Molecular breeding technique for creating super-high-yield high-quality c4 crop and use thereof

Abstract

A molecular breeding technique for creating a super-high-yield high-quality C4 crop and the use thereof. For the first time, the combined use of the GIF1 gene and the PHO1;2 gene being very effective for the improvement of traits of C4 crops is disclosed, the trait improvement comprising: (i) maintaining stalks green so as to increase the biomass of stalks for silage; (ii) increasing the saccharide content of stalks and reducing the phosphorus content of stalks; (iii) reducing the phosphorus content of grains; (iv) reducing the phytic acid content of grains; or (v) increasing the starch content of grains. In addition, the combined use of the GIF1 gene and the PHO1;2 gene can improve the yield or biomass traits of C4 crops, said improvement comprising: (a) greatly improving the yield; (b) greatly improving the biomass; (c) improving the utilization rate of phosphorus; and (d) improving the photosynthetic efficiency.

Description

Molecular breeding technology and its application for developing super high-yield and high-quality C4 crops

[0001] This application claims priority to patent application No. CN 202310789613.7, filed on June 29, 2023; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the fields of botany and molecular biology. More specifically, the present invention relates to a molecular breeding technology for creating super-high-yield and high-quality C4 crops and its application. Background Art

[0003] C4 plants, often abbreviated as C4 plants, absorb carbon dioxide from the air during their growth process and initially synthesize compounds containing four carbon atoms, such as malic acid or aspartic acid. Plants such as corn and sugarcane, on the other hand, primarily synthesize three-carbon molecules, such as phosphoglyceric acid, and are considered C3 plants. C4 plants have many advantages over C3 plants, including faster growth, higher carbon dioxide utilization rates, and lower water requirements.

[0004] Approximately 300 species of economic grasses are C4 plants. Cultivating new varieties that resemble C4 plants is of great significance for improving food security worldwide.

[0005] Corn (Zea mays L.), a C4 plant, is a widely cultivated primary cash crop and food staple worldwide. Its seeds can be used as food for humans and livestock feed, or processed into industrial raw materials such as starch and brewing. The germ can also be refined into artificial vegetable oil, while the stems and leaves can be used as silage or dry feed for livestock. With population growth, improving corn traits and enhancing its quality are currently key goals in corn breeding. However, improvements in corn traits, quality, and yield remain limited, and understanding of corn remains insufficient.

[0006] In production, the improvement effect contributed by the application of a single gene is often very limited due to the influence of material background and environment. Usually, the effective aggregation of multiple genes is required to improve one or more target traits. This is gene pyramiding breeding.

[0007] However, not all optimal gene combinations produce ideal additive effects or phenotypes; many may experience no effect or even negative effects. For example, combining NADH-GOGAT with NAD-IDH or NADH-GDH did not improve plant phenotypes or nitrogen utilization, indicating that this strategy cannot further improve the C / N balance in plants (Canas et al., 2020) and that the process of combining different genes is relatively complex.

[0008] Therefore, studying how to further improve or modify the yield, quality and other excellent traits of corn is of great significance for significantly increasing corn yield.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide a molecular breeding technology for creating super high-yield and high-quality C4 crops and its application.

[0011] In a first aspect of the present invention, a method for improving the grain or stalk traits of C4 crops or preparing C4 crops with improved grain or stalk traits is provided, comprising: jointly upregulating the expression or activity of GIF1 and PHO1;2 in C4 crops; the trait improvements include: (i) maintaining the green color of the stalks and increasing the biomass of the stalks for ensilage; (ii) increasing the sugar content of the stalks and reducing the phosphorus content of the stalks; (iii) reducing the phosphorus content of the grains; (iv) reducing the phytic acid content of the grains or (v) increasing the starch content of the grains.

[0012] In another aspect of the present invention, a method for improving the yield or biomass traits of C4 crops is provided, comprising: jointly upregulating the expression or activity of GIF1 and PHO1;2 in C4 crops; the improved yield or biomass traits include: (a) significantly increasing (50% or more) yield; (b) increasing biomass; (c) improving phosphorus utilization; and (d) improving photosynthetic efficiency.

[0013] In one or more embodiments, the increasing of yield (including yield per plant) or biomass (including biomass per plant) includes: increasing plant height, increasing root system, increasing yield per ear, increasing number of grains per ear, increasing 100-grain weight of grains, increasing stem diameter, increasing leaves, and increasing ear length; preferably, increasing the number of grains per ear includes: increasing the number of rows of grains and increasing the number of grains in each row.

[0014] In one or more embodiments, the upregulation of GIF1 and PHO1;2 expression or activity comprises: overexpressing GIF1 and PHO1;2 in C4 crops; preferably, comprising: introducing GIF1 and PHO1;2 genes or expression cassettes containing said genes (such as contained in an expression construct or expression vector) into C4 crops; introducing gene editing reagents that increase the expression or activity of GIF1 and PHO1;2 genes in C4 crops into C4 crops; increasing the expression of GIF1 and PHO1;2 genes in C4 crops, or increasing their expression in specific tissues, by expressing an enhanced promoter or a tissue-specific promoter; increasing the expression of GIF1 and PHO1;2 genes in C4 crops by using enhancers; or, targeted screening of varieties with high expression of GIF1 and PHO1;2 genes in different C4 crops, and obtaining offspring with improved traits through hybrid breeding.

[0015] In another aspect of the present invention, a combination of GIF1 and PHO1;2 or their up-regulated molecules is provided for improving the traits of C4 crops, or preparing C4 crops with improved traits; wherein the improved traits include: (i) maintaining the green color of the stems and increasing the biomass of the stems for silage; (ii) increasing the sugar content of the stems and reducing the phosphorus content of the stems; (iii) reducing the phosphorus content of the grains; (iv) reducing the phytic acid content of the grains or (v) increasing the starch content of the grains.

[0016] In another aspect of the present invention, a combination of GIF1 and PHO1;2 or their up-regulating molecules is provided for improving the yield or biomass traits of C4 crops; wherein the improved yield or biomass traits include: (a) increasing yield; (b) increasing biomass; (c) improving phosphorus utilization; and (d) improving photosynthetic efficiency.

[0017] In one or more embodiments, the up-regulating molecules include: expression cassettes for expressing GIF1 and PHO1;2 (such as contained in an expression construct or expression vector); gene editing reagents that increase the expression or activity of GIF1 and PHO1;2 genes in C4 crops; or up-regulating molecules that interact with GIF1 and PHO1;2, thereby increasing their expression or activity.

[0018] In another aspect of the present invention, a C4 crop cell is provided, which expresses exogenous GIF1 and PHO1;2 expression cassettes; preferably, it comprises: expression cassette 1, which comprises: a promoter, a gene encoding GIF1, and a terminator; and expression cassette 2, which comprises: a promoter, a gene encoding PHO1;2, and a terminator; preferably, the expression cassettes are contained in an expression construct or an expression vector.

[0019] In one or more embodiments, the C4 crop is or the GIF1 or PHO1;2 is from: a cereal C4 crop; preferably, the cereal C4 crop includes (but is not limited to): corn (Zea mays), sorghum (Sorghum bicolor), millet (Setaria italica), etc.

[0020] In one or more embodiments, the PHO1;2 encoding gene includes a cDNA sequence, a genomic sequence, or an artificially optimized or modified sequence based thereon.

[0021] In one or more embodiments, the GIF1 encoding gene includes a cDNA sequence, a genomic sequence, or an artificially optimized or modified sequence based thereon.

[0022] In one or more embodiments, the amino acid sequence of the PHO1;2 polypeptide is selected from the following group: (i) a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 2 or 4; (ii) a polypeptide having the function of regulating the trait, which is formed by replacing, deleting or adding one or several (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues of the amino acid sequence as shown in SEQ ID NOs: 1, 2 or 4, and derived from (i); (iii) a polypeptide having an amino acid sequence with ≥80% (preferably ≥85%, ≥90%, ≥95% or ≥98%) homology to the amino acid sequence as shown in SEQ ID NOs: 1, 2 or 4, and having the function of regulating the trait; or (iv) a polypeptide having a tag sequence or an enzyme cleavage site sequence added to the N- or C-terminus of the polypeptide having the amino acid sequence as shown in SEQ ID NOs: 1, 2 or 4, or a signal peptide sequence added to its N-terminus.

[0023] In one or more embodiments, the amino acid sequence of the polypeptide of GIF1 is selected from the following groups: (i) a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO: 3 or 5; (ii) a polypeptide having the function of regulating the trait and derived from (i) formed by replacing, deleting or adding one or several (such as 1-20, 1-10, 1-5, 1-3) amino acid residues in the amino acid sequence as shown in SEQ ID NO: 3 or 5; (iii) a polypeptide having an amino acid sequence with a homology of ≥80% (preferably ≥85%, ≥90%, ≥95% or ≥98%) to the amino acid sequence as shown in SEQ ID NO: 3 or 5, and having the function of regulating the trait; or (iv) a polypeptide formed by adding a tag sequence or an enzyme cleavage site sequence to the N- or C-terminus of the polypeptide having the amino acid sequence as shown in SEQ ID NO: 3 or 5, or adding a signal peptide sequence to its N-terminus.

[0024] In another aspect of the present invention, there is provided a use of the GIF1 gene and the PHO1;2 gene or the proteins encoded thereby, as molecular markers for identifying traits of C4 crops, or as molecular markers for targeted screening of C4 crops; the traits include: (i) stalk color, stalk biomass for ensilage; (ii) stalk sugar content, stalk phosphorus content; (iii) grain phosphorus content; (iv) grain phytic acid content or (v) increased grain starch content.

[0025] In one or more embodiments, the expression levels of GIF1 gene and PHO1;2 gene or the activities of GIF1 protein and PHO1;2 protein in C4 crops are analyzed to determine the traits of C4 crops or to perform targeted screening.

[0026] In another aspect of the present invention, a method for identifying traits of C4 crops is provided, comprising: analyzing the activities of GIF1 and PHO1;2 genes or proteins encoded thereby in the C4 crops; if the activities of GIF1 and PHO1;2 genes or proteins encoded thereby in the C4 crops to be tested are equal to or higher than the average values ​​of the C4 crops of that type, it indicates that the crops have excellent traits, wherein the excellent traits are selected from the group consisting of: (i) maintaining stalk greenness and increasing stalk biomass for silage; (ii) increasing stalk sugar content and decreasing stalk phosphorus content; (iii) decreasing grain phosphorus content; (iv) decreasing grain phytic acid content; or (v) increasing grain starch content; if the expression level of the PHO1;2 gene or the activity of the PHO1;2 protein in the C4 crops to be tested is lower than the average values ​​of the C4 crops of that type, the crops have unsatisfactory traits.

[0027] In another aspect of the present invention, a method for targeted selection of C4 crops with improved traits is provided, the method comprising: analyzing the activities of the GIF1 gene and the PHO1;2 gene or the proteins encoded therein in the C4 crop; if the activities of the GIF1 gene and the PHO1;2 gene or the proteins encoded therein in the C4 crop to be tested are equal to or higher than the average value of the C4 crop of that type, it indicates that the crop has excellent traits, wherein the excellent traits are selected from the group consisting of: (i) maintaining the green color of the stems and increasing the biomass of the stems for silage; (ii) increasing the sugar content of the stems and reducing the phosphorus content of the stems; (iii) reducing the phosphorus content of the grains; (iv) reducing the phytic acid content of the grains; or (v) increasing the starch content of the grains; and the crop is used as a C4 crop for improved traits.

[0028] In one or more embodiments, the selected C4 crop has high expression of GIF1 gene and PHO1;2 gene or high activity of GIF1 protein and PHO1;2 protein. Preferably, the high expression or high activity means that the expression or activity is statistically significantly increased compared with the average value of expression or activity of the same type or species of C4 crops.

[0029] In one or more embodiments, the increase, enhancement, promotion, improvement or enhancement represents a significant increase, enhancement, promotion, improvement or enhancement, such as an increase, enhancement, promotion, improvement or enhancement of 20%, 40%, 60%, 80%, 90% or more.

[0030] In one or more embodiments, the reduction refers to a significant reduction, such as a reduction of 20%, 40%, 60%, 80%, 90% or less.

[0031] In another aspect of the present invention, a method for screening substances (potential substances) for improving C4 crop traits is provided, the method comprising: (1) adding a candidate substance to a system expressing GIF1 and PHO1;2; (2) detecting the system, observing the expression or activity of GIF1 and PHO1;2, and if the expression or activity is increased, it indicates that the candidate substance is a substance that can be used to improve C4 crop traits; wherein the improved C4 crop traits include substances selected from the group consisting of: (i) maintaining stem greenness and increasing the biomass of stems for silage; (ii) increasing stem sugar content and reducing stem phosphorus content; (iii) reducing grain phosphorus content; (iv) reducing grain phytic acid content; or (v) increasing grain starch content.

[0032] In one or more embodiments, the improved traits further include those selected from the group consisting of: (vi) substantially increased yield (e.g., increased by 50% or more); (vii) increased biomass; (viii) increased phosphorus utilization; and (ix) increased photosynthetic efficiency.

[0033] In one or more embodiments, a control group is further included to clearly distinguish the difference in expression or activity of GIF1 and PHO1;2 in the test group and the control group.

[0034] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules designed for GIF1 and PHO1;2 genes or their encoded proteins or their upstream or downstream proteins or genes (such as upregulators, small molecule compounds, gene editing constructs, etc.).

[0035] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Overexpression of ZmPHO1;2 driven by AJ and Ubi promoters increases maize yield.

[0037] Figure 2 Overexpression of ZmPHO1;2 driven by AJ and 35S promoters increases maize yield.

[0038] Figure 3 AD and qRT detection of expression levels of ZmPHO1;2a and ZmPHO1;2b overexpressing strains.

[0039] Fig. 4 Overexpression of ZmGIF1 driven by AG and 35S promoters.

[0040] Figure 5 The F1 hybrids produced by hybridization of AG, B104, and C01 exhibited significant heterosis.

[0041] Figure 6 Hybrid corn with the dual genes AF, ZmGIF1, and ZmPHO1;2a significantly increased yield.

[0042] Figure 7 Hybrid maize synthesized by AF, 35S-ZmGIF1, and Ubi-ZmPHO1;2 significantly increased biomass.

[0043] Figure 8. Hybrid corn with AF, 35S-ZmGIF1, and 35S-ZmPHO1;2 polymerization significantly increased biomass.

[0044] Figure 9: Hybrid corn synthesized by AD, 35S-ZmGIF1, and Ubi-ZmPHO1;2 showed significant yield increase.

[0045] Figure 10: Hybrid corn with AD, 35S-ZmGIF1 and 35S-ZmPHO1;2 polymerization significantly increased yield.

[0046] Figure 11 Hybrid corn with AF, ZmGIF1 and ZmPHO1;2 significantly enhances the photosynthetic efficiency of C4 crops.

[0047] Figure 12 AG, ZmGIF1, and ZmPHO1;2 hybrid corn reduce grain phosphorus content.

[0048] Figure 13 AD, ZmPHO1;2 and ZmGIF1 hybrid corn enhances corn kernel quality.

[0049] Figure 14 AF, ZmPHO1;2, and ZmGIF1 hybrid corn can be used as excellent silage.

[0050] Figure 15. AE. Polymerization of SbPHO1;2 and SbGIF1 in sorghum increases sorghum grain weight and yield. DETAILED DESCRIPTION

[0051] After in-depth research, the inventors have revealed for the first time that the combined use of the GIF1 gene and the PHO1;2 gene is highly effective in improving C4 crop traits, including: (i) maintaining stem greenness and increasing silage stem biomass; (ii) increasing stem sugar content and reducing stem phosphorus content; (iii) reducing grain phosphorus content; or (iv) reducing grain phytic acid content. Furthermore, the combined use of the GIF1 gene and the PHO1;2 gene can improve C4 crop yield or biomass traits, including: (a) significantly increasing yield; (b) increasing biomass; (c) improving phosphorus utilization; and (d) improving photosynthetic efficiency. This invention provides a new approach for improving C4 crops.

[0052] GIF1 and PHO1;2

[0053] As used herein, the term "PHO1;2 gene or PHO1;2 protein (polypeptide)" includes PHO1;2 genes or proteins from C4 crops, such as corn or sorghum. In corn, there are two types of PHO1;2, including ZmPHO1;2a and ZmPHO1;2b. In sorghum, there is SbPHO1;2.

[0054] As used herein, the "GIF1 gene or GIF1 protein (polypeptide)" refers to the GIF1 gene or GIF1 protein from C4 crops, such as corn or sorghum.

[0055] In the present invention, the GIF1 protein or PHO1;2 protein also includes fragments, derivatives and analogs thereof.

[0056] As used herein, the terms "fragment," "derivative," and "analog" refer to protein fragments that substantially retain the same biological function or activity as the polypeptide described herein, and may be (i) proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins having a substituent group in one or more amino acid residues, or (iii) proteins formed by fusing an additional amino acid sequence to this protein sequence. These fragments, derivatives, and analogs are within the scope of those skilled in the art, as defined herein. Biologically active fragments of the GIF1 or PHO1;2 proteins described herein can be applied to the present invention.

[0057] In the present invention, the term "PHO1;2 protein" refers to a protein having the sequence of any one of SEQ ID NOs: 1, 2, or 4, which has the trait-improving activity described above in the present invention, and also includes variants of SEQ ID NOs: 1, 2, or 4 that have the same function as these polypeptides. The term "GIF1 protein" refers to a protein having the sequence of SEQ ID NOs: 3 or 5, which has the trait-improving activity described above in the present invention, and also includes variants of SEQ ID NOs: 3 or 5 that have the same function as these polypeptides. These variants include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5), and additions or deletions of one or more amino acids (usually within 20, preferably within 10, and more preferably within 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter protein function. For another example, adding or deleting one or more amino acids at the C-terminus and / or N-terminus generally does not alter protein function.

[0058] In the present invention, the "GIF1" or "PHO1;2" also includes its homologs in C4 crops. It should be understood that although the present invention prefers GIF1 or PHO1;2 obtained from a specific C4 species, corn, other proteins from C4 crops that have high homology to the GIF1 or PHO1;2 protein polypeptide (such as 80% or higher homology to the polypeptide sequence shown in SEQ ID NO: 1, 2 or 4; more preferably 85% or higher homology, such as 90%, 95%, 98% or 99% homology) and have the same function are also included in the present invention. Methods and tools for comparing sequence identity 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 based on the percentage of identical positions.

[0059] The present invention also includes polynucleotides (genes) encoding the polypeptides, which may be natural genes from crops or their degenerate sequences.

[0060] The vector containing the coding sequence, and the host cell produced by genetic engineering using the vector or polypeptide coding sequence are also included in the present invention. Methods well known to those skilled in the art can be used to construct appropriate expression vectors.

[0061] Host cells are typically plant cells. Plant transformation can generally be achieved using methods such as Agrobacterium transformation or biolistic transformation, such as the leaf disc method or embryonic embryo transformation method; the Agrobacterium method is preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits relative to wild-type plants.

[0062] As used herein, the term "crop" refers to plants that have economic value in agriculture and industry, such as grain, cotton, and oilseed production. This value can be found in useful parts of the plant, such as seeds, fruits, roots, stems, and leaves. Crops include, but are not limited to, dicotyledons and monocotyledons. Preferred crops are C4 crops, with corn and sorghum being more preferred.

[0063] In the present invention, the crops include plants expressing GIF1 and PHO1;2, preferably C4 cereal crops. Preferably, the cereal crops have grains.

[0064] application

[0065] Corn is an important economic and food crop worldwide. Further improving the yield of corn with high photosynthetic efficiency, C4, is crucial for meeting the rapidly growing demand for corn-derived food, feed, and fuel. High crop yields depend on a well-balanced relationship between "source, sink, and flow." In production, breeders typically enhance the yield of corn inbred lines and even hybrids by aggregating superior genetic resources to enhance the desirable characteristic of large ears. In the present invention, two key grain-filling genes, PHO1;2 (encoding an inorganic phosphorus transporter) and GIF1 (encoding a cell wall sucrose invertase), were analyzed to enhance the source and sink strength of crops and promote photosynthesis in C4 crops. The two genes were transferred into different inbred lines (C01 and B104) to produce overexpression strains. The PHO1;2 and GIF1 overexpressing transgenic lines from different sources were further hybridized and polymerized through hybrid breeding. Analysis revealed that the hybrid corn had extremely significant improvements in the following traits, which were caused by the synergistic effect of the combined application of the two genes: (i) maintaining the green color of the stems and increasing the biomass of the stems for ensilage; (ii) increasing the sugar content of the stems and reducing the phosphorus content of the stems; (iii) reducing the phosphorus content of the grains; (iv) reducing the phytic acid content of the grains; (or) (v) increasing the starch content of the grains. At the same time, the combined application of the two genes can significantly improve the photosynthetic efficiency of C4 crops by about 30%, and the final yield is increased by about 60%; the biomass increases by about 80%, and the sugar content in the stems and leaves increases, which can be used as excellent silage; in addition, the phosphorus content and phytic acid content in the grains are significantly reduced, and the content of nutrients such as starch and sugar is increased, thereby enhancing the quality of corn grains. The present invention also found that the improved hybrid corn can maintain high-yield traits under low-fertilizer conditions and has a high fertilizer utilization rate. The high-yield breeding target gene and hybrid corn produced by the present invention provide new methods and ideas for significantly increasing the yield of C4 crops such as corn, sorghum, and millet, improving grain quality, reducing fertilizer use, and improving the soil environment.

[0066] In previous studies, the present inventors cloned two key positive regulatory genes for grain filling, GIF1 and OsPHO1;2, in rice. GIF1 encodes a cell wall sucrose invertase that converts sucrose into glucose and fructose. Overexpression of GIF1 significantly increased the activity of the cell wall sucrose invertase, ultimately increasing rice yield (Wang et al., 2008). OsPHO1;2 encodes an inorganic phosphorus transporter of the PHO1 family with specific efflux and influx activities. It regulates the balance of Pi in endosperm cells during grain filling, affects the activity of AGPase, and ultimately regulates filling. Overexpression of OsPHO1;2 can reduce the accumulation of Pi in grains, promote grain filling and rice yield, maintain high-yield traits under low-P conditions, and has high P utilization efficiency (Ma et al., 2021). Importantly, these two key grain-filling genes are highly conserved in Poaceae crops, and their gene functions are also conserved. In maize, the deletion of homologous genes of GIF1 and OsPHO1;2 also produces severe grain-filling defect phenotypes (Kang et al., 2009; Ma et al., 2021), indicating that PHO1;2 and GIF1 in maize also regulate grain filling.

[0067] The inventors discovered that the two grain-filling genes, PHO1;2 and GIF1, regulate the grain-filling process in cereals from different perspectives. PHO1;2 promotes phosphorus distribution to leaves to support photosynthesis, generating more carbon assimilates that are then transported to the grains to promote grain filling, while also maintaining a steady-state level of inorganic phosphorus accumulation in the grains during the filling period. GIF1, on the other hand, is responsible for unloading sucrose from the grains. Sucrose transported from the source to the grains via vascular tissue is broken down into monosaccharides by GIF1, which are used to synthesize starch, replenishing the sink and promoting grain filling. This invention addresses both the source and sink perspectives by overexpressing the two grain-filling genes in different inbred lines. These two overexpressing lines from different backgrounds are then crossed to produce hybrid corn lines with both genes overexpressed. The yield and other superior traits of the improved hybrid corn are then analyzed. Field fertilizer utilization efficiency of the high-yield hybrid corn is also evaluated, demonstrating the potential for high-yield, high-quality, and low-fertilizer hybrid corn. Furthermore, improvements in various other traits are also achieved.

[0068] Based on the inventors' novel discovery, a method for improving plants is provided, comprising: upregulating the expression or activity of PHO1;2 in the plant; wherein the improved traits include: (i) maintaining stem greenness and increasing the biomass of stems for silage; (ii) increasing the sugar content of the stems and reducing the phosphorus content of the stems; (iii) reducing the phosphorus content of the grains; (iv) reducing the phytic acid content of the grains (or) (v) increasing the starch content of the grains.

[0069] The present invention also provides a method for improving the yield or biomass traits of C4 crops, comprising: jointly upregulating the expression or activity of GIF1 and PHO1;2 in C4 crops; the improved yield or biomass traits include: (a) significantly improving yield; (b) significantly improving biomass; (c) improving phosphorus utilization; and (d) improving photosynthetic efficiency.

[0070] It should be understood that based on the experimental data and regulatory mechanisms provided by the present invention, various methods well known to those skilled in the art can be used to regulate the expression of GIF1 and PHO1;2, and these methods are all included in the present invention.

[0071] In the present invention, substances that upregulate the expression or activity of GIF1 and PHO1;2 in plants include promoters, agonists, activators, and upregulators. The "upregulation", "increase", and "promotion" mentioned above include "upregulation", "promotion" of protein activity or "upregulation", "increase", and "promotion" of protein expression. Any substance that can increase the activity of GIF1 and PHO1;2 proteins, increase the stability of GIF1 and PHO1;2 genes or the proteins encoded by them, upregulate the expression of GIF1 and PHO1;2 genes, or increase the effective action time of GIF1 and PHO1;2 proteins can be used in the present invention as substances useful for upregulating GIF1 and PHO1;2 genes or the proteins encoded by them. They can be compounds, small chemical molecules, or biological molecules. The biological molecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0072] As another embodiment of the present invention, a method for upregulating the expression of GIF1 and PHO1;2 genes or the proteins encoded by them in plants is also provided, the method comprising: transferring the expression constructs or vectors of GIF1 and PHO1;2 into plant tissues, organs or tissues to obtain plant tissues, organs or seeds transformed with the polynucleotides encoding GIF1 and PHO1;2; and regenerating the obtained plant tissues, organs or seeds transformed with the exogenous polynucleotides encoding GIF1 and PHO1;2 into plant plants.

[0073] Other methods for increasing the expression of GIF1 and PHO1;2 genes, or their homologs, are well known in the art. For example, expression of PHO1;2 genes, or their homologs, can be enhanced by driving them with a strong promoter. Alternatively, expression of PHO1;2 genes can be enhanced by using enhancers (such as the first intron of the Actin gene), or gene editing can be used to increase the expression and protein activity of GIF1 and PHO1;2 genes, or their homologs. Strong promoters suitable for the methods of the present invention include, but are not limited to, the 35S promoter and the Ubi promoter of maize.

[0074] The methods described can be carried out using any suitable conventional means, including reagents, temperatures, pressure conditions, and the like.

[0075] Now that the functions of the GIF1 and PHO1;2 genes are known, they can be used as molecular markers for targeted plant screening. This new discovery can also be used to screen for substances or potential substances that modulate this mechanism to regulate plant architecture, yield, organelles, or the cell cycle. GIF1 and PHO1;2, or the proteins they encode, can also be used as tracking markers for the progeny of genetically transformed plants.

[0076] Therefore, the present invention provides a method for targeted selection or identification of plants, which comprises: identifying the expression or activity of GIF1 and PHO1;2 genes in a test plant; if the GIF1 and PHO1;2 proteins of the test plant are highly expressed or highly active, then the plant: (i) maintains the green color of the stems and increases the biomass of the stems for silage; (ii) increases the sugar content of the stems and reduces the phosphorus content of the stems; (iii) reduces the phosphorus content of the grains; (iv) reduces the phytic acid content of the grains; (or) (v) increases the starch content of the grains; otherwise, the plant's traits are undesirable.

[0077] When evaluating the test plant, the expression level or mRNA level of GIF1 and PHO1;2 can be measured to understand whether the expression or mRNA level in the test plant is higher than the average value of such plants. If it is significantly higher, it has improved traits.

[0078] The present invention provides a method for screening for regulating plant architecture traits, yield traits, organelles or cell cycles, the method comprising: adding a candidate substance to a system expressing GIF1 and PHO1;2; detecting the system to observe the expression or activity of GIF1 and PHO1;2, and if the expression or activity is increased, it indicates that the candidate substance is a substance that can be used to improve C4 crop traits; wherein the improved C4 crop traits include substances selected from the following groups: (i) maintaining stem greenness and increasing the biomass of stems for ensilage; (ii) increasing the sugar content of the stems and reducing the phosphorus content of the stems; (iii) reducing the phosphorus content of the grains; (iv) reducing the phytic acid content of the grains; (or) (v) increasing the starch content of the grains.

[0079] Methods for screening for substances that act on proteins or genes, or specific regions thereof, as targets are well known in the art and can be used in the present invention. The candidate substances can be selected from the group consisting of peptides, polymeric 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 will appreciate how to select an appropriate screening method.

[0080] To detect protein-protein interactions and the strength of the interactions, various techniques well known to those skilled in the art can be used, such as GST pull-down technique (GST-Pull Down), bimolecular fluorescence complementation assay, yeast two-hybrid system or immunoprecipitation technique.

[0081] After large-scale screening, a class of potential substances that specifically act on GIF1 and PHO1;2 and have a regulatory effect on the plant traits can be obtained.

[0082] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0083] Materials and Methods

[0084] 1. Construction of transgenic corn materials

[0085] The genomic and coding region sequences of the three genes used in this study, ZmPHO1;2a (Zm00001d051945), ZmPHO1;2b (Zm00001d018445), and ZmGIF1 (Zm00001d003776), were obtained from the NCBI database. The three genes were cloned using PCR amplification of gDNA or cDNA from the B73 maize inbred line. Two different strong promoter overexpression systems, Ubi and 35S, were constructed for ZmPHO1;2a and ZmPHO1;2b (using pCambia3301 as the expression vector). All transgenic constructs were transformed into C01 or B104 inbred lines using Agrobacterium tumefaciens EHA105-mediated genetic transformation.

[0086] 2. Phenotype and yield trait analysis

[0087] All maize and sorghum accessions were grown twice annually in transgenic greenhouses in Songjiang, Shanghai (spring and summer), and once annually in Sanya, Hainan (winter). Each generation was self-pollinated using a strict bagging method for three consecutive generations, and homozygous lines were selected for phenotypic analysis. The F1 hybrid maize generation did not require bagging and was naturally pollinated. At the late grain filling stage, measurements were taken of plant height, functional leaf size, leaf number, ear position, and number of tassel branches. At maturity, biomass (aboveground part of the ear), yield, number of kernel rows, number of kernels per row, ear length, ear inner diameter, number of kernels per ear, and 100-kernel weight were analyzed.

[0088] 3. Gene Expression Analysis

[0089] Corn leaves, seeds and other tissues were collected in 2 mL imported EP tubes (with steel balls added in advance) and quickly frozen in liquid nitrogen. Grind into powder using a grinder at 40 Hz for 50 seconds, and extract total RNA using the TRIzol (Invitrogen) method. 2 μg of total RNA was reverse transcribed according to the instructions of the Weizan reverse transcription kit, and the cDNA product was used for qPCR analysis. The detection instrument used was a Bio-Rad real-time fluorescence quantitative PCR instrument. Premix Ex Taq TM (2×) (Takara).

[0090] The reaction used a two-step amplification procedure: 95℃ pre-denaturation for 30s, 95℃ denaturation for 10s, 60℃ annealing and extension for 30s, 40 cycles, and additional melting curve analysis. -ΔΔCT The relative gene expression was analyzed by the method.

[0091] 4. Determination of soluble sugar and total starch content in tissues

[0092] Corn leaves or seeds (0.40 g) were thoroughly ground with liquid nitrogen and placed in a 2 mL centrifuge tube. 1 mL of MilliQ water was added, and the tube was uncapped and incubated in a 100°C waterbath for 15–20 min. The tube was then transferred to a 10 mL centrifuge tube and made up to 5–10 mL with deionized water, depending on the sample weight. The tube was centrifuged at 10,000 g for 10 min, and the supernatant was filtered through a 0.45 μm filter. The clarified sample solution was then loaded manually or automatically into a sampling vial (0.6 mL sample) for analysis of glucose, fructose, and sucrose on an ion chromatograph (ICS-3000, DIONEX) using a CarboPac™ PA1 column. The mobile phase consisted of 200 mM NaOH at a flow rate of 1.5 mL / min, using an electrochemical detector.

[0093] Corn seeds were ground and passed through a 0.5 mm sieve. The ground sample (accurately weighed 100 mg) was added to a test tube (16 x 120 mm), ensuring that the entire sample was at the bottom of the tube. 0.2 mL of 80% v / v ethanol was added to moisten the sample to aid dispersion and mixed using a vortex mixer. The total starch content of the sample was determined using a Megazyme K-TSTA kit.

[0094] 5. Phosphorus content determination

[0095] (1) Determination of inorganic phosphorus content

[0096] Take 0.5 g of sample, add 10 mL of extract (12.5% ​​TCA, 25 mM MgCl2), shake overnight at 4°C, centrifuge at 10,000 g for 15 minutes at 4°C, collect 5 mL of supernatant, and determine the phosphorus content using the ammonium vanadium molybdate colorimetric method. Repeat three times for each sample. Most samples can be transferred to an ELISA plate for analysis. Preparation of phosphorus standard solution and plotting the working curve: Dry potassium dihydrogen phosphate at 105°C for 1 hour, cool in a desiccator, weigh 0.2195 g, dissolve in water, transfer to a 1000 mL volumetric flask, add 3 mL of nitric acid, make up to volume with deionized double-distilled water, and shake well to prepare a 50 μg / mL phosphorus standard solution. Accurately transfer 0.0, 1.0, 2.0, 4.0, 8.0, and 16.0 mL of P standard solution into a 50 mL volumetric flask, add 10 mL of ammonium vanadium molybdate color developer (containing 100 g / L ammonium molybdate, 2.35 g / L ammonium vanadate, and 165 mL / L 65% nitric acid) to each flask, dilute to the scale with deionized double distilled water, shake well, and let stand at room temperature for 10 min. Use 0.0 mL of P standard solution as a control and measure the absorbance of various P standard solutions at a wavelength of 400 nm using a 751 spectrophotometer. Plot a working curve (GB / T 6437-2002) with Pi content as the abscissa and absorbance as the ordinate.

[0097] (2) Determination of total phosphorus content

[0098] 10 mg of samples from different tissues were added to microwave digestion tubes, and then 1 mL of 65% concentrated HNO3 was added to each tube. The samples were digested for about 4-5 h using a Microwave 3000 (Anton PAAR, Graz, Austria) microwave digestion system (a boiling water bath for 4 h can also be used instead).

[0099] After digestion, open the lid of the microwave digestion tube and place it in an acid remover or a fume hood at 160°C to remove the acid (about 1-1.5 hours); the remaining volume should be 1.0 mL, and then add deionized water to make up to 14 mL.

[0100] Digested samples were used to determine phosphorus (P), sulfur (S), and various trace element concentrations. Phosphorus content was determined using an inductively coupled plasma optical emission spectrometer (ICP-OES) (PerkinElmer, USA) on our instrument platform. Six biological replicates were performed for each sample.

[0101] 6. Determination of phytic acid content

[0102] Approximately 0.3 g of tissue sample was taken and 10 mL of 0.6 M HCl was added to a 50 mL centrifuge tube. The tube was mixed by inversion and then dissolved in boiling water for 30 min. The dissolved sample was removed, cooled to room temperature, and centrifuged at 10,000 g for 15 min at 4°C. 500 μL of the supernatant was aspirated and diluted 10-fold to 5 mL with deionized water. The diluted sample was passed through an IC-RP column, an IC-H column, and a 0.22 μm microporous filter membrane in sequence to remove hydrophobic substances, alkaline earth metal ions, carbonate ions, metal ions, and macromolecular substances in the sample.

[0103] The prepared samples were analyzed by ion exchange chromatography ICS-2000, where the separation column and guard column were selected as IonPac AS11-HC (250 mm × 4 mm) and IonPac AG11-HC (50 mm × 4 mm), respectively. The eluent was 50 mM KOH (prepared with deionized water to reduce ion interference), and the elution rate was set to 1 mL min -1 The signal was detected at an inhibitory current of 124 mA, and the standard sample was prepared with sodium phytate (P-3168, Sigma). Each sample was repeated three times.

[0104] 7. Photosynthesis measurement

[0105] The steady-state photosynthetic rate (A net ) was measured. 2 Light saturation (1500 μmol m -2 s -1 ) Net CO2 assimilation rate (Anet). A was measured under the following additional settings net : Ambient relative humidity, 400 ppm reference CO2, flow rate 500 μmol s -1 All steady-state photosynthesis measurements were conducted in the field between 9:00 AM and 11:30 AM on sunny days.

[0106] 8. Gene protein sequence

[0107] ZmPHO1;2a (SEQ ID NO: 1)

[0108] ZmPHO1;2b (SEQ ID NO: 2)

[0109] ZmGIF1 (SEQ ID NO: 3)

[0110] SbPHO1;2 (SEQ ID NO: 4)

[0111] SbGIF1 (SEQ ID NO: 5)

[0112] Example 1: Overexpression of ZmPHO1;2 increases corn yield

[0113] There are two homologous genes of OsPHO1;2 in maize, namely ZmPHO1;2a (Zm00001d051945) and ZmPHO1;2b (Zm00001d018445). In order to evaluate the phenotype of overexpression of ZmPHO1;2 in maize and its mechanism of regulating maize grain filling, the present inventors constructed an overexpression system of the full-length genomic sequence (gDNA) of the two genes in the maize C01 inbred line and expressed them under different overexpression promoters. The constructs were driven by maize Ubiquitin (Ubi), namely Ubi-ZmPHO1;2a-OE and Ubi-ZmPHO1;2b-OE, and 35S-ZmPHO1;2a-OE and 35S-ZmPHO1;2b-OE, driven by the strong promoter pCaMV35S of tobacco mosaic virus. They were transformed into the C01 inbred line through the Agrobacterium EHA105-mediated genetic transformation system to produce different independent overexpression strains.

[0114] Phenotypic analysis and statistics of stable, independent transgenic lines were performed at the T2 and T3 generations. The results showed that the Ubi promoter-driven ZmPHO1;2a overexpressing lines performed similarly to wild-type C01. Yield traits, including the number of rows, number of grains per row, 100-grain weight, and yield per ear, showed increasing trends but no significant differences (Figures 1A-F). In contrast, the Ubi promoter-driven ZmPHO1;2b overexpressing lines performed better than wild-type C01 overall. Two lines (Ubi-ZmPHO1;2b#3 and Ubi-ZmPHO1;2b#4) showed significantly larger ears, with significant increases in the number of rows, number of grains per row, 100-grain weight, and yield per ear (Figures 1B, GJ).

[0115] In addition, analysis of the ZmPHO1;2 overexpression lines driven by the 35S promoter revealed that the ZmPHO1;2a overexpression lines all exhibited a significantly larger ear phenotype, with the number of grain rows, number of grains per row, and 100-grain weight significantly higher than that of the wild-type C01, and the final yield per ear significantly increased (Figure 2A, CF); the ZmPHO1;2b overexpression lines had larger ears, increased number of grains per ear and grain weight, and the final weight per ear was also higher than that of the wild-type (Figure 2B, GJ).

[0116] These results indicate that ectopic overexpression of ZmPHO1;2 driven by either the Ubi promoter or the 35S promoter can increase maize yield to a certain extent, among which the 35S promoter has a better effect.

[0117] To analyze the differences generated by different promoters, the inventors performed qPCR gene expression analysis on two promoter-driven overexpression lines. They found that the Ubi promoter had relatively low levels of overexpression, especially Ubi-ZmPHO1;2a, which showed only a 100-400-fold upregulation, while the 35S-driven overexpression of both genes was upregulated by more than several thousand-fold (Figures 3A-D). This indicates that the gene upregulation factor is closely correlated with gene expression and phenotype.

[0118] Example 2: Effect of Overexpression of ZmGIF1 on Corn Yield

[0119] To investigate the regulatory role of the ZmGIF1 gene in maize grain filling and its contribution to yield, the inventors overexpressed the full-length CDS coding region of ZmGIF1, driven by the 35S promoter, in the maize B104 inbred line. Independent overexpression lines were generated and phenotypes were analyzed repeatedly in the T2 and T3 generations. In two randomly selected lines, expression levels were significantly upregulated, and yield at maturity showed an increasing trend in the number of grains per row and per ear, with ears also tending to become larger (Figures 4A-G).

[0120] Therefore, ZmGIF1 also has a certain yield-increasing effect when driven by a strong promoter. This suggests that the yield increase from overexpression of the single genes ZmPHO1;2 and ZmGIF1 is relatively small. The fundamental reason is that there is a lack of coordination between the "source" and "sink." Unilaterally increasing either the "source" or the "sink" cannot achieve maximum utilization.

[0121] Example 3: Hybrid corn overexpressing both ZmPHO1;2 and ZmGIF1 genes significantly increases corn biomass. The present inventors discovered that overexpressing both ZmPHO1;2 and ZmGIF1 genes alone showed potential to increase corn yield, but only modestly. Considering the coordination between the "source-sink" function, a crucial factor in increasing crop yield, they hypothesized that the source and sink of single-gene overexpression were not fully utilized. Simultaneously overexpressing both genes could enhance and bridge both the "source" and "sink" of corn. First, the present inventors investigated the feasibility of hybridization between two parents. Considering the B104 background, which has more pollen but a lower ear position, and the C01 background, which has less pollen but a more appropriate ear position, the B104 background was selected as the male parent and the C01 background as the female parent, and hybridization was performed to produce F1 hybrids. Analysis of the F1 hybrids revealed significant heterosis in biomass, plant vigor, ear length, number of kernels per ear, kernel weight, and yield (Figures 5A-G).

[0122] Building on the heterosis between the parents, the inventors further hybridized ZmPHO1;2 (C01) and ZmGIF1 (B104) from different backgrounds with stable homozygous overexpression in the T2-T3 generations to produce improved F1 hybrids. Hybrid seeds overexpressing both ZmGIF1 and ZmPHO1;2a driven by 35S were obtained in Shanghai in the spring of 2022. A total of three independent lines were obtained, and the F1 hybrids of wild-type B104 and C01 served as controls. Results showed that after harvest at maturity, the F1 generation clearly carried heterosis, with F1 corn ears significantly larger than those of wild-type C01 or B104. More importantly, compared with the wild-type hybrid B104*C01F1, the F1 combination overexpressing ZmGIF1*ZmPHO1;2a exhibited highly significant ear size. Statistical analysis revealed significant increases in the number of kernel rows, kernels per row, kernels per ear, and 100-kernel weight, ultimately increasing yield per ear by approximately 50% (Figures 6A-F). This indicates that the hybrid of ZmGIF1 and ZmPHO1;2a can significantly increase corn yield.

[0123] To further validate the yield-increasing effect of enhanced heterosis brought about by dual-gene aggregation, the inventors generated F1 hybrids of ZmPHO1;2a and ZmPHO1;2b driven by the Ubi promoter and the 35S promoter, respectively, with ZmGIF1. Four independent lines were selected for each combination, three of which were used for analysis. The F1 hybrids were planted in Hainan during the winter. Hybrid maize derived from the ZmGIF1 and ZmPHO1;2 dual-gene aggregation exhibited superior phenotypes, including significantly thicker stalks and larger leaves, during the seedling stage. Photographs of plant shape, root systems, and female ears during the mid-grain filling phase revealed significant heterosis between the two backgrounds. The root systems of the hybrid maize hybrids were significantly more developed and heavier than those in the control, with significantly increased plant height and larger and longer leaves. Comparison of biomass during the grain-filling phase revealed a significant increase in biomass (Figures 7A-F). In the hybrid maize of ZmGIF1 and 35S-driven ZmPHO1;2a and ZmPHO1;2b, both ZmPHO1;2a and ZmPHO1;2b showed significantly developed root systems compared with the control, significantly increased plant height, larger and longer leaves, and a highly significant increase in biomass of about 80% (Figures 8A-F).

[0124] In summary, the hybrid corn produced by the aggregation of the two filling genes ZmGIF1 and ZmPHO1;2 can greatly improve the field growth traits of hybrid corn, including biomass, which is especially important when used to prepare silage corn.

[0125] Example 4: Hybrid corn synthesized by polymerizing ZmPHO1;2 and ZmGIF1 significantly increases corn yield

[0126] Yield analysis at maturity revealed that the improved hybrid maize showed significant increases in ear length, number of ear rows, number of kernels per row, and number of kernels per ear compared to the control (B104*C01). The number of ear rows increased by 2-3, and the number of kernels per ear increased by approximately 50% (Figures 9A-D, 10A-D). 100-kernel weight and yield per plant also significantly increased, with kernel weight increasing by 12% and final yield increasing by 40-68% (Figures 9A-D, 10A-D). The yield-enhancing effect of the Ubi promoter-driven ZmPHO1;2 gene and ZmGIF1 gene combinations was weaker than that of the 35S promoter-driven ZmPHO1;2 gene and ZmGIF1 combinations (Figures 9A-D, 10A-D), due to the lower expression level of the Ubi-driven ZmPHO1;2 gene.

[0127] In summary, hybrid corn that combines the two key grain-filling genes ZmGIF1 and ZmPHO1;2 can further significantly enhance the hybrid vigor of crops and achieve a substantial increase in yield of crops such as corn.

[0128] Example 5: Hybrid corn synthesized from ZmPH01;2 and ZmGIF1 significantly improves photosynthesis in C4 crops

[0129] Typically, plant photosynthesis is divided into two major processes: the light reaction and the dark reaction. The light reaction uses light energy to generate ATP and NADPH, which are consumed to produce triose phosphates for further carbohydrate synthesis in the dark reaction. Although the carbon fixation process in plant photosynthesis requires the participation of Rubisco, C3 and C4 plants fix carbon differently. C3 crops, such as rice, use ribulose diphosphate (C5) to fix carbon dioxide. The resulting six-carbon molecule (C6) after binding to CO2 is very unstable and immediately splits into two three-carbon compounds, 3-phosphoglycerate. This process requires the carboxylation of Rubisco. In addition to carboxylation, Rubisco also has an oxygenation function, namely, binding O2, thereby reducing the photosynthetic rate. C4 crops, such as corn, lack Rubisco in their mesophyll cells. Instead, phosphoenolpyruvate (PEP) directly fixes CO2 to produce malate or aspartate, a four-carbon acid, which then undergoes the same reaction in the bundle sheath cells as in C3 crops, but without the oxygenation of Rubisco. Furthermore, PEP's CO2 binding capacity is much greater than that of RuBP. The inventors believe that during photosynthesis, efficient and stable photosynthetic rates are often limited by three factors: Rubisco activity, photosynthetic electron transport, and triose phosphate utilization. All three processes require phosphorus. Therefore, overall, high crop photosynthetic rates are often affected by phosphorus limitation, ultimately impacting yield. C4 crops inherently have higher photosynthetic efficiencies than C3 crops. Therefore, relieving the phosphorus limitation in C4 crops could further enhance their high photosynthetic efficiency.

[0130] The aggregation of two grain-filling genes, ZmPHO1;2 and ZmGIF1, can significantly increase corn yield. In terms of its mechanism of action, it should be addressed from both the source and sink aspects. In hybrid corn, the photosynthesis of C4 crops was first measured. At the mid-grain-filling stage (20 days ago), the net photosynthetic rate (A) of the control (B104*C01) F1 and the hybrid corn with ZmPHO1;2 and ZmGIF1 was measured in the field. net ) and the original parental inbred lines B104 and C01 were also tested. The results showed that the F1 produced by the polymerization of ZmGIF1 with ZmPHO1;2a and ZmGIF1 with ZmPHO1;2b significantly increased photosynthesis. In particular, the polymerization of ZmPHO1;2a and ZmPHO1;2b with ZmGIF1 driven by the 35S promoter increased photosynthesis by 30% (Figures 11A-D). The fundamental reason for this is that the action of ZmPHO1;2 optimizes the distribution of Pi in the plant, increases Pi accumulation in the leaves of photosynthetic tissues, alleviates the occurrence of phosphorus limitation, promotes the metabolic process of photosynthesis, and promotes high photosynthetic efficiency, thereby producing more photosynthetic products (sucrose) (Figures 11E-F). This significantly increases the accumulation of maize sources, which is the basis for ensuring high crop yields. The polymerization of ZmPHO1;2 with ZmGIF1 driven by the Ubi promoter can increase photosynthesis by approximately 15%-20%.

[0131] Based on its own photosynthetic structure, C4 crops have higher photosynthetic efficiency and CO2 fixation efficiency compared to C3 crops. Especially under high light and high temperature environments, the improvement of photosynthetic efficiency of C3 crops is prone to reaching a bottleneck or upper limit and is prone to phosphorus limitation under high temperature and high light. The photosynthetic efficiency of C3 crops is affected by the activity of Rubisco enzyme, thereby limiting further increase in yield. Although the function of PHO1 can alleviate phosphorus limitation, its function in C4 crops is stronger than that in C3 crops. On this basis, the present invention further improves the photosynthetic efficiency of C4 crops such as corn, which is of great significance for the improvement of high photosynthetic efficiency, especially under high temperature and high light conditions, where crops have a large demand for Pi. The polymerization of ZmPHO1;2 and ZmGIF1 can significantly alleviate phosphorus limitation and ensure the high photosynthetic rate of C4 crops.

[0132] In summary, hybrid corn with overexpression of the ZmPHO1;2 and ZmGIF1 genes can significantly improve the photosynthesis of C4 crop corn, which is the basis for ensuring a substantial increase in hybrid corn yield.

[0133] Example 6: ZmGIF1 and ZmPHO1;2 hybrid corn reduces grain phytic acid content

[0134] In cereal grains, reducing the phytic acid content is also one of the very important target traits for crop improvement. This is because phytic acid is an anti-nutritional factor. The increase of phytic acid will reduce the absorption of other nutrients. After animals and poultry consume it, they cannot degrade phytic acid due to the lack of phytase in their bodies, and the phytic acid is excreted from the body, causing eutrophication of the environment, thereby limiting the promotion and application of crop varieties.

[0135] To examine the effects of ZmPHO1;2 and ZmGIF1 overexpression on grain phosphorus content in hybrid maize, the present inventors measured grain phosphorus content at maturity. Pi content results showed that the 35S-driven ZmPHO1;2a and ZmPHO1;2b overexpression lines had significantly lower Pi content compared to wild-type C01; while the ZmGIF1 overexpression line had no difference in Pi content compared to wild-type B104 ( Figures 12A-C ). Inorganic phosphorus (Pi), total phosphorus, and phytic acid were also measured in the hybrid maize.

[0136] The results showed that the improved hybrid corn could increase corn yield while significantly reducing the phosphorus content of corn kernels, including Pi, total phosphorus, and the anti-nutritional factor phytic acid (Figure 12D-G).

[0137] These results indicate that the improved hybrid maize with the combination of ZmPHO1;2 and ZmGIF1 significantly improved phosphorus utilization efficiency and phytic acid content in grains.

[0138] Example 7: Improved Kernel Quality of ZmGIF1 and ZmPHO1;2 Hybrid Corn

[0139] The above evidence shows that hybrid corn can significantly improve photosynthesis and corn yield, but whether the increase in yield will reduce the quality of the grains also needs to be weighed. The quality of the corn grains was then measured and analyzed. The results showed that compared with the control, the total sugar content and sucrose content in the improved hybrid corn grains in which ZmGIF1 and ZmPHO1;2 were polymerized were significantly increased (Figure 13A-C), indicating that the improved hybrid corn significantly increased the storage capacity and promoted the metabolism and unloading capacity of sugars. The inventors found that phosphorus plays an important role in the grain filling process, especially in the initial step of starch synthesis, where 1-phosphate glucose (G1P) is used to synthesize ADP-Glucose (ADPG, a key substrate for starch synthesis) under the catalysis of AGPase enzyme. This step is also an important rate-limiting step in starch synthesis. During this process, ZmGIF1 decomposes sucrose to produce glucose, which serves as the raw material for starch synthesis, while ZmPHO1;2 regulates the phosphorus balance during ADPG synthesis, prompting the enzyme reaction to proceed in the forward direction, producing a continuous supply of substrate for starch synthesis, promoting grain filling, and ultimately significantly increasing the total starch content of the grain (Figure 13A-C). As a result, the quality and starch content of corn kernels in hybrid corn are both improved.

[0140] In addition, the protein content in corn kernels was further determined, and it was found that the protein content of the improved hybrid corn kernels was lower than that of the control F1, but the decrease ratio was low, and there was little difference between the two lines and the control (Figure 13D).

[0141] These results indicate that the improved hybrid corn with the combination of ZmGIF1 and ZmPHO1;2 has improved corn kernel quality.

[0142] Example 8: ZmGIF1 and ZmPHO1;2 hybrid corn can be used as silage corn

[0143] The above evidence shows that hybrid corn improved by ZmPHO1;2 and ZmGIF1 can significantly increase corn biomass, with thicker stalks, larger leaves, and increased plant height (Figures 7 and 8). It is worth noting that the control begins to age and turn yellow when approaching maturity, while the improved hybrid corn still has relatively green stalks (Figure 14A), thus hybrid corn is suitable as silage after harvesting the ears.

[0144] Therefore, the inventors analyzed the nutrients in the stalks of hybrid corn. The results showed that the improved hybrid corn stalks had significantly increased soluble total sugar and sucrose content, while the Pi content in the stalks was significantly reduced (Figures 14B-D). Further statistical observations in the field showed that the improved hybrid corn significantly increased stalk diameter, which also plays an important role in the lodging resistance of corn (Figures 14E-F).

[0145] The above results indicate that the improved hybrid corn can not only increase the biomass of silage, but also improve the nutritional level of silage, and can be used as an excellent silage feed.

[0146] Example 9: Polymerization of SbPHO1;2 and SbGIF1 significantly increases sorghum yield

[0147] To further verify the improvement effect of the source-sink synergy theory based on PHO1;2 and GIF1 genes in other C4 crops, the present invention further cloned the endogenous sorghum genes SbPHO1;2 and SbGIF1 in the sorghum model variety background BT×623, constructed the two genes in the same overexpression vector (pCambia1301-35S), achieved synchronous overexpression of the two genes, transformed the sorghum Wheatland background, and obtained positive transgenic strains.

[0148] Further phenotypic analysis of the transgenic sorghum at maturity revealed that the grains of the positive lines were significantly larger than those of the wild-type Wheatland (Figure 15A). Statistical analysis showed that the transgenic sorghum grain length, width, thickness, and weight were significantly increased (Figures 15B-E). This indicates that the combined and synergistic improvement of SbPHO1;2 and SbGIF1 significantly promoted the grain filling process in sorghum, ultimately increasing grain weight and sorghum yield. This further demonstrates the applicability of the present invention's creation of super-high-yield crops based on the PHO1;2 and GIF1 genes in multiple grass C4 crops.

[0149] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. A method for improving the seed or stalk traits of a C4 crop or preparing a C4 crop with improved seed or stalk traits, comprising: GIF1 and PHO1 are jointly upregulated in C4 crops; 2; the trait improvements include: (i) maintaining the green color of the stems and increasing the biomass of the stems for ensilage; (ii) increasing the sugar content of the stems and reducing the phosphorus content of the stems; (iii) reducing the phosphorus content of the grains; (iv) reducing the phytic acid content of the grains; and (v) increasing the starch content of the grains.

2. A method for improving the yield or biomass traits of a C4 crop, comprising: GIF1 and PHO1 are jointly upregulated in C4 crops; 2 expression or activity; The yield or biomass-enhancing trait includes: (a) increasing yield; (b) increasing biomass; (c) increasing phosphorus utilization; and (d) increasing photosynthetic efficiency.

3. The method according to claim 2, characterized in that The method of increasing yield or biomass includes: increasing plant height, increasing root system, increasing yield per ear, increasing number of grains per ear, increasing 100-grain weight, increasing stem diameter, increasing leaves, and increasing ear length; preferably, increasing number of grains per ear includes: increasing number of rows of grains, and increasing number of grains per row.

4. The method according to any one of claims 1 to 3, characterized in that: The described upregulation of GIF1 and PHO1; 2. The expression or activity of GIF1 and PHO1 in C4 crops; 2. Preferably, the ... Introducing GIF1 and PHO1;2 genes or expression cassettes containing said genes into C4 crops; Introducing gene editing agents into C4 crops that increase the expression or activity of GIF1 and PHO1;2 genes in C4 crops; Using expression-enhancing promoters or tissue-specific promoters to increase the expression of GIF1 and PHO1;2 genes in C4 crops, or to increase their expression in specific tissues; Using enhancers to increase the expression of GIF1 and PHO1;2 genes in C4 crops; or Targeted screening of varieties with high expression of GIF1 and PHO1;2 genes in different C4 crops was conducted, and offspring with improved traits were obtained through hybrid breeding.

5. A combination of GIF1 and PHO1;2 or their up-regulated molecules for improving the traits of C4 crops, or preparing C4 crops with improved traits; wherein, The improved traits include: (i) maintaining the green color of the stems and increasing the biomass of the stems for ensilage; (ii) increasing the sugar content of the stems and reducing the phosphorus content of the stems; (iii) reducing the phosphorus content of the grains; (iv) reducing the phytic acid content of the grains; and (v) increasing the starch content of the grains.

6. A combination of GIF1 and PHO1;2 or their up-regulated molecules for improving C4 crop yield or biomass traits; wherein, The yield or biomass-enhancing trait includes: (a) increasing yield; (b) increasing biomass; (c) increasing phosphorus utilization; and (d) increasing photosynthetic efficiency.

7. The use according to claim 5 or 6, characterized in that The up-regulating molecules include: expression boxes for expressing GIF1 and PHO1;2; gene editing reagents for increasing the expression or activity of GIF1 and PHO1;2 genes in C4 crops; or up-regulating molecules that interact with GIF1 and PHO1;2 to increase their expression or activity.

8. A C4 crop cell expressing exogenous GIF1 and PHO1; 2 expression cassette; Preferably, it comprises: expression cassette 1, which comprises: a promoter, a gene encoding GIF1, and a terminator; and expression cassette 2, which comprises: a promoter, a gene encoding PHO1; 2, and a terminator; preferably, the expression cassette is contained in an expression construct or an expression vector.

9. The method, use or cell according to any one of claims 1 to 8, characterized in that The C4 crop is or the GIF1 or PHO1;2 is from: cereal C4 crops; preferably, the cereal C4 crops include: corn (Zea mays), sorghum (Sorghum bicolor), millet (Setaria italica) and the like.

10. A GIF1 gene and a PHO1;2 gene Or the use of the protein encoded by it as a molecular marker for identifying the traits of C4 crops, or as a molecular marker for targeted screening of C4 crops; the traits include: (i) Stalk color and stalk biomass for ensilage; (ii) stem sugar content and stem phosphorus content; (iii) grain phosphorus content; (iv) grain phytic acid content or (v) increase grain starch content.

11. A method for identifying traits of a C4 crop, comprising: Analysis of GIF1 gene and PHO1 in C4 crops; 2 gene or the activity of the protein encoded by it; if the activity of the GIF1 gene and the PHO1;2 gene or the protein encoded by them in the C4 crop to be tested is equal to or higher than the average value of the C4 crop of this type, it indicates that the crop has excellent traits, and the excellent traits are selected from: (i) maintaining the greenness of the stems and increasing the biomass of the stems for silage; (ii) increasing the sugar content of the stems and reducing the phosphorus content of the stems; (iii) reducing the phosphorus content of the grains; (iv) reducing the phytic acid content of the grains or (v) increasing the starch content of the grains; if the expression level of the PHO1;2 gene or the activity of the PHO1;2 protein in the C4 crop to be tested is lower than the average value of the C4 crop of this type, the crop has unsatisfactory traits.

12. A method for targeted selection of C4 crops with improved traits, the method comprising: Analysis of GIF1 gene and PHO1 in C4 crops; 2 genes or the activities of the proteins encoded by them; if the activities of the GIF1 gene and the PHO1;2 genes or the proteins encoded by them in the C4 crop to be tested are equal to or higher than the average value of the C4 crop of this type, it indicates that the crop has excellent traits, and the excellent traits are selected from: (i) maintaining the green color of the stems and increasing the biomass of the stems for ensilage; (ii) increasing the sugar content of the stems and reducing the phosphorus content of the stems; (iii) reducing the phosphorus content of the grains; (iv) reducing the phytic acid content of the grains or (v) increasing the starch content of the grains; and the crop is used as a C4 crop with improved traits.

13. A method for screening a substance for improving C4 crop traits, the method comprising: (1) adding candidate substances to the expression of GIF1 and PHO1; 2 system; (2) detecting the system, observing the expression or activity of GIF1 and PHO1;2, and if the expression or activity thereof is increased, it indicates that the candidate substance is a substance that can be used to improve the traits of C4 crops; wherein the improved traits of C4 crops include substances selected from the group consisting of: (i) maintaining the green color of the stems and increasing the biomass of the stems for silage; (ii) increasing the sugar content of the stems and reducing the phosphorus content of the stems; (iii) reducing the phosphorus content of the grains; (iv) reducing the phytic acid content of the grains or (v) increasing the starch content of the grains; Preferably, the improved traits also include those selected from the group consisting of: (vi) increased yield; (vii) increased biomass; (viii) increased phosphorus utilization; and (ix) increased photosynthetic efficiency.

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