Method for improving field growth performance of corn and application

Abstract

The invention provides a method for improving field growth performance of corn and application. The application of the GLK1 gene or the G2 gene disclosed by the invention can be used for remarkably improving the characters of gramineous plants for the first time, including stalk greening and / or stalk thickening, plant height increasing, biomass increasing, starch content increasing, soluble sugar content and / or yield increasing, and / or photosynthetic ability improving. The invention provides a new way for improvement of gramineous plants.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of botany and molecular biology, and more particularly, the present application relates to a method for improving the field growth performance of maize and applications. BACKGROUND

[0002] It is predicted that the yield of major crops needs to increase by about 2.4% per year until 2050 to meet the demand of population growth and social development without increasing the amount of arable land. However, due to factors such as species itself and natural environment, the growth of crops under field conditions often cannot reach the ideal state, and how to improve the field growth performance of crops has always been an important research direction to promote high yield and stable yield.

[0003] Maize, as an important food crop, in addition to the edible value of its seeds, its stems also have forage value, industrial value, and ecological value, etc. On the one hand, maize seeds contain high-quality fat, protein, crude fiber, trace elements, and carbohydrates, etc.; on the other hand, maize stalks are one of the important sources of livestock feed, and the crushed maize stalks can also be used as edible fungus cultivation base material or greenhouse soilless cultivation substrate, or they can be returned to the field to improve soil structure and increase soil fertility; and, the maize stalks can also be used to produce clean bioenergy such as biogas and alcohol, for microbial fermentation and pharmaceutical industry raw materials, or for the manufacture of paper, boards, and packaging materials. The above-mentioned multi-level value-added maize stalk resources can reduce environmental pollution and increase farmers' income.

[0004] However, the correlation regulation of leaf photosynthesis, stem growth, and biomass of grasses, especially crops such as maize which is a C4 plant, is still less known, and it is necessary to further effectively improve the related field growth performance by exploring and utilizing suitable means and methods. SUMMARY

[0005] The purpose of the present application is to provide a method for improving the field growth performance of maize and applications.

[0006] In a first aspect of the present application, a method for improving traits of a grass plant or preparing a grass plant with improved traits is provided, comprising: up-regulating the expression or activity of GLK1 or G2 in the grass plant; the improvement of the traits includes: (i) stem greening and / or stem thickening (including increasing the (biological) mass of the stem for ensiling), (ii) increasing plant height, (iii) increasing biomass, (iv) increasing starch content, soluble sugar content and / or yield, and / or (v) improving photosynthetic capacity.

[0007] In one or more embodiments, the up-regulation of the expression or activity of GLK1 or G2 includes overexpressing GLK1 or G2 in the plant.

[0008] In one or more embodiments, the up-regulating GLK1 or G2 expression or activity comprises: introducing a GLK1 or G2 gene or an expression cassette containing the gene (e.g., contained in an expression construct or expression vector) into a plant; introducing a gene editing reagent that enhances GLK1 or G2 gene expression or activity into a plant; enhancing GLK1 or G2 gene expression with an expression-enhancing promoter or a tissue-specific promoter, or enhancing its expression in a particular tissue; enhancing GLK1 or G2 gene expression in a plant with an enhancer; or, in different plants, directionally screening for varieties with high GLK1 or G2 gene expression, and obtaining a trait-improved offspring through hybridization breeding.

[0009] In another aspect of the present application, there is provided a use of GLK1 or G2 or an up-regulating molecule thereof for trait improvement of a plant in the family Poaceae, or for preparing a trait-improved plant in the family Poaceae; wherein the trait improvement comprises: (i) stem greening and / or stem thickening (including increasing the (bio)mass of the stem for silage), (ii) increasing plant height, (iii) increasing biomass, (iv) increasing starch content, soluble sugar content and / or yield, and / or (v) enhancing photosynthetic capacity.

[0010] In one or more embodiments, the up-regulating molecule comprises: an expression cassette expressing GLK1 or G2 (e.g., contained in an expression construct or expression vector); a gene editing reagent that enhances GLK1 or G2 gene expression or activity in a plant in the family Poaceae; or an up-regulating molecule that interacts with GLK1 or G2, thereby enhancing its expression or activity.

[0011] In one or more embodiments, the plant in the family Poaceae is or the GLK1 or G2 is from: a cereal plant in the family Poaceae.

[0012] In one or more embodiments, the cereal plant in the family Poaceae is a C4 plant (C4 crop).

[0013] In one or more embodiments, the cereal grasses include, but are not limited to, Zea mays, Oryza sativa, Sorghum bicolor, Setaria italica, Hordeum vulgare, Zizania latifolia, Leersia japonica Makino, Triticum aestivum, Setaria italica, Panicum miliaceum, Secale cereale, Avena sativa L., Brachypodium distachyum.

[0014] In one or more embodiments, the GLK1-encoding gene includes a cDNA sequence, a genomic sequence, or a sequence artificially optimized or engineered based thereon.

[0015] In one or more embodiments, the G2-encoding gene includes a cDNA sequence, a genomic sequence, or a sequence artificially optimized or engineered based thereon.

[0016] In one or more embodiments, the cereal grass is maize.

[0017] In one or more embodiments, the amino acid sequence of the G2 polypeptide is selected from the group consisting of: (i) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; (ii) a polypeptide derived from (i) by substitution, deletion, or addition of one or several (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues, which has the function of the regulatory trait; (iii) a polypeptide having an amino acid sequence with a homology of >80% (more preferably >85%, >90%, >95%, or >98%) to the amino acid sequence set forth in SEQ ID NO: 2, which has the function of the regulatory trait; or (iv) a polypeptide formed by adding a tag sequence or a restriction site sequence to the N or C terminus of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, or adding a signal peptide sequence to the N terminus thereof.

[0018] In one or more embodiments, the amino acid sequence of the polypeptide of GLK1 is selected from the group consisting of: (i) a polypeptide having any of the amino acid sequences of SEQ ID NO: 4; (ii) a polypeptide derived from (i) by substitution, deletion, or addition of one or several (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues to the amino acid sequence of SEQ ID NO: 4, which has the function of the modulated trait; (iii) a polypeptide having an amino acid sequence with a homology of >80% (preferably >85%, >90%, >95%, or >98%) to the amino acid sequence of SEQ ID NO: 4, which has the function of the modulated trait; or (iv) a polypeptide formed by adding a tag sequence or a restriction site sequence to the N- or C-terminus of the polypeptide of the amino acid sequence of SEQ ID NO: 4, or adding a signal peptide sequence to the N-terminus thereof.

[0019] In another aspect of the present application, there is provided a use of GLK1 gene or G2 gene or the encoded protein thereof as a molecular marker for identifying a trait of a plant in the family Poaceae, or as a molecular marker for directed selection of a plant in the family Poaceae; the trait includes: (i) a stem greenness trait and / or a stem thickness trait, (ii) a plant height trait, (iii) a biomass trait, (iv) a starch content, soluble sugar content trait, and / or a yield trait, and / or (v) a photosynthetic capacity trait.

[0020] In one or more embodiments, the trait of a plant in the family Poaceae is identified or the plant in the family Poaceae is subjected to directed selection by analyzing the expression level of GLK1 gene or G2 gene or the activity of GLK1 protein or G2 protein in the plant in the family Poaceae.

[0021] In another aspect of the present application, there is provided a method for identifying a trait of a plant in the family Poaceae or for directed selection of a plant in the family Poaceae with improved traits, comprising: analyzing the expression or activity of GLK1 gene or G2 gene or the encoded protein thereof in the plant in the family Poaceae; if the expression or activity of GLK1 gene or G2 gene or the encoded protein thereof in the plant to be tested is higher than the average value of the same type of plant in the family Poaceae, it indicates that the plant has an excellent trait, and the excellent trait is selected from: (i) stem greenness and / or stem thickness, (ii) increased plant height, (iii) increased biomass, (iv) increased starch content, soluble sugar content, and / or yield, and / or (v) improved photosynthetic capacity; and the plant is selected as a plant with improved traits.

[0022] In one or more embodiments, the selected plant in the family Poaceae has high expression of G2 gene or GLK1 gene or high activity of G2 protein or GLK1 protein, preferably, the high expression or high activity refers to a statistically significant increase in expression or activity compared to the average expression or activity of the same type or species of plant in the family Poaceae.

[0023] In one or more embodiments, the increase, enhancement, promotion, improvement or augmentation means a significant increase, enhancement, promotion, improvement or augmentation, such as an increase, enhancement, promotion, improvement or augmentation of 20%, 40%, 60%, 80%, 90% or more.

[0024] In another aspect of the application, there is provided a method of screening a substance (potential substance) for improving a trait of a plant of the family Poaceae, the method comprising: (1) adding a candidate substance to a system expressing GLK1 or G2; (2) detecting the system to observe the expression or activity of GLK1 or G2 therein, and if the expression or activity of GLK1 or G2 is improved, it indicates that the candidate substance is a substance that can be used to improve a trait of a plant of the family Poaceae; wherein the trait of a plant of the family Poaceae is selected from the group consisting of: (i) stem greening and / or stem thickening, (ii) increase in plant height, (iii) increase in biomass, (iv) increase in starch content, soluble sugar content and / or yield, and / or (v) increase in photosynthetic capacity.

[0025] In one or more embodiments, a control group is further included to clearly distinguish the difference in the expression or activity of GLK1 or G2 in the test group from that of the control group.

[0026] In one or more embodiments, the candidate substance includes (but is not limited to) a regulatory molecule (such as an up-regulator, a small molecule compound, a gene editing construct, etc.) designed against GLK1 or G2 gene or the protein encoded thereby or an upstream or downstream protein or gene thereof.

[0027] In another aspect of the application, there is provided a plant cell, tissue or organ of the family Poaceae expressing an expression cassette of GLK1 or G2 exogenously; preferably, the expression cassette comprises: a promoter, a coding gene of GLK1 or G2, and a terminator; preferably, the expression cassette is comprised in an expression construct or an expression vector.

[0028] In one or more embodiments, the plant cell, tissue or organ does not have the ability of propagation, is not a plant propagation material, and cannot be directly propagated into a plant.

[0029] Other aspects of the application will be apparent to those skilled in the art from consideration of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Plasmid map for making overexpression corn plants ZmUBIpro:ZmGLK1 (UBI promoter driving ZmGLK1 expression) (top panel) and for making overexpression corn plants ZmUBIpro:ZmG2 (UBI promoter driving ZmG2 expression) (bottom panel).

[0031] Figure 2quantification of the field growth phenotype of the overexpression maize materials.

[0032] (A, D) Copy number analysis of ZmUBIpro:ZmGLKl (A) and ZmUBIpro:ZmG2 (D).

[0033] (B, E) Local phenotype of field grown ZmUBIpro:ZmGLKl (B) and ZmUBIpro:ZmG2 (E) plants, red arrows indicate greener and thicker stems of ZmUBIpro:ZmGLKl and ZmUBIpro:ZmG2, respectively.

[0034] (C) Phenotype of field grown ZmUBIpro:ZmGLKl and ZmUBIpro:ZmG2 plants.

[0035] Figure 3 quantification of the field growth phenotype of the overexpression maize materials.

[0036] (A, D) Expression level detection of ZmUBIpro:ZmGLKl (A) and ZmUBIpro:ZmG2 (D).

[0037] (B, C, E, F) Measurement of plant height (B), leaf chlorophyll content (C), stem thickness (E) and net photosynthetic rate (F) of field grown ZmUBIpro:ZmGLKl and ZmUBIpro:ZmG2 plants.

[0038] Figure 4 staining of starch in leaves of overexpression maize materials ZmUBIpro:ZmGLKl and ZmUBIpro:ZmG2.

[0039] Figure 5 staining of starch in bracts of overexpression maize materials ZmUBIpro:ZmGLKl and ZmUBIpro:ZmG2.

[0040] Figure 6 determination of soluble sugar and starch content in leaves of overexpression maize materials ZmUBIpro:ZmGLKl and ZmUBIpro:ZmG2. Leaf soluble sugar content (A) and leaf starch content (B) of field grown ZmUBIpro:ZmGLKl and ZmUBIpro:ZmG2 plants. DETAILED DESCRIPTION

[0041] The present inventors have made intensive studies and for the first time disclosed that the application of GLK1 gene or G2 gene is very effective for the improvement of traits of plants in the family Poaceae, including: green stem and / or thick stem, increased plant height, increased biomass, increased starch content, soluble sugar content and / or yield, and / or improved photosynthetic capacity. The present application provides a new approach for the improvement of plants in the family Poaceae.

[0042] Target gene

[0043] As used herein, the terms "improved traits" and "improved growth performance" are used interchangeably, unless otherwise specified. The term "improved growth performance" includes "improved field growth performance".

[0044] As used herein, the term "G2 gene or G2 protein (polypeptide)" refers to a G2 gene or G2 protein from a plant in the family Poaceae, such as corn.

[0045] As used herein, the term "GLK1 gene or GLK1 protein (polypeptide)" refers to a GLK1 gene or GLK1 protein from a plant in the family Poaceae, such as corn.

[0046] In the present application, the GLK1 protein or G2 protein also includes fragments, derivatives and analogs thereof. As used herein, the terms "fragment", "derivative" and "analog" refer to a protein fragment that substantially maintains the same biological function or activity of the polypeptide, which can be (i) a protein with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) a protein with a substituent group in one or more amino acid residues, or (iii) a protein formed by fusion of an additional amino acid sequence to the protein sequence, etc. These fragments, derivatives and analogs are within the scope known to those skilled in the art according to the definition herein. The biologically active fragments of the GLK1 or G2 protein can be applied to the present application.

[0047] In the present application, the term "G2 protein" refers to a protein having a sequence as set forth in any one of SEQ ID NO: 2, which has the trait improvement activity as previously described in the present application, and also includes variants of the sequence of SEQ ID NO: 2 which have the same function as these polypeptides. The term "GLK1 protein" refers to a protein having a sequence as set forth in SEQ ID NO: 4, which has the trait improvement activity as previously described in the present application, and also includes variants of the sequence of SEQ ID NO: 4 which have the same function as these polypeptides. These variants include, but are not limited to, deletion, insertion and / or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, still more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with similar or identical amino acids usually does not change the function of the protein. For another example, addition or deletion of one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein.

[0048] In the present application, the "GLK1" or "G2" also includes its homologs in the grass family. It should be understood that, although GLK1 or G2 obtained from a specific species, maize, is preferred in the present application, other proteins from the grass family which have high homology with the GLK1 or G2 protein polypeptides described (such as 80% or more homology with the polypeptide sequence set forth in SEQ ID NO: 2; more preferably 85% or more homology, such as 90%, 95%, 98% or 99% homology) and have the same function are also included in the present application. Methods and tools for comparing sequence homology are also well known in the art, such as BLAST. "Homology" refers to the level of similarity (i.e. sequence similarity or identity) between two or more nucleic acids or polypeptides in terms of percentage of positions that are identical.

[0049] The present application also includes polynucleotides (genes) encoding the polypeptides, which can be natural genes from crops or degenerate sequences thereof.

[0050] Vectors containing the coding sequences, and host cells genetically engineered with the vectors or polypeptide coding sequences are also included in the present application. Methods well known to those skilled in the art can be used to construct suitable expression vectors.

[0051] The host cell is usually a plant cell. The transformed plant can be generally obtained by using Agrobacterium transformation or biolistic transformation, such as leaf disc method, immature embryo transformation method, etc.; preferably Agrobacterium method. The transformed plant cells, tissues or organs can be regenerated into plants by conventional methods, thereby obtaining plants with altered traits relative to wild type.

[0052] As used herein, the term "crop" refers to plants of economic value in agriculture and industry, such as grain, cotton, oil, etc., the economic value of which can be embodied in useful parts of the plant, such as seeds, fruits, roots, stems, leaves, etc. Crops include, but are not limited to, dicotyledonous or monocotyledonous plants. Preferred crops are grasses, more preferably corn, sorghum, etc.

[0053] In the present application, the crop includes plants expressing GLK1 or G2; preferably C4 cereal crops. Preferably, the cereal crop is one having grain.

[0054] Applications

[0055] Corn is an important economic and food crop worldwide, and further improvement of corn traits is of great significance to meet the rapidly growing demand for corn-derived food, feed and fuel, etc. In the present application, two key regulatory genes, G2 or GLK1, are explained, which can achieve significant trait improvement.

[0056] Plants use light energy to absorb CO2 to synthesize carbohydrates through photosynthesis. Crops such as rice and wheat perform C3 photosynthesis, and after absorbing CO2, they first synthesize compounds containing three carbon atoms such as triphosphate glycerol, which does not operate in a sufficiently efficient manner due to the presence of photorespiration. Crops such as corn and sorghum have evolved specialized and efficient C4 photosynthesis. Compared with C3 rice, C4 corn has functionally differentiated chloroplasts and specifically distributed metabolic enzymes in the bundle sheath (BS) cells and mesophyll (M) cells, respectively. After CO2 enters the M cells, it is first converted into a four-carbon product (such as malate or aspartate), which is then transported to the BS cells to release CO2, and CO2 is concentrated around Rubisco, thereby effectively reducing photorespiration and facilitating efficient CO2 assimilation (CO2 concentration mechanism). The present inventors have found that in C4 plants such as corn and sorghum, transcription factors G2 and GLK1 are relatively enriched in BS and M cells, respectively, and regulate chloroplast development and affect photosynthesis.

[0057] The genes described in the present application can increase the degree of stem thickness, increase plant height, and improve biomass, which is of great significance in the production of silage and the recycling of straw. The genes can increase starch content and soluble sugar content, which has significant significance in improving yield and nutritional value of plants.

[0058] Based on the new findings of the present inventor, a method for improving plants is provided, which comprises up-regulating the expression or activity of GLK1 or G2 in a plant of the family Poaceae; and the trait improvement comprises: stem greening and / or stem thickening (including increasing the (bio)mass of the stem for silage), increasing plant height, increasing biomass, increasing starch content, soluble sugar content and / or yield, and / or improving photosynthetic capacity.

[0059] As understood by those skilled in the art, based on the experimental data and regulatory mechanisms provided by the present application, various methods well known to those skilled in the art can be used to regulate the expression of GLK1 or G2, which are included in the present application.

[0060] In the present application, the substances that up-regulate the expression or activity of GLK1 or G2 in plants include promoters, agonists, activators, up-regulators. The "up-regulation", "increase", "promotion" include "up-regulation", "promotion" of protein activity or "up-regulation", "increase", "promotion" of protein expression. Any substance that can increase the activity of GLK1 or G2 protein, increase the stability of GLK1 or G2 gene or the protein encoded by it, up-regulate the expression of GLK1 or G2 gene, or increase the effective action time of GLK1 or G2 protein can be used in the present application as a substance useful for up-regulating GLK1 or G2 gene or the protein encoded by it. They can be compounds, chemical small molecules, biological molecules. The biological molecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

[0061] As another embodiment of the present application, a method for up-regulating the expression of GLK1 or G2 gene or the protein encoded by it in a plant is also provided, which comprises: transforming a GLK1 or G2 expression construct or vector into plant tissue, organ or tissue, obtaining plant tissue, organ or seed transformed with the coding polynucleotide of GLK1 or G2; and regenerating a plant plantlet from the obtained plant tissue, organ or seed transformed with the coding polynucleotide of GLK1 or G2.

[0062] Other methods for increasing the expression of GLK1 or G2 gene or its homologous gene are well known in the art. For example, the expression of GLK1 or G2 gene or its homologous gene can be enhanced by driving it with a strong promoter. Alternatively, the expression of the G2 gene can be enhanced by enhancers (such as the first intron of Actin gene, etc.), or the expression of GLK1 or G2 gene or its homologous gene and protein activity can be increased by gene editing means. Suitable strong promoters for the method of the present application include but are not limited to: 35S promoter, Ubi promoter of maize, etc.

[0063] Any appropriate conventional means, including reagents, temperature, pressure conditions, etc. can be used to implement the method.

[0064] Having the function of GLK1 or G2 gene known, one can use it as a molecular marker to screen plants. One can also screen for substances or potential substances that can regulate this mechanism to direct the plant's plant type traits, yield traits, organelles or cell cycle. One can also use GLK1 or G2 or the protein encoded by it as a tracking marker for genetically transforming the offspring of a plant.

[0065] Therefore, the present application provides a method for selecting or identifying a plant, which comprises: identifying the expression or activity of GLK1 or G2 gene in a test plant; if the GLK1 or G2 protein of the test plant is highly expressed or active, it has: green stem and / or thick stem, increased plant height, increased biomass, increased starch content, soluble sugar content and / or yield, and / or improved photosynthetic capacity; otherwise, it has undesirable traits.

[0066] When evaluating a test plant, one can determine the expression or mRNA amount of GLK1 or G2 to see if it is higher than the average of such plants, if it is significantly higher, it has improved traits.

[0067] The present application provides a method for screening substances that can regulate plant type traits, yield traits, organelles or cell cycle, which comprises: adding a candidate substance to a system expressing GLK1 or G2; detecting the system to observe the expression or activity of GLK1 or G2 therein, if the expression or activity thereof is improved, it indicates that the candidate substance is a substance that can be used to improve the traits of a plant in the family Poaceae; wherein improving the traits of a plant in the family Poaceae includes one or more of the following: green stem and / or thick stem, increased plant height, increased biomass, increased starch content, soluble sugar content and / or yield, and / or improved photosynthetic capacity.

[0068] Methods for screening substances that act on a target protein or gene or a specific region thereof are well known to those skilled in the art, and these methods can be used in the present application. The candidate substance can be selected from the group consisting of peptides, poly-peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules and nucleic acid sequences. Depending on the type of substance to be screened, those skilled in the art know how to choose the appropriate screening method.

[0069] Various techniques known to those skilled in the art can be used to detect the interaction between proteins and the strength of the interaction, such as GST precipitation (GST-Pull Down), bi-molecular fluorescence complementation, yeast two-hybrid system or immunoprecipitation technique.

[0070] Through large-scale screening, a class of potential substances that specifically act on GLK1 or G2 and have a regulatory effect on the plant traits can be obtained.

[0071] The application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, such as the conditions described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Science Press, or the conditions suggested by the manufacturers.

[0072] Materials and methods

[0073] Vector construction and production of transgenic plants

[0074] The transgenic materials of corn overexpressing ZmG2 and ZmGLK1 were entrusted to Biocytogen (Jiangsu) Co., Ltd. for genetic transformation, and the background material used was corn inbred line KN5585.

[0075] The vector used for transgenesis was PSC310 vector, the bacterial resistance was kanamycin resistance, and the plant screening marker was Bar resistance.

[0076] Using the Gateway system, the CDS sequences of ZmG2 and ZmGLK1 were respectively connected to the vector, and the expression of ZmG2 and ZmGLK1 was started using the ZmUBI promoter on the vector. The map of the established recombinant expression plasmid is as follows: Figure 1 .

[0077] Growth conditions of transgenic lines

[0078] The corn overexpressing GLK transcription factors was cultured in an artificial climate chamber and in the field, respectively. The corn planted in the artificial climate chamber was first germinated in a small hole tray, and after the seedlings were identified, the transgenic positive materials were selected and transplanted into 6L round pots for culture.

[0079] The soil culture conditions of corn: 60% black soil and 20% vermiculite were mixed.

[0080] The artificial climate chamber culture conditions: light time: 6:00-20:00, light intensity: 400-600 μmol m -2 s -1 , CO2 is at atmospheric level, temperature 27℃ (constant temperature), humidity: 65%-70% (R.H.).

[0081] The plant materials were watered every other day and fertilized every other week.

[0082] Field culture conditions: corn was planted in Shanghai Songjiang Farm (121.4530°E, 31.0428°N) from March to July.

[0083] Transgenic line copy number identification

[0084] (1). Solution preparation for membrane transfer process

[0085] ①. 20x SSC solution (transfer solution): 3M NaCl, 300mM Na-citrate, adjust pH to 7.0 with concentrated HC1.

[0086] ②. Depurination reagent: 25mL concentrated HC1, add water to 1000mL.

[0087] ③. Denaturation solution: 1.5M NaCl, 0.5M NaOH.

[0088] ④. Neutralization solution: 0.5M Tris-HCl, 1.5M NaCl, adjust pH to 7.5 with concentrated HC1.

[0089] (2). Solution preparation for hybridization process

[0090] ①. Hybridization solution: prepare as Table 1, sterilize and aliquot (prepared according to the instruction of Roche kit, kit number 11796895001).

[0091] Table 1

[0092]

[0093] ②. Maleic acid solution: 0.1M Maleic acid, 0.15M NaCl, adjust pH to 7.5 with NaOH. The solution needs to be sterilized.

[0094] ③. Detection buffer: 0.1M Tris-HCl, 0.1M NaCl, adjust pH to 9.5 with concentrated HC1. The solution needs to be sterilized.

[0095] ④. Blocking Regent stocking solution (10x stock solution): 10g Blocking Regent is added to 100mL sterilized maleic acid solution; it can be heated in a 65℃ water bath to form a turbid solution, if it cannot be dissolved, the pH value needs to be checked. After confirming that the Blocking Regent is completely dissolved, it is stored at 4℃ for preparing Blocking solution.

[0096] ⑤. Blocking solution: 10mL Blocking Regent stocking solution is dissolved in 90mL maleic acid solution, and it is prepared immediately before use.

[0097] (6) Wash solution I: 2xSSC solution, 0.1% SDS, solution needs to be sterilized.

[0098] (7) Wash solution II: 0.5xSSC solution, 0.1% SDS, solution needs to be sterilized.

[0099] (8) Membrane washing solution: add 3 mL Tween20 to 1 L maleic acid solution.

[0100] (2) Extract the genomic DNA of the plant (see Method 2.3)

[0101] (3) Enzymatic digestion of the genomic DNA

[0102] ①. Digest 10 μg of DNA in a 50 μL system, try to shorten the digestion time, which should not exceed 16 hours. If the time is too long, the DNA will be degraded and dispersed, which will affect the hybridization effect. Generally, the enzyme digestion is overnight, and the enzyme digestion system can be increased accordingly to shorten the enzyme digestion time.

[0103] ②. Precipitate the enzyme digestion product: add 1 / 10 volume of 3 M NaAc and 2.5 volumes of anhydrous ethanol, precipitate overnight at -20°C, centrifuge at 12000 rpm, 4°C, for 10 min, wash once with anhydrous ethanol, then centrifuge to remove the anhydrous ethanol, and add 36 μL water to dissolve. Make sure that the ethanol is completely blown dry, otherwise it will not be possible to add the gel well after dissolving with water.

[0104] (4) Electrophoresis

[0105] ①. Use a conical flask without nucleic acid dye contamination to melt a whole plate of 0.8% gel as thick as possible. All the utensils used need to be cleaned, including combs, gel plates, electrophoresis tanks, etc., and new electrophoresis liquid needs to be replaced.

[0106] ②. Loading: in addition to the enzyme digestion product, DIG label marker needs to be added.

[0107] ③. Running the gel: run the gel at 20 V overnight, or at 100 V for about 2 h, until the bromophenol blue runs to the edge of the gel.

[0108] (5) Pre-treatment of the gel before transferring the membrane

[0109] ①. Depurination

[0110] If the DNA fragment in the gel is greater than 5 kb, use the depurination reagent to treat the gel for 8-10 min, keep the gel shaking, until the bromophenol blue changes from blue to yellow. Wash twice with sterilized water, pour out the water, and proceed to the next step. (This operation should not exceed 20 min).

[0111] ②. Denaturation

[0112] Use denaturing liquid to treat the gel after deprotonation, 15 minutes twice, keep the gel shaking, and find that the bromophenol blue turns blue again. Wash twice with sterilized water, and proceed to the next step.

[0113] ③. Neutralization

[0114] Use neutralization liquid to treat the gel after denaturation, 15 minutes twice, keep the gel shaking. Wash twice with sterilized water, and proceed to the next step.

[0115] ④. 20×SSC treatment: Place the gel in 20×SSC for 10 minutes.

[0116] (6). Salt bridge preparation and membrane transfer

[0117] ①. First wash a clean porcelain plate, pour 1L 20×SSC solution into it, then place a glass plate on the porcelain plate, place a 25cm×30cm filter paper on the glass plate, and make sure that the filter paper is completely immersed in the SSC solution at both ends, the glass plate direction is perpendicular to the porcelain plate direction, and the filter paper is parallel to the porcelain plate direction.

[0118] ②. Place a 15cm×15cm filter paper on the 25cm×30cm filter paper, then place two 12cm×12cm filter papers (note that there should be no air bubbles, and a glass rod should be used to chase air bubbles).

[0119] ③. Then place the gel, with the gel holes facing down. After placing, cut off the gel holes to ensure that the gel surface is flat, making it easy to place parafilm and plastic wrap later, and use a glass rod to chase air bubbles.

[0120] ④. Place the film on the gel. Before placing the film, immerse it in deionized water, then place it in 20×SSC for 5 minutes. Note that only the corners of the film can be clamped with tweezers, as clamping too far in will affect subsequent observation. After placing the film, do not chase air bubbles with a glass rod, but instead absorb 20×SSC solution onto the film, and cut a small triangle in the upper right corner for subsequent observation. Then place two 12cm×12cm filter papers on the film. At this time, you can use a glass rod to chase air bubbles.

[0121] ⑤. Use a Parafilm film of appropriate length and width to seal around the salt bridge, then use plastic wrap to seal the four corners of the gel to prevent 20×SSC from evaporating. Then place an absorbent paper on top and press a glass plate on it. Fill a 500g medicine bottle with water and press it on the glass plate.

[0122] ⑥. Change the absorbent paper every 30 minutes to 1 hour, repeat 3-4 times, then do not change the absorbent paper and transfer the membrane overnight.

[0123] ⑦. Put the membrane on a piece of filter paper pre-soaked with 2xSSC, with the side of the gel facing up, cross-link under UV light at 254nm for 3min, take out the membrane and transfer it to a dry piece of filter paper, let it dry naturally (about 90min or so), and proceed to the next step.

[0124] (7). Pre-hybridization and hybridization

[0125] ①. PCR primer-labeled probe (synthesized according to Roche PCR DIG Probe Synthesis Kit)

[0126] ②. Pre-hybridization: Pour 15mL of Roche DIG Easy Hyb Granules hybridization solution into the hybridization tube in advance, take out the previously air-dried membrane and put it into the hybridization tube. Note: Make sure to pour the hybridization solution in advance, then put in the membrane, otherwise it will leave marks on the membrane. Use blank hybridization solution for pre-hybridization at 42°C for 3-4 hours. The membrane should be tightly attached to the tube wall without air bubbles.

[0127] ③. Hybridization: Take 8μL of probe (PCR synthesis product) + 100μL of sterile water in an EP tube, seal the tube opening with parafilm, denature in boiling water for 10min, and immediately place in an ice-water mixture. The probe can also be hybridized with the recovered probe hybridization mixture, denature at 68°C in a water bath for 10min, and immediately place on ice. Mix the probe and hybridization solution, discard the pre-hybridization solution, and immediately add the probe-containing hybridization solution to the hybridization tube, hybridize at 42°C for 16h. (The probe-containing hybridization solution can be stored at -20°C, and before use, denature at 68°C in a water bath for 10min, and can be reused three times.)

[0128] ④. Washing: The next day, recover the probe and freeze it in the refrigerator, add washing solution I, and wash at room temperature or 42°C in the hybridization oven for two times, 7min each time. Pour out the washing solution I, add washing solution II, and place in the hybridization oven at 65°C for 15min, and wash twice. The above two steps remove excess probe.

[0129] ⑤. Take out the membrane, transfer it to a lunch box containing washing solution, and gently rinse for 5min.

[0130] ⑥. Blocking: Place the membrane in blocking solution, block for 30min / times, twice.

[0131] ⑦. Discard the blocking solution, replace it with new blocking solution, add DIG antibody at a ratio of 1:10000, and immunohybridize for 30min.

[0132] ⑧. Pour out the blocking solution, elute the antibody with elution solution, and rinse twice, 15min each time.

[0133] (8) Development

[0134] Discard the eluent, put the membrane into the detection solution for 5 min; then discard the detection solution, put the membrane with the front side up on a transparent plastic bag, take out the CSPD-ready-to-use (substrate, 1 mL / 100 cm2of the membrane amount, about 1 mL can be used), add 1 mL of the CSPD solution, drop it on the surface of the membrane, use the gun head to suck the flowing CSPD solution, repeatedly evenly smear and the surface of the membrane, fully react for 5 min, develop for about 45 min, and can be observed under 2000 s.

[0135] Measurement of plant height, stem diameter and chlorophyll content

[0136] Plant height measurement: Take the height of the aboveground material as the height of the plant material (the height from the soil surface to the top of the male spike), measure 6 plant materials for each strain as 6 biological replicates.

[0137] Stem diameter measurement: Take the circumference of the stem closest to the ground (according to) as the stem diameter of the plant, measure 6 plant materials for each strain as 6 biological replicates.

[0138] Use SPAD 502Plus Chlorophyll Meter (Konica Minolta Sensing, Japan) to measure the chlorophyll content of the plant.

[0139] Take 6 different positions in the middle of the leaves of each material for chlorophyll content measurement, take the average of 6 chlorophyll contents as the chlorophyll content of the plant material, measure 12 plant materials for each strain as 12 biological replicates.

[0140] Measurement of photosynthetic gas exchange rate

[0141] Measurement instrument: LI-6400 portable photosynthesis meter (Li-Cor, Inc., USA). Use LI-6400 to measure the CO2 response curve and light response curve of the material planted in Shanghai Songjiang farm

[0142] Measurement of CO2 response curve: The temperature, relative humidity and light intensity in the leaf chamber are set to 28℃, 60% and 1200 μmol m -2 s -1 respectively. The CO2 concentration changes according to the gradient of 400, 200, 50, 100, 150, 200, 300, 400, 500, 600, 800, 1000, 1200 ppm. Adapt for 2 minutes and perform "Auto match" operation before recording photosynthetic rate.

[0143] Measurement of light response curve: The temperature, relative humidity, and CO2 concentration in the leaf chamber were set to 28°C, 60%, and 400 ppm, respectively. The light intensity was changed in a gradient of 1800, 1600, 1400, 1200, 1100, 1000, 900, 700, 500, 300, 150, 75, 50, 20, 10, and 0 μmol m-2s-1. The photosynthetic rate was recorded after 2 minutes of adaptation and "Auto match" operation. -2 s -1

[0144] Starch staining of corn leaves

[0145] (1) Fixation: A sharp blade was used to take the upper middle part of a young fully expanded mature leaf, which was further trimmed into a small piece of tissue with a size of 1 cm x 1 cm, and then placed in a fixing solution (ethanol: glacial acetic acid = 3:1). Vacuum was applied for 20 min, 10 min at a time, and then the sample was fixed at room temperature for 3-5 h or at 4°C overnight.

[0146] (2) Dehydration: In the experiment, four ethanol concentrations of 70%, 80%, 90%, and 100% were used for gradient dehydration, with a two-hour interval between each gradient.

[0147] (3) Decolorization: In the experiment, histoclear, which is less toxic, was used instead of xylene as a decolorizing agent. Four gradients of 25% / 75% histoclear / ethanol, 50% / 50% histoclear / ethanol, 75% / 25% histoclear / ethanol, and 100% histoclear were used for decolorization, with a two-hour interval between each gradient.

[0148] (4) Wax immersion: The decolorized tissue was immersed in a mixture of transparent agent (histoclaer) and paraffin (mixing ratio 1:1, 4 mL each was used in the experiment), and the proportion of paraffin was gradually increased until the transparent agent in the tissue was completely replaced by paraffin. The specific operation was to immerse the sample in a sample bottle containing 100% hitoclear (65°C oven), and replace the paraffin every three hours (4 mL), at least six times.

[0149] (5) Embedding: After the sample was immersed in paraffin, the internal gap was completely occupied by paraffin, and at this time, the same hardness of paraffin was needed to embed the wax block for subsequent sectioning. The specific operation of the experiment was carried out on the embedding machine, and attention should be paid to the direction of the sample placement during the embedding process. The positioning of the sample in the wax block can be changed according to the specific experimental requirements. After embedding, the wax block can be placed on a 4°C freezing table for 30 minutes to allow it to fully solidify.

[0150] ​(6). Sectioning: After obtaining the wax block, the block needs to be trimmed to ensure that the sample is in a square area with four parallel sides and an appropriate area. The bottom edge of the wax block should also be parallel to the knife edge after being mounted on the fixation device. This will ensure the formation of a continuous and flat wax ribbon during sectioning. Then use a microtome (Leica, RM2125RTS) to section the wax block. During sectioning, a sharp and thin pair of tweezers can be used to assist in supporting the wax ribbon to help it extend and avoid overlapping and curling. Place an appropriate number of wax ribbons on the surface of a glass slide soaked in water, and observe the wax ribbons during placement to ensure that the shiny side is facing down.

[0151] (7). Baking: On a 42°C baking platform, the wax ribbon will quickly spread on the water surface and become flat. The water layer will also be evaporated after a few minutes, causing the wax ribbon to adhere to the glass surface. After 2 hours of baking, the wax ribbon and glass are firmly attached and can proceed to the next step. The baking time can be extended or the glass can be stored at 4°C for later use.

[0152] (8). I2-KI staining: After sectioning and baking, the following steps such as dewaxing can be performed, or I2-KI can be used directly for staining. Take 2g of potassium iodide and dissolve it in 5mL of distilled water by heating. Then add 1g of iodine and dilute with water to 300mL. Pour the solution into a brown bottle and store it in the dark. Use a dropper to add the prepared I2-KI solution to the sample surface (about 2-3 drops according to the number of wax ribbons on the glass slide). Then gently cover with a cover glass and place it in the dark for 10-15 minutes to allow the starch granules to be fully stained. Then add a small amount of 95% ethanol to the side of the glass to wash away the excess I2-KI solution. Observe the starch staining under a regular optical microscope and take photos for record.

[0153] Method for determining the soluble sugar content and starch content of leaf:

[0154] Method for determining the soluble sugar content of leaf:

[0155] Weigh the leaf (fresh weight 0.1g) and grind it into fine powder with liquid nitrogen.

[0156] (1) Add 4mL of 80% (v / v) ethanol solution to the ethanol solution containing the leaf powder and transfer it to a 15mL centrifuge tube.

[0157] (2) Incubate at 85°C for 2h, shake well twice during the incubation. After cooling to room temperature, centrifuge at 4000g for 10min at room temperature. Transfer the supernatant to a new 15mL centrifuge tube.

[0158] (3) Repeat steps (2) and (3) 2 times. Combine the supernatants obtained by centrifugation 3 times, and dilute the combined supernatant to 12 mL with 80% (v / v) ethanol solution. The resulting solution is the leaf soluble sugar extract.

[0159] (4) Prepare anthrone reagent: weigh 0.2 g anthrone, add 100 mL of 80% sulfuric acid solution, and dissolve to obtain 0.2% anthrone reagent (freshly prepared before use).

[0160] (5) Prepare 1 mg / mL glucose stock solution. Take 0, 10, 20, 40, 60, 80, and 100 uL of the glucose stock solution, respectively, and add them to 7 test tubes each containing 900 uL of distilled water. Add distilled water to make up to 1 mL.

[0161] (6) Take 50 uL of each of the above 7 glucose standard solutions of different concentrations into 2 mL centrifuge tubes, and add 200 uL of 0.2% anthrone reagent. Mix well, and then place the 2 mL centrifuge tubes in boiling water for 10 min. After boiling, place the 2 mL centrifuge tubes on ice to cool to room temperature.

[0162] (7) Take 200 uL of the reaction solution and place it in an enzyme-labeled plate. Take a sample without adding glucose stock solution as a blank control. Use an enzyme-labeled instrument to detect the optical density (OD 620) at 620 nm.

[0163] (8) Prepare a standard curve with OD 620 as the vertical coordinate and glucose concentration as the horizontal coordinate.

[0164] (9) Take 50 uL of the leaf soluble sugar extract obtained in (4), and take 50 uL of distilled water instead of the sample as a blank control. Add 200 uL of 0.2% anthrone reagent, mix well, and then place the 2 mL centrifuge tubes in boiling water for 10 min. After boiling, place the 2 mL centrifuge tubes on ice to cool to room temperature. Determine the OD 620 value according to the standard curve prepared in step (8) above. Note that the leaf soluble sugar extract can be appropriately diluted with 80% (v / v) alcohol to make the OD 620 measurement value within the range of minimum error (0.2-0.8).

[0165] (10) Find the sugar concentration corresponding to the sample OD 620 value on the glucose standard curve to obtain the total sugar concentration C of the sample (ug / mL). T

[0166] (11) Calculate the total soluble sugar (mainly including free glucose, fructose, and glucose and fructose produced by hydrolysis of sucrose) content SC (ug·g -1 FW) in the leaf using the following formula:

[0167] SC = C T ​X V ÷ F W X D

[0168] In the formula, V is the total volume of the leaf soluble sugar extraction solution (mL), FW is the fresh weight of the leaf (g), and D is the dilution multiple.

[0169] Leaf starch content determination:

[0170] According to the operating instructions of the starch content determination kit K-TSTA (Megazyme, Amyloglucosidase / α-Amylasemethod), the following steps are taken:

[0171] (1) The precipitate obtained by centrifuging the 15 mL centrifuge tube in step (3) above, which is insoluble in 80% (v / v) ethanol solution, is dried at 75°C.

[0172] (2) The dried precipitate in the 15 mL centrifuge tube is crushed and ground finely, 3 mL of 0.1M sodium acetate and 20U of thermostable α-amylase are added, and the mixture is mixed well by vigorous shaking with a vortex shaker.

[0173] (3) The 15 mL centrifuge tube is placed in a boiling water bath for 12 min, and the centrifuge tube is taken out at 4, 8 and 12 min and mixed well by vigorous shaking.

[0174] (4) After cooling to room temperature, 20U of amyloglucosidase is added. Mix well and incubate at 50°C for 30 min.

[0175] (5) 4000g, centrifuge at room temperature for 10 min, transfer the supernatant to a new 5 mL centrifuge tube. Add water to 4 mL and mix well for analysis.

[0176] (6) Take 10uL of sample solution, glucose standard solution and distilled water (blank control) respectively, add 300uL of GOPOD solution, and incubate at 50°C for 20 min.

[0177] (7) The water sample is used as a blank control, and the optical density (OD 510) is detected at 510 nm using an enzyme marker.

[0178] (8) Starch content calculation formula:

[0179] Starch, % = ΔA X F X EV / 0.01 X D X 1 / 1000 X 100 / W X 162 / 180

[0180] Wherein, AA is the optical density at 510 nm read relative to the blank control, F is the conversion of absorbance value to glucose concentration (10 pg glucose per absorbance value of 10 pg glucose measured by GOPOD), EV is the total volume of sample extraction (mL), 0.01 is the sample analysis volume (mL), D is the further sample dilution factor, 1 / 1000 represents the conversion from ug to mg, 100 / W represents the conversion to 100 mg sample, W is the sample weight (mg), 162 / 180 represents the conversion factor for converting the measured free glucose to anhydrous glucose present in starch.

[0181] Gene expression analysis

[0182] (1). Take a certain amount of sample into a grinding tube with small steel balls, freeze in liquid nitrogen, put the sample into the grinder to sample (60 Hz, 45 s, twice), grind the sample into powder, add 1 mL lysis solution (RNAiso Plus, Takara), vortex to mix, and stand at room temperature for 5 min.

[0183] (2). 4°C, 12000 rpm, centrifuge for 5 min, transfer 800 pL of supernatant to a new centrifuge tube, add 160 pL of chloroform (1 / 5 volume of supernatant) to each centrifuge tube, mix well and stand at room temperature for 5 min.

[0184] (3). 4°C, 12000 rpm, centrifuge for 15 min, transfer 400 pL of supernatant to a new centrifuge tube, add an equal volume of isopropanol to each centrifuge tube, stand at room temperature for 15 min.

[0185] (4). 4°C, 12000 rpm, centrifuge for 15 min, pour out the solution in the tube and add 1 mL of 75% ethanol, flick the bottom of the centrifuge tube with your fingers to dislodge the precipitate, and wash the precipitate thoroughly.

[0186] (5). 4°C, 12000 rpm, centrifuge for 5 min, discard the supernatant, and dry at room temperature for 10 min. Add an appropriate amount of DEPC water to the centrifuge tube, dissolve the resulting precipitate, which is RNA, and measure the concentration and quality of the RNA using NanoDrop. Store the RNA at -80°C.

[0187] (6). Take 1 pg of RNA, according to the instructions of the reverse transcription kit (YEASEN, 11141ES60), reverse the RNA to cDNA, and store at -20°C.

[0188] (7). The product after reverse transcription is detected by real-time fluorescent quantitative PCR to detect the expression amount of related genes. The reaction system is prepared according to the instructions of the fluorescent quantitative PCR kit (YEASEN, 11184ES08), and the detection of the expression amount of related genes is completed on the fluorescent quantitative PCR instrument according to the reaction procedure in the instructions. The primers used for related genes are shown in Table 2.

[0189] Table 2, primers involved in the examples

[0190]

[0191] Related gene and protein sequence information

[0192] ZmG2 gene CDS region nucleotide sequence (1386bp) (SEQ ID NO: 1):

[0193]

[0194] The amino acid sequence of the protein translated from the ZmG2 gene (SEQ ID NO: 2):

[0195] MLEVSTLRGPTSSGSKAEQHCGGGGGFVGDHHVVFPTSGDCFAMVDDNLLDYIDFSCDVPFFDADGDILPDLEVDTTELLAEFSSTPPADDLLAVAVFGADDQPAAAVAQEKPSSSLEQTCGDDKGVAVAAARRKLQTTTTTTTTEEEDSSPAGSGANKSSASAEGHSSKKKSAGKNSNGGKRKVKVDWTPELHRRFVQAVEQLGIDKAVPSRILEIMGTDCLTRHNIASHLQKYRSHRKHLMAREAEAATWAQKRHMYAPPAPRTTTTTDAARPPWVVPTTIGFPPPRFCRPLHVWGHPPPHAAAAEAAAATPMLPVWPRHLAPPRHLAPWAHPTPVDPAFWHQQYSAARKWGPQAAAVTQGTPCVPLPRFPVPHPIYSRPAMVPPPPSTTKLAQLHLELQAHPSKESIDAAIGDVLVKPWLPLPLGLKPPSLDSVMSELHKQGVPKIPPAAATTTGATG*

[0196] The nucleotide sequence of the CDS region of the ZmGLK1 gene (1431 bp) (SEQ ID NO: 3):

[0197]

[0198] The amino acid sequence of the protein translated from the ZmGLKl gene (SEQ ID NO:4):

[0199] MLAVSPSPVRCADAEECGGGGASKEMEETAVGPVSDSDLDFDFTVDDIDFGDFFLRLDDGDDALPGLEVDPAEIVFADFEAIATAGGDGGVTDQEVPSVLPFADAAHIGAVDPCCGVLGEDNDAACADVEEGKGECDHADEVAAAGNNNSDSGEAGCGGAFAGEKSPSSTASSSQEAESRRKVSKKHSQGKKKAKVDWTPELHRRFVQAVEELGIDKAVPSRILEIMGIDSLTRHNIASHLQKYRSHRKHMLAREVEAATWTTHRRPMYAAPSGAVKRPDSNAWTVPTIGFPPPAGTPPRPVQHFGRPLHVWGHPSPTPAVESPRVPMWPRHLAPRAPPPPPWAPPPPADPASFWHHAYMRQGPAAHMPDQVAVTPCVAVPMAAARFPAPHVRGSLPWPPPMYRPLVPPALAGKSQQDALFQLQIQPSSESIDAAIGDVLTKPWLPLPLGLKPPSVDSVMGELQRQGVANVPQACG

[0200] Example 1, Field growth phenotype of transgenic plants

[0201] Transgenic plants ZmUBIpro:ZmGLKl (ZmGLKl expression driven by UBI promoter) and transgenic plants ZmUBIpro:ZmG2 (ZmG2 expression driven by UBI promoter) were prepared as described above.

[0202] Three independent single copy transgenic lines with different insertion sites were picked for the experiment by Southern Blot identification. Figure 2 A).

[0203] The expression levels of ZmG2 or ZmGLKl in these transgenic plants were detected, and it was found that the expression levels of ZmG2 and ZmGLKl were significantly higher than those of wild type (A, D). Figure 2 A,D).

[0204] The plants growing in the field were observed. The results showed that the corn stalks overexpressing ZmGLK1 and ZmG2 were obviously greener; and the overall growth of the plants was far superior to that of the wild type. According to the preliminary observation, the overexpression lines were thicker in stem, higher in plant height, and increased in biomass compared with the wild type. Figure 2 B, C, E).

[0205] Example 2, Phenotype analysis of overexpression materials growing in the field: photosynthetic performance analysis

[0206] The chlorophyll content and net photosynthetic rate of the corn overexpressing ZmGLK1 Figure 3 A) and ZmG2 Figure 3 D) growing in the field were measured.

[0207] The results showed that the chlorophyll content of the corn leaves overexpressing ZmGLK1 and ZmG2 was significantly increased Figure 3 C).

[0208] Leaves are the main place for corn to carry out photosynthesis, and chloroplasts are the organelles for photosynthesis. The increase in chlorophyll content to some extent implies the increase in the photosynthetic capacity of corn.

[0209] The net photosynthetic rate of the corn overexpressing ZmGLK1 and ZmG2 growing in the field was measured. The results showed that the net photosynthetic rate of different lines of ZmUBIpro:ZmGLK1 and ZmUBIpro:ZmG2 was significantly improved, especially ZmUBIpro:ZmGLK1-2, ZmUBIpro:ZmGLK1-3 and ZmUBIpro:ZmG2-2 Figure 3 F).

[0210] Example 3, Phenotype analysis of overexpression materials growing in the field: growth-related phenotypes

[0211] The plant height and stem diameter of the corn plants overexpressing ZmGLK1 Figure 3 A) and ZmG2 Figure 3 D) growing in the field were measured.

[0212] The plant height measurement results showed that the plant height of the corn overexpressing ZmGLK1 and ZmG2 was significantly higher than that of the wild type corn Figure 3 B).

[0213] The stem diameter of the corn overexpressing ZmGLK1 and ZmG2 growing in the field was measured. The results showed that the stem diameter of the overexpression materials was significantly higher than that of the wild type plants Figure 3E), overexpression of ZmGLK1 and ZmG2 increases the plant height and stem diameter of maize, not only improves the phenotype of maize in the field, but also improves the potential forage value and ecological value of maize.

[0214] Therefore, overexpression of ZmGLK1 and ZmG2 can increase the biomass of the stem for silage, thereby producing high-yield silage.

[0215] Example 4, measurement of soluble sugar and starch content in leaves of overexpression material

[0216] For overexpression lines ZmUBIpro:ZmGLK1 and ZmUBIpro:ZmG2, the inventors performed other performance analysis. Surprisingly, the inventors found that the accumulation of soluble sugar and starch in the leaves changed.

[0217] The inventors obtained the leaves of overexpression lines, and detected the starch in the leaves with wild type as control. The results of starch staining showed that the starch content in the leaves of ZmUBIpro:ZmGLK1 and ZmUBIpro:ZmG2 was significantly increased, especially the maize overexpressing ZmGLK1 ( Figure 4 ).

[0218] The results of measurement of soluble sugar and starch content in leaves showed that, compared with wild type, the soluble sugar and starch content in the leaves of ZmUBIpro:ZmGLK1 and ZmUBIpro:ZmG2 was significantly increased ( Figure 6 ).

[0219] Example 5, measurement of bract starch content of overexpression material

[0220] When phenotyping the maize growing in the field, in addition to finding that the plant height, stem diameter and leaf chlorophyll content of ZmUBIpro:ZmGLK1 and ZmUBIpro:ZmG2 were significantly higher than those of wild type, when observing the plant material in the later growth stage, the inventors found that the bracts of ZmUBIpro:ZmGLK1 and ZmUBIpro:ZmG2 were significantly greener ( Figure 2 C).

[0221] Further, the inventors performed starch staining on the bracts of wild type and ZmUBIpro:ZmGLK1 and ZmUBIpro:ZmG2 and observed.

[0222] The results showed that ZmUBIpro:ZmGLK1 and ZmUBIpro:ZmG2 had obvious accumulation of starch in the bracts relative to wild type, and the change in starch accumulation in the bracts was more significant than that in the leaves ( Figure 5 ).

[0223] The above embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but should not be understood as a limitation on the patent scope of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims. Meanwhile, all the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a separate reference.

Claims

1. A method of improving a trait of a grass plant or producing a grass plant with an improved trait, comprising: Upregulating the expression or activity of GLK1 or G2 in a plant of the family Poaceae; the trait improvement comprises: (i) greener and / or thicker stems, (ii) increased plant height, (iii) increased biomass, (iv) increased starch content, soluble sugar content and / or yield, and / or (v) improved photosynthetic capacity.

2. The method of claim 1, wherein, The upregulating the expression or activity of GLK1 or G2 comprises overexpressing GLK1 or G2 in a plant.

3. The method of claim 1, wherein, The upregulating the expression or activity of GLK1 or G2 comprises introducing a GLK1 or G2 gene or an expression cassette containing the gene into a plant; introducing a gene editing reagent that improves the expression or activity of a GLK1 or G2 gene into a plant; improving the expression of a GLK1 or G2 gene with an expression-enhancing promoter or a tissue-specific promoter, or improving its expression in a specific tissue; improving the expression of a GLK1 or G2 gene in a plant with an enhancer; or, in different plants, screening for varieties with high expression of GLK1 or G2 genes, and obtaining offspring with trait improvement through hybrid breeding.

4. Use of GLK1 or G2 or an upregulator thereof for trait improvement in a plant of the family Poaceae, or for the manufacture of a plant of the family Poaceae with improved traits; wherein, The trait improvement comprises: (i) greener and / or thicker stems, (ii) increased plant height, (iii) increased biomass, (iv) increased starch content, soluble sugar content and / or yield, and / or (v) improved photosynthetic capacity.

5. Use according to claim 4, characterized in that, The upregulating molecule comprises: an expression cassette expressing GLK1 or G2; a gene editing reagent that improves the expression or activity of a GLK1 or G2 gene in a plant of the family Poaceae; or an upregulating molecule that interacts with GLK1 or G2 to improve its expression or activity.

6. The method according to any one of claims 1 to 3 or the use according to any one of claims 4 to 5, wherein The plant of the family Poaceae is or the GLK1 or G2 is from: a cereal plant of the family Poaceae; preferably, the cereal plant of the family Poaceae is a C4 plant; preferably, the cereal plant of the family Poaceae comprises: corn (Zea mays), rice (Oryza sativa), sorghum (Sorghum bicolor), millet (Setaria italica), barley (Hordeum vulgare), wild rice (Zizania latifolia), Leersia japonica Makino, wheat (Triticum aestivum), millet (Setaria italica), millet (Panicum miliaceum), rye (Secale cereale), oat (Avena sativa L.), Brachypodium distachyum.

7. Use according to claim 5 or 6, characterized in that, The amino acid sequence of the polypeptide of G2 is selected from the group consisting of: (i) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2; (ii) a polypeptide derived from (i) by substitution, deletion, or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, which has the function of regulating the trait; and (iii) a polypeptide having the function of regulating the trait and having at least 70% sequence identity to the amino acid sequence shown in SEQ ID NO:

2. (iii) a polypeptide having an amino acid sequence with a homology of > 80% to the amino acid sequence shown in SEQ ID NO: 2, which has the function of the modulated trait; or (iv) a polypeptide formed after adding a tag sequence or a restriction site sequence to the N or C terminus of the polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or adding a signal peptide sequence to the N terminus thereof. the amino acid sequence of the polypeptide of the GLK1 is selected from the group consisting of: (i) a polypeptide having any one of the amino acid sequences shown in SEQ ID NO: 4; (ii) a polypeptide derived from (i) by substitution, deletion, or addition of one or several amino acid residues to the amino acid sequence shown in SEQ ID NO: 4, which has the function of the modulated trait; (iii) a polypeptide having an amino acid sequence with a homology of > 80% to the amino acid sequence shown in SEQ ID NO: 4, which has the function of the modulated trait; or (iv) a polypeptide formed after adding a tag sequence or a restriction site sequence to the N or C terminus of the polypeptide having the amino acid sequence shown in SEQ ID NO: 4, or adding a signal peptide sequence to the N terminus thereof.

8. Use of a GLK1 gene or a G2 gene or a protein encoded thereby as a molecular marker for identifying a trait in a plant of the family Poaceae, or as a molecular marker for directed selection of a plant of the family Poaceae; said trait comprising: (i) a stem greenness trait and / or a stem thickness trait, (ii) a plant height trait, (iii) a biomass trait, (iv) a starch content, soluble sugar content trait and / or yield trait, and / or (v) a photosynthetic capacity trait.

9. A method of identifying a trait in a grass plant or of directed selection for improved traits in a grass plant, comprising: analyzing the expression or activity of GLK1 gene or G2 gene or the protein encoded by the gene in a plant of the Poaceae family; if the expression or activity of GLK1 gene or G2 gene or the protein encoded by the gene in the plant of the Poaceae family to be tested is higher than the average of the plants of the Poaceae family, it indicates that the plant has excellent traits selected from the group consisting of: (i) stem greening and / or stem thickening, (ii) increased plant height, (iii) increased biomass, (iv) increased starch content, soluble sugar content and / or yield, and / or (v) improved photosynthetic capacity; and the plant is used as a plant for trait improvement.

10. A method for screening a substance for improving traits of a plant of the Poaceae family, the method comprising: (1) adding a candidate substance to a system expressing GLK1 or G2; (2) detecting the system to observe the expression or activity of GLK1 or G2 therein, and if the expression or activity is improved, it indicates that the candidate substance is a substance that can be used to improve traits of a plant of the Poaceae family; wherein improving traits of a plant of the Poaceae family includes traits selected from the group consisting of: (i) stem greening and / or stem thickening, (ii) increased plant height, (iii) increased biomass, (iv) increased starch content, soluble sugar content and / or yield, and / or (v) improved photosynthetic capacity.

11. A cell, tissue or organ of the Gramineae expressing an expression cassette for GLKl or G2; preferably, the expression cassette comprises: a promoter, a gene encoding GLK1 or G2, and a terminator; preferably, the expression cassette is comprised in an expression construct or an expression vector. a promoter, a gene encoding GLK1 or G2, and a terminator; preferably, the expression cassette is comprised in an expression construct or an expression vector.

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