarboxyl group: aspartic acid and glutamic acid; 6) basic amino acids containing two amino-one carboxyl group: lysine and arginine. Aromatic amino acids include phenylalanine and tyrosine. Heterocyclic amino acids include tryptophan, histidine, and proline.

[0023] In some embodiments, the amino acid substitution is substitution of aspartic acid at position 240 with alanine.

[0024] In another aspect, the present application provides a protein sequence for regulating plant height, the aspartic acid at position 240 of the protein is substituted with alanine to disrupt the salt bridge between the amino acid at position 233 and the amino acid at position 240.

[0025] In some embodiments, the nucleotide sequence encoding the original protein is shown as SEQ ID NO: 4.

[0026] In some embodiments, the plant is rice.

[0027] In some embodiments, the plant is indica rice.

[0028] In some embodiments, the plant is Zhonghua 11.

[0029] In another aspect, the present application provides an isolated nucleotide sequence encoding the protein sequence as described above.

[0030] In another aspect, the present application provides an expression vector comprising a nucleotide sequence encoding the protein sequence as described above.

[0031] In some embodiments, the expression vector is a plant expression vector.

[0032] In another aspect, the present application provides a host cell comprising the expression vector as described above.

[0033] In another aspect, the present application provides a method for modulating plant plant height, comprising the step of introducing a nucleotide sequence encoding the protein sequence as described above or an expression vector or a host cell into a plant cell or tissue.

[0034] In another aspect, the present application provides use of the protein sequence as described above or a nucleotide sequence encoding the protein sequence in breeding a plant with increased plant height.

[0035] Definitions

[0036] Open conformation: NAL1 protein presents a split-open pattern of the double ring with one side as the axis.

[0037] Closed conformation: NAL1 protein presents a closed axisymmetric pattern of the double ring. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 shows the purification of Japonica and Indica rice NAL1 recombinant proteins. (A) 10% SDS-PAGE detects the induced amount of Japonica and Indica NAL1 in total protein and the protein amount in supernatant component; (B) Ni-NTA affinity chromatography, S: supernatant, FT: flow-through component, W: washing component, E: 250 mM imidazole elution component, Ec: addition of Usp-2 to remove N-terminal tag; (C) purification results of anion exchange chromatography column; (D) purification results of hydrophobic interaction chromatography column; (E) Indica and Japonica NAL1 proteins purified by molecular exclusion chromatography, the bands were identified by mass spectrometry as NAL1 protein itself.

[0039] Figure 2 shows negative staining electron micrographs of Japonica and Indica NAL1 recombinant proteins. NAL1 recombinant proteins were treated with negative staining, and the protein particle morphology under 120 kV electron microscope. Left panel: Japonica NAL1; right panel: Indica NAL1.

[0040] Figure 3 shows the closed and open states of Japonica NAL1 protein. Blue is the electron density of the closed state NAL1 molecule; yellow is the electron density of the open state NAL1 molecule. Japonica NAL1 protein has both closed and open states.

[0041] Figure 4 shows that indica NAL1 protein only exists in closed state. The figure shows the electron density of closed indica NAL1 molecule.

[0042] Figure 5 shows the position of amino acid at position 233 of japonica and indica NAL1 protein. The figure shows the region of amino acid at position 233 of the double ring contact surface of NAL1 structure. The left figure shows the salt bridge and hydrogen bond length related to histidine at position 233 of japonica NAL1 (NAL1); the right figure shows the salt bridge and hydrogen bond length related to arginine at position 233 of indica NAL1 (nal1).

[0043] Figure 6 shows the conformation of indica NAL1 cryo-EM protein particle after mutation of aspartic acid at position 240 to alanine. The red box highlights that the indica NAL1 has obtained open conformation after modification.

[0044] Figure 7A shows the results of phenotype comparison of plant height of rice materials of Zhonghua 11 (ZH11) and ZH11 NAL1 deletion mutant ZH11-CR, into which indica NAL1 gene (ZH11-CR / nal1), japonica NAL1 gene (ZH11-CR / NAL1) or indica nal1 D240A modified gene (ZH11-CR / nal1 D240A ) is introduced.

[0045] Figure 7B shows the results of data statistics of plant height of rice materials of Zhonghua 11 (ZH11) and ZH11 NAL1 deletion mutant ZH11-CR, into which indica NAL1 gene (ZH11-CR / nal1), japonica NAL1 gene (ZH11-CR / NAL1) or indica nal1 D240A modified gene (ZH11-CR / nal1 D240A ) is introduced.

[0046] Figure 8 shows the results of comparison of degradation ability of TPR2 protein by japonica NAL1, indica NAL1 (nal1) and indica NAL1 modified version nal1 D240A . DETAILED DESCRIPTION

[0047] In order to make the objects, technical solutions and advantages of the present application clearer, the present application is further described in detail below with reference to specific embodiments and the accompanying drawings.

[0048] Gene source: The NAL1 gene of rice is selected as the direct research object in the present application, and recombinant prokaryotic expression is carried out in Escherichia coli.

[0049] Strains used: E. coli; vector construction transformation using DH5α; protein induction expression using BL21(DE3).

[0050] The empty plasmid vector used and its name: pHUE

[0051] The expression vector used in the present application: NAL1-pHUE; nal1-pHUE

[0052] pHUE vector construction: The pHUE plasmid vector can make the N-terminal of the protein with His affinity tag and can completely remove the ubiquitin fusion protein by Usp2-45 protease, so as to finally realize the tag-free purification of the target protein.

[0053] Construction strategy: using the sequence information of the pHUE vector, designing recombinant connection upstream and downstream primers containing homologous arms on gene cloning, and the primer sequence information is as follows:

[0054] Upstream primer: tgttgcgcctccgcggtggaATGAAGCCTTCGGACGATAAG (SEQ ID NO: 5)

[0055] Downstream primer: cagccggatctaagcttgcgTCATTTCTCCAGGTCAAGGCT (SEQ ID NO: 6)

[0056] The vector pHUE is linearized using restriction endonuclease KpnI. The above recovered vector is connected with the PCR fragment to obtain the target recombinant expression plasmid.

[0057] Example 1 E. coli induced expression related to NAL1

[0058] The NAL1 recombinant expression strain taken out at-80℃ was streaked on an ampicillin (ampicillin concentration 100 mg / L) resistant LB solid plate in a clean bench, and incubated at 37℃ overnight. The next day, single colonies were picked into 2 mL sterile EP tubes (previously added ampicillin concentration 100 mg / L) resistant liquid LB medium), and incubated at 37℃, 200 rpm, for 4 h. Pour 80 mL (ampicillin concentration 100 mg / L) ampicillin resistant liquid LB sterile flask, and incubate at 37℃, 200 rpm, for 5 h. Add ampicillin antibiotic (v / v) to the sterilized 1 L LB liquid medium at 1:1000 (ampicillin concentration 100 mg / L), and add 10 mL of bacterial solution using a 5 mL pipette. Incubate at 37℃, 200 rpm, for 2.5 h, and take samples to measure OD value above 0.6 (total 6 L). Add 100 μL of 1M IPTG to each bottle, and incubate at 16℃, 200 rpm, overnight. The next day, collect the bacteria at 6000 rpm for 10 min.

[0059] Example 2 E. coli cell sonication and protein solution treatment

[0060] The collected cell pellet was suspended in 80 mL lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole and 1 mM phenylmethylsulfonyl fluoride PMSF) (using a 50 mL centrifuge tube, tighten the lid, mix well on a vortex shaker), pre-cooled to below 6°C on ice. Take a foam box full of crushed ice, insert the beaker completely into the ice, and use the ultrasonic cell disrupter to break the cell. The total ultrasonic time is 5 min, 5 sec each time, interval 40 sec, set the temperature not to exceed 15°C. Pre-cool the centrifuge and rotor to 4°C. After ultrasonic, pour the bacterial solution into a 50 mL centrifuge tube, pair up, 4°C, 20000 rpm, centrifuge for 40 min. Prepare two clean beakers, pre-cool on ice. Pour the supernatant after centrifugation into beaker one, use a clean syringe and 0.22 μm filter to filter the supernatant into beaker two.

[0061] Example 3 NAL1 protein purification steps

[0062] In order to avoid obtaining false protein components, solve the problem that the sample protein particle morphology is not uniform, so that it cannot be used for image acquisition of cryo-EM in the end, the inventors of the present application found through a large number of experiments that the following purification method can obtain high-purity NAL1 protein with uniform state.

[0063] First step: use Ni-NTA affinity chromatography column

[0064] 4°C, 20000 rpm, 50 min. The supernatant was directly applied to a 3 mL Ni-NTA agarose column (pre-equilibrated with one column volume of lysis buffer) and the flow-through was collected as FT1. The column was then washed with two column volumes of lysis buffer and the flow-through was collected as W1. The column was then washed with two column volumes of lysis buffer containing 25 mM imidazole and the flow-through was collected as W2. Finally, the protein was eluted using lysis buffer containing 250 mM imidazole and collected as FT2. The protein was enriched in FT2 as detected by SDS-PAGE. Preparation of dialysis bag (gloves on): Dialysis bags were boiled for 10 min in 2% (w / v) sodium bicarbonate and 1 mM EDTA (pH 8.0). They were rinsed three times with distilled water. They were boiled for 10 min in 1 mM EDTA (pH 8.0) and stored in clean 1 mM EDTA (pH 8.0) solution at 4°C after cooling. The dialysis bags were washed with distilled water inside and outside before use. The protein was dialyzed against buffer A with the addition of protease Usp2-45 (purified in our laboratory, the amino acid sequence is shown in SEQ ID NO: 1) at 4°C overnight. Dialysis: The protein solution was added to the dialysis bag using a 5 mL pipette. The dialysis solution was buffer A at 4°C with low speed stirring using a magnetic stirrer overnight or more than 4 h. The dialyzed protein solution was filtered using a 0.22 μm filter before column loading.

[0065] The following protein purification buffers (A-E) were prepared. After preparation, all buffers were filtered through a 0.22 μm filter and sonicated in an ultrasonic cleaner for more than 3 min to remove air bubbles.

[0066] Buffer A: 30 mM Tris-HCl, pH 7.5, 30 mM NaCl, 1 mM EDTA

[0067] Buffer B: 30 mM Tris-HCl, pH 7.5, 1 M NaCl, 1 mM EDTA

[0068] Buffer C: 20 mM MOPS / NaOH, pH 7.2, 100 mM NaCl

[0069] Buffer D: 20 mM Tris-HCl, pH 7.5, 1 M (NH4)2SO4,

[0070] Buffer E: 20 mM Tris-HCl, pH 7.5

[0071] MOPS: 3-(morpholinopropanesulfonic acid)

[0072] Second step: Anion exchange column Q

[0073] Connect Q column (5 mL x 2) to GE AKTA protein purification system. Perform pump washing with super-pure water using suction filtration and ultrasonic to remove air bubbles. Further replace ethanol in the column with 1.5 column volume of super-pure water. At the end, flush B pump with buffer B and flush A pump with buffer A. Set flow rate to 3 mL / min, equilibrate column volume to 25-30 mL, and pressure limit to 0.3 MPa. Load: adjust flow rate to 1.5 mL / min, and pressure limit to 0.3 MPa, load filtrate in beaker two to column, and use clean beaker to collect flow-through. At the end of loading, put A pump head back to buffer A, and set program:

[0074] Check the placement of the collector and the used tubes, and adjust to tube position No. 1. Discard the liquid in the waste tank, and use after flushing. Turn on the UV lamp, and perform the set program. At the end, select the eluted sample tube number according to the experienced conductivity range (3-7 mS / cm) and the peak shape of the UV curve, take 10 μL sample for SDS-PAGE detection. Concentrate the protein solution in the selected collector tube to a beaker, and add ammonium sulfate to a final concentration of 0.5 M, and dissolve and mix using a magnetic stirrer at 4 °C. Wash the used Q column with 2 M NaCl (suction filtration and ultrasonic through 0.22 μm filter) for two column volumes, set flow rate to 3 mL / min, flush volume to 50 mL, and pressure limit to 0.3 MPa. Then put A pump and B pump in ddH2O, and perform pump washing program. At the end, set flow rate to 3 mL / min, flush volume to 30 mL, and pressure limit to 0.3 MPa to perform water washing of the column. Then put A pump and B pump in 20% ethanol (suction filtration and ultrasonic through 0.22 μm filter), and perform pump washing program. Set flow rate to 3 mL / min, flush volume to 20 mL, and pressure limit to 0.3 MPa to replace water in the column. At this point, the Q column is used up.

[0075] Third step: use phenyl-sepharose hydrophobic interaction column

[0076] After dismounting the Q column, connect the phenyl hydrophobic column (5 mL x 2) to the AKTA protein purification system, and perform the pump washing operation using the ultra-pure water that is filtered and degassed by ultrasonic. Then, use the above-mentioned ultra-pure water to replace the ethanol in the column by 1.5 times the column volume. Place the A pump in buffer D and the B pump in buffer E, and perform the pump washing program. Note that the B pump is washed first, and then the A pump, so as to ensure that the A pump pipeline in the system does not stay in buffer E, preventing the negative impact on the sample column during sample loading. Set the flow rate to 1.5 mL / min, the volume to 30 mL, and the pressure limit to 0.3 MPa to balance the column. Use buffer D to clean the sample loading column (30 mL). Filter the above-mentioned protein solution dissolved in 0.5 M ammonium sulfate using a 0.22 μm filter. Load: adjust the flow rate to 1.5 mL / min, and the pressure limit to 0.3 MPa, and load the above-mentioned filtrate into the column, and use a clean beaker to collect the flow-through (to prevent air bubbles in the sample loading column from entering the protein purification column), and the pressure limit is 0.3 MPa. Set the program:

[0077] Check the placement of the collector and the test tubes used, and adjust to the position of tube No. 1. Discard the liquid in the waste tank, and use it after flushing. Turn on the ultraviolet lamp, and perform the set program. After completion, select the sample tube number for elution according to the experience interval of the conductivity (experience conductivity interval 3-7 mS / cm) and the peak shape of the UV curve, and take 10 μL of the sample for SDS-PAGE polyacrylamide gel electrophoresis detection. Place the A pump and the B pump in ddH2O, and perform the pump washing program. After completion, set the flow rate to 1 mL / min, the flushing volume to 30 mL, and the pressure limit to 0.3 MPa to perform water cleaning of the column. Then, place the A pump and the B pump in 30% isopropanol (filtered by 0.22 μm filter and ultrasonic), and perform the pump washing program. Set the flow rate to 1 mL / min, the flushing volume to 30 mL, and the pressure limit to 0.3 MPa. Then, place the A pump and the B pump in 20% ethanol (filtered by 0.22 μm filter and ultrasonic), and perform the pump washing program. Set the flow rate to 1 mL / min, the flushing volume to 30 mL, and the pressure limit to 0.3 MPa.

[0078] Fourth step: using molecular sieve Superdex 200 (24 ml)

[0079] Concentrate the collected target protein solution using an ultrafiltration concentration tube (100 kD molecular weight cutoff), 5500 rpm, 4°C, to a volume of less than 2 mL. After cleaning the A pump with buffer C, balance the Superdex 200 (120 mL) connected to the AKTA protein purification system at a flow rate of 0.7 mL / min and a pressure limit of 0.3 MPa. Check to ensure that the AKTA is in the "load" state, and slowly push the concentrated protein sample into the 2 mL sample loading ring using a syringe. Set the program:

[0080] Check the placement of the collector and the used test tubes, adjust to the 1st tube position. Discard the liquid in the waste cylinder, rinse and use. Turn on the UV lamp, execute the set program. Clean the used molecular sieve, set the flow rate 0.8 mL / min, rinse volume 130 mL, pressure limit 0.3 MPa. Then set the A pump in ddH2O, execute the pump cleaning program. After completion, set the A pump and B pump in 20% ethanol (filtered through 0.22 μm filter and ultrasonic), execute the pump cleaning program. Set the flow rate 1 mL / min, rinse volume 130 mL, pressure limit 0.3 MPa to replace the water in the column. Use SDS-PAGE polyacrylamide gel electrophoresis to detect the protein in the collection tube, collect the protein solution with a purity of about 90% or more, use ultrafiltration concentration tube (cut-off molecule 50 kD), 5500 rpm, 4°C, concentrate to a volume of less than 2 mL.

[0081] Example 4 Protein quantification and purity detection

[0082] Use distilled water and lens cleaning paper to clean the nucleic acid protein quantifier metal platform, take 1-1.5 μL of protein dissolved buffer C and drop it on the metal platform, cover the cover, and adjust to zero. Dry the metal platform, take 1-1.5 μL of the protein to be tested and drop it on the metal platform, cover the cover, and click to measure. Divide the measured value by the protein absorbance to get the approximate protein concentration.

[0083] The purity of the protein is detected by SDS-PAGE polyacrylamide gel electrophoresis.

[0084] Example 5 Protein freeze sample preparation, sample screening and data collection and processing

[0085] The particle morphology of NAL1 recombinant protein is evaluated by negative staining electron microscopy technology.

[0086] Freeze sample preparation and sample screening of NAL1 oligomers: hydrophilic treatment of the support grid by glow discharge, semi-automatic control by Vitrobot equipment, and completion of the glassy quick-freezing of the sample using liquid ethane. Observation of the glassy ice layer thickness and cleanliness of the sample on a 200 kV cryo-EM. Collection of electron micrographs of the sample on a 200 kV or 300 kV cryo-EM.

[0087] Electron microscopy data collection and analysis: Electron microscopy data of oligomers of full-length NAL1 of indica and japonica were collected on cryo-electron microscope. As many electron images as possible were collected. The particles were picked up automatically by the program of Relion, and then combined with manual picking up according to the actual situation. The particles with complete morphology were picked up from all electron images according to the classification results of two-dimensional classification, and two-dimensional classification was performed again. On the basis of two-dimensional classification, the good particle groups were selected for three-dimensional classification to obtain the initial structure model. Through repeated picking up of protein particles and repeated iterative optimization of structure density map, the cryo-electron structure of the protein particle was finally obtained.

[0088] Experimental results

[0089] NAL1, nal1 and nal1-D240A were subjected to multi-step chromatography to obtain purified proteins. The proteins were detected by 10% SDS-PAGE, and the results are shown in Figure 1, which shows that the present application obtains high-purity NAL1 protein with uniform state through multi-step chromatography, which is used for cryo-electron microscopy.

[0090] NAL1 recombinant protein stained with uranyl acetate was observed by 120kV electron microscope for protein particle morphology. Overall, indica NAL1 showed better and more uniform protein particles, and the results are shown in Figure 2.

[0091] Through 200kV cryo-electron microscopy data collection and analysis, the protein electron density of japonica NAL1 and indica NAL1 was obtained. Japonica NAL1 showed two states of opening and closing, and the results are shown in Figure 3. Indica NAL1 only showed a closed state, and the results are shown in Figure 4.

[0092] After model building and iterative optimization, the protein structures of japonica and indica NAL1 were finally analyzed. It is a double-ring hexamer structure, and the important differentiation site between japonica and indica at the 233rd amino acid is located on the contact surface of the double ring, which participates in the formation of hydrogen bonds and salt bridges between the double rings. Indica NAL1 adopts arginine at the 233rd position, which forms a salt bridge with the aspartic acid at the 240th position of the adjacent ring with higher interaction strength. In japonica NAL1, the 233rd amino acid is histidine, and the interaction is in the form of hydrogen bond. The comparison of bond length shows that the distance between the 233rd and 240th amino acids of the double ring in indica NAL1 is closer, and the results are shown in Figure 5.

[0093] In order to verify that the interaction between the rings has a regulating function on the "closed-opening" conformation of the hexamer, the 240th amino acid of indica NAL1 was mutated to alanine which does not form hydrogen bonds and salt bridges. The purified protein was subjected to cryo-electron microscopy data collection and processing, and the results of 2D classification directly showed that the modified indica NAL1 had an open conformation (Figure 6).

[0094] Phenotypic comparison of plant height was performed in the field for rice materials of Zhonghua 11 (ZH11), and ZH11-CR with a deletion mutation of NAL1 of ZH11 (ZH11-CR / nal1), japonica NAL1 gene (ZH11-CR / NAL1) or indica nal1 D240A ZH11-CR / nal1 D240A The plant height data statistics further showed that the average plant height of ZH11-CR / nal1 was 74.6 cm, the average plant height of ZH11-CR / NAL1 was 95.1 cm, and the average plant height of ZH11-CR / nal1 D240A ZH11 reached 93.4 cm, indicating that the indica nal1 D240A ZH11-CR / nal1 conferred the function of positive regulation of plant height by indica nal1 (Figure 7B).

[0095] It has been reported that japonica NAL1 can degrade TPR2 protein of rice (Li et al., 2023). Comparison of the degradation ability of japonica NAL1, indica NAL1 (nal1) and indica NAL1 modified version nal1 D240A TPR2 protein showed that the site-directed mutation of D240A enhanced the degradation ability of indica NAL1 to TPR2 (Figure 8), thereby relieving the inhibitory effect of the latter on plant type regulation, which has important application value for realizing the improvement of rice plant type.

[0096] Sequence

[0097] SEQ ID NO:1 Usp2-45 protease amino acid sequence

[0098] SEQ ID NO:2 pHUE plasmid vector sequence

[0099] SEQ ID NO:3 DNA coding sequence of NAL1

[0100] SEQ ID NO:4 DNA coding sequence of nal1

[0101] Reference

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[0106] Wang, Q. X., Xie, W. B., Xing, H. K., Yan, J., Meng, X. Z., Li, X. L., Fu, X. K., Xu, J. Y., Lian, X. M., Yu, S. B., Xing, Y. Z., and Wang, G. W. (2015). Genetic Architecture of Natural Variation in Rice Chlorophyll Content Revealed by a Genome-Wide Association Study. Molecular plant 8, 946-957.

[0107] Xu, J. L., Wang, Y., Zhang, F., Wu, Y., Zheng, T. Q., Wang, Y. H., Zhao, X. Q., Cui, Y. R., Chen, K., Zhang, Q., Lin, H. X., Li, J. Y., and Li, Z. K. (2015). SS1 (NAL1)- and SS2-Mediated Genetic Networks Underlying Source-Sink and Yield Traits in Rice (Oryza sativa L.). PloS one 10.

[0108] Zhang, G. H., Li, S. Y., Wang, L., Ye, W. J., Zeng, D. L., Rao, Y. C, Peng, Y. L, Hu, J., Yang, Y. L, Xu, J., Ren, D. Y., Gao, Z. Y., Zhu, L., Dong, G. J., Hu, X. M., Yan, M. X., Guo, L. B., Li, C. Y., and Qian, Q. (2014). LSCHL4 from Japonica Cultivar, Which Is Allelic to NAL1, Increases Yield of Indica Super Rice 93-11. Molecular plant 7, 1350-1364.

[0109] The above-described embodiments of the present application are merely specific embodiments, and the purpose, technical solutions and beneficial effects of the present application are further described in detail. It should be understood that the above-described embodiments are merely specific embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for conformational identification of a rice NAL1 protein, characterized in that, The method comprises the following steps: a. expressing the rice NAL1 protein; b. purifying the rice NAL1 protein, the purifying comprising the following steps: first step, using a Ni-NTA affinity column; second step, using an anion exchange column; third step, using a phenyl sepharose hydrophobic interaction column; fourth step, using a molecular sieve; c. completing the glassy quick-freezing of the sample using liquid ethane; d. collecting electron microscopy data of the NAL1 protein on a cryo-EM; e. collecting and processing the electron microscopy data to obtain the conformation of the NAL1 protein.

2. A method of changing the conformation of NAL1 protein of indica type rice, characterized in that, The method comprises destroying the salt bridge between the 233rd amino acid and the 240th amino acid by amino acid substitution, thereby changing the conformation of indica rice NAL1 protein, the nucleotide sequence of which is shown as SEQ ID NO:

4.

3. The method of claim 2, wherein, The amino acid substitution is replacing the 240th aspartic acid with alanine.

4. A protein sequence for regulating plant plant height, characterized in that, The 240th aspartic acid of the protein is replaced with alanine to destroy the salt bridge between the 233rd amino acid and the 240th amino acid, and the nucleotide sequence encoding the original protein is shown as SEQ ID NO:

4.

5. An isolated nucleotide sequence, characterized in that, The nucleotide sequence encodes the protein sequence according to claim 4.

6. An expression vector, characterized in that, The expression vector comprises the nucleotide sequence according to claim 5, and optionally, the expression vector is a plant expression vector.

7. A host cell characterized in that, The host cell comprises the expression vector according to claim 6.

8. A method for modulating plant plant height, characterized in that, The method comprises the step of introducing the host cell according to claim 7 into a plant cell or tissue.

9. Use of the protein sequence according to claim 4 or the nucleotide sequence according to claim 5 in cultivating plants with increased plant height.

10. The protein sequence according to claim 4 or the method according to claim 8 or the use according to claim 9, characterized in that, The plant is rice, preferably the plant is indica rice, and preferably the plant is Zhonghua 11.

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