Maize translational control tumor protein ZmTCTP and application thereof

Abstract

The invention discloses a corn translational control tumor protein ZmTCTP and an application thereof. Belongs to the technical field of gene engineering. Results of the invention show that PcENG3 protein of pratylenchus coffee interacts with corn ZmTCTP, growth and development of corn are inhibited by influencing energy synthesis of corn, and ZmTCTP as an important regulatory factor plays an important role in regulating disease resistance, cell differentiation and proliferation of corn. Research results provide a new basis for analysis of infection and pathogenic mechanisms of pratylenchus coffee on corn, provide a theoretical basis for corn root rot nematode resistance breeding, and provide potential candidate prevention and control targets for research and development of novel nematode prevention and control drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and more specifically to a maize translationally controlled tumor protein ZmTCTP and its applications. Background Art

[0002] Pratylenchus coffeae is a plant pathogenic nematode that is of great economic harm in agricultural production. It causes serious damage to the roots of various crops, resulting in significant economic losses to global agricultural production. Pratylenchus coffeae infects the root tissues, leading to the inhibition of crop root development and root rot, causing crop growth retardation and plant death. The mechanical damage caused by the nematode stylet to the crop roots makes the plants more susceptible to other pathogens, such as bacteria, fungi, and viruses, thereby increasing the risk of plants suffering from other diseases, which poses a greater challenge to the current global food crisis. Therefore, in-depth research on the infection mechanism and prevention and control of Pratylenchus coffeae has great theoretical guiding and practical production significance. In recent years, rich research results have been achieved in the existing research on plant parasitic nematodes, which have deepened the understanding of the infection and pathogenesis of Pratylenchus coffeae from many aspects. However, the infection mechanism of Pratylenchus coffeae is still unclear, and no relevant research on the interaction with the host to regulate the host plant immune response to promote parasitism has been reported. Summary of the Invention

[0003] In view of this, the present invention provides a maize translationally controlled tumor protein ZmTCTP and its applications.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A maize translationally controlled tumor protein ZmTCTP, whose amino acid sequence is shown in SEQ ID No.1.

[0006] The above-mentioned maize translationally controlled tumor protein ZmTCTP, whose nucleotide sequence is shown in SEQ ID No.2.

[0007] The above-mentioned maize translationally controlled tumor protein ZmTCTP is used in regulating insect resistance, disease resistance, proliferation and differentiation, and sugar metabolism of maize.

[0008] Further,

[0009] The insect resistance is resistance to Pratylenchus coffeae;

[0010] The disease resistance is resistance to Ustilago maydis;

[0011] The proliferation and differentiation are root tissue development and root cell proliferation and differentiation;

[0012] The sugar metabolism is glycolysis, tricarboxylic acid cycle, and pentose phosphate pathway.

[0013] Furthermore, the regulation is positive regulation.

[0014] Furthermore, the maize translationally controlled tumor protein ZmTCTP interacts with the Pratylenchus coffeae PcENG3 protein;

[0015] The amino acid sequence of the Pratylenchus coffeae PcENG3 protein is shown in SEQ ID No. 3.

[0016] Application of silencing maize translationally controlled tumor protein ZmTCTP in preparing a maize model with weakened resistance to Pratylenchus coffeae.

[0017] It can be seen from the above technical solutions that, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0018] In the present invention, the parasitic and pathogenicity of Pratylenchus coffeae are significantly reduced by virus-mediated silencing of the PcENG3 gene of Pratylenchus coffeae, indicating that PcENG3 plays an important role in the process of infection and pathogenesis. By screening the PcENG3 interacting proteins through a maize yeast library, multiple maize candidate interacting proteins are obtained. Through yeast one-to-one double hybridization, luciferase complementation imaging, bimolecular fluorescence complementation and GST pull-down verification, it is confirmed that there is an interaction between PcENG3 and ZmTCTP. By virus-mediated VIGS silencing of maize ZmTCTP, it is found that the main roots of maize are significantly shortened, the number of lateral roots is significantly reduced, and symptoms such as poor development of tassels and malformation of ears appear in the later growth stage, and the resistance to Pratylenchus coffeae is significantly weakened. In addition, after silencing maize ZmTCTP and inoculating Ustilago maydis, the number of maize galls increases significantly, and the resistance to Ustilago maydis is significantly weakened. In addition, ZmTCTP affects the expression of multiple key genes including glycolysis, tricarboxylic acid cycle and pentose phosphate pathway in the process of maize sugar metabolism. The results of the present invention show that PcENG3 interacts with maize ZmTCTP, inhibits maize growth and development by affecting maize energy synthesis, and ZmTCTP, as an important regulatory factor, plays an important role in regulating maize disease resistance, cell differentiation and proliferation.

[0019] The present invention reveals that during infection, the ENG protein of Pratylenchus coffeae inhibits host immune responses, affects host growth and development, and promotes nematode parasitism by performing multiple functions. The research results provide a new basis for analyzing the infection and pathogenic mechanism of Pratylenchus coffeae on maize, provide a theoretical basis for maize breeding against root rot nematodes, and at the same time provide potential candidate control targets for the development of new control drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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 the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0021] Figure 1 This is the virus-mediated RNAi-PcENG3 in Example 1 of the present invention. Among them, A represents the phenotype of virus-mediated RNAi-PcENG3, TRV2-PcENG3 represents maize transformed with pTRV2-PcENG3, TRV2-GFP represents maize transformed with pTRV2-GFP, WT represents wild-type maize, TRV2-ZmPDS represents maize transformed with pTRV2-ZmPDS, 10 dpa; B represents the PCR detection of positive seedlings of TRV2-PcENG3, and the 4 bands are 4 positive plants.

[0022] Figure 2 This is the influence of RNAi-PcENG3 on the pathogenicity of Pratylenchus coffeae in Example 1 of the present invention. Among them, A represents the silencing expression level of PcENG3 of P. coffeae; B represents the fresh weight of the above-ground part of maize at 60 dpi; C represents the fresh weight of maize roots at 60 dpi; D represents the total number of P. coffeae in the rhizosphere of maize at 60 dpi; WT represents wild-type maize, TRV2-empty represents maize transformed with the empty plasmid pTRV2, TRV2-PcENG3 represents maize transformed with pTRV2-PcENG3, and TRV2-GFP represents maize transformed with pTRV2-GFP.

[0023] Figure 3 This is the staining of Pratylenchus coffeae in maize roots in Example 1 of the present invention. Among them, A represents nematodes in the roots of maize treated with TRV2-GFP; B represents nematodes in the roots of maize treated with water; C represents nematodes in the roots of maize treated with TRV2-empty; D represents nematodes in the roots of maize treated with TRV2-PcENG3; Scale bar: 100 μm.

[0024] Figure 4 This is the screening of PcENG3 interacting proteins in Example 1 of the present invention. Among them, A represents the screening of positive yeast monoclonal clones; B represents the PCR detection of positive yeast monoclonal bacterial solutions.

[0025] Figure 5 This is the yeast interaction verification of PcENG3 protein and ZmTCTP in Example 1 of the present invention. Among them, SD-WL represents a synthetic dropout medium without tryptophan and leucine; SD-WLHA represents a synthetic dropout medium without tryptophan, leucine, histidine, and adenine; AD-T + BD-Lam represents a negative control; AD-T + BD-53 represents a positive control.

[0026] Figure 6 Pull-down verification of PcENG3 protein and ZmTCTP in Example 1 of the present invention;

[0027] Figure 7 Luciferase complementation imaging verification of the interaction between ZmTCTP and PcENG3 in Example 1 of the present invention;

[0028] Figure 8 Bimolecular fluorescence complementation interaction verification of PcENG3 and ZmTCTP in Example 1 of the present invention, where Merged represents the merged channel; YFP represents the yellow fluorescence channel; Bright represents the bright field; Scale bar: 50 μm;

[0029] Figure 9 Represents the effect of silencing the ZmTCTP gene on the growth and development of maize. Among them, A represents the morphology of maize seedlings after ZmTCTP silencing; B represents the normal growth morphology of maize seedlings; C represents the normal growth morphology of maize during the growth period; D represents the normal tassel and ear of maize during the growth period; E represents the growth period morphology of maize with ZmTCTP silencing; F represents the tassel (without ear) of maize during the growth period after silencing;

[0030] Figure 10 Represents the resistance determination of coffee root-lesion nematode by TRV-mediated maize RNAi-ZmTCTP. Among them, A represents the detection of ZmTCTP gene expression level in maize; B represents the fresh weight of the above-ground part of maize at 60 days after inoculation; C represents the total amount of coffee root-lesion nematodes in the rhizosphere of maize at 60 days after inoculation; D represents the fresh weight of maize roots at 60 days after inoculation; WT represents wild-type maize, TRV2-empty represents maize transformed with the empty pTRV2 plasmid, TRV2-ZmTCTP represents maize transformed with pTRV2-ZmTCTP, and TRV2-GFP represents maize transformed with pTRV2-GFP;

[0031] Figure 11 Resistance determination of RNAi-ZmTCTP of different maize varieties to Ustilago maydis in Example 1 of the present invention. Among them, A represents wild-type Zhengdan 958; B represents wild-type Yudan 959; C represents wild-type Yudan 8348; D represents Zhengdan 958; E represents Yudan 959; F represents Yudan 8348; Scale bar = 2 cm;

[0032] Figure 12 Effect of RNAi-ZmTCTP of different maize varieties on the number of disease tumors in Example 1 of the present invention. After T-test, the asterisk represents significant difference (**, P < 0.01);

[0033] Figure 13In Example 1 of the present invention, ZmTCTP in maize is involved in maize cell proliferation and differentiation. Among them, A represents the root development after ZmTCTP silencing treatment, with the healthy control on the left and the silencing treatment on the right; B represents the root length after ZmTCTP silencing treatment, with the WT healthy control and the RNAi-TCTP silencing treatment group; C and E represent the development of the root cap and root tip meristem of maize roots after ZmTCTP silencing treatment; D and F represent the development of the root cap and root tip meristem of maize roots in the control group.

[0034] Figure 14 In Example 1 of the present invention, the effects of RNAi-ZmTCTP on key genes in the glycolysis metabolic pathway of maize were examined. After T-test, the asterisks represent significant differences (*, P<0.05; **, P<0.01).

[0035] Figure 15 In Example 1 of the present invention, the effects of RNAi-ZmTCTP on genes related to the tricarboxylic acid cycle and pentose phosphate metabolic pathway in maize were examined. Among them, A represents the relative expression level of genes in the pentose phosphate metabolic pathway; B represents the relative expression level of ZmCS1 in the tricarboxylic acid cycle. After T-test, the asterisks represent significant differences (*, P<0.05; **, P<0.01). Detailed implementation mode

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0037] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods, which will not be elaborated here one by one.

[0038] Example 1

[0039] 1 Materials

[0040] 1.1 Tested nematode population

[0041] The coffee root-lesion nematode used in the experiment was cultured and preserved in our laboratory.

[0042] 1.2 Tested plant materials

[0043] Nicotiana benthamiana: Cultured in a greenhouse at 25°C until 4 weeks old;

[0044] Maize: The variety is Zhengdan 958, cultured in a greenhouse at 28°C and cultivated to the three-leaf and one-core stage at the seedling stage for standby.

[0045] 1.3 Test Strains and Vectors

[0046] Strains: Escherichia coli DH5α and BL21(DE3), Agrobacterium tumefaciens GV3101, GV3101(pSoup-19) and C58C1, yeast strains Y2H Gold and YTK12. All the above strains are stored in the -80 °C ultra-low temperature freezer in the laboratory;

[0047] Vectors: Gateway vectors (PGWC and PEG102), VIGs vectors (pTRV1 and pTRV2), Y2H vectors (pGBKT7 and pGADT7), pSUC2, protein expression vectors pet-28a, pet-32a and pGEX-4t-1, etc. The vector plasmids are stored in the -20 °C refrigerator in the laboratory.

[0048] 1.4 Test Reagents

[0049] Kit types: Micro RNA extraction kit, DNA extraction kit, DNA gel extraction kit and purification kit, purchased from Beijing Zhuangmeng Biotechnology Co., Ltd.; Plasmid large-scale and small-scale extraction kits are purchased from Novoprotein Scientific Inc. Reverse transcription kit, apoptosis detection kit and SDS-PAGE gel preparation kit are purchased from Yeasen Biotechnology Co., Ltd. (Shanghai).

[0050] Reagent types: Trizol (invitrogen), acetosyringone (solarbio), diethyl pyrocarbonate (DEPC), 10×PBS, salmon sperm DNA, Tween 20, DNA Marker, isopropanol and other reagents are all purchased from Zhengzhou Shijianzhishang Trading Co., Ltd.; RNase and DNase1 are purchased from Beijing solarbio company; 2×Taq PCR StarMix, T4 DNA Ligase, SYBR fluorescence quantitative reaction kit are all purchased from Yeasen Biotechnology Co., Ltd. (Shanghai); DNA Marker, nucleic acid dye, restriction endonuclease, proteinase K (20 mg / mL), etc. are purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0051] 1.5 Preparation of Main Solutions and Media

[0052] (1) Nematode fixing solution: Measure 50 mL of 3.7% paraformaldehyde into a large beaker, slowly add 50 mL of 1×PBS buffer solution in the fume hood, and store it at room temperature in the dark;

[0053] (2) LB liquid medium: Weigh 5 g of Tryptone (purchased from OXOID), 5 g of NaCl, and 5 g of Yeast extract (purchased from OXOID) into a beaker, add 400 mL of sterile water, adjust the pH to 7.4 with 5 M NaOH, make up the volume to 500 mL, sterilize at 121 °C and 0.1 MPa for 30 min, and then store for later use;

[0054] (3) Acetosyringone (AS): Weigh 1 g of AS, dissolve it in 2.5 mL of DMSO in a laminar flow hood, dispense it in aliquots, and store it in the dark at 4 °C;

[0055] (4) MMA injection solution: 10 mM CaCl2, 10 mM MES, 100 μM acetosyringone, make up the volume to 200 mL with sterile deionized water, and store it in the dark at 4 °C;

[0056] (5) 70% glycerol: Prepare 70% (w / w) glycerol with sterile water, sterilize it by autoclaving at 121 °C and 0.1 MPa for 20 min, and then store it for later use;

[0057] (6) 1 M CaCl2: Weigh 14.7 g of CaCl2·2H2O into an Erlenmeyer flask, make up the volume to 100 mL with distilled water, sterilize it by autoclaving, and then store it;

[0058] (7) Ampicillin solution (Amp): Weigh 5 g of sodium ampicillin, dissolve it in sterile deionized water to make a 100 mg / mL solution, filter sterilize it, dispense it in aliquots, and store it at -20 °C. The working concentration is 100 μg / mL;

[0059] (8) Kanamycin solution (Kana): Weigh 2.5 g of kanamycin, dissolve it in 50 mL of sterile deionized water in a laminar flow hood, filter sterilize it by suction filtration, dispense it in aliquots, and store it in the dark at -20 °C;

[0060] (9) Rifampicin solution (Rif): Weigh 1 g of rifampicin, dissolve it in 20 mL of DMSO in a laminar flow hood, filter sterilize it by suction filtration, dispense it in aliquots, and store it in the dark at 4 °C;

[0061] (10) IPTG solution: Weigh 1 g of IPTG (isopropyl-β-D-thiogalactoside), dissolve it in 5 mL of sterile deionized water to make a 200 mg / mL solution, filter sterilize it, dispense it in aliquots, and store it at -20 °C;

[0062] (11) 1 M Tris-HCL buffer (pH = 8.0): Weigh 6.05 g of Tris into a beaker, add 40 mL of deionized water to dissolve it, adjust the pH to 8.0 with concentrated hydrochloric acid, make up the volume to 500 mL, sterilize it by autoclaving at 15 min, and then store it at room temperature;

[0063] (12) 10% SDS: Weigh 10 g of SDS powder into a beaker, add 80 mL of sterile deionized water, heat and dissolve it in a water bath at 42 °C, adjust the pH to 7.2, make up the volume to 100 mL, and store it at room temperature;

[0064] (13) M9 Buffer: Weigh (measure) 3 g of KH2PO4, 6 g of Na2HPO4, 5 g of NaCl, and 1 mL of MgSO4 (1 M) into a beaker respectively, add 800 mL of DEPC water to dissolve and mix well, then make up the volume to 1 L, sterilize it by high-pressure moist heat, and store it at room temperature;

[0065] (14) Nematode fixation and lysis buffer: The concentration of proteinase K in M9 Buffer is 0.5 mg / mL;

[0066] (15) 1× Protein electrophoresis buffer: Weigh 1.6 g of Tris-base, 7.2 g of glycine, and 0.55 g of SDS respectively, dissolve them in deionized water, and make up the volume to 500 mL;

[0067] (16) Coomassie blue staining solution: Weigh 1 g of Coomassie brilliant blue R-250 into a beaker, add 450 mL of methanol, 450 mL of distilled water, and 100 mL of glacial acetic acid in the fume hood successively, then mix well and keep it in the dark for standby;

[0068] (17) Coomassie blue decolorizing solution: Measure 100 mL of methanol, 800 mL of deionized water, and 100 mL of glacial acetic acid successively, mix them well in the fume hood, and keep it in the dark for standby;

[0069] (18) Salmon sperm DNA (heat denaturation): Place it in boiling water and heat for 5 min, then quickly ice-bath it in an ice bath for 5 min, and repeat once;

[0070] (19) 10× TAE buffer: Weigh 48.2 g of Tris-HCl, 11.44 g of glacial acetic acid, and 7.44 g of Na2EDTA·2H2O respectively, make up the volume to 1 L with sterile water.

[0071] 2 Experimental methods

[0072] 2.1 Nematode culture and plant sample treatment

[0073] Prepare carrot callus. The tested population of Pratylenchus coffeae is disinfected overnight with 0.3% streptomycin sulfate solution, washed several times with sterile water, and then inoculated onto a culture dish containing carrot callus in a laminar flow hood, and cultured in the dark at 25 °C.

[0074] Separate Pratylenchus coffeae in diseased soil by the Baermann funnel method (Reise et al., 1987).

[0075] The isolation of root nematodes refers to the method of Kaplan et al. (Kaplan et al., 1997).

[0076] Maize seeds were surface-sterilized with 75% ethanol and then placed in a petri dish containing sterile wet filter paper for 2 days of germination. The seedlings were transplanted into flower pots containing soil and substrate (1:1), and the light was set as light:dark = 16 h:8 h, and the temperature was 28 °C.

[0077] Nicotiana benthamiana was grown at 25 °C under a 16-h photoperiod.

[0078] 2.2 Sample RNA extraction

[0079] RNA of Pratylenchus coffeae was extracted. The centrifuge tubes and pipette tips used in the experiment were soaked in 0.1% DEPC for 24 h, autoclaved and dried before use. The experimental steps are as follows:

[0080] (1) The washed fresh nematode samples were quickly frozen in liquid nitrogen for 30 s and then thoroughly crushed with a grinder;

[0081] (2) Add 600 - 800 μL of Trizol extraction solution, mix well by vigorous shaking and then let it stand on an ice box for 2 - 5 min;

[0082] (3) Add 100 μL of chloroform pre-cooled to -20 °C, mix well by vigorous shaking and then let it stand in an ice bath for 10 min;

[0083] (4) Centrifuge at 13000 rpm for 8 - 10 min at a constant temperature of 4 °C in a high-speed refrigerated centrifuge;

[0084] (5) Transfer the upper liquid to a new centrifuge tube, add 50 μL of isopropanol pre-cooled to -20 °C, invert and mix well and then let it stand in an ice bath for 8 min;

[0085] (6) Centrifuge at 13000 rpm for 5 min at 4 °C, discard the supernatant and then add 75% ethanol pre-cooled to -20 °C and gently pipette the precipitate;

[0086] (7) Centrifuge at 13000 rpm for 5 min at 4 °C, discard the supernatant and then place it in a vacuum dryer at 30 °C for 15 min of drying;

[0087] (8) After adding 30 μL of RNase-free water for dissolution, store it in an ultra-low temperature refrigerator for standby.

[0088] 2.3 RT-qPCR and reaction system

[0089] After detecting the purity and concentration of the sample RNA with an ultraviolet spectrophotometer, according to the kit Instructions for AdvanceFast One-step RT-gDNA Digestion SuperMix for qPCR (All operations are carried out on ice). Refer to the kit instructions for setting the reaction program. The reaction system is as follows:

[0090] (1) Reverse transcription reaction system:

[0091] Table 1 Reverse transcription reaction system

[0092] Reagent Dosage 4×Hifair SuperMix 5 μL gDNA Remover Mix 2 μL RNA 50 pg - 5 μg RNase-free water Up to 20 μL

[0093] (2) qRT-PCR reaction system:

[0094] Table 2 qRT-PCR reaction system

[0095] Reagent Dosage PCR SYBR Green Master Mix 10 μL Primer (qPE3-F / qPE3-R) (10 μM) 0.5 μL each DNA template 1 μL RNase-free water Up to 20 μL

[0096] The primer sequences are as follows:

[0097] qPE3-F: 5'-TAATGAACAGACTGTCAAATGC-3', SEQ ID No.4;

[0098] qPE3-R: 5'-GCGGCTTTGTCCACTTGCTGAT-3', SEQ ID No.5.

[0099] 2.4 Paraffin sections of maize roots

[0100] Paraffin section is a widely used method for plant pathological tissue analysis. After washing the maize roots infected with Pratylenchus coffeae clean, cut them into small segments about 1 cm long and place them in a wet box for later use. The specific steps are as follows:

[0101] (1) Immediately soak the samples in FAA fixative after sampling and place them in a 4°C refrigerator for cold treatment for 48 h;

[0102] (2) After taking out, rinse them clean with ultrapure water and soak them in PBS buffer for 30 min;

[0103] (3) Gradient dehydrate the samples in 50%, 75%, 95% and 100% ethanol successively, with each dehydration duration being 2 h;

[0104] (4) Transparently process the samples (ethanol:xylene = 1:1 for 1 h, and then xylene alone for 1 h);

[0105] (5) Treat overnight with xylene:paraffin = 1:1 and then impregnate with wax;

[0106] (6) After embedding treatment with a paraffin embedding machine, trim the excess wax blocks and place them on a paraffin slicer for sectioning (3-4 μm thick).

[0107] (7) Float the sections on the warm water (40 °C) of a spreading machine to flatten the tissue, and lift the tissue with a glass slide.

[0108] (5) Let it stand at room temperature for 1-5 min and record the color change.

[0109] 2.5 Establishment of a virus-mediated gene silencing system

[0110]

[0111] The experimental method for virus-mediated silencing of ZmTCTP in maize is the same as above. The primer sequences are TRV2-ZmTCTP-F (5'-GTGAGTAAGGTTACCGAATTCCCTGTGGGAGGTCGAGGG-3', SEQ ID No.10) / TRV2-ZmTCTP-R (5'-CGTGAGCTCGGTACCGGATCCAGGTCCTTCAGCTTGCTAAGGA-3', SEQ ID No.11). A specific silencing segment with a sequence length of 300 bp was screened (CCTGTGGGAGGTCGAGGG AAAGTGGGTCACCCAAGGTCCTGTTGATGTGGACATTGGTGCCAATCCATCCGCCGAGGGTGGTGAGGACGAAAGCGTTGATGACACAGCTGTGAAGGTGGTTGATATTGTTGACACATTCCGTCTACAGGAGCAACCTCCTTTTGACAAGAAATCATTTGTGTCTTACATCAAAAAATACATCAAGAATCTCACTGCTGTGTTGGAGCCAGAGAAAGCGGATGAGTTCAAAAAGGGTGTCGAGGGTGCAACCAAGTTTCTCCTTAGCAAGCTGAAGGACCT, SEQ ID No.12).

[0112] (1) Vector linearization

[0113] The pTRV2 plasmid was digested with EcoRΙ and BamHΙ. The reaction system is as follows:

[0114] Table 3 Reaction system

[0115] Reagent Dosage 10×buffer 5 μL EcoRΙ and BamHΙ 2.5 μL each Plasmid DNA 2 μg <![CDATA[ddH2O]]> Up to 50 μL

[0116] Incubate in a 37°C water bath for 5 - 8 h, gently mix during this period, and store in a -20°C refrigerator after the digestion is completed.

[0117] (2) Vector construction

[0118] Use specific primers with homologous arms to amplify the sample cDNA template to obtain a DNA fragment with a vector adapter. The DNA fragment was seamlessly ligated to the pTRV2 linearized vector to obtain a recombinant expression vector. The recombinant expression vector with correct sequencing was transformed into Escherichia coli and Agrobacterium tumefaciens C58C1 competent cells. Empty pTRV1 and pTRV2 Agrobacterium were used as blank controls, and pTRV2-ZmPDS was used as a positive control.

[0119] (3) Agrobacterium-mediated infection of maize

[0120] 1) Try to select plump maize seeds. After treating with 75% ethanol for 10 min, rinse them with sterile water 5 - 8 times, and soak them overnight in sterile water for 8 - 10 h.

[0121] 2) Use a dissecting needle or fine forceps to remove the maize seed coat, and then insert a sharp inoculation needle obliquely into the embryo at a 45° angle on both sides of the germ (1 - 2 mm), and let it stand at room temperature for later use.

[0122] 3) Incubate the Agrobacterium tumefaciens C58C1 liquid at 28 °C and 200 rpm overnight until OD 600 ≥1.0;

[0123] 4) Adjust the OD of the pTRV1 and pTRV2 liquid to 1.0 separately with sterile water, then mix the two plasmid - carrying liquids in equal volumes and add 200 μM acetosyringone.

[0124] 5) Add the maize seeds with the seed coat removed to a tissue culture flask until just submerged by the liquid. Incubate at 15 °C and 60 rpm for 12 - 16 h, then rinse with sterile water 10 times to remove the residual Agrobacterium liquid.

[0125] 6) Place the rinsed maize seed embryos upward on absorbent paper fully moistened with sterile water, and germinate them in the dark at 15 °C and moisturized for 3 d.

[0126] 7) Transplant them into nutrient pots and place them in a constant - temperature incubator at 18 °C / 15 °C (photoperiod: 16 h light / 8 h dark).

[0127] (4) Detection of the silencing efficiency of nematode PcENG3 and pathogenicity determination

[0128] Inoculate the Pratylenchus coffeae suspension on the roots of maize at the three - leaf and one - core stage after Agrobacterium - mediated infection. After 15 d, take out the maize roots, rinse them clean, and use the Trizol liquid nitrogen grinding method to extract the RNA of maize roots in different treatment groups to detect the silencing efficiency of the target gene. There are at least 20 maize plants in each treatment group. After 60 d of inoculation, count the growth of maize (root weight, shoot weight, and rhizosphere nematode population).

[0129] (5) Acid fuchsin staining

[0130] The main steps of acid fuchsin staining of root tissues are as follows:

[0131] 1) Wash the maize roots to be stained, and treat them with 10% NaClO for 5 - 10 min (determine the bleaching time according to the tenderness of the roots);

[0132] 2) Rinse the roots with sterile water 5 - 10 times, blot dry, and add 20 mL of 3.5% acid fuchsin solution.

[0133] 3) After heating the solution to boiling, place the root system in it, perform heat treatment for 3 min, and then let it cool naturally at room temperature;

[0134] 4) Rinse the residual acid fuchsin on the root system with sterile water. After blotting dry the water, add acid glycerol to the beaker;

[0135] 5) Heat it to boiling in a microwave oven and then let it cool naturally. Let it stand at 28 °C and change the glycerol once every 5 h. After 48 h, the nematodes can be seen dyed red.

[0136] 2.6 Screening of maize library by Mating method for two-hybrid

[0137] The method for screening yeast library by Mating method is as follows:

[0138] 1) Pick a fresh yeast clone of Y2H Gold containing Bait plasmid with a size of 2 - 3 mm and transfer it to 50 mL of SD / -Trp liquid medium;

[0139] 2) Incubate it overnight (16 - 22 h) at 30 °C on a shaker with a shaking speed of 250 rpm until OD 600 = 0.8;

[0140] 3) Centrifuge at 1000 g for 5 min and discard the supernatant;

[0141] 4) Resuspend the cells with 5 mL of SD / -Trp liquid medium;

[0142] 5) Melt 1 mL of maize library AD bacterial liquid at room temperature and add it to 5 mL of BD bacterial liquid in a 2.5 L sterilized conical flask;

[0143] 6) Add 45 mL of 2×YPDA (containing 50 μg / mL Kan + ) liquid medium. At the same time, rinse the AD bacterial liquid with 1 mL of 2×YPDA and transfer it to the conical flask;

[0144] 7) Incubate it at 30 °C on a shaker with a low shaking speed of 30 - 60 rpm for 20 - 24 h (vigorous shaking will reduce the binding efficiency, and too low rotation speed will cause the cells to deposit at the bottom and also reduce the binding efficiency);

[0145] 8) After 20 h, observe under a 40× microscope whether the hybrid solution shows zygotes in the shape of Mickey Mouse head or clover;

[0146] 9) After observing the zygotes, centrifuge at 1000 g for 10 min and discard the supernatant to collect the bacterial cells;

[0147] 10) Resuspend the bacterial cells with 50 mL of 0.9% NaCl (containing 50 μg / mL Kan + ) and measure the total volume of the sample;

[0148] 11) Spread the bacterial solution evenly on SD / -Trp / -Leu / -His / X-α-gal / AbA plates with a diameter of 150 mm, about 10 - 20 plates, and culture at 30 °C for 3 - 5 days.

[0149] 12) Transfer the blue clones on the TDO / X / A low-stringency screening plates to the high-stringency screening plates SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA for further screening;

[0150] 13) Perform PCR detection and analysis on the positive clones on SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA and verify them one by one.

[0151] 2.7 Interaction verification

[0152] (1) Agrobacterium infiltration of Nicotiana benthamiana

[0153] 1) Transform the recombinant vector into Agrobacterium tumefaciens GV3101 and culture in an incubator at 28 °C;

[0154] 2) Select the correct monoclonal Agrobacterium and shake it overnight;

[0155] 3) Centrifuge at 2000 rpm for 5 min at room temperature, discard the supernatant, resuspend and vortex until OD 600 = 0.5 - 0.8;

[0156] 4) Select the back of the leaves of Nicotiana benthamiana at the four-leaf stage for infiltration;

[0157] 5) Observe and take pictures with a confocal microscope 2 days later.

[0158] (2) Subcellular localization detection

[0159] 1) Streak the Agrobacterium strain on LB plates with Kan + and Rif resistance and culture in an inverted position at 28 °C for 3 days;

[0160] 2) Use a sterile pipette tip to pick Agrobacterium into 5 mL LB medium (Kan + and Rif resistance) and culture with shaking overnight at 28 °C;

[0161] 3) Centrifuge at 2000 rpm for 5 min, discard the supernatant, resuspend with MMA solution (10 mM MgCl2, 10 mM MES and 0.1 mM acetosyringone) and vortex until OD 600 = 0.8 - 1.0;

[0162] 4) Let it stand at room temperature for 3 h, use a sterile needleless syringe to inject the back of the leaves, and punch holes for detection 2 days later.

[0163] (3) GST pull-down

[0164] After purifying the GST-ZmTCTP bait protein sample, add it to the prepared GST-tagged protein purification magnetic beads. Invert and mix well, then place the centrifuge tube on a rotary mixer and incubate overnight at 4°C in a refrigerator with inversion.

[0165] 1) Washing away impurities

[0166] Place the centrifuge tube on a magnetic separator and let it stand for 1 min to separate the magnetic beads. Add 4 volumes of washing buffer (PBS, pH = 7.4), then repeatedly pipette to discard the impurities. Repeat the above steps 3 times.

[0167] 2) Binding of target protein to bait protein-magnetic bead complex

[0168] Add the purified HIS-PcENG3 sample to the treated GST-ZmTCTP complex, invert and mix well. Place the centrifuge tube on a rotary mixer and incubate overnight at 4°C in a refrigerator with inversion (the time can be adjusted according to the binding effect).

[0169] 3) Washing away impurities

[0170] After HIS-PcENG3 binds to GST-ZmTCTP, completely denature the protein sample and store it at -20°C for WB detection.

[0171] 4) Elution of target protein

[0172] Add 3 volumes of elution buffer (50 mM Tris-HCl, 20 mM reduced glutathione, pH = 8.0) equal to the volume of the magnetic beads to the above centrifuge tube. Pipette 5 times, then place it on a rotary mixer at room temperature for 5 - 10 min, then place it on a magnetic separator and let it stand for 1 min. After the solution becomes clear, aspirate the supernatant and collect the elution fraction, that is, obtain the target protein and target protein complex. Repeat the operation two more times, collect the elution fractions respectively, and perform WB detection after complete denaturation.

[0173] (4) Luciferase complementation imaging to verify in vivo interaction

[0174] PcENG3 and ZmTCTP were respectively inserted into the pCambia1300-cLuc and pCambia1300-nLuc vectors to obtain the PcENG3-cLuc and ZmTCTP-nLuc recombinant plasmids. The positive controls (SAR-cLuc and SGF-nLuc plasmids) were obtained by the same method. These plasmids were respectively transformed into competent Agrobacterium tumefaciens GV3101 cells. After culturing and induction, the Agrobacterium tumefaciens carrying PcENG2-cLuc was mixed with the Agrobacterium tumefaciens carrying ZmTCTP-nLuc and SGF-nLuc in equal amounts. The mixed Agrobacterium tumefaciens cultures were infiltrated into the leaves of Nicotiana benthamiana alone. After treatment in an artificial climate chamber at 20 °C for 48 h, the leaves were infiltrated again with 0.2 mM luciferin solution, and the luciferase activity was detected by photographing. PcENG3-cLuc and SGF-nLuc were used as negative controls.

[0175] 2.8 Establishment of an indoor inoculation system for Ustilago maydis

[0176] Artificial inoculation and resistance evaluation of Ustilago maydis were carried out in accordance with the Technical Specification for Identification of Maize Resistance to Ustilago maydis (NY / T1248.12 - 2016).

[0177] 2.9 Inoculation method for Ustilago maydis

[0178] The test pathogen was the Ustilago maydis strain. The glycerol bacteria stored in the ultra-low temperature refrigerator were thawed in an ice bath in advance. The bacterial solution was dipped with an inoculation loop and streaked on the PS solid medium to activate the strain. It was cultured upside down in an incubator at 25 °C for 3 d. The activated colonies were picked and inoculated into the PS liquid medium, and cultured with shaking at 25 °C and 200 rpm for 24 h.

[0179] The injection method was used for pathogen inoculation as follows:

[0180] 1) The concentration of the bacterial solution was detected using an ultraviolet spectrophotometer and adjusted to OD 600 = 1.0;

[0181] 2) 0.01% Tween 20 was added;

[0182] 3) The bacterial solution was aspirated with a 5 mL sterile syringe with a needle and whipped to mix evenly;

[0183] 4) The needle was obliquely inserted into the middle of the maize stem, and 2 mL was injected into each plant until the bacterial solution overflowed from the core leaves.

[0184] 2.10 Data processing and analysis

[0185] In this invention, the significance analysis of two groups of data for processing and analysis was performed using an independent samples T-test (Student's ttest), and the significance analysis of multiple groups of data was performed using analysis of variance (Analysis of Variance, abbreviated as ANOVA).

[0186] 3 Results and Analysis

[0187] 3.1 PcENG3 gene silencing reduces the pathogenicity and parasitism of nematodes

[0188] Previously in the laboratory, PcENG3 was silenced by soaking Pratylenchus coffeae with synthetic dsRNA-PcENG3. It was found that PcENG3 is related to the infection of Pratylenchus coffeae. Due to reasons such as the short duration of continuous silencing of dsRNA-PcENG3 and its easy degradation, it is restricted from persistently silencing the target gene. Virus-mediated gene silencing specifically recognizes and degrades the mRNA of the pathogenic target gene, thereby interfering with the transcription and translation of the target gene, and is suitable for RNAi research of obligate biotrophic pathogens.

[0189] In the virus-mediated PcENG3 silencing experiment, maize transformed with the empty plasmid pTRV2 and pTRV2-GFP was used as the negative control, and maize transformed with pTRV2-ZmPDS was used as the positive control. The recombinant plasmid was transformed into Agrobacterium tumefaciens C58C1 for infecting maize seeds. The results showed that obvious "photobleaching" occurred after the germination of maize transformed with pTRV2-ZmPDS, and no obvious growth defect characteristics were observed in the negative control and pTRV2-PcENG3 maize( Figure 1 A), and the TRV2-PcENG3 seedlings were confirmed to be positive plants by PCR detection( Figure 1 B). After Pratylenchus coffeae infected the pTRV2-PcENG3 maize, the expression level of PcENG3 decreased significantly by about 50%, and there was no significant difference in the expression level of PcENG3 in the roots of maize in the WT, pTRV2-empty and pTRV2-GFP groups( Figure 2 A). 60 days after inoculating Pratylenchus coffeae, the fresh weight of the above-ground part and the fresh weight of the roots of the maize plants transformed with TRV2-PcENG3 increased significantly( Figure 2 B, Figure 2 C), and the number of P. coffeae in the rhizosphere of maize decreased significantly by about 30%( Figure 2 D, Figure 3 ), indicating that the pathogenicity of nematodes to maize roots was weakened, and there was no significant difference in the growth of maize in the WT, pTRV2-empty and pTRV2-GFP groups.

[0190] 3.2 Screening of the PcENG3-maize interaction library

[0191] The constructed BD-PcENG3 plasmid was transformed into yeast Y2H Gold competent cells. After 3 days, white yeast single colonies appeared. The yeast colonies on the screening medium plate were picked into a liquid medium (SD / -Trp) for expansion culture. The AD yeast solution of the maize library was expanded through 2×YPDA liquid medium and then co-cultured for 24 h. The bacterial solution was evenly spread on the SD / -Trp / -Leu / -His / -Ade / X-α-gal / AbA plate, and the blue colonies were screened for PCR bacterial solution detection to obtain the candidate interacting protein sequence information. Sequencing and comparative analysis of the positive monoclonal clones found that five positive clones ( Figure 4 ) might have an interaction relationship with PcENG3. The proteins were: maize translationally controlled tumor protein, maize senescence-related protein, maize ubiquitin-conjugating enzyme, maize translationally controlled tumor protein C, and maize heat shock protein-70 (Table 4).

[0192] Table 4 PcENG3 candidate interacting proteins

[0193]

[0194] 3.3 Verification of the interaction between PcENG3 and maize ZmTCTP

[0195] To explore the interaction mechanism between PcENG3 and maize, the yeast interaction library was screened using the Mating method. Using PcENG3 as the bait protein, multiple clones were screened. After PCR detection, verification, and sequencing, it was found that ZmTCTP might interact with PcENG3. In the yeast two-hybrid experiment, AD-T and BD-53 were used as positive controls, and AD-T and BD-Lam were used as negative controls. The recombinant plasmids AD-ZmTCTP and BD-PcENG3 were co-transformed into Y2H Gold yeast competent cells and spread on the solid media of the double-deficient medium (SD-LW) and the quadruple-deficient medium (SD-LWHA), respectively, and cultured in an incubator for 5 days. The results showed that AD-ZmTCTP and BD-PcENG3, as well as AD-T and BD-53, grew normally on SD-LWHA and showed blue on the plate supplemented with X-α-gal. AD-T and BD-Lam grew normally on SD-LW and could not grow on SD-LWHA ( Figure 5 ). The results of the yeast interaction verification experiment showed that PcENG3 and ZmTCTP interacted in vitro.

[0196] 3.4 GST pull-down verification of the in vitro interaction between PcENG3 and ZmTCTP

[0197] PcENG3 and ZmTCTP were respectively constructed into the prokaryotic expression vectors PET-32a and pGEX-4T-1 for induced expression. After gel enrichment and desalting through an adsorption column, Western blot detection was carried out. The results showed that GST was detected using anti-GST for the protein eluted and enriched by co-incubating PcENG3-HIS with GST, and protein bands of PcENG3-HIS and ZmTCTP-GST were detected using anti-GST for the protein eluted and enriched by co-incubating PcENG3-HIS with ZmTCTP-GST( Figure 6 ), demonstrating the in vitro interaction between PcENG3-HIS and ZmTCTP-GST.

[0198] 3.5 Verification of the in vivo interaction between PcENG3 and ZmTCTP by luciferase complementation imaging

[0199] In this study, PcENG3-cLuc and nLuc-SGF were used as negative controls, and SAR-cLuc and nLuc-SGF were used as positive controls. The results showed that fluorescence signals could be observed after co-expression of PcENG3-cLuc and nLuc-ZmTCTP in Nicotiana benthamiana leaves 2 days after infiltration with Agrobacterium, while no fluorescence signal was observed for the co-expression of nLuc-ZmTCTP and SAR-cLuc( Figure 7 ). LCA research indicated the interaction between PcENG3 and ZmTCTP in Nicotiana benthamiana in vivo.

[0200] 3.6 Verification of the in vivo interaction between PcENG3 and ZmTCTP by bimolecular fluorescence complementation

[0201] A recombinant fusion expression vector was constructed by connecting the N-terminus of PcENG3 with nYFP, and a fusion expression vector was constructed by connecting the C-terminus of ZmTCTP with nYFP. Agrobacterium carrying nYFP-PcENG3 and nYFP-ZmTCTP were mixed in equal volumes and infiltrated into Nicotiana benthamiana leaves for transient expression to verify the interaction relationship. As Figure 8 shown, there was no yellow fluorescence in the control group, and yellow fluorescence appeared in the cytoplasm and nucleus for nYFP-PcENG3 + nYFP-ZmTCTP. Research indicated the interaction between PcENG3 and ZmTCTP in the cytoplasm and nucleoplasm.

[0202] 3.7 Silencing of maize ZmTCTP promotes nematode infection

[0203] In the virus-mediated maize ZmTCTP silencing experiment, ZmPDS was used as a positive control. The results showed that after ZmTCTP silencing, maize seedlings grew slowly, with narrow leaves, weakened elongation of the main maize roots, and reduced number of lateral roots( Figure 9 A), and obvious reproductive defects appeared in maize at the later growth stage, with male inflorescences aborting and not producing pollen( Figure 9 E), and female inflorescences developing abnormally(Figure 9 F), there was no obvious change in the control treatment group ( Figure 9 B– Figure 9 D). There was no significant difference in the expression level of maize ZmTCTP among the WT, pTRV2-empty, and pTRV2-GFP groups, and the silencing efficiency of ZmTCTP in RNAi-ZmTCTP maize was 39% Figure 10 A), with a significant difference (P < 0.05). Sixty days after inoculation with Pratylenchus coffeae, the nematode population in the rhizosphere of maize and the related growth parameters of maize were counted for each group. The results showed that the fresh weight of the aboveground part of the RNAi-ZmTCTP maize plants increased ( Figure 10 B), and the fresh weight of the roots of the RNAi-ZmTCTP maize plants decreased significantly ( Figure 10 D), and the number of nematodes in the maize rhizosphere increased significantly by about 23% ( Figure 10 C). The pathogenicity of nematodes to maize increased, and there was no significant difference in the growth of maize among the WT, pTRV2-empty, and pTRV2-GFP groups.

[0204] 3.8 Silencing of maize ZmTCTP promotes the formation of plant galls

[0205] Ustilago maydis mainly harms the leaves, stems, tassels, and ears of maize, characterized by tumor-like protrusions with various shapes. Indoor resistance assays for Ustilago maydis showed that Yudan 959 and Yudan 8348 of maize were highly resistant to Ustilago maydis varieties ( Figure 11 B, Figure 11 C), and Zhengdan 958 of maize was a susceptible variety ( Figure 11 A). Maize Zhengdan 958, Yudan 959, and Yudan 8348 were respectively subjected to ZmTCTP silencing treatment. Ustilago maydis was inoculated by injection 15 days after germination, and the number of galls was investigated 15 days after inoculation. The results of the resistance assay showed that the number of galls of maize Zhengdan 958, Yudan 959, and Yudan 8348 after ZmTCTP silencing treatment ( Figure 11 D, Figure 11 E, Figure 11 F, Figure 12 ) increased significantly (P < 0.01), and the resistance of maize to Ustilago maydis was significantly weakened.

[0206] 3.9 Maize ZmTCTP is involved in maize cell proliferation and differentiation

[0207] The maize seeds treated with Agrobacterium were rinsed clean and then germinated in the dark with moisture retention. The root length was measured 4 days after germination. The results showed that the root system development was slow after ZmTCTP silencing treatment ( Figure 13 A), and the average length of the main root was 3.91 cm, which was significantly lower than that of the WT ( Figure 13B). Samples were taken at 1 cm above the root tip and embedded in paraffin for safranin-fast green staining. It was found that compared with the control group, the development of the root cap and root tip meristem of maize roots was slow after ZmTCTP silencing, and tissues such as root vascular bundles and vessels were not fully developed. Figure 13 C– Figure 13 F). The results showed that ZmTCTP was involved in the development of maize root tissues and the proliferation and differentiation of root cells.

[0208] 3.10 Effects of ZmTCTP silencing on the expression of genes in the sugar metabolism pathway in maize

[0209] To clarify the effect of ZmTCTP on the sugar metabolism pathway in maize, the differential expression of key enzyme genes in the glycolysis, tricarboxylic acid cycle, and pentose phosphate pathway of maize was measured and analyzed to screen the key genes ZmHXK1, ZmHXK2, ZmPK1, ZmPK2, ZmPFK1, ZmPFK2, and ZmPFK3 in the glycolysis pathway, the tricarboxylic acid cycle-citrate synthase gene ZmCS1, and the pentose phosphate pathway-glucose-6-phosphate dehydrogenase genes ZmG6PD1 and ZmG6PD2. ZmActin was used as an internal reference. The results of RT-qPCR detection showed that ZmTCTP regulated the expression of key enzyme genes in the glycolysis, tricarboxylic acid cycle, and pentose phosphate pathway during the seedling stage of maize, affecting energy supply, mainly manifested in inhibiting the expression of ZmHXK1, ZmHXK2, ZmPK1, ZmPK2, and ZmPFK2 in the glycolysis pathway. Figure 14 ); hindering the expression of ZmG6PD2 in the pentose phosphate pathway Figure 15 (A) and the expression level of the tricarboxylic acid cycle gene ZmCS1 Figure 15 (B).

[0210] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. The same or similar parts among the embodiments can be referred to each other.

[0211] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A maize translation controlled tumor protein ZmTCTP, characterized in that: Its amino acid sequence is shown in SEQ ID No.1; MLVYQDLLSGDELLSDSFTYKELENGVLWEVEGKWVTQGPVDVDIGANPSAEGG EDESVDDTAVKVVDIVDTFRLQEQPPFDKKSFVSYIKKYIKNLTAVLEPEKADEFKKGV EGATKFLLSKLKDLQFFVGESMKDEASVVFAYYKDGATNPTFLYFSHGLKEIKC, SEQ ID No.

1.

2. The maize translation controlled tumor protein ZmTCTP according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID No.2; ATGTTGGTTTATCAGGATCTCCTATCTGGCGACGAGCTCCTGTCGGATTCATTCACCTACAAGGAGCTCGAGAACGGCGTCCTGTGGGAGGTCGAGGGAAAGTGGGTCACCCAAGGTCCTGTTGATG TGGACATTGGTGCCAATCCATCCGCCGAGGGTGGTGAGGACGAAAGCGTTGATGACACAGCTGTGAAGGTGGTTGATATTGTTGACACATTCCGTCTACAGGAGCAACCTCCTTTTGACAAGAAATC ATTTGGTCTTACATCAAAAAATACATCAAGAATCTCACTGCTGTGTTGGAGCCAGAGAAAGCGGATGAGTTCAAAAAGGGTGTCGAGGGTGCAACCAAGTTTCTCCTTAGCAAGCTGAAGGACCTT CAATTTTTTGTTGGTGAGAGCATGAAGGACGAAGCTTCCGTGGTATTCGCCTATTACAAGGATGGTGCCACTAATCCGACATTCCTCTATTTCTCTCATGGTCTTAAGGAGATCAAGTGCTAG, SEQ ID No.

2.

3. Use of the maize translation controlled tumor protein ZmTCTP according to claim 1 in regulating maize insect resistance, disease resistance, proliferation and differentiation, and sugar metabolism.

4. The use according to claim 3, characterized in that The insect resistance is resistance to Pratylenchus coffee nematode; The disease resistance is resistance to Ustilago maydis; The proliferation and differentiation includes root tissue development and root cell proliferation and differentiation; The sugar metabolism includes glycolysis, tricarboxylic acid cycle and pentose phosphate pathway.

5. The use according to claim 3, characterized in that The regulation is positive regulation.

6. The use according to claim 3, characterized in that The maize translation control tumor protein ZmTCTP interacts with the coffee nematode PcENG3 protein; The amino acid sequence of the coffee nematode PcENG3 protein is shown in SEQ ID No. 3; MSSIFLLVSLLIPFLGLANGQTTPTTPTTPKSPTTPTSPKTPVTPSADGGCCPHGKLAVKGTQLVGADGTPVQLRGMSLFWSNFPEGAPFYNEQTVKCLKCNWDANIVRAAMGVENDKGYLQNPDAEYAKMEAVVDAAMKNCMYVLVDWHYTSATAYPEKAADFFDKISKKCAGKCNCLY etc ID No.

3.

7. Application of silencing maize translation-controlled tumor protein ZmTCTP in preparing a maize model with reduced resistance to coffee nematode.

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