A new gene for increasing grain yield of gramineae and application thereof

Abstract

The application provides a new gene for increasing grain yield of plants in the family Poaceae and application thereof. A new GRAS11 and downstream molecule EXPB15 related to regulation of plant yield traits or endosperm quality are disclosed, which are positive regulation genes for yield or endosperm quality of plants in the family Poaceae, and loss of function thereof leads to decrease of yield and quality, but has no influence on other traits of plants. The application provides a new way for trait improvement of plants.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and botany, and more specifically, this invention relates to a new gene for increasing the grain yield of grass plants and its application. Background Technology

[0002] Among grasses, maize exhibits stronger drought resistance, cold resistance, tolerance to poor soil conditions, and excellent environmental adaptability compared to rice and wheat, resulting in a high proportion of its planting area. However, maize's yield per unit area is significantly lower than that of rice and other crops, indicating considerable potential for advancing maize germplasm resource research and developing high-yield maize varieties. The theoretical yield per maize plant is determined by both the number of kernels per ear and kernel weight, with kernel weight further determined by four indicators: kernel length, width, thickness, and kernel fullness. Due to the dense arrangement and mutual compression of kernels in maize ears, the three performance indicators—kernel length, width, and thickness—are closely linked and heavily influenced by the number of kernels per ear. Therefore, progress in determining yield-related QTLs for maize has been relatively slow compared to that for rice. In 2019, Huazhong Agricultural University used a tropical small-grain maize variety (SK) as material and applied PacBio sequencing technology to assemble three generations of genomes. They also located a quantitative trait locus, qHKW1, that simultaneously controls grain shape and weight through recombinant inbred lines, and cloned the ZmBAM1d gene, which encodes a CLV1 / BAM receptor kinase protein that positively regulates grain shape and weight (Yang et al., 2019). In 2020, Huazhong Agricultural University cloned the quantitative trait locus qKW9, which encodes a DYW motif pentapeptide repeat protein involved in the CU editing of the chloroplast NADH dehydrogenase-like complex subunit ndhB, affecting maternal photosynthesis and thus grain filling (Huang et al., 2020).

[0003] The resources of maize kernel weight-related genes obtained through QTL mapping are relatively limited. Improving maize kernel yield through transgenic methods is another approach. Currently, improving maize yield through transgenic methods mainly utilizes two types of genes: 1. genes that enhance the efficiency of carbohydrate transport into the kernel, such as overexpression of ZmGIF1 / Mn1 (Li et al., 2013); 2. genes that enhance the starch synthesis capacity of maize endosperm, such as overexpression of AGPase large and small subunits or E. coli AGPase (Boehlein et al., 2014; Tuncel and Okita, 2013).

[0004] Endosperm generally refers to the trophoblast formed after the fusion of sperm and polar nuclei during double fertilization in angiosperms; it is also called the endosperm. This tissue is neither a gametophyte nor a sporophyte, and its chromosome ploidy is generally triploid; it is an important component of the seeds of many plants (such as grasses).

[0005] Currently, the genetic resources utilized in transgenic research mainly focus on carbohydrate transport and storage. However, from the perspective of endosperm development, the increase in endosperm cell volume is a passive process. Synergistic regulation of endosperm cell expansion and the transport and synthesis of storage substances is beneficial for obtaining higher-yielding germplasm resources. Therefore, this field urgently needs to consider this starting point and explore target genes that can be effectively regulated. Summary of the Invention

[0006] The purpose of this invention is to provide a new gene for increasing the grain yield of grass plants and its application.

[0007] In a first aspect of the invention, a method for improving the yield or endosperm quality of grass plants is provided, comprising: upregulating the expression or activity of GRAS11 or its downstream molecule EXPB15 in the plant.

[0008] In one or more embodiments, the GRAS11 includes its homologs, preferably its homologs in grasses.

[0009] In one or more embodiments, the EXPB15 includes its homologs, preferably its homologs in grasses.

[0010] In one or more embodiments, the GRAS11 comprises a protein or a gene.

[0011] In one or more embodiments, the EXPB15 comprises a protein or a gene.

[0012] In one or more embodiments, GRAS11 indirectly regulates the downstream molecule EXPB15 through transcription factors that can interact with it.

[0013] In one or more embodiments, the improvement of the yield of grass plants includes: increasing grain weight, increasing grain width, and increasing grain thickness.

[0014] In one or more embodiments, improving the endosperm quality of grass plants includes: increasing endosperm thickness and / or width, and promoting endosperm cell expansion and / or enlargement (including increased width).

[0015] In one or more embodiments, the "promoting endosperm cell expansion and / or enlargement" includes the advance of cell expansion and / or enlargement in time.

[0016] In one or more embodiments, the "promoting endosperm cell expansion and / or enlargement" includes cell enlargement during or after plant maturation.

[0017] In one or more embodiments, the “endosperm cell expansion and / or enlargement” refers to endosperm cell expansion and / or enlargement that begins during the sap filling stage.

[0018] In one or more embodiments, the “endosperm cell expansion and / or enlargement” results in increased seed size (including width or thickness) and / or increased seed weight after plant maturity.

[0019] In one or more embodiments, the upregulation includes: expressing exogenous GRAS11 or its downstream molecule EXPB15 in plants; preferably, expressing it with an endosperm-specific promoter; more preferably, the endosperm specificity includes (but is not limited to): a 27-kD γ-prolactin promoter, a 10-kD δ-prolactin promoter, etc.

[0020] In one or more embodiments, the upregulation includes: increasing the efficiency of transcription or translation of GRAS11 or its downstream molecule EXPB15 in plants.

[0021] In one or more embodiments, the upregulation includes: regulation with an upregulating molecule that interacts with GRAS11 or its downstream molecule EXPB15, thereby increasing the expression or activity of GRAS11 or its downstream molecule EXPB15.

[0022] In one or more embodiments, after upregulating the expression or activity of GRAS11 or its downstream molecule EXPB15 in plants, the method further includes: self-pollinating, hybridizing, or testcrossing the obtained plants to obtain progeny plants.

[0023] In another aspect of the invention, there is provided the use of GRAS11, its downstream molecule EXPB15, or their upregulated molecules for: (a) increasing the yield of grass plants, or (b) improving the endosperm quality of grass plants.

[0024] In one or more embodiments, (a) includes: increasing kernel weight (kernel weight), increasing kernel width (kernel width), and increasing kernel thickness (kernel thickness).

[0025] In one or more embodiments, (b) includes: increasing the thickness and / or width of the endosperm, promoting the expansion and / or enlargement of endosperm cells (including an increase in width).

[0026] In one or more embodiments, the upregulated molecule includes: an expression cassette or expression construct (including an expression vector) that expresses GRAS11 or its downstream molecule EXPB15; an expression cassette or expression construct that improves the translation efficiency of GRAS11 or its downstream molecule EXPB15; or an upregulated molecule that interacts with GRAS11 or its downstream molecule EXPB15 to improve its expression or activity.

[0027] In another aspect of the invention, an expression construct is provided, comprising an operatively linked endosperm-specific expression promoter, GRAS11, and / or its downstream molecule EXPB15; preferably, the expression construct comprises an operatively linked endosperm-specific expression promoter, GRAS11, and its downstream molecule EXPB15; more preferably, the endosperm-specific expression promoter comprises (but is not limited to): a 27-kD γ-prolysin promoter or a fragment thereof, a 10-kD δ-prolysin promoter, etc.

[0028] In one or more embodiments, the 27-kD γ-prolysin promoter comprises the nucleotide sequence of SEQ ID NO:5 or positions 1-453 therein.

[0029] In another aspect of the invention, a gramineous plant cell, tissue, or organ is provided, wherein an exogenous upregulating GRAS11 protein molecule is provided, said upregulating molecule being the expression construct described above.

[0030] In one or more embodiments, the plant cells, tissues or organs described herein do not have reproductive capacity, are not plant propagation materials, and cannot be directly propagated into plants.

[0031] In one or more embodiments, the grass is a plant that expresses GRAS11 or its downstream molecule EXPB15 or their homologs; preferably, the grass includes (but is not limited to): maize, sorghum, rice, barley, wheat, oats, rye, and bulrush.

[0032] In one or more embodiments, the grass plant is a grass crop.

[0033] In one or more embodiments, the grass is a seed-bearing plant, the seed having an endosperm structure.

[0034] In one or more embodiments, the grass plant is a plant in which GRAS11 or its downstream molecule EXPB15 is expressed at relatively low levels (e.g., significantly lower than the expression level of the gene in the plant) or not expressed at all, thereby increasing the yield or endosperm quality of the plant by increasing the expression or activity of GRAS11 or its downstream molecule EXPB15.

[0035] In one or more embodiments, the amino acid sequence of the GRAS11 polypeptide is selected from the group consisting of: (i) a polypeptide with the amino acid sequence shown in SEQ ID NO:2; (ii) a polypeptide derived from (i) having the polypeptide function, formed by substituting, deleting, or adding one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues of the amino acid sequence shown in SEQ ID NO:2; (iii) a polypeptide having the regulatory trait function, with an amino acid sequence homology ≥70% (e.g., 70% to 99.5% homology; preferably ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, ≥98%, or ≥99%) with homology to the amino acid sequence shown in SEQ ID NO:2; (iv) an active fragment of a polypeptide with the amino acid sequence shown in SEQ ID NO:2; or (v) a polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:2, or by adding a signal peptide sequence to its N end.

[0036] In one or more embodiments, the amino acid sequence of the EXPB15 polypeptide is selected from the group consisting of: (i) a polypeptide with the amino acid sequence shown in SEQ ID NO:4; (ii) a polypeptide derived from (i) having the polypeptide function, formed by substituting, deleting, or adding one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues of the amino acid sequence shown in SEQ ID NO:4; (iii) a polypeptide having the regulatory trait function, with an amino acid sequence homology ≥70% (e.g., 70% to 99.5% homology; preferably ≥75%, ≥80%, ≥85%, ≥90%, ≥95%, ≥98%, or ≥99%) with homology to the amino acid sequence shown in SEQ ID NO:4; (iv) an active fragment of a polypeptide with the amino acid sequence shown in SEQ ID NO:4; or (v) a polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:4, or by adding a signal peptide sequence to its N end.

[0037] In another aspect of the invention, the use of GRAS11 or its downstream molecule EXPB15 (including protein or gene) is provided as a molecular marker for identifying traits in grasses, including yield traits or endosperm traits.

[0038] In another aspect of the present invention, a method for targeted selection or identification of high-yielding and high-endosperm-quality gramineous plants is provided, comprising: identifying the expression or activity of GRAS11 or its downstream molecule EXPB15 in a test plant; if the expression or activity of GRAS11 or its downstream molecule EXPB15 in the test plant is higher than or equal to the average expression or activity of GRAS11 or its downstream molecule EXPB15 in the same type of plant (control plant), then the plant is a high-yielding and high-endosperm-quality gramineous plant.

[0039] In one or more embodiments, the yield traits include: grain weight, grain width, and grain thickness. Preferably, if GRAS11 or its downstream molecule EXPB15 is highly expressed or highly active (e.g., significantly higher than the average expression or activity of similar or plant-like species), then the grain weight, grain width, and grain thickness are high; if GRAS11 or its downstream molecule EXPB15 is lowly expressed or lowly active (e.g., significantly lower than the average expression or activity of similar or plant-like species), then the grain weight, grain width, and grain thickness are low.

[0040] In one or more embodiments, the endosperm traits include: endosperm thickness and / or width, and the ability to promote endosperm cell expansion and / or enlargement. Preferably, if GRAS11 or its downstream molecule EXPB15 is highly expressed or highly active (e.g., significantly higher than the average expression or activity of similar or identical plants), then its endosperm thickness and / or width is large, and its endosperm cell expansion and / or enlargement ability is high; if GRAS11 or its downstream molecule EXPB15 is lowly expressed or low active (e.g., significantly lower than the average expression or activity of similar or identical plants), then its endosperm thickness and / or width is small, and its endosperm cell expansion and / or enlargement ability is low.

[0041] In one or more embodiments, high expression or high activity means a statistically significant increase in expression or activity compared to the average expression or activity of similar or identical plants, such as an increase of 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0042] In one or more embodiments, the low expression or low activity refers to a statistically significant reduction in expression or activity compared to the average expression or activity of similar or identical plants, such as a reduction of 10%, 20%, 40%, 60%, 80%, 90%, or lower.

[0043] In one or more embodiments, "high quantity" / "high quality (or good)" means that the quantity is statistically significantly higher than that of similar or plant species, such as 10%, 20%, 40%, 60%, 80%, 90% or higher.

[0044] In one or more embodiments, "low amount" means that it is statistically lower than that of the same type or plant, such as 10%, 20%, 40%, 60%, 80%, 90% or lower.

[0045] In another aspect of the present invention, a method is provided for screening substances (potential substances) that promote the improvement of traits in gramineous plants, wherein the trait improvement includes: high yield and high endosperm quality. The method includes: (1) adding the candidate substance to a system expressing GRAS11 or its downstream molecule EXPB15; (2) detecting the system and observing the expression or activity of GRAS11 or its downstream molecule EXPB15 therein. If its expression or activity is increased (significantly increased, such as by 10%, 20%, 40%, 60%, 80%, 90% or higher), it indicates that the candidate substance is a substance that promotes the improvement of traits in gramineous plants.

[0046] In one or more embodiments, the method further includes setting up a control group in which the candidate substance is not added, thereby clearly distinguishing the difference in expression or activity of GRAS11 or its downstream molecule EXPB15 in the test group from that in the control group.

[0047] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules (such as upregulators, small molecule gene editing constructs, etc.) designed for the GRAS11 or its downstream molecule EXPB15 or its upstream or downstream proteins or genes.

[0048] In another aspect of the invention, a method for identifying overexpressed plant material is provided, the overexpressed plant material comprising a 27-kD γ-glucosinolate promoter and GRAS11 or EXPB15 expressed therethrough, the method comprising identifying the presence of the nucleotide sequence shown in SEQ ID NO:6 or SEQ ID NO:9 in the overexpressed material.

[0049] In one or more embodiments, the presence of the nucleotide sequence shown in SEQ ID NO:6 (including the GRAS11 sequence) in the overexpression material is identified using primers of SEQ ID NO:7 and SEQ ID NO:8.

[0050] In one or more embodiments, the presence of the nucleotide sequence shown in SEQ ID NO:9 (including the EXPB15 sequence) in the overexpression material is identified using primers SEQ ID NO:10 and SEQ ID NO:11.

[0051] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0052] Figure 1 Cell proliferation and expansion during maize endosperm development. (A) Freehand section of W64A kernel, scale bar: 2 mm. Arrow: embryo sac, En: endosperm, asterisk: embryo. Double arrows on 14DAP and 24DAP endosperm represent areas for cell counting in the endosperm. (B) Observation of semi-thin sections of W64A endosperm at different stages after pollination. UEC: cells to be expanded, MEC: moderately expanded cells. Scale bar: 50 μm.

[0053] Figure 2 1. Statistical analysis of cell proliferation and expansion during maize endosperm development. (A) Freehand section of W64A kernel, scale bar: 2 mm. Arrow: embryo sac, En: endosperm, asterisk: embryo. Double arrows on 14DAP and 24DAP endosperm represent areas for cell counting in the endosperm. (B) Observation of semi-thin sections of W64A endosperm at different stages after pollination. UEC: cells to expand, MEC: moderately expanded cells, CEC: fully expanded cells. Scale bar: 50 μm.

[0054] Figure 3 Co-expression analysis identified the key gene ZmGRAS11 for endosperm expansion. (A) Based on a Pearson correlation coefficient (PCC) threshold of 0.6, 34 transcription factors were identified that are associated with starch synthesis and zein encoding genes. Yellow circles indicate indicator genes, and green circles indicate transcription factors. Gray lines indicate the positive correlation between indicator genes and TFs. (B) The ZmGRAS11 gene is associated with 12 guide genes.

[0055] Figure 4 Amino acid sequence alignment of several DELLA proteins. Black shading indicates identical amino acid residues, while gray shading indicates similar amino acid residues. The SLR, SLRL1, and SLRL2 genes are derived from the rice (Oryzasativa L.) genome. The RGA gene is derived from Arabidopsis thaliana. SLRL1, SLRL2, and ZmGRAS11 do not contain DELLA, LEQLE, or TVHYNP domains at their N-terminus.

[0056] Figure 5 Subcellular localization of ZmGRAS11 protein. Subcellular localization of ZmGRAS11-GFP. DAPI was used as a nuclear dye.

[0057] Figure 6Structure of ZmGRAS11 (A) and detection of its self-activation ability (B). Detection of ZmGRAS11 self-activation in yeast. Using the endosperm-specific transcription factor O2 as a positive control, neither the full-length ZmGRAS11 protein nor the half-length ZmGRAS11 protein showed self-activation ability.

[0058] Figure 7 1. Expression pattern analysis of maize DELLA genes. (A) Expression pattern analysis of seven maize DELLA genes in public databases. The color labels on the right represent the differences in expression levels. S0-S38 represent maize kernels at different stages from 0 to 38 days after pollination; Em10-Em38 represent embryos from 10 to 38 days after pollination; En6-En38 represent endosperm from 10 to 38 days after pollination. (B) Relative quantitative PCR detection results of D8, D9, and ZmGRAS11 in different maize tissues. All genes were normalized with the SGT1 gene, and each sample group used 5 biological replicates, and the mean was used for plotting. The relative expression level of D8 was calculated using the expression level of D8 at 24 days after pollination as 1, the relative expression level of D9 was calculated using the expression level of D9 at 24 days after pollination as 1, and for ZmGRAS11, the expression level of ZmGRAS11 in the root was calculated as 1.

[0059] Figure 8 In situ hybridization of ZmGRAS11. In situ hybridization of ZmGRAS11 was performed 12 days after pollination of maize endosperm. (A) Hybridization using an antisense probe; (B) Hybridization using a sense probe.

[0060] Figure 9 Schematic diagram of ZmGRAS11 editing sites and detection at the RNA and protein levels. (A) Schematic diagram of CRISPR / Cas9-mediated zmgras11 editing events. The zmgras11-C gene editing mutant has a cytosine deletion in the LHRI domain, while the zmgras11+A gene editing mutant has an adenine insertion. (B) RT-qPCR validation of ZmGRAS11 expression levels in wild-type and gene-edited mutants. The mean of 5 biological replicates was used for plotting, and the p-value was calculated using Student's t-test two-tailed test. (C) Total protein was extracted from the endosperm of wild-type and mutants 18 days after pollination. Immunohistochemistry was performed using ZmGRAS11 antibody, with ACTIN antibody used as a control.

[0061] Figure 10Phenotypic analysis of zmgras11. (A) Ear phenotypes of KN5585 (WT), zmgras11-C (-C), zmgras11+A (+A), and zmgras11-C×zmgras11+A (-C×+A). Scale bar = 5 cm. (B) Grain phenotype analysis of KN5585 (WT), zmgras11-C (-C), and zmgras11+A (+A). Scale bar = 1 cm. (C) Statistical comparison of grain weight between wild type and zmgras11 mutant. (D) Statistical comparison of grain length between wild type and zmgras11 mutant. (E) Statistical comparison of grain width between wild type and zmgras11 mutant. (F) Statistical comparison of grain thickness between wild type and zmgras11 mutant. All statistics were performed using Student's t-test, with 7-9 biological replicates for statistical plotting.

[0062] Figure 11 Phenotypic analysis of zmgras11 (Sanya). (A) From left to right: ear phenotypes of KN5585 (WT), zmgras11-C (-C), and zmgras11+A (+A) (3 each). Scale bar = 5 cm. (B) From top to bottom: grain phenotype analysis of KN5585 (WT), zmgras11-C (-C), and zmgras11+A (+A). Scale bar = 1 cm. (C) Statistical comparison of grain weight between wild type and zmgras11 mutant. (D) Statistical comparison of grain length between wild type and zmgras11 mutant. (E) Statistical comparison of grain width between wild type and zmgras11 mutant. (F) Statistical comparison of grain thickness between wild type and zmgras11 mutant. All statistics were performed using Student's t-test, with 6 biological replicates for plotting.

[0063] Figure 12 Starch and protein were determined in wild-type and zmgras11 gene-edited mutants. (A) Total starch was determined in wild-type and zmgras11 gene-edited mutants, with six biological replicates for each sample. (B) SDS-PAGE analysis of gliadin and non-gliadin was performed in wild-type and zmgras11 gene-edited mutants, with three samples from three different ears. 27γ, 27-kDγ gliadin; 22α, 22-kDα gliadin; 19α, 19-kDα gliadin; 16γ, 16-kDγ gliadin; 15β, 15-kDβ gliadin; 10δ, 10-kDδ gliadin.

[0064] Figure 13Internal replication of zmgras11 in the endosperm. Blue squares represent the proportion of different ploidies in the endosperm of the 18-DAP wild-type (expressed as a percentage of the total number of nuclei), purple squares represent zmgras11-C, and orange squares represent zmgras11+A. Data are presented as mean and were measured using six biological replicates. p-values ​​were determined using a two-tailed Student's t-test. An asterisk indicates a p-value less than 0.05.

[0065] Figure 14 The zmgras11 mutant exhibits phenotypes of smaller grain width and smaller cells. (A) Measurements and observations of freehand sections of wild-type (KN5585) and mutant materials. En represents endosperm, scale bar = 2 mm. (B) Statistical analysis of endosperm width (mm), cell number, and cell width (μm) in wild-type and mutant materials. All data are expressed as mean values. Endosperm thickness and mean cell width were measured using 6-25 biological replicates; cell number was measured using 4-16 biological replicates.

[0066] Figure 15 The zmgras11 mutant exhibits a delayed cell expansion phenotype. (A) Measurements and observations of half-thin sections of wild-type (KN5585) and mutant cells. Scale bar = 5 μm. (B) Statistical analysis of cell width (μm) in wild-type and mutant materials at 14 and 24 days post-pollination. Data are expressed as mean values, with 5-10 biological replicates; UEC: cells to be expanded; MEC: moderately expanded cells; CEC: fully expanded cells.

[0067] Figure 16 Phenotypic images of ZmGRAS11 overexpression materials. (A) Phenotypic images of wild-type ears (KN5585) and ZmGRAS11 overexpression ears (OE-1 and OE-2). Scale bar = 2 cm. (B) Phenotypic images of wild-type and overexpression grains. Scale bar = 1 cm. (C) Validation of relative quantification of wild-type and overexpression grains 18 days after pollination. All samples were normalized with the SGT1 gene, and all data were plotted using 5 biological replicates and averages. (D) Total protein was extracted from the endosperm of wild-type and overexpression materials 18 days after pollination. Immunohistochemistry was performed using ZmGRAS11 antibody, with ACTIN antibody hybridization as a control. (E) Statistical comparison of grain weight between wild-type and overexpression materials. (F) Statistical comparison of grain length between wild-type and overexpression materials. (G) Statistical comparison of grain width between wild-type and overexpression materials. (H) Statistical comparison of grain thickness between wild-type and overexpression materials. All statistics were performed using Student's t-test, with 4-6 biological replicates used for plotting.

[0068] Figure 17 Enhanced endosperm starch cell expansion in ZmGRAS11 overexpression materials. (A) Semi-thin sections of wild-type and ZmGRAS11 overexpression grains 18 days post-pollination. Scale bar = 50 μm. (B) Statistical analysis of endosperm width, cell number, and cell width in wild-type and overexpression materials. Endosperm width and mean cell width were calculated using 15-25 biological replicates, and cell number was calculated using 5-6 biological replicates, with the mean value used for plotting. P-values ​​for all statistics were calculated using the two-tailed Student's t-test. (C) Single-cell width measurements of wild-type and overexpression grains 18 days post-pollination. All statistics were calculated using Student's t-test, with 5-6 biological replicates used for plotting.

[0069] Figure 18 ZmGRAS11 regulates genes related to cell expansion. (A) Differential gene enrichment analysis (GO) was performed on wild-type and mutant samples 18 days post-pollination. (B) Expression levels of differentially expressed genes related to cell wall synthesis were analyzed using RNA-seq. (C) RT-qPCR validation was performed on the differentially expressed genes related to cell wall synthesis selected by RNA-seq using wild-type, zmgras11+A, and overexpression materials. All expression levels were normalized using the SGT1 gene.

[0070] Figure 19 1. The activation of EXPB15 and β-glucosidase by ZmGRAS11 was verified using a dual-fluorescence system. (A) Schematic diagram of the dual-fluorescence reporter system. 35S: cauliflower mosaic virus 35S promoter; Ter: terminator. (B) Left side shows the transcriptional activation values ​​of maize endosperm, and right side shows the transcriptional activation values ​​of Arabidopsis protoplasts. Transcriptional activation driven by EXPB15 as the promoter is represented by the left y-axis, and transcriptional activation driven by BG as the promoter is represented by the right y-axis. All statistics were performed using Student's t-test, with 5 biological replicates and the mean values ​​used for plotting.

[0071] Figure 20 Relative quantification of the EXPB-15 gene in maize endosperm. All expression levels were normalized using the SGT1 gene. All statistics were performed using Student's t-test, with five biological replicates and the mean values ​​used for plotting.

[0072] Figure 21Enhanced endosperm starch cell expansion in p27::EXPB15 overexpression materials. (A) Semi-thin sections of wild-type and p27::EXPB15 overexpression grains 24 days post-pollination. Scale bar = 100 μm. (B) Statistics on endosperm width, cell number, and cell width in wild-type and overexpression materials. Endosperm width and mean cell width were calculated using 15-25 biological replicates, and cell number was calculated using 5-6 biological replicates, with the mean value used for plotting. P-values ​​for all statistics were calculated using the two-tailed Student's t-test. (C) Single cell width measurements of sections of wild-type and overexpression grains 24 days post-pollination. All statistics were calculated using Student's t-test, with 5-6 biological replicates used for plotting. Detailed Implementation

[0073] Through extensive research, the inventors have revealed a novel gene, GRAS11, and its downstream molecule EXPB15, which are associated with regulating plant yield traits or endosperm quality. These genes are positive regulators of grain yield or endosperm quality in grasses; their loss of function leads to decreased yield and quality, but has no effect on other plant traits. This invention provides a new approach for improving plant traits.

[0074] the term

[0075] As used herein, "plant" refers to a plant with an embryo and endosperm structure. Those skilled in the art know that plant embryos and endosperm have similar compositions, and plants with embryos or endosperm structures share common characteristics, including numerous conserved genes or regulatory elements in their genomes that regulate gene transcription and expression, such as a series of elements that regulate the formation of the embryo or endosperm. In this invention, the "plant" expresses GRAS11, its downstream molecule EXPB15 (including their homologs), or contains GRAS11, its downstream molecule EXPB15, and the signaling pathways they participate in. Based on knowledge in the art, plants expressing GRAS11 and its downstream molecule EXPB15 inherently possess the mechanism of action claimed in this invention, and can achieve the technical effects claimed in this invention. In some preferred embodiments, the plant is a crop, preferably a cereal crop, which is a crop with grains (ears). The "cereal crop" can be a grass (Poaceae). Preferably, the grasses mentioned include: corn, rice, barley, wheat, oats, rye, sorghum, and short-stalked grass.

[0076] As used herein, the terms “enhancement,” “improvement,” or “enhancement” are interchangeable and, in their application, should mean an increase of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25% or 30%, compared to the control plant as defined herein.

[0077] Regarding "control plants," selecting appropriate control plants is a routine part of experimental design. These can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties of the same species or class as the plant being evaluated. Control plants can also be individuals from transgenic plants that have lost their transgenic components due to segregation. As used in this article, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0078] As used in this article, “grain” refers to the fruit or seed of a plant, and is also called ear grain in crops such as corn, rice, wheat, and barley.

[0079] As used herein, a “promoter” or “promoter region” refers to a nucleic acid sequence that is typically located upstream (5' end) of the coding sequence of a target gene and guides the transcription of the nucleic acid sequence into mRNA. Generally, a promoter or promoter region provides recognition sites for RNA polymerase and other factors necessary for proper transcription initiation. In this document, the promoter or promoter region includes variants of the promoter, obtained through insertion or deletion of regulatory regions, random or site-directed mutagenesis, etc. Gene transcription regulated by tissue- or organ-specific promoters generally occurs only in certain specific organs or tissues. A “promoter fragment” refers to a segment of the promoter that possesses the necessary sites for initiating transcription and the transcription start point; that is, the “promoter fragment” retains the basic function of its corresponding promoter. Optionally, the promoter fragment can be a basic promoter or a core promoter.

[0080] As used herein, “operationally linked” or “operationally connected” refers to a functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region is placed at a specific position relative to the nucleic acid sequence of a target gene, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby “operationally linked” to the nucleic acid sequence.

[0081] As used herein, “target gene” refers to the novel target protein / target gene GRAS11 or its downstream molecule EXPB15 that can be applied to regulate plant traits, as indicated in this invention.

[0082] As used herein, the term "specific expression" refers to the expression of a target gene at a specific time and / or in a specific tissue. "Tissue specificity," also known as "organ specificity," refers to the phenomenon where, under the regulation of certain regulatory elements, genes are often expressed only in specific organs or tissues, exhibiting their associated developmental regulatory characteristics. In this invention, "tissue specific expression" refers to specific expression in plant endosperm. Generally, if mRNA is expressed in a tissue or organ at a level at least 1.2, 1.5, 2, 3, or 4 times higher than in other tissues or organs, preferably at least 5 times higher, more preferably at least 10 times higher, and most preferably at least 100 times higher, the expression of the relevant gene is considered tissue- or organ-specific.

[0083] As used in this article, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a target gene sequence is not naturally occurring, then the promoter is exogenous to the target gene. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0084] As used in this article, “non-reproductive material” refers to a biological material that does not have the characteristic of using photosynthesis to synthesize carbohydrates and proteins from inorganic substances such as water, carbon dioxide and inorganic salts to sustain its life.

[0085] Positive regulatory molecules

[0086] This invention, through studying the development process of plant seed endosperm, found that in the early stages of endosperm development, endosperm size is determined by both cell number and cell size; while in the middle and late stages of endosperm development, the key factor determining endosperm or seed size is cell size, which is also the critical period for endosperm filling. This invention, through co-expression network analysis, screened 34 transcription factors highly expressed during the endosperm filling period. Overexpression of GRAS11 or its downstream gene EXPB15 increased endosperm cell volume, ultimately increasing seed width and weight.

[0087] In this invention, unless otherwise specified, GRAS11 or EXPB15 includes its homologs (homologous proteins / homologous genes). In some embodiments, the GRAS11 protein is a protein (peptide) having the amino acid sequence shown in SEQ ID NO:2; this invention also includes sequence variants having the same function as the GRAS11 protein. The EXPB15 protein is a protein (peptide) having the amino acid sequence shown in SEQ ID NO:4; this invention also includes sequence variants having the same function as the EXPB15 protein.

[0088] Sequence variations of the protein include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 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 (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the GRAS11 / EXPB15 protein (e.g., 70% or higher homology to the polypeptide sequence shown in SEQ ID NO:2 or SEQ ID NO:4; preferably 80% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and having the same function as the GRAS11 / EXPB15 protein is also included in this invention.

[0089] In this invention, polypeptides derived from species other than maize that share high homology with the sequences shown in SEQ ID NO:2 or SEQ ID NO:4, or that play the same or similar roles in the same or similar signaling pathways, are also included.

[0090] It should be understood that although GRAS11 or EXPB15 obtained from a specific species is preferred in this invention, other polypeptides or genes obtained from other species, especially grasses, that are highly homologous to GRAS11 or EXPB15 (e.g., having more than 70%, more particularly 80%, 85%, 90%, 95%, or even more than 98% sequence identity) are also within the scope of this invention.

[0091] The present invention also provides isolated proteins, which are fragments of GRAS11 or EXPB15 or formed by adding other proteins or tags at both ends.

[0092] This invention also relates to a polynucleotide sequence encoding GRAS11 or EXPB15 of the present invention, or a sequence variant thereof. The polynucleotide may be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to or a degenerate variant of the coding region sequence shown in SEQ ID NO:1 or SEQ ID NO:3. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO:2 or SEQ ID NO:4, but differing from the coding region sequence shown in SEQ ID NO:1 or SEQ ID NO:3. This invention also relates to variants (variants) of the aforementioned polynucleotide that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of the present invention.

[0093] The present invention also relates to a vector containing the aforementioned polynucleotide, and a host cell genetically engineered using the aforementioned vector or polypeptide to encode nucleic acids.

[0094] In this invention, the polynucleotide sequence encoding the polypeptide of this invention can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable in the host. An important characteristic of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements. Preferably, the expression vector may also selectively contain resistance elements, selection elements, or reporter gene elements, such as Bar or GUS.

[0095] When the aforementioned polynucleotide is expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. An enhancer is a cis-acting factor of DNA, typically consisting of approximately 10 to 300 base pairs, that acts on the promoter to enhance gene transcription.

[0096] Transforming host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. Plant transformation can be performed using methods such as Agrobacterium-mediated transformation or gene gun transformation, including spraying, leaf disc transformation, and embryo transformation.

[0097] Application of plant improvement

[0098] Through extensive systematic research, the inventors cloned the full-length sequence of GRAS11 and identified its biological function. This gene plays an important role in the development of grain endosperm.

[0099] First, using maize as the research object, the inventors studied the dynamic changes in cell proliferation and expansion during the endosperm development of the kernel. Maize kernel size is controlled by two important factors: cell number and cell size. A maize kernel consists of the endosperm, embryo, and seed coat, with the endosperm accounting for 90% of the total seed weight and playing a decisive role in kernel size. To investigate the dynamic changes in cell number and cell size during maize kernel growth and development, freehand sections, fixed-embedded sections, and semi-thin sections of W64A kernels from 0 to 30 days after pollination were observed. Simultaneously, the dynamic changes in endosperm thickness, cell size, and cell number were statistically analyzed. It was found that in the early stages of maize endosperm development, endosperm size is jointly determined by cell number and cell size; while in the middle and late stages of endosperm development, the key factor determining endosperm or kernel size is cell size, which is also the critical period for endosperm filling.

[0100] Secondly, the inventors conducted research on the identification of highly expressed transcription factors during the maize endosperm grain-filling stage. Since the endosperm grain-filling process is coupled with the post-expansion of endosperm cells, genes involved in the synthesis of endosperm storage substances were used as guide genes. Co-expression analysis was employed to identify transcription factors regulating post-expansion of endosperm cells. ZmGRAS11, belonging to the GRAS gene family, was one of the identified transcription factors and is specifically expressed during the endosperm grain-filling stage, potentially participating in the process of endosperm cell expansion and storage substance synthesis. Gene-edited mutant materials were created using CRISPR-Cas9 technology in the KN5585 genetic background, and two mutant lines were selected for further research. The deletion mutant lines showed reductions in both grain width and thickness, with a greater reduction in grain thickness. RNA-seq and RT-qPCR were used in combination to identify interacting proteins and downstream genes of ZmGRAS11, establishing a gene regulatory network for post-expansion of the endosperm. Using various in vivo and in vitro methods, EXPANSIN B-15 (EXPB15, Zm00001d017493) was identified as a downstream gene of ZmGRAS11, which may be involved in the post-endosperm expansion process.

[0101] Furthermore, the inventors used an endosperm-specific expression promoter to drive the overexpression of the ZmGRAS11 gene / ZmEXPB15, and found that the p27::ZmGRAS11 overexpression material showed significant improvements in grain weight, grain width, and grain thickness, and the endosperm cell size of the overexpression material was significantly increased compared to the wild-type material. The endosperm cell size of the p27::ZmEXPB15 overexpression material was significantly increased compared to the wild-type material.

[0102] Based on the above, this invention reveals the dynamic changes in cell proliferation and expansion during maize kernel endosperm development, and proposes for the first time that: in the early stages of maize endosperm development, endosperm size is determined by both cell number and cell size; while in the middle and late stages of endosperm development, the key factor determining endosperm or kernel size is cell size, which is also the critical period for endosperm filling. This invention identifies highly expressed transcription factors during maize endosperm filling and studies the regulatory network of endosperm cell expansion. In this invention, ZmGRAS11 gene-edited mutant materials were created using CRISPR-Cas9 technology, confirming that ZmGRAS11 regulates the post-endosperm cell expansion pathway, and also demonstrating that EXPANSIN B-15 (EXPB15, Zm00001d017493) is a downstream gene of ZmGRAS11, participating in the post-endosperm expansion process. Therefore, ZmGRAS11 / ZmEXPB15 positively regulates kernel weight by regulating the post-endosperm cell expansion pathway, and is an excellent gene resource for improving maize yield per plant.

[0103] Based on the above-mentioned new discovery of the inventors, a use is provided for GRAS11, its downstream molecule EXPB15, or its regulatory molecule, for: regulating the yield traits and endosperm traits of grass plants.

[0104] Meanwhile, the present invention also provides a method for regulating yield traits or endosperm traits of grass plants, comprising: regulating the expression or activity of GRAS11 or EXPB15 in plants; wherein, GRAS11 or EXPB15 includes its homologs.

[0105] It should be understood that, after learning about the role of GRAS11 or EXPB15 in the regulation of yield and endosperm traits in grasses, various methods well known to those skilled in the art can be used to regulate the expression or activity of GRAS11 or EXPB15 as needed, and these methods are all included in this invention.

[0106] The activity of GRAS11 or EXPB15 can be upregulated using upregulators of its expression or activity. These upregulators include promoters, agonists, and activators. The terms "upregulation" and "promotion" include both upregulation and promotion of protein activity and protein expression. Any substance that can increase the activity of GRAS11 or EXPB15, improve the stability of the GRAS11 or EXPB15 gene or protein, upregulate GRAS11 or EXPB15 gene expression, or increase the effective duration of action of the GRAS11 or EXPB15 protein can be used in this invention as a useful substance for upregulating the GRAS11 protein or its encoded protein. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0107] In a preferred embodiment, a method for upregulating the expression of GRAS11 and / or EXPB15 in plants is provided, the method comprising: introducing an expression construct or vector expressing GRAS11 and / or EXPB15 into the plant.

[0108] Preferably, a method for preparing transgenic plants is provided, comprising: (1) using exogenous GRAS11 and / or EXPB15 encoding nucleic acid primers to obtain plant tissues or organs transformed with the polypeptide encoding nucleic acid; and (2) regenerating plant plants from the plant tissues or organs obtained in step (1) that have been transformed with the exogenous polypeptide encoding nucleic acid of the present invention.

[0109] As a preferred example, the method includes the steps of: (s1) providing Agrobacterium carrying an expression vector containing nucleic acids encoding GRAS11 and / or EXPB15; (s2) contacting a plant tissue or organ with the Agrobacterium in step (s1) to transfer and integrate the nucleic acid encoding the polypeptide into the chromosome of a plant cell; (s3) selecting plant tissues or organs incorporating the nucleic acid encoding GRAS11 and / or EXPB15; and (s4) regenerating the plant tissue or organ from step (s3) into a plant.

[0110] Given that the regulatory effects of GRAS11 and / or EXPB15 in this invention occur in the endosperm of the grain, therefore, as a preferred embodiment of this invention, the expression of GRAS11 and / or EXPB15 is driven by an endosperm-specific expression promoter, thereby...

[0111] The present invention also includes plants obtained using any of the foregoing methods, said plants comprising: transgenic plants that have incorporated the coding nucleic acid of the polypeptide.

[0112] It should be understood that by combining multiple methods to simultaneously improve maize endosperm filling—source, sink, and flow—it is possible to obtain higher-yielding maize germplasm resources.

[0113] Applications as molecular markers

[0114] Based on the inventors' new findings, this invention provides molecular markers, namely GRAS11 and / or EXPB15, suitable for identifying plants with high yield and high endosperm quality. This invention also relates to specific molecular markers designed for said GRAS11 and / or EXPB15, and identification strategies.

[0115] Therefore, the present invention provides a method for targeted selection or identification of plants with regulated agronomic traits, comprising: identifying the expression or activity of GRAS11 and / or EXPB15 in the test plant; if the expression or activity of GRAS11 and / or EXPB15 in the test plant is higher than the average expression or activity of GRAS11 and / or EXPB15 in the same type of plant (control plant), then it is a plant with high yield and good endosperm quality; or, if the expression or activity of GRAS11 and / or EXPB15 in the test plant is lower than the average expression or activity of GRAS11 and / or EXPB15 in the same type of plant (control plant), then it is a plant with low yield and unsatisfactory endosperm quality.

[0116] Based on the novel findings of this invention, those skilled in the art can employ any of the various techniques known in the art or under development to analyze nucleic acid sequences, and these techniques are all included in this invention. The methods described include, but are not limited to: sequencing, PCR amplification, probe methods, hybridization, restriction enzyme digestion analysis, allele polymorphism analysis (such as melting curve analysis) for nucleic acid sequence identification, etc.

[0117] The identification method of this invention only requires PCR reaction and / or agarose gel electrophoresis. By determining the length of the corresponding PCR product, the phenotype or yield of the sample can be accurately and rapidly determined. It is low-cost, suitable for large-scale identification, and requires very little sample. If needed, those skilled in the art can design primers for identifying the molecular markers.

[0118] Methods for obtaining DNA from the sample to be tested are well-known to those skilled in the art, such as the traditional phenol / chloroform / isoamyl alcohol method, or commercially available DNA extraction kits. Polymerase chain reaction (PCR) is also well-known to those skilled in the art; its basic principle is the in vitro enzymatic synthesis of specific DNA fragments. The method of this invention can be performed using conventional PCR techniques.

[0119] This invention has promising applications in molecular design breeding and crop variety improvement using genetic engineering technology.

[0120] After understanding the functions of GRAS11 and / or EXPB15, 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 can target and regulate yield and endosperm quality by modulating this mechanism.

[0121] This invention provides a method for screening substances (potential substances) that promote the improvement of traits in grass plants, wherein the trait improvement includes: high yield and high endosperm quality; the method includes: (1) adding the candidate substance to a system expressing GRAS11 and / or EXPB15; (2) detecting the system and observing the expression or activity of GRAS11 and / or EXPB15 therein, and if the expression or activity is increased, it indicates that the candidate substance is a substance that promotes the improvement of plant traits.

[0122] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0123] The detection of protein-protein interactions and their strength can be achieved using a variety of techniques well-known to those skilled in the art, such as GST-Pull Down, bimolecular fluorescence complementation assays, yeast two-hybrid systems, or immunoprecipitation techniques.

[0124] Through large-scale screening, a class of substances that specifically act on GRAS11 and / or EXPB15 and have a regulatory effect on the improvement of plant traits can be obtained.

[0125] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or according to the manufacturer's recommendations.

[0126] sequence material

[0127] ZmGRAS11 (Zm00001d009646) protein sequence (SEQ ID NO:2):

[0128] MDAHTTHIVARKNTSLSFTSNELMDHFPFAWLPMDPFTAMALSGTLSPSFSTTSTNDTMYNLNPALYASLVPPIRKPATPTTLEVGRQCKEEEDASIRLVHLLITCTSAIETGDYSIAQGNLSEARKILGEIPTSTGIGRVGKHFIDALVQRLFPAYPHAAPPSPSPSTSIDLHNNFYDAGPYLKFAYSTANQAILKAIKGYNHVHIIDFSLMQGLQWPALMDVFSAREGGPPKLRITGIGPNPIGGRDELHEVGIRLAKYAHSVGIDFTFQGVCVDQLDRLCDWMLLKPIKGEAVAINSILQLHRLLVDPDANPVVPAPIDILLKLVIKINPMIFTVVEHEADHNRPPLLERFTNALFHYATMFDSLEAMHRCTSGRDITDSLTEVYLRGEIFDIVCGEGSARTERHELFGHWRERLTYAGLTQVWFDPDEVDTLKDQLIHVTSLSGSGFNILVCDGSLALAWHNRPLYVATAWCVTGGNAASSMVGNICKGTNDSRRKENRNGPME*

[0129] ZmGRAS11 cDNA sequence (SEQ ID NO:1):

[0130] atggatgctcacaccactcacattgttgcaagaaagaacacttcgctttcattcacctcaaatgagct catggatcattttcccttcgcttggttacccatggaccccttcacagccatggcgctcagcggtaccctctcaccc tcattctctaccacatctaccaatgataccatgtacaaccttaacccagcgttgtatgcatccctcgtgccaccaa taaggaaacctgcaaccccgaccacactagaggtagggaggcaatgcaaggaggaggaggatgcatccattcgtct ggtgcacctgctta tcacttgcaccagtgccattgagactggtgactactcgatcgcacaaggaaacttgtctgaggcacgcaagattcttggggaaatcccaacttcgaccgggatt ggccgtgttgggaaacacttcatcgacgcactag ttcaacgcctcttcccggcatatccgcacgcagcaccaccttctccatctccttctacatcgatcgatctgcacaa caatttttatgatgcaggtccctacctaaagttcgcctactccactgccaaccaagcaatcctcaaagcaatcaaaggatacaaccatgtacacataattgacttttccctgatgcaaggtctccagtggccggcactcatggatgtcttctccgcccgtgagggtgggccaccaaagctccgaatcacaggcattggcccgaacccaataggtggccgtgacgagctccatgaagtgggaattcgcctcgccaagtatgcacactcggtgggtatcgacttcactttccagggagtctgtgtcgatcagcttgataggttgtgcgattggatgcttctcaaaccaatcaaaggagaggcagttgccataaactccatcctacagctccatcgcctcctcgttgacccagatgcaaacccagtggtgcccgcaccaatagatatcctcctcaaattggtcatcaagataaaccccatgatcttcacggtggttgagcatgaggcagatcacaacagaccaccactactagagaggttcactaatgccctcttccactatgcgaccatgtttgactctttggaggccatgcatcgttgtaccagtggtagagacatcaccgactcactcacagaggtgtaccttcgaggtgagatttttgacattgtctgcggcgagggcagtgcacgcaccgaacgtcatgagttgtttggtcactggagggagaggctcacctatgctgggctaactcaagtgtggttcgaccccgatgaggttgacacgctaaaagaccagttgatccatgtgacatccttatctggctctgggttcaacatcctagtgtgtgatggcagccttgcactagcgtggcataatcgcccgttatatgtggcaacagcttggtgtgtgacaggaggaaatgctgccagttccatggttggcaacatctgtaagggtacaaatgatagtagaagaaaggaaaaccgtaatggacccatggagtag

[0131] ZmEXPB15 (Zm00001d017493) Protein Sequence (SEQ ID NO:4):

[0132] MPSGDRVYVCTSVFPCRWCMRVPERRRPAAVLVHDRRRQSIHLPERQGLRRLLSGEMHRARVVLRQPGDRGPHRRVPRRRVPGRARALRPERHGVRSHGEGRPGGPAPRRRTPQDPVHSGAVQLARAGHRVQGGRRLEPELLRGAHRVRVRGRRAGVRGAHAARRRRRGGVGADAAVVGRGVAVQLRRHPAGALLRPPHLRLRRDGRRQQRHPRGVDARRHVPLGRQLRQLTRTDGRTEITARPPARPPAGRRDGRERRGAIAFSL*

[0133] ZmEXPB15 cDNA Sequence (SEQ ID NO:3):

[0134] atgccttccggggaccgggtatacgtgtgtacgtcggtttttccgtgcaggtggtgcatgcgggtaccagaacgccgtcgaccagccgccgttctcgtccatgatcgccgccggcagtccatccatcttccagaacggcaagggctgcggcgcctgctatcaggtgaaatgcaccgggcacgcgtcgtgctccggcagcccggtgaccgtggtcctcaccgacgagtgccccggcggcgcgtgcctggacgagcccgtgcacttcgacctgagcggcacggcgttcggagccatggcgaaggacggccaggcggaccagctccgcggcgccggacacctcaagatccagtacactcgggtgccgtgcaactggcgagggctggacatcgcgttcaaggtggacgccggctcgaacccgaactacttcgcggtgctcatcgagtacgagtccggggacggcgagctggagtccgtggagctcatgcagcgcggcggcggcggcgggggggcgtgggcgccgatgcagcagtcgtggggcgcggtgtggcggtacaactccggcgacaccctgcaggcgcccttctccgtccgcctcacctccggctccggcgggacggtcgtcgccagcaacgtcatccccgcggggtggacgcccggcgccacgtaccgctcggtcgtcaacttcgacaactaacgaggacggacggacggaccgaaataacggcacgcccgcccgcccgcccgccggccggccgccgggatggccgtgaaaggcgtggcgctatagcttttagtttatga

[0135] Sequence of the 27-kD γ-gliadin promoter (SEQ ID NO:5):

[0136] ttataatcaacccgcactcttataatctcttctctactactataataagagagtttatgtacaaaata aggtgaaattatgtataagtgttctggatattggttgttggctccatattcacacaacctaatcaatagaaaacat atgttttattaaaacaaaatttatcatatatcatatatatatatataaaccgtagcaatgcacgggcatataacta gtgcaacttaatacatgtgtgtattaagatgaataagagggtatccaaataaaaaacttgttcgcttacgtctgga tcgaaaggggttggaaacgattaaatctcttcctagtcaaaattgaatagaaggagatttaatctctcccaatccc cttcgatcatccaggtgcaaccgtataagtcctaaagtggtgaggaacacgaaacaaccatgcattggcatgtaaa gctccaagaatttgttgtatccttaacaactcacagaacatcaaccaaaattgcacgtcaagggtattgggtaagaaacaatcaaacaaatcctctctgtgtgcaaagaaacacggtgagtcatgccgagatcatactcatctgatatacatgc ttacagctcacaagacattacaaacaactcatattgcattacaaagatcgtttcatgaaaaataaaataggccggacaggacaaaaatccttgacgtgtaaagtaaatttacaacaaaaaaaaagccatatgtcaagctaaatctaattc gttttacgtagatcaacaacctgtagaaggcaacaaaactgagccacgcagaagtacagaatgattccagatgaaccatcgacgtgctacgtaaagagagtgacgagtcatatacatttggcaagaaaccatgaagctgcctacagccg tctcggtggcataagaacacaagaaattgtgttaattaatcaaagctataaataacgctcgcatgcctgtgcacttctccatcaccaccactgggtcttcagaccattagctttatctactccagagcgcagaagaacccgatcgacacc

[0137] Example 1: Dynamic changes in cell proliferation and cell expansion during maize kernel endosperm development.

[0138] The size of maize kernels is controlled by two important factors: cell number and cell size. A maize kernel consists of the endosperm, embryo, and seed coat, with the endosperm accounting for 90% of the total seed weight and playing a decisive role in kernel size. To investigate the dynamic changes in cell number and cell size during maize kernel growth and development, the inventors observed freehand sections, fixed-embedded sections, and semi-thin sections of W64A kernels from 0 to 30 days post-pollination (DAP); simultaneously, they statistically analyzed the dynamic changes in endosperm thickness, cell size, and number.

[0139] Statistical observations of freehand sections show that ( Figure 1(AB) Immediately after pollination, the endosperm thickness was only 0.13±0.009 mm, but reached 2.56±0.13 mm 10 days after pollination. During this period, the endosperm cell count showed that the endosperm grew from one cell to 47.6±3.7 cells; this rapid cell proliferation corresponds to the rapid mitosis of endosperm cells 4 to 8 days after pollination. Subsequently, the cells entered a plateau phase of division, followed by the synthesis and storage of large amounts of storage substances such as starch and protein. 30 days after pollination, the endosperm thickness reached 4.11±0.28 mm, an increase of 61% compared to the endosperm 10 days after pollination; however, at the same time, the number of endosperm cells reached 54.4±1.6, an increase of only 14% compared to the endosperm 10 days after pollination. This result reveals that during the endosperm filling stage, the key factor determining the size of the endosperm or grain is cell size, that is, the cell expansion process.

[0140] Simultaneously, the average size of each cell (endosperm thickness / cell number) was calculated, revealing a high correlation between the width of a single cell and the width of the endosperm, with similar patterns. In the early stages (0-10 days post-pollination), cell width and cell number growth patterns were similar, while in the later stages of endosperm development (10-30 days post-pollination), no correlation was found. Figure 2 A).

[0141] To investigate changes in maize endosperm cell expansion, the size of individual endosperm cells was statistically analyzed at 14 and 24 days post-pollination. Figure 2 B). The endosperm was symmetrically longitudinally sectioned along the center of the embryo, and the thickness and individual cell size of the thickest part of the endosperm in the upper half were counted. The results showed that the thickest part of the maize endosperm at 14 days had 51 cell layers, while at 24 days it had 55 cell layers. Based on the degree of endosperm cell expansion, the inventors divided them into three categories: (1) under-expanded cells (UEC, containing aleurone layer and sub-aleurone layer, cell expansion has not yet started during cell division; (2) medium-expanded cells (MEC), cells are in a rapid expansion period; (3) completely expanded cells (CEC), cell expansion has been completed. In the statistical process, it was also found that the path of cell expansion is closely related to the path of endosperm filling. These two processes start from the central endosperm (CEC) and extend to the peripheral area (MEC and UEC); as the grain filling process proceeds, the size of UEC cells and MEC cells in 24DAP endosperm is significantly higher than that in 14DAP endosperm; however, possibly due to the dehydration effect or accumulation of stored substances in the later stage of grain development leading to an increase in cell turgor pressure, the CEC cells in 24DAP endosperm are significantly smaller than those in 14DAP endosperm cells. Figure 2 B).

[0142] The above results indicate that endosperm expansion after 10DAP is mainly determined by cell size, and the endosperm filling process is coupled with the cell expansion process.

[0143] Example 2: Discovery of transcription factors regulating cell expansion during the maize endosperm filling stage

[0144] Since the endosperm filling process is coupled with the post-expansion of endosperm cells, the inventors aimed to use endosperm storage substance synthesis genes as guide genes and employ co-expression analysis to identify transcription factors regulating post-expansion of endosperm cells. Thirty-two endosperm storage substance synthesis genes highly expressed were selected as guide genes, mainly including starch synthase genes and prolamins. Simultaneously, 134 transcription factors highly expressed during the endosperm filling period were screened using public databases. Point-to-point co-expression pattern analysis was performed on these 134 transcription factors and the 32 guide genes, and the Pearson Correlation Coefficient (PCC) was calculated.

[0145] Transcription factors with a PCC greater than 0.6 and associated with more than 10 guide genes were selected as candidate genes. Figure 3 A) 34 were screened. Through in-depth research and analysis, a GRAS family transcription factor, ZmGRAS11 (GRMZM2G023872, also known as Zm00001d009646), was identified. This factor is associated with 12 guide genes and may be involved in the process of endosperm cell expansion and the synthesis of storage substances. Figure 3 B).

[0146] GRAS transcription factors are plant-specific transcription factors, named by the Arabidopsis GAI (gibberellin-insensitive), RGA (repressor of ga1-3), and SCARECROW (SCR) genes, all of which have a variable amino terminus and a highly conserved carboxyl terminus containing five recognizable motifs. Comparison of the ZmGRAS11 amino acid sequence with sequences of the GRAS family revealed that ZmGRAS11 belongs to the DELLA subfamily of GRAS transcription factors. Unlike other DELLA proteins, ZmGRAS11 lacks the DELLA domain characteristic of DELLA proteins, containing only a conserved carboxyl terminus; this situation is also observed in the rice SLRL1 and SLRL2 genes. Previous reports have indicated that the DELLA domain plays a crucial role in the corresponding GA signaling of DELLA proteins. The presence of DELLA proteins lacking the DELLA domain, unique to gramineous crops, suggests that this subfamily of genes has a different mode of function than traditional DELLA genes, representing an atypical class of DELLA proteins. Figure 4 ).

[0147] To investigate the subcellular localization of ZmGRAS11, enhanced green fluorescent protein (eGFP) was ligated to the N-terminus of the ZmGRAS11 protein, and the fusion protein was transformed into tobacco (N. benthamiana) leaves. Under laser confocal microscopy, ZmGRAS11 protein was observed to be expressed in the cell nucleus (…). Figure 5 Meanwhile, the yeast GAL4 system was used to investigate whether ZmGRAS11 exhibits self-activation. It was found that ZmGRAS11 did not exhibit self-activation in either the full-length protein or proteins with truncated N-termini or C-termini removed. Figure 6 AB).

[0148] Example 3: ZmGRAS11 is a member of the DELLA family that is specifically highly expressed in maize endosperm.

[0149] According to the fourth edition of the maize genome annotation, seven DELLA genes were identified in the maize genome, three of which are expressed in the endosperm: DWARF8 (D8, ZmGRAS54, Zm00001d033680), DWARF9 (D9, ZmGRAS12, Zm00001d013465), and ZmGRAS11 (Zm00001d009646). ZmGRAS11 is highly expressed during the endosperm filling stage, while the classic DELLA genes D8 and D9 are expressed in the early stages of endosperm development. Figure 7 A).

[0150] The results of relative quantitative PCR on three genes expressed in the endosperm were similar to those of RNA-seq. Figure 7 B).

[0151] To further investigate the spatial expression pattern of ZmGRAS11, in situ hybridization was performed on the endosperm 12 days after pollination. The results of the in situ hybridization showed that ZmGRAS11 was mainly expressed at the outer edge of the endosperm, particularly concentrated at the elongated tip of the endosperm. Figure 8 AB).

[0152] Example 4: The zmgras11 deletion mutant produces smaller seeds with less weight.

[0153] To investigate the function of ZmGRAS11, gene-editing mutants were created in the maize KN5585 genetic background using CRISPR-Cas9 technology. The protein structure of ZmGRAS11 was analyzed, and the LHRI domain was selected as the target site for the sgRNA. Eight independent editing events were obtained, and the editing types were detected. Finally, two single plants with cytosine deletion (-C) at 353 bp and adenine insertion (+A) at 352 bp were selected for further research. Figure 9 Both editing events (A) lead to frameshifting of ZmGRAS11, resulting in premature termination. RNA was extracted and reverse transcribed from wild-type and the two prematurely terminated materials, and then analyzed by relative quantitative PCR to investigate the expression level of ZmGRAS11 in different materials. The results showed that the mRNA level of the ZmGRAS11 gene was significantly reduced in both prematurely terminated materials. Figure 9 B). Immunoblotting analysis of the three materials revealed that ZmGRAS11 protein was undetectable in the two materials that were terminated prematurely. Figure 9 C) indicates that both gene editing events resulted in the ZmGRAS11 gene deletion mutant: zmgras11.

[0154] To investigate the phenotype of zmgras11, wild-type and two mutant types of materials were planted at the Sanya Cotton Research Institute's Damao Base and the Shanghai Songjiang Farm in 2019 and 2020, respectively. After pollination and harvest, ears with normal seed setting rates from the three genotypes were selected for seed testing. Observation of the materials from Shanghai Songjiang revealed that although the mature ears did not show obvious phenotypes, after threshing and photographing the kernels arranged, it could be seen that the kernel width and thickness of zmgras11 were reduced, with a greater reduction in kernel thickness. Figure 10 A); After a detailed analysis of the threshed grains, using the ear of grain as the unit, it can be clearly seen that, compared with the wild type, the grain weight of both mutants shows a slight decreasing trend. Figure 10 B), the statistical results also show that this reduction in particle weight is significant. Figure 10 C), grain length, grain width, and grain thickness were significantly reduced. Figure 10 DF). The materials in Sanya also underwent the same changes ( Figure 11 AF).

[0155] The protein and starch content of wild-type and mutant seeds were tested, and no significant difference was found in either protein or starch content. Figure 12 (AB) indicates that the ZmGRAS11 gene has no effect on the synthesis of storage substances such as starch and protein.

[0156] Cell ploidy of the endosperm 18 days after pollination was analyzed by flow cytometry, and no significant difference was found. Figure 13This indicates that the deletion of the ZmGRAS11 gene did not affect the intracellular replication pathway.

[0157] Example 5: The zmgras11 gene-edited mutant affected the grain width during the grain-filling stage.

[0158] To investigate what factors led to the reduced grain weight and width of zmgras11, the number and size of cells in wild-type and mutant cells at different developmental stages were observed and statistically analyzed.

[0159] After observation and measurement of freehand sections, it was found that the wild-type had smaller grains than the mutant in the early stage of endosperm development; however, after entering the endosperm filling stage, whether at 18 or 24 days after pollination, the wild-type had larger endosperm width and cell width than the mutant; but there was no significant difference in cell number between the wild-type and the mutant. Figure 14 AB).

[0160] After longitudinally sectioning the seeds 14 and 24 days post-pollination, measuring the width of 30 cells from the outer periphery of the endosperm inwards, it was found that the mutant had significantly more MEC cells than the wild type, and the mutant cells were much smaller than those of the wild type at the same cell layer. Figure 15 AB).

[0161] These results indicate that the reduced endosperm thickness in the zmgras11 mutant is due to decreased cell expansion during the sap filling stage.

[0162] Example 6: Overexpression of ZmGRAS11 can increase the cell size and grain weight of seeds.

[0163] To determine the function of ZmGRAS11 from multiple perspectives, the pTF102 binary expression vector was used to overexpress the ZmGRAS11 gene in maize (KN5585 background) using the endosperm-specific 27-kDγ-prolysin promoter as a potent promoter, and the overexpression material p27::ZmGRAS11 was prepared.

[0164] Using primers p27F3 ​​5'-atgcttacagctcacaagac-3' (SEQ ID NO:7) and 366R 5'-gagtaggcgaactttaggtaggg-3' (SEQ ID NO:8), a 1023bp band can be amplified. After sequencing, the PCR product contains a partial 27kD promoter sequence and a partial ZmGRAS11 cDNA sequence (SEQ ID NO:6).

[0165]

[0166] Nine overexpression events were identified, and two of them (OE-1 and OE-2) were selected as subjects for subsequent research. Figure 16 AB). RT-qPCR confirmed that the mRNA levels of ZmGRAS11 significantly increased in both overexpression events. Figure 16 C); and in terms of protein, immunoassay also demonstrated a significant increase in ZmGRAS11 protein levels in both overexpression events (C); Figure 16 D).

[0167] Statistical analysis of mature ears of grain revealed that the overexpression material significantly improved grain weight, grain width, and grain thickness. Figure 16 EH).

[0168] Observation of semi-thin sections revealed no significant difference in cell number between wild-type and overexpression materials 18 days after pollination. However, the overexpression material showed a significant increase in endosperm thickness and average cell width compared to the wild-type material. Figure 17 A). However, for individual cells, the number and size of cells to be expanded from the overexpressed material were not significantly different from those of the wild type. Figure 17 B), but in the overexpression material, MEC cells were much larger than wild-type cells (B). Figure 17 C).

[0169] As discussed earlier, for cells that have completed expansion, because they are compressed in the middle of the endosperm, CEC cells 24 days after pollination are smaller than those 14 days after pollination. Figure 2 For overexpression materials, OE-1 endosperm cells are more easily compressed compared to wild-type. Figure 17 ).

[0170] Example 7: ZmGRAS11 regulates cell expansion by modulating EXPANSIN B-15.

[0171] To identify the downstream regulatory network of the ZmGRAS11 gene, RNA-seq analysis was performed on wild-type and zmgras11-C endosperm 18 days after pollination. By comparing the gene expression levels of zmgras11-C and wild-type, using parameters of P < 0.05 and fold change > 2.0, 2318 differentially expressed genes (DEGs) were identified, of which 1010 genes were downregulated and 1308 genes were upregulated. GO (Gene Ontology) enrichment analysis of the downregulated differentially expressed genes revealed enrichment in the GA signaling pathway, cell wall synthesis, and immune response-related pathways. Figure 18A). DELLA protein, under GA induction, affects cell wall synthesis by regulating related genes and cell expansion factors, thereby influencing cell expansion. Further, combined with GO enrichment analysis, the inventors identified six potential downstream target genes from the differentially expressed genes. Figure 18 B); RT-qPCR validation of these six genes using mutants and overexpression materials revealed that only β-glucosidase (BG, Zm00001d015231) and EXPANSIN B-15 (EXPB15, Zm00001d017493) showed a consistent trend, i.e., downregulated in mutants and upregulated in overexpression materials. Figure 18 C).

[0172] The transcriptional activation of downstream genes BG and EXPB15 by ZmGRAS11 was detected using a dual-fluorescence reporter system. Using a gene gun, the EXPB15 promoter-driven LUC vector and the 35S-driven ZmGRAS11 vector were co-transformed into maize endosperm. Figure 19 A) After cultivation, the activation value of the ZmGRAS11-added vector was found to be approximately twice that of the unadded vector. Simultaneously, the inventors repeated the above experiment in Arabidopsis leaf protoplasts and found similar results. However, the LUC vector driven by the BG promoter was not activated by ZmGRAS11 in either maize endosperm or Arabidopsis leaf protoplasts. Figure 19 B).

[0173] EXPB is a cell wall extension protein that plays a role in cell expansion during cell growth. EXPB15 is highly expressed in maize endosperm, and its expression is significantly downregulated in the zmgras11 mutant during the grain-filling stage. Figure 20 The presence of this information suggests that ZmGRAS11 plays a crucial role in regulating endosperm cell size. Since ZmGRAS11 itself does not possess direct transcriptional activation capabilities, this implies that the ZmGRAS11 protein indirectly regulates EXPB15 through transcription factors that interact with it.

[0174] Example 8: Overexpression of ZmEXPB15 can expand the volume of endosperm cells.

[0175] To determine the function of ZmEXPB15 during the endosperm expansion stage, the pTF102 binary expression vector was used to overexpress the ZmEXPB15 gene in maize (KN5585 background) using the endosperm-specific 27-kDγ-prolysin as a potent promoter, and the overexpression material p27::ZmEXPB15 was prepared.

[0176] Genomic DNA was extracted from the transgenic biological material, and a 1169bp band was amplified using primers p27F3 ​​5'-atgcttacagctcacaagac-3' (SEQ ID NO:7) and W1023 5'-ccgtccgtcctcgttagttgtc-3' (SEQ ID NO:10). Sequencing of the PCR product revealed a partial 27kD promoter sequence and a partial ZmEXPB15 cDNA sequence (SEQ ID NO:9).

[0177] 5'atgcttacagctcacaagacattacaaacaactcatattgcattacaaagatcgtttcatgaaaaataaaataggccggacaggacaaaaatccttgacgtgtaaagtaaa tttacaacaaaaaaaaagccatatgtcaagctaaatctaattcgttttacgtagatcaacaacctgtagaaggcaacaaaactgagccacgcagaagtacagaatgattccaga tgaaccatcgacgtgctacgtaaagagagtgacgagtcatatacatttggcaagaaaccatgaagctgcctacagccgtctcggtggcataagaacacaagaaattgtgttaat taatcaaagctataaataacgctcgcatgcctgtgcacttctccatcaccaccactgggtcttcagaccattagctttatctactccagagcgcagaagaacccgatcgacacc ATGGGATC-3'

[0178] Seventeen T0 overexpression events were obtained. By detecting the Bar gene copy number, three single-copy insertion lines were selected and backcrossed into the wild type KN5585. Backcross ears were sampled 24 days after pollination, and single kernels were identified for genotyping and fixed with FAA. Kernels with WT and OE genotypes were semi-thin-embedded and sectioned, and the number and size of cells on the endosperm thickness were counted.

[0179] The results showed that there was no significant difference in cell number between wild-type and overexpression materials 24 days after pollination, but the overexpression material had significantly higher endosperm thickness and average cell width than the wild-type material. Figure 21AB). Regarding individual cell size, layers 2-7 of the overexpression material were significantly larger than wild-type cells, including UEC and some MEC cells (AB). Figure 21 C). The above data demonstrate that ZmEXPB15 is indeed involved in the post-cell expansion pathway of maize endosperm cells.

[0180] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences <120> A new gene that increases grain yield in grasses and its application <130> 216349 <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1527 <212> DNA <213> Corn (Zea mays L.) <400> 1 atggatgctc acaccactca cattgttgca agaaagaaca cttcgctttc attcacctca 60 aatgagctca tggatcattt tcccttcgct tggttaccca tggacccctt cacagccatg 120 gcgctcagcg gtaccctctc accctcattc tctaccacat ctaccaatga taccatgtac 180 aaccttaacc cagcgttgta tgcatccctc gtgccaccaa taaggaaacc tgcaaccccg 240 accacactag aggtagggag gcaatgcaag gaggaggagg atgcatccat tcgtctggtg 300 cacctgctta tcacttgcac cagtgccatt gagactggtg actactcgat cgcacaagga 360 aacttgtctg aggcacgcaa gattcttggg gaaatcccaa cttcgaccgg gattggccgt 420 gttgggaaac acttcatcga cgcactagtt caacgcctct tcccggcata tccgcacgca 480 gcaccacctt ctccatctcc ttctacatcg atcgatctgc acaacaattt ttatgatgca 540 ggtccctacc taaagttcgc ctactccact gccaaccaag caatcctcaa agcaatcaaa 600 ggatacaacc atgtacacat aattgacttt tccctgatgc aaggtctcca gtggccggca 660 ctcatggatg tcttctccgc ccgtgagggt gggccaccaa agctccgaat cacaggcatt 720 ggcccgaacc caataggtgg ccgtgacgag ctccatgaag tgggaattcg cctcgccaag 780 tatgcacact cggtgggtat cgacttcact ttccagggag tctgtgtcga tcagcttgat 840 aggttgtgcg attggatgct tctcaaacca atcaaaggag aggcagttgc cataaactcc 900 atcctacagc tccatcgcct cctcgttgac ccagatgcaa acccagtggt gcccgcacca 960 atagatatcc tcctcaaatt ggtcatcaag ataaacccca tgatcttcac ggtggttgag 1020 catgaggcag atcacaacag accaccacta ctagagaggt tcactaatgc cctcttccac 1080 tatgcgacca tgtttgactc tttggaggcc atgcatcgtt gtaccagtgg tagagacatc 1140 accgactcac tcacagaggt gtaccttcga ggtgagattt ttgacattgt ctgcggcgag 1200 ggcagtgcac gcaccgaacg tcatgagttg tttggtcact ggagggagag gctcacctat 1260 gctgggctaa ctcaagtgtg gttcgacccc gatgaggttg acacgctaaa agaccagttg 1320 atccatgtga catccttatc tggctctggg ttcaacatcc tagtgtgtga tggcagcctt 1380 gcactagcgt ggcataatcg cccgttatat gtggcaacag cttggtgtgt gacaggagga 1440 aatgctgcca gttccatggt tggcaacatc tgtaagggta caaatgatag tagaagaaag 1500 gaaaaccgta atggacccat ggagtag 1527 <210> 2 <211> 508 <212> PRT <213> Zea mays L. <400> 2 Met Asp Ala His Thr Thr His Ile Val Ala Arg Lys Asn Thr Ser Leu 1 5 10 15 Ser Phe Thr Ser Asn Glu Leu Met Asp His Phe Pro Phe Ala Trp Leu 20 25 30 Pro Met Asp Pro Phe Thr Ala Met Ala Leu Ser Gly Thr Leu Ser Pro 35 40 45 Ser Phe Ser Thr Thr Ser Thr Asn Asp Thr Met Tyr Asn Leu Asn Pro 50 55 60 Ala Leu Tyr Ala Ser Leu Val Pro Pro Ile Arg Lys Pro Ala Thr Pro 65 70 75 80 Thr Thr Leu Glu Val Gly Arg Gln Cys Lys Glu Glu Glu Asp Ala Ser 85 90 95 Ile Arg Leu Val His Leu Leu Ile Thr Cys Thr Ser Ala Ile Glu Thr 100 105 110 Gly Asp Tyr Ser Ile Ala Gln Gly Asn Leu Ser Glu Ala Arg Lys Ile 115 120 125 Leu Gly Glu Ile Pro Thr Ser Thr Gly Ile Gly Arg Val Gly Lys His 130 135 140 Phe Ile Asp Ala Leu Val Gln Arg Leu Phe Pro Ala Tyr Pro His Ala 145 150 155 160 Ala Pro Pro Ser Pro Ser Pro Ser Thr Ser Ile Asp Leu His Asn Asn 165 170 175 Phe Tyr Asp Ala Gly Pro Tyr Leu Lys Phe Ala Tyr Ser Thr Ala Asn 180 185 190 Gln Ala Ile Leu Lys Ala Ile Lys Gly Tyr Asn His Val His Ile Ile 195 200 205 Asp Phe Ser Leu Met Gln Gly Leu Gln Trp Pro Ala Leu Met Asp Val 210 215 220 Phe Ser Ala Arg Glu Gly Gly Pro Pro Lys Leu Arg Ile Thr Gly Ile 225 230 235 240 Gly Pro Asn Pro Ile Gly Gly Arg Asp Glu Leu His Glu Val Gly Ile 245 250 255 Arg Leu Ala Lys Tyr Ala His Ser Val Gly Ile Asp Phe Thr Phe Gln 260 265 270 Gly Val Cys Val Asp Gln Leu Asp Arg Leu Cys Asp Trp Met Leu Leu 275 280 285 Lys Pro Ile Lys Gly Glu Ala Val Ala Ile Asn Ser Ile Leu Gln Leu 290 295 300 His Arg Leu Leu Val Asp Pro Asp Ala Asn Pro Val Val Pro Ala Pro 305 310 315 320 Ile Asp Ile Leu Leu Lys Leu Val Ile Lys Ile Asn Pro Met Ile Phe 325 330 335 Thr Val Val Glu His Glu Ala Asp His Asn Arg Pro Pro Leu Leu Glu 340 345 350 Arg Phe Thr Asn Ala Leu Phe His Tyr Ala Thr Met Phe Asp Ser Leu 355 360 365 Glu Ala Met His Arg Cys Thr Ser Gly Arg Asp Ile Thr Asp Ser Leu 370 375 380 Thr Glu Val Tyr Leu Arg Gly Glu Ile Phe Asp Ile Val Cys Gly Glu 385 390 395 400 Gly Ser Ala Arg Thr Glu Arg His Glu Leu Phe Gly His Trp Arg Glu 405 410 415 Arg Leu Thr Tyr Ala Gly Leu Thr Gln Val Trp Phe Asp Pro Asp Glu 420 425 430 Val Asp Thr Leu Lys Asp Gln Leu Ile His Val Thr Ser Leu Ser Gly 435 440 445 Ser Gly Phe Asn Ile Leu Val Cys Asp Gly Ser Leu Ala Leu Ala Trp 450 455 460 His Asn Arg Pro Leu Tyr Val Ala Thr Ala Trp Cys Val Thr Gly Gly 465 470 475 480 Asn Ala Ala Ser Ser Met Val Gly Asn Ile Cys Lys Gly Thr Asn Asp 485 490 495 Ser Arg Arg Lys Glu Asn Arg Asn Gly Pro Met Glu 500 505 <210> 3 <211> 801 <212> DNA <213> Maize (Zea mays L.) <400> 3 atgccttccg gggaccgggt atacgtgtgt acgtcggttt ttccgtgcag gtggtgcatg 60 cgggtaccag aacgccgtcg accagccgcc gttctcgtcc atgatcgccg ccggcagtcc 120 atccatcttc cagaacggca agggctgcgg cgcctgctat caggtgaaat gcaccgggca 180 cgcgtcgtgc tccggcagcc cggtgaccgt ggtcctcacc gacgagtgcc ccggcggcgc 240 gtgcctggac gagcccgtgc acttcgacct gagcggcacg gcgttcggag ccatggcgaa 300 ggacggccag gcggaccagc tccgcggcgc cggacacctc aagatccagt acactcgggt 360 gccgtgcaac tggcgagggc tggacatcgc gttcaaggtg gacgccggct cgaacccgaa 420 ctacttcgcg gtgctcatcg agtacgagtc cggggacggc gagctggagt ccgtggagct 480 catgcagcgc ggcggcggcg gcgggggggc gtgggcgccg atgcagcagt cgtggggcgc 540 ggtgtggcgg tacaactccg gcgacaccct gcaggcgccc ttctccgtcc gcctcacctc 600 cggctccggc gggacggtcg tcgccagcaa cgtcatcccc gcggggtgga cgcccggcgc 660 cacgtaccgc tcggtcgtca acttcgacaa ctaacgagga cggacggacg gaccgaaata 720 acggcacgcc cgcccgcccg cccgccggcc ggccgccggg atggccgtga aaggcgtggc 780 gctatagctt ttagtttatg a 801 <210> 4 <211> 266 <212> PRT <213> Zea mays L. <400> 4 Met Pro Ser Gly Asp Arg Val Tyr Val Cys Thr Ser Val Phe Pro Cys 1 5 10 15 Arg Trp Cys Met Arg Val Pro Glu Arg Arg Arg Pro Ala Ala Val Leu 20 25 30 Val His Asp Arg Arg Arg Gln Ser Ile His Leu Pro Glu Arg Gln Gly 35 40 45 Leu Arg Arg Leu Leu Ser Gly Glu Met His Arg Ala Arg Val Val Leu 50 55 60 Arg Gln Pro Gly Asp Arg Gly Pro His Arg Arg Val Pro Arg Arg Arg 65 70 75 80 Val Pro Gly Arg Ala Arg Ala Leu Arg Pro Glu Arg His Gly Val Arg 85 90 95 Ser His Gly Glu Gly Arg Pro Gly Gly Pro Ala Pro Arg Arg Arg Thr 100 105 110 Pro Gln Asp Pro Val His Ser Gly Ala Val Gln Leu Ala Arg Ala Gly 115 120 125 His Arg Val Gln Gly Gly Arg Arg Leu Glu Pro Glu Leu Leu Arg Gly 130 135 140 Ala His Arg Val Arg Val Arg Gly Arg Arg Ala Gly Val Arg Gly Ala 145 150 155 160 His Ala Ala Arg Arg Arg Arg Arg Gly Gly Val Gly Ala Asp Ala Ala 165 170 175 Val Val Gly Arg Gly Val Ala Val Gln Leu Arg Arg His Pro Ala Gly 180 185 190 Ala Leu Leu Arg Pro Pro His Leu Arg Leu Arg Arg Asp Gly Arg Arg 195 200 205 Gln Gln Arg His Pro Arg Gly Val Asp Ala Arg Arg His Val Pro Leu 210 215 220 Gly Arg Gln Leu Arg Gln Leu Thr Arg Thr Asp Gly Arg Thr Glu Ile 225 230 235 240 Thr Ala Arg Pro Pro Ala Arg Pro Pro Ala Gly Arg Arg Asp Gly Arg 245 250 255 Glu Arg Arg Gly Ala Ile Ala Phe Ser Leu 260 265 <210> 5 <211> 1051 <212> DNA <213> Zea mays L. <400> 5 ttataatcaa cccgcactct tataatctct tctctactac tataataaga gagtttatgt 60 acaaaataag gtgaaattat gtataagtgt tctggatatt ggttgttggc tccatattca 120 cacaacctaa tcaatagaaa acatatgttt tattaaaaca aaatttatca tatatcatat 180 atatatatat aaaccgtagc aatgcacggg catataacta gtgcaactta atacatgtgt 240 gtattaagat gaataagagg gtatccaaat aaaaaacttg ttcgcttacg tctggatcga 300 aaggggttgg aaacgattaa atctcttcct agtcaaaatt gaatagaagg agatttaatc 360 tctcccaatc cccttcgatc atccaggtgc aaccgtataa gtcctaaagt ggtgaggaac 420 acgaaacaac catgcattgg catgtaaagc tccaagaatt tgttgtatcc ttaacaactc 480 acagaacatc aaccaaaatt gcacgtcaag ggtattgggt aagaaacaat caaacaaatc 540 ctctctgtgt gcaaagaaac acggtgagtc atgccgagat catactcatc tgatatacat 600 gcttacagct cacaagacat tacaaacaac tcatattgca ttacaaagat cgtttcatga 660 aaaataaaat aggccggaca ggacaaaaat ccttgacgtg taaagtaaat ttacaacaaa 720 aaaaaagcca tatgtcaagc taaatctaat tcgttttacg tagatcaaca acctgtagaa 780 ggcaacaaaa ctgagccacg cagaagtaca gaatgattcc agatgaacca tcgacgtgct 840 acgtaaagag agtgacgagt catatacatt tggcaagaaa ccatgaagct gcctacagcc 900 gtctcggtgg cataagaaca caagaaattg tgttaattaa tcaaagctat aaataacgct 960 cgcatgcctg tgcacttctc catcaccacc actgggtctt cagaccatta gctttatcta 1020 ctccagagcg cagaagaacc cgatcgacac c 1051 <210> 6 <211> 1023 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(1023) <223> Fusion sequence of promoter and cDNA <400> 6 atgcttacag ctcacaagac attacaaaca actcatattg cattacaaag atcgtttcat 60 gaaaaataaa ataggccgga caggacaaaa atccttgacg tgtaaagtaa atttacaaca 120 aaaaaaaagc catatgtcaa gctaaatcta attcgtttta cgtagatcaa caacctgtag 180 aaggcaacaa aactgagcca cgcagaagta cagaatgatt ccagatgaac catcgacgtg 240 ctacgtaaag agagtgacga gtcatataca tttggcaaga aaccatgaag ctgcctacag 300 ccgtctcggt ggcataagaa cacaagaaat tgtgttaatt aatcaaagct ataaataacg 360 ctcgcatgcc tgtgcacttc tccatcacca ccactgggtc ttcagaccat tagctttatc 420 tactccagag cgcagaagaa cccgatcgac accatggatg gatgctcaca ccactcacat 480 tgttgcaaga aagaacactt cgctttcatt cacctcaaat gagctcatgg atcattttcc 540 cttcgcttgg ttacccatgg accccttcac agccatggcg ctcagcggta ccctctcacc 600 ctcattctct accacatcta ccaatgatac catgtacaac cttaacccag cgttgtatgc 660 atccctcgtg ccaccaataa ggaaacctgc aaccccgacc acactagagg tagggaggca 720 atgcaaggag gaggaggatg catccattcg tctggtgcac ctgcttatca cttgcaccag 780 tgccattgag actggtgact actcgatcgc acaaggaaac ttgtctgagg cacgcaagat 840 tcttggggaa atcccaactt cgaccgggat tggccgtgtt gggaaacact tcatcgacgc 900 actagttcaa cgcctcttcc cggcatatcc gcacgcagca ccaccttctc catctccttc 960 tacatcgatc gatctgcaca acaattttta tgatgcaggt ccctacctaa agttcgccta 1020 ctc 1023 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(20) <223> Primer <400> 7 atgcttacag ctcacaagac 20 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(23) <223> Primer <400> 8 gagtaggcga actttaggta ggg 23 <210> 9 <211> 1169 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(1169) <223> Fusion sequence of promoter and cDNA <400> 9 atgcttacag ctcacaagac attacaaaca actcatattg cattacaaag atcgtttcat 60 gaaaaataaa ataggccgga caggacaaaa atccttgacg tgtaaagtaa atttacaaca 120 aaaaaaaagc catatgtcaa gctaaatcta attcgtttta cgtagatcaa caacctgtag 180 aaggcaacaa aactgagcca cgcagaagta cagaatgatt ccagatgaac catcgacgtg 240 ctacgtaaag agagtgacga gtcatataca tttggcaaga aaccatgaag ctgcctacag 300 ccgtctcggt ggcataagaa cacaagaaat tgtgttaatt aatcaaagct ataaataacg 360 ctcgcatgcc tgtgcacttc tccatcacca ccactgggtc ttcagaccat tagctttatc 420 tactccagag cgcagaagaa cccgatcgac accatgggat ccatgccttc cggggaccgg 480 gtatacgtgt gtacgtcggt ttttccgtgc aggtggtgca tgcgggtacc agaacgccgt 540 cgaccagccg ccgttctcgt ccatgatcgc cgccggcagt ccatccatct tccagaacgg 600 caagggctgc ggcgcctgct atcaggtgaa atgcaccggg cacgcgtcgt gctccggcag 660 cccggtgacc gtggtcctca ccgacgagtg ccccggcggc gcgtgcctgg acgagcccgt 720 gcacttcgac ctgagcggca cggcgttcgg agccatggcg aaggacggcc aggcggacca 780 gctccgcggc gccggacacc tcaagatcca gtacactcgg gtgccgtgca actggcgagg 840 gctggacatc gcgttcaagg tggacgccgg ctcgaacccg aactacttcg cggtgctcat 900 cgagtacgag tccggggacg gcgagctgga gtccgtggag ctcatgcagc gcggcggcgg 960 cggcgggggg gcgtgggcgc cgatgcagca gtcgtggggc gcggtgtggc ggtacaactc 1020 cggcgacacc ctgcaggcgc ccttctccgt ccgcctcacc tccggctccg gcgggacggt 1080 cgtcgccagc aacgtcatcc ccgcggggtg gacgcccggc gccacgtacc gctcggtcgt 1140 caacttcgac aactaacgag gacggacgg 1169 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(22) <223> primer <400> 10 ccgtccgtcc tcgttagttg tc 22

Claims

1. A method for improving the yield and endosperm quality of grass plants, comprising upregulating GRAS11 in the plants; wherein, The improvement of the yield of grass plants includes: increasing grain weight, increasing grain width, and increasing grain thickness; the improvement of the endosperm quality of grass plants includes: increasing endosperm thickness and width, and promoting endosperm cell expansion and enlargement. The upregulation includes the expression of exogenous GRAS11 in plants driven by an endosperm-specific promoter, which is the 27-kD γ-prolyzin promoter; The amino acid sequence of the polypeptide of GRAS11 is shown in SEQ ID NO: 2; the grass plant is maize.

2. The method as described in claim 1, characterized in that, The method comprises: (1) using exogenous GRAS11-encoding nucleic acid primers on plant organs or tissues to obtain plant tissues or organs transformed with the polypeptide encoding nucleic acid; and (2) regenerating plant plants from the plant tissues or organs transformed with the exogenous polypeptide encoding nucleic acid of the present invention obtained in step (1).

3. The method as described in claim 2, characterized in that, The method includes: (s1) providing Agrobacterium carrying an expression vector containing a nucleic acid encoding GRAS11; (s2) contacting a plant tissue or organ with the Agrobacterium from step (s1) to transfer and integrate the nucleic acid encoding the polypeptide into the chromosome of a plant cell; (s3) selecting plant tissues or organs into which the nucleic acid encoding GRAS11 has been introduced; and (s4) regenerating the plant tissue or organ from step (s3) into a plant.

4. The method as described in claim 1, characterized in that, After upregulating the expression or activity of GRAS11 in plants, the process also includes: self-pollinating, hybridizing, or testcrossing the obtained plants to obtain progeny plants.

5. The method as described in claim 1, characterized in that, The GRAS11 regulates its downstream molecule EXPB15, the amino acid sequence of which is shown in SEQ ID NO: 4; EXPB15 increases the endosperm thickness and average cell width of grass endosperm cells, and expands the volume of endosperm cells.

6. A method for increasing the endosperm thickness and average cell width of endosperm cells in grass plants and expanding the volume of endosperm cells, comprising upregulating the expression or activity of EXPB15, a downstream molecule of GRAS11, in the plant; the amino acid sequence of the polypeptide of GRAS11 is shown in SEQ ID NO: 2; the amino acid sequence of the polypeptide of EXPB15 is shown in SEQ ID NO: 4; EXPB15 expands the volume of endosperm cells; the grass plant is maize.

7. An application of GRAS11 for: (a) increasing the yield of grasses and (b) improving the endosperm quality of grasses; (a) comprising: Increase kernel weight, increase kernel width, and increase kernel thickness; (b) includes: increasing the thickness and width of the endosperm, promoting the expansion and enlargement of endosperm cells; the amino acid sequence of the polypeptide of GRAS11 is shown in SEQ ID NO: 2; the grass is maize.

8. Uses of GRAS11 and its downstream molecule EXPB15 as molecular markers for identifying traits in grasses; The traits include: Yield traits and endosperm traits; wherein, the yield traits include: grain weight, grain width and grain thickness; the endosperm traits include: endosperm thickness and width, and endosperm cell expansion and enlargement; the amino acid sequence of the polypeptide of GRAS11 is shown in SEQ ID NO: 2; the amino acid sequence of the polypeptide of EXPB15 is shown in SEQ ID NO: 4; the grass plant is maize.

9. A method for targeted selection or identification of high-yielding gramineous plants with high endosperm quality, comprising: The expression or activity of GRAS11 and its downstream molecule EXPB15 in the test plant was identified. If the expression or activity of GRAS11 and its downstream molecule EXPB15 in the test plant was higher than the average expression or activity of GRAS11 and its downstream molecule EXPB15 in this type of plant, then it was a high-yielding gramineous plant with high endosperm quality. The high yield included: increased grain weight, increased grain width, and increased grain thickness. The high endosperm quality included: increased endosperm thickness and width, and promoted endosperm cell expansion and enlargement. The amino acid sequence of the GRAS11 polypeptide is shown in SEQ ID NO:

2. The amino acid sequence of the EXPB15 polypeptide is shown in SEQ ID NO:

4. The gramineous plant is maize.

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