Molecular breeding technology for creating ultra-high-yield high-quality C4 crops and application of molecular breeding technology

Abstract

The invention provides a molecular breeding technology for creating super-high-yield high-quality C4 crops and application of the molecular breeding technology. GIF1 gene and PHO1 are disclosed for the first time; the combined application of the gene 1, 2 is very effective for the improvement of C4 crop traits, and the improvement of the traits comprises the following steps: (i) keeping the stalks green and increasing the biomass of the stalks for silage; (ii) the sugar content of the stalks is increased, and the phosphorus content of the stalks is reduced; (iii) reducing the phosphorus content of the grains; (iv) the phytic acid content of the grains is reduced or (v) the starch content of the grains is increased. In addition, a GIF1 gene and a PHO1; the combined application of the gene 2 can improve the yield or biomass character of C4 crops, including: (a) greatly improving the yield; (b) the biomass is greatly improved; (c) the utilization rate of phosphorus is improved; (d) the photosynthetic efficiency is improved. The invention provides a new way for improvement of C4 crops.

Description

[0001] This application claims priority to patent application filed on June 29, 2023, with application number CN 202310789613.7; the entire contents of which are incorporated herein. Technical Field

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

[0003] C4 plants are often written as C4 plants. Plants that absorb carbon dioxide from the air during their growth process and first synthesize compounds containing four carbon atoms such as malic acid or aspartic acid, such as corn and sugarcane. Crops such as wheat and rice first synthesize three-carbon atom molecules such as phosphoglyceric acid, which are C3 plants. C4 plants have many advantages over C3 plants, such as strong growth ability, high carbon dioxide utilization rate, and less water requirement.

[0004] Among the economic plants of the Gramineae family, about 300 species belong to C4 plants. Cultivating new varieties similar to C4 plants is of great significance to improving the food situation around the world.

[0005] The C4 plant corn (Zea mays L.) is a major cash crop and food crop widely grown worldwide. The seeds of cultivated corn can be used as food for humans and livestock feed or processed into industrial raw materials such as starch and brewing. The germ part can also be used to refine oil and make artificial vegetable oil. The stems and leaves can be used as silage or dry feed for livestock. With the growth of population, improving corn traits and improving quality are important goals of current corn breeding. However, the improvement of corn traits, quality or yield is still limited, and people's understanding of corn is still 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. It is usually necessary to effectively aggregate multiple genes to improve one or more target traits. This is gene pyramiding breeding.

[0007] However, not all excellent gene aggregations can produce ideal additive effects or phenotypes. Most of them will have no effect or even negative effects. For example, the aggregation of NADH-GOGAT with NAD-IDH or NADH-GDH did not improve the phenotype and nitrogen utilization of plants, indicating that the C / N balance in plants cannot be further improved by this strategy (Canas et al., 2020), which also shows that the aggregation process of different genes is relatively complex.

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

[0009] 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.

[0010] 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.

[0011] 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) substantially increasing (50% or more) yield; (b) increasing biomass; (c) improving phosphorus utilization; and (d) improving photosynthetic efficiency.

[0012] In one or more embodiments, the increasing yield (including single plant yield) or biomass (including single plant biomass) includes: increasing plant height, increasing root system, increasing yield per ear, increasing the 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 grain rows, and increasing the number of grains in each row.

[0013] In one or more embodiments, the upregulation of the expression or activity of GIF1 and PHO1;2 includes: overexpressing GIF1 and PHO1;2 in C4 crops; preferably, including: introducing GIF1 and PHO1;2 genes or expression boxes containing the genes (such as contained in an expression construct or an expression vector) into C4 crops; introducing gene editing agents 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 enhanced promoters or tissue-specific promoters; increasing the expression of GIF1 and PHO1;2 genes in C4 crops by enhancers; or, directed 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.

[0014] In another aspect of the present invention, a combination of GIF1 and PHO1;2 or the use of 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 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.

[0015] In another aspect of the present invention, a combination of GIF1 and PHO1;2 or the use of their up-regulated 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.

[0016] 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 to increase their expression or activity.

[0017] In another aspect of the present invention, a C4 crop cell is provided, which expresses 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.

[0018] In one or more embodiments, the C4 crop is or the GIF1 or PHO1;2 is from: cereal C4 crops; preferably, the cereal C4 crops include (but are not limited to): corn (Zea mays), sorghum (Sorghumbicolor), millet (Setaria italica) and the like.

[0019] 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.

[0020] 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.

[0021] In one or more embodiments, the amino acid sequence of the PHO1;2 polypeptide is selected from the following groups: (i) a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO: 1, 2 or 4; (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: 1, 2 or 4; (iii) a polypeptide having the function of regulating the trait and 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: 1, 2 or 4; 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: 1, 2 or 4, or adding a signal peptide sequence to its N terminus.

[0022] 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 more (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.

[0023] In another aspect of the present invention, there is provided a use of GIF1 gene and PHO1;2 gene or the proteins encoded by them, which are used as molecular markers for identifying the traits of C4 crops, or as molecular markers for targeted screening of C4 crops; the traits include: (i) stem color, biomass of stem for ensilage; (ii) stem sugar content, stem phosphorus content; (iii) grain phosphorus content; (iv) grain phytic acid content or (v) increased grain starch content.

[0024] In one or more embodiments, the traits of C4 crops are identified or targeted screening is performed by analyzing the expression levels of GIF1 gene and PHO1;2 gene or the activities of GIF1 protein and PHO1;2 protein in C4 crops.

[0025] In another aspect of the present invention, a method for identifying the traits of C4 crops is provided, comprising: analyzing the activities of GIF1 gene and PHO1;2 gene or proteins encoded by them in C4 crops; if the activities of GIF1 gene and PHO1;2 gene or 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 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.

[0026] In another aspect of the present invention, a method for directed selection of C4 crops with improved traits is provided, the method comprising: analyzing the activities of GIF1 gene and PHO1;2 gene or the proteins encoded by them in C4 crops; if the activities of GIF1 gene and PHO1;2 gene or the proteins encoded by them in the C4 crops to be tested are equal to or higher than the average value of the C4 crops of this type, it indicates that the crops have 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 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; using the crops as C4 crops with improved traits.

[0027] 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.

[0028] 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.

[0029] In one or more embodiments, the reduction refers to a significant reduction, such as a reduction of 20%, 40%, 60%, 80%, 90% or less.

[0030] 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 therein, and if the expression or activity thereof is increased, it indicates that the candidate substance is a substance that can be used to improve C4 crop traits; wherein improving C4 crop traits includes being selected from the following groups: (i) maintaining stem greenness, increasing the biomass of stems for silage; (ii) increasing stem sugar content, reducing stem phosphorus content; (iii) reducing grain phosphorus content; (iv) reducing grain phytic acid content or (v) increasing grain starch content;

[0031] In one or more embodiments, the improved traits also 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.

[0032] In one or more embodiments, a control group is also included to clearly distinguish the difference in expression or activity of GIF1 and PHO1;2 in the test group and that in the control group.

[0033] 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.).

[0034] 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

[0035] Figure 1 AJ and Ubi promoter-driven ZmPHO1;2 overexpression increases corn yield.

[0036] Figure 2 Overexpression of ZmPHO1;2 driven by AJ and 35S promoters increases maize yield.

[0037] Figure 3 AD and qRT were used to detect the expression levels of ZmPHO1;2a and ZmPHO1;2b overexpression strains.

[0038] Figure 4 Overexpression of ZmGIF1 driven by AG and 35S promoters.

[0039] Figure 5 The F1 produced by the hybridization of AG, B104 and C01 has obvious hybrid vigor.

[0040] Figure 6 Hybrid corn with the dual genes AF, ZmGIF1 and ZmPHO1;2a significantly increased yield.

[0041] Figure 7 Hybrid maize in which AF, 35S-ZmGIF1 and Ubi-ZmPHO1;2 were synthesized significantly increased biomass.

[0042] Figure 8 Hybrid maize in which AF, 35S-ZmGIF1 and 35S-ZmPHO1;2 were synthesized had significantly increased biomass.

[0043] Fig. 9 Hybrid corn synthesized by AD, 35S-ZmGIF1 and Ubi-ZmPHO1;2 significantly increased yield.

[0044] Fig.10 The hybrid corn with AD, 35S-ZmGIF1 and 35S-ZmPHO1;2 significantly increased yield.

[0045] Fig.11 Hybrid corn with AF, ZmGIF1 and ZmPHO1;2 significantly enhances the photosynthetic efficiency of C4 crops.

[0046] Fig.12 AG, ZmGIF1 and ZmPHO1;2 hybrid corn reduced grain phosphorus content.

[0047] Fig.13 AD, ZmPHO1;2 and ZmGIF1 hybrid corn enhances corn grain quality.

[0048] Fig.14 AF, ZmPHO1;2 and ZmGIF1 hybrid corn can be used as excellent silage.

[0049] Fig.15 The polymerization of SbPHO1;2 and SbGIF1 in AE and sorghum increased sorghum grain weight and yield. DETAILED DESCRIPTION

[0050] 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 very effective for improving the traits of C4 crops, including: (i) maintaining the greenness 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; or (iv) reducing the phytic acid content of the 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, including: (a) significantly increasing the yield; (b) increasing the biomass; (c) improving the phosphorus utilization rate; (d) improving the photosynthetic efficiency. The present invention provides a new approach to the improvement of C4 crops.

[0051] GIF1 and PHO1;2

[0052] As used herein, the "PHO1;2 gene or PHO1;2 protein (polypeptide)" includes PHO1;2 gene or PHO1;2 protein from C4 crops, such as corn or sorghum. In corn, there are two PHO1;2, including ZmPHO1;2a and ZmPHO1;2b. In sorghum, it includes SbPHO1;2.

[0053] 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.

[0054] In the present invention, the GIF1 protein or PHO1;2 protein also includes their fragments, derivatives and analogs. As used herein, the terms "fragment", "derivative" and "analog" refer to protein fragments that substantially maintain the same biological function or activity as the polypeptide, and may be (i) a protein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein with a substitution group in one or more amino acid residues, or (iii) a protein formed by the fusion of an additional amino acid sequence to this protein sequence, etc. According to the definition herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art. The biologically active fragments of the GIF1 or PHO1;2 protein can be applied to the present invention.

[0055] In the present invention, the term "PHO1;2 protein" refers to a protein having a sequence as shown in any one of SEQ ID NO: 1, 2 or 4 with the aforementioned trait-improving activity of the present invention, and the term also includes variants of the sequences of SEQ ID NO: 1, 2 or 4 with the same functions as these polypeptides. The term "GIF1 protein" refers to a protein having a sequence as shown in SEQ ID NO: 3 or 5 with the aforementioned trait-improving activity of the present invention, and the term also includes variants of the sequences of SEQ ID NO: 3 or 5 with the same functions 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, and even more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or more amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein.

[0056] 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 with the GIF1 or PHO1;2 protein polypeptide (such as 80% or higher homology with 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.

[0057] The present invention also includes polynucleotides (genes) encoding the polypeptides, which may be natural genes from crops or their degenerate sequences.

[0058] The vector containing the coding sequence, and the host cell produced by genetic engineering using the vector or the polypeptide coding sequence are also included in the present invention. Methods well known to those skilled in the art can be used to construct a suitable expression vector.

[0059] Host cells are usually plant cells. Plants can generally be transformed using methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, young embryo transformation method, etc.; Agrobacterium method is preferred. Transformed plant cells, tissues or organs can be regenerated into plants using conventional methods to obtain plants with altered traits relative to the wild type.

[0060] As used herein, the term "crop" refers to plants with economic value in agriculture and industry such as grain, cotton, and oil, and its economic value can be reflected in the useful parts of the plant such as seeds, fruits, roots, stems, and leaves. Crops include, but are not limited to: dicotyledonous plants or monocotyledonous plants. Preferred crops are C4 crops, and more preferably corn, sorghum, etc.

[0061] In the present invention, the crop includes plants expressing GIF1 and PHO1;2, preferably C4 cereal crops. Preferably, the cereal crops have grains.

[0062] application

[0063] Corn is an important economic and food crop worldwide. Further improving the yield of corn with high photosynthetic efficiency of C4 is of great significance to meet the rapidly growing demand for corn-derived food, feed and fuel. The high yield of crops depends on the good coordination and balance between "source-sink-flow". In production, breeders usually increase the yield of corn inbred lines and even hybrid corn by aggregating some excellent genetic resources to increase the ideal characteristics of large ears. In the present invention, by analyzing two key filling genes PHO1;2 (encoding inorganic phosphorus transporter) and GIF1 (encoding cell wall sucrose invertase), it is possible to respectively enhance the source strength and sink strength of crops and open up the photosynthesis of C4 crops, and the two genes are respectively transferred into different inbred lines (C01 and B104) to produce overexpression strains, and further hybridize the PHO1;2 and GIF1 overexpression transgenic lines from different sources by hybrid breeding. Analysis shows that hybrid corn has extremely significant improvements in the following traits, which are caused by the synergistic effect of the combined application of the two genes: (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. 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 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, which enhances 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 efficiency of fertilizer utilization. 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.

[0064] In previous studies, the inventors cloned two key grain filling positive regulatory genes GIF1 and OsPHO1;2 in rice. GIF1 encodes a cell wall sucrose invertase, which converts sucrose into glucose and fructose. Overexpression of GIF1 significantly increased the activity of cell wall sucrose invertase, ultimately increasing rice yield (Wang et al., 2008); OsPHO1;2 encodes an inorganic phosphorus transporter of the PHO1 family, which has specific efflux activity and influx activity. It regulates the balance of Pi in endosperm cells during grain filling, affects the activity of AGPase enzyme, and ultimately regulates filling. Overexpression of OsPHO1;2 can reduce the accumulation of Pi in grains, promote grain filling and rice yield, and maintain high-yield traits under low-phosphorus conditions, with high phosphorus utilization (Ma et al., 2021). Importantly, these two key grain filling genes are highly conserved in Poaceae crops, and their gene functions are also conserved. The loss of homologous genes of GIF1 and OsPHO1;2 in maize 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.

[0065] The inventors found that the two filling genes PHO1;2 and GIF1 regulate the filling process of grains from different perspectives. PHO1;2 can promote the distribution of phosphorus to leaves to support photosynthesis, produce more carbon assimilates, and further transport them to the grains to promote grain filling, while maintaining the steady-state level of inorganic phosphorus accumulation in the grains during the filling period; while GIF1 is responsible for the unloading of sucrose in the grains. The sucrose transported from the source to the grains through the vascular tissue is decomposed into monosaccharides under the action of GIF1, which is used to synthesize starch and enrich the sink, thereby promoting grain filling. From the perspective of source and sink, the present invention will transform different inbred line backgrounds respectively through the overexpression of two filling genes, and then further hybridize the overexpression strains of two different backgrounds to produce hybrid corn with simultaneous overexpression of two genes, and analyze the yield traits and other excellent traits of the improved hybrid corn. At the same time, the field fertilizer utilization rate of high-yield hybrid corn is evaluated, and high-yield, high-quality, low-fertilizer hybrid corn can be achieved. At the same time, improvements in many other traits are also formed.

[0066] Based on the new discovery of the present inventors, a method for improving plants is provided, the method comprising: up-regulating the expression or activity of PHO1;2 in the plant; 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 (or) (v) increasing the starch content of the grains.

[0067] 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 the yield; (b) significantly improving the biomass; (c) improving phosphorus utilization; and (d) improving photosynthetic efficiency.

[0068] It should be understood that according to the experimental data and regulatory mechanism provided by the present invention, a variety of 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.

[0069] In the present invention, substances that up-regulate the expression or activity of GIF1 and PHO1;2 in plants include promoters, agonists, activators, and up-regulators. The "up-regulation", "increase", and "promotion" mentioned above include "up-regulation", "promotion" of protein activity or "up-regulation", "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 proteins encoded by them, up-regulate the expression of GIF1 and PHO1;2 genes, and increase the effective action time of GIF1 and PHO1;2 proteins can be used in the present invention as substances useful for up-regulating GIF1 and PHO1;2 genes or proteins encoded by them. They can be compounds, small chemical molecules, and biological molecules. The biological molecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0070] As another embodiment of the present invention, a method for up-regulating 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 encoding polynucleotides of GIF1 and PHO1;2; and regenerating the obtained plant tissues, organs or seeds transformed with the exogenous encoding polynucleotides of GIF1 and PHO1;2 into plant plants.

[0071] Other methods for increasing the expression of GIF1 and PHO1;2 genes or their homologous genes are well known in the art. For example, the expression of PHO1;2 genes or their homologous genes can be enhanced by driving with a strong promoter. Alternatively, the expression of the PHO1;2 gene can be enhanced by an enhancer (such as the first intron of the Actin gene, etc.), or the expression of GIF1 and PHO1;2 genes or their homologous genes and protein activity can be increased by gene editing. Strong promoters suitable for the method of the present invention include, but are not limited to, 35S promoter, Ubi promoter of corn, etc.

[0072] The methods described may be carried out using any suitable conventional means, including reagents, temperatures, pressure conditions, and the like.

[0073] After knowing the functions of GIF1 and PHO1;2 genes, they can be used as molecular markers for targeted screening of plants. Based on this new discovery, it is also possible to screen for substances or potential substances that can regulate plant type traits, yield traits, organelles or cell cycles by regulating this mechanism. GIF1 and PHO1;2 or their encoded proteins can also be used as tracking markers for the offspring of a gene-transformed plant.

[0074] Therefore, the present invention provides a method for directional selection or identification of plants, the method comprising: 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 it: (i) maintains the green color of the stems and increases the biomass of the stems for ensilage; (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, its traits are not ideal.

[0075] When evaluating the plants to be tested, the expression or mRNA levels of GIF1 and PHO1;2 can be measured to understand whether the expression or mRNA levels in the plants to be tested are higher than the average value of such plants. If they are significantly higher, they have improved traits.

[0076] The present invention provides a method for screening and regulating plant type 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, observing the expression or activity of GIF1 and PHO1; 2 therein, 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 following groups: (i) maintaining the green color of stems and increasing the biomass of stems for silage; (ii) increasing the sugar content of stems and reducing the phosphorus content of stems; (iii) reducing the phosphorus content of grains; (iv) reducing the phytic acid content of grains; (or) (v) increasing the starch content of grains.

[0077] Methods for screening substances acting on a protein or gene or a specific region thereof as a target are well known to those skilled in the art, and all of these methods can be used in the present invention. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, organic small molecules, inorganic small molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art will know how to select an appropriate screening method.

[0078] The interaction between proteins and the strength of the interaction can be detected by various techniques well known to those skilled in the art, such as GST-Pull Down, bimolecular fluorescence complementation experiment, yeast two-hybrid system or immunoprecipitation technology.

[0079] 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.

[0080] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0081] Materials and Methods

[0082] 1. Construction of transgenic corn materials

[0083] The genome sequences and coding region sequences of the three genes ZmPHO1; 2a (Zm00001d051945), ZmPHO1; 2b (Zm00001d018445) and ZmGIF1 (Zm00001d003776) used in the present invention are all derived from the NCBI database, and the three genes are cloned by amplifying the gDNA or cDNA of the B73 corn inbred line by PCR. For ZmPHO1; 2a and ZmPHO1; 2b, two different strong promoter overexpression systems, Ubi and 35S, were constructed (using pCambia3301 as an expression vector), and all transgenic constructions were transformed into C01 or B104 inbred line backgrounds by Agrobacterium EHA105-mediated genetic transformation.

[0084] 2. Phenotype and yield trait analysis

[0085] All maize and sorghum materials are planted twice a year in the transgenic greenhouse in Songjiang, Shanghai (spring and summer), and once a year in Sanya, Hainan (winter). Each generation adopts a strict bagging self-pollination method for continuous self-pollination for 3 generations, and then the homozygous lines are selected for phenotypic analysis. The F1 generation of hybrid maize does not need to be bagged, and natural pollination is sufficient. In the late filling period, plant height, functional leaf size, leaf number, ear position, number of male ear branches, etc. are measured; in the mature stage, biomass (other parts outside the female ear in the aboveground part), yield, number of kernel rows, number of kernels per row, ear length, ear inner diameter, number of kernels per ear, and 100-kernel weight are analyzed.

[0086] 3. Gene expression analysis

[0087] Corn leaves, seeds and other tissues were collected in 2 mL imported EP tubes (with steel beads added in advance) and quickly frozen in liquid nitrogen. Grind into powder using a grinder at 40 Hz for 50 s, 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).

[0088] The reactions were performed using a two-step amplification procedure: 95°C pre-denaturation for 30 s, 95°C denaturation for 10 s, 60°C annealing and extension for 30 s, 40 cycles, and additional melting curve analysis. -△△CT The relative gene expression was analyzed by the method.

[0089] 4. Determination of soluble sugar and total starch content in tissues

[0090] Take corn leaves or seeds (0.40g), grind them thoroughly with liquid nitrogen, put them into a 2mL centrifuge tube, add 1mL MillQ water, open the centrifuge tube cap, treat them in a 100℃ water bath for 15-20min, transfer them into a 10mL centrifuge tube, dilute them to 5-10mL with deionized water according to the sample weight, centrifuge them at 10000g for 10min, take the supernatant and filter it with a 0.45μm filter membrane; use the filtered clarified sample solution to load the sample manually or load it into a sampling bottle for automatic injection (0.6mL sample), and analyze glucose, fructose and sucrose on an ion chromatograph (ICS-3000, DIONEX) CarboPacTM PA1 column. The mobile phase is 200mM NaOH solution, the flow rate is 1.5mL / min, and the electrochemical detector is used.

[0091] Corn seeds were ground and passed through a 0.5 mm sieve. The ground samples (accurately weighed 100 mg) were added to a test tube (16 × 120 mm), ensuring that all samples were located at the bottom of the test tube. 0.2 mL of ethanol solution (80% v / v) was added to wet the sample to help disperse it and mixed with a vortex mixer. The total starch content of the sample was determined using a Megazyme K-TSTA kit.

[0092] 5. Determination of phosphorus content

[0093] (1) Determination of inorganic phosphorus content

[0094] Take 0.5g of sample, add 10mL of extract (12.5% ​​TCA, 25mM MgCl2), shake overnight at 4℃, centrifuge at 10000g for 15min at 4℃, take 5mL of supernatant, and determine the P content by ammonium vanadium molybdate colorimetric method. Repeat 3 times for each sample. Most samples can be transferred to ELISA plate for determination. Preparation of P standard solution and drawing of working curve: Dry potassium dihydrogen phosphate at 105℃ for 1h, cool in a desiccator, weigh 0.2195g and dissolve in water, transfer to a 1000mL volumetric flask, add 3mL of nitric acid, make up to volume with deionized double distilled water, shake well, and make 50μg / mL P 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. Using 0.0 mL of P standard solution as a control, measure the absorbance of various P standard solutions at a wavelength of 400 nm using a 751 spectrophotometer, and draw a working curve (GB / T 6437-2002) with Pi content as the horizontal coordinate and absorbance as the vertical coordinate.

[0095] (2) Determination of total phosphorus content

[0096] 10 mg samples of 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 (it can also be replaced by a boiling water bath for 4 h);

[0097] After digestion, open the lid of the microwave digestion tube and place it in an acid remover or in a fume hood at 160°C to remove 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.

[0098] The digested samples were used to determine the concentrations of P, S and various trace elements. The phosphorus content was determined using an inductively coupled plasma optical emission spectrometer (ICP-OES) (PerkinElmer, USA) on our instrument platform. Six biological replicates were set for each sample.

[0099] 6. Determination of phytic acid content

[0100] Take about 0.3g of tissue sample, add 10mL of 0.6M HCl to a 50mL centrifuge tube, mix thoroughly by inversion, and dissolve in boiling water for 30min; take out the dissolved sample, cool it to room temperature, centrifuge it at 10000g for 15min at 4℃, take 500μL of supernatant and dilute it 10 times to 5mL with deionized water; pass the diluted sample through IC-RP column, IC-H column, and 0.22μm microporous filter membrane in sequence to remove hydrophobic substances, alkaline earth metal ions, carbonate ions, metal ions, macromolecular substances, etc. in the sample.

[0101] The prepared samples were analyzed by ion exchange chromatography ICS-2000, where the separation column and guard column were IonPac AS11-HC (250 mm × 4 mm) and IonPac AG11-HC (50 mm × 4 mm) respectively, and the eluent was 50 mM KOH (needed to be prepared with deionized water to reduce ion interference), and the elution rate was set to 1 mL min -1 The signal was detected at 124 mA inhibition current, and the standard sample was prepared with sodium phytate (P-3168, Sigma). Each sample was repeated 3 times.

[0102] 7. Photosynthesis measurement

[0103] The steady-state photosynthetic rate (A net ) was measured. 2 Light saturation (1500 μmol m -2 s -1 ) Net CO2 assimilation rate (A net ). A was measured with 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 and 11:30 am on sunny days.

[0104] 8. Gene protein sequence

[0105] ZmPHO1;2a (SEQ ID NO:1)

[0106] MVKFSREYEASIIPEWKAAFVDYRCLKKLVKRIKIARRDATREAGGGSSSDATSSYGFSVLDPVRALAAHFASATPPAVSPANGDEEEEDDDAESLEPDSGELVVVATAKHEREFLEKAGEELEKVNKFYAAQEADMLARGAALVEQLRILADVKRILANHAVASRRSRPAGDSPPPPPSADGRSNSGRHLLSSSSPFLQASPQSMLDGSAELPQARVAEEVMAALERNGVSFVGSGLGSKAKKDGGGKQLTGRAAALPATVRIDVPPTSPGRAALKVWEELVNVLRKDGADPAAAFVHRKKVQHAEKSIRDAFLALYRGLDLLNKFSSLNVKAFTKILKKFVKVSEQQRKTDLFSEKVKRSPFSSSDKVLQLADEVECIFSRHFAGNDRKVAMKYLKPQQPRNTHMITFLVGLFTGTFVSLFIIYSVLAHVAGIFSSTGNTAYMEIVYHVFSMFALISLHVFLYGCNLLAWKSSRISHNFIFDFSPSTALTHRDAFLLSASIMCTVVAALVVNLFLSNAGATYANALPGALLLLSAAALFCPFNVFYRSTRYCFMRVMRNIMLSPFYKVLMADFFMADQLTSQIALLRHLEFTGCYFMAGTFTTHAYGSCTSSSQYKNLAYVLSFLPYYWRAMQCLRRYLEEGHDIDQLANAGKYISAMVAAAVRFKYAAAPTPFWMWMVIVSSTGATIYQLYWDFVMDWGFLDLRSKNRWLRDQLILKNKPIYYASMMLNLVLRLAWAESVMKLRLGMVESRLLDFSLASLEIIRRGHWNFYRLENEHLNNAGKFRAVKTVPLPFRELETD

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

[0108] MVKFSREYEASIIPEWKAAFVDYKGLKKLVKRIKIARRDRAARSTSNDHDDATTTTYGFSVLDPVRALASHFNNATPPASPEGGSDDALRSLESDSGELVRATDKHEQEFVERADEELEKVNKFYAAQEADMLARGDALIEQLRILADVKRILADHAAASSRRGRARLARTGGNSSPPSVDGSNSGRHLLSSPFVASSPQSMSDGSVQLQQARVAEGAAVAEEVMAALERNGVSFVGGGLGKAKKDGSGKQLMGRAALLQLPATVRIDIPPTSPGRAALKVWEELVNVLRKDGADPAAAFVHRKKVQHAEKSIRDAFLALYRGLDLLKKFSSLNVKAFTKILKKFVKVSEQHRKGDLFSEKVKRSPFSSSDKVLQLADEVECIFLRHFAGNDRKVAMKYLKPQQPRNTHMVTFLVGLFTGTFVSLFIIYSVLAHVAGIFSSTGNTAYMEIVYHVLSMFALISLHVFLYGCNLSMWKGTRINHNFIFDFSSTALTHRDAFLMSASIMCTVVAALVVNLFLRNAGATYANALPGALLLLSAGVLFCPFNIFYRSTRFCFMRVMRNIVLSPFYKVLMADFFMADQLTSQIPLLRHLEFTGCYFMAETFRTHAYGSCTSSSQYKNLAYVLSFLPYYWRAMQCLRRYLEEGHDMNQLANAGKYVSAMVAAAVRFKYAATPTPFWMWMVIASSTGATIYQLYWDFVMDWGFLNPKSKNFWLRDQLILKNKSIYYASMMLNLVLRLAWAESVMKLRLGMVESRLLDFSLASLEIIRRGHWNFYRLENEHLNNAGKFRAVKTVPLPFRELETD

[0109] ZmGIF1(SEQ ID NO:3)

[0110] MRALVVVSFASACLLLLLQLAGASHVVYNYKDLEAEAAAATDQVPPSIVNPLLRTGYHFQPPKNWINDPNAPMYYKGWYHFFYQYNPKGAVWGNIVWAHSVSRDLINWVALEPALRPSIPGDRYGCWSGSATVLPDGGGPVIMYTGVDHPDINYQVQNVAYPKNVSDPLLREWVKPSHNPVIVPEGGINATQFRDPTTAWRGPGPEQWRLLVGSAAGSSPRGVAYVYRSRDFRRWRRVRRPLHSAATGMWECPDFYPVSKGGAPRAGLETSVPPGPRVKHVLKNSLDLRRYDYYTVGTYHPRAERYVPDDPAGDEHRLRYDYGNFYASKTFYDPAKRRRILWGWANESDSAADDVAKGWAGIQAIPRTVWLDPSGKQLLQWPIEEVEALREKSVTLKNRLIKAGHHVEVTGIQTAQADVEVSFEVSPAALAGAETLDPALAYDAEKLCGVKRADVRGGVGPFGLWVLASANRKERTAVFFRVFKPAAGSDKPVVLMCTDPTKSSLNPNLYRPTFAGFVDTDISNGKISLRSLIDRSVVESFGAGGKTCILSRVYPSLAIGKDARLYVFNNGRAHVKVSRLTAWEMKKPVMNGA

[0111] SbPHO1;2(SEQ ID NO:4)

[0112] MVKFSREYEASIIPEWKAAFVDYKGLKKLVKRIKIARRDAAPLLAAGAGGGRRSSDASSGSYGFSVLDPVRALAAHFAATASPTTQVGDDDGDSDSGLESDSGELVRATDKHEQEFLEKADEELEKVNKFYAAQEADMLARGEALIEQLRILADVKRILADHAAASRRGRSRLARTAASSPPPSVNGSNSGRHLLSSPFVVASPQSMSDGSVELQQARVAEGAAVAEEVMAALERNGVSFVGGGLGKAKKDGSGKQLMGRAALLQLPATVRIDIPPTSPGRAALKVWEELVNVLRKDGADPAAAFVHRKKVQHAEKNIRDAFLALYRGLELLKKFSSLNVKAFTKILKKFVKVSEQQRKTDLFSEKVKRSPFSSSDKVLQLADEVESIFLRHFAGNDRKVAMKYLKPQQPRNTHMITFLVGLFTGTFVSLFIIYSVLAHVAGIFSSTGNTAYMEIVYHVFSMFALISLHVFLYGCNLFMWKSTRISHNFIFDFSSSTALTHRDAFLMSASIMCTVVAALVINLFLRNAGATYANALPGALLLLSAVVLFCPFNVFYRSTRYCFMRVMRNIMLSPFYKVLMADFFMADQLTSQIPLLRHLEFTGCYFMAGTFRTHEYGSCTSSSLYKNLAYVLSFLPYYWRAMQCLRRYLEEGHDLNQLANAGKYISAMVAAAVRFKYAATPTPFWMWMVIISSTGATIYQLYWDFVMDWGFLNPKSKNFWLRDQLILKNKSVYYASMMLNLVLRLAWAQSVMKLHLGMVESRLLDFSLASLEIIRRGHWNFYRLENEHLTNAGKFRAVKTVPLPFRELETD

[0113] SbGIF1(SEQ ID NO:5)

[0114] MRVVGMVAWACIWLLLLQLAGASHVVYDENLEVEAAAAAVPPSIVDPLLRTGYHFQPRKNWINDPNAPLYYKGWYHIFYQYNPKGAVWGNIVWGHSVSRDLINWVALKPAIEPSIPSDKYGCWSGSATTLPDGTPAIMYTGVNRPDVN YQVQNIAYPRNKSDPLLREWVKPSHNPIIVPKAGVNATQFRDPTTAWRHADGGGHWRLLIGSLEGAARGVAYVYRSRDFKRWTRVRRPLHSAATGMWECPDFYPLSTAGRRMGVETSSSSAAAAGSRRRQANKYVLKNSLDLRRYDYYT IGTYDPAAERYVPDDPAGDERHLRYDYGNFYASKTFYDPAKRRRILWGWANESDTAADDVAKGWAGIQAIPRTVWLDPSGKQLLQWPIEEVEALRGKSVTLKNRVIKPGQHVEVTGIQTAQADVEVSFEVPSLAGAEALDDPALAGDA QRLCGARGAAVEGGVGPFGLWVLASANREERTAVFFRVFRPARGGGKPVVLMCTDPCKSSLDPNLYQPTFAGFVDTDISNGKISLRRSLIDRSVVESFGAGGKTCILSRVYPSLAIGKNARLYVFNNGKADVKVSSLTAWEMKKPLMNGA

[0115] Example 1: Overexpression of ZmPHO1;2 increases corn yield

[0116] 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 filling, the 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 produced under the drive of 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, and were transformed into the C01 inbred line through the Agrobacterium EHA105-mediated genetic transformation system to produce different independent overexpression strains.

[0117] The phenotypes of stable and independent transgenic lines were analyzed and counted in the T2 and T3 generations. The results showed that the overall performance of the Ubi promoter-driven ZmPHO1;2a overexpression line was similar to that of the wild-type C01. Although the yield traits including the number of rows of grains, the number of grains per row, the 100-grain weight and the yield per ear showed an increasing trend, there was no significant difference ( Figure 1 AF); while the Ubi promoter-driven ZmPHO1;2b overexpression lines performed better than the wild-type C01 overall. The ears of the two lines (Ubi-ZmPHO1;2b#3 and Ubi-ZmPHO1;2b#4) were significantly larger, and the number of grain rows, number of grains per row, 100-grain weight, and yield per ear were also significantly increased ( Figure 1 B, GJ).

[0118] In addition, analysis of the ZmPHO1;2 overexpression lines driven by the 35S promoter revealed that the ZmPHO1;2a overexpression lines all showed a significantly larger ear phenotype, with significantly higher grain rows, grains per row, and 100-grain weight than the wild-type C01, and a significantly increased final yield per ear ( Figure 2 The ears of the ZmPHO1;2b overexpressing strains became larger, the number of grains per ear and the grain weight increased, and the final weight per ear was also higher than that of the wild type ( Figure 2 B, GJ).

[0119] These results indicate that ectopic overexpression of ZmPHO1;2 driven by either the Ubi promoter or the 35S promoter can increase corn yield to a certain extent, among which the 35S promoter has a better effect.

[0120] In order to analyze the differences produced by different promoters, the inventors performed qRCR gene expression detection on the overexpression strains driven by the two promoters and found that the overexpression level of the Ubi promoter was relatively low, especially Ubi-ZmPHO1;2a, which was only upregulated by 100-400 times, while the overexpression of the two genes driven by 35S was upregulated by more than thousands of times ( Figure 3 AD). It can be seen that the up-regulation fold of a gene is closely related to the expression of the gene and the phenotype.

[0121] Example 2: Effect of overexpression of ZmGIF1 on corn yield

[0122] In order to investigate the regulatory role of ZmGIF1 gene in corn grain filling and its contribution to yield, the inventors transferred the overexpression construct of ZmGIF1 full-length CDS coding region sequence driven by 35S promoter into corn B104 inbred line, obtained independent overexpression strains, and repeated the phenotype analysis in T2 and T3 generations. In two randomly selected strains, the expression level was significantly upregulated, and the yield traits at maturity showed that the number of grains per row and per ear had an increasing trend, and the ears also had a tendency to become larger ( Figure 4 AG).

[0123] Therefore, ZmGIF1 also has a certain yield-increasing effect under the drive of a strong promoter. It can be seen that the yield-increasing amplitude of single gene ZmPHO1;2 and ZmGIF1 overexpression is small. The fundamental reason is that there is no coordination between "source-sink". Unilateral increase of "source" or "sink" cannot achieve maximum utilization.

[0124] Example 3: Hybrid corn with overexpression of ZmPHO1;2 and ZmGIF1 genes significantly increases corn biomass

[0125] The inventors found that the overexpression of single genes ZmPHO1;2 and ZmGIF1 both showed the potential to promote corn yield, but the yield could only be increased slightly. Considering the coordination between the "source-sink" factors necessary for crop yield increase, it is speculated that the source and sink of single gene overexpression have not been fully utilized, and the simultaneous overexpression of the two genes can simultaneously enhance and open up the "source" and "sink" of corn. First, the inventors verified the possibility of hybridization between the two parents. In the selection of the male and female parents, considering that the B104 background has more pollen but a lower ear position, while the C01 background has less pollen but a suitable ear position, therefore, the B104 background was selected as the male parent and the C01 background as the female parent, and the hybrid F1 was produced by hybridization. Analysis of F1 found that it showed obvious hybrid vigor in biomass, plant growth, ear length, number of grains per ear, grain weight and yield ( Figure 5 AG).

[0126] On the basis of the hybrid vigor between the parents, the inventors further hybridized and polymerized the T2-T3 generations of stably homozygous overexpressed ZmPHO1; 2 (C01) and ZmGIF1 (B104) of different backgrounds to produce F1 improved hybrids. In the spring of 2022 in Shanghai, hybrid seeds with overexpression of ZmGIF1 and ZmPHO1; 2a driven by 35S were obtained, a total of 3 independent strains, and the F1 hybrid of wild-type B104 and C01 was used as a control. The results showed that after harvesting at maturity, the F1 generation clearly carried hybrid vigor, that is, the F1 corn cob was significantly larger than that of wild-type C01 or B104. More importantly, compared with the wild-type hybrid B104*C01 F1, the ear type of the F1 combination overexpressing ZmGIF1*ZmPHO1; 2a was significantly larger. Statistical analysis found that the number of grain rows, the number of grains per row, the number of grains per ear, and the 100-grain weight were significantly increased, and the final yield per ear increased by about 50% ( Figure 6 AF). This indicates that the hybrid of ZmGIF1 and ZmPHO1;2a can significantly increase corn yield.

[0127] In order to further verify the yield-increasing effect of enhanced hybrid vigor brought by dual gene aggregation, the inventors respectively generated F1s of ZmPHO1;2a and ZmPHO1;2b and ZmGIF1 driven by Ubi promoter and 35S promoter, and selected 4 independent strains for each combination, of which 3 strains were used for analysis, and F1s were planted in Hainan in winter. At the seedling stage, it was found that the hybrid corns with dual gene aggregation of ZmGIF1 and ZmPHO1;2 showed excellent phenotypes such as significantly thicker stalks and larger leaves; in the middle of grain filling, the plant type, root system and female ears were photographed and observed, and it was found that the hybrid vigor of the two backgrounds was very obvious; the root system of the aggregated hybrid corn was significantly more developed and heavier than the control, the plant height was significantly increased, the leaves became larger and longer, and the whole plant was harvested during the grain filling period to compare the biomass. It was found that the biomass of the hybrid corn was significantly increased ( Figure 7 AF). In the hybrid maize of ZmGIF1 and 35S driven ZmPHO1;2a and ZmPHO1;2b, both ZmPHO1;2a and ZmPHO1;2b showed significantly more developed root systems than the control, significantly increased plant height, larger and longer leaves, and extremely significant increase in biomass by about 80% ( Figure 8 AF).

[0128] 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.

[0129] Example 4: Hybrid corn produced by polymerizing ZmPHO1;2 and ZmGIF1 greatly increases corn yield

[0130] The yield trait analysis at maturity showed that the improved hybrid corn had significantly increased ear length, ear row number, kernel number per row, and kernel number per ear compared with the control (B104*C01). The ear row number increased by 2-3 rows, and the kernel number per ear increased by about 50% ( Fig. 9 AD, Fig.10 AD); 100-grain weight and single-plant yield also increased significantly, with grain weight increasing by 12% and final yield increasing by 40-68% ( Fig. 9 AD, Fig.10 AD). Among them, the polymerization effect of ZmPHO1;2 gene driven by Ubi promoter and ZmGIF1 was weaker than that of ZmPHO1;2 gene driven by 35S promoter and ZmGIF1 ( Fig. 9 AD, Fig.10 AD), which is due to the low expression level of ZmPHO1;2 driven by Ubi.

[0131] In summary, hybrid corn that combines the two key 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.

[0132] Example 5: Hybrid corn synthesized by polymerizing ZmPHO1;2 and ZmGIF1 greatly improves photosynthesis of C4 crops

[0133] Generally, photosynthesis of plants is divided into two major processes: light reaction and dark reaction. The ATP and NADPH produced by light energy in the light reaction are consumed and triose phosphate is produced for further carbohydrate synthesis in the dark reaction. Although the carbon fixation process of plant photosynthesis requires the participation of Rubisco enzyme, the carbon fixation methods of C3 plants and C4 plants are different: C3 crops such as rice use ribulose diphosphate (C5) to fix carbon dioxide. The 6-carbon molecule (C6) generated after combining with CO2 is very unstable and immediately splits into two compounds containing 3 carbon atoms, 3-phosphoglycerate. This process requires carboxylation of Rubisco enzyme. In addition to the carboxylation function, Rubisco enzyme also has an oxygenation function, that is, it combines with O2 to reduce the photosynthetic rate; C4 crops such as corn do not have Rubisco enzyme in the mesophyll cells. Phosphoenolpyruvate (PEP) directly fixes CO2 to generate malic acid or aspartic acid with 4 carbon atoms, and then goes to the vascular bundle sheath cells to carry out the same reaction process as C3. This process does not involve the oxygenation of Rubisco, and the binding ability of PEP with CO2 is much greater than that of RuBP. The inventors believe that in the process of photosynthesis, the efficient and stable photosynthetic rate is often limited by three factors, namely Rubisco activity, photosynthetic electron transfer and triose phosphate utilization, and these three processes all require the participation of "phosphorus". Therefore, from an overall perspective, the high photosynthetic rate of crops is often affected by phosphorus limitation, which ultimately affects the yield. The photosynthetic efficiency of C4 crops themselves is higher than that of C3 crops. Therefore, if the phosphorus limitation of C4 crops can be released, the high photosynthetic efficiency of C4 crops can be further improved.

[0134] 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 started from both the source and the sink. In hybrid corn, the photosynthesis of C4 crops was first measured. At the middle of grain filling (20DAP), 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 parent B104 and C01 inbred lines were measured at the same time. The results showed that the F1 produced by the polymerization of ZmGIF1 and ZmPHO1;2a and ZmGIF1 and ZmPHO1;2b can significantly increase photosynthesis, especially the polymerization of ZmPHO1;2a, ZmPHO1;2b and ZmGIF1 driven by the 35S promoter can increase photosynthesis by 30% ( Fig.11 AD), the fundamental reason is that ZmPHO1;2 optimizes the distribution of Pi in the plant, increases the accumulation of Pi in the leaves of photosynthetic tissues, slows down the production of phosphorus limitation, promotes the metabolic process of photosynthesis, and promotes the high efficiency of photosynthesis, thereby producing more photosynthetic products (sucrose) ( Fig.11EF), thus significantly increasing the accumulation of maize sources, which is the basis for ensuring high crop yields. The polymerization of ZmPHO1;2 and ZmGIF1 driven by the Ubi promoter can increase photosynthesis by about 15%-20%.

[0135] Based on its own photosynthetic structure, C4 crops have higher photosynthetic efficiency and CO2 fixation efficiency compared with C3 crops. Especially under high light and high temperature environment, the improvement of photosynthetic efficiency of C3 crops is easy to reach bottleneck or upper limit and phosphorus limitation is easy to occur under high temperature and high light. The photosynthetic efficiency of C3 crops is affected by the activity of Rubisco enzyme, which limits the further increase of 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 light efficiency, especially under high temperature and high light conditions, the large demand of crops for Pi. The polymerization of ZmPHO1;2 and ZmGIF1 can significantly alleviate phosphorus limitation and ensure the high photosynthetic rate of C4 crops.

[0136] In summary, hybrid corn with overexpression of 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.

[0137] Example 6: Hybrid corn ZmGIF1 and ZmPHO1;2 reduces grain phytic acid content

[0138] 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, thus limiting the promotion and application of crop varieties.

[0139] In order to detect the effect of overexpression of ZmPHO1;2 and ZmGIF1 on the phosphorus content in the grains in hybrid corn, the inventors measured the phosphorus content of the grains at maturity. The Pi content results showed that the Pi content of the 35S-driven ZmPHO1;2a and ZmPHO1;2b overexpression lines was significantly reduced compared with the wild type C01; in the ZmGIF1 overexpression line, the Pi content was no different from that of the wild type B104 ( Fig.12 AC). In hybrid maize, inorganic phosphorus (Pi), total phosphorus, and phytic acid were determined.

[0140] 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 ( Fig.12 DG).

[0141] These results indicate that the improved hybrid maize with ZmPHO1;2 and ZmGIF1 significantly improved phosphorus utilization and phytic acid content in grains.

[0142] Example 7: Improvement of grain quality in hybrid corn of ZmGIF1 and ZmPHO1;2

[0143] 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 kernels also needs to be considered. The quality of the corn kernels 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 kernels of ZmGIF1 and ZmPHO1;2 were significantly increased ( Fig.13 AC), indicating that the improved hybrid corn has 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, in which 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. In this process, the glucose produced by the decomposition of sucrose by ZmGIF1 is the raw material for starch synthesis, while ZmPHO1;2 regulates the balance of phosphorus in the ADPG synthesis process, prompting the enzyme-linked reaction to proceed in the forward direction to produce a continuous supply of starch synthesis substrates, promoting grain filling, and ultimately significantly increasing the total starch content of the grain ( Fig.13 AC). As a result, in hybrid corn, the quality and starch content of corn kernels are improved.

[0144] 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 were two lines with little difference compared with the control ( Fig.13 D).

[0145] These results indicate that the improved hybrid corn with ZmGIF1 and ZmPHO1;2 has improved corn kernel quality.

[0146] Example 8: ZmGIF1 and ZmPHO1;2 hybrid corn can be used as silage corn

[0147] The above evidence shows that hybrid corn improved by ZmPHO1;2 and ZmGIF1 can significantly increase corn biomass, make stems thicker, leaves larger, and plant height higher ( Figure 7 , Figure 8 ), and it is worth noting that the control corn begins to age and turn yellow near maturity, while the improved hybrid corn still has relatively green stalks ( Fig.14A), so that hybrid corn can be suitable for silage after the ears are harvested.

[0148] Therefore, the inventors measured and analyzed the nutrients in the stalks of hybrid corn. The results showed that the soluble total sugar content and sucrose content in the improved hybrid corn stalks were significantly increased, while the Pi content in the stalks was significantly reduced ( Fig.14 BD). Further statistical observations in the field showed that the improved hybrid corn significantly increased stem thickness, which also played an important role in the lodging resistance of corn ( Fig.14 EF).

[0149] 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.

[0150] Example 9: Polymerization of SbPHO1;2 and SbGIF1 significantly increases sorghum yield

[0151] In order 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 genes SbPHO1;2 and SbGIF1 of sorghum 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.

[0152] Further analysis of the phenotype of transgenic sorghum at maturity revealed that the grain size of the positive strains was significantly larger than that of the wild-type Wheatland. Fig.15 A), statistical analysis showed that the grain length, width, thickness and weight of transgenic sorghum increased significantly ( Fig.15 BE), which shows that the aggregation and synergistic improvement of SbPHO1;2 and SbGIF1 significantly promoted the grain filling process of sorghum, and finally increased the grain weight and sorghum yield. This further shows that the creation of super high-yield crops based on PHO1;2 and GIF1 genes described in the present invention is applicable to multiple Gramineae C4 crops.

[0153] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

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 (Setariaitalica) 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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