CN112513275B

CN112513275B - Application of miR396 or mutant of encoding gene thereof in regulation and control of agronomic traits of plants

Info

Publication number: CN112513275B
Application number: CN202080003128.7A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Shandong Shunfeng Biotechnology Co Ltd
Current assignee: Shandong Shunfeng Biotechnology Co Ltd
Priority date: 2019-07-05
Filing date: 2020-06-23
Publication date: 2023-01-10
Anticipated expiration: 2040-06-23
Also published as: CN112251434A; CN112513275A; WO2021004269A1

Abstract

Provides application of miR396 or a mutant of a coding gene thereof in regulation and control of plant agronomic traits. Also provided are the use of a GRF gene or protein promoter encoding the same, a method of improving an agronomic trait in a plant, a composition for improving an agronomic trait in a plant under low nitrogen conditions and the use thereof, a method of making a gene edited plant tissue or plant cell and a method of making a gene edited plant.

Description

Application of miR396 or mutant of encoding gene thereof in regulation and control of agronomic traits of plants

Technical Field

The invention relates to the field of crop genetics, in particular to application of miR396 or a mutant of a coding gene thereof in regulation and control of plant agronomic traits.

Background

Rice is one of the most important grain crops in the world and is also the first large grain crop in China. The dwarfing breeding from the end of the 50 th to the beginning of the 60 th of the 20 th century and the successful application of the three-line hybrid indica rice in the 70 th make two major leaps on the rice yield per unit in China and make great contribution to meeting the food self-sufficiency in China. In the past decade, with the increasing population of China, the requirement on the total production of rice is gradually increased. In the modern agricultural production process, people play a very important role in promoting the development of agricultural production by using chemical fertilizers, particularly applying nitrogen fertilizers. However, when people apply a large amount of nitrogen fertilizer, a lot of problems and influences are brought, which not only causes huge waste to economy and resources, but also causes serious threat to ecological environment, and excessive use of nitrogen fertilizer causes soil acidification, secondary salinization, greenhouse effect, ozone layer holes, eutrophication of water bodies and the like. Therefore, breeding a new variety that can maintain or improve the yield per unit of existing rice under low nitrogen conditions is an important issue for genetic breeders.

microRNAs (miRNAs) are a class of non-coding single-stranded small RNA molecules of 20-24 nucleotides in length that bind to the mRNA of a target gene through base pairing, thereby causing degradation or translational inhibition of the mRNA. miRNA controls the growth, development, stress tolerance and the like of plants by regulating and controlling the accumulation of genes encoding proteins in-situ cells in the plants.

However, the current miRNA targets have limited ability to improve agronomic traits in plants.

There is therefore an urgent need in the art to develop a method capable of significantly improving agronomic traits in plants.

Disclosure of Invention

The invention aims to provide a method capable of obviously improving the agronomic traits of plants.

In a first aspect of the invention there is provided the use of a nucleic acid construct, or a mutant of a gene encoding the same, for modulating an agronomic trait in a plant under low nitrogen conditions or for the manufacture of a composition or formulation for modulating an agronomic trait in a plant under low nitrogen conditions, wherein the agronomic trait in the plant is selected from one or more of the group consisting of:

(a) Yield and/or biomass;

(b) Development of panicle and/or grain types;

(c) Size, weight and/or number of fruits and/or seeds;

(d) Grain length;

(e) Grain width;

(f) Ear length;

(g) Thousand seed weight;

(h) Leaf length;

(i) Leaf width;

(j) Effective tillering number;

(k) The number of fruit pods;

wherein the nucleic acid construct has a structure of formula I from 5 'to 3':

X1-X2-X3 (I)

wherein X1 is absent or selected from positions 1-12 of SEQ ID No. 1 or positions 1-9 of SEQ ID No. 2;

x2 is selected from a mature sequence/a conserved sequence of miR 396;

x3 is none or selected from positions 44-184 of SEQ ID NO. 1 or positions 41-176 of SEQ ID NO. 2;

and, each "-" is a bond or a nucleotide linking sequence.

In another preferred example, the modulating an agronomic trait of a plant comprises:

(a) Increasing yield and/or biomass;

(b) Promoting development of panicle type and/or granule type;

(c) Increasing the size, weight and/or number of fruits and/or seeds;

(d) The grain length is increased;

(e) The grain width is increased;

(f) The ear length is increased;

(g) Increasing the thousand seed weight;

(h) The leaf length is increased.

In another preferred example, the mature/conserved sequences of miR396 comprise mature/conserved sequences of miR396a, miR396b, miR396c, miR396d, miR396e, miR396f, miR396g, and/or miR396h.

In another preferred example, the mature sequence/conserved sequence of miR396 comprises a mature sequence/conserved sequence of miR396e, and/or miR396f.

In another preferred example, the mature sequence of miR396 is the mature sequence of miR396e and/or the mature sequence of miR396f.

In another preferred example, the conserved sequence of the miR396 is selected from positions 13-43 of SEQ ID NO. 1 or positions 10-40 of SEQ ID NO. 2.

In a further preferred embodiment of the present invention,the low nitrogen condition refers to the nitrogen content N of the culture medium or the field growth condition _L The ratio of nitrogen content N0 in the whole culture medium or under field growth conditions (N) _L N0) is 0 to 1, preferably 0.01 to 0.9, preferably 0.1 to 0.9, preferably 0.3 to 0.8, more preferably 0.5 to 0.8, more preferably 0.6 to 0.8.

In another preferred embodiment, the low nitrogen condition is that the amount of nitrogen fertilizer applied is lower than the conventional amount of fertilizer applied during the growth of the crop, or that no additional nitrogen fertilizer is applied to the soil, the conventional amount of fertilizer applied is known to the skilled person or farmer to ensure stable and high quality of the crop, such as 150-350 kg per hectare for rice, wheat and corn, and the amount of fertilizer applied is different for different areas of different crops, and the conventional amount can be determined by the skilled person or farmer's planting experience, and in the present invention, the amount of nitrogen fertilizer applied is preferably 210-270 kg per hectare.

In another preferred embodiment, the low nitrogen condition is that no additional nitrogen fertilizer is applied in the growing environment of the plant, and the total nitrogen content in the soil is about 0.5-3.0g/kg soil under the condition of no additional nitrogen fertilizer; in a different embodiment, the total nitrogen content of the soil is 1-2.5g/kg soil, preferably 1.2-2.2g/kg soil, without applying additional nitrogen fertilizer.

In another preferred embodiment, the nucleic acid construct is selected from the group consisting of:

(a) Has a nucleotide sequence shown as

SEQ ID NO

1 or 2;

(b) A polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95%) to a sequence represented by SEQ ID No. 1 or 2;

(c) A polynucleotide comprising 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides which is truncated or added to the 5 'end and/or the 3' end of the polynucleotide shown in SEQ ID NO. 1 or 2.

In another preferred embodiment, the sequence of the nucleic acid construct is as shown in SEQ ID NO. 1 or 2.

In another preferred embodiment, the coding gene encodes the nucleic acid construct described above.

In another preferred example, the encoding genes include MIR396a, MIR396b, MIR396c, MIR396d, MIR396e, MIR396f, MIR396g, and/or MIR396h.

In another preferred embodiment, the encoding gene comprises MIR396e and/or MIR396f.

In another preferred embodiment, the nucleic acid construct or gene encoding the same is derived from one or more plants selected from the group consisting of: arabidopsis, rice, cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, rape, spinach, lettuce, cucumber, garland chrysanthemum, water spinach, celery and leaf lettuce.

In another preferred embodiment, the nucleic acid construct or gene encoding the same is derived from rice.

In another preferred embodiment, the nucleic acid construct or mutant of the gene encoding the nucleic acid construct is derived from one or more plants selected from the group consisting of: arabidopsis, rice, cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, rape, spinach, lettuce, cucumber, garland chrysanthemum, water spinach, celery and leaf lettuce.

In another preferred embodiment, the nucleic acid construct or mutant of the gene encoding the nucleic acid construct is derived from rice.

In another preferred example, the nucleic acid construct or a mutant of the encoding gene thereof comprises a mutant of miR396a, miR396b, miR396c, miR396d, miR396e, miR396f, miR396g and/or miR396h.

In another preferred example, the nucleic acid construct or mutant of the gene encoding it comprises a mutant of miR396e and/or miR396f.

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

In another preferred embodiment, the composition comprises (a) a miR396 inhibitor; and (b) an agronomically acceptable carrier.

In another preferred embodiment, the composition or formulation is in a dosage form selected from the group consisting of: a solution, an emulsion, a suspension, a powder, a foam, a paste, a granule, an aerosol, or a combination thereof.

In another preferred embodiment, the plant includes angiosperms and gymnosperms.

In another preferred embodiment, the gymnosperm is selected from the group consisting of: cycadaceae (Cycadaceae), podocarpaeaceae (podocarpaeceae), araucaceae (araucaceae), pinaceae (Pinaceae), cedaceae, cypress, cephalotaxaceae, taxaceae, ephedra, gnetaceae, monotype, welchidaceae, or combinations thereof.

In another preferred embodiment, the plant includes a monocotyledon and a dicotyledon.

In another preferred embodiment, the plant includes herbaceous plants and woody plants.

In another preferred embodiment, the herbaceous plant is selected from the group consisting of: solanaceae, poaceae, leguminous plants, or combinations thereof.

In another preferred embodiment, the woody plant is selected from the group consisting of: actinidiaceae, rosaceae, moraceae, or their combination.

In another preferred embodiment, the plant is selected from the group consisting of: cruciferous plants, gramineae, leguminous plants, solanaceae, actinidiaceae, malvaceae, paeoniaceae, rosaceae, liliaceae, or combinations thereof.

In another preferred embodiment, the plant is selected from the group consisting of: arabidopsis thaliana, rice, cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, rape, spinach, lettuce, cucumber, garland chrysanthemum, water spinach, celery, leaf lettuce or a combination thereof.

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

In another preferred embodiment, the nucleic acid construct or mutant of the gene encoding it is natural or synthetic.

In another preferred embodiment, the mutant of the nucleic acid construct or the gene encoding the same comprises a substitution, insertion, and/or deletion, preferably a deletion of a large fragment, of a base of the nucleic acid construct or the gene encoding the same.

In another preferred embodiment, the expression or activity of the nucleic acid construct or a mutant of the gene encoding it is reduced by more than or equal to 50%, preferably more than or equal to 70%, more preferably more than or equal to 90% or 100% compared to the expression or activity of the wild-type nucleic acid construct or the gene encoding it.

In another preferred embodiment, when the ratio of the activity E1 of the nucleic acid construct or a mutant of the gene encoding it to the background activity E0 of the wild-type nucleic acid construct or a mutant of the gene encoding it is ≦ 1/2, preferably ≦ 1/5, more preferably ≦ 1/10, still more preferably 0.

In another preferred embodiment, the nucleic acid construct or mutant of the gene encoding the nucleic acid construct reduces, silences or loses regulatory effect on the target gene.

In another preferred embodiment, the target gene comprises a GRF gene.

In another preferred embodiment, the GRF gene is selected from the group consisting of: GRF4, GRF6, GRF8, or a combination thereof.

In another preferred embodiment, the GRF gene comprises GRF8.

In another preferred embodiment, the mutation site of the nucleic acid construct or gene encoding the nucleic acid construct comprises at least a portion of the mature sequence region or the conserved sequence region.

In another preferred embodiment, the mutation site of the nucleic acid construct or gene encoding the nucleic acid construct is in the mature sequence region or in the conserved sequence region.

In a second aspect, the present invention provides the use of a GRF gene or protein promoter encoding the same for modulating agronomic traits in plants under low nitrogen conditions or for the manufacture of a composition or formulation for modulating agronomic traits in plants under low nitrogen conditions, wherein the agronomic traits in plants are selected from one or more of the group consisting of:

(a) Yield and/or biomass;

(b) Development of panicle and/or grain types;

(c) Size, weight and/or number of fruits and/or seeds;

(d) Grain length;

(e) The grain width is wide;

(f) Ear length;

(g) Thousand seed weight;

(h) Leaf length;

(i) Leaf width;

(j) Effective tillering number;

(k) And (4) the number of the fruit pods.

In another preferred embodiment, the GRF gene comprises GRF4, GRF6 and/or GRF8.

In another preferred embodiment, the GRF gene comprises a GRF protein-encoding gene and a GRF gene conserved region sequence.

In another preferred embodiment, the GRF gene includes a wild-type GRF gene and a mutant GRF gene.

In another preferred embodiment, the mutant form comprises a mutant form in which the function of the encoded protein is not altered after mutation (i.e., the function is the same or substantially the same as the wild-type encoded protein).

In another preferred embodiment, the polypeptide encoded by said mutant GRF gene is the same or substantially the same as the polypeptide encoded by a wild-type GRF gene.

In another preferred embodiment, the mutant GRF gene comprises a polynucleotide having a homology of 80% or more (preferably 90% or more, more preferably 95% or more) to a wild type GRF gene.

In another preferred embodiment, said mutant GRF gene comprises a polynucleotide truncated or supplemented with 1-60 (preferably 1-30, more preferably 1-10) nucleotides at the 5 'and/or 3' end of the wild type GRF gene.

In another preferred embodiment, the GRF gene is selected from the group consisting of: a cDNA sequence, a genomic sequence, or a combination thereof.

In another preferred embodiment, the nucleotide sequence of the GRF4 gene is selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 3 or 4;

(b) A polynucleotide having a sequence as set forth in SEQ ID No. 5;

(c) Polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95%) with the sequence shown in SEQ ID No. 5;

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

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

In another preferred embodiment, the nucleotide sequence of the GRF4 gene is shown as SEQ ID No. 5.

In another preferred embodiment, the nucleotide sequence of the GRF6 gene is selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as shown in SEQ ID No. 6;

(b) A polynucleotide having a sequence as set forth in SEQ ID No. 7;

(c) Polynucleotide having homology of more than or equal to 75% (preferably more than or equal to 85%, more preferably more than or equal to 90% or more than or equal to 95%) with the sequence shown in SEQ ID NO. 7;

(d) A polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or the 3' end of the polynucleotide shown in SEQ ID No. 7;

In another preferred embodiment, the nucleotide sequence of the GRF6 gene is shown as SEQ ID No. 7.

In another preferred embodiment, the nucleotide sequence of the GRF8 gene is selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 8;

(b) A polynucleotide having a sequence as set forth in SEQ ID No. 9;

(c) A polynucleotide having a nucleotide sequence homology of 75% or more (preferably 85% or more, more preferably 90% or more or 95%) to a sequence represented by SEQ ID No. 9;

(d) A polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or the 3' end of the polynucleotide shown in SEQ ID No. 9;

In another preferred example, the nucleotide sequence of the GRF8 gene is shown as SEQ ID No. 9.

In another preferred embodiment, the amino acid sequence of said GRF4 protein is selected from the group consisting of:

(i) A polypeptide having an amino acid sequence as set forth in SEQ ID No. 3 or 4;

(ii) (ii) a polypeptide derived from (i) in which the amino acid sequence shown in SEQ ID NO. 3 or 4 is substituted, deleted or added with one or more (e.g., 1-10) amino acid residues; or

(iii) The homology of the amino acid sequence and the amino acid sequence shown in SEQ ID NO. 3 or 4 is more than or equal to 80 percent (preferably more than or equal to 90 percent, more preferably more than or equal to 95 percent or more than or equal to 98 percent), and the polypeptide has the GRF4 activity.

In another preferred embodiment, the amino acid sequence of the GRF4 protein is shown in SEQ ID No. 3 or 4.

In another preferred embodiment, the amino acid sequence of said GRF6 protein is selected from the group consisting of:

(i) A polypeptide having an amino acid sequence shown in SEQ ID No. 6;

(ii) (ii) a polypeptide derived from (i) wherein the amino acid sequence shown in SEQ ID NO. 6 is substituted, deleted or added with one or more (e.g. 1-10) amino acid residues; or

(iii) The homology of the amino acid sequence and the amino acid sequence shown in SEQ ID NO. 6 is more than or equal to 80 percent (preferably more than or equal to 90 percent, more preferably more than or equal to 95 percent or more than or equal to 98 percent), and the polypeptide has the GRF6 activity.

In another preferred example, the amino acid sequence of the GRF6 protein is shown as SEQ ID No. 6.

In another preferred embodiment, the amino acid sequence of said GRF8 protein is selected from the group consisting of:

(i) A polypeptide having an amino acid sequence shown in SEQ ID No. 8;

(ii) (ii) a polypeptide derived from (i) wherein the amino acid sequence shown in SEQ ID NO. 8 is substituted, deleted or added with one or more (e.g. 1-10) amino acid residues; or

(iii) The homology of the amino acid sequence and the amino acid sequence shown in SEQ ID NO. 8 is more than or equal to 80 percent (preferably more than or equal to 90 percent, more preferably more than or equal to 95 percent or more than or equal to 98 percent), and the polypeptide has the GRF8 activity.

In another preferred example, the amino acid sequence of the GRF8 protein is shown as SEQ ID No. 8.

In another preferred embodiment, the GRF gene or protein-encoded promoter thereof comprises a substance that promotes expression or activity of the GRF gene or protein-encoded thereof.

In another preferred embodiment, the expression or activity of the GRF gene or its encoded protein means that the expression or activity of the GRF gene or its encoded protein is increased by 10% or more, preferably 20% or more, preferably 50% or more, more preferably 70% or more.

In another preferred embodiment, the GRF gene or protein promoter encoding same is selected from the group consisting of: small molecule compounds, nucleic acid molecules, enzymes, or combinations thereof.

In another preferred embodiment, the GRF gene or protein encoding the same is derived from one or more plants selected from the group consisting of: arabidopsis thaliana, rice, cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, rape, spinach, lettuce, cucumber, crowndaisy chrysanthemum, water spinach, celery and leaf lettuce.

In another preferred embodiment, the GRF gene or the protein encoding the same is derived from rice.

In another preferred embodiment, the composition comprises (a) an enhancer of the GRF gene or protein encoded thereby; and (b) an agronomically acceptable carrier.

In another preferred embodiment, the plant is selected from the group consisting of: arabidopsis, rice, cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, rape, spinach, lettuce, cucumber, garland chrysanthemum, water spinach, celery, lettuce or a combination thereof.

In a third aspect of the invention, there is provided a method of improving an agronomic trait in a plant comprising:

under low nitrogen conditions, the expression or activity of miR396 in the plant is reduced or the expression or activity of GRF gene or protein encoded by the GRF gene in the plant is improved.

In another preferred example, the reduction of the expression or activity of miR396 can be achieved by any one of the following means:

I. mutating a precursor sequence of miR396 so as to reduce the expression or activity of a mature sequence of miR 396;

II. The mature sequence of miR396 is mutated to reduce its expression or activity.

In another preferred example, the miR396 is miR396e and/or miR396f.

In another preferred embodiment, the GRF gene comprises GRF8.

In another preferred example, the reduction of the expression or activity of miR396 in the plant is achieved by:

(1) Mutating miR396 in plants to obtain the nucleic acid construct mutant, and/or

(2) Introducing an inhibitor of miR396 into said plant.

In another preferred example, the method comprises the steps of:

(i) Providing a plant or plant cell; and

(ii) Introducing an inhibitor of miR396 into the plant or plant cell, thereby obtaining a plant or plant cell with miR396 expression down-regulated.

In another preferred example, the inhibitor of miR396 comprises an inhibitor of miR396a, miR396b, miR396c, miR396d, miR396e, miR396f, miR396g, and/or miR396h.

In another preferred example, the inhibitor of miR396 comprises an inhibitor of miR396e and/or miR396f.

In another preferred embodiment, the inhibitor of miR396 is selected from the group consisting of: a small molecule compound, an antisense nucleic acid, a microRNA, a siRNA, an RNAi, a Crispr agent, or a combination thereof.

In another preferred example, the "decrease" means that the decrease in expression or activity of miR396 meets the following condition:

the ratio of A1/A0 is less than or equal to 80 percent, preferably less than or equal to 60 percent, more preferably less than or equal to 40 percent, and most preferably 0 to 30 percent; wherein A1 is the expression or activity of miR396 in the plant; a0 is the expression or activity of the same miR396 in wild type plants of the same type.

In another preferred embodiment, said reduction means that the expression level E1 of MIR396 in said plant is 0-80%, preferably 0-60%, more preferably 0-40%, more preferably 0-30% of wild type compared to the expression level E0 of wild type MIR 396.

In another preferred embodiment, said reducing the expression or activity of miR396 in the plant is achieved by a method selected from the group consisting of: gene mutation, gene knockout, gene disruption, RNA interference techniques, criprpr techniques, or a combination thereof.

In another preferred example, the reduction of expression or activity of miR396 in the plant is achieved by gene editing of miR396 with 1 or more sgRNA-mediated Cas9 nucleases.

In another preferred embodiment, the method comprises administering to a plant an enhancer of the GRF gene or a polypeptide encoded thereby.

In another preferred embodiment, the method comprises introducing into the plant an exogenous GRF gene or protein encoding the same.

In another preferred example, the method comprises the steps of:

(i) Providing a plant or plant cell; and

(ii) Introducing a GRF gene sequence into said plant or plant cell, thereby obtaining a plant or plant cell having upregulated GRF gene expression.

In another preferred example, the method comprises the steps of:

(a) Providing agrobacterium carrying an expression vector of a GRF gene sequence;

(b) Contacting a plant cell or tissue or organ with the agrobacterium of step (a) such that the GRF gene sequence is transferred into the plant cell and integrated into the chromosome of the plant cell;

(c) Selecting plant cells or tissues or organs into which the GRF gene sequence has been transferred; and

(d) Regenerating the plant cell or tissue or organ of step (c) into a plant.

In another preferred example, the improved plant agronomic trait comprises:

(a) Increasing yield and/or biomass;

(b) Promoting development of panicle type and/or granule type;

(c) Increasing the size, weight and/or number of fruits and/or seeds;

(d) The grain length is increased;

(e) The grain width is increased;

(f) The ear length is increased;

(g) Increasing the thousand seed weight;

(h) The leaf length is increased;

(i) Increase the effective tillering number.

In a fourth aspect, the present invention provides a composition for improving agronomic traits of plants under low nitrogen conditions, comprising:

(i) A miR396 inhibitor, and/or a promoter for GRF gene or its encoded protein; and

(ii) An agronomically acceptable carrier.

In another preferred embodiment, the composition comprises an agricultural composition.

In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, or combinations thereof.

In another preferred embodiment, the composition comprises component (a) in an amount of 0.0001 to 99wt%, preferably 0.1 to 90wt%, based on the total weight of the composition.

In another preferred example, the improved plant agronomic trait comprises:

(a) Increasing yield and/or biomass;

(b) Promoting development of panicle type and/or granule type;

(c) Increasing the size, weight and/or number of fruits and/or seeds;

(d) The grain length is increased;

(e) The grain width is increased;

(f) The ear length is increased;

(g) Increasing the thousand seed weight;

(h) The leaf length is increased.

In a fifth aspect, the present invention provides a use of the composition of the fourth aspect for improving agronomic traits of plants under low nitrogen conditions.

In a sixth aspect, the present invention provides a method for preparing a gene-edited plant tissue or plant cell, comprising the steps of:

reducing the expression or activity of miR396 in the plant tissue or plant cell, and/or increasing the expression or activity of a GRF gene or protein encoding thereof in the plant tissue or plant cell, under low nitrogen conditions, thereby obtaining a gene-edited plant tissue or plant cell.

The seventh aspect of the present invention provides a method for preparing a gene-edited plant, comprising the steps of:

regenerating the gene-edited plant tissue or plant cell produced by the method according to the sixth aspect of the present invention into a plant body, thereby obtaining a gene-edited plant.

In an eighth aspect, the present invention provides a gene-edited plant produced by the method of the seventh aspect.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

FIG. 1 shows the effect of mir396e mutation type on seed type.

FIG. 2 shows the difference in seed size between wild type and mir396e mutant.

FIG. 3 shows the growth situation of plants under different nitrogen conditions.

FIG. 4 shows the effect of mutation of miR396ef on seed type, thousand kernel weight under low nitrogen conditions.

Wherein, (a) the influence of miR396ef mutation on grain length and grain width;

(b) Influence of miR396ef mutation on grain thickness;

(c) Effect of mutation of miR396ef on thousand kernel weight.

Fig. 5 shows a slice view of the seeds.

Wherein, (a) the effect of miR396ef mutation on glume cell length;

(b) Influence of miR396ef mutation on the number of palea and palea cells.

FIG. 6 shows the effect of mir396ef mutation on ear length, prime, ear size.

Wherein, (a) the effect of miR396ef mutation on ear length;

(b) Influence of mutation of miR396ef on the number of main branches;

(c) Influence of mutation of miR396ef on grain number per ear.

FIG. 7 shows the effect of mir396ef mutation on rice yield.

Wherein, (a) the yield of the miR396ef mutant under the non-low nitrogen condition;

(b) Yield of miR396ef mutant under low nitrogen conditions.

FIG. 8 shows the effect of miR396ef mutation on rice leaf length.

FIG. 9 shows the effect of mir396ef mutation on rice plant height.

FIG. 10 shows the effect of mir396ef mutation on rice biomass.

FIG. 11 shows miR396ef regulating the kernel and panicle type development via miR396-GRF4/6/8-GIF1/2/3 pathway.

Wherein (a) mir396 targets the resistance gene sequence;

(b) np is the influence of rGRF4, np is rGRF6 and np is rGRF8 on the grain type of the grains;

(c) np rGRF6 and np rGRF8 on ear length;

(d) GRE-GIF interaction;

(e) Plant type and seed breeding of gif1 mutants.

Detailed Description

The inventor discovers or synthesizes a novel nucleic acid construct shown in formula I of miR396 and family members thereof or mutants of coding genes thereof for the first time through extensive and intensive research. The nucleic acid construct or the mutant of the coding gene thereof can obviously improve the agronomic traits of plants under the low nitrogen condition, and comprises the following components: (a) increased yield and/or biomass; (b) promoting development of panicle and/or grain types; (c) Size, weight and/or number of fruits and/or seeds; (d) grain length; (e) grain width; (f) ear length; (g) thousand kernel weight; (h) leaf length; (i) leaf width; (j) effective tiller number; (k) number of pods, etc. Furthermore, the inventors have surprisingly found that increasing the expression or activity of the GRF (such as GFR4, 6, and/or 8) gene or protein encoding it significantly improves agronomic traits in plants under low nitrogen conditions, including: (a) increasing yield and/or biomass; (b) promoting development of panicle and/or grain types; (c) The size, weight and/or number of fruits and/or seeds; (d) grain length; (e) grain width; (f) ear length; (g) thousand kernel weight; (h) leaf length; (i) leaf width; (j) effective tiller number; (k) Pod number, and in addition, it has been discovered for the first time that the nucleic acid constructs of the invention, or mutants of the genes encoding same, affect plant yield, biomass, through GRF (e.g., GRF 4/6/8) under low nitrogen conditions; development of panicle and/or grain types; size, weight and/or number of fruits and/or seeds; grain length; grain width; ear length; thousand seed weight; leaf length; leaf width; effective tillering number; pod number and the like. On this basis, the present inventors have completed the present invention.

GRF gene

As used herein, the terms "GRF gene" and "gene of the invention" are used interchangeably and refer to a gene of the invention that modulates an agronomic trait in a plant.

In a preferred embodiment, the gene of the present invention comprises GRF4, GRF6, GRF8, and preferably, the gene of the present invention comprises GRF8. More preferably, the GRF gene of the present invention is derived from rice.

GRF, collectively known as growth-regulating factors, is a plant-specific class of transcription factors. It can form a functional complex by the interaction of the N-terminal QLQ structural domain and the SNH structural domain in the GIF (GRF-interaction factor) protein, and participate in the expression regulation of downstream genes together. The number of members of different species is slightly different, 9 in Arabidopsis, 12 in rice and 14 in maize.

The GRF gene of the present invention may be in a DNA form or an RNA form. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The genomic DNA may be identical to the sequences shown in SEQ ID NO. 5, 7, 9 or degenerate variants. The DNA of the present invention may be single-stranded or double-stranded, and the DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequences shown in SEQ ID NO. 5, 7, 9 or may be a degenerate variant.

As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences which encode a protein having the sequence of SEQ ID No. 3, 4, 6 or 8, but which differ from the sequence of the coding region as set forth in SEQ ID No. 5, 7, 9.

A polynucleotide encoding a mature polypeptide of SEQ ID No. 3, 4, 6 or 8 comprising: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

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

The present invention also relates to polynucleotides which hybridize to the above-described sequences and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" refer to (1) hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃ etc. at the time of hybridization; or (3) hybridization only when the identity between two sequences is at least 90% or more, preferably 95% or more. And, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID No. 2.

The invention also relates to nucleic acid fragments hybridizing with the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR) to determine and/or isolate polynucleotides encoding polypeptides associated with thermotolerant properties.

Polypeptide coded by GRF gene

As used herein, the terms "polypeptide of the invention", "polypeptide encoded by a GRF gene", "protein encoded by a GRF gene" and "GRF polypeptide" are used interchangeably and refer to a polypeptide of the invention that has the ability to modulate agronomic traits in plants under low nitrogen conditions.

In a preferred embodiment, the polypeptide of the invention comprises GRF4, GRF6 and/or GRF8. More preferably, the polypeptide of the present invention is derived from rice.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the invention can be naturally purified products, or chemically synthesized products, or using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptides of the invention may or may not also include an initial methionine residue.

The invention also includes fragments, derivatives and analogs of the GRF polypeptides. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as a native GRF polypeptide of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (i) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are well within the skill of those in the art in light of the teachings herein.

In a preferred embodiment, the polypeptide of the invention refers to a polypeptide having a sequence of SEQ ID No. 3, 4, 6 or 8 that modulates agronomic traits in plants under low nitrogen conditions. Also included are variants of the sequences of SEQ ID No. 3, 4, 6 or 8 having the same function as the GRF polypeptide. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of GRF polypeptides.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA for the SPL polypeptide, and polypeptides or proteins obtained using antisera directed against GRF polypeptides. The invention also provides other polypeptides, such as fusion proteins comprising a GRF polypeptide or fragment thereof. In addition to almost full-length polypeptides, the invention also encompasses soluble fragments of GRF polypeptides. Typically, the fragment has at least about 10 contiguous amino acids, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the GRF polypeptide sequence.

The invention also provides GRF polypeptides or analogs thereof. These analogs may differ from the native GRF polypeptide by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Such polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, the term "conservative variant GRF polypeptide" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids having similar or similar properties as compared to the amino acid sequence shown in SEQ ID NO. 3, 4, 6 or 8. In such proteins, substitutions with amino acids that are similar or analogous in properties will not generally alter the function of the protein, nor will the addition of one or more amino acids at the C-terminus and/or \ terminus. These conservative variants are preferably produced by amino acid substitutions according to the following table.

Initial residue(s)	Representative substitutions	Preferred substitutions
			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	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；Ala	Leu

Promoter for GRF gene or its coded protein

In the present invention, the promoter of GRF gene or protein encoded thereby includes a substance capable of increasing the expression and/or activity of GRF gene or protein encoded thereby.

In the present invention, the promotion of the GRF gene or its encoded protein is not particularly limited as long as it can promote expression of GRF or enhance GRF protein activity, and is within the scope of the present invention.

In a preferred embodiment, the promoter of the GRF gene or its encoded protein comprises a small molecule compound, a nucleic acid, an enzyme, or the like.

miR396

The miR396 is a non-coding single-stranded small RNA molecule with the length of 20-24 nucleotides, is mainly positioned at the 5' end of a coding sequence, and the 5' end and the 3' end matching part are subjected to base pairing to form a neck ring structure, and comprises 8 members of miR396a, miR396b, miR396c, miR396d, miR396e, miR396f, miR396g and miR396h. The mRNA of a target gene is combined through base pairing, so that the degradation or translation inhibition of the mRNA is caused, and the important regulation effect is played in the growth and development process of plants.

In a preferred embodiment, the precursor sequence of miR396e is shown in SEQ ID NO. 1, and the precursor sequence of miR396f is shown in SEQ ID NO. 2.

mature sequence/conserved sequence of miR396

In the present invention, mature/conserved sequence refers to a truly functional sequence formed by cleavage of an RNA precursor sequence.

Specifically, in the invention, miR396 represents an RNA precursor sequence; the mature sequence is an RNA sequence formed by shearing an RNA precursor sequence; the conserved sequence refers to an RNA fragment with consistent sequence among different species, the mature sequence can be identical to the conserved sequence, the mature sequence and the conserved sequence can have cross, and the conserved sequence can be longer than the mature sequence. MIR396 represents the DNA sequence encoding MIR 396.

Among the miR396 family, the mature sequence/conserved sequence similarity between subtypes a, b, c, d, e, f, g, h of the miR396 family is very high. Mature sequences of the miR396 family are well known in the art, for example, the mature sequence of miR396e is uucacagcuuuuuuguaacug (SEQ ID NO.:1, positions 12-33), and the mature sequence of miR396f is cuccacaggcuuuuuuuuguaacug (SEQ ID NO.:2, positions 9-30).

Nucleic acid constructs of the invention or mutants of the genes encoding them

The invention provides a nucleic acid construct or a mutant of a coding gene thereof, which is used for regulating and controlling the agronomic traits of plants under the condition of low nitrogen.

In the present invention, the nucleic acid constructs of the invention have 5'-3' of the structure of formula I:

X1-X2-X3 (I)

x2 is selected from a mature sequence/a conserved sequence of miR 396;

and, each "-" is a bond or a nucleotide connecting sequence.

In the invention, the nucleic acid construct or the mutant of the encoding gene thereof comprises miR396a, miR396b, miR396c, miR396d, miR396e, miR396f, miR396g and/or miR396h mutant.

In the present invention, the mutant of the nucleic acid construct of the present invention or the gene encoding the same may be a single mutation, may be a double mutation, may be a multiple mutation, preferably a single mutation or a double mutation, more preferably a mutation of miR396e and/or miR396f.

The various elements used in the constructs of the invention are either known in the art or can be prepared by methods known to those skilled in the art.

The vector of the present invention is constructed by inserting a mutant of the gene encoding the construct of the present invention or the GRF gene into a foreign vector, particularly a vector suitable for the manipulation of transgenic plants.

The vector of the invention is used for transforming plant cells so as to mediate the vector of the invention to integrate plant cell chromosomes, and the transgenic plant cells are prepared.

The transgenic plant cell of the present invention is regenerated into a plant body, thereby obtaining a transgenic plant.

The mutant of the coding gene of the above-mentioned nucleic acid construct constructed according to the present invention can be introduced into a plant cell by a conventional genetic transformation technique (e.g., agrobacterium transformation technique), thereby obtaining a plant cell carrying the mutant of the coding gene of the nucleic acid construct (or a vector carrying the mutant of the coding gene of the nucleic acid construct), or obtaining a plant cell having the mutant of the coding gene of the nucleic acid construct integrated into its genome.

Inhibitor of miR396

The invention also provides an inhibitor for miR396, and the inhibitor for miR396 can inhibit the expression or activity of miR 396. In the present invention, the inhibitor of miR396 is selected from the group consisting of: a small molecule compound, an antisense nucleic acid, a microRNA, a siRNA, an RNAi, a Crispr agent, or a combination thereof.

Use of

The invention also provides application of the nucleic acid construct shown in the formula I or a mutant of the coding gene thereof, the miR396 inhibitor and/or the GRF gene or the coding protein promoter thereof, which are used for controlling the agronomic traits of plants under the low-nitrogen condition. In the invention, the nucleic acid construct shown in the formula I or the mutant of the coding gene is derived from rice.

In the invention, the expression or activity of miR396 can be inhibited by gene mutation, gene knockout, gene interruption, RNA interference technology, crispr technology and the like.

In a preferred embodiment, miR396 can be genetically edited by Cas9 nuclease mediated with 1 or more sgrnas.

Improvement of plants (such as rice)

The present invention also provides a method of modifying a plant (e.g., rice), the modification comprising: (a) increasing yield and/or biomass; (b) promoting development of panicle and/or grain types; (c) increasing grain length; (d) increasing the grain width; (e) increasing the ear length; (f) increasing thousand seed weight; (g) increasing leaf length; (h) increasing leaf width, comprising the steps of: reducing the expression or activity of miR396 in the plant under low nitrogen conditions, administering an inhibitor of miR396, a nucleic acid construct as defined in the first aspect of the invention or a mutant of the encoding gene thereof, and/or increasing the expression or activity of a GRF gene or the encoding protein thereof in the plant.

As known to those skilled in the art, the same trait in different kinds of crops can be characterized by different terms, or the same trait can be characterized by other traits with different terms, such as in wheat, rice and other crops, and the yield can be reflected by the shape of spike length, spike grain number, grain type and the like; in soybean, the yield can be reflected by the number of pods and the grain type; in tomato, the yield can be reflected by fruit size and number; therefore, the improvement of the traits of crops of the present invention is not limited to the traits of the present invention, and includes other traits of the same concept as the traits of the present invention in other crops not described in the present invention.

In the present invention, other substances capable of regulating plant traits may be further treated with conventional methods to the plant or plant seed, thereby improving the traits of the corresponding plant.

The main advantages of the present invention include:

(1) The invention discovers for the first time that a new nucleic acid construct shown in formula I of miR396 and family members thereof or a mutant of a coding gene thereof, particularly miR396e and/or miR396f subtype mutants, can obviously improve plant traits under the condition of low nitrogen, and comprises the steps of (a) increasing yield and/or biomass; (b) promoting development of panicle and/or grain types; (c) increasing grain length; (d) increasing the grain width; (e) increasing spike length; (f) increasing thousand seed weight; (g) increasing leaf length; (h) increasing leaf width.

(2) The invention discovers for the first time that the nucleic acid construct shown in formula I or the mutant of the coding gene can up-regulate GRF8 gene expression.

(3) The invention firstly discovers that the nucleic acid construct shown in the formula I or the mutant of the coding gene thereof regulates the development of the kernel and spike types through miR396-GRF4/6/8-GIF1/2/3 pathways.

(4) The invention discovers for the first time that the nucleic acid construct shown in the formula I or the mutant of the coding gene thereof can reduce the use amount of a nitrogen fertilizer, improve the utilization rate of the nitrogen fertilizer, improve the biomass energy accumulation of rice, improve the yield and biomass of the rice and reduce the environmental pollution of the nitrogen fertilizer.

(5) The present invention has for the first time found that increasing the expression or activity of GRF (including GRF4, 6, and/or 8, especially GRF 8) genes or their encoded proteins in plants can significantly improve plant traits under low nitrogen conditions, including (a) increasing yield and/or biomass; (b) promoting development of panicle and/or grain types; (c) increasing grain length; (d) increasing the grain width; (e) increasing spike length; (f) increasing thousand kernel weight; (g) increasing leaf length; (h) increasing leaf width.

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

Unless otherwise specified, all reagents and materials used in examples of the present invention are commercially available products.

In the present invention, the medium used in the present invention is commercially available Mucuna B medium, and the total nutrient condition is that the commercially available Mucuna B medium of the present invention contains various components required for plant growth, wherein the nitrogen content is (0.034 g/L); the nitrogen-free condition is that nitrogen is not contained but other whole components are contained on the basis of the commercially available Mucun B medium used in the present invention.

The low nitrogen condition of the invention is that the amount of nitrogen fertilizer used is lower than the conventional amount of fertilizer applied during the growth of crops or no additional nitrogen fertilizer is applied to the soil, and the conventional amount of fertilizer is the amount of fertilizer applied which is known to a person skilled in the art or a farmer to ensure the stable and high quality of crops. In the field operation of the embodiment of the invention, the low-nitrogen condition is that no additional nitrogen fertilizer is applied in the plant growth environment, and the total nitrogen content in the field is 0.5-3.0g/kg soil under the condition that no additional nitrogen fertilizer is applied; in various embodiments, the total nitrogen content in the field may be 1-2.5g/kg soil, 1.2-2.2g/kg soil without additional application of nitrogen fertilizer.

The conventional amount of nitrogen fertilizer, such as 150-350 kg per hectare for rice, wheat and corn, varies from crop to crop, and can be determined by those skilled in the art or by the farmer's planting experience, and in the present invention, it is preferable that the nitrogen fertilizer is applied at 210-270 kg per hectare.

Example 1 under low nitrogen conditions, single mutation of miR396e can increase rice yield

1. Gene editing site design

To design a gene editing vector for miR396e, simultaneous targeting of other family members of miR396 is avoided. Specifically, a target site is selected from a stem-loop sequence of a miR396 precursor, and a sgRNA sequence is designed aiming at the target site: GCUCAUGUUGGGAUUGUGGU (SEQ ID NO.: 10).

2. Construction of CRISPR-Cas9 gene editing tool

A)ddH ₂ Dissolving the primers to 10. Mu.M with O, adding 8. Mu.l of annealing solution (an annual buffer: TE buffer plus 50mM NaCl) to 1. Mu.l of each of the forward and reverse primers, and mixing;

b) Running an annealing program on the uniformly mixed primers, heating the mixed primers to 95 ℃ by using a PCR instrument, keeping the temperature for 5min, then reducing the temperature by 0.1 ℃ every 1s, and reducing the temperature to 16 ℃;

c) Digesting the CRISPR-Cas9 vector by Bsa I enzyme, and recovering the vector fragment for later use;

d) Connecting gRNA and Crispr-Cas9 carrier,

adding water to 10 μ l, and connecting at 16 deg.C for 2hr.

E) Transforming Escherichia coli, selecting monoclonal M13F sequencing verification fragment and successfully connecting into vector.

3. Genetic transformation of vectors

A) The plasmid constructed above is directly transformed into agrobacterium EHA105:

1. plasmid DNA is added into agrobacterium tumefaciens competent cells, then the cells are subjected to ice bath for 30min, then the cells are put into liquid nitrogen for 1min, and then the cells are immediately put into a water bath kettle at 37 ℃ for water bath for 2min.

2. Taking out the centrifuge tube, adding LB culture medium, and shaking culturing for 3-5 hr.

3. Taking out the bacterial liquid and coating the plate on an LB culture medium plate containing corresponding antibiotics, and carrying out inverted culture in an incubator. Colonies were visible around 2 days.

B) Rice transgenosis:

1. inducing callus, soaking the seed after shelling in NaClO for disinfection, washing with sterile water, inoculating in NB induction medium, and culturing in incubator for 10-15 days.

2. Subculturing the callus, cutting the induced callus with a single-sided knife, placing into a subculture medium, and culturing under the same conditions.

3. And (3) carrying out agrobacterium infection and resistant callus screening, carrying out propagation on the agrobacterium EH105 strain transferred into the target vector, and then soaking the callus with a better state.

4. Sucking out or pouring out the bacterial liquid, and culturing the callus in a dark box for 48-72h.

5. The callus after the end of co-cultivation was washed by soaking in Carbenicill-resistant sterile water to remove Agrobacterium.

6. The callus was blotted dry and inoculated on a selection medium containing antibiotics and cultured under light for two weeks.

7. Differentiating and culturing the callus, selecting the callus with vigorous growth (resistant callus) and transferring the selected callus to a differentiation culture medium containing antibiotics. Most of the callus rapidly grows within one week, and green spots appear on the callus, and seedlings are rapidly differentiated from the green callus.

4. Plant culture and mutant screening

A) Transferring the differentiated robust seedlings to a rooting culture medium containing antibiotics for rooting culture for one week, hardening the seedlings at room temperature for 2-3 days, cultivating the seedlings in a greenhouse matrix for 15-20 days, and transplanting the seedlings to a field.

B) Taking leaves of each plant, extracting genome DNA, and designing primers at two sides of the target site. The amplified fragments were subjected to Sanger sequencing to determine the genotype of each plant.

C) And (3) detecting miR396e gene mutation types, screening a series of mutation types in a miR396e mature region in the T0 generation, and continuously expanding propagation through 4-5 generations to continuously increase mutation type populations.

D) And (3) detecting the accumulation level of the mature miR396e in the miR396e/f mutant by Northern blot hybridization by using the miR396e as a detection probe.

5. Results of the experiment

Multiple mutation types aiming at single mutation of miR396e are detected, and observation and analysis show that the miR396e mutant phenotype is expressed in the way of influencing the rice grain type (grain length, grain width and/or grain weight) (figure 1).

Further analysis showed that mir396e mutant plants had a 4.77% increase in seed length and a 5.58% increase in grain width over wild type plants (FIG. 2).

6. Conclusion of the experiment

The miR396e single mutation can change the grain type of rice seeds, particularly can increase the grain length, the grain width and/or the grain thickness, and finally improves the yield of rice through the change of the grain type.

Example 2 under the condition of low nitrogen, the miR396ef double mutant can improve the yield of rice

Obtaining different types of mutants such as miR396ef and miR396abcef by adopting the method in the experimental example 1; different gRNAs are designed, and miR396ef double mutants and other different types of mutants such as miR396abcef are obtained.

(1) Influence of growth situation of miR396ef mutant (miR 396ef double mutant) under nitrogen-free condition

Observations of the mutant and wild type seedlings grown under different nitrogen conditions revealed that the mir396ef mutant had hardly any difference from the wild type under holo-element (nitrogen content 0.034 g/L) culture conditions; however, in the medium without nitrogen (nitrogen content 0 g/L), the growth potential of the three mir396e/f mutants was significantly stronger than that of the wild type (FIG. 3). The experimental result preliminarily shows that miR396e/f can participate in the process of responding to the external nitrogen deficiency nutrition stress and has important influence on the growth potential of rice.

(1) Influence of miR396ef on grain type under low nitrogen condition

Under the field environment and the low-nitrogen condition (no nitrogen fertilizer is applied, the total nitrogen content of the soil is about 1.5g/kg of soil), the different mutation types of miR396ef can increase the grain length of rice by 8.46%, the grain width by 7.95%, the grain thickness by 8.16% and the thousand-grain weight by 26.51% (shown in figure 4, a-c). The mir396abcef mutant has the same grain type as the mir396ef mutant, and no obvious statistical difference exists.

Section observation is carried out on wild type seeds and mutant seeds, and the change of the grain type is mainly reflected in the increase of the number of glume cells and the increase of the volume of the glume cells of the seeds. (FIG. 5, a-b)

(2) Under the condition of low nitrogen, miR396ef mutation can increase the grain number per ear of rice

Under field conditions and low nitrogen conditions (no nitrogen fertilizer is applied), mir396ef ears are long (20.55 +/-1.83cm VS 17.93 +/-1.64 cm) and have more main branches (8.8 +/-1.34VS 7.33 +/-1.56) compared with wild type. Longer ear lengths and more ear branches are generally associated with more grains, and ear numbers for mir396ef mutant are higher than wild type (77.82 ± 8.89vs 72.35 ± 10.96). (FIG. 6, a-c)

(3) Under the condition of low nitrogen, miR396ef mutation can increase the yield of rice

Our field yield trials showed that in a field where 270kg ha-1 nitrogen fertilizer was applied under normal cultivation conditions (2 m.times.2 m), the mir396ef mutant increased grain yield by about 4% over the wild type.

Under low nitrogen conditions (no nitrogen fertilizer applied), the mir396ef mutant rice showed a significant increase in grain yield of about 15% over the wild type. (FIG. 7, a-b)

In conclusion, under the condition of low nitrogen, the mir396ef double mutation has more obvious influence on phenotypes such as rice grain type, grain number per ear, plant height and the like compared with the normal cultivation condition, and finally the yield of rice can be improved through the influence on the phenotypes.

Example 3 miR396ef mutant can improve biomass of rice under low nitrogen condition

(1) miR396ef mutation can increase rice leaf length

In the field condition, in the middle stage of grouting, selecting three tillers with main effects for each single plant, and selecting 20 single plants in total; shearing the leaf sheaths at the positions of the flag leaf sheaths of the three tillers of each selected single plant; the distance from the blade tip to the sheath was measured with a graduated scale. Mir396ef double mutant flag leaves were 32.8% longer compared to wild type flag leaves (fig. 8).

(2) miR396ef mutation can increase rice plant height

Under the field condition, in the later stage of grouting, 20 single plants are selected; the distance from the ground surface to the ear of each individual plant was measured with a graduated scale. The results show that the mir396ef mutant (74.13 + -3.54 cm) is higher than the wild type (69.33 + -3.20 cm), but the tiller number is similar to the wild type. (FIG. 9)

(3) miR396ef mutation can increase biomass accumulation of overground part of rice

Under field conditions, after rice is mature, selecting 20 individual plants; cutting the single plants from the ground plane; putting all the plant tissues on the cut overground part into a 60 ℃ oven, and baking for two weeks; the dry matter weight of each individual plant was weighed. The mir396ef mutant increased 25% of the aerial dry biomass per plant under low nitrogen cultivation conditions compared to the wild plant (fig. 10). Detection analysis shows that the photosynthetic rate of flag leaf per leaf area has no significant difference compared with the wild type. Therefore, the mutant plant height is increased; the flag leaf area is increased, the total photosynthetic rate is increased, and more photosynthetic products can be accumulated; the nitrogen absorption and utilization rate is increased, and the factors become favorable factors for increasing the dry matter of the mir396ef mutant plants.

Example 4miR396ef regulates grain and panicle development via miR396-GRF4/6/8-GIF1/2/3 pathway

All 12 GRF transcription factor genes in rice carry miR396 target sites. We found that only OsGRF4, osGRF6 and OsGRF8 were up-regulated in mir396ef mutant plants compared to wild type. RLM-race (5' RNA ligase-mediated rapid amplification of cDNA ends) analysis shows that miR396 can directly cut mRNA of OsGRF4 and OsGRF6 at a specific position of miR396 pairing region in vivo.

To study mir 396-mediated seed and ear development regulated by OsGRF4, osGRF6 and OsGRF8, we constructed mir 396-targeted resistance gene sequences of OsGRF4, osGRF6 and OsGRF8 (designated np: rGRF4, np: rGRF6 and np: rGRF8, respectively) (FIG. 11 a) and transferred them into rice under the control of their own promoters. We have found that: (1) The np: rGRF4, np: rGRF6 and np: rGRF8 seeds were larger than the wild type plants, with np: rGRF4 having the largest grain size increase and np: rGRF6 having the smallest grain size increase (FIG. 11 b). (2) Both np: rGRF6 and np: rGRF8 increased in ear length (FIG. 11 c), and np: rGRF4 had ear length and branch number similar to wild type. These results indicate that mir396ef molecule regulates seed and ear development by regulating its target genes OsGRF4, osGRF6 and OsGRF 8.

GRF has been shown to interact with the transcriptional co-activator GIFs. To find GIFs that interact with GRF4 and GRF6, we performed a yeast two-hybrid screen. OsGIF1, osGIF2 and OsGIF3 were found to interact with OsGRF4, and OsGIF3 also interacted with OsGRF6 (FIG. 11 d). GIF1 mutants were generated with CRISPR/Cas9 technology and the expected short leaf phenotype was observed. The gif1 mutants also showed smaller plant and aborted seed phenotypes compared to the wild type (fig. 11 e). These results indicate that the development of the plant type and the grain type of rice is regulated by the miR396EF-GRF4/6/8-GIF1/2/3 module.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shunheng Biotech Co., ltd

<120> P2020-1164

<130> application of miR396 or mutant of encoding gene thereof in regulation and control of plant agronomic traits

<150> CN201910606371.7

<151> 2019-07-05

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 184

<212> RNA

<213> Artificial sequence (artificial sequence)

<400> 1

gcgggcaugc uuuccacagg cuuucuugaa cugugaacuc gugggggugu augugcucau 60

guugggauug uggucggugg ccuccaauuc ucugaaaaga aagcugaauu gucgagcucc 120

ccguucuguc uuuggucguc ucuaccuguu gaugguucaa gaaagcccau ggaaaccaug 180

ccgc 184

<210> 2

<211> 176

<212> RNA

<213> Artificial sequence (artificial sequence)

<400> 2

gccaugcucu ccacaggcuu ucuugaacug ugaacucgug ugugcaugcu ccucauauau 60

uguucuagau cccaugcaug augcauaucg aucgaucuga ucugaauuag gucaucgaug 120

cgcaucugga uccccaucuu guugauaguu caagaaaguc cuuggaaaac auggug 176

<210> 3

<211> 422

<212> PRT

<213> Rice (Oryza sativa)

<400> 3

Met Pro Pro Cys Leu Arg Arg Trp Pro Thr Thr Ala Arg Pro Arg Gln

1 5 10 15

Pro Arg Pro Pro Pro Ser Ser Pro Ser Ala Ala Pro Pro Arg Ser Pro

20 25 30

Arg Lys Gln Arg Glu Pro Ala Ala Thr Thr His Phe Leu Gly Ser Ser

35 40 45

Gly Ala Cys Asp Asn Thr Val Arg Arg Cys Val Trp Val Gly Gly Cys

50 55 60

Arg Gly Gly Gly Gly Val Ala Met Gly Glu Asp Ala Pro Met Thr Ala

65 70 75 80

Arg Trp Pro Pro Ala Ala Ala Ala Arg Leu Pro Pro Phe Thr Ala Ala

85 90 95

Gln Tyr Glu Glu Leu Glu Gln Gln Ala Leu Ile Tyr Lys Tyr Leu Val

100 105 110

Ala Gly Val Pro Val Pro Pro Asp Leu Val Leu Pro Ile Arg Arg Gly

115 120 125

Leu Asp Ser Leu Ala Ala Arg Phe Tyr Asn His Pro Ala Leu Gly Tyr

130 135 140

Gly Pro Tyr Phe Gly Lys Lys Leu Asp Pro Glu Pro Gly Arg Cys Arg

145 150 155 160

Arg Thr Asp Gly Lys Lys Trp Arg Cys Ser Lys Glu Ala Ala Pro Asp

165 170 175

Ser Lys Tyr Cys Glu Arg His Met His Arg Gly Arg Asn Arg Ser Arg

180 185 190

Lys Pro Val Glu Thr Gln Leu Val Ala Gln Ser Gln Pro Pro Ser Ser

195 200 205

Val Val Gly Ser Ala Ala Ala Pro Leu Ala Ala Ala Ser Asn Gly Ser

210 215 220

Ser Phe Gln Asn His Ser Leu Tyr Pro Ala Ile Ala Gly Ser Asn Gly

225 230 235 240

Gly Gly Gly Gly Arg Asn Met Pro Ser Ser Phe Gly Ser Ala Leu Gly

245 250 255

Ser Gln Leu His Met Asp Asn Ala Ala Pro Tyr Ala Ala Val Gly Gly

260 265 270

Gly Thr Gly Lys Asp Leu Arg Tyr Thr Ala Tyr Gly Thr Arg Ser Leu

275 280 285

Ala Asp Glu Gln Ser Gln Leu Ile Thr Glu Ala Ile Asn Thr Ser Ile

290 295 300

Glu Asn Pro Trp Arg Leu Leu Pro Ser Gln Asn Ser Pro Phe Pro Leu

305 310 315 320

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

325 330 335

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

340 345 350

Asn Asp Tyr Ala Ala Val Asp Ser Val Lys Gln Glu Asn Gln Thr Leu

355 360 365

Arg Pro Phe Phe Asp Glu Trp Pro Lys Gly Arg Asp Ser Trp Ser Asp

370 375 380

Leu Ala Asp Glu Asn Ala Asn Leu Ser Ser Phe Ser Gly Thr Gln Leu

385 390 395 400

Ser Ile Ser Ile Pro Met Ala Ser Ser Asp Phe Ser Ala Ala Ser Ser

405 410 415

Arg Ser Thr Asn Gly Asp

420

<210> 4

<211> 394

<212> PRT

<213> Rice (Oryza sativa)

<400> 4

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

1 5 10 15

Ser Ser Pro Ala Ala Ala Thr Ala Ser Leu Leu Pro Phe Cys Arg Ser

20 25 30

Thr Pro Leu Ser Ala Gly Gly Gly Gly Val Ala Met Gly Glu Asp Ala

35 40 45

Pro Met Thr Ala Arg Trp Pro Pro Ala Ala Ala Ala Arg Leu Pro Pro

50 55 60

Phe Thr Ala Ala Gln Tyr Glu Glu Leu Glu Gln Gln Ala Leu Ile Tyr

65 70 75 80

Lys Tyr Leu Val Ala Gly Val Pro Val Pro Pro Asp Leu Val Leu Pro

85 90 95

Ile Arg Arg Gly Leu Asp Ser Leu Ala Ala Arg Phe Tyr Asn His Pro

100 105 110

Ala Leu Gly Tyr Gly Pro Tyr Phe Gly Lys Lys Leu Asp Pro Glu Pro

115 120 125

Gly Arg Cys Arg Arg Thr Asp Gly Lys Lys Trp Arg Cys Ser Lys Glu

130 135 140

Ala Ala Pro Asp Ser Lys Tyr Cys Glu Arg His Met His Arg Gly Arg

145 150 155 160

Asn Arg Ser Arg Lys Pro Val Glu Thr Gln Leu Val Ala Gln Ser Gln

165 170 175

Pro Pro Ser Ser Val Val Gly Ser Ala Ala Ala Pro Leu Ala Ala Ala

180 185 190

Ser Asn Gly Ser Ser Phe Gln Asn His Ser Leu Tyr Pro Ala Ile Ala

195 200 205

Gly Ser Asn Gly Gly Gly Gly Gly Arg Asn Met Pro Ser Ser Phe Gly

210 215 220

Ser Ala Leu Gly Ser Gln Leu His Met Asp Asn Ala Ala Pro Tyr Ala

225 230 235 240

Ala Val Gly Gly Gly Thr Gly Lys Asp Leu Arg Tyr Thr Ala Tyr Gly

245 250 255

Thr Arg Ser Leu Ala Asp Glu Gln Ser Gln Leu Ile Thr Glu Ala Ile

260 265 270

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

275 280 285

Pro Phe Pro Leu Ser Ser Tyr Ser Gln Leu Gly Ala Leu Ser Asp Leu

290 295 300

Gly Gln Asn Thr Pro Ser Ser Leu Ser Lys Val Gln Arg Gln Pro Leu

305 310 315 320

Ser Phe Phe Gly Asn Asp Tyr Ala Ala Val Asp Ser Val Lys Gln Glu

325 330 335

Asn Gln Thr Leu Arg Pro Phe Phe Asp Glu Trp Pro Lys Gly Arg Asp

340 345 350

Ser Trp Ser Asp Leu Ala Asp Glu Asn Ala Asn Leu Ser Ser Phe Ser

355 360 365

Gly Thr Gln Leu Ser Ile Ser Ile Pro Met Ala Ser Ser Asp Phe Ser

370 375 380

Ala Ala Ser Ser Arg Ser Thr Asn Gly Asp

385 390

<210> 5

<211> 3825

<212> DNA

<213> Rice (Oryza sativa)

<400> 5

aaagcaccat tactaaagac cgcggcgtgt gcttgcgttg cgagcgagcg agagcgagag 60

agagattgag agagagagag ggaagggatg gcgatgccgt atgcctccct gtctccggcg 120

gtggccgacc accgctcgtc cccggcagcc gcgaccgcct ccctcctccc cttctgccgc 180

tccaccccgc tctccgcgta agcaacgcga acccgcggct acaacccatt ttcttggctc 240

cagtggtgca tgtgacaaca cggtgagacg ttgtgtgtgg gtgggtgggt gcaggggcgg 300

tggtggcgtc gcgatggggg aggacgcgcc gatgaccgcg aggtggccgc cggcggcggc 360

ggcgaggctg ccgccgttca ccgcggcgca gtacgaggag ctggagcagc aggcgctcat 420

atacaagtac ctggtggcag gcgtgcccgt cccgccggat ctcgtgctcc ccatccgccg 480

cggactcgac tccctcgccg cccgcttcta caaccatccc gcccgtacgt cgtgttccta 540

tttcttgcct ctcctctacc atcgctgcat tgcttttgga tgcttgttta gtgtcggctt 600

ctttgtttat tccgatcagg cgtactttgc ttccatttgt taattggctc cgggtcattt 660

gttaatccgg gttacgcgat tcaagaaaca tgcgtgtgtg tttttatgct atcctccgga 720

tttggtaata aaaaggcttg tttttaaatc caaaactcgt gctcgcttca cgattagcgc 780

atcatttttt ttttttgggg gggggggggg ggaagtttgc ccatcattct gtctctgttt 840

gatctgatag aggacgtgca cacgctcttg tctgaaataa aatcttttgt ttatcagtat 900

gcccatggga taagccattt tctctgtgaa ccaacaccct ggcaaactgt ttttttgctc 960

gccatttttg agcgattgct aagaacagat aactatgccc tgcatatgga tcggatatgg 1020

acttctcaaa tattcaaatg ccattctatt aggaactcaa aatgcattac caacaaatgc 1080

attcttgtgt gtaacacggt tgctacgatg tgcctgtttt tgtacagttg gatatggtcc 1140

gtacttcggc aagaagctgg acccagagcc agggcggtgc cggcgtacgg acggcaagaa 1200

atggcggtgc tcgaaggagg ccgcgccgga ttccaagtac tgcgagcgcc acatgcaccg 1260

cggccgcaac cgttcaagaa agcctgtgga aacgcagctg gtcgcccagt cccaaccgcc 1320

ctcatctgtt gtcggttctg cggcggcgcc ccttgctgct gcctccaatg gcagcagctt 1380

ccaaaaccac tctctttacc ctgctattgc cggcagcaat ggcgggggcg gggggaggaa 1440

catgcccagc tcatttggct cggcgttggg ttctcagctg cacatggata atgctgcccc 1500

ttatgcagct gttggtggtg gaacaggcaa agatctcagg tgattgttca tttctttttt 1560

tttaatcaaa cgccatattt acttgtttag cactgtcttg aatcatgata tgtatccttc 1620

cgttgtctaa aaaaaaggtg ccatgctcta actgattggt gtcaggtgga tgcagttatg 1680

aatctgtatt tttcattgtg atcggttaat aactgtgtcc catttgtttg cattggtggc 1740

aatcgaatca gctgtccatg ctcagtagta ctacttcgat ttggtgctgc aatcactgaa 1800

agtctgaaac tttactctct gcactgcaaa aatttgtgtt atgtttaggt ttccagagtg 1860

ctgcctcttt gcccttccca tactttctgg tatcagtttt cagccccaga agccggggac 1920

agtctccata agagatttct gctcaggtga aactggggtg cagggtctta acatggcttt 1980

ggcccagtag tttgaaacat gtactgtcca taaagatgat actactacat atttgtgtct 2040

gccctcgcag tgcttgtgcc tgctggtagc tgatcatggc ttcccttggc atttactcca 2100

cttctttatt cctccacaga atccagttgt ttctgtctct gctcttcagg ggcagtcaat 2160

tatttggccc ttgcaaaata ctgtctctga agatgtctca ccgatcacca ctatacctga 2220

aacattttcc agtggccagc gtgagctgca tgatgctcca agtcaactct atactcatcc 2280

aatgttgatg attagatttt aacaatgcaa ctctttgatt tatcttccct acaaaaaaaa 2340

aggaactctt tgatttatct tcggtgaatc tcagtctgac cttagtacct agcctcatta 2400

tttacttcac caaatgtata actctacagt gcttgttcgt gttgatttgg tttagtttag 2460

ttattgaatt attcggtcac cttagtcttt gattgttttt ttctttctgc tcttgtcatc 2520

aactgtttag ggttcagctg acttgctgct gcaactaaac tgtcttctgg ttttactgca 2580

aaatagaatg tttcttgggc catgatctgc tgctatatat gattagttaa accatggttc 2640

tatgttttct tatatgaatt catgacaaga atactaactt ttggaaaagg taattttatt 2700

ttttttgtat gataataatg ctttggattc tttctagttt atctgtcgga cttaggttaa 2760

ctacatttcc tccggtacat ggatttattt cattcttaca attgagccct tatgaatatt 2820

ttcttcctaa ttctgttcta aaaagttaga attgacatat tttcgatagg tacatgccta 2880

gcacttgcat tcgtgtttcc tactaattcc caatcactgt atcttctcaa attcaggtat 2940

actgcttatg gcacaagatc tttggcggat gagcagagtc aactcattac tgaagctatc 3000

aacacatcta ttgaaaatcc atggcggctg ctgccatctc agaactcgcc atttcccctt 3060

tcaagctatt ctcagctggg ggcactaagt gaccttggtc agaacacccc cagctcactt 3120

tcaaaggttc agaggcagcc actttcgttc tttgggaacg actatgcggc tgtcgattct 3180

gtgaagcaag agaaccagac gctgcgtccc ttctttgatg agtggccaaa gggaagggat 3240

tcatggtcag acctcgctga tgagaatgct aatctttcgt cattctcagg cacccaactg 3300

tcgatctcca taccaatggc atcctctgac ttctcggcgg ccagttctcg atcaactaat 3360

ggtacgacta cttgatctcc ccccaattac ttcgtgcgtg tttatgtctg tatcctgcaa 3420

tgtctgaaga tttcttactg aaaacgtcat ctggtctgtg tgcaggtgac tgaatgctgc 3480

gtggatgatg atcctgctgc ccagtgaact catactggcc ttgttgctgt cttgctctgc 3540

gattttctgc ctgctcgcgc ccaccgtacg atagtagcaa aacattctat gcttctgtaa 3600

tttaccagtg ttcccctgtc agatttgcgt gtgaaatcga tcaaactccg tggtctcctt 3660

tggacgaagg gagatgtcaa cgttttcctt gatgtttact gctagtaaca tcttattact 3720

tcccaaatgc tgatcagcct ctgcttgcta tgctctcctg tttgtcgact caacagtgcc 3780

gtcaacatca gtgcagcaac gtgaggctca tgctttttaa ggtca 3825

<210> 6

<211> 356

<212> PRT

<213> Rice (Oryza sativa)

<400> 6

Met Leu Ser Ser Ser Pro Ser Ala Ala Ala Pro Gly Ile Gly Gly Tyr

1 5 10 15

Gln Pro Gln Arg Gly Ala Ala Val Phe Thr Ala Ala Gln Trp Ala Glu

20 25 30

Leu Glu Gln Gln Ala Leu Ile Tyr Lys Tyr Leu Val Ala Gly Val Pro

35 40 45

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

50 55 60

Ala Thr Tyr Ser Phe Ala Asn Pro Ala Ala Ala Pro Phe Tyr His His

65 70 75 80

His His His Pro Ser Leu Ser Tyr Tyr Ala Tyr Tyr Gly Lys Lys Leu

85 90 95

Asp Pro Glu Pro Trp Arg Cys Arg Arg Thr Asp Gly Lys Lys Trp Arg

100 105 110

Cys Ser Lys Glu Ala His Pro Asp Ser Lys Tyr Cys Glu Arg His Met

115 120 125

His Arg Gly Arg Asn Arg Ser Arg Lys Pro Val Glu Ser Lys Thr Ala

130 135 140

Ala Pro Ala Pro Gln Ser Gln Pro Gln Leu Ser Asn Val Thr Thr Ala

145 150 155 160

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

165 170 175

Thr His Gly Leu Ser Leu Gly Gly Ala Gly Ser Ser Gln Phe His Val

180 185 190

Asp Ala Pro Ser Tyr Gly Ser Lys Tyr Ser Leu Gly Ala Lys Ala Asp

195 200 205

Val Gly Glu Leu Ser Phe Phe Ser Gly Ala Ser Gly Asn Thr Arg Gly

210 215 220

Phe Thr Ile Asp Ser Pro Thr Asp Ser Ser Trp His Ser Leu Pro Ser

225 230 235 240

Ser Val Pro Pro Tyr Pro Met Ser Lys Pro Arg Asp Ser Gly Leu Leu

245 250 255

Pro Gly Ala Tyr Ser Tyr Ser His Leu Glu Pro Ser Gln Glu Leu Gly

260 265 270

Gln Val Thr Ile Ala Ser Leu Ser Gln Glu Gln Glu Arg Arg Ser Phe

275 280 285

Gly Gly Gly Ala Gly Gly Met Leu Gly Asn Val Lys His Glu Asn Gln

290 295 300

Pro Leu Arg Pro Phe Phe Asp Glu Trp Pro Gly Arg Arg Asp Ser Trp

305 310 315 320

Ser Glu Met Asp Glu Glu Arg Ser Asn Gln Thr Ser Phe Ser Thr Thr

325 330 335

Gln Leu Ser Ile Ser Ile Pro Met Pro Arg Cys Gly Ser Pro Ile Gly

340 345 350

Pro Arg Leu Pro

355

<210> 7

<211> 3275

<212> DNA

<213> Rice (Oryza sativa)

<400> 7

ccccctctcc tctccctctc acactcacac gctgcagcag cagcagcagc agcagctttc 60

ccaccgactc ctccccctcc tccattaatg gccgccacca agaaccctcc aacccccacg 120

tgacctcctc ctcccctccc cctccccctc cccctcccga cctcgccgcc ggcgacctcc 180

cttcttcttc ctgcttgcct gctcgcttgc ctgcctggtt cgaccgatgc tgagctcgtc 240

gccctcggcg gcggcgccgg ggataggagg gtaccagccg cagcgcgggg cggcggtctt 300

cacggcggcg cagtgggcgg agctggagca gcaggcgctc atttacaagt acctcgtcgc 360

cggtgtcccc gtcccgggcg atctcctcct cccaatccgc ccccactcct ccgccgccgc 420

cacctactcc ttcgccaacc ccgccgccgc gcccttctac caccaccacc accacccctc 480

tcgtaagctc tctctccatc ttttttccac aaatggtcca tctcttgttt gcttcatgct 540

tgggtattca aatctgagaa aaatttatat atgtgtgcgc gtgtgctttc ttgggacttt 600

ttcttttttt tttgtttctt ctttcaggac aggatctctt tgctgccctg ctcattggga 660

ttgatttgct attgctctca cgatttattg atagatgaac gtacacggat ctttgcttat 720

agtatgtccg tttaagctgt tcgattgatt ctttgctcac tcttatatcc taagcaaatt 780

aagcatatag tagttattac cattaccaac tttgcattgg gttgatgaaa tgttgaagtg 840

gtgcattttg atctagtttt aatatgaaca atgatgaatg ctgatatgga ttcaatgtgc 900

ctgtgctcat gtcactgcag tgagctatta tgcctactat ggcaagaagc ttgaccctga 960

gccgtggcgt tgccgccgca ccgacggcaa gaagtggcgg tgctccaagg aggcgcaccc 1020

cgactccaag tactgcgagc gccacatgca ccgtggccgc aaccgttcaa gaaagcctgt 1080

ggaatccaag accgctgccc ctgcgcccca gtcgcagccc cagctgtcca atgtcacgac 1140

cgcgactcac gacaccgatg cgcctctccc gtcactcact gtgggtgcta aaacccacgg 1200

tctgtccctt ggtggtgctg gctcgtcgca gttccatgtc gacgcaccat cgtacggcag 1260

caagtatccc ctctaatctc attgactctg tgttgaatgc ttatttgaat taagcttgcc 1320

tagattgatt gcatattatg ctggaataga gctgatctgg tagctttctt aaaagggttg 1380

agacactcaa aatagtattt gagctctaat gttgttgatg cttattgctc aagtagtgaa 1440

cctaccctca ttccagtaat tataagtgtg tgaggctgcc taagaatttg tcgatcagag 1500

cttattcaat gagttagtca gatgagtgat actgccaatt tacatgtgac cataacccca 1560

actaaaaatt ttgtacctgg attatgttag ttaaatctgt gtattgcatt ccatccatcc 1620

ttgatgctac aaacctctgt gggcacaatg tcataagcat atgtctgttg cttgtgcctt 1680

tagccctatt atactgttgc tttttgcagt aatattgtac gcttttccag atcacctaag 1740

ccctagctga ttttgacctt tattgcccta actcctgatt atgttttgag aaatagtgtt 1800

aacagttgtc atgttctggg gtgtttttgg tccatgccct ctaaaatctt ggtaccgtct 1860

tttttatgct ggaaaatttt gcttgtgtca tcttttttgt gtgccatacc ttagtttagt 1920

atttcctcct ctgctagcaa tcacaaaata aaaatattgt accttcgaca ccatccttat 1980

gcttgtgctg catgcatggc atatgttcta gtttcgtgtc attttgcatt tgcatcttgc 2040

tagatgactg acaaagctta ttttgctcct tcgactattt ttcgttcttt ttcctttttt 2100

aactatagta ggttgtctta ttatcgtttt ggtagcattt ttcctatgca gctgctttta 2160

tgtcctcccc ttgagttgta gaaatccctt agctactgtg aacctgtaat catcccatta 2220

tattccattt gatgctgcgc aaatctacat gtactgtgtc tgagtctggt gtctggtcct 2280

aattttcatg tatcaatggc tatgcaggta ctctcttgga gctaaagctg atgtgggtga 2340

actgagcttc ttctcaggag catcaggaaa caccaggggc ttcaccattg attctccaac 2400

agatagctca tggcattcac tgccttccag tgtaccccca tacccgatgt caaagccaag 2460

ggactctggc ctcctaccag gtgcctactc ctactcccac cttgaacctt cacaggaact 2520

tggccaggtc accatcgcct cgctgtccca agagcaggag cgccgctctt ttggtggtgg 2580

agcggggggg atgctaggaa atgtgaagca cgagaaccag ccgctgaggc ctttcttcga 2640

tgagtggcct gggaggcgag actcgtggtc ggagatggat gaggagaggt ccaaccagac 2700

ctccttctcg acaacccagc tctcgatctc catcccgatg cccagatgtg ggtcccctat 2760

cggtccgcgt ctaccttgag catcccttca ccaacatttc tctcacacaa ttcattccat 2820

tttctttgat gatgcaggtg attgagaact ttgctgcttg tggcagcggg gtggacctct 2880

accccgcatt ttaccgctgc tagtgagttg gatcagtgat tgcgcctccc ctggttcttt 2940

gttcaattgt atcgtgctat gaactagtta agagaaccct actttttttt tctagtagaa 3000

gagacagaaa actcttatcc atcatcatgt tttaagattc cacgatgttt tgtacctgca 3060

acccacgacc tgccggctgc tggaagttac tggctgtctg taatgtttgt agtagatgat 3120

ctatgatgta tcatgtatct atctacttgt tccgaattgc ggaaaccaaa gccgataatc 3180

tggcatgtgc caacgtcctc cttaaagctc ttgtgctatg tattttgctt ttggtgggaa 3240

aaaagaagaa aagaaggttc catttttctt aataa 3275

<210> 8

<211> 409

<212> PRT

<213> Rice (Oryza sativa)

<400> 8

Met Leu Ser Ser Cys Gly Gly His Gly His Gly Asn Pro Arg Ser Leu

1 5 10 15

Gln Glu Glu His His Gly Arg Cys Gly Glu Gln Gln Gly Gly Gly Gly

20 25 30

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

35 40 45

Arg Ala Ser Pro Pro Ser Pro Ser Ser Ser Ser Phe Leu Gly Ser Thr

50 55 60

Ser Ser Ser Cys Ser Gly Gly Gly Gly Gly Gly Gln Met Leu Ser Phe

65 70 75 80

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

85 90 95

Met Gln Gly Val Leu Ala Arg Val Arg Gly Pro Phe Thr Pro Thr Gln

100 105 110

Trp Met Glu Leu Glu His Gln Ala Leu Ile Tyr Lys His Ile Ala Ala

115 120 125

Asn Val Ser Val Pro Ser Ser Leu Leu Leu Pro Ile Arg Arg Ser Leu

130 135 140

His Pro Trp Gly Trp Gly Ser Phe Pro Pro Gly Cys Ala Asp Val Glu

145 150 155 160

Pro Arg Arg Cys Arg Arg Thr Asp Gly Lys Lys Trp Arg Cys Ser Arg

165 170 175

Asp Ala Val Gly Asp Gln Lys Tyr Cys Glu Arg His Ile Asn Arg Gly

180 185 190

Arg His Arg Ser Arg Lys His Val Glu Gly Arg Lys Ala Thr Leu Thr

195 200 205

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

210 215 220

His Thr Val Ala Arg Gln Lys Gln Val Lys Gly Ser Ala Ala Thr Val

225 230 235 240

Ser Asp Pro Phe Ser Arg Gln Ser Asn Arg Lys Phe Leu Glu Lys Gln

245 250 255

Asn Val Val Asp Gln Leu Ser Pro Met Asp Ser Phe Asp Phe Ser Ser

260 265 270

Thr Gln Ser Ser Pro Asn Tyr Asp Asn Val Ala Leu Ser Pro Leu Lys

275 280 285

Leu His His Asp His Asp Glu Ser Tyr Ile Gly His Gly Ala Gly Ser

290 295 300

Ser Ser Glu Lys Gly Ser Met Met Tyr Glu Ser Arg Leu Thr Val Ser

305 310 315 320

Lys Glu Thr Leu Asp Asp Gly Pro Leu Gly Glu Val Phe Lys Arg Lys

325 330 335

Asn Cys Gln Ser Ala Ser Thr Glu Ile Leu Thr Glu Lys Trp Thr Glu

340 345 350

Asn Pro Asn Leu His Cys Pro Ser Gly Ile Leu Gln Met Ala Thr Lys

355 360 365

Phe Asn Ser Ile Ser Ser Gly Asn Thr Val Asn Ser Gly Gly Thr Ala

370 375 380

Val Glu Asn Leu Ile Thr Asp Asn Gly Tyr Leu Thr Ala Arg Met Met

385 390 395 400

Asn Pro His Ile Val Pro Thr Leu Leu

405

<210> 9

<211> 3601

<212> DNA

<213> Rice (Oryza sativa)

<400> 9

gttggctagt ccaggactag agggtgcagt gcattcaatt gcttgcttcc tcttcctccc 60

ctcctccttc cccaaagcag caaggccagc ctgtgtttcc caaacaccca cagccatcac 120

ctcctcttct tcctctctgc agtaggggtg ctaggctagg gtagctagct agctaccatc 180

atcatgagct caatgccaca agaagccatt gctccccatc cttcctaacc ttcctgctgg 240

ttttgcaaac atcccacaca cacacaaagc agtgacagtg agtgccaatg ctgagctctt 300

gtggtggcca tggccatgga aatccaagaa gcttgcaaga agaacaccat ggcagatgtg 360

gtgagcagca aggtggagga ggaggaggag ggcaagagca agagcaagat gggttcttgg 420

tgagagaggc aagggcatcc ccaccatctc catcttcttc atcatttctt ggatccacaa 480

gctcttcttg ttctggagga ggaggaggag ggcagatgtt gagcttctcc tcccccaatg 540

gaacagcagg tgagatgaac tgatgatgct gatgctgcag tgcaaagaac cagggaaaaa 600

aagatttatt tgcttttttt tttagtcttc tctggtgaat gtactgttgc atccgtggtg 660

tgtgtgtgtc tgtggggttt gatcgatccc cagctggtga ttgtttttgc ccatcatggt 720

ctgagagtct ctcatggcat catcctgcaa aagcccctgc ctttggggtc ttgtcaatag 780

caaaaggagg gctcttctct tgttattccc ctccacacac actcttttgc ttttcttgca 840

acccacctcc acctcaagga tgtgtcttgt ccccaaagat gtgagctttt tcttccccct 900

tcatcccaag aaaaaataaa gctaaagaag gcagcagcag cagcaagcac cgacttgtgt 960

tgtgctgctg tttccttaaa atcttgcatg tgttggaacc aggaaaccat acaccacatg 1020

ctcatgggca tctttggcat caggcctatt tgctcttctt ggctttagta gtgagtactt 1080

catcatttgt gctcatcagt tttgcttgtg ttgtgatgaa gggttgggct tgagctcagg 1140

aggaagcatg cagggggtct tggcaagggt cagggggccg ttcaccccaa cacagtggat 1200

ggagctggag caccaggcac tgatctacaa gcacattgct gcaaatgttt ctgtcccttc 1260

cagcttgctc ctccccatca ggagaagcct ccatccatgg ggtactacct tttttattca 1320

gctcatgtga ttgttattct gttctgcgtc atatcccctt tctgttgtaa atagctcaag 1380

ataggaataa aggaataagt acttagaaaa tatgaaagaa gaaaatgcag gtcatatggt 1440

tgcagttgcc tgttagtttc agatgtataa aggtgtccca gattggggtt ctgttgtgtt 1500

gaggttggta gttatatctt gttgtctttc ttgggacagg atggctttgt gattaattag 1560

tttgtaaagg cagtaatcga agtccctttc tggtactaaa agcagtattg gctgcaaatc 1620

taggcattat aatgatttga agattgcatt gatactagaa atgtactgct tttggatgtt 1680

tgcatgatgc tcatgtgcta atatttccag ttaccaatgt ggtgcttctt gagtttagat 1740

ataaaactct tctcttacat tgggattctg attttttttt agttgtgcct ctagtctttt 1800

ccacaacaag atctccaaat gtagtcataa agtgttctga ttccctttaa ctggcgtcat 1860

gtttggtatg tacaaactaa gttcttatgc agacctggga ggaatgttgc tgaaatagtg 1920

caagaatata tcgttttgag gtggtttata tatttctgac atgaaatggg cattctattt 1980

tgcaccagga tggggatcat tccctcctgg ctgtgctgat gtagaaccca gaagatgccg 2040

ccgcacagac ggcaagaagt ggcggtgctc cagagatgct gttggggatc agaagtattg 2100

tgagcgacac ataaaccgtg gtcgccatcg ttcaagaaag catgtggaag gccgaaaggc 2160

gacactcacc attgcagaac catccacggt tattgctgct ggtgtatcat ctcgcggcca 2220

cactgtggct cggcagaagc aggtgaaagg ctcagctgct actgtctctg atcctttctc 2280

gagacaatcc aacaggtgaa gttgcctgat cctacatgaa tatgcatttt gtgcttcttg 2340

catgatgtgt ggacactgtg aggtggtctt tgtcaaaata aatgacatct gttgccgata 2400

cagtaattta gtccagcatg tggtaatgga ggtgccaaat cctaacaacc tataagtaat 2460

ttatgtatgt atcaggattc taggcagcat aataaaacat ctaacaaata gatattgtac 2520

agaaaagtta agatgaattt gaccaagcta gtgatatcaa tatacagtag gaaagtcctc 2580

acacaattat ttagtttgat tatactccag tttctgtgat tgcattagtc attcatttaa 2640

ttatgagtta atgtccctac aatgaactcc attttttctg ctctctctct ctctctcctt 2700

tgtaatgaag gaaattcatt tcttctttaa cctgctgcaa ttaacagaag gaaaatttgc 2760

acattgttat ctgaactaaa gatctgactc tactttgcag gaaatttctg gagaaacaga 2820

acgttgtcga ccaattgtct cccatggatt catttgattt ctcatccaca caatcttctc 2880

caaactatga caatgtagca ttgtcaccac tgaagttgca ccatgatcat gatgaatctt 2940

acatcgggca tggagcaggc agttcatcag aaaaaggcag tatgatgtac gaaagtcggt 3000

taacagtctc taaggaaaca cttgatgatg gacctttagg tgaagttttc aaaagaaaga 3060

attgccaatc agcttctaca gaaatcttaa ctgaaaaatg gactgagaac cccaacttac 3120

attgcccatc tggaatccta caaatggcta ctaagttcaa ttcaatttcc agcggcaaca 3180

cagtaaatag tggtggcacc gcagtggaga atcttatcac tgataatgga tatcttactg 3240

caagaatgat gaatcctcat attgtcccaa cacttctcta aggctgtgtt tgaaagttca 3300

tgttggattc gcaaatgatt gaaagtacgt ttatggatgg ttctataagt tcctgttgtt 3360

tccttcgtta tgtgttcttg tgttcctcac ctttttatct tttggttgag gttggtatgt 3420

tgtaattttc tcctgtgcta ccttgtaata tgctgaagtt aaatgctctt ctaaaaattt 3480

ctgttgcaat ccaagattca ggatttatgt gtccaattct gaattttaat gaatcatgcc 3540

ccttaaatgt aaagggaatt gtatatttca ttaatttgac aaaaacgttg ccttctgctc 3600

c 3601

<210> 10

<211> 20

<212> RNA

<213> Artificial sequence (artificial sequence)

<400> 10

gcucauguug ggauuguggu 20

Claims

1. A method of improving an agronomic trait in a plant comprising:

reducing the expression or activity of miR396 in the plant under a low nitrogen condition; the miR396 is miR396e and/or miR396f;

the agronomic trait of the plant is selected from one or more of the group consisting of:

(a) Yield and/or biomass;

(b) Development of panicle and/or grain types;

(c) Size, weight and/or number of fruits and/or seeds;

(d) Grain length;

(e) Grain width;

(f) Ear length;

(g) Thousand seed weight;

(h) Leaf length;

(i) Leaf width.

2. Use of a composition comprising: (i) a miR396 inhibitor; and (ii) an agronomically acceptable carrier; it is characterized in that it is used for improving the agronomic characters of plants under the condition of low nitrogen; the miR396 is miR396e and/or miR396f;

(a) Yield and/or biomass;

(b) Development of panicle and/or grain types;

(c) Size, weight and/or number of fruits and/or seeds;

(d) Grain length;

(e) The grain width is wide;

(f) Ear length;

(g) Thousand seed weight;

(h) Leaf length;

(i) Leaf width.