Application of ZmGLK63 and its encoding gene in increasing yield and resisting lodging

Abstract

The present invention discloses the application of ZmGLK63 and its encoding gene in increasing yield and lodging resistance, belonging to the field of genetic engineering technology. The present invention has found that the ZmGLK63 gene can regulate the yield, nitrogen utilization ability, and lodging resistance of corn. After knocking out the ZmGLK63 gene using gene editing technology, the ZmGLK63 gene-edited strain showed higher nitrogen utilization efficiency and lodging resistance compared to wild-type corn, and was able to significantly increase corn yield. The present invention provides a new genetic resource for the creation of new high-yield, lodging-resistant corn varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to application of ZmGLK63 and its encoding gene in increasing yield and resisting lodging. Background Art

[0002] Maize (Zea mays L.) is an important food crop. High-volume nitrogen fertilizer application is the primary method for increasing maize yield. However, long-term excessive nitrogen fertilizer application not only reduces nitrogen fertilizer use efficiency and pollutes the environment, but also easily causes crop lodging. Maize lodging directly impacts yield stability and mechanized production efficiency. Lodging is primarily caused by insufficient stalk strength, defective root architecture, or imbalanced plant morphology, and is particularly pronounced under high-density planting and extreme climate conditions.

[0003] Improving maize's nitrogen use efficiency and lodging resistance can significantly reduce nitrogen fertilizer use, increase maize yields and mechanized production efficiency, and reduce environmental pollution. This is a primary means of achieving reduced-yield production and agricultural mechanization. Therefore, identifying superior maize genetic resources with high nitrogen use efficiency and lodging resistance, and developing new maize varieties with these properties, is crucial for food security and sustainable agricultural development. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of ZmGLK63 and its encoding gene in improving yield and lodging resistance, so as to solve the problems existing in the above-mentioned prior art. The present invention has found that the ZmGLK63 gene can regulate the yield, nitrogen utilization ability and lodging resistance of corn. After knocking out the ZmGLK63 gene using gene editing technology, the ZmGLK63 gene-edited strain showed higher nitrogen utilization efficiency and lodging resistance compared with wild-type corn, and was able to significantly increase corn yield, providing a new gene resource for the creation of new high-yield and lodging-resistant corn varieties.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides applications of biomaterials related to the ZmGLK63 protein or its encoding gene, wherein the application is any of the following:

[0007] A1) Increase plant yield;

[0008] A2) Improve plant nitrogen utilization efficiency;

[0009] A3) Improve the plant's resistance to lodging;

[0010] A4) Cultivate high-yielding plants;

[0011] A5) Cultivate plants with high nitrogen utilization efficiency;

[0012] A6) Cultivate lodging-resistant plants;

[0013] The ZmGLK63 protein is a protein having an amino acid sequence as shown in SEQ ID NO. 2, or a protein having the same function as the amino acid sequence shown in SEQ ID NO. 2 after one or more amino acid residues are substituted and / or deleted and / or added.

[0014] Furthermore, the relevant biological material is any one of the following:

[0015] B1) a ZmGLK63 gene encoding the ZmGLK63 protein;

[0016] B2) sgRNA targeting the ZmGLK63 gene described in B1);

[0017] B3) a recombinant vector for knocking out the ZmGLK63 gene described in B1);

[0018] B4) A recombinant microorganism comprising the recombinant vector described in B3).

[0019] Furthermore, the improvement method described in any one of A1) to A3) is to reduce the content or activity of the ZmGLK63 protein by inhibiting or knocking down the expression level of the ZmGLK63 protein encoding gene, so as to improve plant yield, nitrogen utilization efficiency or lodging resistance.

[0020] Optionally, the plant is corn.

[0021] The present invention also provides an sgRNA targeting the ZmGLK63 gene, the sequence of the sgRNA is shown in SEQ ID NO.5; the nucleotide sequence of the ZmGLK63 gene is shown in SEQ ID NO.1.

[0022] The present invention also provides a recombinant vector for knocking out the ZmGLK63 gene, wherein the recombinant vector contains the above-mentioned sgRNA.

[0023] The present invention also provides a recombinant microorganism with a ZmGLK63 gene knocked out, wherein the recombinant microorganism comprises the recombinant vector.

[0024] The present invention also provides a method for improving plant yield, nitrogen utilization ability and lodging resistance, comprising introducing the above-mentioned recombinant vector or the above-mentioned recombinant microorganism into a plant to inhibit or knock down the expression of the ZmGLK63 gene, thereby improving plant yield, nitrogen utilization ability and lodging resistance;

[0025] The nucleotide sequence of the ZmGLK63 gene is shown in SEQ ID NO.1.

[0026] Optionally, the plant is corn.

[0027] The present invention also provides a method for cultivating high-yield plants, high nitrogen utilization rate plants or lodging-resistant plants, comprising introducing the above-mentioned recombinant vector or the above-mentioned recombinant microorganism into a plant, inhibiting or knocking down the expression level of the ZmGLK63 gene, and constructing high-yield plants, high nitrogen utilization rate plants or lodging-resistant plants;

[0028] The nucleotide sequence of the ZmGLK63 gene is shown in SEQ ID NO.1.

[0029] Optionally, the plant is corn.

[0030] The present invention also provides a ZmGLK63 mutant gene obtained by the above method, wherein the nucleotide sequence of the ZmGLK63 mutant gene is shown as SEQ ID NO.6 or SEQ ID NO.7.

[0031] The present invention also provides an application of the ZmGLK63 mutant gene, wherein the application is any one of the following:

[0032] C1) Increase plant yield;

[0033] C2) Improve plant nitrogen utilization efficiency;

[0034] C3) Improve plant resistance to lodging.

[0035] Optionally, the plant is corn.

[0036] The present invention discloses the following technical effects:

[0037] The present study found that the ZmGLK63 gene can regulate corn yield, nitrogen utilization, and lodging resistance. Using gene editing technology to knock out the ZmGLK63 gene, ZmGLK63-edited strains exhibited higher nitrogen utilization efficiency and lodging resistance compared to wild-type corn, significantly increasing corn yield. This research provides new genetic resources for the development of new high-yield, lodging-resistant corn varieties, offering new technical support for cultivating high-yield, lodging-resistant varieties, overcoming the shortcomings of traditional breeding, and accelerating the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0039] Figure 1 Schematic diagram of the structure of the ZmGLK63 gene knockout vector;

[0040] Figure 2 Schematic diagram of the target location structure of ZmGLK63 gene knockout;

[0041] Figure 3 Schematic diagram of the mutation types of ZmGLK63 editing materials (Zmglk63-1 and Zmglk63-2);

[0042] Figure 4 Figures 1 and 2 show the field phenotype observation and thrust measurement results of the ZmGLK63 gene-edited material; a shows the field phenotype of the ZmGLK63 gene-edited material and the wild-type material; b shows the thrust of the ZmGLK63 gene-edited material and the wild-type material at the ear position; c shows the thrust of the ZmGLK63 gene-edited material and the wild-type material at 40 cm from the ground; d shows the thrust of the ZmGLK63 gene-edited material and the wild-type material at 20 cm from the ground.

[0043] Figure 5 Figures 1 and 2 show the root phenotype observation and aerial root angle measurement results of the ZmGLK63 gene-edited material; a shows the root phenotype of the ZmGLK63 gene-edited material and the wild-type material; b shows the aerial root angle measurement results of the ZmGLK63 gene-edited material and the wild-type material;

[0044] Figure 6 ZmGLK63 gene editing material 15 The results of the N isotope detection experiment are shown in Figure 1. Among them, a is the ZmGLK63 gene editing material and the wild type material. 15 N-labeled nitrate absorption rate; b is the ZmGLK63 gene-edited material and the wild-type material 15 N-labeled nitrate transport capacity;

[0045] Figure 7 This is an analysis of the yield and yield traits of wild-type maize B104 and ZmGLK63 gene-edited lines under normal nitrogen field and low nitrogen field conditions; among them, a and c are the ear phenotypes of ZmGLK63 gene-edited materials and wild-type materials under normal nitrogen field (NN) and low nitrogen field (LN) conditions, respectively; b and d are the measurement results of ear length and ear weight of ZmGLK63 gene-edited materials and wild-type materials under normal nitrogen field (NN) and low nitrogen field (LN), respectively. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are all commercially available reagents and materials unless otherwise specified.

[0052] Example 1: Obtaining ZmGLK63 gene editing material

[0053] 1. Construction of CRISPR / Cas9 gene knockout vector for ZmGLK63

[0054] Based on the ZmGLK63 gene (Zm00001eb077410, whose nucleotide sequence is shown in SEQ ID NO.1 and whose amino acid sequence is shown in SEQ ID NO.2), two target sequences spanning exons were selected and sgRNAs were designed. The sgRNAs were ligated into the pCPB vector using homologous recombination technology to construct a ZmGLK63 gene knockout vector named pCPB-Ubi::hspCas9. The structure of the ZmGLK63 gene knockout vector is shown in the figure. Figure 1 The target location is shown in Figure 2The sequences are as follows:

[0055] Target sequence 1: GAATTCGGATACGAGGGAAC (SEQ ID NO. 3).

[0056] Target sequence 2: GAATTTGCTGACTCTCAACA (SEQ ID NO. 4).

[0057] sgRNA:

[0058] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO. 5).

[0059] SEQ ID NO.1:

[0060]

[0061] SEQ ID NO.2:

[0062] MNLQSLPVATGAPYPASMAPSSSAFSTVTTHGCFPYSTPSTAHPSLSGILPCNNNMISYSVLPEEPSGGIFSGQSPEGCADPGDIDYRVESQQIPGPGRTVDESDNRDEWFVPTDITWNWHQMAESAPVPSYPNSKSWQHDEQHMVHESVSVPSEEPQQLC PTVSPLATTNNAHCPKRAKARMRWTMEMHDRFVDAVNLLGGCESAKPKAILDIMNVEGLTRDQVKSHFQKYKLQVKQHPSEVPGTSVEMTMRSEAIPSDVQKHIQDYALQVQVEFQKKLHDMVESTMLEIRRSLLENHVMSLHELEQRQSSYRGSDDAGA.

[0063] 2. Genetic transformation of maize plants

[0064] The ZmGLK63 gene knockout vector pCPB-Ubi::hspCas9 was genetically transformed into maize plants (maize inbred line B104) using Agrobacterium-mediated transfection. The resulting maize plants were identified by PCR and Sanger sequencing, ultimately yielding two homozygous mutant plants, named Zmglk63-1 and Zmglk63-2. The gene mutation types of Zmglk63-1 and Zmglk63-2 plants are as follows: Figure 3 The mutation type of Zmglk63-1 is a frameshift mutation caused by a 298bp deletion between target sites 1 and 2; the mutation type of Zmglk63-2 is a frameshift mutation caused by a 294bp insertion at target site 1 and a 300bp deletion between target sites 1 and 2. The nucleotide sequence of Zmglk63-1 is shown in SEQ ID NO. 6, and the nucleotide sequence of Zmglk63-2 is shown in SEQ ID NO. 7.

[0065] SEQ ID NO.6:

[0066]

[0067] SEQ ID NO.7:

[0068]

[0069] Example 2 Field phenotypic observation and thrust determination of ZmGLK63 gene-edited materials

[0070] The gene-edited lines Zmglk63-1 and Zmglk63-2, along with the wild-type line (CK), were planted in Langfang, Hebei Province, in the summer of 2024. The phenotypes of each line were observed in the field, and the stalk thrust at the ear, 40 cm above the ground, and 20 cm above the ground were measured using a stalk thrust meter.

[0071] The phenotypes of each strain are as follows Figure 4 As shown in a, it can be seen that the stems of Zmglk63-1 and Zmglk63-2 are thicker than those of the wild type. Figure 4 As shown in the figure (bd), the three thrusts of Zmglk63-1 and Zmglk63-2 were significantly higher than those of the wild-type material. These results indicate that editing the ZmGLK63 gene can make maize stalks thicker and improve its lodging resistance.

[0072] Example 3 Root phenotype and aerial root angle determination of ZmGLK63 gene-edited materials

[0073] To verify whether ZmGLK63 is related to lodging resistance, the roots of the gene-edited materials Zmglk63-1 and Zmglk63-2 and the wild-type material (CK) were dug out in the field for phenotypic observation and aerial root angle measurement.

[0074] Root phenotypes such as Figure 5 As shown in a, it can be seen that compared with the wild type, the roots of Zmglk63-1 and Zmglk63-2 are significantly developed and have more lateral roots. Figure 5 As shown in (b), the gene-edited material has a larger aerial root angle. These results indicate that editing the ZmGLK63 gene leads to more developed roots in maize and increases the angle of the aerial roots, thereby improving maize lodging resistance.

[0075] Example 4 ZmGLK63 gene editing material 15 N isotope detection experiment

[0076] In order to explore whether the ZmGLK63 gene is related to efficient nitrogen use, the gene-edited materials Zmglk63-1 and Zmglk63-2 and the wild-type material (WT) were subjected to laboratory 15N isotope labeling experiment. Select corn seeds of uniform size and germinate them on germination paper. After germination, culture them in clear water and then change to normal nutrient solution until they reach the three-leaf and one-heart stage (activating nitrogen-induced gene expression in corn seedlings). Then, transfer the corn seedlings to nitrogen-free culture medium and culture them in the absence of nitrogen for three days (to remove the influence of residual nitrogen). After three days of culture in the absence of nitrogen, replace it with 5mM 15 After incubating for 5 hours in a solution of N-labeled potassium nitrate, the corn seedlings were removed and rinsed three times with a calcium sulfate solution, each rinse lasting 1 minute. The aboveground and underground parts were then separated and immediately placed in a 100°C oven for 1 hour. The seeds were then dried at 65°C, ground with a grinder, passed through a 100-mesh sieve, and sent to the Analytical and Testing Center of the Institute of Environmental Science and Engineering, Chinese Academy of Agricultural Sciences, for isotope content determination.

[0077] The formula of normal nutrient solution is: 4mM KNO3, 0.75mM K2SO4, 0.65mM MgSO4, 0.1mM KCl, 0.25mM KH2PO4, 0.001mM H3BO3, 0.001mM MnSO4·H2O, 0.0001mM CuSO4·5H2O, 0.0001mM ZnSO4·7H2O, 0.000005mM (NH4)6Mo7O 24 and 0.2 mM Fe-EDTA.

[0078] The formula of nitrogen-free nutrient solution is: 0.75 mM K2SO4, 0.65 mM MgSO4, 0.1 mM KCl, 0.25 mM KH2PO4, 0.001 mM H3BO3, 0.001 mM MnSO4·H2O, 0.0001 mM CuSO4·5H2O, 0.0001 mM ZnSO4·7H2O, 0.000005 mM (NH4)6Mo7O 24 and 0.2 mM Fe-EDTA.

[0079] The results are as follows Figure 6 As shown, Zmglk63-1 and Zmglk63-2 15 The absorption and transport capacity of N-labeled nitrate was significantly higher than that of the wild type. This indicates that editing the ZmGLK63 gene can improve the absorption and transport capacity of nitrate in maize, thereby improving the nitrogen use efficiency of maize.

[0080] Example 5 Yield traits of ZmGLK63 gene-edited materials in low nitrogen fields and normal nitrogen fields

[0081] To investigate whether the ZmGLK63 gene is associated with efficient nitrogen use efficiency, the gene-edited accessions Zmglk63-1 and Zmglk63-2, along with the wild-type accession (WT), were subjected to normal and low nitrogen treatments in Langfang, Hebei Province. The normal nitrogen treatment (NN) included 191 kg / ha of urea (approximately 88 kg of pure nitrogen / ha), 750 kg / ha of superphosphate, and 135 kg / ha of potassium chloride applied before sowing. An additional 200 kg / ha of urea (approximately 92 kg of pure nitrogen / ha) was applied during the jointing stage. The low nitrogen treatment (LN) followed the same fertilization schedule as the normal nitrogen treatment, except for the omission of nitrogen fertilizer. Maize cobs were harvested and yield was determined.

[0082] The results are as follows Figure 7 As shown, the per-plant yield of Zmglk63-1 and Zmglk63-2 was significantly higher than that of the wild type under both low- and normal-nitrogen conditions. Further testing revealed that the ear length and weight of Zmglk63-1 and Zmglk63-2 were significantly higher than those of the wild type. These results suggest that editing the ZmGLK63 gene can increase corn yield under low-nitrogen conditions in the field, thereby improving nitrogen use efficiency and reducing fertilizer application.

[0083] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of ZmGLK63 protein or its encoding gene related biomaterials, characterized in that: The application is any of the following: A1) Increase plant yield; A2) Improve plant nitrogen utilization efficiency; A3) Improve the plant's resistance to lodging; A4) Cultivate high-yielding plants; A5) Cultivate plants with high nitrogen utilization efficiency; A6) Cultivate lodging-resistant plants; The method for improving in any one of A1) to A3) is to reduce the content or activity of the ZmGLK63 protein by inhibiting or knocking down the expression of the encoding gene, thereby improving plant yield, nitrogen utilization efficiency or lodging resistance; The method of cultivating according to any one of A4) to A6) is to cultivate high-yield plants, plants with high nitrogen utilization efficiency or plants resistant to lodging by inhibiting or knocking down the expression of the encoding gene; The ZmGLK63 protein is a protein with an amino acid sequence as shown in SEQ ID NO.2; The relevant biological material is any one of the following: B1) a ZmGLK63 gene encoding the ZmGLK63 protein; B2) sgRNA targeting the ZmGLK63 gene described in B1); B3) a recombinant vector for knocking out the ZmGLK63 gene described in B1); B4) a recombinant microorganism comprising the recombinant vector described in B3); The plant is corn.

2. A method for improving plant yield, nitrogen utilization ability and lodging resistance, characterized in that: A recombinant vector or a recombinant microorganism that knocks out the ZmGLK63 gene is introduced into a plant to inhibit or knock down the expression of the ZmGLK63 gene, thereby improving plant yield, nitrogen utilization ability, and lodging resistance; The nucleotide sequence of the ZmGLK63 gene is shown in SEQ ID NO.1; The recombinant vector contains an sgRNA targeting the ZmGLK63 gene; the sequence of the sgRNA is shown in SEQ ID NO.5; the recombinant microorganism contains the recombinant vector; The plant is corn.

3. A method for cultivating high-yield plants, high nitrogen utilization efficiency plants or lodging-resistant plants, characterized in that: A recombinant vector for knocking out the ZmGLK63 gene or a recombinant microorganism for knocking out the ZmGLK63 gene is introduced into a plant to inhibit or knock down the expression level of the ZmGLK63 gene, thereby constructing a high-yield plant, a high nitrogen utilization efficiency plant, or a lodging-resistant plant; The nucleotide sequence of the ZmGLK63 gene is shown in SEQ ID NO.1; The recombinant vector contains an sgRNA targeting the ZmGLK63 gene; the sequence of the sgRNA is shown in SEQ ID NO.5; the recombinant microorganism contains the recombinant vector; The plant is corn.

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