CN110241130B - GSN1 Gene, Encoded Protein and Application for Controlling Plant Grain Number and Grain Weight - Google Patents

Abstract

The invention relates to a GSN1 gene for controlling the grain number and the grain weight of a plant, an encoded protein and application thereof, and particularly provides application of a substance, wherein the substance is the GSN1 gene or the encoded protein thereof, or a mutant protein thereof, or an accelerant or an inhibitor thereof, and is used for regulating and controlling the plant characters or preparing a composition for regulating and controlling the plant characters, and the characters comprise one or more characters selected from the following group: (i) a pellet form; (ii) ear size; (iii) setting percentage. The invention also discovers for the first time that plant traits can be improved by modulating the expression level and/or activity of the GSN1 protein in said plants.

Description

GSN1 Gene, Encoded Protein and Application for Controlling Plant Grain Number and Grain Weight

technical field

The invention relates to the field of agronomy, in particular, the invention relates to the GSN1 gene, encoded protein and application thereof for controlling the grain number and grain weight of plants.

Background technique

With the continuous growth of the worldwide population and the gradual deterioration of the global ecological environment, food shortage has become an increasingly serious worldwide problem. It is estimated that the global population will reach 9 billion by 2050, which requires a large output of food crops in the world. Amplitude increased. Therefore, traditional methods of genetic breeding can no longer meet this demand. Using modern molecular genetics theory to study the molecular mechanism of crop yield formation can help us maximize crop yield and meet the urgent needs of population growth. Rice is one of the most important food crops in the world, and it has also been developed into an important model plant in plant science research. The traits of grains per panicle and grain weight of rice seeds are two of the three main factors that determine rice yield (i.e. effective panicles per plant, grains per panicle and grain weight), while grain weight is determined by the Determined by grain length, grain width and grain thickness. Generally speaking, increasing the number of grains per panicle of rice will reduce the thousand-grain weight of rice, on the contrary, increasing the number of grains per panicle of rice will usually reduce the number of grains per panicle of rice. Although the number of grains per panicle and grain weight are both carbon pools, there is competition for the source during development, which makes the two traits check each other. The same inverse correlation between seed size and seed number has also been observed during plant evolution. Therefore, it is of great biological significance to reveal the molecular regulation mechanism between grain number per panicle and grain weight by molecular genetic methods, and it can provide a technical method for genetic improvement of crop yield.

So far, no breakthrough has been achieved in the cloning and research of genes controlling rice grain number and grain weight in this field. Therefore, there is an urgent need in this field to carry out functional research on genes related to controlling rice grain shape and grain weight, so as to improve plant traits.

Contents of the invention

The object of the present invention is to provide related genes for controlling the grain type, grain weight and grain number of plants such as rice and the application thereof.

The first aspect of the present invention provides a use of a substance, the substance is the GSN1 gene or its encoded protein, or its mutant protein, or its promoter or inhibitor, for regulating the traits of plants or for preparing and regulating plant Compositions of traits comprising one or more traits selected from the group consisting of:

(i) grain type;

(ii) the number of grains per ear;

(iii) Seed setting rate.

In another preferred example, the traits also include one or more traits selected from the following group:

(iv) plant height;

(v) number of tillers;

(vi) Yield per plant;

(vii) Number of primary branches;

(viii) Number of secondary branches;

(ix) siliques;

(x) Ear length.

In another preferred example, the grain type includes grain length, grain width, and/or thousand grain weight.

In another preferred example, the composition is an agricultural composition.

The second aspect of the present invention provides a use of a substance, the substance is the GSN1 gene or its encoded protein, the GSN1 mutant protein with enhanced activity, or its promoter, to regulate the traits of plants or to prepare a combination for regulating the traits of plants Objects, wherein the traits include one or more traits selected from the group consisting of:

(i) reduced particle size;

(ii) increase the number of grains per ear;

(iii) Increase seed setting rate.

In another preferred example, the activity is the dephosphorylation activity of GSN1.

In another preferred example, the activity enhancement refers to the ratio of the dephosphorylation activity A1 of the mutein to the dephosphorylation activity A0 of the wild-type GSN1 protein ≥ 1.2, preferably ≥ 1.5.

In another preferred embodiment, the substance is the GSN1 gene or its encoded protein, or its promoter, which is also used to regulate one or more of the following traits of plants:

(iv) increasing the number of tillers in grasses;

(v) lead to increased branching in Arabidopsis;

(vi) results in silique shortening in Arabidopsis.

In another preferred example, the promoter is a small molecular compound that promotes the expression of the GSN1 gene or its encoded protein.

In another preferred embodiment, the accelerator is selected from the group consisting of small molecule compounds, nucleic acid molecules, or combinations thereof.

In another preferred example, the plants include crops, forestry plants, vegetables, fruits, flowers, pastures (including turfgrass); preferably include grasses, leguminous plants and cruciferous plants, more preferably include rice, Corn, sorghum, wheat, soybean or Arabidopsis.

In another preferred example, the plants include: rice, wheat, corn, sorghum, and cruciferous plants (cabbage).

In another preferred embodiment, the rice is selected from the group consisting of indica rice, japonica rice, or a combination thereof.

In another preferred example, the GSN1 gene is selected from the group consisting of cDNA sequence, genome sequence, or a combination thereof.

In another preferred example, the GSN1 gene is from a Poaceae plant.

In another preferred embodiment, the GSN1 gene is from one or more plants selected from the group consisting of rice, corn, sorghum, wheat, soybean or Arabidopsis, millet, alfalfa, Brachypodium distachys, tomato , Populus trichocarpa, peach, columbine.

In another preferred example, the amino acid sequence of the GSN1 protein is selected from the following group:

(i) a polypeptide having the amino acid sequence shown in SEQ ID NO.:2;

(ii) The amino acid sequence shown in SEQ ID NO.: 2 is formed by substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and has the function of regulating agronomic traits or has A polypeptide derived from (i) that is dephosphorylated; or

(iii) The amino acid sequence has a homology of ≥80% (preferably ≥90%, more preferably ≥95% or ≥98%) with the amino acid sequence shown in SEQ ID NO.:2, a polypeptide having the GSN1 activity .

In another preferred example, the nucleotide sequence of the GSN1 gene is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.:2;

(b) a polynucleotide whose sequence is as shown in SEQ ID NO.:1;

(c) a polynucleotide whose nucleotide sequence has a homology of ≥75% (preferably ≥85%, more preferably ≥90% or ≥95%) to the sequence shown in SEQ ID NO.:1;

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

(e) A polynucleotide complementary to the polynucleotide described in any one of (a)-(d).

The third aspect of the present invention provides a use of a substance, the substance is a GSN1 mutant protein or an inhibitor thereof, and the mutant protein is a GSN1 mutant protein whose activity is reduced compared with the wild-type GSN1 protein, and is used for regulating plant One or more of the following traits:

(i) increasing grain size;

(ii) reduce the number of grains per ear;

(iii) Reduce seed setting rate.

In another preferred embodiment, the substance is a mutant protein with reduced GSN1 activity.

In another preferred example, the activity is the dephosphorylation activity of GSN1.

In another preferred example, the decrease in activity means that the ratio of the dephosphorylation activity A1 of the mutant protein to the dephosphorylation activity A0 of the wild-type GSN1 protein is ≤0.8, preferably ≤0.6.

In another preferred example, the GSN1 mutein is an activity-losing mutein.

In another preferred example, the substance is also used to regulate one or more of the following traits of plants:

(iv) reducing the number of tillers in grasses;

(v) results in reduced branching in Arabidopsis;

(vi) leads to elongated siliques in Arabidopsis.

In another preferred example, the amino acid sequence of the mutein is selected from the following group:

(i) a polypeptide having the amino acid sequence shown in SEQ ID NO.:3;

(ii) The amino acid sequence shown in SEQ ID NO.: 3 is formed by substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and has the function of regulating agronomic traits, a polypeptide derived from (i); or

(iii) Amino acid sequence has a homology of ≥80% (preferably ≥90%, more preferably ≥95% or 98%) with the amino acid sequence shown in SEQ ID NO.:3, and has decreased GSN1 protein activity peptide.

In another preferred example, the nucleotide sequence encoding the mutein is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.:3;

(b) a polynucleotide whose sequence is as shown in SEQ ID NO.:4;

(c) a polynucleotide whose nucleotide sequence is ≥75% (preferably ≥85%, more preferably ≥90%) homologous to the sequence shown in SEQ ID NO.:4;

(d) truncating or adding 1-60 (preferably 1-30, more preferably 1-10) cores at the 5' end and/or 3' end of the polynucleotide shown in SEQ ID NO.:4 polynucleotides of nucleotides;

(e) A polynucleotide complementary to the polynucleotide described in any one of (a)-(d).

In another preferred embodiment, the inhibitor is selected from the group consisting of small molecule compounds, antisense nucleic acids, microRNA, siRNA, RNAi, Crispr reagents, or combinations thereof.

In another preferred example, when the substance is the GSN1 gene or its encoded protein (including mutant proteins with increased activity), or its promoter, the substance is also used for:

(a) Dephosphorylation modification of MPK1 protein.

In another preferred example, when the substance dephosphorylates the MPK1 protein, the traits of the plant are as follows: the grain size becomes smaller and/or the number of grains per panicle increases.

In another preferred example, when the substance is a mutant protein with reduced GSN1 activity or a GSN1 inhibitor, the substance is also used for:

(a) Inhibition of dephosphorylation modification of MPK1 protein.

In another preferred example, when the substance (such as a mutant protein with reduced GSN1 activity or a GSN1 inhibitor) inhibits the dephosphorylation modification of the MPK1 protein, the traits of the plant are: the grain size becomes larger, and/or The number of grains per spike is reduced.

The fourth aspect of the present invention provides a method for improving plant traits, comprising the steps of:

(i) Regulating the expression level and/or activity of the GSN1 protein in the plant, thereby improving plant traits.

In another preference, in step (i), the relative expression of wild-type GSN1 protein and mutant gns1 protein is regulated, thereby improving plant traits.

In another preferred example, the dephosphorylation activity of the mutant gns1 protein is reduced or absent.

In another preferred embodiment, in step (i), the expression level and/or activity of the wild-type GSN1 gene is up-regulated.

In another preferred embodiment, in step (i), the expression level and/or activity of the wild-type GSN1 gene is down-regulated.

In another preferred example, the amino acid sequence of the wild-type GSN1 protein is shown in SEQ ID NO.:2.

In another preferred example, the nucleotide sequence of the GSN1 gene is shown in SEQ ID NO.:1.

In another preferred example, the amino acid sequence of the mutant gsn1 protein is shown in SEQ ID NO.:3.

In another preferred example, the nucleotide sequence of the gsn1 gene is shown in SEQ ID NO.:4.

In another preferred example, when the ratio of the activity E1 of GSN1 in the plant to the background activity E0 of the wild-type GSN in the plant is ≥2 times, preferably ≥5 times, more preferably ≥10 times, The improved traits of the plant are smaller grain shape and/or increased grain number per panicle.

In another preferred example, when the ratio of the activity E1 of GSN1 in the plant to the background activity E0 of the wild-type GSN in the plant is ≤1/2, preferably ≤1/5, more preferably ≤1/ At 10 o'clock, the traits of the plant were improved as the grain shape became larger and/or the number of grains per panicle decreased.

The fifth aspect of the present invention provides a method for dephosphorylating MPK1 protein, comprising the steps of:

The GSN1 protein is contacted with the MPK1 protein, thereby dephosphorylating the MPK1 protein.

In another preferred example, the GSN1 protein includes wild-type GSN1 protein or its mutant protein.

In another preferred example, the mutein includes a mutein with enhanced activity.

In another preferred example, the source of the MPK1 protein is selected from one or more plants of the following group: rice, corn, sorghum, wheat, soybean or Arabidopsis, millet, alfalfa, Brachypodium distachys, tomato , Populus trichocarpa, peach, columbine.

The sixth aspect of the present invention provides an isolated mutant GSN1 protein, the mutant GSN1 protein is a non-natural protein, and the mutant GSN1 protein has the activity of regulating plant traits, and the traits are selected from one of the following groups One or more traits:

(i) grain type;

(ii) the number of grains per ear;

(iii) Seed setting rate.

In another preferred example, the mutant GSN1 protein includes a mutant protein with increased activity or a mutant protein with decreased activity.

In another preferred example, the mutant GSN1 protein is a mutant protein with reduced activity.

In another preferred example, the mutant GSN1 protein is mutated at the core amino acid of the wild-type GSN1 protein corresponding to SEQ ID NO.:2 selected from the following group:

Serine (S) at position 146.

In another preferred example, the amino acid at position 146 is mutated to phenylalanine (F).

In another preferred example, the amino acid sequence of the mutant GSN1 protein is shown in SEQ ID NO.:3.

In another preferred example, except for the mutation (such as amino acid 146), the remaining amino acid sequence of the mutant GSN1 protein is identical or substantially identical to the sequence shown in SEQ ID NO.:2.

In another preferred embodiment, the substantially identical is at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acid differences, wherein, The difference includes amino acid substitution, deletion or addition, and the mutant GSN1 protein still has dephosphorylation activity (such as dephosphorylation activity of MPK1 protein).

In another preferred example, the homology with the sequence shown in SEQ ID NO.: 2 is at least 80%, preferably at least 85% or 90%, more preferably at least 95%, and most preferably at least 98%.

In another preferred example, the mutant GSN1 protein additionally contains auxiliary elements selected from the group consisting of signal peptide, secretory peptide, tag sequence (such as 6His), or a combination thereof.

In another preferred example, the mutant GSN1 protein has the activity of regulating plant traits, and the traits are selected from one or more traits of the following group:

(i) The particle shape becomes larger;

(ii) the number of grains per ear decreases;

(iii) Decreased seed setting rate.

The seventh aspect of the present invention provides a polynucleotide encoding the mutant GSN1 protein described in the sixth aspect of the present invention.

In another preferred embodiment, the polynucleotide is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.:3;

(b) a polynucleotide whose sequence is as shown in SEQ ID NO.:4;

(c) A polynucleotide whose nucleotide sequence has a homology of ≥80% (preferably ≥90%) to the sequence shown in SEQ ID NO.:4 and encodes the polypeptide shown in SEQ ID NO.:3;

(d) A polynucleotide complementary to the polynucleotide described in any one of (a)-(c).

In another preferred embodiment, the polynucleotide is selected from the group consisting of DNA sequence, RNA sequence, or a combination thereof.

The eighth aspect of the present invention provides a vector containing the polynucleotide described in the seventh aspect of the present invention.

In another preferred example, the vectors include expression vectors, shuttle vectors, and integration vectors.

The ninth aspect of the present invention provides a host cell containing the vector of the eighth aspect of the present invention, or the polynucleotide of the seventh aspect of the present invention integrated in its genome.

In another preferred embodiment, the host cells are eukaryotic cells, such as yeast cells or plant cells.

In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

The tenth aspect of the present invention provides a method for producing the mutant GSN1 protein described in the sixth aspect of the present invention, comprising the steps of:

Under conditions suitable for expression, culturing the host cell according to the ninth aspect of the present invention, so as to express the mutant GSN1 protein; and

The mutant GSN1 protein was isolated.

The eleventh invention of the present invention provides an enzyme preparation comprising the mutant GSN1 protein described in the sixth aspect of the present invention.

The twelfth aspect of the present invention provides a method for preparing transgenic plants, comprising the steps of:

The polynucleotide encoding the protein described in the sixth aspect of the present invention is introduced into plant cells, and the plant cells are cultured to regenerate plants.

In another preferred embodiment, the method includes the steps of:

(s1) providing an Agrobacterium carrying an expression vector containing a polynucleotide encoding the protein described in the sixth aspect of the present invention;

(s2) contacting the plant cell, tissue or organ with the Agrobacterium in step (s1), so that the polynucleotide encoding the protein described in the sixth aspect of the present invention is transferred into the plant cell, tissue or organ;

(s3) Screening the plant cells or tissues or organs transfected with the polynucleotide encoding the protein described in the sixth aspect of the present invention; and

(s4) regenerating the plant cells or tissues or organs in step (s3) into plants.

The thirteenth aspect of the present invention provides a method for identifying large-grained or small-grained plants, comprising the steps of:

(i) Identify whether the plant sample has the polypeptide or its coding gene of the amino acid sequence shown in SEQ ID NO.: 2 or 3; if it has the polypeptide or its coding gene of the amino acid sequence shown in SEQ ID NO.: 2, it is small grain type A plant, such as a polypeptide having an amino acid sequence shown in SEQ ID NO.: 3 or a gene encoding it, is a large-grained plant.

In another preferred example, in step (i), it is determined by sequencing whether the plant sample has the nucleotide sequence shown in SEQ ID NO.:1 or SEQ ID NO.:4.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

The following drawings are used to illustrate specific embodiments of the present invention, but not to limit the scope of the present invention defined by the claims.

Figure 1 shows the phenotypic characteristics of gsn1 mutants.

(A) Plant type of wild-type FAZ1 (abbreviation of "Fengaizhan 1", an indica rice variety) and gsn1; (B) Grain type of FAZ1 and gsn1; (C) Grains of FAZ1 and gsn1 after shelling; (D ) panicle type of FAZ1 and gsn1; (E) pistil of FAZ1 and gsn1; (F) anther of FAZ1 and gsn1; (G-Q) phenotype comparison of FAZ1 and gsn1, including plant height (G), tiller number (H), Grain length (I), grain width (J), thousand-grain weight (K), number of grains per ear (L), seed setting rate (M), yield per plant (N), number of primary branches (O), number of secondary branches ( P) and panicle length (Q).

Figure 2 shows the positional cloning and genetic complementation verification of the GSN1 gene.

(A) Map-based cloning of GSN1; (B) Sequence alignment of conserved regions of GSN1 proteins in different species; (C-D) panicle type (C) and grain type (D) of FAZ1, gsn1 and complementary plant gGSN1com; (E-F) FAZ1 And the panicle type (E) and grain type (F) of the RNAi plant GSN1RNAi; (G-H) FAZ1 and the panicle type (G) and grain type (H) of the overexpression plant UBI::GSN1; (I-J) FAZ1 and the RNAi plant GSN1RNAi Comparison of grain length (I) and number of grains per ear (J) in (K-L) comparison of grain length (K) and number of grains per ear (L) of FAZ1, gsn1 and overexpression plants UBI::GSN1.

Figure 3 shows the phenotypic characteristics of the japonica rice variety 95-22 and the near isogenic line NIL(gsn1).

(A) Plant type of 95-22 and near isogenic line NIL(gsn1); (B) panicle type of 95-22 and near isogenic line NIL(gsn1); (C) 95-22 and near isogenic line NIL (gsn1) grain shape; (D) 95-22 and near isogenic line NIL (gsn1) grain shape; (E-G) 95-22 and near isogenic line NIL (gsn1) grain length (E), Comparison of grain width (F) and grain number per panicle (G).

Figure 4 shows that inhibition of GSN1 leads to increased grain shape and decreased grain number per panicle in rice.

(A) Plant type, grain type and panicle type phenotype of japonica rice variety ZH11 and GSN1RNAi plants; (B) Plant type, grain type and panicle type phenotype of ZH11 and GSN1CRISPR plants; (C-F) grain length of ZH11 and GSN1RNAi ( C), grain width (D), number of grains per ear (E), and relative expression of GSN1 gene in vivo (F); (G-J) grain length (G), grain width (H), and number of grains per ear of ZH11 and GSN1 CRISPR (I), and the GSN1 gene CRISPR/Cas9 gene editing site (J).

Figure 5 shows that overexpression of GSN1 derived from the gsn1 mutant in the ZH11 background has a dominant negative inhibitory effect.

(A) Plant type, grain type and panicle type of japonica rice ZH11 and overexpressed GSN1FAZ1 (derived from FAZ1) plant UBI::GSN1FAZ1; (B) Japonica rice ZH11 and overexpressed GSN1S146F (derived from gsn1) plant UBI::GSN1S146F Type, grain type and panicle type; (C-F) Grain length (C), grain width (D), grain number per panicle (E) and relative expression level of GSN1 (F) of ZH11 and UBI::GSN1FAZ1; (G-J) ZH11 and UBI::GSN1S146F grain length (G), grain width (H), grain number per panicle (I) and relative expression level of GSN1 (J).

Figure 6 shows that the overexpression of GSN1 resulted in an increase in the number of grains per panicle and a decrease in grain shape.

(A) Plant type, grain type and panicle type of japonica rice ZH11 and overexpressed GSN1ZH11 (derived from ZH11) plant UBI::GSN1ZH11; (B) Plant type, grain type and panicle type of japonica rice ZH11 and overexpressed GSN1ZH11 plant 35S::GSN1ZH11 panicle type; (C-F) grain length (C), grain width (D), grain number per panicle (E) and relative expression level of GSN1 (F) of (C-F) ZH11 and UBI::GSN1ZH11; (G-J) ZH11 and 35S::GSN1ZH11 Grain length (G), grain width (H), grain number per panicle (I) and relative expression level of GSN1 (J).

Figure 7 shows that mutations in GSN1 lead to accelerated cell division without affecting cell expansion or elongation.

(A) glumes of FAZ1 and gsn1 at heading stage; (B) cross-section of glumes of FAZ1 and gsn1; (C) partial enlargement of the cross-section of glumes; (D) comparison of cross-sections of FAZ1 and gsn1 anthers; (E) glume Comparison of the number and size of parenchyma cells in the cross-section of the shell; (F) the comparison of the area of the septum vascular bundles and the number of cells in the cross-section of the anther; (G) scanning electron microscope observation of papillae on the epidermis of the glume of FAZ1 and gsn1; (H) the glume of FAZ1 and gsn1 Scanning electron microscope observation of epidermal cells in the shell; (I) Comparison of papillae spacing in the outer glume of FAZ1 and gsn1; (J) Comparison of the size of epidermal cells in the glume of FAZ1 and gsn1; (K) Flow of glume cells in FAZ1 and gsn1 young panicles Cell analysis; (L) Comparison of the cell cycle distribution of FAZ1 and gsn1 young spike glume cells.

Figure 8 shows that GSN1 encodes a dual-specificity phosphatase localized in the cytoplasm, and the mutation of serine at position 146 to phenylalanine results in the loss of dephosphorylation activity of the GSN1 protein.

(A-B) PNPP (A) and OMFP (B) were used as substrates to detect the enzyme activity of GSN1 and GSN1S146F; (C) The kinetic curve of GSN1 enzyme activity; (D) GSN1 was localized in the cytoplasm of tobacco leaf epidermal cells; ( E) GSN1 is localized in the cytoplasm of rice protoplasts; (F-G) Expression patterns of GSN1 at seedling (F) and reproductive growth (G) stages.

Figure 9 shows that GSN1 and OsMPK1 specifically interact and dephosphorylate OsMPK1.

(A) Yeast two-hybrid experiment verified the interaction between GSN1 and OsMPK1 in yeast cells; (B) BiFC experiment verified the interaction between GSN1 and OsMPK1 in tobacco leaf epidermal cells; (C) co-IP experiment proved the interaction between GSN1 and OsMPK1 (D) GSN1 can dephosphorylate OsMPK1, but GSN1S146F cannot dephosphorylate OsMPK1.

Figure 10 shows that GSN1 is a negative regulator of the rice OsMKKK10-OsMKK4-OsMPK1 cascade pathway.

(A) Spike type of FAZ1, gsn1, OsMPK1RNAi and gsn1/OsMPK1RNAi double mutation; (B) Grain type of FAZ1, gsn1, OsMPK1RNAi and gsn1/OsMPK1RNAi double mutation; (C) FAZ1, gsn1, OsMKK4CRISPR and gsn1/OsMKK4CRISPR double mutation (D) Grain type of FAZ1, gsn1, OsMKK4CRISPR and gsn1/OsMKK4CRISPR double spike; (E) FAZ1, gsn1, OsMKKK10CRISPR and gsn1/OsMKKK10CRISPR double spike type; (F) FAZ1, gsn1, OsMKKK10CRISPR and gsn1 /OsMKKK10CRISPR double process grain type; (G) comparison of grain length of FAZ1, gsn1, OsMPK1RNAi and gsn1/OsMPK1RNAi double process; (H) comparison of grain length of FAZ1, gsn1, OsMKK4CRISPR and gsn1/OsMKK4CRISPR double process; (I) FAZ1 , gsn1, OsMKKK10CRISPR and gsn1/OsMKKK10CRISPR doublet grain length comparison.

Figure 11 shows that the overexpression of the rice GSN1 gene in Arabidopsis resulted in more branches and shorter siliques. It shows that GSN1 also plays a role in other species and has functional conservation. Among them, CK is the control, #1 and #2 are two independent lines of 35S::GSN1 overexpression transgenic plants, the scale bar is 5cm.

Detailed ways

After extensive and in-depth research, the present inventor unexpectedly discovered a plant (such as rice) GSN1 gene or its encoded protein, or its mutant protein, or its promoter for the first time by studying a large number of plant trait loci Or inhibitors, for regulating the traits of plants, said traits include one or more traits selected from the group: (i) grain type; (ii) ear grain number; (iii) seed setting rate. In addition, the present inventors also unexpectedly found for the first time that mutating the 146th amino acid (serine) of the GSN1 protein in plants to phenylalanine can significantly increase the grain shape and reduce the number of grains per panicle. In addition, the present inventors unexpectedly discovered for the first time that the traits of plants can be improved by regulating the expression activity of GSN1 protein in plants. Experimental results show that the wild-type GSN1 protein of the present invention can interact with OsMPK1 and dephosphorylate OsMPK1, while a mutant gsn1 protein that lacks activity cannot dephosphorylate phosphorylated GST-OsMPK1 grooming. On this basis, the present inventors have completed the present invention.

the term

As used herein, the term "AxxB" means that amino acid A at position xx is changed to amino acid B, for example "D308N" means that amino acid D at position 308 is mutated to N, and so on.

Mutant protein of the present invention and encoding nucleic acid thereof

As used herein, the terms "mutant protein", "mutant protein of the present invention", "mutant GSN1 protein", and "mutant GSN1 protein of the present invention" are used interchangeably, and all refer to a non-naturally occurring GSN1 mutein, and the The mutant protein is an artificially modified protein based on the protein shown in SEQID NO.: 2, wherein the mutant protein contains core amino acids related to the activity of regulating plant traits (such as grain shape, ear grain weight), and the core amino acids At least one of them is artificially modified; and the mutant protein of the present invention has the activity of increasing grain shape and/or reducing the number of grains per panicle.

The term "core amino acid" refers to an amino acid that is based on SEQ ID NO.:2 and that is at least 80% homologous to SEQ ID NO.:2, such as 84%, 85%, 90%, 92%, 95%, 98%. In the sequence, the corresponding position is the specific amino acid described herein, such as based on the sequence shown in SEQ ID NO.: 2, the core amino acid is: Serine (S) at position 146;

Moreover, the mutant protein with reduced activity obtained by mutating the above-mentioned core amino acid has the activity of increasing grain shape and/or reducing the number of grains per panicle.

Preferably, in the present invention, the core amino acid of the present invention is mutated as follows: the 146th serine (S) is mutated to phenylalanine (F), which can lead to a significant decrease or even loss of the activity of the GSN1 protein.

It should be understood that the numbering of the amino acids in the mutant protein of the present invention is based on SEQ ID NO.: 2. When the homology of a specific mutant protein to the sequence shown in SEQ ID NO.: 2 reaches 80% or more, the amino acid number of the mutant protein The amino acid numbering may have a misplacement relative to the amino acid numbering of SEQ ID NO.: 2, such as 1-5 positions to the N-terminal or C-terminal of the amino acid, and using conventional sequence alignment techniques in the art, those skilled in the art usually It can be understood that such dislocations are within a reasonable range, and should not cause homology up to 80% (such as 90%, 95%, 98%), having the same or similar increased grain shape, And/or active muteins that reduce the number of grains per ear are not within the scope of the muteins of the present invention.

The mutein of the present invention is a synthetic protein or a recombinant protein, that is, it may be a product of chemical synthesis, or produced from a prokaryotic or eukaryotic host (eg, bacteria, yeast, plant) using recombinant technology. Depending on the host used in the recombinant production protocol, muteins of the invention may be glycosylated, or may be non-glycosylated. The muteins of the invention may or may not include an initial methionine residue.

The present invention also includes fragments, derivatives and analogs of said muteins. As used herein, the terms "fragment", "derivative" and "analogue" refer to a protein that substantially retains the same biological function or activity of the mutein.

The mutein fragments, derivatives or analogs of the present invention may be (i) muteins having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a mutein with a substitution group in one or more amino acid residues, or (iii) a mature mutein combined with another compound (such as an elongated mutein A mutein formed by fusion of a compound with a half-life, such as polyethylene glycol), or (iv) an additional amino acid sequence fused to the mutein sequence (such as a leader sequence or secretory sequence or used to purify the mutein The sequence or protein sequence, or the fusion protein formed with the antigen IgG fragment). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein. In the present invention, the conservatively substituted amino acid is preferably produced by performing amino acid substitution according to Table I.

Table I

initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

The mutein with reduced activity of the present invention has the activity of increasing grain shape and/or reducing the number of grains per panicle.

An activity-deficient GSN1 mutant protein is shown in SEQ ID NO.:3. It should be understood that, compared with the sequence shown in SEQ ID NO.: 3, the mutant protein of the present invention generally has higher homology (identity), preferably, the mutant protein and the sequence shown in SEQ ID NO.: 3 The homology of the indicated sequences is at least 80%, preferably at least 85%-90%, more preferably at least 95%, most preferably at least 98%.

In addition, the muteins of the present invention can also be modified. Modified (usually without altering primary structure) forms include: chemically derivatized forms of muteins such as acetylation or carboxylation in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications during the synthesis and processing of the mutein or during further processing steps. This modification can be accomplished by exposing the mutein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are muteins that have been modified to increase their resistance to proteolysis or to optimize solubility.

The present invention also provides polynucleotide sequences encoding GSN1 polypeptides, proteins or variants thereof. A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include: DNA, genomic DNA, or synthetic DNA, and DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide can be the same as the sequence of the coding region shown in SEQ ID NO.: 4 or a degenerate variant.

The term "polynucleotide encoding a mutein" may include a polynucleotide encoding a mutein of the present invention, or may also include additional coding and/or non-coding sequences. In the present invention, a preferred polynucleotide sequence encoding the mutein gsn1 is shown in SEQ ID NO.:4.

The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or muteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering its encoded mutein. Function.

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and 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 hybridizable under stringent conditions (or stringent conditions) to the polynucleotides of the present invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%.

The muteins and polynucleotides of the present invention are preferably provided in an isolated form, more preferably, purified to homogeneity.

It should be understood that although the GSN1 gene of the present invention is preferably from rice, other plants that are highly homologous (such as having more than 80%, such as 85%, 90%, 95% or even 98% sequence identity) to the rice GSN1 gene from other plants Genes are also contemplated by the present invention. Methods and tools for aligning sequence identities are also well known in the art, such as BLAST.

The full-length polynucleotide sequence of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. 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 relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the polynucleotide of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, And can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

Wild-type GSN1 protein and gene

As used herein, "wild-type GSN1 protein" and "wild-type GSN1 protein controlling rice grain number and grain weight" are used interchangeably, and refer to a naturally occurring, unmodified GSN1 protein whose nucleotides can be passed through It is obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and its amino acid sequence can be deduced from the nucleotide sequence.

In the present invention, the gene of GSN1 includes genome gene and cDNA gene.

For rice, the amino acid sequence of a typical wild-type GSN1 protein is shown in SEQ ID NO.:2. A typical cDNA nucleotide sequence encoding wild-type GSN1 protein is shown in SEQ ID NO.: 1, and the accession number of the rice GSN1 genome sequence is LOC_Os05g02500.

Representative GSN1 homologs of other species include (but are not limited to): the GSN1 gene of rice (OsGSN1), the GSN1 homolog of maize (GRMZM2G005350 and rice similarity 84.4%), the GSN1 homolog of sorghum (Sobic .009G017400 and rice similarity 89.5%), wheat GSN1 homologous gene (A genome Traes_1AS_73C2E93D4 and rice similarity 87%; B genome Traes_1BS_90AEC8678 and rice similarity 84.6%; D genome Traes_1DS_4C0964710 and rice similarity 88.1%), millet GSN1 homologous gene (Seita.3G061100 and rice similarity 80.4%), Brachypodium distachyon GSN1 homologous gene (Bradi2g37450 and rice similarity 83.4%), soybean GSN1 homologous gene (Glyma.02G095100 and rice similarity 62.4%), Arabidopsis GSN1 homolog (AT3G55270 and rice similarity 59.7%), alfalfa GSN1 homolog (Medtr7g023250 and rice similarity 65.7%), tomato GSN1 homolog (Solyc05g054700 and rice similarity 53.1%), the GSN1 homologous gene of Populus trichocarpa (Potri.008G049900 and rice similarity 58.8%), the GSN1 homologous gene of peach (Prupe.2G228100 and rice similarity 63.4%), the GSN1 homologous gene of Aquilegia (64.5% similarity between Aqcoe1G474900 and rice).

Expression vector

The present invention also relates to vectors containing the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors of the present invention or the mutant protein coding sequences of the present invention, and methods for producing the polypeptides of the present invention through recombinant techniques.

The polynucleotide sequences of the present invention can be used to express or produce recombinant muteins by conventional recombinant DNA techniques. Generally speaking, there are the following steps:

(1). Use the polynucleotide (or variant) encoding the mutein of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

The present invention also provides a recombinant vector comprising the gene of the present invention. As a preferred manner, the downstream of the promoter of the recombinant vector contains multiple cloning sites or at least one restriction site. When the target gene of the present invention needs to be expressed, the target gene is linked into a suitable multiple cloning site or restriction site, so that the target gene is operably linked to the promoter. As another preferred mode, the recombinant vector includes (from 5' to 3' direction): a promoter, a target gene, and a terminator. If necessary, the recombinant vector may also include elements selected from the group consisting of: 3' polynucleotide signal; non-translated nucleic acid sequence; transport and targeting nucleic acid sequence; resistance selectable marker (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); enhancers; or operons.

In the present invention, the polynucleotide sequence encoding the mutant protein can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding muteins of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Those of ordinary skill in the art can use well-known methods to construct expression vectors containing the genes described in the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. When using the gene of the present invention to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before the transcription initiation nucleotide.

An expression cassette or vector comprising the gene of the present invention can be used to transform an appropriate host cell such that the host expresses the protein. The host cells can be prokaryotic cells, such as Escherichia coli, Streptomyces, and Agrobacterium; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Those of ordinary skill in the art will know how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism (such as Escherichia coli), it can be treated with CaCl ₂ or electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Transformation of plants can also use methods such as Agrobacterium transformation or gene gun transformation, such as leaf disk method, immature embryo transformation method, flower bud soaking method and the like. Transformed plant cells, tissues or organs can be regenerated into plants by conventional methods, so as to obtain transgenic plants.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (eg rice cells).

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The main advantages of the present invention include:

(1) The GSN1 of the present invention or its encoded protein, or its mutant protein, or its accelerator, or its inhibitor can regulate the agronomic traits of plants (eg, grain shape, grain number per ear, seed setting rate, etc.).

(2) Mutating the 146th amino acid (serine) of the GSN1 protein in the plant to phenylalanine, which can significantly improve the plant's traits (such as increasing the grain shape, reducing the number of grains per ear, etc.).

(3) The GSN1 protein of the present invention has dephosphorylation activity, while the mutated GSN1 ^S146F protein loses the dephosphorylation ability.

(4) The present invention finds for the first time that the complete OsMKKK10-OsMKK4-OsMPK1 cascade signaling pathway is involved in the development of rice grain shape and panicle shape.

(5) The present invention finds for the first time that GSN1 is a negative regulator of the OsMKKK10-OsMKK4-OsMPK1 signaling pathway, and the number of grains per panicle and grain type (grain weight) of rice can be balanced by precise regulation of the signaling pathway.

(6) The present invention finds for the first time that the plant OsMKKK10-OsMKK4-OsMPK1 cascade signaling pathway is a negative regulatory factor involved in plant development.

(7) The present invention discovers, for the first time, the molecular genetic mechanism based on GSN1 that regulates the developmental balance of plant seed size and seed number.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions. Unless otherwise specified, the materials and reagents used in the examples are all commercially available products.

general method

1. Experimental materials and positional cloning

In the invention, the indica rice variety FAZ1 is used for EMS mutagenesis, then the panicle type and grain type mutant gsn1 is screened, and the background is purified by backcrossing it with FAZ1. In order to locate the gsn1 mutant gene, it was crossed with the japonica rice variety ZH11 to generate an F ₁ population, and then an F ₂ segregated population for subsequent mapping. First, 682 rice plants were used to initially locate GSN1 at the short arm of chromosome 5, between molecular markers G05220 and G05591. Then, 13792 rice plants were used to fine-map it between molecular markers G05333 and G05340, about 17.5kb. There are two candidate genes LOC_Os05g02490 and LOC_Os05g02500 in this interval. We found that the 437th base in the coding region of the candidate gene LOC_Os05g02500 was mutated from C to T by primer GSN1 Genotyping sequencing, which led to the mutation of 146th serine to phenylalanine. Therefore, it is speculated that the candidate gene LOC_Os05g02500 may be GSN1.

The 5' end oligonucleotide primer sequence of G05220 is:

5'-ATAGCTAGAGAGAGCGGTCA-3' (SEQ ID NO.:5)

The 3' end primer sequence is:

5'-GAGAGTAACTGAAGCAGCAAG-3'(SEQ ID NO.:6)

The 5' end oligonucleotide primer sequence of G05591 is:

5'-TGACTTTAAGGGCCTGTTT-3' (SEQ ID NO.: 7)

The 3' end primer sequence is:

5'-GGGGTATTTCAGACAATTCA-3' (SEQ ID NO.:8)

The 5' end oligonucleotide primer sequence of G05333 is:

5'-CTGGATATACATGTGTCTAATTCT-3' (SEQ ID NO.:9)

The 3' end primer sequence is:

5'-GCATATCCAGGTTAATTTATAGC-3'(SEQ ID NO.:10)

The 5' end oligonucleotide primer sequence of G05340 is:

5'-TGGCTAAAATAATATCGTATCG-3'(SEQ ID NO.:11)

The 3' end primer sequence is:

5'-AGGATCTCAGTTGCAGTAATCT-3' (SEQ ID NO.: 12)

The 5' end oligonucleotide primer sequence for GSN1 Genotyping is:

5'-GGGGGTTTCTTCTACTTTTGTTTTT-3' (SEQ ID NO.: 13)

The 3' end primer sequence is:

5'-CTCCACATCAAGTACGCTATCACC-3' (SEQ ID NO.: 14)

2. Genetic complementation verification, overexpression, RNA interference and CRISPR/Cas9 gene editing

In order to further verify the candidate gene LOC_Os05g02500, the full-length genome sequence of LOC_Os05g02500 derived from the wild-type FAZ1 was constructed on the pCOMBIA1300 complementary vector, and then genetically transformed by the rice immature embryo transformation method mediated by Agrobacterium tumefaciens EHA105, and the transgenic positive lines were screened . In addition, the GSN1 gene derived from FAZ1 was constructed on the pCOMBIA1301 vector, and the GSN1 gene was overexpressed under the ubiquitin promoter. In order to interfere with the expression of GSN1 and OsMPK1, artificial small interfering RNA and antisense RNA were designed respectively to inhibit the expression of GSN1 and OsMPK1. CRISPR/Cas9 technology is used for gene editing. According to experimental needs, CRISPR/Cas9 gene knockout vectors were designed for several target genes (GSN1, OsMKK4, OsMKKK10) for knockout of target genes. Similar to genetic complementation, the genetic transformation was carried out through the rice immature embryo transformation method mediated by Agrobacterium tumefaciens EHA105, and the transgenic positive lines were screened, planted in the field, and the phenotype was investigated in the transgenic _T2 generation.

The 5' end oligonucleotide primer sequence constructed by the pCOMBIA1300 complementary vector is:

5'-GATCAATAAAAGATGGTCATCAGTG-3' (SEQ ID NO.: 15)

The 3' end primer sequence is:

5'-GGAGATAGGAAAGGTGGTGATGT-3' (SEQ ID NO.: 16)

The 5' end oligonucleotide primer sequence constructed by the pCOMBIA1301 overexpression vector is:

5'-CACCATGGCCACGCCCGACGACGGCG-3'(SEQ ID NO.:17)

The 3' end primer sequence is:

5'-ACGAGCATTCAGCACTTCCAAAAGAT-3' (SEQ ID NO.: 18)

The 5' end oligonucleotide primer sequence constructed by the GSN1 artificial small interfering RNA vector is:

5'-TCGGTACCCAGCAGCAGCCACAGCAAAA-3'(SEQ ID NO.:19)

The 3' end primer sequence is:

5'-TCTCTAGAGCTGCTGATGCTGATGCCAT-3' (SEQ ID NO.:20)

The 5' end oligonucleotide primer sequence constructed by the OsMPK1 artificial antisense RNA vector is:

5'-CCCGGATCCGAGCTCATGGACGCCGGGGCGCAGCC-3'(SEQ ID NO.:21)

The 3' end primer sequence is:

5'-GGGGGTACCACTAGTTTGTACTTGGCGGTGACCTC-3' (SEQ ID NO.: 22)

The 5' end oligonucleotide primer sequence constructed by CRISPR/Cas9 knockout GSN1 vector is:

5'-GGCAGTTCTGGCGCTCCGCGTCG-3'(SEQ ID NO.:23)

The 3' end primer sequence is:

5'-AAACCGACGCGGAGCGCCAGAAC-3'(SEQ ID NO.:24)

The 5' end oligonucleotide primer sequence constructed by CRISPR/Cas9 knockout OsMKK4 vector is:

5'-GGCAGCGACGTGAGGTCCCGCTG-3'(SEQ ID NO.:25)

The 3' end primer sequence is:

5'-AAACCAGCGGGACCTCACGTCGC-3'(SEQ ID NO.:26)

The 5' end oligonucleotide primer sequence constructed by CRISPR/Cas9 knockout OsMKKK10 vector is:

5'-GGCACCTCATCGATACATTTCAT-3' (SEQ ID NO.:27)

The 3' end primer sequence is:

5'-AAACATGAAATGTATCGATGAGG-3'(SEQ ID NO.:28)

3. GSN1 enzyme activity detection

GSN1 encodes a dephosphorylase with dephosphorylation ability. The present invention speculates that GSN1 ^S146F in the background of the gsn1 mutant has lost the ability to dephosphorylate. Therefore, MBP-GSN1 and MBP-GSN1 ^S146F fusion proteins were purified in vitro, and then phosphate PNPP (p-nitrophenyl phosphate) and OMFP (3-o-methylfluoresceinphosphate) were used as substrates, respectively, at absorbance values of 405nm and 477nm. Assay to assess the dephosphorylation capacity of MBP-GSN1 and MBP-GSN1 ^S146F . Purified MBP protein can be used as a negative control. Consistent with the speculation, GSN1 ^S146F did lose its dephosphorylation activity.

The 5' end oligonucleotide primer sequence constructed by MBP-GSN1 and MBP-GSN1 ^S146F protein expression vector pMAL-c5x is:

5'-CGGGATCGAGGGAAGGATTTCAATGGCCACGCCCGA-3' (SEQ ID NO.:29)

The 3' end primer sequence is:

5'-AGCTTATTTAATTACCTGCAGGTTAACGAGCATTCAGC-3' (SEQ ID NO.:30)

4. Cytology detection

Compared with wild-type FAZ1, the gsn1 mutant has significantly increased grain length and grain width, so a series of observations were carried out to study the cytological basis of rice grain size enlargement. Rice glumes that were about to head at the big bract stage were selected and fixed in FAA, part of which was used for paraffin-embedded semi-thin sections, and part of which was used for scanning electron microscope observation of the outer and inner epidermis of the glumes. Paraffin-embedded semi-thin sections were used to observe and count the size and number of parenchyma cells in the cross-section of glume, which reflected cell elongation or division. Carry out scanning electron microscope observation after the sample dehydration that FAA is fixed, epidermis in glume is mainly to observe the size of inner epidermis cell and the number of cells per unit area; Epidermis in glume is mainly to observe the distance between surface papilla There is a papillae in the middle of the cells, based on which the average size of the cells can be judged). Generally speaking, the number of tetraploid cells is relatively increased in cells in vigorous division phase, and the distribution of cell cycle is also changed. Accordingly, the young rice spikes and glumes were selected, the nuclei were extracted and stained with DAPI, and the cell ploidy of each sample was analyzed by flow cytometry to compare the relative rates of cell division of FAZ1 and gsn1 samples .

5. Protein interaction detection

In order to find the interaction protein and dephosphorylation substrate of GSN1 protein, the in vitro protein interaction verification was carried out by yeast two-hybrid technology. The coding sequences of the GSN1 and OsMPK1 genes were fused and constructed on the PGBKT7 and PGADT7 vectors, and then the yeast Y2HGOLD strain was co-transfected and allowed to grow on the three-deficient or four-deficient SD medium. After 3 days, it was judged according to the size and color of the plaques Whether the two target proteins GSN1 and OsMPK1 interact. Based on the results of yeast two-hybrid, it was found that GSN1 and OsMPK1 interacted. Therefore, we further verified in vivo by using bimolecular fluorescence complementary BiFC technology. The coding regions of GSN1 and OsMPK1 genes were respectively constructed at the N-terminal and C-terminal of YFP, that is, nYFP-GSN1 and cYFP-OsMPK1, and then co-transformed into tobacco through the mediation of Agrobacterium, cultured in the dark for 48 hours, and then used laser confocal microscopy Make observations. BiFC experiments found that GSN1 and OsMPK1 do interact. This result was further confirmed by co-immunoprecipitation co-IP technology. That is, GSN1 was fused to a carrier containing a Flag tag, and OsMPK1 was fused to a carrier containing a Myc tag, and then co-transfected tobacco was mediated by Agrobacterium, and the total protein of tobacco was extracted after culturing for 48 hours. Next, incubate with the magnetic beads of the affinity Flag tag protein and the total tobacco protein, so that the protein with the Flag tag is bound to the magnetic beads, and after the magnetic beads are eluted, the Flag and Myc antibodies are used for detection respectively. The co-IP experiment also verified the interaction between GSN1 and OsMPK1 in vivo.

The 5' end oligonucleotide primer sequence constructed by the yeast two-hybrid vector PGBKT7-GSN1 is:

5'-ATGGCCATGGAGGCCGAATTCATGGCCACGCCCGA-3'(SEQ ID NO.:31)

The 3' end primer sequence is:

5'-TGCGGCCGCTGCAGGTCGTGCGGCCGCTGCAGGTCG-3'(SEQ ID NO.:32)

The 5' end oligonucleotide primer sequence constructed by the yeast two-hybrid vector PGADT7-GSN1 is:

5'-ATGGCCATGGAGGCCAGTGAAATGGCCACGCCCGA-3'(SEQ ID NO.:33)

The 3' end primer sequence is:

5'-TGCAGCTCGAGCTCGATGGATCGGCCGCTGCAGGTCG-3'(SEQ ID NO.:34)

The 5' end oligonucleotide primer sequence constructed by the yeast two-hybrid vector PGBKT7-OsMPK1 is:

5'-ATGGCCATGGAGGCatggacgccggggcgcagccgc-3'(SEQ ID NO.:35)

The 3' end primer sequence is:

5'-TGCGGCCGCTGCAGGTctactggtaatcagggttgaacgcaa-3' (SEQ ID NO.:36)

The 5' end oligonucleotide primer sequence constructed by the yeast two-hybrid vector PGADT7-OsMPK1 is:

5'-ATGGCCATGGAGGCCatggacgccggggcgcagccgc-3'(SEQ ID NO.:37)

The 3' end primer sequence is:

5'-TGCAGCTCGAGCTCctactggtaatcagggttgaacgcaa-3' (SEQ ID NO.:38)

The 5' end oligonucleotide primer sequence constructed by the BiFC vector nYFP-GSN1 is:

5'-GATTTCTGAGGAGGATCTTCCCGGGGCCACGCCCGA-3'(SEQ ID NO.:39)

The 3' end primer sequence is:

5'-AAGCAGGGCATGCCTGCAGGTCGACTTAACGAGCATT-3'(SEQ ID NO.:40)

The 5' end oligonucleotide primer sequence constructed by the BiFC vector cYFP-GSN1 is:

5'-CGACTCTAGGAGCTCGGTACCCGGGATGGCCACGCC-3' (SEQ ID NO.:41)

The 3' end primer sequence is:

5'-GAACATCGTATGGGTACATACTAGTACGAGCATTCA-3' (SEQ ID NO.:42)

The 5' end oligonucleotide primer sequence constructed by the BiFC vector nYFP-OsMPK1 is:

5'-GATTTCTGAGGAGGATCTTCCCGGGgacgccggggcgc-3' (SEQ ID NO.:43)

The 3' end primer sequence is:

5'-AAGCAGGGCATGCCTGCAGGTCGACctactggtaatcaggg-3' (SEQ ID NO.:44)

The 5' end oligonucleotide primer sequence constructed by the BiFC vector cYFP-OsMPK1 is:

5'-CGACTCTAGGAGCTCGGTACCCGGGatggacgccggggcg-3'(SEQ ID NO.:45)

The 3' end primer sequence is:

5'-GAACATCGTATGGGTACATACTAGTctggtaatcagggttga-3' (SEQ ID NO.: 46)

The 5' end oligonucleotide primer sequence constructed by the Co-IP vector pCOMBIA1306-Flag-GSN1 is:

5'-AGAGAACACGGGGGACGAGCTCGGTACCATGGCCAC-3' (SEQ ID NO.:47)

The 3' end primer sequence is:

5'-ATCCAAGGGCGAATTGGTCGACTCTAGAACGAGCATT-3' (SEQ ID NO.:48)

The 5' end oligonucleotide primer sequence constructed by the Co-IP vector pCOMBIA1301-Myc-OsMPK1 is:

5'-tttggagagaacacgggggactTCTAGAatggacgccggggcgcagccgc-3' (SEQ ID NO.:49)

The 3' end primer sequence is:

5'-tcggagattagcttttgttcGGATCCctggtaatcagggttgaacgca-3' (SEQ ID NO.:50)

6. Phosphorylation and dephosphorylation detection

After proving the interaction between GSN1 and OsMPK1 proteins through in vitro and in vivo experiments, it is necessary to verify whether GSN1 can dephosphorylate OsMPK1 in vitro. MBP-GSN1, MBP-GSN1 ^S146F , MBP-OsMKK4 ^CA and GST-OsMPK1 fusion proteins were purified respectively. First, the purified fusion protein MBP-OsMKK4 ^CA was used to phosphorylate GST-OsMPK1. This modification occurred on the TEY motif of GST-OsMPK1, which could be detected by phosphorylation antibody phos-p42/44. Then, the purified fusion protein MBP-GSN1, MBP-GSN1 ^S146F and phosphorylated modified GST-OsMPK1 were incubated together, and the phosphorylated antibody phos-p42/44 was used to detect the degree of dephosphorylation of phosphorylated GST-OsMPK1.

The 5' end oligonucleotide primer sequence constructed by the GST-OsMPK1 protein expression vector is:

5'-CCGGAATTCCCATGGCCACGCCCGACGACGGCG-3' (SEQ ID NO.:51)

The 3' end primer sequence is:

5'-ATAAGAATGCGGCCGCTTAACGAGCATTCAGCACTTCC-3'(SEQ ID NO.:52)

The 5' end oligonucleotide primer sequence constructed by the MBP-OsMKK4 ^CA protein expression vector is:

5'-CGGGATCGAGGGAAGGATTTCAATGCGACCGGGCGG-3' (SEQ ID NO.:53)

The 3' end primer sequence is:

5'-AGCTTATTTAATTACCTGCAGGTCATGACGGAGGCGG-3' (SEQ ID NO.:54)

Example 1 Through the EMS mutagenesis of the indica rice variety Fengaizhan No. 1 (FAZ1), a mutant gsn1 with significantly larger grain size and reduced number of grains per panicle was obtained, and GSN1 that controlled its phenotype was cloned Gene

The grain length, grain width and 1000-grain weight of the gsn1 mutant increased significantly; the number of grains per panicle decreased significantly; flower organs increased and fertility decreased. Therefore, the gsn1 mutant is a good material for studying the interaction between grain number per panicle and grain weight (as shown in Figure 1). The candidate gene GSN1 was successfully mapped and cloned using the gsn1 mutant and the F ₂ segregating population of the japonica rice variety Zhonghua 11. Studies have shown that the 437th nucleotide C of the GSN1 allele from the gsn1 mutant background is mutated to T, thereby causing the 146th serine of the encoded protein to be mutated to phenylalanine. The candidate gene GSN1 was confirmed by transgenic genetic complementation validation, and it was its mutation that caused the gsn1 phenotype. In addition, it was found that reducing (down-regulating) the expression of GSN1 in the wild-type FAZ1 background can increase the size of rice grains and reduce the number of grains per panicle; on the contrary, enhancing (increasing) the expression of GSN1 can reduce the number of grains and increase the number of grains per panicle (as shown in Figure 2 shown). In addition, the mutant gsn1 was backcrossed with japonica rice variety 95-22 for multiple generations, and then a near-isogenic line NIL (gsn1) containing only the gsn1 mutation site was obtained in BC ₆ F ₂ . The near-isogenic line showed a very similar phenotype to the gsn1 mutant: larger grains and fewer grains per panicle, indicating that the gene regulates grain weight and grain number in both indica and japonica rice. Therefore, the GSN1 gene is a very potential genetic improvement site for balancing the number of grains per panicle and grain shape of rice (as shown in Figure 3).

Example 2 Overexpression of mutant GSN1 under the background of japonica rice variety ZH11 has a dominant negative inhibitory effect

GSN1 is a positive regulator of grain number per panicle, but a negative regulator of grain weight and grain type at the same time. Reducing the expression of GSN1 in the japonica rice ZH11 background can reduce the number of grains per panicle and increase the size of rice grains. On the contrary, the overexpression of the allele GSN1 from ^ZH11 and the allele GSN1 of FAZ1 in their background, respectively, could increase the number of grains per panicle and reduce the number of grains. Accordingly, overexpressing the GSN1 allele from the gsn1 mutant in the ZH11 background revealed that the rice grain size became larger and the number of grains per panicle decreased, which was basically consistent with the phenotype after GSN1 was inhibited. Therefore, it was inferred that the overexpression of the GSN1 mutant gene in the ZH11 background had a dominant negative inhibitory effect (as shown in Figure 4, Figure 5 and Figure 6).

Example 3 Mutations in GSN1 cause specific effects on cell division, but do not affect cell expansion or elongation

Since the grain length and grain width of the gsn1 mutant increased, the floral organs also increased accordingly, which confirmed that GSN1 was involved in the regulation of cell division at the cytological level, but did not affect cell elongation. Transverse sectioning of glumes and anthers of FAZ1 and gsn1 at the heading stage of rice showed that there was no significant difference in the size of glume parenchyma cells and septum vascular bundle cells between FAZ1 and gsn1, but the number of cells in the gsn1 mutant increased significantly. Using scanning electron microscope to observe the outer and inner epidermis of the mature glume, there was no difference in the distance between the papillae on the surface of the outer glume and the cell size of the inner epidermis between FZA1 and gsn1. In addition, the flow cytometric analysis of the young panicle cells of both FAZ1 and gsn1 found that compared with the wild type, the number of cells in the 4C phase of the mutant was significantly increased. These results above indicate that gsn1 mutants have an accelerated cell division rate, resulting in larger organs (as shown in Figure 7).

Example 4 GSN1 encodes a dual-specificity phosphatase localized in the cytoplasm, and the mutation of the 146th serine to phenylalanine results in the loss of dephosphorylation activity of the GSN1 protein

By purifying MBP-GSN1 and MBP-GSN1 ^S146F fusion proteins in vitro, and using the compound OMFP (3-o-methylfluorescein phosphate) as a substrate for enzyme activity experiments, it was found that the fusion protein MBP-GSN1 had obvious dephosphorylation activity, while The mutant MBP-GSN1 ^S146F lost its dephosphorylation activity. Therefore, the conserved serine at position 146 is very important for the complete biological function of GSN1 protein. In addition, studies using tobacco leaf cells and rice protoplasts respectively found that GSN1 protein was localized in the cytoplasm. The qRT-PCR results showed that GSN1 was expressed in tissues at various stages, but it was mainly expressed at the young panicle stage and spikelet (as shown in Figure 8).

Example 5 GSN1 and OsMPK1 interact specifically, and GSN1 dephosphorylates OsMPK1

Through the yeast two-hybrid technique, it was found that GSN1 and OsMPK1 could interact in vitro. Then, this result was confirmed using BiFC bimolecular fluorescence complementation experiments and co-IP co-immunoprecipitation. Together, these in vivo results show that GSN1 and OsMPK1 can indeed interact. In order to study whether OsMPK1 can be dephosphorylated after the interaction between GSN1 and OsMPK1, fusion proteins MBP-GSN1, MBP-GSN1 ^S146F , GST-OsMPK1 and constitutively activated MBP-MKK4 ^CA (T238D and S244D) were expressed, respectively. Perform in vitro phosphorylation and dephosphorylation experiments. The results showed that the fusion protein MBP-OsMKK4 ^CA could phosphorylate GST-OsMPK1, and then the phosphorylated GST-OsMPK1 could be dephosphorylated by MBP-GSN1. However, MBP-GSN1 ^S146F cannot dephosphorylate the phosphorylated GST-OsMPK1, which is consistent with the expectation of the present invention. Using specific phosphorylation antibody phos-p42/44 and mass spectrometry detection, it was found that both phosphorylation and dephosphorylation occurred on the activation ring TEY motif of GST-OsMPK1 (as shown in Figure 9).

Example 6 Rice OsMKKK10-OsMKK4-OsMPK1 cascade reaction pathway participates in the development and balance of grain number per panicle and grain weight (grain size)

At present, the complete MAPK cascade involved in the development process in rice has not been reported. In the wild-type FAZ1 background, OsMKKK10 and OsMKK4 were knocked out by CRISPR/Cas9 gene knockout technology. : The rice panicle shape becomes compact, the number of grains per panicle increases and the grain shape becomes smaller (grain weight decreases). Although the knockout of OsMPK1 by CRISPR/Cas9 technology is lethal, it is found that the transgenic lines with suppressed (down-regulated expression) of OsMPK1 have increased number of grains per panicle and reduced grain size by RNAi technology to interfere with the expression of OsMPK1. /Cas9 knockout OsMKKK10 and OsMKK4 have obvious phenotype consistency. In addition, overexpression of constitutively activated OsMKK4 ^CA (T238D and S244D) in the FAZ1 background resulted in larger grain size (increased grain weight) and decreased number of grains per panicle. Therefore, the research of the present invention shows that the OsMKKK10-OsMKK4-OsMPK1 cascade reaction pathway controls the development of rice panicle type by participating in the development of grain number and grain size (grain weight) per panicle (as shown in Figure 10).

Example 7 GSN1 is a negative regulator of rice OsMKKK10-OsMKK4-OsMPK1 cascade reaction pathway

The gsn1 mutant exhibited sparse panicles (decreased grain number) and large grains, whereas the components of the inhibited OsMKKK10-OsMKK4-OsMPK1 cascade pathway each exhibited dense panicles (increased grain number) and small grains. Therefore, the present invention introduced mutations or suppressed cascade components in the gsn1 mutant background, and found that inhibiting OsMKKK10-OsMKK4-OsMPK1 cascade components can restore the phenotype of gsn1. These studies demonstrate that the phenotype of gsn1 mutants is dependent on the activation of components of the OsMKKK10-OsMKK4-OsMPK1 cascade. Therefore, GSN1 is a negative regulator of this cascade signaling pathway (as shown in Figure 10).

Example 8

Arabidopsis experiments

In this example, considering the functional conservation of GSN1, the rice GSN1 gene was overexpressed in Arabidopsis thaliana, a cruciferous plant, to understand the genetic function of the gene in Arabidopsis thaliana, and to provide a basis for GSN1 in other species. The application in provides the basis.

The results are shown in FIG. 11 , and the results indicated that overexpression of wild-type GSN1 derived from rice in Arabidopsis thaliana resulted in increased branches of Arabidopsis thaliana and shortened siliques.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

<120> GSN1 Gene, Encoded Protein and Application for Controlling Plant Grain Number and Grain Weight

<130> P2018-0007

<160> 54

<170> PatentIn version 3.5

<210> 1

<211> 2295

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<213> Oryza sativa

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atggccacgc ccgacgacgg cggagggggt gtcggcggcg gcgcggggaa gaagttctgg 60

cgctccgcgt cgtggtccgc ctcccgggac acgccgcccg atgcggcgac gcctgcgggg 120

gcgggcggcg gcggcggcgc gggggcaggg caggcgcgcc gcatcccgcc gccgccgcct 180

ctgaccccgc gcggcgggaa ggggcggtcc tgcctcccgc cgctgcagcc gctcaacatc 240

acccgccgga gcctcgacga gtggccgcgg gcgggctccg acgacgtggg cgagtggccc 300

aacccgacca cccccggcgc ctccaaggcg gaaggcgccg gctcggccaa gcccggggag 360

ggcctccgct tggacctctc atccctccgg tcgcagggtc gcaaagacca gatcgccttc 420

ttcgacaagg agtgctccaa ggttgctgac cacgtctacc ttggaggcga cgccgttgcc 480

aagaaccgcg acatcctgcg caagaacggg atcacccacg tgctcaattg cgtcggcttc 540

gtttgtcccg agtacttcaa gtcggacctc gtctaccgga cgctttggct tcaggacagc 600

ccgacggagg acatcaccag catcttgtat gatgtgtttg attactttga ggacgtcagg 660

gagcagggtg ggcgtgtctt cgtacattgt tgccagggggg tatcaaggtc tacatcgctg 720

gtgatagcgt acttgatgtg gagggaaggg cagagctttg acgatgcgtt ccagcttgtg 780

aaagctgccc gggggatcgc aaatccaaat atggggttcg cttgccagct actccagtgc 840

cagaagcggg ttcatgcgat tccgctatct ccaaactcgg ttctgcggat gtaccggatg 900

gcacctcact caccttatgc gccgctgcat ttagtaccca aaatgctgaa cgaaccctca 960

cctgctgccc tggactctag gggtgcattc atcgttcatg tgctctcatc gatctatgtt 1020

tgggttggaa tgaagtgtga tcaagtaatg gagaaggatg caagagcagc ggcgttccag 1080

gtggtgaggt atgagaaggt gcaggggcat atcaaagttg tcagggaagg attggagcta 1140

ccagaatttt gggacgcctt ttcaagtgca cctgttaatt cagacagcaa tacaaagatt 1200

agcaaggatc agattgattc agcatccaag actggcccgg ggaaccgtag ggtggaatct 1260

tatgatgctg attttgagct tgtatacaaa gcaatcactg ggggtgttgt gccagcattc 1320

tcttcttcag gtgctggtga tgagacccat cttccagcaa gagaaagtac ctggagttca 1380

ctaaggcgca agttcatctc taggtcgtta gctcgagtct attcagattc tgctttaatc 1440

agggacttgg atccgcgggt ggaccgagta caacacctgg ctgcagaggc atcaacatca 1500

ccacctttcc tatctccgag ctctttgtca tcagattcaa gcatcagttc aaagtatagt 1560

tcagactcac cctcactgtc accttcaact agctcgccaa catcacttgg tctttcgccc 1620

gcttcatcta atttttccca cactttggtg ccatcatcta ggtcccccct tcaccaatca 1680

tctaacgagg aaccttcaaa atctggcctg gggtcaatac gctctccctc caagacctct 1740

tctatagcag aaagaagagg agggttttca tctctgaagt taccatcatt ccaaaaggat 1800

ctagtattac caccaagggt accgaccagc cttcgcagag aagaggaagt cacagataag 1860

agtaataata atagtgtaaa acagcttact ggtgtgtgtt gcccagagaa atgcactggc 1920

aatacttcaa cagtgcacac taaaactgga ataactgagc gtactgacag tatctcagag 1980

gcttgcggta atttacaact attagtctac cgttggccca gcaaggaaaa gctaactact 2040

ttcactcgca aggatcttga cccaaaatca gttttgattt ttgttactcc tgaagacagc 2100

agaagtgaag cagttaaaac ggtacacatt tgggtagggag gtgaatatga gagcagcaaa 2160

tgtgttgaca ctgttgattg gcagcaagtt gttggtgatt tttttcatct aaaggagctc 2220

ggcaatactc ttcctgttaa ggttttcaaa gagcatgaaa cggagaatct tttggaagtg 2280

ctgaatgctc gttaa 2295

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Met Ala Thr Pro Asp Asp Gly Gly Gly Gly Val Gly Gly Gly Ala Gly

1 5 10 15

Lys Lys Phe Trp Arg Ser Ala Ser Trp Ser Ala Ser Arg Asp Thr Pro

20 25 30

Pro Asp Ala Ala Thr Pro Ala Gly Ala Gly Gly Gly Gly Gly Ala Gly

35 40 45

Ala Gly Gln Ala Arg Arg Ile Pro Pro Pro Pro Pro Leu Thr Pro Arg

50 55 60

Gly Gly Lys Gly Arg Ser Cys Leu Pro Pro Leu Gln Pro Leu Asn Ile

65 70 75 80

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

85 90 95

Gly Glu Trp Pro Asn Pro Thr Thr Pro Gly Ala Ser Lys Ala Glu Gly

100 105 110

Ala Gly Ser Ala Lys Pro Gly Glu Gly Leu Arg Leu Asp Leu Ser Ser

115 120 125

Leu Arg Ser Gln Gly Arg Lys Asp Gln Ile Ala Phe Phe Asp Lys Glu

130 135 140

Cys Ser Lys Val Ala Asp His Val Tyr Leu Gly Gly Asp Ala Val Ala

145 150 155 160

Lys Asn Arg Asp Ile Leu Arg Lys Asn Gly Ile Thr His Val Leu Asn

165 170 175

Cys Val Gly Phe Val Cys Pro Glu Tyr Phe Lys Ser Asp Leu Val Tyr

180 185 190

Arg Thr Leu Trp Leu Gln Asp Ser Pro Thr Glu Asp Ile Thr Ser Ile

195 200 205

Leu Tyr Asp Val Phe Asp Tyr Phe Glu Asp Val Arg Glu Gln Gly Gly

210 215 220

Arg Val Phe Val His Cys Cys Gln Gly Val Ser Arg Ser Thr Ser Leu

225 230 235 240

Val Ile Ala Tyr Leu Met Trp Arg Glu Gly Gln Ser Phe Asp Asp Ala

245 250 255

Phe Gln Leu Val Lys Ala Ala Arg Gly Ile Ala Asn Pro Asn Met Gly

260 265 270

Phe Ala Cys Gln Leu Leu Gln Cys Gln Lys Arg Val His Ala Ile Pro

275 280 285

Leu Ser Pro Asn Ser Val Leu Arg Met Tyr Arg Met Ala Pro His Ser

290 295 300

Pro Tyr Ala Pro Leu His Leu Val Pro Lys Met Leu Asn Glu Pro Ser

305 310 315 320

Pro Ala Ala Leu Asp Ser Arg Gly Ala Phe Ile Val His Val Leu Ser

325 330 335

Ser Ile Tyr Val Trp Val Gly Met Lys Cys Asp Gln Val Met Glu Lys

340 345 350

Asp Ala Arg Ala Ala Ala Phe Gln Val Val Arg Tyr Glu Lys Val Gln

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Gly His Ile Lys Val Val Arg Glu Gly Leu Glu Leu Pro Glu Phe Trp

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Asp Ala Phe Ser Ser Ala Pro Val Asn Ser Asp Ser Asn Thr Lys Ile

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Ser Lys Asp Gln Ile Asp Ser Ala Ser Lys Thr Gly Pro Gly Asn Arg

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Arg Val Glu Ser Tyr Asp Ala Asp Phe Glu Leu Val Tyr Lys Ala Ile

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Thr Gly Gly Val Val Pro Ala Phe Ser Ser Ser Ser Gly Ala Gly Asp Glu

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Thr His Leu Pro Ala Arg Glu Ser Thr Trp Ser Ser Leu Arg Arg Lys

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Phe Ile Ser Arg Ser Leu Ala Arg Val Tyr Ser Asp Ser Ala Leu Ile

465 470 475 480

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

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Ala Ser Thr Ser Pro Pro Phe Leu Ser Pro Ser Ser Leu Ser Ser Asp

500 505 510

Ser Ser Ile Ser Ser Lys Tyr Ser Ser Asp Ser Pro Ser Leu Ser Pro

515 520 525

Ser Thr Ser Ser Pro Thr Ser Leu Gly Leu Ser Pro Ala Ser Ser Asn

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Phe Ser His Thr Leu Val Pro Ser Ser Arg Ser Pro Leu His Gln Ser

545 550 555 560

Ser Asn Glu Glu Pro Ser Lys Ser Gly Leu Gly Ser Ile Arg Ser Pro

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Ser Lys Thr Ser Ser Ser Ile Ala Glu Arg Arg Gly Gly Phe Ser Ser Leu

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Lys Leu Pro Ser Phe Gln Lys Asp Leu Val Leu Pro Pro Arg Val Pro

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Thr Ser Leu Arg Arg Glu Glu Glu Val Thr Asp Lys Ser Asn Asn Asn

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Ser Val Lys Gln Leu Thr Gly Val Cys Cys Pro Glu Lys Cys Thr Gly

625 630 635 640

Asn Thr Ser Thr Val His Thr Lys Thr Gly Ile Thr Glu Arg Thr Asp

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Ser Ile Ser Glu Ala Cys Gly Asn Leu Gln Leu Leu Val Tyr Arg Trp

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Pro Ser Lys Glu Lys Leu Thr Thr Phe Thr Arg Lys Asp Leu Asp Pro

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Lys Ser Val Leu Ile Phe Val Thr Pro Glu Asp Ser Arg Ser Glu Ala

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Val Lys Thr Val His Ile Trp Val Gly Gly Glu Tyr Glu Ser Ser Lys

705 710 715 720

Cys Val Asp Thr Val Asp Trp Gln Gln Val Val Gly Asp Phe Phe His

725 730 735

Leu Lys Glu Leu Gly Asn Thr Leu Pro Val Lys Val Phe Lys Glu His

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Glu Thr Glu Asn Leu Leu Glu Val Leu Asn Ala Arg

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Met Ala Thr Pro Asp Asp Gly Gly Gly Gly Val Gly Gly Gly Ala Gly

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Lys Lys Phe Trp Arg Ser Ala Ser Trp Ser Ala Ser Arg Asp Thr Pro

20 25 30

Pro Asp Ala Ala Thr Pro Ala Gly Ala Gly Gly Gly Gly Gly Ala Gly

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Ala Gly Gln Ala Arg Arg Ile Pro Pro Pro Pro Pro Leu Thr Pro Arg

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Gly Gly Lys Gly Arg Ser Cys Leu Pro Pro Leu Gln Pro Leu Asn Ile

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

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Gly Glu Trp Pro Asn Pro Thr Thr Pro Gly Ala Ser Lys Ala Glu Gly

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Ala Gly Ser Ala Lys Pro Gly Glu Gly Leu Arg Leu Asp Leu Ser Ser

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Leu Arg Ser Gln Gly Arg Lys Asp Gln Ile Ala Phe Phe Asp Lys Glu

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Cys Phe Lys Val Ala Asp His Val Tyr Leu Gly Gly Asp Ala Val Ala

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Lys Asn Arg Asp Ile Leu Arg Lys Asn Gly Ile Thr His Val Leu Asn

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Cys Val Gly Phe Val Cys Pro Glu Tyr Phe Lys Ser Asp Leu Val Tyr

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Arg Thr Leu Trp Leu Gln Asp Ser Pro Thr Glu Asp Ile Thr Ser Ile

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Leu Tyr Asp Val Phe Asp Tyr Phe Glu Asp Val Arg Glu Gln Gly Gly

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Arg Val Phe Val His Cys Cys Gln Gly Val Ser Arg Ser Thr Ser Leu

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Val Ile Ala Tyr Leu Met Trp Arg Glu Gly Gln Ser Phe Asp Asp Ala

245 250 255

Phe Gln Leu Val Lys Ala Ala Arg Gly Ile Ala Asn Pro Asn Met Gly

260 265 270

Phe Ala Cys Gln Leu Leu Gln Cys Gln Lys Arg Val His Ala Ile Pro

275 280 285

Leu Ser Pro Asn Ser Val Leu Arg Met Tyr Arg Met Ala Pro His Ser

290 295 300

Pro Tyr Ala Pro Leu His Leu Val Pro Lys Met Leu Asn Glu Pro Ser

305 310 315 320

Pro Ala Ala Leu Asp Ser Arg Gly Ala Phe Ile Val His Val Leu Ser

325 330 335

Ser Ile Tyr Val Trp Val Gly Met Lys Cys Asp Gln Val Met Glu Lys

340 345 350

Asp Ala Arg Ala Ala Ala Phe Gln Val Val Arg Tyr Glu Lys Val Gln

355 360 365

Gly His Ile Lys Val Val Arg Glu Gly Leu Glu Leu Pro Glu Phe Trp

370 375 380

Asp Ala Phe Ser Ser Ala Pro Val Asn Ser Asp Ser Asn Thr Lys Ile

385 390 395 400

Ser Lys Asp Gln Ile Asp Ser Ala Ser Lys Thr Gly Pro Gly Asn Arg

405 410 415

Arg Val Glu Ser Tyr Asp Ala Asp Phe Glu Leu Val Tyr Lys Ala Ile

420 425 430

Thr Gly Gly Val Val Pro Ala Phe Ser Ser Ser Ser Gly Ala Gly Asp Glu

435 440 445

Thr His Leu Pro Ala Arg Glu Ser Thr Trp Ser Ser Leu Arg Arg Lys

450 455 460

Phe Ile Ser Arg Ser Leu Ala Arg Val Tyr Ser Asp Ser Ala Leu Ile

465 470 475 480

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

485 490 495

Ala Ser Thr Ser Pro Pro Phe Leu Ser Pro Ser Ser Leu Ser Ser Asp

500 505 510

Ser Ser Ile Ser Ser Lys Tyr Ser Ser Asp Ser Pro Ser Leu Ser Pro

515 520 525

Ser Thr Ser Ser Pro Thr Ser Leu Gly Leu Ser Pro Ala Ser Ser Asn

530 535 540

Phe Ser His Thr Leu Val Pro Ser Ser Arg Ser Pro Leu His Gln Ser

545 550 555 560

Ser Asn Glu Glu Pro Ser Lys Ser Gly Leu Gly Ser Ile Arg Ser Pro

565 570 575

Ser Lys Thr Ser Ser Ser Ile Ala Glu Arg Arg Gly Gly Phe Ser Ser Leu

580 585 590

Lys Leu Pro Ser Phe Gln Lys Asp Leu Val Leu Pro Pro Arg Val Pro

595 600 605

Thr Ser Leu Arg Arg Glu Glu Glu Val Thr Asp Lys Ser Asn Asn Asn

610 615 620

Ser Val Lys Gln Leu Thr Gly Val Cys Cys Pro Glu Lys Cys Thr Gly

625 630 635 640

Asn Thr Ser Thr Val His Thr Lys Thr Gly Ile Thr Glu Arg Thr Asp

645 650 655

Ser Ile Ser Glu Ala Cys Gly Asn Leu Gln Leu Leu Val Tyr Arg Trp

660 665 670

Pro Ser Lys Glu Lys Leu Thr Thr Phe Thr Arg Lys Asp Leu Asp Pro

675 680 685

Lys Ser Val Leu Ile Phe Val Thr Pro Glu Asp Ser Arg Ser Glu Ala

690 695 700

Val Lys Thr Val His Ile Trp Val Gly Gly Glu Tyr Glu Ser Ser Lys

705 710 715 720

Cys Val Asp Thr Val Asp Trp Gln Gln Val Val Gly Asp Phe Phe His

725 730 735

Leu Lys Glu Leu Gly Asn Thr Leu Pro Val Lys Val Phe Lys Glu His

740 745 750

Glu Thr Glu Asn Leu Leu Glu Val Leu Asn Ala Arg

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<213> Oryza sativa

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atggccacgc ccgacgacgg cggagggggt gtcggcggcg gcgcggggaa gaagttctgg 60

cgctccgcgt cgtggtccgc ctcccgggac acgccgcccg atgcggcgac gcctgcgggg 120

gcgggcggcg gcggcggcgc gggggcaggg caggcgcgcc gcatcccgcc gccgccgcct 180

ctgaccccgc gcggcgggaa ggggcggtcc tgcctcccgc cgctgcagcc gctcaacatc 240

acccgccgga gcctcgacga gtggccgcgg gcgggctccg acgacgtggg cgagtggccc 300

aacccgacca cccccggcgc ctccaaggcg gaaggcgccg gctcggccaa gcccggggag 360

ggcctccgct tggacctctc atccctccgg tcgcagggtc gcaaagacca gatcgccttc 420

ttcgacaagg agtgcttcaa ggttgctgac cacgtctacc ttggaggcga cgccgttgcc 480

aagaaccgcg acatcctgcg caagaacggg atcacccacg tgctcaattg cgtcggcttc 540

gtttgtcccg agtacttcaa gtcggacctc gtctaccgga cgctttggct tcaggacagc 600

ccgacggagg acatcaccag catcttgtat gatgtgtttg attactttga ggacgtcagg 660

gagcagggtg ggcgtgtctt cgtacattgt tgccagggggg tatcaaggtc tacatcgctg 720

gtgatagcgt acttgatgtg gagggaaggg cagagctttg acgatgcgtt ccagcttgtg 780

aaagctgccc gggggatcgc aaatccaaat atggggttcg cttgccagct actccagtgc 840

cagaagcggg ttcatgcgat tccgctatct ccaaactcgg ttctgcggat gtaccggatg 900

gcacctcact caccttatgc gccgctgcat ttagtaccca aaatgctgaa cgaaccctca 960

cctgctgccc tggactctag gggtgcattc atcgttcatg tgctctcatc gatctatgtt 1020

tgggttggaa tgaagtgtga tcaagtaatg gagaaggatg caagagcagc ggcgttccag 1080

gtggtgaggt atgagaaggt gcaggggcat atcaaagttg tcagggaagg attggagcta 1140

ccagaatttt gggacgcctt ttcaagtgca cctgttaatt cagacagcaa tacaaagatt 1200

agcaaggatc agattgattc agcatccaag actggcccgg ggaaccgtag ggtggaatct 1260

tatgatgctg attttgagct tgtatacaaa gcaatcactg ggggtgttgt gccagcattc 1320

tcttcttcag gtgctggtga tgagacccat cttccagcaa gagaaagtac ctggagttca 1380

ctaaggcgca agttcatctc taggtcgtta gctcgagtct attcagattc tgctttaatc 1440

agggacttgg atccgcgggt ggaccgagta caacacctgg ctgcagaggc atcaacatca 1500

ccacctttcc tatctccgag ctctttgtca tcagattcaa gcatcagttc aaagtatagt 1560

tcagactcac cctcactgtc accttcaact agctcgccaa catcacttgg tctttcgccc 1620

gcttcatcta atttttccca cactttggtg ccatcatcta ggtcccccct tcaccaatca 1680

tctaacgagg aaccttcaaa atctggcctg gggtcaatac gctctccctc caagacctct 1740

tctatagcag aaagaagagg agggttttca tctctgaagt taccatcatt ccaaaaggat 1800

ctagtattac caccaagggt accgaccagc cttcgcagag aagaggaagt cacagataag 1860

agtaataata atagtgtaaa acagcttact ggtgtgtgtt gcccagagaa atgcactggc 1920

aatacttcaa cagtgcacac taaaactgga ataactgagc gtactgacag tatctcagag 1980

gcttgcggta atttacaact attagtctac cgttggccca gcaaggaaaa gctaactact 2040

ttcactcgca aggatcttga cccaaaatca gttttgattt ttgttactcc tgaagacagc 2100

agaagtgaag cagttaaaac ggtacacatt tgggtagggag gtgaatatga gagcagcaaa 2160

tgtgttgaca ctgttgattg gcagcaagtt gttggtgatt tttttcatct aaaggagctc 2220

ggcaatactc ttcctgttaa ggttttcaaa gagcatgaaa cggagaatct tttggaagtg 2280

ctgaatgctc gttaa 2295

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

atagctagag agagcggtca 20

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

gagagtaact gaagcagcaa g 21

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

tgactttaag ggcctgttt 19

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

ggggtatttc agacaattca 20

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

ctggatatac atgtgtctaa ttct 24

<210> 10

<211> 23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 10

gcatatccag gttaatttat agc 23

<210> 11

<211> 22

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 11

tggctaaaat aatatcgtat cg 22

<210> 12

<211> 22

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 12

aggatctcag ttgcagtaat ct 22

<210> 13

<211> 24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 13

gggggttctt ctacttttgt tttt 24

<210> 14

<211> 24

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 14

ctccacatca agtacgctat cacc 24

<210> 15

<211> 25

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 15

gatcaataaa agatggtcat cagtg 25

<210> 16

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 16

ggagatagga aaggtggtga tgt 23

<210> 17

<211> 26

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 17

caccatggcc acgcccgacg acggcg 26

<210> 18

<211> 26

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 18

acgagcattc agcacttcca aaagat 26

<210> 19

<211> 28

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 19

tcggtaccca gcagcagcca cagcaaaa 28

<210> 20

<211> 28

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 20

tctctagagc tgctgatgct gatgccat 28

<210> 21

<211> 35

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 21

cccggatccg agctcatgga cgccggggcg cagcc 35

<210> 22

<211> 35

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 22

gggggtacca ctagtttgta cttggcggtg acctc 35

<210> 23

<211> 23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 23

ggcagttctg gcgctccgcg tcg 23

<210> 24

<211> 23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 24

aaaccgacgc ggagcgccag aac 23

<210> 25

<211> 23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 25

ggcagcgacg tgaggtcccg ctg 23

<210> 26

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 26

aaaccagcgg gacctcacgt cgc 23

<210> 27

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 27

ggcacctcat cgatacattt cat 23

<210> 28

<211> 23

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 28

aaacatgaaa tgtatcgatg agg 23

<210> 29

<211> 36

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 29

cgggatcgag ggaaggattt caatggccac gcccga 36

<210> 30

<211> 38

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 30

agcttattta attacctgca ggttaacgag cattcagc 38

<210> 31

<211> 35

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 31

atggccatgg aggccgaatt catggccacg cccga 35

<210> 32

<211> 36

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 32

tgcggccgct gcaggtcgtg cggccgctgc aggtcg 36

<210> 33

<211> 35

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 33

atggccatgg aggccagtga aatggccacg cccga 35

<210> 34

<211> 37

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 34

tgcagctcga gctcgatgga tcggccgctg caggtcg 37

<210> 35

<211> 36

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 35

atggccatgg aggcatggac gccggggcgc agccgc 36

<210> 36

<211> 42

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 36

tgcggccgct gcaggtctac tggtaatcag ggttgaacgc aa 42

<210> 37

<211> 37

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 37

atggccatgg aggccatgga cgccggggcg cagccgc 37

<210> 38

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 38

tgcagctcga gctccctactg gtaatcaggg ttgaacgcaa 40

<210> 39

<211> 36

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 39

gatttctgag gaggatcttc ccggggccac gcccga 36

<210> 40

<211> 37

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 40

aagcagggca tgcctgcagg tcgacttaac gagcatt 37

<210> 41

<211> 36

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 41

cgactctagg agctcggtac ccgggatggc cacgcc 36

<210> 42

<211> 36

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 42

gaacatcgta tgggtacata ctagtacgag cattca 36

<210> 43

<211> 38

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 43

gatttctgag gaggatcttc ccggggacgc cggggcgc 38

<210> 44

<211> 41

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 44

aagcagggca tgcctgcagg tcgacctact ggtaatcagg g 41

<210> 45

<211> 40

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 45

cgactctagg agctcggtac ccgggatgga cgccggggcg 40

<210> 46

<211> 42

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 46

gaacatcgta tgggtacata ctagtctggt aatcagggtt ga 42

<210> 47

<211> 36

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 47

agagaacacg ggggacgagc tcggtaccat ggccac 36

<210> 48

<211> 37

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 48

atccaagggc gaattggtcg actctagaac gagcatt 37

<210> 49

<211> 50

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 49

tttggagaga acacggggga cttctagaat ggacgccggg gcgcagccgc 50

<210> 50

<211> 48

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 50

tcggagatta gcttttgttc ggatccctgg taatcagggt tgaacgca 48

<210> 51

<211> 33

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 51

ccggaattcc catggccacg cccgacgacg gcg 33

<210> 52

<211> 38

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 52

ataagaatgc ggccgcttaa cgagcattca gcacttcc 38

<210> 53

<211> 36

<212>DNA

<213> Artificial sequence (artificial sequence)

<400> 53

cgggatcgag ggaaggattt caatgcgacc gggcgg 36

<210> 54

<211> 37

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 54

agcttattta attacctgca ggtcatgacg gaggcgg 37

Claims (19)

1. The use of a substance which isGSN1A gene or its encoded protein, or an enhancer thereof, for modulating a trait in a plant or for the preparation of a composition for modulating a trait in a plant, wherein the trait comprises one or more traits selected from the group consisting of:

(i) Reducing the particle size;

(ii) Increasing the grain number of the spikes;

(iii) Increase the seed setting rate ofGSN1The amino acid sequence of the protein is shown as SEQ ID NO.2, and the plant is rice.

2. Use according to claim 1, for modulating one or more of the following traits in a plant:

(v) Causes increased branching in Arabidopsis;

(vi) Leading to silique shortening in arabidopsis thaliana.

3. The use of a substance which isGSN1A mutein, orGSN1Genes or their codingsAn inhibitor of a protein, said mutein being a GSN1 mutein having reduced activity compared to the wild-type GSN1 protein, for modulating one or more of the following traits in a plant:

(i) Increasing the grain type;

(ii) The grain number of the grains per spike is reduced;

(iii) Reducing the fruit set rate, wherein the mutant protein is mutated at a core amino acid of a wild-type GSN1 protein shown as SEQ ID NO.2, wherein the core amino acid is selected from the group consisting of:

serine (S) at position 146, the amino acid at position 146 being mutated to phenylalanine (F), theGSN1The amino acid sequence of the protein is shown in SEQ ID NO.2, and the plant is rice.

4. Use according to claim 3, wherein the substance is also used for modulating one or more of the following traits in a plant:

(v) Results in reduced branching in Arabidopsis;

(vi) In Arabidopsis thaliana, the silique is caused to lengthen.

5. The use according to claim 3, wherein the amino acid sequence of the mutein is represented by SEQ ID No. 3.

6. A method of improving a trait in a plant comprising the steps of:

(i) Modulating the expression level and/or activity of GSN1 protein in said plant, thereby improving plant traitsGSN1The amino acid sequence of the protein is shown as SEQ ID NO.2, the plant is rice, and when the ratio of the activity E1 of GSN1 in the plant to the background activity E0 of wild GSN in the plant is more than or equal to 2 times, the character of the plant is improved to be that the grain type is reduced and/or the grain number per ear is increased;

when the ratio of the activity E1 of GSN1 in the plant to the background activity E0 of wild-type GSN in the plant is less than or equal to 1/2, the character of the plant is improved to be that the grain type is enlarged and/or the grain number per ear is reduced.

7. The method of claim 6, wherein the trait of said plant is improved by reduced grain type and/or increased grain number per ear when the ratio of E1 activity of GSN1 in said plant to E0 background wild type GSN activity in said plant is greater than or equal to 5 fold.

8. The method of claim 6, wherein the trait of said plant is improved by increased grain size and/or decreased grain number per ear when the ratio of E1 activity of GSN1 in said plant to background wild type GSN activity E0 in said plant is ≦ 1/5.

9. A method for dephosphorylating an MPK1 protein, comprising the steps of:

contacting the GSN1 protein with the MPK1 protein to dephosphorylate the MPK1 proteinGSN1The amino acid sequence of the protein is shown as SEQ ID NO.2, and the MPK1 protein is derived from rice.

10. An isolated mutant GSN1 protein, wherein said mutant GSN1 protein is a non-native protein and said mutant GSN1 protein has activity in modulating a trait in a plant, said trait being selected from the group consisting of one or more traits selected from the group consisting of:

(i) Grain type;

(ii) The grain number of the ears;

(iii) Setting percentage;

the mutant GSN1 protein is mutated in the core amino acid of the wild GSN1 protein shown in SEQ ID NO.2, wherein the core amino acid is selected from the following group:

serine (S) at the 146 th site, the amino acid (S) at the 146 th site is mutated into phenylalanine (F), and the plant is rice.

11. The mutant GSN1 protein of claim 10, wherein said mutant GSN1 protein has the amino acid sequence shown in SEQ ID No. 3.

12. A polynucleotide encoding the mutant GSN1 protein of claim 10.

13. A vector comprising the polynucleotide of claim 12.

14. A host cell comprising the vector of claim 13, or having the polynucleotide of claim 12 integrated into its genome.

15. A method of producing the mutant GSN1 protein of claim 10 comprising the steps of:

culturing the host cell of claim 14 under conditions suitable for expression, thereby expressing a mutant GSN1 protein; and

isolating the mutant GSN1 protein.

16. An enzyme preparation comprising a mutant GSN1 protein according to claim 10.

17. A method of producing a transgenic plant comprising the steps of:

introducing a polynucleotide encoding the protein of claim 10 into a plant cell, culturing said plant cell, and regenerating a plant, said plant being rice.

18. A method of identifying a large or small grain plant comprising the steps of:

(i) Identifying whether the plant sample has the polypeptide of the amino acid sequence shown in SEQ ID NO.2 or 3 or the coding gene thereof; if the polypeptide with the amino acid sequence shown in SEQ ID NO.2 or the coding gene thereof is a small-grain plant, if the polypeptide with the amino acid sequence shown in SEQ ID NO.3 or the coding gene thereof is a large-grain plant, the plant is rice.

19. The method of claim 18, wherein in step (i) it is determined by sequencing whether the plant sample has a nucleotide sequence as set forth in SEQ ID No.1 or SEQ ID No. 4.

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