Application of ZmTCP20 gene in regulation and control of growth, development and yield of crops

Abstract

The invention discloses application of a ZmTCP20 gene in regulation and control of crop growth, development and yield, and belongs to the technical field of gene engineering. It is found that the corn TCP family member ZmTCP20 has the effect of directly and positively regulating the nitrogen utilization efficiency of crops, nitrogen absorption of the crops can be promoted through overexpression of the ZmTCP20 gene, the biomass and total nitrogen accumulation of plants can be remarkably increased, and the nitrate absorption capacity of root systems is improved; meanwhile, two-year field trials further show that the overexpression gene ZmTCP20 can significantly improve the plant height and biomass of crops, increase the nitrogen accumulation amount in the plants, enable crop clusters to be lengthened and thickened and further improve the yield of the clusters, and after the gene ZmTCP20 is knocked out, the contrary is that the overexpression gene ZmTCP20 is knocked out. The functional analysis of the gene ZmTCP20 provides important gene resources and theoretical guidance for the cultivation of new crop varieties with efficient nitrogen utilization.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a... ZmTCP20 The application of genes in regulating crop growth, development and yield. Background Technology

[0002] Corn is one of the most important food crops in my country and even globally, playing a vital role in feed and industrial raw materials. However, to ensure continuous increases in corn yield, large amounts of nitrogen fertilizer are used in corn production, leading to a decrease in overall nitrogen fertilizer utilization efficiency and causing serious non-point source pollution. Therefore, research on the molecular mechanisms by which corn improves nitrogen absorption and utilization is of great significance for improving nitrogen fertilizer utilization efficiency, ensuring corn food security, and reducing agricultural non-point source pollution.

[0003] The TCP family is a group of plant-specific transcription factors. TCP proteins are characterized by a conserved atypical domain near the N-terminus, composed of approximately 60 amino acid residues with a helical-loop helical structure, similar to eukaryotic bHLH transcription factors. Studies have found that TCPs can regulate various biological processes in plants. In maize, TCP has also been reported to participate in regulating the growth and development of male inflorescences. However, current research on TCP family genes in maize is still limited, and the exploration of their biological functions is relatively weak. Furthermore, transcription factors that effectively regulate nitrogen-efficient use in maize are rarely reported. Therefore, further research on the regulatory role of TCP family genes in maize growth is of great significance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: to provide a ZmTCP20 The application of genes in regulating crop growth, development and yield can solve the technical problems of low nitrogen uptake and poor growth in crops under low nitrogen conditions.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a ZmTCP20 Applications of genes in improving crop tolerance to low nitrogen, regulating crop growth and development, and yield. ZmTCP20 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0006] Based on the above technical solution, the present invention can be further improved as follows: further, ZmTCP20 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

[0007] Furthermore, it can be applied through knockout or overexpression. ZmTCP20 Genes enable the regulation of crop tolerance to low nitrogen, growth and development, and yield.

[0008] Furthermore, by knocking out ZmTCP20 Genes that reduce crop tolerance to low nitrogen levels, inhibit crop growth and development, and reduce yield; through overexpression ZmTCP20 Genes can improve crop tolerance to low nitrogen levels, promoting crop growth, development, and yield.

[0009] Furthermore, the traits of growth and development are plant height, root biomass, plant biomass, ear length, and ear width; the trait of yield is yield per ear.

[0010] Furthermore, by knocking out ZmTCP20 Genes that reduce crop tolerance to low nitrogen levels result in decreased nitrogen accumulation and a slower rate of nitrate uptake; reduced plant height, root biomass, and overall plant biomass; shorter and thinner ears; and lower yield per ear. Overexpression of these genes can reduce crop tolerance to low nitrogen levels, leading to decreased nitrogen accumulation and a slower rate of nitrate uptake. ZmTCP20 Genes enhance crop tolerance to low nitrogen levels, increase nitrogen accumulation and nitrate absorption rates; increase plant height, root biomass, and overall plant biomass; and result in longer, thicker ears and increased yield per ear.

[0011] Furthermore, the crops are monocotyledonous plants.

[0012] Furthermore, the crop is corn.

[0013] The present invention also discloses a method comprising the above. ZmTCP20 Recombinant gene expression vectors.

[0014] The present invention also discloses a method comprising the above. ZmTCP20 Engineered bacteria that express genes or recombinant expression vectors.

[0015] The present invention also discloses a formulation for improving crop tolerance to low nitrogen, regulating crop growth and development and yield, comprising the above-mentioned recombinant expression vector or engineered bacteria.

[0016] The beneficial effects of this invention are: This invention discovers members of the corn TCP family. ZmTCP20 It has a direct positive regulatory effect on crop nitrogen use efficiency, through overexpression ZmTCP20 The gene can promote nitrogen uptake in crops, significantly increasing plant biomass and total nitrogen accumulation, and enhancing the root system's ability to absorb nitrate. Furthermore, two years of field trials have shown that overexpression of the gene... ZmTCP20 It can significantly increase crop plant height and biomass, increase nitrogen accumulation in the plant, make crop ears longer and thicker, and thus increase ear yield, by knocking out genes. ZmTCP20 The opposite is true afterwards. Genes ZmTCP20 The functional analysis provides important genetic resources and theoretical guidance for cultivating new crop varieties with high nitrogen utilization efficiency. Attached Figure Description

[0017] Figure 1 for ZmTCP20 Electrophoresis results of full-length PCR products of the gene; Figure 2 for ZmTCP20 A graph showing gene expression levels in different maize tissues; Figure 3 Phylogenetic tree diagram of TCP family proteins; Figure 4 for ZmTCP20 Gene amino acid sequence analysis and protein domain mapping; Figure 5 This is a schematic diagram of the structure of the P-super promoter::ZmTCP20-GFP recombinant vector; Figure 6 Subcellular localization map of ZmTCP20 protein; Figure 7 This is a schematic diagram of the structure of the GST-ZmTCP20 recombinant plasmid; Figure 8 This is a Western blot image of the GST-ZmTCP20 protein; Figure 9 for ZmTCP20 Image showing the identification results of gene knockout material types; Figure 10 for ZmTCP20 Gene expression level map of gene overexpression material; Figure 11 for ZmTCP20 Phenotypic diagram of nitrogen hydroponic treatment on transgenic materials; Figure 12 for ZmTCP20 Phenotypic diagram of the first leaf of the transgenic material after nitrogen hydroponic treatment; Figure 13 for ZmTCP20 Biomass of transgenic materials after nitrogen hydroponics treatment; Figure 14 for ZmTCP20 A graph showing the nitrogen accumulation of genetically modified materials after hydroponic nitrogen treatment. Figure 15 for ZmTCP20 Graph of nitrate uptake rate of transgenic materials after nitrogen hydroponic treatment; Figure 16 for ZmTCP20 NO3 in root tips of transgenic materials after nitrogen hydroponics treatment - Flow velocity diagram; Figure 17 for ZmTCP20 Phenotypic diagram of transgenic material plants at the tasseling stage treated with nitrogen fertilizer in the field; Figure 18 for ZmTCP20Plant height of transgenic material at the tasseling stage after nitrogen fertilizer treatment in the field; Figure 19 for ZmTCP20 Biomass of transgenic material plants at the tasseling stage after nitrogen fertilizer treatment in the field; Figure 20 for ZmTCP20 A graph showing the nitrogen accumulation of transgenic materials during the tasseling stage after nitrogen fertilizer treatment in the field. Figure 21 for ZmTCP20 Phenotypic diagram of the spike of transgenic material at harvest after nitrogen fertilizer treatment in the field; Figure 22 for ZmTCP20 Image showing the length of the fruit spike at harvest after nitrogen fertilizer treatment of transgenic material in the field; Figure 23 for ZmTCP20 Image of ear diameter of transgenic material at harvest after nitrogen fertilizer treatment in the field; Figure 24 for ZmTCP20 Figure showing the yield per ear of transgenic material at harvest after nitrogen fertilizer treatment in the field. Detailed Implementation

[0018] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0019] ZmTCP20 The nucleotide sequence of the gene is as follows: ATGGACCCCAAGTTCCCCCCACCCCCACCGCTAAACAAAACGGAGCCCACCACCGCGACGACCACCACCACCTCGACCGCGCAGCAGCAGCAGCAGCAGCTGGATCCTAAGGACTACCAGCAGCAGCAGCAGCAGCCGGCGCAGCACCTGCAAATCCAAATCCACCAGTCGCAGCAGGACGGAGGCGGCGGAGGGAAGGAGCAGCAGCAGCTGCAGGTGGTGGCGCAGCCCGGGGAGAGGAGGCAGCAGGCGCTCGCGCCCAAGCGGAGCTCCAACAAGGACCGACACACCAAGGTCGACGGCAGGGGCCGGCGGATCCGGATGCCCGCGCTCTGCGCCGCGCGGATCTTCCAGCTCACGCGGGAACTCGGCCACAAGTCCGACGGCGAGACCGTCCAGTGGCTGCTGCAGCAGGCCGAGCCGGCCATCGTCGCCGCCACCGGCACGGGCACCATACCGGCGTCCGCGCTCGCCTCCGTCGCGCCCTCGCTCCCGTCGCCCACCTCCGGGCTCGCCAGGCCGCACCACCACATGTGGGCGCCGTCCGCCGGCTTCTCCTCGCCCTCCTTCCTGAACTCTGCCGCCGCGGGCACGGGCGATGCCGCCGGTATCATGCAGCGGATGGGGATCCCCGCGGGCTTCGAGCTGCCGGGAGCCTCCGCCGCCGGAGCCACCCTCGGCGCCGGCGGCCACATCGGCTTTGCGCCCATGTTCGCTGGACACGCCGCCGCCATGCCGGGGCTCGAGCTCGGGCTATCGCAGGACGGCCACATCGGCGTGCTCGCCGCGCAGTCGATCAGCCAGTTCTACCACCAGGTGGGTGCTGCCGCCGGCGGCGGCGGCCAGATGCATCACGCGCACGGGCACCATCATCACCATCACCAGCAGCAGGAGGACGGGGAGGACGACCGCGAGGACGGCGAGTCCGATGACGAGTCTGGGCAGTAG(SEQ ID NO:1)。

[0020] ZmTCP20The amino acid sequence of the protein encoded by the gene is as follows: MDPKFPPPPPLNKTEPTTATTTTTSTAQQQQQQLDPKDYQQQQQQPAQHLQIQIHQSQQDGGGGGKEQQQLQVVAQPGERRQQALAPKRSSNKDRHTKVDGRGRRIRMPALCAARIFQLTRELGHKSDGETVQWLLQQAEPAIVAATGTGTIPASALAS VAPSLPSPTSGLARPHHHMWAPSAGSSPSFLNSAAAGTGDAAGIMQRMGIPAGFELPGASAAGATLGAGGHIGFAPMFAGHAAAMPGLELLGLSQDGHIGVLAAQSISQFYHQVGAAAGGGGQMHHAHGHHHHHHQQQEDGEDDREDGESDDESGQ (SEQ ID NO: 2).

[0021] The maize variety ND101 used has been publicly disclosed in the following literature: 1.Feng T, Wang Y, Zhang M, Zhuang J, Zhou Y*,Duan L*. ZmSCE1a positively regulates drought tolerance by enhancing the stability of ZmGCN5. Plant Journal. 2024, 120(5): 2101-2112. 2.Wang X, Ren Z, Xie S, Li Z, Zhou Y*,Duan L*. Jasmonate mimicmodulates cell elongation by regulating antagonistic bHLH transcription factors via brassinosteroid signaling. Plant Physiology. 2024,295(4): 2712-2726. Example 1 ZmTCP20 Cloning of genes 1. RNA was extracted from maize (ND101) leaves using the Adley Reverse Transcription Kit (purchased from Beijing Adley Biotechnology Co., Ltd.), and then first-strand cDNA was synthesized using the M-MLV Reverse Transcription Kit (purchased from Takara). The first-strand cDNA product was diluted 5-fold and used as a template for amplification. ZmTCP20The full-length gene was amplified using the Novizan Planta® Max Super-Fidelity DNA Polymerase kit. ZmTCP20 The full-length gene was used. The 10 μL PCR reaction system included: 5 μL of 2×Phanta Max Buffer, 0.2 μL of dNTP Mix (10 mM), 1 μL of cDNA, 0.2 μL of Phanta Max Super-Fidelity DNA Polymerase, 0.4 μL of upstream primer F1, 0.4 μL of downstream primer R1, and 2.8 μL of ddH2O. The PCR amplification program was: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 30 s, for 35 cycles; and a final extension at 72℃ for 5 min (a total of 20 reaction systems).

[0022] according to ZmTCP20 The gene sequence was used to design two specific primers (upstream primer F1 and downstream primer R1) for PCR amplification to obtain the PCR product. The sequences of the upstream primer F1 and the downstream primer R1 used are as follows: Upstream primer F1: ATGGACCCCAAGTTCCCCCC (SEQ ID NO: 3); Downstream primer R1: CTACTGCCCAGACTCGTCAT (SEQ ID NO: 4).

[0023] The PCR products were electrophoresed on a 1.5% agarose gel, and the results are as follows. Figure 1 As shown. The product was recovered and purified using an agarose gel DNA recovery kit (purchased from Kangwei Biotechnology Co., Ltd.), ultimately yielding the purified fragment. The recovered fragment was sequenced (performed by Beijing BGI Sequencing Co., Ltd.) to obtain the full-length gene CDS, i.e. ZmTCP20 Gene sequence. Sequencing results show that... ZmTCP20 The gene sequence is 948 bp in length and encodes a complete ORF reading frame of 315 amino acids.

[0024] 2. Genes ZmTCP20 Different organizational expression modes RNA was extracted from different maize tissues (roots, leaves, leaf sheaths, stems, silks, young ears, pedicels, and anthers) using the Adley Reverse Transcription Kit (purchased from Beijing Adley Biotechnology Co., Ltd.). First-strand cDNA was then synthesized using the M-MLV Reverse Transcription Kit (purchased from Takara). The first-strand cDNA product was diluted 5-fold and used as a template for amplification. ZmTCP20 Gene expression levels were determined. Real-time quantitative PCR was performed using the upstream primer qPCR-ZmTCP20F and the downstream primer qPCR-ZmTCP20R to detect gene expression. ZmTCP20 Expression levels in different tissues, detection results as follows Figure 2 As shown. ZmTCP20 Genes are expressed in various tissues, with higher expression in the roots, spikelets, and pedicels, providing the basis for their biological functions.

[0025] The sequences of the upstream primer qPCR-ZmTCP20F and the downstream primer qPCR-ZmTCP20R used are as follows: Upstream primer qPCR-ZmTCP20F: GCAGCAGCAGCAGCTGG (SEQ ID NO: 5); Downstream primer qPCR-ZmTCP20R: CCACCTGCAGCTGCTGCTGC (SEQ ID NO: 6).

[0026] Example 2: Purification of ZmTCP20 protein 1. ZmTCP20 protein sequence analysis The full-length protein sequence of gene ZmTCP20 was analyzed using the NCBI website, and phylogenetic analysis of its protein sequence was performed using MEGA. The results are as follows: Figure 3 As shown, genes ZmTCP20 The encoded protein sequence is similar to that in Arabidopsis thaliana. AtTCP20 Protein and rice OsTCP3 The protein showed high homology. The amplified full-length gene sequence was translated into amino acid sequences and entered into the NCBI website for CD-Search analysis. Figure 4 The prediction shows that the protein is 315 amino acids long in total, with amino acid sequences from position 90 to position 187 being conserved domains of TCP family transcription factors, which contain helical-loop helical amino acid residues and typical transcription factor DNA binding site domains.

[0027] 2. Subcellular localization of ZmTCP20 protein (1) The subcellular localization of ZmTCP20 protein was studied using a tobacco leaf expression system. Based on the multiple cloning site of the expression vector P-super1300-GFP and... ZmTCP20 Gene coding region sequence was used to design an amplified gene. ZmTCP20 The upstream primer GFP-ZmTCP20-F1 and the downstream primer GFP-ZmTCP20-R1 of the entire coding region were used to obtain the P-super promoter::ZmTCP20-GFP recombinant vector. The specific method was as follows: using the primer obtained in Example 1... ZmTCP20 Using the gene as a template, PCR amplification was performed using the upstream primer GFP-ZmTCP20-F1 and the downstream primer GFP-ZmTCP20-R1 to obtain the gene containing... ZmTCP20The product of the homologous arm of the gene and the vector; the vector P-super1300-GFP was digested with XbaI and purified to obtain the vector frame; the PCR-purified product and the vector frame were ligated to obtain the P-super promoter::ZmTCP20-GFP recombinant vector ( Figure 5 The P-super1300-GFP vector is cleaved at the XbaI restriction site and then ligated. ZmTCP20 The recombinant vector was obtained by transing the gene sequence while keeping the other sequences of P-super1300-GF unchanged.

[0028] Upstream primer GFP-ZmTCP20-F1: 5'-CACCAAATCGACTCTAGAATGGACCCCAAGTTCCCCCC-3' (SEQ ID NO: 7); Downstream primer GFP-ZmTCP20-R1: 5'-ATAGGTACCCGGGCTCTAGACTGCCCAGACTCGTCAT-3' (SEQ ID NO: 8).

[0029] (2) Agrobacterium preparation: The constructed P-super promoter::ZmTCP20-GFP recombinant vector was transformed into Agrobacterium GV3-101. Successfully transformed Agrobacterium single clones were identified and used for tobacco injection. The specific transformation method is as follows: Agrobacterium competent cells stored at -80℃ were placed at room temperature and partially thawed (ice-water mixture) before being placed on ice. 1 μg of plasmid DNA (P-super promoter::ZmTCP20-GFP recombinant vector) was added to every 100 μL of competent cells. The mixture was stirred by hand at the bottom of the tube and then placed on ice for 5 min, liquid nitrogen for 5 min, 37℃ water bath for 5 min, and ice bath for 5 min in sequence. 700 μL of antibiotic-free YEP liquid medium was added and cultured at 28℃ with shaking for 2 h. Subsequently, the bacteria were collected by centrifugation at 6000 rpm for 1 min. About 100 μL of supernatant was collected, and the bacterial blocks were gently resuspended by pipetting and spread on YEP plates containing antibiotics (kanamycin and rifampin). The plates were incubated at 28℃ for 3 days.

[0030] (3) Planting tobacco: Plant tobacco seeds in soil, and take them out for use after about 30 days when the seedlings have grown to 4-5 leaves.

[0031] (4) Tobacco injection: Transformed Agrobacterium single clones were picked and placed in 10 mL of YEP medium containing antibiotics (50 mg / L kanamycin and 25 mg / L rifampin) and cultured at 28°C and 250 rpm for 36 h on a shaker. Then, the cells were collected by centrifugation at 4000 rpm for 10 min at room temperature and resuspended in 250 mL of resuspension buffer (2.5 mL 1 M MgCl2; 500 μL 0.1 M AS; 5 mL 0.5 M MES; pH=5.7) to OD. 600 =0.6, and let stand in the dark for 3 hours. Take the prepared tobacco leaves, draw the bacterial solution into a syringe, remove the needle, and use your finger to hold the front of the leaf to allow the bacterial solution to penetrate from the back of the leaf. After injection, incubate in the dark for 12 hours, and then move the tobacco to the light for 48 hours.

[0032] (5) Observation: Gently cut tobacco leaves (the part injected with Agrobacterium) with scissors, place them on a coverslip, and add a small amount of deionized water to adhere the slide. Place the prepared slide under a laser confocal microscope to observe the expression of GFP. The results showed that ( Figure 6 GFP green fluorescence signal was observed in the cell nuclei of tobacco leaves, and the ZmTCP20 protein was located in the cell nucleus.

[0033] 3. Purification of ZmTCP20 protein The one obtained in Example 1 ZmTCP20 Using the gene as a template, PCR amplification was performed using upstream primer F2 and downstream primer R2 to obtain a sample containing... ZmTCP20 The product of gene and vector homologous arms; the product was recovered and purified by digesting the vector pGEX-4T-1 with EcoRI and BamHI. ZmTCP20 Gene fragments and the pGEX-4T-1 linear vector were used; the gene fragments and the linear vector were ligated to obtain the GST-ZmTCP20 recombinant plasmid, as shown below. Figure 7 As shown, the DNA fragment between the BamHI and EcoRI restriction sites of pGEX-4T-1 is replaced. ZmTCP20 The recombinant plasmid was obtained by preserving the pGEX-4T-1 gene and other sequences. The sequences of the upstream primer F2 and the downstream primer R2 used are as follows: Upstream primer F2: 5'-CTGGTTCCGCGTGGATCCATGGACCCCAAGTTCCCCCC-3' (SEQ ID NO: 9); Downstream primer R2: 5'- GAGTCGACCCGGGAATTCCTGCCCAGACTCGTCAT-3' (SEQ ID NO: 10).

[0034] The GST-ZmTCP20 recombinant plasmid was transformed into *E. coli* BL21 using the heat shock transformation method. After overnight culture at 37°C, positive single colonies were picked and inoculated into 2 mL of LB broth containing 100 mg / L ampicillin antibiotic, and cultured overnight at 37°C and 200 rpm. The bacterial culture was then inoculated into 100 mL of LB broth containing ampicillin antibiotic and cultured at 37°C and 200 rpm until OD (dose expiratory rate) was reached. 600 To a final concentration of 0.8, transfer the bacterial culture to a 28°C shaker and cool for 15 min. Then add IPTG (isopropyl-β-D-thiopyranoside, final concentration 0.8 mM) and induce at 28°C and 100 rpm for 6 h. Collect the bacterial culture in a 50 mL round-bottom tube and centrifuge at 4°C and 4000 rpm for 15 min. Discard the supernatant and collect the bacterial cells. Resuspend the cells in 10 mL of protein lysis buffer (2 mL 1 M NaH₂PO₄; 360 μL 1 M K₂HPO₄; 270 μL 2 M NaCl; 5.6 mL 5 M NaCl, diluted to 200 mL with water). Add lysin (final concentration 100 µg / mL) and protease inhibitor (final concentration 0.1 mg / mL), mix well, and incubate on ice for 15 min. Transfer the round-bottom tube to an ice box for heat conduction during ultrasonic disruption. Set the ultrasonic disruption program to: 30% power, 10 min, with 5 s intervals for 5 s of ultrasonication. After sonication, the beads were centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was transferred to a clean 15 mL centrifuge tube, and 100 μL of GST-beads equilibrated with protein lysis buffer was added. Triton X-100 was added to bring the final solution to a Triton X-100 volume fraction of 0.5%. The mixture was then incubated at 4 °C for 4 h. After incubation, the beads were centrifuged at 4,000 rpm for 5 min, the supernatant was discarded, and the beads were washed three times with lysis buffer. The collected beads were centrifuged and transferred to a clean 1.5 mL EP tube. An appropriate amount of elution buffer (0.36 g Maltose to 100 mL of lysis buffer) was added, and the beads were eluted at room temperature for 10-15 min or at 4 °C for 2-3 h. The supernatant was then transferred to a clean 1.5 mL EP tube, and 5 μL was used for protein electrophoresis and Western blot. The experimental results are as follows: Figure 8 As shown, the remaining purified protein was added to a final concentration of 15% glycerol, mixed well, and then aliquoted into centrifuge tubes and stored at -80°C.

[0035] Example 3 ZmTCP20 Obtaining transgenic plants 1. ZmTCP20 Obtaining knockout materials The transgenic materials were obtained from the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University and identified. The steps for obtaining the transgenic materials are as follows: (1) After the transformation vector was constructed, Agrobacterium (GV3101) was used for transformation; (2) Embryo preparation: Select Agrobacterium-mediated transformation technology and select suitable ND101 embryos for subsequent transformation; (3) Infection: Invert the 2 mL round-bottom centrifuge tube containing the embryos 3 times, ensuring that the embryos do not stick to the wall, and heat shock at 45°C for 3 min. After removing the infection solution with a pipette tip, transfer the embryos to a 50 mL centrifuge tube containing Agrobacterium infection solution with a long spoon, gently shake the centrifuge tube 20 times, immerse the embryos vertically, and incubate in the dark at 22°C for 30 min. Infection is complete. (4) Co-culture: Aspirate the bacterial culture, transfer the embryonic embryos to a co-culture medium lined with two layers of sterile filter paper, and turn the embryos over with the back of a No. 15 scalpel. Seal the culture dish with breathable sealing film and incubate in the dark at 22°C for 36 hours; (5) Screening (first screening, second screening): After co-culture, the immature embryos are transferred to induction-selection medium, evenly arranged, and cultured in the dark at 28℃ for 14-16 days. After the first screening, the medium is changed, and the second screening is performed after 14-16 days; (6) Predifferentiation: Transfer the callus with the radicle removed to the predifferentiation medium, place 16 callus per dish, and culture in a low light cycle of 16h light + 8h darkness at 25℃ for 14 days to obtain mature callus. (7) Differentiation: Transfer mature callus to differentiation medium, with 8 mature callus per dish. Culture conditions are 25℃ and 80-100 μE / mL. 2 With a light intensity of / s, and after 30 days, three-leaf seedlings differentiated, resulting in genetically transformed seedlings; (8) Rooting: Transfer the rootless seedlings to a rooting medium and culture them under light. New roots will begin to grow after one week. (9) Transplanting: When the seedlings in the rooting bottle have grown more than 3 roots, T1 genetically transformed seedlings are obtained.

[0036] The above steps are used to obtain T2 generation or higher transgenic materials. zmtcp20 #Remove materials and ZmTCP20 (overexpression material).

[0037] For T2 generation and above zmtcp20 #Knockout materials were used, and DNA was extracted from the plants using the CTAB method for identification. Primers were designed near the target gRNA site, and the target band was amplified using the upstream primer CR-ZmTCP20F and the downstream primer CR-ZmTCP20R. The products were then sent to the company for sequencing (sequencing was performed by Beijing BGI Sequencing Co., Ltd.). The identification results are as follows: Figure 9 As shown, two mutation types were identified, namely mutants. zmtcp20 #1 and mutants zmtcp20 #2. Mutant zmtcp20#1 is a 14-base knockout mutation that results in a frameshift mutation in protein expression; mutant zmtcp20 #2 represents a single-base insertion mutation, resulting in a frameshift mutation in protein expression. The sequences of the upstream primer CR-ZmTCP20F and the downstream primer CR-ZmTCP20R used for detection are as follows: Upstream primer CR-ZmTCP20F: 5'-ATGGACCCCAAGTTCCCCCC-3' (SEQ ID NO: 11); Downstream primer CR-ZmTCP20R: 5'-CACTGGACGGTCTCGCCGTCGG-3' (SEQ ID NO: 12).

[0038] 2. ZmTCP20 Obtaining overexpression plants For T2 generation and above ZmTCP20 RNA was extracted from maize leaves using the overexpression materials (ZmTCP20OE1 and ZmTCP20OE2) using an Adley kit (purchased from Beijing Adley Biotechnology Co., Ltd.), and first-strand cDNA was synthesized using an M-MLV reverse transcription kit (purchased from Takara). The obtained first-strand cDNA was used as a template for real-time quantitative PCR using upstream primer OE-ZmTCP20F and downstream primer OE-ZmTCP20R to detect the gene. ZmTCP20 The expression level, the detection results are as follows Figure 10 As shown, genes in overexpressed materials ZmTCP20 The expression level far exceeded that of the donor material ND101. The sequences of the upstream primer OE-ZmTCP20F and the downstream primer OE-ZmTCP20R are as follows: Upstream primer OE-ZmTCP20F: 5'-GCAGCAGCAGCAGCAGCTGG-3' (SEQ ID NO: 13); Downstream primer OE-ZmTCP20R: 5'-CCACCTGCAGCTGCTGCTGC-3' (SEQ ID NO: 14).

[0039] Example 4 ZmTCP20 Experiment on nitrogen hydroponic treatment of transgenic plants 1. ZmTCP20 Sensitivity of transgenic materials to nitrogen stress right ZmTCP20 Knockout mutant plants ( zmtcp 20#1 and zmtcp 20#2) ZmTCP20Overexpressing plants (ZmTCP20OE1 and ZmTCP20OE2) and ND101 control plants were subjected to controlled nitrogen hydroponic treatment. The hydroponic treatment was divided into a normal nitrogen treatment (control treatment) group and a hydroponic solution containing NO3. - The concentration of NO3 in the hydroponic solution was 4 mM / L. Low nitrogen stress treatment (low nitrogen treatment) group: - The concentration is 0.05 mM / L. For example... Figure 11 As shown, compared with the ND101 control plants, ZmTCP20 Knockout mutant plants ( zmtcp 20#1 and zmtcp 20#2) is more sensitive to low nitrogen stress. ZmTCP20 Overexpressing plants (ZmTCP20OE1 and ZmTCP20OE2) were less sensitive. This was observed in the first leaf... Figure 12 It was learned that under low-nitrogen treatment, ZmTCP20 The leaves of the knockout mutant plants aged and dried out. ZmTCP20 Overexpression makes the leaves of plants more tolerant to low nitrogen treatment.

[0040] After 7 days of low-nitrogen treatment, the roots of corn plants were collected, dried with absorbent paper, placed in envelopes, and dried in a 75℃ oven for 2 days. The dry weight of the roots was then measured. The results showed that ( Figure 13 Under normal nitrogen treatment and low nitrogen stress, compared with the control group ND101, ZmTCP20 Plants that overexpress the gene have higher root biomass. ZmTCP20 Conversely, the knockout mutant plants have a lower root biomass than ND101.

[0041] 2. After nitrogen treatment ZmTCP20 Determination of total nitrogen accumulation in transgenic materials Experimental plants were selected under normal nitrogen treatment and low nitrogen stress treatment, respectively. zmtcp 20#1、 zmtcp Nine maize seedlings of uniform size were randomly selected under each treatment (20#2, ZmTCP20OE1, ZmTCP20OE2, and ND101), with three seedlings per replicate, for a total of three biological replicates. The sample surface was cleaned with deionized water, and the underground root system was collected. After blotting with absorbent paper, the sample was placed in an envelope and placed in an oven at 105℃ for 30 min to kill the green color, followed by drying at 75℃ to constant weight. The sample was then weighed, ground, and passed through a 200-mesh sieve. 0.1 g of the ground sample was added to a digestion tube, along with a nitrogen analyzer and 5 mL of concentrated sulfuric acid. The tube was digested at 410℃ for 1 h, then cooled to room temperature and analyzed using a Kjeldahl nitrogen analyzer. The specific method for total nitrogen content was described in Bremner (1982). Blank values ​​and recoveries were determined using the same weight of sucrose and ammonium sulfate as the test sample for each determination.

[0042] ; ; ; Where a is the recovery rate (%), A is the actual nitrogen content (mg / g), b is the nitrogen content of the plant sample (mg / g), V2 is the volume of hydrochloric acid used in the sample titration (mL), V1 is the volume of hydrochloric acid used in the blank titration (mL), C is the concentration of the hydrochloric acid standard solution (mol / L), M is the mass of the plant sample (g), c is the nitrogen accumulation of the plant (mg), and d is the biomass of the plant sample (g).

[0043] The results are as follows Figure 14 As shown, under both normal and low nitrogen treatments, compared to ND101, ZmTCP20 Overexpressing plants have higher nitrogen accumulation levels. ZmTCP20 Knockout mutant plants accumulate less nitrogen.

[0044] 3. After nitrogen treatment ZmTCP20 Measurement of nitrate absorption rate of transgenic materials Plants with uniform growth after 6 days of nitrogen treatment were placed in a 0.1 mM CaSO4 solution for 1 min to equilibrate, and then transferred to a solution containing 0.05 mM and 4 mM Ca(II)2. 15 The roots were placed in a 299% (atom) solution of NO3 for 30 minutes. After treatment, the roots were rinsed in deionized water for 1 minute to remove residual Ca2+ on the surface. 15 NO3) 2. After absorbing the water from the root surface, place it in a 120℃ oven to dry for 30 minutes, then dry it at 75℃ to constant weight. Next, grind the sample, pass it through a 100-mesh sieve, and accurately weigh 0.02 mg of the sieved sample. Wrap it in a tin cup and analyze it using a stable isotope mass spectrometer (Vario PYRO cube ISOprime 100, CheadleHulme, UK). 15 Nitrogen content determination.

[0045] The results are as follows Figure 15 As shown, compared with ND101, under different nitrogen treatments ZmTCP20 The roots of overexpressing the gene can absorb more nutrients per unit time. 15 NO3 - ,and ZmTCP20 The root system of knockout mutant plants absorbs nutrients per unit time. 15 NO3 - less.

[0046] 4. After nitrogen treatment ZmTCP20 NO3 in the root tip of genetically modified material -Measurement of flow rate Determination of NO3 in root samples using non-destructive ion micrometer (NMT) - Flow rate, measured at a distance of 700 µm from the root tip for NO3 - The absorption rate was measured. The specific procedure was as follows: The root tips of nitrogen-treated maize seedlings were cut and placed in a equilibration solution (0.1 mM NH4NO3, 0.1 mM KCl, 0.1 mM CaCl2, 0.3 mM MES, pH=6.0) for 10-20 min. The equilibrated root tips were then fixed in a new petri dish. 5 mL of test buffer (4 mM Ca(NO3)2 and 0.05 mM Ca(NO3)2) was added. The sample was placed under a microscope, and the microscope and microelectrode were adjusted so that the tip of the microelectrode was approximately 5 mm from the test surface. Measurements were then taken, and readings were started after the data stabilized. The test duration was 3-5 min, with NO3 recorded every 6 seconds. - Flow rate, calculated by taking 5 values ​​read within 30 seconds, average flow rate, NO3 - Flow velocity data were analyzed using imFluxes software (imfluxes.com, Xuyue (Beijing) Sci.&Tech. Co., Ltd., Beijing, China).

[0047] The results are as follows Figure 16 As shown, compared with ND101, under different nitrogen treatments ZmTCP20 NO3 knockout mutant plant roots - A lower flow rate indicates a weaker ability to absorb nitrate ions; ZmTCP20 Overexpression of NO3 in plant roots - The flow rate is relatively high, and the root system has a strong absorption capacity.

[0048] Example 5 ZmTCP20 Field planting trials of nitrogen treatment in transgenic plants 1. ZmTCP20 Growth status of genetically modified materials Through the ZmTCP20 Knockout mutant plants ( zmtcp 20#1 and zmtcp 20#2) ZmTCP20Field trials were conducted on overexpressing plants (ZmTCP20OE1 and ZmTCP20OE2) and ND101 control plants at the Wuqiao Experimental Station of China Agricultural University, Hebei Province (37°36′N, 116°28′E) to investigate field phenotypic and yield results. The field fertilization program followed a nitrogen fertilizer application protocol (control group: nitrogen content 225 kg / ha; low nitrogen treatment group: nitrogen content 75 kg / ha), with other fertilizers applied consistently. Fertilizer was applied as a single basal fertilizer application. Sowing plots were established in different nutrient plots, with each plot having a row length of 2.5 m, a row spacing of 0.6 m, and a plant spacing of 0.25 m. Single and double seeds were sown, and manual thinning was performed at the three-leaf stage to ensure 10 plants per row. Three replicates were established in each nutrient plot. Field phenotypic observations and statistical analysis were conducted at the tasseling stage and harvest stage.

[0049] Field trial results during the tasseling stage of the plant are as follows: Figure 17 As shown, under normal nitrogen fertilizer and low nitrogen fertilizer treatments, compared with ND101, ZmTCP20 Overexpression results in better growth of the above-ground parts of the plant and slower senescence of the lower leaves; ZmTCP20 The above-ground parts of the knockout mutant plants are smaller, and the bottom leaves age and fall off prematurely, but there is no difference in the growth period.

[0050] 2. ZmTCP20 Plant height and biomass of transgenic materials after nitrogen treatment in the field The plant height of maize at the tasseling stage was measured. Thirty maize plants were randomly selected from each treatment group for height measurement. The results are as follows: Figure 18 As shown, under normal nitrogen fertilizer and low nitrogen fertilizer treatments, ZmTCP20 The height of the overexpressing plants was significantly higher than that of the control plants ND101, and at the same time, ZmTCP20 The height of the knockout mutant plants was significantly lower than that of the control plant ND101.

[0051] After the maize reached the tasseling stage, six maize plants were randomly selected from each treatment. The samples were dried in an oven at 70℃ before biomass was measured. The experimental results are as follows: Figure 19 As shown, under normal nitrogen fertilizer and low nitrogen fertilizer treatments, compared with ND101, ZmTCP20 Overexpression plants have greater biomass, and at the same time, ZmTCP20 The biomass of the knockout mutant plants was significantly lower than that of the control plant ND101.

[0052] 3. ZmTCP20 Determination of total nitrogen accumulation in transgenic materials after field nitrogen treatment The total nitrogen accumulation of maize plants during the tasseling stage was determined using the same method as in Example 4. The results are as follows: Figure 20 As shown, under normal nitrogen treatment and low nitrogen treatment, compared with ND101, ZmTCP20 Overexpression plants had higher total nitrogen accumulation. ZmTCP20 Knockout mutant plants accumulate less nitrogen.

[0053] 4. ZmTCP20 Field yield of genetically modified materials after nitrogen treatment After the corn ears were dried at harvest time, photos were taken and the ear length, ear width, and ear yield were recorded. Thirty corn ears were randomly selected from each treatment for measurement and data analysis.

[0054] The phenotypic results of the ear are as follows Figures 21-23 As shown, under different nitrogen treatments, compared with ND101, ZmTCP20 Overexpression resulted in larger, longer, and thicker fruit clusters in the plants. ZmTCP20 The phenotype is reversed after knocking out the fruit; the ears become shorter and thinner.

[0055] The yield per ear of maize in each treatment group was calculated, and the results are as follows: Figure 24 As shown, compared to ND101, ZmTCP20 The yield per ear of maize in overexpressing plants was significantly increased; ZmTCP20 Knocking out corn significantly reduced the yield per ear.

Claims

1. A kind ZmTCP20 The application of genes in improving crop tolerance to low nitrogen, regulating crop growth and development, and yield is characterized by, The ZmTCP20 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The application involves knockout or overexpression. ZmTCP20 Genes enable the regulation of crop tolerance to low nitrogen, growth and development, and yield.

3. The application according to claim 2, characterized in that, By knocking ZmTCP20 Genes that reduce crop tolerance to low nitrogen levels, inhibit crop growth and development, and reduce yield; through overexpression ZmTCP20 Genes can improve crop tolerance to low nitrogen levels, promoting crop growth, development, and yield.

4. The application according to claim 1 or 2, characterized in that, The growth and development traits are plant height, root biomass, plant biomass, ear length, and ear width; the yield trait is yield per ear.

5. The application according to claim 1 or 2, characterized in that, By knocking ZmTCP20 Genes that reduce crop tolerance to low nitrogen levels result in decreased nitrogen accumulation and a slower rate of nitrate uptake; reduced plant height, root biomass, and overall plant biomass; shorter and thinner ears; and lower yield per ear. Overexpression of these genes can reduce crop tolerance to low nitrogen levels, leading to decreased nitrogen accumulation and a slower rate of nitrate uptake. ZmTCP20 Genes enhance crop tolerance to low nitrogen levels, increase nitrogen accumulation and nitrate absorption rates; increase plant height, root biomass, and overall plant biomass; and result in longer, thicker ears and increased yield per ear.

6. The application according to claim 1, characterized in that, The crop in question is a monocotyledonous plant.

7. The application according to claim 6, characterized in that, The crop in question is corn.

8. A recombinant expression vector, characterized in that, Includes the claims 1 ZmTCP20 Gene.

9. An engineered bacterium, characterized in that, Includes the claims 1 ZmTCP20 The gene or the recombinant expression vector as described in claim 8.

10. A formulation for improving crop tolerance to low nitrogen, regulating crop growth and yield, characterized in that, It comprises the recombinant expression vector of claim 8 or the engineered bacteria of claim 9.

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