CN114763375A

CN114763375A - Gene for regulating and controlling quality of rice grains and application thereof

Info

Publication number: CN114763375A
Application number: CN202011626461.1A
Authority: CN
Inventors: 韩斌; 陈二旺; 侯青青; 顾周琳; 刘坤; 戴冰馨
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2022-07-19
Anticipated expiration: 2040-12-31
Also published as: CN114763375B

Abstract

The invention relates to a gene for regulating and controlling rice grain quality and application thereof. The invention provides a method for regulating and controlling the agronomic traits and/or the grain quality of plants, which comprises regulating the expression of a GS10 gene in the plants.

Description

Gene for regulating and controlling quality of rice grains and application thereof

Technical Field

The invention belongs to the field of biotechnology and botany; more specifically, the invention relates to a regulatory gene of rice grain quality and application thereof.

Background

Rice is one of the most important food crops for human beings. It is estimated that by 2050, the world population will reach 90 billion. The growing food demand of the rapid population, the reduction of arable area, and the deterioration of farming environment pose serious challenges to human food safety. Therefore, in limited land resources, the cultivation of a new green rice variety with high safety, high yield and high efficiency is an important scientific problem faced by contemporary molecular breeders.

The yield of rice is regulated and controlled by various important complex agronomic traits, such as plant type (plant height, spike shape, leaf inclination angle and the like), ion nutrition absorption, disease resistance, insect resistance, environmental stress resistance and the like. At present, methods for cloning rice related genes mainly comprise QTL, mutant localization and whole Genome association analysis (GWAS). The grain yield of rice is mainly determined by four factors, namely the number of ears per plant or the effective tiller number, the number of ears, the grain weight and the proportion of full grains. In contrast to the most common dicotyledonous model plant Arabidopsis embryos, which occupy most of the space of the mature seed, in rice seeds the endosperm occupies most of the space of the mature seed. In a mature seed, a large fraction of storage compounds, mainly starch, relatively small amounts of storage proteins and lipids and traces of other substances are contained. The rice grain type character is one of important agronomic characters of rice and is an important factor forming the rice yield, so that the excavation and analysis of the rice complex character related genes have certain guiding significance for future rice breeding. In the past research, as more and more rice grain type QTLs are discovered, such as GW2, GS3, GS5 and the like, molecular control network diagrams among the QTLs are continuously perfected, so that more favorable conditions are provided for molecular marker breeding. The regulation mechanism of the granule type molecule mainly comprises ubiquitination mediated proteasome degradation, phytohormones, G-protein signal transduction and the like.

Tandem Repeat proteins such as Armadillo repeats associating protein (ARM protein), Tetratricopeptide Repeat (TPR protein) and WD40 protein are proteins that are commonly present in animals and plants and play important roles in various vital activities. Among them, the Armadillo (ARM) catenin family is a family that is widely found in prokaryotes and eukaryotes and consists of multiple tandem repeat proteins, each repeat consisting of approximately 42 amino acids. First, each repeat forms three α -helical building blocks, and gradually forms a conserved tertiary structure when multiple tandem repeats are formed.

In the past research on rice grain type related genes, more than 20 QTLs for controlling grain types are reported and the mechanism of the QTLs is analyzed one after another, but the regulation network of the grain type gene mechanism and the relation between the grain types and the quality are rarely known. Wild rice is subject to a great deal of spontaneous variation in order to adapt to a variable natural environment, and the gene polymorphism of the wild rice is often more abundant than that of cultivated rice on the genome of the wild rice. Meanwhile, only a few natural variations are fixed in the cultivated rice due to the influence of bottleneck effect in the acclimatization process from the wild rice to the cultivated rice. There are still a large number of variations in the wild rice population, which have great potential for improving the adaptation of cultivation to the natural environment. Few QTLs are known based on current development and utilization of wild rice populations, and the specific function of these environmentally selected mutation sites is largely unknown.

With the discovery of more and more molecular analysis on rice grain type characters, a certain balance mechanism exists between rice grain type and single plant yield. For example, in mutant studies, GSN1 was found to encode a protein that binds OsMKP1 to OsMPK6 and dephosphorylates it, thereby further participating in the cascade signaling of OsMKKK10-OsMKK4-OsMPK 6. There is a balance between GSN1 and GSN1 between grain number per ear and grain size, and the final yield per plant is not necessarily affected. The same balance mechanism can also reflect the balance between grain type, effective tillering, plant height and other agronomic traits, and the plant self has 'source-sink flow'. Although many grain types have been cloned at present, the actual application of the grain type greatly contributes little to the yield.

When the progeny of the hybrid rice population is analyzed for quality-related genes, the most closely related characteristics in the aspect of rice quality regulation are grain type characteristics, particularly aspect ratio, except for the known natural variation sites of wax and talk 5 which mainly regulate the rice quality. In addition, research and grain types affecting rice quality, particularly appearance quality, are the most compact, and currently cloned QTLs mainly comprise GL7/GW7, GLW7, GW8, GS9, GW5 and the like, and the genes show excellent appearance quality when the genes are slender grains, which is probably related to the development and filling rate of glumes. Therefore, to further address the future breeding goal of "high yield and high quality", we need to resort to newer methods and more grain type-related gene cloning and functional studies.

Disclosure of Invention

The invention aims to provide a rice quality related gene and application thereof.

In a first aspect the present invention provides the use of a substance selected from the group consisting of: a GS10 gene or a protein encoding it, or an enhancer or inhibitor thereof, for use in modulating an agronomic trait in a plant selected from one or more of: leaf length, leaf width, single plant yield, cell yield, plant height, tiller number, grain number per ear, ear length, branch number, top awn, heading stage, stem diameter, glume cell width, glume cell length, glume cell unit area, leaf inclination angle, grain length, grain width, grain weight, BR sensitivity, BR signal path, grain transparency, grain starch cell compactness, grain protein content and grain amylose content.

In one or more embodiments, the substance is the GS10 gene or its encoded protein, or its promoter, and the modulating an agronomic trait of a plant is selected from one or more of: reducing the leaf length, increasing the leaf width, reducing the yield of a single plant, reducing the yield of a cell, reducing the plant height, reducing the tillering number, reducing the grain number per spike, reducing the spike length, reducing the number of branches, reducing the awn, shortening the heading stage, increasing the diameter of stalks, increasing the width of glumes cells, reducing the length of glumes cells, increasing the unit area of glumes cells, reducing the inclination angle of leaves, reducing the grain length, increasing the grain width, increasing the grain thickness, increasing the grain weight, increasing the BR sensitivity, negatively regulating and controlling a BR signal path, reducing the transparency of grains, increasing the compactness of starch cells of the grains, reducing the protein content of the grains and reducing the amylose content of the grains.

In one or more embodiments, the substance is an inhibitor of the GS10 gene, and the agronomic trait of the regulatory plant is selected from one or more of: increasing leaf length, reducing leaf width, increasing single plant yield, increasing cell yield, increasing plant height, increasing tiller number, increasing grain number per spike, increasing spike length, increasing branch number, increasing top awn, prolonging heading period, reducing stem diameter, reducing glume cell width, increasing glume cell length, reducing glume cell unit area, increasing leaf inclination angle, increasing grain length, reducing grain width, reducing grain thickness, reducing grain weight, BR sensitivity, positively regulating BR signal path, increasing grain transparency, reducing grain starch cell compactness, increasing grain protein content, and increasing grain amylose content.

In one or more embodiments, the accelerator is selected from the group consisting of: a small molecule compound, a nucleic acid molecule, or a combination thereof. Preferably, the nucleic acid molecule is a nucleic acid construct comprising the coding sequence of GS 10.

In one or more embodiments, the inhibitor is an inhibitory molecule that specifically interferes with the transcription and/or expression of the GS10 gene.

In one or more embodiments, the suppressor molecule targets the GS10 gene or its transcript as a suppressor.

In one or more embodiments, the inhibitory molecule has

SEQ ID NO

1 or 2 as the inhibitory target.

In one or more embodiments, the inhibitory molecule is selected from the group consisting of: (1) a small molecule compound, an antisense nucleic acid, a microRNA, a siRNA, an RNAi, a dsRNA, a sgRNA, an antibody, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1). Preferably, the inhibitory molecule is a dsRNA or construct targeting SEQ ID NO 2 or its transcript for silencing.

In one or more embodiments, the inhibitor is an agent that causes a deletion mutation in an exon of the GS10 gene. Preferably, the inhibitor is an agent that deletes position 569-572 or 1209-1213 of the GS10 coding sequence (e.g., SEQ ID NO: 2).

In one or more embodiments, the inhibitor is an agent, such as an sgRNA, that knocks out the GS10 coding sequence (e.g., SEQ ID NO:2) at positions 569-572 or 1209-1213 using a technique selected from the group consisting of ZFN, TALEN, and CRISPR.

In one or more embodiments, the inhibitor is a sgRNA used to knock out the GS10 coding sequence (e.g., SEQ ID NO:2) at positions 569-572 or 1209-1213.

In one or more embodiments, the sgrnas are as set forth in

SEQ ID NOs

3 or 4.

In one or more embodiments, the inhibitor further comprises a Cas enzyme (e.g., Cas9), its coding sequence, and/or a nucleic acid construct expressing the Cas enzyme.

In one or more embodiments, the plant is a cereal crop.

In one or more embodiments, the plant is a graminaceous plant.

In one or more embodiments, the gramineae is rice, barley, wheat, oats, rye.

In one or more embodiments, the rice comprises indica, japonica, or a combination thereof.

In one or more embodiments, the rice is nipponbare, DJ.

In one or more embodiments, the number of branches includes a primary number of branches and/or a secondary number of branches.

In one or more embodiments, the leaves include xiphoid leaves.

In one or more embodiments, the GS10 gene includes a cDNA sequence, a genomic sequence, or a combination thereof.

In one or more embodiments, the GS10 gene is from a plant of the family poaceae, preferably from rice.

In one or more embodiments, the amino acid sequence of the GS10 gene is selected from the group consisting of:

(a) polypeptide with a sequence shown as SEQ ID NO. 1;

(b) a polypeptide which is formed by substituting, deleting or adding one or more (such as 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the sequence shown in SEQ ID NO. 1, has the functions of the polypeptide (a) and is derived from the polypeptide (a); or

(c) A polypeptide derived from (a) having more than 90% (preferably 93%; more preferably 95% or 98%) homology to the polypeptide sequence of (a) and having the function of the polypeptide of (a).

In one or more embodiments, the nucleic acid sequence of the GS10 gene is selected from the group consisting of:

(1) a polynucleotide encoding a polypeptide as shown in SEQ ID NO. 1;

(2) 2 or a polynucleotide having 80% (preferably 90%; more preferably 95% or 98%) or more homology thereto;

(3) a polynucleotide in which 1 to 60, preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or 3' end of the polynucleotide shown in SEQ ID NO. 2;

(4) a polynucleotide complementary to the polynucleotide of any one of (1) to (3).

In a second aspect, the present invention provides a method of modulating an agronomic trait in a plant, the method comprising: regulating the expression or activity of the GS10 gene in the plant, thereby regulating the agronomic traits of the plant. Preferably, the agronomic trait is selected from one or more of: leaf length, leaf width, single plant yield, cell yield, plant height, tillering number, grain number per spike, spike length, branch number, top awn, heading period, stem diameter, glume cell width, glume cell length, glume cell unit area, leaf inclination angle, grain length, grain width, grain weight, BR sensitivity, BR signal path, grain transparency, grain starch cell compactness, grain protein content and grain amylose content.

In one or more embodiments, the plant is a cereal crop.

In one or more embodiments, the plant is a graminaceous plant.

In one or more embodiments, the gramineae is rice, barley, wheat, oats, rye.

In one or more embodiments, the rice is nipponica.

In one or more embodiments, the amino acid sequence of the GS10 gene is selected from the group consisting of seq id no:

(a) polypeptide with a sequence shown as SEQ ID NO. 1;

(1) a polynucleotide encoding a polypeptide as shown in SEQ ID NO. 1;

(4) a polynucleotide complementary to any one of the polynucleotides described in (1) to (3).

In a preferred embodiment, the method of modulating an agronomic trait in a plant comprises: up-regulating the expression or activity of the GS10 gene in a plant; thereby reducing the leaf length, increasing the leaf width, reducing the yield of a single plant, reducing the yield of a cell, reducing the plant height, reducing the tillering number, reducing the grain number per spike, reducing the spike length, reducing the number of branches and stalks, reducing the awn, shortening the heading stage, increasing the diameter of the stems, increasing the width of glume cells, reducing the length of glume cells, increasing the unit area of glume cells, reducing the leaf inclination angle, reducing the grain length, increasing the grain width, increasing the grain thickness, increasing the grain weight, increasing the BR sensitivity, negatively regulating and controlling the BR signal path, reducing the transparency of grains, increasing the compactness of starch cells of the grains, reducing the protein content of the grains and reducing the amylose content of the grains.

In one or more embodiments, the upregulating expression of the GS10 gene in a plant comprises: the GS10 gene is transferred into plants to obtain transformed plants.

In one or more embodiments, expression of the GS10 gene is driven by the Ubi promoter.

In one or more embodiments, the method of upregulating expression of the GS10 gene in a plant comprises:

(1) providing an Agrobacterium harboring a nucleic acid construct comprising the GS10 gene,

(2) contacting a cell or tissue or organ of a plant with the Agrobacterium of step (1), thereby transferring the nucleic acid construct into the plant tissue or organ.

In one or more embodiments, the nucleic acid construct is an expression vector or a recombinant vector.

In one or more embodiments, the method of upregulating expression of the GS10 gene in a plant further comprises:

(3) selecting plant tissues, organs or seeds into which the GS10 gene is transferred; and

(4) regenerating the plant tissue, organ or seed of step (3) into a plant.

In another preferred embodiment, the method of modulating an agronomic trait in a plant comprises: down-regulating expression or activity of GS10 in a plant; thereby increasing the leaf length, reducing the leaf width, increasing the yield of a single plant, increasing the yield of a cell, increasing the plant height, increasing the tillering number, increasing the grain number per spike, increasing the spike length, increasing the number of branches and stalks, increasing the awn, prolonging the heading period, reducing the diameter of the stalks, reducing the width of glume cells, increasing the length of glume cells, reducing the unit area of glume cells, increasing the inclination angle of the leaves, increasing the grain length, reducing the grain width, reducing the grain thickness, reducing the grain weight, BR sensitivity, positively regulating a BR signal channel, increasing the transparency of grains, reducing the compactness of starch cells of the grains, increasing the protein content of the grains and increasing the amylose content of the grains.

In one or more embodiments, the downregulating expression or activity of a GS10 gene in a plant comprises: an inhibitor that down-regulates GS10 gene transcription, protein expression, or protein activity is transferred into a plant.

In one or more embodiments, the inhibitory molecule has

SEQ ID NO

1 or 2 as the inhibitory target.

In one or more embodiments, the inhibitor is an agent that causes a deletion mutation in an exon of the GS10 gene. Preferably, the inhibitor is an agent that deletes the GS10 coding sequence (e.g., SEQ ID NO:2) at positions 569-572 or 1209-1213.

In one or more embodiments, the inhibitor is an agent, e.g., an sgRNA, that knocks out the GS10 coding sequence (e.g., SEQ ID NO:2) at positions 569-572 or 1209-1213 using a technique selected from the group consisting of ZFNs, TALENs, and CRISPR.

In one or more embodiments, the inhibitor is a sgRNA for the knock-out of the GS10 coding sequence (e.g., SEQ ID NO:2) at positions 569-572 or 1209-1213.

In one or more embodiments, the sgRNA is as set forth in

SEQ ID NOs

3 or 4. In one or more embodiments, the inhibitor further comprises a Cas enzyme (e.g., Cas9), its coding sequence, and/or a nucleic acid construct expressing the Cas enzyme.

In one or more embodiments, the method of down-regulating expression of a GS10 gene in a plant comprises:

(i) providing an agrobacterium carrying a nucleic acid construct that can interfere with gene expression, said nucleic acid construct containing or producing said inhibitor;

(ii) (ii) contacting a cell or tissue or organ of the plant with the Agrobacterium of step (i) thereby transferring the nucleic acid construct into the plant tissue or organ.

In one or more embodiments, the method of down-regulating expression of a GS10 gene in a plant further comprises:

(iii) selecting a plant tissue, organ or seed into which the nucleic acid construct has been transferred; and

(iv) (iv) regenerating the plant tissue, organ or seed of step (iii) into a plant.

In another aspect of the invention, there is provided the use of the GS10 gene as a molecular marker for identifying agronomic traits in plants.

In one or more embodiments, the agronomic trait includes: leaf length, leaf width, single plant yield, cell yield, plant height, tillering number, grain number per spike, spike length, branch number, top awn, heading period, stem diameter, glume cell width, glume cell length, glume cell unit area, leaf inclination angle, grain length, grain width, grain weight, BR sensitivity, BR signal path, grain transparency, grain starch cell compactness, grain protein content and grain amylose content.

In one or more embodiments, the plant is a cereal crop.

In one or more embodiments, the plant is a graminaceous plant.

In one or more embodiments, the gramineae is rice, barley, wheat, oats, rye.

In one or more embodiments, the rice is nipponica.

In one or more embodiments, the leaves include xiphoid leaves.

In one or more embodiments, the GS10 gene comprises a cDNA sequence, a genomic sequence, or a combination thereof.

(a) polypeptide with a sequence shown as SEQ ID NO. 1;

(1) a polynucleotide encoding a polypeptide as shown in SEQ ID NO. 1;

(3) 2 at the 5 'end and/or 3' end of the polynucleotide shown in SEQ ID NO. 2, or 1-60, preferably 1-30, more preferably 1-10) nucleotides;

The invention also provides an expression cassette for expressing the GS10 gene, wherein the expression cassette comprises the following elements from 5 'to 3': a 5' UTR region, an ORF sequence of the GS10 gene, and a terminator,

in one or more embodiments, the ORF sequence of the GS10 gene encodes:

(a) polypeptide with a sequence shown as SEQ ID NO. 1;

In one or more embodiments, the ORF sequence of the GS10 gene comprises a nucleic acid sequence selected from the group consisting of:

(1) a polynucleotide encoding a polypeptide as shown in SEQ ID NO. 1;

The invention also provides nucleic acid constructs comprising an expression cassette as described herein or a complement thereof.

The invention also provides a host cell (1) comprising a nucleic acid construct comprising an expression cassette described herein, or a complement thereof, or (2) having an expression cassette described herein integrated into the chromosome.

In one or more embodiments, the host cell is a plant cell, preferably a graminaceous plant cell, more preferably a rice cell.

In another aspect of the invention, an inhibitor targeting the GS10 gene is provided.

In one or more embodiments, the inhibitor is selected from the group consisting of: (1) a small molecule compound, an antisense nucleic acid, a microRNA, a siRNA, an RNAi, a dsRNA, a sgRNA, a specific antibody, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1). The inhibitor specifically interferes with the transcription and/or expression of the GS10 gene. Preferably, the inhibitor is an inhibitor targeting

SEQ ID NO

1 or 2.

In one or more embodiments, the sgrnas are as set forth in

SEQ ID NOs

3 or 4.

The present invention also provides the use of an expression cassette as described herein for improving an agronomic trait in a crop selected from the group consisting of:

reducing the leaf length, increasing the leaf width, reducing the yield of a single plant, reducing the yield of a cell, reducing the plant height, reducing the tillering number, reducing the grain number per spike, reducing the spike length, reducing the number of branches, reducing the awn, shortening the heading stage, increasing the diameter of stalks, increasing the width of glumes cells, reducing the length of glumes cells, increasing the unit area of glumes cells, reducing the inclination angle of leaves, reducing the grain length, increasing the grain width, increasing the grain thickness, increasing the grain weight, increasing the BR sensitivity, negatively regulating and controlling a BR signal path, reducing the transparency of grains, increasing the compactness of starch cells of the grains, reducing the protein content of the grains and reducing the amylose content of the grains; or

Increasing leaf length, reducing leaf width, increasing single plant yield, increasing cell yield, increasing plant height, increasing tiller number, increasing grain number per spike, increasing spike length, increasing branch number, increasing top awn, prolonging heading period, reducing stem diameter, reducing glume cell width, increasing glume cell length, reducing glume cell unit area, increasing leaf inclination angle, increasing grain length, reducing grain width, reducing grain thickness, reducing grain weight, BR sensitivity, positively regulating BR signal path, increasing grain transparency, reducing grain starch cell compactness, increasing grain protein content, and increasing grain amylose content.

The invention also provides the use of the promoter element of the GS10 gene for the space-time specific expression of foreign proteins, wherein the space-time specific expression refers to the specific expression in the cytoplasm or cell membrane of cells at seedling and/or young ear stage.

The invention also provides the application of the substance for down-regulating the expression or activity of the GS10 gene and the substance for up-regulating the expression or activity of one or two genes selected from GW5 and GW7(GL7) in stabilizing the grain type, improving the appearance quality of rice and improving the aspect ratio of the rice.

In one or more embodiments, the agent that down-regulates expression or activity of the GS10 gene is an inhibitor of the GS10 gene. Preferably, the inhibitor of the GS10 gene is as described in the first aspect herein.

In one or more embodiments, the substance that upregulates the expression or activity of a gene selected from one or both of GW5 and GW7(GL7) is a gene selected from one or both of GW5 and GW7(GL7) or a protein encoded thereby, or a promoter therefor. The accelerator is selected from the following group: a small molecule compound, a nucleic acid molecule, or a combination thereof.

The invention also provides a complex formed by binding GS10 protein to a protein selected from SnRK1A, OsOFP8 and OSH 1.

In one or more embodiments, the GS10 protein is from rice.

In one or more embodiments, the amino acid sequence of the GS10 protein is selected from the group consisting of:

(a) a polypeptide with a sequence shown in SEQ ID NO. 1;

Drawings

FIG. 1 is a rice grain type QTL mapping chart. A, based on W1943 and GLA4 recombinant inbred line; b, mapping of rice grain type related genes.

FIG. 2 is a LOD plot of rice grain type QTL GS10 location. TGW, thousand kernel weight; GL, grain length; GW, grain width; GS, aspect ratio.

FIG. 3 is a fine mapping and phenotypic analysis of GS 10. A, GLA4 and CSSL 50. Scale, 5 cm. B, comparison of the particle types of GLA4 and CSSL 50. Scale, 1 cm. C, initial positioning of GS 10. D, fine positioning of GS 10. E, the gene structure of GS 10. F-I, GLA4 and NIL-gs10 were compared for differences in grain length (F), grain width (G), thousand kernel weight (H) and grain thickness (I). Values show mean ± standard deviation (n ═ 10), differential significance test was performed using Student's t-test (P <0.05, t test).

FIG. 4 is the identification and screening of the GS10 near isogenic line. Background characterization of A-B, recombinants 2017#44-6-6(A) and NIL-gs10 (B).

FIG. 5 is a comparison of other important agronomic traits for GLA4 and NIL-gs 10. A-J, GLA4 and NIL-gs10 for comparison of differences in ear length (A), first-order branch (B), second-order branch (C), grain per ear (D), plant height (E), sword leaf length (F), sword leaf width (G), effective tiller number (H), individual plant yield (I) and plot yield (J). Values show mean ± standard deviation (n ═ 10), with Student's t-test for differential significance (P <0.05, t test).

FIG. 6 is an analysis of natural variation at the level of GS10 DNA in GLA4 and W1943. Yellow part, CDS. Grey part, UTR.

FIG. 7 is a protein alignment of GS10 in GLA4 and W1943. A-B, comparison of the differences between the GS10 gene sequence (A) and the protein sequence (B).

FIG. 8 shows transgene validation of GS 10. A, GLA4, NIL-GS10, CP-1(pGLA4:: gGS10GLA4), GS10-CR, OX (Ubi:: GS10GLA4) and CP-2(pW1943:: gGS10GLA4) were compared in grain and ear type between different rice lines. Granular Scale bar,1 cm. Spike type Scale bar,5 cm. And B-E, GLA4, NIL-GS10, CP-1, GS10-CR, OX, CP-2 and the like, and the difference statistics of grain length (B), grain width (C), thousand kernel weight (D) and ear length (E) among different rice lines. CP, complementary line; OX, overlay line. The values show the mean ± standard deviation (n ═ 20), with different letters representing significant differences between the 0.05 levels (Duncan's multiple range test).

FIG. 9 shows a comparison of grain width differences between two different complementation lines CP-1 and CP-2. A-B, comparison of grain width differences between two different complementation lines CP-1(A) and CP-2 (B). 10 strains with phenotype were selected from each strain T0 generation and subjected to T1 generation statistics (n-12).

FIG. 10 shows the plant type and expression level analysis of transgenic plants. And comparing the differences of plant types among different rice strains, such as A, GLA4, NIL-gs10, CP-1, OX, CP-2 and the like. B, analyzing the expression quantity among different rice strains such as GLA4, NIL-gs10, CP-1, OX and CP-2.

FIG. 11 shows a comparison of the differences in leaf inclination and flag leaf between strains GLA4, NIL-GS10, GS10-CR and OX. A, GLA4, NIL-GS10, GS10-CR and OX leaf inclination map. Scale, 10 cm. B, GLA4, NIL-GS10, GS10-CR and OX xiphoid map. Scale, 5 cm. C-D, GLA4, NIL-GS10, GS10-CR and OX leaf inclination and flag leaf difference data statistics. The values show the mean ± standard deviation (n ═ 20), with different letters representing significant differences between the 0.05 levels (Duncan's multiple range test).

FIG. 12 is a knockout experiment in Nipponbare using CRISPR-Cas 9. A, NIP and CR strain patterns. Scale, 10 cm. Spike patterns of NIP and CR. Scale, 5 cm. C, particle pattern of NIP and CR. Scale, 1 cm. Statistics of particle length (D) and particle width (E) for D-E, NIP and CR. CR, CRISPR-Cas 9. The values show the mean ± standard deviation (n ═ 10) and the different letters represent significant differences between 0.05 levels (Duncan's multiple range test).

FIG. 13 shows a T-DNA mutant validation experiment for GS 10. Strain diagrams of the A, HY and T-DNA mutants of gs 10. HY, hwayuong. Scale, 10 cm. B, DNA structure diagram of T-DNA mutant gs 10. C, HY, gs10 and the complementary plant HY-CP-1. Scale, 1 cm. The expression level of GS10 in the context of D, HY and GS10 varied. Data statistics for grain length (E) and grain width (F) for E-F, HY, and gs 10. The values show the mean ± standard deviation (n ═ 10), with different letters representing significant differences between the 0.05 levels (Duncan's multiple range test).

FIG. 14 is an analysis of the expression level of GS10 in rice at each stage. PA, pre-anthesis spikelet; FL, xiphoid leaf; LS, leaf sheath; TB, tillering nodules.

FIG. 15 is the subcellular localization of GS 10. Subcellular localization of GS10 in tobacco and rice protoplasts. Rice protoplast ruler, 10 μm; tobacco cell ruler, 10 μm and 20 μm. 35S GFP was used as a control.

Fig. 16 is the co-location of GS10 and GW 5. The rice protoplast scale was 20 μm.

Figure 17 shows that GS10 regulates cell size rather than cell number in glumes. Semi-thin sections of the glumes in the non-flowering spikelets in a, GLA4 and NIL-gs 10. The scale bar is, in order, 0.5cm and 50 μm. B, GLA4 and NIL-gs10 glume cell outer epidermis electron scanning microscope observation. Scale, 1mm (top) and 100 μm (bottom). C, GLA4 and NIL-gs10 statistics on the number of glume cell lengths. D, statistics of cell numbers at the GLA4 and NIL-gs10 grain length levels. E, analyzing the difference of expression quantity of related cell cycle and division genes in GLA4 and NIL-gs10 young ear stage (3-5 cm). OsActin was used as an internal reference gene, and the expression level of GLA4 was set to 1. Values show mean ± standard deviation (n ═ 3), with Student's t-test for differential significance (P <0.05, t test).

FIG. 18 shows a comparison of the protein structures of GS10, arm and CTNNB 1.

FIG. 19 shows the genetic relationship of GS10 and its homologous gene, GSL 1. A, GS10 and its homologous genes. B, GS10 and the knockout strain type of the homologous gene GSL1 in the rice. Scale, 10 cm. C, single and double knockout grain type difference comparison of GS10 and GSL1 based on ZH11 background. Scale, 1 cm. D-E, statistics of grain length and grain width of single-knockout and double-knockout plants of GS10 and GSL1 based on the background of ZH 11. The values show the mean ± standard deviation (n ═ 10) and the different letters represent significant differences between 0.05 levels (Duncan's multiple range test).

FIG. 20 is a graph showing the measurement of the expression levels of the related differentially expressed genes in the young ears of GLA4 and OX. Differential expression detection of genes related to GS10 development regulation in A-J, GLA4 and OX scion. OsActin is used as an internal reference gene, and values are shown as mean ± standard deviation (n ═ 3).

FIG. 21 is a GS10 yeast Sieve library experiment. A, GS10 and BR related gene yeast interaction verification. B, GS10 was validated for yeast interaction with OsGSK2, SnRK1A, GS9 and OsOFP 8.

FIG. 22 is a genetic analysis of plant type and grain type between GS10 and OsGSK2 transgenic lines. A, plant type comparison of different transgenic plants GS10-CR-ZH11, GS10-OX-ZH11, GS10-OsGSK2-CR, OsGSK2-CR, OsGSK2-OX and OsGSK2-RNAi based on the background of ZH 11. Scale, 10 cm. B, comparing the grain types of different transgenic plants based on the ZH11 background. Scale, 1 cm. C-D, statistics of grain width (C) and grain length (D) among different transgenic plants based on ZH11 background. The values show the mean ± standard deviation (n ═ 10) and the different letters represent significant differences between 0.05 levels (Duncan's multiple range test).

FIG. 23 is a comparison of the difference in leaf inclination between transgenic lines GS10 and OsGSK 2. A, comparison of leaf inclination differences based on the background of ZH11 for GS10-CR-ZH11, GS10-OX-ZH11, GS10-OsGSK2-CR, OsGSK2-CR, OsGSK2-OX and OsGSK 2-RNAi. Scale, 10 cm. B, leaf inclination data statistics of GS10-CR-ZH11, GS10-OX-ZH11, GS10-OsGSK2-CR, OsGSK2-CR, OsGSK2-OX and OsGSK2-RNAi based on the background of ZH 11. Analysis of GS10 expression level in C, ZH11 and GS10-OX-ZH 11. D, plant height comparison of ZH11 and GS10-OX-ZH 11.

FIG. 24 is a demonstration of the in vivo and in vitro interaction of GS10 and OsOFP 8. A, GS10 and OsOFP 8. And B, verifying the interaction of GS10 and OsOFP8 proteins in tobacco bodies based on the YFP system. The scale bars are 20 μm and 5 μm in this order. And C, GS10 and OsOFP8 protein interactive verification in tobacco bodies based on the YFP system. D, GS10 and OsOFP8 protein validation of Co-IP level in plants.

FIG. 25 is the co-localization of GS10 and OsOFP8 in plants. A, co-localization of GS10 and OsOFP8 in tobacco bodies. Scale, 40 nm. B, co-localization of rice protoplasts GS10 and OsOFP 8. Scale, 40 nm.

FIG. 26 shows that GS10 may be involved in negative regulator of rice BR signal. A, GAL4 and NIL-gs10 in the background of 0 and 1mg BL hormone treatment after 3 days of seedling growth status comparison. Scale, 1 cm. Statistics of epicotyls of 0 and 1mg BL hormone treated seedlings against a background of GAL4 and NIL-gs 10. C, GS10 protein degradation in response to BL in OX background. CBB, coomassie brilliant blue staining. Changes in the inclination of posterior leaflet treated with 0 and 1mg BL hormone in the context of D-E, GAL4 and NIL-gs 10. Statistics of the inclination of the posterior leaflet treated with 0. mu.g, 10. mu.g and 1000. mu.g BL hormones against the background of GAL4 and NIL-gs 10. The values show the mean ± standard deviation (n ═ 3). t-test significant differences: p <0.05, P <0.01, P < 0.001; NS, no significant difference.

FIG. 27 is the genetic relationship of gs10 and other major-granule QTLs. And A, comparing the grain types of different polymeric breeding materials. The polymeric breeding materials NIL-gs10/GL7, NIL-gs10/GW5, NIL-GL7/GW5 and NIL-gs10/GW5/GL7 are polymerized from NIL-gs10, NIL-GW5 and NIL-GL7/GW7 respectively. B-D, NIL-gs10, NIL-GW5, NIL-GL7/GW7, NIL-gs10/GL7, NIL-gs10/GW5, NIL-GL7/GW5 and NIL-gs10/GW5/GL7 (n-10). The values show the mean ± standard deviation, with different letters representing significant differences between the 0.05 levels (Duncan's multiple range test).

Fig. 28 shows that the polymerization of gs10 and GW5 can greatly improve the appearance quality of rice kernels.

Fig. 29 is a comparison of grain taste value, protein, fatty acid and amylose content differences based on gs10 and GW5 polymeric materials. A, GLA4, NIL-gs10, NIL-GW5 and NIL-gs10/GW5 are observed by electronic scanning of endosperm cells of different materials. Scale, 10 μm. B-E, GLA4, NIL-gs10, NIL-GW5 and NIL-gs10/GW5, the food taste value (B), the protein content (C), the amylose content (D) and the fatty acid content (E) are respectively taken as data statistics of the intrinsic quality of different material seeds. The values show the mean ± standard deviation, with different letters representing significant differences between the 0.05 levels (Duncan's multiple range test).

Fig. 30 is CRISPR-Cas9 knock-out experiment of GS10 based on Dongjing background. Genotypes of A, DJ and CR plants. B, DJ and CR. Scale, 10 cm. Spike type of C, DJ and CR. Particle type comparison of D, DJ and CR. Scale, 1 cm. Statistics of grain length (E) and grain width (F) for E-F, DJ and CR. CR, CRISPR-Cas 9. The values show the mean ± standard deviation (n ═ 10), with different letters representing significant differences between the 0.05 levels (Duncan's multiple range test).

Detailed Description

The inventor firstly discloses that the agronomic traits of plants can be obviously regulated by directionally regulating the expression level of GS10 gene in cereal plants (crops), thereby achieving the purposes of improving the quality of the cereal crops, increasing the yield and the like. The agronomic trait is selected from one or more of the group consisting of: leaf length, leaf width, single plant yield, cell yield, plant height, tiller number, grain number per ear, ear length, branch number, top awn, heading stage, stem diameter, glume cell width, glume cell length, glume cell unit area, leaf inclination angle, grain length, grain width, grain weight, BR sensitivity, BR signal path, grain transparency, grain starch cell compactness, grain protein content and grain amylose content.

As used herein, a "cereal crop" may be a graminaceous plant or a miscanthus (crop). Preferably, the gramineous plant is rice, barley, wheat, oats, rye. Miscanthus sinensis refers to a plant having needles on the seed husk. As used herein, the term "crop" or "crop" is not particularly limited, including but not limited to: rice, wheat, barley, etc.

As used herein, the polypeptide encoded by the GS10 gene is designated "GS 10". In the present invention, the term "GS 10" refers to a polypeptide of SEQ ID NO. 1 having GS10 activity. The term also includes variants of the sequence of SEQ ID NO:1 having the same function as GS 10. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, 1 to 20, 1 to 10, 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Amino acids with similar properties are often referred to in the art as families of amino acids with similar side chains, which are well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, lactic acid, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Also, for example, the addition of one or more amino acids at the amino-and/or carboxy-terminus will not generally alter the function of the polypeptide or protein. Conservative amino acid substitutions for many commonly known non-genetically encoded amino acids are known in the art. Conservative substitutions of other non-coding amino acids may be determined based on a comparison of their physical properties with those of genetically coded amino acids.

Variants of the polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants.

Any polypeptide having high homology to GS10 (e.g., 70% or greater homology to the sequence shown in SEQ ID NO: 1; preferably 80% or greater homology; more preferably 90% or greater homology, e.g., 95%, 98% or 99%) and having a similar or identical function to GS10 is also encompassed by the present invention. The same or similar functions are mainly used for regulating and controlling the agronomic traits of crops (such as rice).

The invention also includes analogs of the claimed polypeptides. These analogs may differ from the native SEQ ID NO:1 by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other well-known biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the protein in vivo or in vitro such as acetoxylation or carboxylation. Modifications also include glycosylation, such as those performed during protein synthesis and processing. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).

The polypeptide fragment, derivative or analogue of the invention may be: (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent group in one or more amino acid residues; or (iii) a polypeptide formed by fusing the mature polypeptide to another compound, such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol; or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the scope of those skilled in the art as defined herein.

In addition, any biologically active fragment of GS10 can be used in the present invention. Herein, a biologically active fragment of GS10 is meant to be a polypeptide that still retains all or part of the function of full-length GS 10. Typically, the biologically active fragment retains at least 50% of the activity of full-length GS 10. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length GS 10.

The invention also relates to polynucleotide sequences encoding the GS10 of the invention or variants, analogs, derivatives thereof. The polynucleotide may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in SEQ ID NO. 2 or may be a degenerate variant.

The present invention also relates to variants of the above polynucleotides encoding fragments, analogs and derivatives of the polypeptides having the same amino acid sequence as the present invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby. As used herein, degenerate variants refer in the present invention to nucleic acid sequences which encode a protein having SEQ ID NO. 1, but differ from the sequence of the coding region shown in SEQ ID NO. 2. A "polynucleotide encoding a polypeptide" may be a polynucleotide comprising a sequence encoding the polypeptide, or may further comprise additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" refer to (1) hybridization and elution at a lower ionic strength and a higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. And, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptide of SEQ ID NO. 1.

It is to be understood that although the genes provided in the examples of the present invention are derived from rice, the gene sequences of GS10 derived from other similar plants (particularly plants belonging to the same family or genus as rice) and having a certain homology (e.g., greater than 70%, such as 80%, 85%, 90%, 95%, or even 98% sequence identity) with the sequence of the present invention (preferably, the sequence is shown in SEQ ID NO: 1) are also included in the scope of the present invention, as long as the sequence can be easily isolated from other plants by one skilled in the art after reading the present application, based on the information provided herein. Methods and means for aligning sequence identity are also well known in the art, for example BLAST.

The full-length GS10 nucleotide sequence or its fragment of the present invention can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using a commercially available DNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. Usually, it is cloned into a vector, transferred into a cell, and then isolated from the propagated host cell by a conventional method to obtain the relevant sequence.

In addition, the sequence of interest can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also provides a recombinant vector comprising the gene of the invention. In a preferred embodiment, the promoter downstream of the recombinant vector comprises a multiple cloning site or at least one cleavage site. When it is desired to express the target gene of the present invention, the target gene is ligated into a suitable multiple cloning site or restriction enzyme site, thereby operably linking the target gene with the promoter. As another preferred mode, the recombinant vector comprises (in the 5 'to 3' direction): a promoter, a gene of interest, and a terminator. If desired, the recombinant vector may further comprise an element selected from the group consisting of: a 3' polyadenylation signal; an untranslated nucleic acid sequence; transport and targeting nucleic acid sequences; resistance selection markers (dihydrofolate reductase, neomycin resistance, hygromycin resistance, green fluorescent protein, etc.); an enhancer; or operator. Exemplary vectors are for example pNCGR-OX.

Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. The expression vector may be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In general, any plasmid and vector may be used as long as it can replicate and is stable in the host.

One of ordinary skill in the art can construct expression vectors containing the genes described herein using well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. When the gene of the invention is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added in front of the transcription initiation nucleotide.

Vectors comprising the gene, expression cassette or gene of the invention may be used to transform appropriate host cells to allow the host to express the protein. The host cell may be a prokaryotic cell, such as E.coli, Streptomyces, Agrobacterium; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. It will be clear to one of ordinary skill in the art how to select an appropriate vector and host cell. Transformation of a host cell with recombinant DNA may be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., Escherichia coli), the cells may be treated by the CaCl2 method or may be electroporated. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome encapsulation, etc.). The transformed plant may be transformed by methods such as Agrobacterium transformation or biolistic transformation, for example, leaf disc method, immature embryo transformation, flower bud soaking method, etc. The transformed plant cells, tissues or organs can be regenerated into plants by conventional methods to obtain transgenic plants. When expressed in higher eukaryotic cells, the polynucleotide will provide enhanced transcription when an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on a promoter to increase gene transcription.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

The polypeptides described herein may be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include (but are not limited to): conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof.

Transformation of a host with recombinant DNA may be carried out by conventional techniques well known to those skilled in the art. The transformed plant may be transformed by methods such as Agrobacterium transformation or particle gun transformation, for example, spray method, leaf disk method, rice immature embryo transformation method, etc. The transformed plant tissue or organ can be regenerated into a plant by a conventional method, thereby obtaining a plant with modified traits.

The invention provides application of the GS10 gene to regulation and control of plant agronomic traits; or for screening substances useful for regulating agronomic traits in plants (i.e., the substances regulate agronomic traits in plants by modulating the expression of the GS10 gene). Preferably, the GS10 gene can be used for increasing agronomic traits.

The invention also relates to GS10 up-regulation agents or inhibitors and uses thereof. Because the up-regulator or the inhibitor of GS10 can regulate the expression and/or the activity and the like of GS10, the up-regulator or the inhibitor can also regulate and control the agronomic traits of plants by influencing GS10, thereby achieving the aim of improving the plants.

In one aspect, any substance that increases the activity of GS10, increases its stability, promotes its expression, prolongs its effective duration, or promotes its gene transcription and translation can be used in the present invention as an "enhancer" of the GS10 gene for the regulation of agronomic traits in plants. For example, an expression vector which enhances the transcription, expression or activity of the GS10 gene.

In another aspect, any substance that reduces the activity, reduces the stability, inhibits the expression, reduces the duration of action, or reduces the transcription and translation of GS10 can be used in the present invention as a down-regulator, antagonist, or inhibitor of GS10, such as an interfering molecule that interferes with the expression of the GS10 gene (e.g., an interfering molecule that can form microRNA). The inhibitor, antagonist or inhibitor can be used for regulating and controlling the agronomic traits of plants. Methods for making interfering molecules that interfere with the expression of a particular gene, once the target sequence is known, are well known to those skilled in the art.

Furthermore, to down-regulate GS10 gene expression or activity, a gene knockout vector can be introduced into the cell and/or the gene can be knocked out or knocked down using gene editing techniques such as ZFNs, TALENs, or CRISPR/Cas 9. ZFN, TALEN and CRISPR/Cas9 technologies suitable for use in the present invention are well known in the art. Each technique realizes the knockout of a target gene through the coaction of a DNA recognition domain and an endonuclease.

The invention also relates to a method for modulating an agronomic trait in a plant comprising modulating expression or activity of a GS10 gene in said plant. The up-regulation of the expression or activity of the GS10 gene can reduce the leaf length, increase the leaf width, reduce the yield of a single plant, reduce the cell yield, reduce the plant height, reduce the tillering number, reduce the grain number per spike, reduce the spike length, reduce the stem number, reduce the top awn, shorten the heading period, increase the stem diameter, increase the glume cell width, reduce the glume cell length, increase the unit area of the glume cell, reduce the leaf inclination angle, reduce the grain length, increase the grain width, increase the grain thickness, increase the grain weight, BR sensitivity, negatively regulate and control a BR signal path, reduce the grain transparency, increase the grain starch cell compactness, reduce the grain protein content and reduce the grain amylose content. The expression or activity of the GS10 gene is reduced, so that the leaf length can be increased, the leaf width can be reduced, the yield of a single plant can be increased, the yield of a cell can be increased, the plant height can be increased, the tillering number can be increased, the grain number per spike can be increased, the spike length can be increased, the number of branches can be increased, the awn can be increased, the heading stage can be prolonged, the diameter of a stem can be reduced, the width of glume cells can be reduced, the length of glume cells can be increased, the unit area of the glume cells can be reduced, the inclination angle of the leaves can be increased, the grain length can be increased, the grain width can be reduced, the grain thickness can be reduced, the grain weight can be reduced, the BR sensitivity can be improved, the BR signal channel can be positively regulated, the grain transparency can be increased, the grain starch cell compactness can be reduced, the protein content of the grain can be increased, and the amylose content of the grain can be increased.

In one aspect, the present invention provides a method of modulating an agronomic trait in a plant (such as a crop), the method comprising: over-expressing the GS10 gene in the plant to increase the agronomic trait. After knowing the use of the GS10 gene, various methods well known to those skilled in the art can be used to modulate the expression of the GS10 gene. For example, GS10, which can deliver an expression unit (e.g., an expression vector, a virus, etc.) carrying the GS10 gene to a target and allow expression of the activity, can be achieved by a method known to those skilled in the art.

In one embodiment of the present invention, the GS10 gene is cloned into an appropriate vector by a conventional method, and the recombinant vector containing the foreign gene is introduced into a plant tissue or organ to express the GS10 gene in the plant. Plants overexpressing the GS10 gene can be obtained by regenerating the plant tissues or organs into plants.

In another aspect, the present invention provides another method of modulating an agronomic trait in a plant (e.g., a crop), the method comprising: reducing expression of a GS10 gene in the plant (including making no or low expression of a GS10 gene); thereby reducing the agronomic characters.

Various methods well known to those skilled in the art can be used to reduce or delete the expression of the GS10 gene, such as delivering an expression unit (e.g., expression vector or virus, etc.) carrying the antisense GS10 gene to a target such that the cells or plant tissues do not express or express GS10 is reduced. Alternatively, the GS10 gene can be knocked out by approaches known to those skilled in the art, and/or the GS10 gene can be knocked out or knocked out using gene editing techniques such as ZFNs, TALENs, or CRISPR/Cas 9.

The invention also provides the use of the promoter element of the GS10 gene for the spatiotemporal specific expression of foreign proteins, wherein the spatiotemporal specific expression refers to the specific expression in the cytoplasm or cell membrane of cells at seedling and/or young ear stage.

The invention also provides the application of the substance for down-regulating the expression or activity of the GS10 gene and the substance for up-regulating the expression or activity of one or two genes selected from GW5 and GL7 in stabilizing the grain type, improving the appearance quality of rice and improving the length-width ratio of the rice. In one or more embodiments, the agent that down-regulates the expression or activity of the GS10 gene is an inhibitor of the GS10 gene. Preferably, the inhibitor of the GS10 gene is as described in the first aspect herein. In one or more embodiments, the substance that upregulates the expression or activity of one or both of the genes selected from GW5 and GL7/GW7 is a gene selected from one or both of GW5 and GL7/GW7 or a protein encoded thereby, or a promoter therefor. Methods for up-regulating the expression or activity of one or both of the genes for GW5 and GL7/GW7 are the same as those described elsewhere herein, e.g., vectors for expression of GW5 or GL7/GW7 are transferred or integrated into plants.

The invention also provides a complex formed by binding GS10 protein to a protein selected from SnRK1A, OsOFP8 and OSH 1. In one or more embodiments, the GS10 protein is from rice. In one or more embodiments, the amino acid sequence of the GS10 protein is selected from the group consisting of seq id no: (a) polypeptide with a sequence shown as SEQ ID NO. 1; (b) a polypeptide which is formed by substituting, deleting or adding one or more (such as 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the sequence shown in SEQ ID NO. 1, has the functions of the polypeptide (a) and is derived from the polypeptide (a); or (c) a polypeptide derived from (a) which has more than 90% (preferably 93%; more preferably 95% or 98%) homology to the polypeptide sequence of (a) and which has the function of the polypeptide of (a).

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein. The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Examples

Materials and methods

1 Rice Material

The gene mapping of GS10 in this experiment was from phenotypic studies and data analysis of recombinant Inbred Lines (BILs, Backcross infected Lines) of oryza sativa indica, europa japonica l.ssp GLA4 and oryza sativa (o.rufipogon W1943). Both the primary and fine positioning originate from the single-Segment replacement system CSSL50(CSSLs, Chromosome Segment localization Lines). The basic transformation materials referred to herein are the Asian cultivated rice Nipponbare (Nipponbare), Dongjin and Hwayoung, respectively. Both wild rice and oryza sativa material involved in population genetic analysis were collected, stored and subjected to low abundance sequencing by the inventors.

2 Experimental methods

The DNA extraction methods referred to in this paper are mainly DNA crude extraction methods (TPS method and CTAB method) and plasmid major extraction. The conventional Trizol method was used for RNA extraction, and the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit from TOYOBO was used for RNA inversion into cDNA.

The real-time quantitative PCR instrument selected in the quantitative experiment is an Applied Biosystems Q5 real time PCR instrument, and the reagent is THUNDERBIRD from TOYOBO

The qPCR Mix kit and OsActin in the paper are used as sample internal references.

In the examples, the following experimental methods, paraffin section, seedling hormone treatment, preparation of rice protoplast, PEG-mediated transformation of rice protoplast, RACE experiment, Co-IP experiment, construction and transformation of rice embryo callus transgenic material, statistical measurement of rice glume cell and rice endosperm quality by scanning electron microscopy, subcellular localization experiment, bimolecular fluorescence complementation experiment (BiFC & BiLC), yeast sieve bank and yeast two-hybrid experiment were also used. The above experiments were performed according to conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the laboratory manual, or according to the manufacturer's recommendations.

3 data statistics and analysis

All data analyses were based on setting up multiple replicates and differential test analyses were performed using Duncan's multiple range test and Student's t-test. The mapping software referred to in this paper is mainly Microsoft Excle and Graphpad Prism 8.0. In this, specific SNP calling data were referenced to laboratory published data when analyzing wild and cultivated rice populations for GS10 (Huang et al, 2012). Protein evolutionary tree analysis was generated in MEGA6 software based on NCBI database selection of protein sequences for specific rice genes. The DNA, RNA and protein reference sequences of The relevant genes involved in this study were referenced to IRGSP 1.0(The Rice mutation Project Database) and MSU (Rice Genome mutation Release 7) databases.

Example 1, identification of genotype-associated genes in the recombinant inbred line populations of W1943 and GLA4

Common wild rice o.rufipogon and oryza sativa o.sativa l. are the most typical resources in AA genes in classified populations of rice genera to study rice domestication and evolution (Chen et al, 2019). In the past, polymorphic domestication analysis of 446 wild rice and 950 oryza sativa worldwide located 55 naturally selected domesticated regions (Huang et al, 2012 a). In order to verify and analyze selective sweet, a set of low abundance sequenced wild rice with indica rice background Guangdong dwarf 4(GLA4) and wild rice with japonica rice background (W1943) which are available in laboratories are utilized to generate a recombinant inbred line after hybridization, and a series of QTLs (shown in figure 1 and A) related to the grain type trait are positioned through a whole genome SNP marker. Among these candidate QTLs, GW5/qSW5/GSE5, GL7/GW7, and GL6 were cloned in conventional studies, and among them, GW5, which is the major gene controlling the GRAIN width of CHROMOSOME 5 and has the highest contribution rate, was selected and studied at site GRAIN SIZE ON CHROMOSOME 10(GS10) having a phenotype contribution rate of about 10% (fig. 1, B).

Example 2 Primary and Fine positioning of GS10

In the population of recombinant inbred lines BILs, we initially selected, by examining the grain type traits (grain length, grain width and aspect ratio), a single-fragment replacement line CSSL50 which was replaced by wild rice W1943 only at 16Mb-23.66Mb (IRGSP-1.0) in the context of chromosome 10. In the BIL population, the characteristics such as grain length, grain width and length-width ratio are combined with WinQTL and the like by combining with related molecular markers, mainly insertion and deletion molecular markers (Indel markers), and linkage analysis is carried out, so that a grain type gene locus exists on a chromosome 10, and the association degree between the grain type gene locus and the grain width and thousand grain weight is highest (figure 2). We observed the agronomic traits of CSSL50 rice related grain type, and found that the replacement line showed significant differences in both grain length and grain width compared to the parent GLA4 (FIG. 3, A). Compared to the parental GLA4, CSSL59 increased grain length by 4%, but decreased grain width and thousand kernel weight by about 9% and 10%, respectively (fig. 3, B). In 6 months of 2015, GLA4 is used as a recurrent hybrid male parent and CSSL50 is used as a female parent to obtain hybrid F₁And (4) generation. In 2015 winter, we will F₁Under generation of seed, F is obtained₂Segregating population seeds of generations and seeding F in summer 2016₂Seed generation, and 155 strains F₂The population was initially mapped (FIG. 3, C).

During initial localization, we initially placed one deletion and insertion molecular marker (In-del marker) approximately every 500kb, and finally a total of 17 valid markers. The gene location was located between OP10-25i (19.44Mb) and OP10-11(22.05Mb) by phenotypic and genotypic correlations, still within the selective sweet range (Huang et al, 2012). Subsequently, In-del and SNP marker mapping were continued for the important recombinants 2016#26-1-4, 2016#27-2-3, 2016#27-4-5, and 2016# 27-5-4. Due to the limited number of recombinants and recombination exchange conditions, the genes are preliminarily positioned between 20.576Mb and 21.33Mb, and are deviated from a selection range (selective sweet), and the domesticated genes related to the selective sweet are predicted to be probably not used for controlling the grain width isophenotype. In pair F₂The analysis of the grain phenotype of the population revealed that the genotype was classified into 91 strain GLA4 genotype and 71 strain W1943 genotypeTaking the progeny as an example, the grain type controlled by GS10 has the advantages that the grain length is lengthened by 3%, the grain width is narrowed by 8%, the grain weight is reduced by 9% and the grain thickness is reduced by 4% in wild rice and cultivated rice, and the analysis results show that GS10 is a QTL for negative regulation of the grain type.

After obtaining the precise position of the grain width gene, 5250 effective groups are planted in the summer of 2017 by using a method of using recombinants to assist a large group, and finally the gene is positioned between two important recombinants of 2017#19-3-10 and 2017#26-1-4 by continuously reducing the genetic distance. We annotated at BC by combining RAP-DB IRGSP 1.0(The Rice Annotation Project Database) and MSU genes₂F₄GS10 was further localized in SNP10-6 and SNP10-3, approximately in the range of 10kb, and the gene showed semi-dominant on the grain type (FIG. 3, D). Only one gene in this 10kb range, including the coding region of the gene and the range of about 2kb before the start of transcription, was used as a candidate gene for GS 10. The progeny of two important recombinants, which were planted and the phenotype of the grain type-related trait of the segregating population of the progeny, were not phenotyped, and the result confirmed that the candidate gene was located in the 10kb range. We refer to database analysis that there are only 1 gene annotated locus within the range of candidate genes and this locus encodes a novel unknown armadillo (arm) protein with 6 tandem repeats as candidate genes (fig. 3, E).

Example 3 phenotypic Observation analysis of agronomic traits

In 2017 winter, to further access the small segment near isogenic lines, we screened 4100 individuals in the greenhouse through 288-well seedling culture trays (tray), and screened 14 recombinants using Indel molecular markers G10-20.401, G10-20.576 and G10-20.838 as molecular markers. Among these recombinants, we used G10-20.401 and G10-20.838 as GLA4 type and G10-20.576 as heterozygote type as ideal for the final recombinants, and carried out next generation sequencing with low abundance of 2 individuals containing about 220kb heterozygote type among 14 recombinants (FIG. 4). We planted these two individuals in the greenhouse and performed progeny seed collection, planting and SNP sequencing. Finally, we have obtained a near isogenic line containing approximately the fragment from W1943170 kb, which contains 8 genes and contains GS 10.

Meanwhile, in summer of 2018, a near isogenic line NIL-GS10 containing a GS10 fragment and having an approximate 170kb W1943 homozygous mutation type is selected, wild type GLA4 is used as a contrast, and important agronomic characters such as sword leaf length, sword leaf width, single plant yield, cell yield, plant height, effective tiller number, grain number per spike, spike length, primary branch stalk, secondary branch stalk and the like are observed and counted respectively. Interestingly, NIL-gs10 showed a marked increase in yield per plant but no change in yield per plot compared to GLA4 with top miscanthus, 4 days later in heading, reduced stalk diameter and longer long ear type. The above results indicate that GS10 is a "pleiotropic" QTL (FIG. 5).

Example 4 polymorphism analysis of GS10

After the candidate gene was obtained, the full-length cDNA was then analyzed for polymorphisms. We combined the database predicted sequence comparison analysis and the determination of GS10 transcripts by 5 'RACE and 3' RACE, and found that there were 2 In-del markers and 3 SNP changes In both parental cDNAs, using GLA4 as reference (FIG. 6). The results showed that a deletion of one 3 bases was present in the wild rice 5' UTR, and that there were 2 SNP changes in the Coding region Coding sequence (CDS) of the gene which did not result in amino acid changes, located at positions +432 and +1599, respectively. Notably, in NIL-gs10, there was a five base deletion in the sequence (CCGCC), and protein translation was predicted to cause a frameshift mutation in wild rice (FIG. 7).

Example 5 verification of transgenic experiments

5.1 transgene validation against GLA4

As we did not exclude the influence of promoter region around 2kb on the phenotype of the granule type in the previous mapping, we first constructed the complementary vector CP-1(pGLA4:: gGS 10) in order to further verify whether the candidate gene GS10 is a QTL producing granule type changes or not (pGLA4:: gGS 10)^GLA4) And CP-2(pW1943:: gGS 10)^GLA4) By selecting from different parents GLA4And the Promoter of W1943 were fused to pCAMBIA1300 from genomic DNA derived from GLA4, respectively, and transformed into NIL-gs10 (FIG. 8, A). Each complementary line is at T₀At least 30 plants are obtained in generation, the grain phenotype of each individual plant is preliminarily counted, and 10 individual plants are selected for T₁The agronomic traits were carefully counted and the results showed that both of these complementary lines were able to complement the grain type of NIL plants compared to wild-type GLA4, with lines in the aspect ratio even exceeding the phenotype of GLA4 and appearing as short rounded grains (FIG. 9). In addition, we made statistics on the panicle type and the plant type of CP-1 and CP-2, and the results showed that GS10 shows a tendency to shorten with increasing dose, and the plant type becomes more compact (FIG. 10).

At the same time, we made Over-expression (vector: pNCGR-OX) Over-expression (OX, Ubi:: GS10) with GLA4 as background material^GLA4) And knockout experiments (GS10-CR) (FIG. 8, A). In overexpression experiments, we used ubiquitin (ubi) as a promoter to drive the CDS of the GLA4 coding region at T₀In the generation, 13 plants are obtained. According to phenotype statistics of OX transgenic material offspring, the expression level of GS10 is greatly improved compared with wild type GLA4 (figure 10, B), but the change on the grain type level is weak, the change on the grain length is only 3%, and the plant type and the spike type do not change obviously (figure 8, B). In GS10-CR transgenic experiment, the coding region of the gene designs double targets to obtain large-fragment-deleted homozygous transgenic plants. Compared with the wild-type GLA4, GS10-CR has significant changes in both grain type (grain length and grain width) and plant type levels (FIG. 8, B-E), and particularly GS10-CR can be reduced by about 10% in grain width, which is similar to the grain type and spike type of NIL-GS10 (FIG. 10). In addition, we also performed overexpression experiments with the green fluorescent protein GFP tag in the context of NIL-GS10 Ubi:GS 10-GFP, which obtained transformation of 11 individuals in total, and each individual showed a tendency of short round grain type, indicating that GS10 had a certain dose effect on grain type regulation. As described above, the site where GS10 really functions in regulatory grain type is not involved in promoter and UTR changes but in coding region CDS, and allele from wild rice W1943 causes frame shiftAnd (4) mutation.

We analyzed the characteristics of inclination angle and length of leaves of main sword-shaped spikes after GLA4, NIL-GS10, GS10-CR and OX grouting, and found that the inclination angle of the leaves of NIL-GS10 and GS10-CR transgenic plants is increased in comparison with GLA4 in leaf inclination angle phenotype and is looser in plant type (FIG. 11, A); NIL-GS10 and GS10-CR transgenic plants showed greater length in comparison to GLA4 in relation to the length of the flag leaf (FIG. 11, B). In contrast, over-expressed material OX showed a tendency to decrease in either leaf inclination or flag compared to wild-type GLA 4. Taken together, the above results indicate that GS10 regulates leaf inclination and flag leaf length in addition to yield traits such as grain length, grain width and grain weight, presumably to be involved in plant type regulation (fig. 11, C-D).

5.2 CRISPR-Cas9 knockout validation under Nippon weather background

The CRISPR-Cas9 experiment is carried out on japonica rice material Nip by using the same knockout target point, and the PAM site selected in the experiment is consistent with GS 10-CR. We initially selected 2 independent T₁Homozygous lines NIP-CR-1 and NIP-CR-2 (FIG. 12, A), in which T is expressed₂Phenotype statistics was performed on the progeny of homozygous line, and the results showed that the panicle type of the knocked-out plants showed a tendency to lengthen, which is consistent with the knocking-out results in the background of indica rice (fig. 12, B). On the other hand, both knockout lines showed a tendency of longer grain length, narrower grain width and smaller grain thickness in grain type trait (FIG. 12, C-E). From the above results, we can find that GS10 has great variation in japonica rice material regardless of grain width, thousand grain weight and plant type, and is more obvious than the related phenotype of indica rice material GLA 4.

5.3 reverse complementation verification of T-DNA insertion mutants

We obtained Hwayoung (HY) rice mutants of T-DNA in the T-DNA (transfer DNA) mutant pool of the laboratory of professor Ann Korea (Gene An) (FIG. 13, A), and isolated F progeny after crossing the mutants₂Phenotypic studies and linkage analysis were performed to find that the mutant insertion was indeed linked to the grain width phenotype. We design primers at both ends of T-DNA and combine one generationAfter sequencing, the T-DNA was found to be inserted 1480bp upstream of the CDS of GS10 (FIG. 13, B). Compared with wild type HY, GS10 has significant changes in grain length, grain width and plant type, grain length increased by 15%, grain width decreased by 6% and loose plant type shown (FIG. 13, C), which are very similar to GS10-CR and NIL-GS 10. The expression level of GS10 was reduced by 60% in the insertion mutant plants compared to the wild type HY (FIG. 13, D). At the same time, we combined the complementation experiment with pGLA4 with the GLA4 genotype gGS10^GLA4The vector was introduced into a mutant gs10 on a p2300 vector to obtain 15 positive Ts in total₀And (4) single plants. We are at T for complementary materials₁Statistics of the phenotype of transgenic plants in generations revealed that the phenotype of wild type could not be fully restored in both grain length and grain width levels in progeny of transgenic plants represented by HY-CP-1 compared with wild type (FIG. 13, C). The identification and reverse complementation of this T-DNA insertion mutant further confirmed that GS10 plays an important role in both grain and plant type traits (FIG. 13, E-F). By combining a transgenic experiment taking GLA4 as background and a gene knockout experiment in Nipponbare background, we can conclude that GS10 can cause the change of rice grain type and plant type no matter under the regulation of a promoter or a coding region.

Example 6 subcellular localization and expression profiling

To further explore the changes in expression of GS10, we sampled the GLA4 parent material at the 2-week seedling stage of leaf, flag leaf, leaf sheath, scion and stalk node, respectively. After RNA extraction, we then performed qRT-PCR experiments, which showed no specific expression of GS10 from juvenile to reproductive phase. Wherein, the expression quantity of GS10 in the seedling stage, the sword leaf stage and the young ear stage of each stage is higher than that of the leaf sheath, the endosperm and the stalk node. At the same time, we also constructed a transformation vector with the self-promoter from GLA4 driving the reporter gene GUS (beta-glucuronidase, beta-glucuronide) after transformation with ZH11 at T₂After homozygous plants are obtained in generation, GS10 is highly expressed in seedlings and young ears by color development through reaction with substrates in a reaction solution (figure 14).

In subcellular localization experiments, GLA4-GS10-CDS was ligated to the N-and C-termini of pCAMBIA1300-GFP, respectively, and these two vectors were transferred into Agrobacterium GV3101-P19, respectively, and the tobacco leaf lower epidermis was injected, and signals were observed under fluorescence 72 hours later. The experimental results showed that, regardless of whether GS10 was linked to the N-terminus or C-terminus of GFP, GS10 was localized in the cytoplasm or on the cell membrane of tobacco cells without being affected by the difference in GFP protein structure (fig. 15). We also constructed gs10 from W1943 allele, and linked GFP in front of the protein gs10 (GFP-gs10), and found that the protein distribution in plant leaves is diffuse, and the position of protein expression cannot be traced.

Protein structure prediction showed no transmembrane protein domain, and we combined with its homologous protein position to predict that GS10 might be localized inside the cell membrane, as well as binding to cytoskeletal or intracellular membrane proteins as the homologous protein in animal cells. We also verified that GS10 fused to the N-terminus of GFP in rice protoplasts, and marked NLS-RFP as a nuclear localization marker, indicating that GS10 might localize in the cytoplasm. We then co-localized with the key granulocytic gene GW5, which is known to be localized to the cell membrane. In experimental design, GW5 fused RFP protein and GS10 fused GFP protein were introduced into rice protoplasts together, and it was shown that GS10 and GW5 had membrane localization signals partially overlapping and expression signals were present in the cytoplasm (fig. 16).

Example 7 GS10 influences the granulotype by regulating cell shape rather than cell number

Previous grain type studies have found that changes in grain type are primarily limited by changes in the size resulting from glume cell division and changes in the number resulting from cell proliferation. To further clarify how GS10 was regulated at the glume cell level, we selected non-flowering rice grains from young ears 1 day before ear emergence and performed semi-thin section experiments in GLA4 and NIL-GS10, respectively. As shown, there was no significant difference in the number of cell layers of the palea compared to NIL-gs10 for GLA4, whereas the cells of GLA4 were larger than those of NIL-gs10 at the cell size level (FIG. 17, A). We preliminarily speculate that the main cause of the changes in grain type may be due to the fact that the cell width and thus the size of the cell is further altered in NIL without altering the number of palea cells.

Based on this hypothesis, we first performed scanning electron microscope observations on the cells of the palea and lemma of the mature grain glume cells, and in the statistics we used the cells at the longest transverse axis and the widest longitudinal axis of the grain length as statistical criteria, and the results showed that there was no difference in the cell numbers of NIL-gs10 and GLA4, but there was a difference in the length and width of the cells, both at the grain length and grain width levels (fig. 17, B-D). We combined the horizontal and vertical axis of the cells and found that GLA4 cells had a larger unit area than NIL-gs 10. To further validate the correlation with cell size, we selected some genes closely related to cell division, such as: CDC20, CDK family genes, E2F2, H1, KN, and the like. We select 1-3cm young ears and combine qRT-PCR experiments, and found that the expression amounts of genes such as CDKA2, CYCA2-3, CYCB2-1, CYCD4, CYCLA, H1, KN, MCM3 and the like are remarkably different between GLA4 and NIL-gs10 (figure 17, E). These more diverse genes were expressed in the NIL at a lower level than GLA4, and we preliminarily speculated that GS10 mainly positively regulates some genes involved in cell division during ear development and thus participates in changes in the granulometry.

Example 8 homologous protein and evolutionary Tree analysis

GS10 encodes 6 ARM repeats of armadillo, and a gene family was found to have 158 members in total in studies of their homologous genes (Tewari et al, 2010). In the past research on Arabidopsis thaliana, the gene family with ARM domain may be involved in different pathways in plants depending on whether the motif carried before or after, such as U-box, kinesin, LRR, etc. In the rice research report, the Spotted leaf 11(spl11) encodes a protein carrying U-box and ARM repeat domain, and participates in ubiquitination and protein-protein binding processes (Zeng et al, 2004), respectively. The frame shift mutation of the spl11 protein can cause cell death and weakened defense function of plants, thereby being more susceptible to diseases. It was found to be more similar to the β -Catenin (β -Catenin) family protein by comparing homologous proteins between similar species, as in human cell studies comparing extensive Catenin beta 1(CTNNB1) and arm in drosophila cells, but the mechanism of β -Catenin in rice and arabidopsis is rarely reported (fig. 18). In the Wnt/beta-catenin signaling pathway, GSK3 and AtBIN2/OsGSK2 in plant cells are homologous proteins (Sharma et al, 2014a), which play an important regulatory role in downstream pathways, and we speculate that GSK3 beta phosphorylated beta-catenin may also phosphorylate GS10 in plant cells by OsGSK 2.

Through the evolutionary tree analysis of related homologous PROTEINs of rice GS10, LOC _ Os03g02580(GSL1, GS10-LIKE1 PROTEIN) is found to be the most recent related PROTEIN in the family, and the functions of most genes in the family are not excavated (FIG. 19, A). GSL1 also encoded 6 ARM tandem repeats in MSU gene annotation, which we randomly knocked out in the ZH11 background (fig. 19, B). The results showed that there was no significant difference in plant type between GSL1 and GS10, and there was a difference in plant height and inclination of flag leaf (fig. 19, B). In the regulation of grain shape, GSL1-CR and GSL1-GS10-CR have no significant difference in grain length and grain width, and both can make the grain shape thin and long (FIG. 19, C-D). Combining the above results with the changes in grain and plant types of GS10-CR-ZH11, we speculate that GS10 may have some redundancy with GSL1 in protein functions, and they may play the same role in gene pathways.

Example 9, GS10 RNA-seq analysis

To further explore the changes in GS10 at the transcriptome level. We selected three kinds of materials such as GLA4, GS10-CR and OX as the flowering stage of the sword leaf to sample and extract the total RNA for RNA sequencing analysis. During the experimental design, we set up 2 biological replicates of each different combination, totaling 9 transcriptome data. Analysis results show that in comparison with GLA4, 2476 Differentially Expressed Genes (DEGs) are found in total, 1838 genes are up-regulated, and 638 genes are down-regulated. In contrast, CR found 346 differentially expressed genes compared to GLA4, of which 177 were up-regulated and 169 were down-regulated. The GO (gene ontology) analysis of the two types of differentially expressed genes shows that GS10 is mainly involved in certain metabolic processes, particularly catalysis and hydrolysis reaction, so as to regulate and control stress reaction in life activities, such as cell wall synthesis and certain hydrolytic metabolic processes. These results are consistent with the previous researches on the ARM tandem repeat protein family gene participating in stress reaction processes such as disease resistance and stress resistance. Because fewer DEGs were produced in GS10-CR compared to GLA4, we mined downstream regulatory genes in the data for OX and GLA 4.

As shown by the analysis of the differentially expressed genes of OX and WT, GS10 regulates some genes related to the development of flower organs, grain types and plant types, such as OsMADS1, LAX2, GL7, DTH7, OsBZR1, OsIAA family genes, OsJAZ family genes and the like. We speculate that GS10 may be involved in the regulation of BR as well as its homologous gene TUD1, thereby affecting changes in grain and plant types. It is worth noting that we preliminarily selected some reported known genes related to the grain type and the plant type, and combined with qRT-PCR, re-detected the expression level of these DEGs. As a result, it was found that GS10 can negatively regulate the granule type gene GL7 and the BR pathway receptor gene OsBR1, and positively regulate OsMADS1, BG1, LAX2, OsBZR1, OsAP2-39, OsGA2OX2, DTH7, OsGA2OX5, GH3.8 and the like. (FIG. 20) by combining the above results, it can be concluded that GS10 shows "pleiotropic effect" in its agronomic trait, which is likely to participate in and regulate multiple gene pathways of rice.

Example 10 GS10 binding to OsOFP8 regulates Rice grain and plant type

OsGSK2 is a most important transkinase in the BR signal transduction pathway, and many kernel-type-associated nuclear genes, such as GRF4, OFP8 and GS9 (Tong and Chu,2018), have been regulated in past studies. The overexpression plant OsGSK2-OX (go) of the negative regulatory factor GSK2 of BR shows that the plant is short, compact and small in grain shape on phenotype; and the RNAi plant OsGSK2-RNAi (Gi) shows that the plant is thick, the plant type is loose and the grain type is large. We wanted to go further to explore whether GS10 and OsGSK2 have a connection on the BR pathway. In previous studies, the subcellular localization of OsGSK2 was either cytoplasmic or nuclear, whereas GS10 was expressed only cytoplasmic. To further explore how GS10 participates in and exerts regulatory BR signals in the cytoplasm, we initially used the yeast two-hybrid screening library approach. In later experimental analysis, the method for screening the interacting protein by the yeast bank is not smooth, and only the gene SnRK1A is screened by the two screening results. Past researches show that SnRK1A is mainly involved in ABA signals and hardly involved in the research of rice yield regulation such as grain type and the like. Meanwhile, GS10 is connected to a yeast BD vector, and some key proteins related to BR signal transduction are selected and connected to an AD vector, wherein the key proteins comprise receptor proteins OsBRI1 and OsBAK1, GW5 inhibiting GSK2 activity, and transcription factors regulated by OsGSK2 at the downstream of BR, including DLT, GRF4, BZR1, OSH1, OFP8, OFP14, GS9 and the like. Experimental results show that GS10 is able to interact with SnRK1A, OsOFP8 and OSH1 in vitro (fig. 21, a).

We first ligated OsGSK2 to AD vector and GS10 to BD vector, respectively, by yeast double-hybrid experiments, using two-plate-lacking control and three-plate-lacking and four-plate-lacking experiments to show that there was no interaction between them (FIG. 21, B).

We also combined some genetic experiments to verify whether there is a certain relation between GS10 and OsGSK2, an important transgene in BR pathway. Interestingly, we designed a double knockout experiment of OsGSK2 and GS10 with ZH11 as a background, and used Go (OsGSK2-OX) and Gi (OsGSK2-RNAi) as controls to obtain transgenic lines such as GS10-CR-ZH11, GS10-OX-ZH11, GS10-OsGSK2-CR and OsGSK2-CR (FIG. 22, A). T in these transgenic plants₂As shown by the examination of grain type and plant type in the generation homozygous plants, the grain type trait of GS10-OsGSK2-CR is compared with that of OsGSK2-CR and GS10-CR-ZH11, and OsGSK2 can cause grains to be enlarged without changing grain width; while GS10-OsGSK2-CR and GS10-CR-ZH11 do not have any difference in grain length and grain width; this suggests that even though OsGSK2 does not interact with GS10, they all affect grain type in BR pathway regulation, and that GS10 has some epistasis in BR pathway to OsGSK2 (fig. 22, B-D).

In the above transgenic plant types, GS10-OsGSK2-CR and GS10-CR showed looser plant types and a tendency of increased inclination angle of flag leaf compared with ZH11 (FIG. 23, A-B); however, OsGSK2-CR did not show the same drastic BR phenotype as OsGSK2-RNAi, indicating that some redundancy may exist in the rice GSK family (FIG. 23, A). Meanwhile, related overexpression experiments of GS10 in ZH11 are combined, and it can be seen that after the expression level of GS10 in japonica rice material ZH11 is greatly improved (fig. 23, C), the plant type is more compact, the stems are thickened, the inclination angles of the sword leaves are upright, the spike is thickened and shortened, and the number of grains without spikes is not significantly changed (fig. 23, A). It was noted that its plant height showed a decrease as low as OsGSK2, and the degree of change could reach around 25% (FIG. 23, D).

OsOFP8 is phosphorylated by OsGSK2, and the protein is phosphorylated to cytoplasm and is aggregated and finally degraded by protease, which is a transfer factor for positively regulating BR signals (Yang et al, 2016). It is used as a more important positive regulatory factor in BR signal path, and under the condition of overexpression, the inclination angle of plant leaf is increased, and the grain shape is narrowed and lengthened. We preliminarily predicted that OsOFP8 may act as a mediating protein of GS10 in response to BR signaling to regulate expression of downstream BR-associated genes. First, we performed BiFC experiments in tobacco, and by fusing GS10 and OsOFP8 to the N-terminus and C-terminus of yellow fluorescent protein YFP, respectively, the experimental results showed that GS10 and OsOFP8 are interactive in plants and cytoplasmic. Meanwhile, the verification is carried out in tobacco bodies by combining the BiLC experiment, GS10 and OsOFP8 are respectively fused with the N end and the C end of the fluorescein protein luciferase, and the same experiment result is also obtained by combining the results of a positive control and a negative control. We used Co-IP method to verify protein interaction at protein level, and the 35S::6xMyc-OsOFP8 and no-load 35S::6xMyc and 35S::3Flag-GS10 were transformed to express protein transiently in tobacco. After the protein extracts were adsorbed by Flag beads for 3-4h, the presence of in vivo interaction was checked by the western blotting experiment using Flag and Myc antibodies. Final experimental results showed that GS10 and OsOFP8 did interact in vivo (fig. 24).

OsOFP8 acts as a transcription factor downstream of OsGSK2, is phosphorylated by the latter and degraded in the cytoplasm. But we found that the region where OsOFP8 and GS10 interact is located in the cytoplasm, how do GS10 affect the role of OsOFP 8? We first performed co-localization experiments in plants (tobacco and rice protoplasts), using OsOFP8 fused with mCherry as a control, GS10 fused with GFP and then transferring GS10-GFP and OsOFP8-mCherry into tobacco and rice protoplasts together, and the results showed that GS10 indeed caused OsOFP8 coring (FIG. 25). Meanwhile, the qRT-PCR experiments are combined to find that the expression level of OsOFP8 is reduced in young ears of GS10-OX-ZH11 compared with wild type ZH11, which shows that GS10 has a certain inhibition effect on OsOFP8 at the transcription level.

Example 11, GS10 is involved in BR Signal transduction

In the verification of the transgenic experiment, GS10 is found to regulate and control the plant type besides the grain type and the panicle type. In the background of GLA4, NIL-gs10 showed significant differences in plant height and in the degree of looseness of plant type compared to wild type. Then, statistics is carried out on the leaf inclination angle of the transgenic material, and the result shows that NIL-gs10 and CR are both larger than GLA4, and the plant types are scattered; compared with NIL-gs10, CP-1, CP-2 showed a tendency to become smaller, and the plant type was more compact. This indicates that GS10 is a positive regulator in the regulation of rice plant type, especially leaf inclination.

In past studies, it was found that only one homologous gene TUD1 is currently cloned from members of the ARM family, which binds to RGA1(D1) and regulates changes in grain and plant types by participating in signal transduction between G-protein and BR (Hu et al, 2013). Meanwhile, in protein prediction, GS10 was found to encode 6 armadillo reptiles, which have certain homology with CTNNB1 in human and arm in Drosophila. The latter two proteins are mainly involved in the classical Wnt/beta-catenin signal transduction pathway. Therefore, we preliminarily predicted that GS10 may also have similar function of BR signaling.

BR signals are mainly used in rice research to regulate grain type and plant type (plant height and leaf inclination) (Tong and Chu, 2018). To further study the gene function of GS10 in rice, we first processed it with brassinolide response to see if it responds to BR. We grown them by treating GLA4 and NIL-gs10 (seeds soaked in advance until white) in 0.3% agar medium containing 0. mu.g, 500. mu.g, 1mg of the brassinolide analogue 2,4-epiBL, respectively, protected from light in a 30 ℃ incubator. The results of the treatments showed that the epicotyls became longer with increasing hormone concentrations in NIL plants compared to GLA4 under the same conditions (FIG. 26, A-B). We also used protein-tagged HA-bearing OX material against GLA4 as treatment material, with nodes every 30min and 1mM BL and MG132 as treatment conditions, respectively. The experimental results show that GS10 was gradually degraded over time (fig. 26, C). We also added different concentrations of BL to GLA4 and NIL plants at their leaf inclination at the 10-day seedling stage, followed by treatment with light at 30 degrees for 2 days, and the results showed that CR plants and WT showed a greater change in leaf inclination compared to WT under the 1mg concentration treatment (FIG. 26, D-F). It was demonstrated that mutant GS10 could increase sensitivity of BR, and GS10 could negatively regulate BR signaling during normal life activities.

Example 12 relationship of GS10 to other major granule type genes

In previous studies, it was found that there is a large correlation between the traits related to grain type and quality, for example, grain length, grain width, and aspect ratio (Gong et al, 2017). The appearance quality is enhanced by the long and narrow grains which are finally produced when the aspect ratio of the rice grain type is increased, and the appearance quality is deteriorated (Wang et al, 2012; Wang et al, 2015 b; Liu et al, 2018). To further explore the relationship of GS10 to other grain type genes, we purposefully combined three major grain type genes, GW5, GL7/GW7 and GS10, located in this set of recombinant inbred lines by means of molecular polymerization (fig. 27, a). Among them, GW5 and GL7/GW7 have been reported as excellent genes for excellent quality control and grain type. GW5 from wild rice W1943 controls elongated granules, with a phenotype very similar to GS10 on granule type, and regulates granule type by binding protein function and inhibiting kinase activity of OsGSK 2. GL7/GW7 is a QTL site with a relatively major effect on grain length, interestingly, GW8 binds to a promoter region of GW7 and inhibits the expression of the promoter region so as to regulate grain type, and the two genes also have certain improvement on rice quality.

In the progeny seeds bred by the polymeric materials, compared with NIL-GW5/gs10, NIL-GL7/gs10 and NIL-gs10, gs10 can increase the grain length of GW5 and GL7 in the grain length property and has a certain additive effect (figure 27, B). In the grain width trait, gs10 polymerized with either GW5 or GL7, also made the progeny seeds finer (fig. 27, C). The above results indicate that gs10 has some additive effect on the grain type trait for both major genes, GW5 and GL 7. It is noteworthy that the polymeric material NIL-GW5/GL7/gs10 is wider than the latter two in terms of particle width as compared to NIL-GW5/GL7 and NIL-GL7/gs 10. The above results further demonstrate that the aggregation of two major genes, GW5 and GL7, can greatly change the particle width, and that after being incorporated into gs10, a balance exists in the particle shape, and the particle shape is not drastically changed.

At the thousand kernel weight level we can see a tendency for both GW5 and gs10 to reduce the kernel weight, in addition to the significant contribution of GL7 to the kernel weight (fig. 27, D). NIL-GW5/GL7 did not show any significant difference in thousand kernel weight compared to NIL-GW 5. The thousand kernel weight of NIL-GW5/gs10 decreased when GW5 polymerized gs10, the same result was also seen in NIL-GL7/gs 10. While when the three genes were aggregated together, thousand grain weight of NIL-GW5/GL7/gs10 was lowest compared to the other materials, indicating that gs10 had a negative regulatory effect at the grain weight level (FIG. 27, D).

Example 13 polymerization of gs10 with GW5 improved the appearance and quality of rice kernels

It is noted that GLA4 is a high-yielding and poor-looking base material, and it is clear that the appearance clarity in brown rice is improved somewhat in NIL-gs10 and NIL-GW5 as compared to wild-type GLA 4. NIL-GW5/gs10 showed a great improvement in seed transparency, enabling a substantial reduction in chalkiness. Meanwhile, the scanning of an electron microscope is carried out on the arrangement degree of the starch at the fault level of the rice grains, and the result shows that the arrangement of the starch at the fault level of GLA4 is looser, and NIL-gs10 is followed, while the arrangement of starch cells of NIL-GW5/gs10 and NIL-GW5 is tighter, which is consistent with the result on the appearance quality of the grains (figure 29, A). The above results indicate that gs10 can improve the appearance quality of rice seeds by affecting the close arrangement of starch cells, and further, by fusing with GW5, the rice quality can be greatly improved in future molecular breeding (fig. 28).

We further measured the intrinsic quality of rice kernels for the above polymeric materials by a rice kernel taste instrument. As a result, it was found that NIL-gs10 and NIL-GW5 were only slightly increased compared to the wild-type GLA4, both in protein and amylose content. But if they are aggregated together, the intrinsic quality is greatly improved. It is noted that NIL-GW5 increased the fatty acid content in the fatty acid content, and these results indicate that gs10 is an allelic variation that does not combine quality with grain weight, and that the aggregation of GW5 and gs10 significantly improved the rice appearance quality. If the quality is pursued, the application can greatly improve the rice quality in future breeding (figure 29, B).

Example 14 analysis of Natural variation and acclimatization of GS10

By combining the low-abundance sequencing data of wild rice and cultivated rice in a laboratory, the polymorphism of the region is analyzed after SNP calling is carried out on 20.1-20.5Mb (IRGSP 1.0) of a No. 10 chromosome of rice, and the polymorphism of the wild rice is higher than that of the cultivated rice, and the polymorphism of the indica is higher than that of the japonica. This indicates that during the evolution, this region was strongly selected and that GS10, which is normally protein functional, was gradually immobilized in oryza sativa. As can be seen from our information on haplotyping of wild rice, GS10 still has a great deal of variation in the wild rice population, which may be due to partial deletion of the base and thus may also generate the same frame shift mutation and also bring about variation in grain type and plant type as allele of W1943. In which we used Indel3+569 as an example, GS10 deleted four bases of CGGC in part of wild rice and cultivated rice. In order to create similar genotypes in rice, a PAM locus as GS10-CR is designed and verified by Dongjing (DJ) of japonica rice background, so that two transgenic lines DJ-CR-1 and DJ-CR-2 are obtained. The results are shown in FIG. 30.

In both transgenic lines, they generated deletion mutations of a partial base and both contained Indel3+569(-CGGC) as the mutation site. At T₂In the offspring, the homozygous plant phenotype is statistically analyzed, and the ears of DJ-CR-1 and DJ-CR-2 are longer and the ears of the plants are second-level compared with DJThe tendency of reduction in the number of peduncles was consistent with gene knockouts in NIP. In addition, the grain shape traits of the three materials are analyzed, and DJ-CR-1, DJ-CR-2 and DJ show the tendency that the plant types of plants become loose, the inclination angle of the sword leaves becomes larger, and the grain shape becomes thinner and longer compared with DJ. Especially at the grain width level, CRISPR-produced mutants can cause up to 15% changes. We speculate that other alleles produced by wild rice also produce a long-grain phenotype in rice due to mutations in the gene, as does W1943.

Summary of the invention

Ordinary wild rice (Oryza rufipogon) and Oryza sativa (Oryza sativa L.) are the most typical genetic resource materials for studying rice domestication and evolution in the AA genome of the classified population of Oryza. The grain type is a complex agronomic shape closely related to the yield and quality of rice, and is controlled by multiple genes. Meanwhile, with the rise of hybrid rice breeding and the great increase of rice yield, the rice quality is more and more paid attention by breeders and consumers. At present, research on Quantitative Trait Loci (QTLs) of grain types in natural populations has been greatly advanced, but QTLs that regulate quality and are grain types are still poorly understood in the evolution process. The scientific problem concerned by the research is to excavate and analyze new genes influencing yield and quality and an evolution mechanism thereof in wild rice with abundant genetic variation, perfect a granular gene molecular regulation network and evaluate the agricultural production application value of the genes.

A set of low-abundance sequenced wild rice material (W1943) with indica rice background, Guang-Lu-ai 4(GLA4) and ordinary wild rice material with a non-glutinous rice background, which are constructed in a laboratory, are hybridized to generate a recombinant inbred line, so that QTLs (quantitative trait loci) related to yield traits (grain type, plant type and seed setting rate) are positioned. In this study, we selected the recombinant inbred line and mapped a GRAIN SIZE candidate gene GS10(GRAIN SIZE ON CHROMOSOME 10) by combining phenotypic study and map-based cloning. This gene encodes a novel armadillo (arm) subfamily protein with 6 tandem repeats. The gs10 derived from wild rice has a frame shift mutation due to deletion of 5 nucleotides, resulting in loss of protein function, resulting in a narrow and long grain shape, a reduced grain thickness, a reduced thousand grain weight, and a larger leaf inclination angle, but the appearance quality of the grains is excellent. The agronomic trait regulatory analysis of GLA4 and the near isogenic line NIL-GS10 revealed that GS10 has a pleiotropic effect and that mutated GS10 did not affect individual and cell yields. Transgenic experiments such as complementation, overexpression, gene knockout and the like further prove that GS10 can positively regulate grain width, but negatively regulate properties such as grain length, panicle length and the like. In addition, by integrating the size and number statistical analysis of the glume cells of the mature seeds and the observation of the semi-thin slices of the immature glumes, the fact that the gene mainly influences the size of the glume cells is revealed, and the number is not influenced, so that the grain type change is regulated. Experiments such as RNA-seq and yeast two-hybrid combination prove that GS10 can be combined with OsOFP8 to participate in BR signal path, but does not interact with OsGSK2 in protein function but is positioned in OsGSK2 in grain type character. Meanwhile, the aggregate breeding of gs10 and GW5 with the main effect of controlling grain type and quality under the background of GLA4 can further improve the aspect ratio in cultivated rice and further improve the appearance quality. Based on the sequencing of the coding regions of 114 parts of wild rice and 997 parts of oryza sativa GS10, 16 haplotypes were obtained, and these results indicate that the allelic variation site of GS10, which functions normally in wild rice and has compact plant type and increased thousand-kernel weight, is essentially fixed in the oryza sativa population. Notably, gs10 from W1943 still has many allelic variations in the wild rice population that produce frameshift mutations in the coding region, and a few in the aus and aromatic populations, suggesting that dysfunctional gs10 has great potential for future rice quality improvement.

Sequences as referred to herein

GS10 protein	SEQ ID NO:1
		GS10 Gene coding sequence	SEQ ID NO:2
U3-sgRNA	SEQ ID NO:3
		U6a-sgRNA	SEQ ID NO:4

Sequence listing

<110> prominent innovation center of molecular plant science of Chinese academy of sciences

<120> gene for regulating and controlling quality of rice grains and application thereof

<130> 20A631

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 560

<212> PRT

<213> Artificial Sequence

<400> 1

Met Ser Leu His Cys Cys Pro Ala Ala Gly Asp Pro Pro Ala Pro Ala

1 5 10 15

Gly Thr Ala Glu Glu Leu Leu Glu Arg Ala Arg Ser Leu Val Pro Ala

20 25 30

Ala Leu Asp Ala Ala Arg Ala Ala Thr Gly Phe Gly Gly Arg Trp Lys

35 40 45

Val Ile Ala Ala Arg Leu Glu Arg Val Pro Pro Cys Leu Ser Asp Leu

50 55 60

Ser Ser His Pro Cys Phe Ser Lys Asn Ser Leu Cys Arg Glu Leu Leu

65 70 75 80

Gln Ser Val Ala Ala Thr Leu Ala Glu Ala Ala Glu Leu Gly Ala Arg

85 90 95

Cys Arg Glu Pro Pro Arg Ala Gly Lys Leu Gln Met Gln Ser Asp Leu

100 105 110

Asp Ala Leu Ala Gly Lys Leu Asp Leu Asn Leu Arg Asp Cys Ala Leu

115 120 125

Leu Ile Lys Thr Gly Val Leu Ser Asp Ala Thr Val Pro Pro Val Ala

130 135 140

Pro Ala Ala Glu Ala Ala Ala Gln Thr Asp Val Arg Glu Leu Leu Ala

145 150 155 160

Arg Leu Gln Ile Gly His Ala Glu Ala Lys His Arg Ala Val Asp Gly

165 170 175

Leu Leu Asp Ala Leu Arg Glu Asp Glu Lys Ser Val Leu Ser Ala Leu

180 185 190

Gly Arg Gly Asn Val Ala Ala Leu Val Gln Leu Leu Thr Ala Thr Ala

195 200 205

Pro Lys Ile Arg Glu Lys Ala Ala Thr Val Leu Cys Leu Leu Ala Glu

210 215 220

Ser Gly Ser Cys Glu Cys Leu Leu Val Ser Glu Gly Ala Leu Pro Pro

225 230 235 240

Leu Ile Arg Leu Val Glu Ser Gly Ser Leu Val Gly Arg Glu Lys Ala

245 250 255

Val Ile Thr Leu Gln Arg Leu Ser Met Ser Pro Asp Ile Ala Arg Ala

260 265 270

Ile Val Gly His Ser Gly Val Arg Pro Leu Ile Asp Ile Cys Gln Thr

275 280 285

Gly Asp Ser Ile Ser Gln Ser Ala Ala Ala Gly Ala Leu Lys Asn Leu

290 295 300

Ser Ala Val Pro Glu Val Arg Gln Ala Leu Ala Glu Glu Gly Ile Val

305 310 315 320

Arg Val Met Val Asn Leu Leu Asp Cys Gly Val Val Leu Gly Cys Lys

325 330 335

Glu Tyr Ala Ala Glu Cys Leu Gln Ser Leu Thr Ser Ser Asn Asp Gly

340 345 350

Leu Arg Arg Ala Val Val Ser Glu Gly Gly Leu Arg Ser Leu Leu Ala

355 360 365

Tyr Leu Asp Gly Pro Leu Pro Gln Glu Ser Ala Val Gly Ala Leu Arg

370 375 380

Asn Leu Val Ser Ser Ala Ile Ser Pro Asp Ser Leu Val Ser Leu Gly

385 390 395 400

Val Leu Pro Arg Leu Val His Val Leu Arg Glu Gly Ser Val Gly Ala

405 410 415

Gln Gln Ala Ala Ala Ala Ala Ile Cys Arg Val Ser Ser Ser Ser Glu

420 425 430

Met Lys Arg Leu Val Gly Glu His Gly Cys Met Pro Leu Leu Val Arg

435 440 445

Leu Leu Glu Ala Lys Ser Asn Gly Ala Arg Glu Val Ala Ala Gln Ala

450 455 460

Val Ala Ser Leu Met Ser Cys Leu Ala Asn Ala Arg Asp Ile Lys Lys

465 470 475 480

Asp Glu Lys Ser Val Pro Asn Leu Val Gln Leu Leu Glu Pro Ser Pro

485 490 495

Gln Asn Thr Ala Lys Lys Tyr Ala Ile Ser Cys Leu Leu Thr Leu Ser

500 505 510

Ala Ser Lys Arg Cys Lys Lys Leu Met Ile Ser His Gly Ala Ile Gly

515 520 525

Tyr Leu Lys Lys Leu Ser Glu Met Asp Val Ala Gly Ala Lys Lys Leu

530 535 540

Leu Glu Lys Leu Glu Arg Gly Lys Leu Arg Asn Leu Phe Ser Arg Lys

545 550 555 560

<210> 2

<211> 1683

<212> DNA

<213> Artificial Sequence

<400> 2

atgagcttgc attgctgtcc ggcggccggc gacccgccgg cgccggccgg gacggcggag 60

gagctgctgg agcgggcgcg gtcgctggtg ccggcggcgc tggacgcggc gcgcgcggcg 120

accggcttcg gcggccggtg gaaggttatc gcggcgaggc tggagagggt gccgccgtgc 180

ctgtccgacc tgtccagcca cccgtgcttc tccaagaact cgctctgccg cgagcttctg 240

cagtcggtgg ccgccacgct cgccgaggcc gccgagctcg gggcgcgctg ccgcgagccg 300

ccgagggccg ggaagctgca gatgcagagt gacctcgacg cgctcgccgg gaagctcgac 360

ctgaacctcc gggattgcgc gctgcttatc aagaccggtg tgctgtccga cgcgaccgtg 420

ccaccggtgg cgccggcggc tgaggcggcg gcgcagacgg atgtgcggga gctgcttgcg 480

aggcttcaga tcgggcacgc ggaggcgaag caccgggcgg tggatgggct cctcgacgcg 540

ctacgcgagg acgagaagag cgtgctgtcg gcgctcggcc gcggcaacgt ggcggcgctg 600

gtgcagctgc tgacggcgac ggcgcccaag atcagggaga aggcggccac cgtcctctgc 660

ttgctggccg agtccggcag ctgcgagtgc ttgctggtgt cggagggcgc gctgccgccg 720

ctcatccggc tggtcgagtc cggcagcctg gtcggccggg agaaggccgt gatcacgctg 780

cagcggctgt ccatgtcgcc cgacattgcc cgcgccatcg tcggccacag cggcgtccgc 840

ccgctgatcg acatctgcca gaccggggac tccatctcgc agtccgcggc ggccggcgcg 900

ctcaagaacc tctccgcggt gcccgaggtg cgccaagcgc tggcggagga agggatcgtg 960

cgcgtcatgg tcaacctgct cgactgcggc gtcgtgctcg gctgcaagga gtacgccgcg 1020

gagtgcctcc agagcctcac gtcgagcaac gacggcctcc gccgcgccgt cgtgtccgag 1080

ggcggcctcc gcagcctgct cgcctacctc gacggcccgc tgccgcagga gtccgccgtg 1140

ggcgcgctcc gcaacctggt gagcagcgcc atctcgccgg acagcctggt gtcgctgggc 1200

gtgctccccc gcctcgtcca cgtgctccgc gagggctccg tgggcgcgca gcaggcggcc 1260

gcggcggcga tctgcagggt gtcgagctcg tcggagatga agcgcctggt cggcgagcac 1320

gggtgcatgc cgctgctggt gcggctgctg gaggcgaagt cgaacggcgc gcgcgaggtg 1380

gcggcgcagg cggtggcgag cctgatgagc tgcctggcga acgccaggga catcaagaag 1440

gacgagaaga gcgtccccaa cctggtgcag ctgctggagc cgagccccca gaacacggcc 1500

aagaagtacg ccatctcctg cctcctcacg ctgtcggcga gcaagcgctg caagaagctg 1560

atgatctcgc acggcgccat tggctacctc aagaagctct ccgagatgga cgtcgccggc 1620

gccaagaagc tgctcgagaa gcttgagcga ggcaagctgc gcaacctctt cagtaggaag 1680

taa 1683

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 3

aagagcgtgc tgtcggcgct cgg 23

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 4

tgatcgacat ctgccagacc gggg 24

Claims

1. Use of a substance for modulating an agronomic trait in a plant, the substance selected from the group consisting of: GS10 gene or its coding protein, or its promoter or inhibitor, wherein the agronomic trait is selected from one or more of the following: leaf length, leaf width, single plant yield, cell yield, plant height, tillering number, grain number per ear, spike length, branch number, top awn, heading period, stem diameter, glume cell width, glume cell length, glume cell unit area, leaf inclination angle, grain length, grain width, grain weight, BR sensitivity, BR signal path, grain transparency, grain starch cell compactness, grain protein content, grain amylose content,

preferably, the plant is a cereal crop.

2. The use according to claim 1,

the GS10 gene codes an amino acid sequence selected from the group consisting of:

(a) polypeptide with a sequence shown as SEQ ID NO. 1;

(b) 1 through one or more amino acid residue substitution, deletion or addition, and (a) derived polypeptide with (a) polypeptide function; or

(c) A polypeptide derived from (a) having more than 90% homology with the polypeptide sequence of (a) and having the function of the polypeptide of (a);

and/or the presence of a gas in the atmosphere,

the nucleic acid sequence of the GS10 gene is selected from the group consisting of:

(1) a polynucleotide encoding a polypeptide as shown in SEQ ID NO. 1;

(2) 2 or a polynucleotide having more than 80% homology with the polynucleotide shown in SEQ ID NO;

(3) 2, truncating or adding 1-60 nucleotides to the 5 'end and/or the 3' end of the polynucleotide shown in SEQ ID NO;

3. The use according to claim 1,

the accelerator is selected from the group consisting of: a small molecule compound, a nucleic acid molecule, or a combination thereof;

the inhibitor is an inhibitor molecule which specifically interferes with the transcription and/or expression of the GS10 gene, and preferably the inhibitor molecule takes the GS10 gene or a transcript or an encoded protein thereof as an inhibition target; more preferably, the inhibitory molecule is selected from the group consisting of: (1) a small molecule compound, an antisense nucleic acid, a microRNA, a siRNA, an RNAi, a dsRNA, a sgRNA, an antibody, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1).

4. A method of modulating an agronomic trait in a plant, the method comprising: modulating expression or activity of a GS10 gene in a plant, thereby modulating an agronomic trait in the plant, the agronomic trait being selected from one or more of the following: leaf length, leaf width, single plant yield, cell yield, plant height, tillering number, grain number per ear, spike length, branch number, top awn, heading period, stem diameter, glume cell width, glume cell length, glume cell unit area, leaf inclination angle, grain length, grain width, grain weight, BR sensitivity, BR signal path, grain transparency, grain starch cell compactness, grain protein content, grain amylose content,

preferably, the method of modulating an agronomic trait in a plant comprises: up-regulating the expression or activity of the GS10 gene in a plant; thereby reducing the leaf length, increasing the leaf width, reducing the yield of a single plant, reducing the yield of a cell, reducing the plant height, reducing the tillering number, reducing the grain number per spike, reducing the spike length, reducing the number of branches and stalks, reducing the awn, shortening the heading stage, increasing the diameter of the stems, increasing the width of glume cells, reducing the length of glume cells, increasing the unit area of glume cells, reducing the leaf inclination angle, reducing the grain length, increasing the grain width, increasing the grain thickness, increasing the grain weight, increasing the BR sensitivity, negatively regulating and controlling a BR signal path, reducing the transparency of grains, increasing the compactness of starch cells of the grains, reducing the protein content of the grains and reducing the amylose content of the grains; more preferably, said up-regulating expression of the GS10 gene in a plant comprises: transferring GS10 gene into plant to obtain transformed plant,

the method for regulating and controlling the agronomic traits of the plants comprises the following steps: down-regulating expression or activity of GS10 in a plant; thereby increasing the leaf length, reducing the leaf width, increasing the yield of a single plant, increasing the yield of a cell, increasing the plant height, increasing the tillering number, increasing the grain number per spike, increasing the spike length, increasing the number of branches and stalks, increasing the awn, prolonging the heading period, reducing the diameter of the stalks, reducing the width of glume cells, increasing the length of glume cells, reducing the unit area of glume cells, increasing the inclination angle of the leaves, increasing the grain length, reducing the grain width, reducing the grain thickness, reducing the grain weight, BR sensitivity, positively regulating a BR signal path, increasing the transparency of grains, reducing the compactness of starch cells of the grains, increasing the protein content of the grains and increasing the amylose content of the grains; more preferably, said down-regulating the expression or activity of the GS10 gene in the plant comprises: an inhibitor that down-regulates GS10 gene transcription, protein expression, or protein activity is transferred into the plant.

5. The method of claim 4, wherein the inhibitor is an inhibitory molecule that specifically interferes with the transcription and/or expression of the GS10 gene,

preferably, the suppressor molecule targets the GS10 gene or its transcript or encoded protein as a suppressor,

more preferably, the inhibitory molecule is selected from the group consisting of: (1) a small molecule compound, an antisense nucleic acid, a microRNA, a siRNA, an RNAi, a dsRNA, a sgRNA, an antibody, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1).

6. Use of the GS10 gene as a molecular marker for identifying agronomic traits in plants, the agronomic traits comprising: leaf length, leaf width, single plant yield, cell yield, plant height, tiller number, grain number per ear, ear length, branch number, top awn, heading stage, stem diameter, glume cell width, glume cell length, glume cell unit area, leaf inclination angle, grain length, grain width, grain weight, BR sensitivity, BR signal path, grain transparency, grain starch cell compactness, grain protein content and grain amylose content.

7. An expression cassette for expressing the GS10 gene, which has the following elements in order from 5 'to 3': the 5' UTR region, the ORF sequence of the GS10 gene, and a terminator.

8. Use of the expression cassette of claim 7 for improving an agronomic trait in a crop selected from the group consisting of: reducing the leaf length, increasing the leaf width, reducing the yield of a single plant, reducing the yield of a cell, reducing the plant height, reducing the tillering number, reducing the grain number per spike, reducing the spike length, reducing the number of branches, reducing the awn, shortening the heading stage, increasing the diameter of stems, increasing the width of glume cells, reducing the length of glume cells, increasing the unit area of glume cells, reducing the leaf inclination angle, reducing the grain length, increasing the grain width, increasing the grain thickness, increasing the grain weight, increasing the BR sensitivity, negatively regulating and controlling a BR signal path, reducing the transparency of grains, increasing the compactness of starch cells of the grains, reducing the protein content of the grains and reducing the amylose content of the grains.

9. A sgRNA targeting the GS10 gene or a nucleic acid construct capable of producing the sgRNA,

preferably, the sgRNA is shown in SEQ ID NO 3 or 4.

10. The application of a substance for down-regulating the expression or activity of GS10 gene and a substance for up-regulating the expression or activity of one or two genes selected from GW5 and GW7(GL7) in stabilizing grain shape, improving appearance quality of rice and improving aspect ratio of rice.