ZmPAR1 gene and application thereof

Abstract

The invention relates to the technical field of corn, in particular to a ZmPAR1 gene and application thereof. A coding gene of nucleoside kinase is found in a corn genome. The nucleoside kinase is obtained by carrying out in-vitro recombinant expression on the coding gene. Tests find that the nucleoside kinase has binding activity to various nucleoside substrates and can catalyze nucleoside to synthesize monophosphate nucleotide. The gene affects the plant height, the leaf included angle, the ear height, the leaf length and the leaf width of corn. A new corn material can be obtained by knocking down the gene or overexpressing the gene, and technical support is provided for cultivation of the new corn material and construction of germplasm resources.

Description

Technical Field

[0001] The present application relates to the technical field of corn, and in particular to the ZmPAR1 gene and its application. Background Art

[0002] Corn is an important dual-purpose crop for food and feed, and also an important industrial raw material. As the density of corn planting increases, the leaves of corn plants block each other, which can easily trigger the shade avoidance syndrome of the plants, leading to increased plant height, thinner stems, narrower leaves, and premature aging. By improving the plant type of corn, including plant height, leaf angle, leaf length, leaf width, etc., the occurrence of shade avoidance syndrome can be reduced to a certain extent. The improvement of corn plant type depends on the cloning of plant type genes. However, the number of known plant type genes is limited, and more new genetic resources are still needed to cultivate varieties with ideal plant types. Summary of the invention

[0003] The inventors of the present application discovered a gene encoding a nucleoside kinase in the maize genome. The gene is located at 189856343-189862099bp on chromosome 1 of the genome, and the reference genome number is Zm-B73-REFERENCE-GRAMENE-4.0 (database (https: / / maizegdb.org / )). The nucleoside kinase was obtained by in vitro recombinant expression of the encoding gene. Tests have found that the nucleoside kinase has binding activity to a variety of nucleoside substrates and can catalyze the synthesis of monophosphate nucleotides from nucleosides.

[0004] In addition, the inventors of the present application also found that the gene affects the plant height, leaf angle, ear height, leaf length and leaf width of corn. New corn materials can be obtained by knocking down or overexpressing the gene, providing technical support for the cultivation of new corn materials and the construction of germplasm resources.

[0005] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0006] In a first aspect, the embodiments disclose an enzyme preparation, which comprises the protein shown in SEQ ID NO: 1 as an active ingredient.

[0007] In a second aspect, the embodiment discloses a method for preparing a ZmPAR1 protein. The method comprises providing an expression vector comprising a nucleotide sequence encoding the ZmPAR1 protein, transforming the expression vector into Escherichia coli to obtain an expression strain, and obtaining the ZmPAR1 protein from the culture fluid of the expression strain. The nucleotide sequence encoding the ZmPAR1 protein is shown in SEQ ID NO:2.

[0008] In a third aspect, the embodiments disclose a method for synthesizing monophosphate nucleotides in vitro, comprising the step of incubating a protein as shown in SEQ ID NO: 1 with nucleosides in vitro.

[0009] In a fourth aspect, the embodiment discloses a CRISPR vector, comprising a guide sequence targeting the maize ZmPAR1 gene, and an expression sequence of Cas9. The ZmPAR1 gene is located at 189856343 to 189862099 bp of chromosome 1 of the maize genome Zm-B73-REFERENCE-GRAMENE-4.0. The guide sequence is shown in SEQ ID NO: 6. The expression sequence of Cas9 is shown in SEQ ID NO: 5.

[0010] In a fifth aspect, the embodiment discloses a method for deleting the ZmPAR1 gene of corn, comprising the step of introducing the CRISPR vector described in the fourth aspect into a wild corn plant. The ZmPAR1 gene is located at position 189856343 to 189862099 bp on chromosome 1 of the corn genome Zm-B73-REFERENCE-GRAMENE-4.0.

[0011] In a sixth aspect, the embodiment discloses a method for improving corn phenotype, comprising the step of introducing the CRISPR vector described in the fourth aspect into a wild corn plant. The improved corn phenotype comprises reducing plant height, increasing leaf angle, reducing ear height, shortening leaf length, and reducing leaf width.

[0012] In a seventh aspect, the embodiments disclose an overexpression vector comprising a nucleic acid molecule as shown in SEQ ID NO:2.

[0013] In an eighth aspect, the embodiments disclose a method for overexpressing the ZmPAR1 gene, comprising the step of introducing the overexpression vector described in the seventh aspect into a wild corn plant.

[0014] In the ninth aspect, the embodiment discloses a method for improving corn phenotype, comprising the step of introducing the overexpression vector described in the first aspect into a wild corn plant. The modified corn phenotype comprises reducing leaf angle, increasing plant height and ear height, and increasing leaf length and leaf width. The ZmPAR1 gene is located at 189856343 to 189862099 bp on chromosome 1 of the corn genome Zm-B73-REFERENCE-GRAMENE-4.0.

[0015] In a tenth aspect, the embodiment discloses the use of the protein as shown in SEQ ID NO: 1 or the nucleic acid molecule as shown in SEQ ID NO: 2. The use is selected from:

[0016] 1) Preparation of nucleoside kinase:

[0017] 2) Preparation of catalysts for in vitro synthesis of monophosphate nucleotides;

[0018] 3) preparing a vector for knocking down the ZmPAR1 gene, wherein the ZmPAR1 gene is located at position 189856343 to 189862099 bp on chromosome 1 of the maize genome Zm-B73-REFERENCE-GRAMENE-4.0;

[0019] 4) Improving the first corn phenotype, wherein the improving the first corn phenotype comprises reducing plant height, increasing leaf angle, reducing ear height, shortening leaf length, and reducing leaf width.

[0020] 5) preparing a vector for overexpressing the ZmPAR1 gene, wherein the ZmPAR1 gene is located at position 189856343 to 189862099 bp on chromosome 1 of the maize genome Zm-B73-REFERENCE-GRAMENE-4.0;

[0021] 6) improving the second corn phenotype, wherein the improving the second corn phenotype comprises reducing the leaf angle, increasing the plant height and ear height, and increasing the leaf length and leaf width;

[0022] 7) Prepare new corn materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The prokaryotic expression results of the ZmPAR1 protein provided in the examples. Figure 1 A is a Coomassie staining image, Crude represents crude extract, Unbound represents flow-through, 5thwash represents the fifth wash, E1 represents the first tube of eluate, E2 represents the second tube of eluate, and the arrow indicates the position of the fusion protein. Figure 1 B is the development picture of Western blot. After development, the target protein is mixed in the crude extract, the flow-through and the second tube of eluate, but not in the fifth washing solution and the first tube of eluate. The arrow indicates the position of the fusion protein after development.

[0024] Figure 2 The LC-MS spectrum of the ZmPAR1 protein provided in the examples as an affinity enzyme test for nucleosides. Figure 2 A is the AMP, ADP and ATP signals detected by LC-MS before the reaction and after the reaction at 30°C for 50 min in the presence of 100 μM ATP using 2 μg PAR1 protein and 50 μM adenosine. The horizontal axis represents the peak time and the vertical axis represents the signal response value. Figure 2 B is the incubation of PAR1 with inosine, and LC-MS detected the IMP, ADP and ATP signals before and after 50 min of reaction. Figure 2C is the incubation of PAR1 with uridine, and LC-MS detects the UMP, ADP and ATP signals before and after 50 min of reaction. Figure 2 D is the incubation of PAR1 with cytidine, and LC-MS detects the CMP, ADP and ATP signals before and after 50 min of reaction. Figure 2 E is the incubation of PAR1 with guanosine, and LC-MS detected the GMP, ADP and ATP signals before and after 50 min of reaction.

[0025] Figure 3 The results of the kinetic constant determination of inosine (A) and uridine (B) provided in the examples. The abscissa represents the substrate concentration, the left ordinate represents the reaction rate, and the right ordinate represents the ratio of substrate to reaction rate. The kinetic curve graph is drawn using the software Graph, with an error of SD, and three biological replicates for each substrate concentration.

[0026] Figure 4 The gene sequence (A) and protein sequence schematic diagram (B) of the cr-1 mutant, cr-2 mutant and wild-type strain (WT) provided in the examples.

[0027] Figure 5 This is a mature phenotype diagram of the cr-1 mutant, cr-2 mutant and wild-type strain (WT) provided in the examples, scale bar, 10 cm.

[0028] Figure 6 The phenotypic measurement statistics of the cr-1 mutant, cr-2 mutant and wild strain (WT) provided in the examples. The phenotypes include: plant height (plantheight), ear height (earheight), leaf length (leaflength), leaf width (leafwidth) and leaf angle (leafangle). Asterisks indicate statistical differences, ***, p<0.001; *, p<0.05; ns, p>0.05.

[0029] Figure 7 Fruit pictures and fruit phenotype statistics of the cr-1 mutant, cr-2 mutant and wild strain (WT) provided in the examples. Phenotypes include ear length and ear diameter. Scale bar, 2 cm. Asterisks indicate statistical differences, ***, p<0.001.

[0030] Figure 8 Western blot detection diagram of the inbred line B104 provided in the examples, and the overexpression transgenic lines PAR1-Flag-24, PAR1-Flag-21, PAR1-Flag-20, PAR1-Flag-8 and PAR1-Flag-5.

[0031] Fig. 9 OE provided for the embodiment 21 strain, OE 24 Fruit pictures and fruit phenotype statistics of strains and wild strains (WT). Phenotypes include: hundred-grain weight, grain length, grain width, ear length and ear diameter, kernel row numbers. Scale bar, 2 cm. Asterisks indicate statistical differences, **, p<0.01; ns, p>0.05. Error is SD. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0033] Preparation of ZmPAR1 protein

[0034] Some embodiments provide a method for preparing a ZmPAR1 protein. The method comprises providing an expression vector comprising a nucleotide sequence encoding the ZmPAR1 protein, transforming the expression vector into Escherichia coli to obtain an expression strain, and obtaining the ZmPAR1 protein from the culture fluid of the expression strain. The nucleotide sequence encoding the ZmPAR1 protein is shown in SEQ ID NO: 1.

[0035] In some embodiments, the expression vector is a pET28a-MBP-T7-6×HIS plasmid (CB113269532, Beijing Solebio) cloned with the coding sequence of ZmPAR1 (shown in SEQ ID NO: 2).

[0036] In some embodiments, the method for preparing ZmPAR1 protein comprises:

[0037] 1) A DNA molecule as shown in SEQ ID NO: 2 was amplified by PCR, and the DNA molecule was connected with the pET28a-MBP-T7-6×HIS restriction fragment by one-step cloning (Clon Express II One Step Cloning Kit, C112-01). The obtained ligated fragment was transformed into Escherichia coli, positive clones were screened, and expression plasmids were extracted from the culture of positive clones.

[0038] 2) The expression plasmid was transferred into RIL cells (BL21-CodonPlus(DE3)-RIL, ZY1025, Shanghai Zeye Biotechnology Co., Ltd.), positive clones were screened, and inoculated into LB liquid culture medium containing Kan antibiotic at a volume ratio of 1:100, and cultured at 37°C and 180 rpm for 4 h. The OD600 value was approximately 0.8-1.0.

[0039] 3) Add the inducer IPTG to the step 2) to a final concentration of 0.2 mM, precool the shaker to 16°C, 180 r / min, and shake and culture for 16-18 h. Centrifuge the fermentation broth at 5000 r / min for 10 min to collect the bacteria, add 20 mL of lysis solution (QC25-05102, Qincheng Bio), shake vigorously to suspend the bacteria, and add the protease inhibitor PMSF at a final concentration of 0.1 mM to prevent protein degradation.

[0040] 4) Use a high-pressure crusher to break the protein. 1000mPa, 4℃, high-pressure crushing 2-3 times. Transfer the bacterial solution to a 50mL centrifuge tube, centrifuge at 14000r / min, 4℃ for 1h, and take the supernatant for use.

[0041] 5) The supernatant obtained in step 4) was purified using an MBP-tagged affinity chromatography column (filler: Amylose Resin High Flow, NEB, E8022S), and the sugar substances therein were removed by chromatography to obtain a ZmPAR1 fusion protein with a C-terminal MBP tag, with an expected size of 86.4 kDa.

[0042] like Figure 1 As shown in A, a protein of the expected size was found in the second eluate (E2).

[0043] like Figure 1 As shown in B, after the MBP antibody (ABclonal, catalog number AE016) was incubated with the ZmPAR1 protein for color development, the target protein was detected in the crude extract (Crude), the flow-through (Unbound) and the E2 lane.

[0044] ZmPAR1 protein as a nucleoside affinity enzyme

[0045] The test example uses nucleosides as substrates to characterize the enzymatic activity of ZmPAR1 protein. In this test example, five different nucleosides (adenosine, inosine, uridine, cytidine and guanosine) are used as substrates. After the ZmPAR1 protein is incubated with the five nucleosides in vitro for 50 minutes, the changes in the reaction products are detected. After the ZmPAR1 protease activity reaction, adenosine monophosphate (AMP), inosine monophosphate (IMP), uridine monophosphate (UMP), cytidine monophosphate (CMP) and guanosine monophosphate (GMP) can be generated accordingly. This enzyme activity test uses ATP to provide energy, consuming one molecule of ATP to produce one molecule of ADP.

[0046] Configuration of buffer stock solution: 1M NH4HCO3 (pH=7.5), 0.1M MgCl2, 0.5M KCl, 1M DTT (dithiothreitol), 10mM ATP.

[0047] Configuration of substrate stock solution: 500 mM inosine, 500 mM uridine, 500 mM cytidine, 500 mM adenosine, 50 mM guanosine.

[0048] After protease was expressed and purified in prokaryotes, the protein concentration was adjusted to 20 ng / μL using the BSA standard curve detection method.

[0049] The enzyme activity test steps:

[0050] Prepare the following components in a 250μL PCR tube: 40mM NH4HCO3, 2mM MgCl2, 10mM KCl, 1mM DTT and 1mM ATP buffer; 2μg protein; 100μM substrate. The total reaction volume is 100μL. Incubate at 30℃ for 50min. Add an equal volume of chloroform / isoamyl alcohol (1:1), mix by inversion or oscillation, and centrifuge at low speed for 10min. Pipette the supernatant and extract again. After high-speed centrifugation for 10min, pipette the supernatant. After diluting twice the volume, dispense 100μL into a mass spectrometer bottle for LC-MS detection.

[0051] like Figure 2 As shown, the monophosphate product AMP ( Figure 2 A) IMP( Figure 2 B) UMP Figure 2 C) CMP Figure 2 D) GMP Figure 2 E) and the product of ATP consumption, ADP ( Figure 2 AE), indicating that ZmPAR1 has phosphorylation activity on all five substrates, further indicating that ZmPAR1 has broad nucleoside substrate activity.

[0052] ZmPAR1 protein has stronger catalytic activity towards inosine and uridine

[0053] Test Example By measuring the kinetic constants of inosine and uridine, it was found that ZmPAR1 has a high catalytic activity for the substrates inosine and uridine.

[0054] For the determination of the kinetic constants of inosine and uridine, the amount of monophosphate nucleotide products was used to reflect the enzyme rate. The concentration and volume of the enzyme reaction buffer remained unchanged, and the substrate concentration was set into six different gradients. Three replicates were set for each gradient. The substrate concentration of inosine was set as follows: 1mM, 2mM, 5mM, 10mM, 15mM and 20mM. The substrate concentration of uridine was set as follows: 5mM, 20mM, 40mM, 60mM, 80mM and 100mM. The reaction buffer included: 40mM NH4HCO3, 2mM MgCl2, 10mM KCl, 1mM DTT and 1mM ATP buffer. First, prepare the reaction solution containing 2μg of protease and precool it on ice. Then add the substrate sample to the RNase-free eight-tube strip (the eight-tube strip was placed on an ice box), and then add the precooled protease reaction solution. The total reaction volume was 100μL. Centrifuge and mix for 1min. Use a PCR instrument to control the temperature, and the reaction time is divided into 3min and 6min. After the reaction is completed, put it on ice immediately to keep it low. After 2min, add phenol-chloroform for the first extraction. Invert it upside down several times to mix, centrifuge it at low speed for 10min, aspirate 80μL of the supernatant, and add an equal volume of phenol-chloroform for the second extraction, centrifuge it at high speed for 10min, and aspirate 60μL of the supernatant. Finally, add an equal volume of double distilled water to dilute it. Take 50~80μL of the diluted sample for LC-MS detection.

[0055] The quantification of the product was determined using the standard curve method. First, the substance to be tested was diluted to the following seven concentrations: 10ppb, 20ppb, 50ppb, 100ppb, 200ppb, 500ppb, 1000ppb and 2000ppb. LC-MS detected the peak area of ​​the corresponding concentration, and the standard curve formula was generated with the peak area as the horizontal axis and the concentration as the vertical axis. R 2 The standard curve was greater than 0.99. The concentration of the substance was then calculated based on the measured peak area.

[0056] like Figure 3 As shown in A, the K of inosine m The value is 1.39mM, and the turnover rate of inosine K cat 0.054s -1 , the catalytic efficiency of inosine K cat / K m 38.84M -1 s -1 .like Figure 3 As shown in B, the K m The value is 11.33mM, and the turnover rate of uridine Kcat 0.171s -1 , the catalytic efficiency K of uridine cat / K m 15.09M -1 s -1 This indicates that ZmPAR1 preferentially binds to inosine and has a higher catalytic efficiency for inosine, but the substrate turnover rate of uridine is higher than that of inosine.

[0057] Application of ZmPAR1 (knockdown)

[0058] 1. Preparation of ZmPAR1 knockdown editing plasmid

[0059] Referring to “High-Throughput CRISPR / Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize,The Plant Cell, Vol. 32: 1397~1413, May 2020”, the sgRNA shown in SEQ ID NO: 6 was cloned into the plasmid pCXB053 (Li, C., Liu, C., Qi, X., Wu, Y., Fei, X., Mao, L., Cheng, B., Li, X., and Xie, C. (2017). RNA-guided Cas9 as an in vivo desired-target mutator in maize. Plant Biotechnol. J. 15: 1566–1576.) by enzyme digestion and ligation to obtain the ZmPAR1 knockdown editing plasmid.

[0060] 2. Construction of ZmPAR1 knockdown maize strains

[0061] The obtained ZmPAR1 knockdown editing plasmid was transformed into Agrobacterium EHA105 (Agrobacterium tumefaciens, Beijing Tianenze Gene Technology Co., Ltd., 140383), and positive clones were screened from the transformants. The positive clones were used to infect the immature embryo callus tissue of corn KN5585 (Weimi Biotechnology), and the regenerated plants were cultured by herbicide screening to obtain transgenic regenerated plants, thereby obtaining cr-1 mutant strains and cr-2 mutant strains.

[0062] 3. Sequencing of cr-1 mutants and cr-2 mutants

[0063] like Figure 4As shown in A and 4B, the cr-1 mutant lacks 2 bases, resulting in premature termination of transcription, ultimately encoding 193 amino acids. The cr-2 mutant has a 1-base insertion, which results in premature termination of transcription, ultimately encoding 194 amino acids. Although both cr-1 and cr-2 lineages result in premature transcription termination, the first domain of the ZmPAR1 protein is partially retained, and both cr-1 and cr-2 mutants lack the D2 domain.

[0064] 4. Phenotypic observation of cr-1 mutant and cr-2 mutant

[0065] like Figure 5 and 6 As shown, the plant height and ear height of the cr-1 mutant were significantly lower than those of the wild type ("WT", KN5585, 24.4%, 25.3%), and the leaf length and leaf width were significantly shorter than those of the wild type (7.4%, 22.2%). The plant height and ear height of the cr-2 mutant were significantly lower than those of the wild type (49.5%, 59.7%), and the leaf length and leaf width were significantly shorter than those of the wild type (37.9%, 38.8%). In addition, the leaf angle of the cr-1 mutant was significantly greater than that of the wild type (12.6%), while the leaf angle of cr-2 did not change significantly compared with the wild type.

[0066] like Figure 7 As shown, the ear length of the cr-1 mutant was significantly lower than that of the wild type ("WT" in the figure, 48.4%), and the ear diameter was significantly lower than that of the wild type (16.1%). The ear length of the cr-2 mutant was significantly lower than that of the wild type (28.2%), and the ear diameter was significantly lower than that of the wild type (12.1%). Since the cr-1 mutant and the cr-2 mutant still contain some functional domains, it is speculated that the cr-1 mutant and the cr-2 mutant may belong to weak mutation alleles.

[0067] It can be seen from this that knocking down ZmPAR1 can reduce corn plant height, increase corn leaf angle, reduce corn cob height, shorten corn leaf length, and reduce corn leaf width. Application of ZmPAR1 (overexpression)

[0068] 1. Construction of ZmPAR1 overexpression vector

[0069] The promoter lac promoter in plasmid pCAMBIA3300 (SnapGene) was replaced with Ubi promoter (SEQ ID NO: 3) to obtain plasmid WMV014.

[0070] A fusion DNA molecule containing maize ubiquitin promoter, ZmPAR1 coding sequence and Flag tag (pUBI::ZmPar1-Flag, SEQ ID NO: 4) was synthesized.

[0071] The fusion DNA molecule was connected to the linearized WMV014 fragment, the connection product was transformed into Escherichia coli, positive clones were screened from the transformants, and the ZmPAR1 overexpression vector was extracted from the culture of the positive clones.

[0072] 2. Construction of ZmPAR1 overexpression maize strains

[0073] The obtained ZmPAR1 overexpression vector was transformed into Agrobacterium tumefaciens (Agrobacterium tumefaciens, Beijing Tianenze Gene Technology Co., Ltd., 140383), and positive clones were screened from the transformants. The positive clones were used to infect the immature embryo callus of corn B104 inbred line (Weimi Biotechnology), and the regenerated plants were cultured and screened with herbicides to obtain transgenic regenerated plants. Five overexpression transgenic lines were obtained: PAR1-Flag-24, PAR1-Flag-21, PAR1-Flag-20, PAR1-Flag-8 and PAR1-Flag-5.

[0074] 3. Western Blot Detection

[0075] The ZmPAR1 protein levels of five overexpressing maize lines were detected by Western Blot.

[0076] like Figure 8 As shown, compared with the maize B104 inbred line, the five overexpression transgenic lines of PAR1-Flag-21 (abbreviated as OE 21 ) and PAR1-Flag-24 (abbreviated as OE 24 ) had significantly increased ZmPAR1 protein level.

[0077] 4. Field experiment

[0078] like Fig. 9 As shown, compared with the wild-type maize strain ("WT", B104), OE 21 Plant height increased by 8.9%, OE 21 The ear height increased by 22.9%, the leaf length of OE21 increased by 7.9%, and the 21 Leaf width increased by 10.3%, OE 21 The leaf angle decreased by 7.2%, OE 21 The ear diameter increased by 5.0%.

[0079] like Fig. 9 As shown, compared with the wild-type maize strain ("WT" in the figure), OE 24 Plant height increased by 7.1%, ear height increased by 10.9% in OE24, and OE 24 Leaf length increased by 3.9%, OE 24 The ear diameter increased by 3.1%.

[0080] It can be seen that by overexpressing the ZmPAR1 gene, reducing the leaf angle, increasing the plant height and ear height, and increasing the leaf length and leaf width, technical support is provided for the breeding of corn varieties.

[0081] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. An enzyme preparation, comprising the protein shown in SEQ ID NO: 1 as an active ingredient.

2. A method for preparing ZmPAR1 protein, comprising: Providing an expression vector comprising a nucleotide sequence encoding the ZmPAR1 protein, wherein the nucleotide sequence encoding the ZmPAR1 protein is shown in SEQ ID NO: 2; Transforming the expression vector into Escherichia coli to obtain an expression strain; The ZmPAR1 protein is obtained from the culture solution of the expression strain.

3. A method for synthesizing monophosphate nucleotides in vitro, comprising the step of incubating the protein shown in SEQ ID NO: 1 with nucleosides in vitro.

4. A CRISPR vector, comprising a guide sequence targeting the maize ZmPAR1 gene and an expression sequence of Cas9; the ZmPAR1 gene is located at 189856343 to 189862099 bp of chromosome 1 of the maize genome Zm-B73-REFERENCE-GRAMENE-4.0; the guide sequence is shown in SEQ ID NO:6; the expression sequence of Cas9 is shown in SEQ ID NO:

5.

5. A method for deleting the maize ZmPAR1 gene, comprising the step of introducing the CRISPR vector of claim 4 into a wild maize plant, wherein the ZmPAR1 gene is located at position 189856343 to 189862099 bp on chromosome 1 of the maize genome Zm-B73-REFERENCE-GRAMENE-4.

0.

6. A method for improving corn phenotype, comprising the step of introducing the CRISPR vector of claim 4 into a wild corn plant, wherein the improved corn phenotype comprises reducing plant height, increasing leaf angle, reducing ear height, shortening leaf length, and reducing leaf width.

7. An overexpression vector comprising the nucleic acid molecule shown in SEQ ID NO:

2.

8. A method for overexpressing the ZmPAR1 gene, comprising the step of introducing the overexpression vector according to claim 7 into wild corn plants.

9. A method for improving corn phenotype, comprising the step of introducing the overexpression vector of claim 7 into wild corn plants, wherein the modified corn phenotype comprises reducing leaf angle, increasing plant height and ear height, increasing leaf length and leaf width, and the ZmPAR1 gene is located at position 189856343 to 189862099 bp on chromosome 1 of the corn genome Zm-B73-REFERENCE-GRAMENE-4.

0.

10. Use of the protein as shown in SEQ ID NO: 1 or the nucleic acid molecule as shown in SEQ ID NO: 2, wherein the use is selected from: 1) preparing nucleoside kinase; 2) Preparation of catalysts for in vitro synthesis of monophosphate nucleotides; 3) preparing a vector for knocking down the ZmPAR1 gene, wherein the ZmPAR1 gene is located at position 189856343 to 189862099 bp on chromosome 1 of the maize genome Zm-B73-REFERENCE-GRAMENE-4.0; 4) Improving the first corn phenotype, wherein the improving the first corn phenotype comprises reducing plant height, increasing leaf angle, reducing ear height, shortening leaf length, and reducing leaf width. 5) preparing a vector for overexpressing the ZmPAR1 gene, wherein the ZmPAR1 gene is located at position 189856343 to 189862099 bp on chromosome 1 of the maize genome Zm-B73-REFERENCE-GRAMENE-4.0; 6) improving the second corn phenotype, wherein the improving the second corn phenotype comprises reducing the leaf angle, increasing the plant height and ear height, and increasing the leaf length and leaf width; 7) Prepare new corn materials.

Images