CN112175966B - New gene NAL8 for controlling plant type, yield and other characters of plant and its application - Google Patents

Abstract

The invention provides a novel gene for regulating plant characters, which is named as NAL8(Narrow Leaf 8), encodes a polypeptide with important biological functions, can regulate plant type characters and yield characters of plants, and is a genetic improvement gene with potential balance of rice Leaf width and grain number per ear. The NAL8 gene also has the ability to regulate the organelles of plant cells and to regulate the cell cycle. The invention also discloses application of the NAL8 as a plant trait regulation target.

Description

New gene NAL8 for controlling plant type, yield and other characters of plant and its application

Technical Field

The invention belongs to the field of botany and molecular biology, and particularly relates to a new gene NAL8 for controlling plant type, yield and other characters of a plant and application thereof.

Background

In the twenty-first century, population is increasing worldwide, economic development and deterioration of ecological environment bring about reduction of cultivated land area, and the embarrassment of balancing population growth and food shortage becomes a worldwide problem. The world population is expected to reach 90 billion in 2050, which presents new challenges for both agricultural product yield and quality. With the expansion of population and the gradual reduction of arable area, how to plant more grains on limited farmland is always the research center of agricultural workers. At present, the traditional breeding method cannot meet the requirement, and various means of molecular biology, molecular marker assisted breeding and the like are comprehensively utilized to help people to improve the crop yield to the maximum extent. Therefore, it is very important work to research means for adjusting the plant type of crops and optimizing the crop planting.

Gramineae, particularly rice, is a major food crop in the world, and rice is also a major food and important export agricultural products for the inhabitants of china. Rice, one of the most important food crops in the world, has recently become an important research material for technologists. The mechanism and the genetic characteristic of rice quality formation are researched from the molecular angle, and theoretical and practical guidance is provided for the breeding of high-quality rice varieties. The leaf morphology is an important photosynthetic efficiency index, and has close relation to the photosynthetic efficiency and the yield of the rice. In general, the longer the leaf length or the wider the leaf width of rice, the higher the photosynthetic efficiency of the plant, the greater the amount of carbon fixation produced, and the higher the quality of the source during transformation of the source bank. The number of seeds per spike of rice is one of three main factors (the effective number of spikes per plant, the number of seeds per spike and the grain weight) for determining the yield of rice. In general, rice has a source-sink relationship, i.e., the wider the leaf width, the higher the yield. The rice grain weight and the grain number per ear also have an antagonistic and balanced relationship.

Therefore, the method has important breeding guiding significance for disclosing the molecular regulation and control mechanism among the plant type, the leaf type and the yield character of the grain crops, and is beneficial to providing a new guiding idea for genetic improvement of the plant type and the yield of the grain crops.

Disclosure of Invention

The invention aims to provide a new gene NAL8 for controlling plant type, yield and other characters of plants, and also provides application of the gene.

In a first aspect of the present invention, there is provided a method of modulating plant type traits, yield traits, organelle development, hormone signaling, or cell cycle in a plant comprising: modulating expression or activity of NAL8 in a plant, thereby modulating plant type traits, yield traits, hormone signalling or organelle development or cell cycle in the plant.

In one preferred embodiment, the method is a method for modulating plant type traits or yield traits in a plant, comprising: (a) up-regulating the expression or activity of NAL8, preferably in plants that express NAL8 at low levels (e.g., at less than 80%, less than 70%, less than 50%, less than 30% or less, or not expressed as wild type NAL 8), such that the plant type or yield traits of the plants are as follows: the plant height is increased, the leaf width is increased, the number of seeds per spike is increased or the seed grain weight (such as thousand grain weight) is increased; or (b) down-regulating the expression or activity of NAL8, so that the plant type character or yield character of the plant is represented as: the plant height is reduced, the leaf width is reduced, the leaf length is increased, the number of seeds per spike is reduced, the seed weight is reduced, the seed width is reduced, the seed setting number is reduced or the yield is reduced.

In another preferred embodiment, the method is a method of modulating the development of a plant organelle comprising: (i) up-regulating the expression or activity of NAL8, preferably in plants that express NAL8 at low levels (e.g., at levels of 80% or less, 70% or less, 50% or less, 30% or less, or none of wild type NAL 8), to stabilize mitochondria, chloroplasts, and develop normally; preferably, the plant organelles are rendered to behave as: chloroplast stacking structure (normal structure and no thinning), chloroplast quantity maintenance (normal quantity), chloroplast thylakoid continuous distribution maintenance or photosynthetic product and lipid product quantity maintenance (normal product quantity); or (ii) down-regulating NAL8 expression or activity, thereby destabilizing chloroplasts or mitochondria of plants; preferably, the organelles of the plant are rendered to exhibit: the stack structure of chloroplast is thinned, the stack number of chloroplast is reduced, the stack structure of chloroplast is discontinuous, the chloroplast is irregular in shape, thylakoid bodies in chloroplast are discontinuously distributed, and the quantity of photosynthetic products and lipid products is reduced.

In another preferred example, the biochemical reaction includes photosynthesis, carbon metabolism, TCA cycle, and the like.

In another preferred embodiment, the method is a method of modulating the cell cycle of a plant comprising: (A) up-regulating NAL8 expression or activity, preferably at low levels Upregulation in plants expressing NAL8 (e.g., expressed at 80% or less, 70% or less, 50% or less, 30% or less, or not expressed as wild type NAL 8) to maintain cell cycle stability; or (B) downregulating the expression or activity of NAL8, thereby modulating the cell cycle and increasing the expression of NAL8 at G₁Cells in stage, decreased cells in the division stage, S stage, or down-regulated cell cycle-associated gene expression.

In another preferred embodiment, the cell cycle related genes include: CycD3, CycD4, CDKA1, CycT1, CDKA2, CycT 1; 2, CycA 2; 1, CycA 2; 2, CycA 2; 3, CycB 2; 1, CycB 2; 2, CDC20, CycD 4; 1.

in another preferred embodiment, the method is a method of modulating plant hormone signaling comprising: (1) up-regulating the expression or activity of NAL8, preferably in plants that have low NAL8 expression, such that the auxin content or cytokinin content of the plant is reduced; or (2) downregulating the expression or activity of NAL8, thereby causing the auxin content of the plant to be increased or the cytokinin content to be decreased.

In another preferred example, upregulating the expression or activity of NAL8 comprises: transferring the gene coding for NAL8 (which may be gDNA or cDNA, and may also contain a promoter) or an expression construct or vector containing the coding gene into a plant; gain-of-function mutation of NAL8, for example, back mutation of the mutation site of NAL8 in which mutation is to occur.

In another preferred example, the down-regulation NAL8 includes: knocking out or silencing a gene encoding NAL8, or inhibiting the activity of NAL8, in a plant; preferably, it comprises: silencing NAL8 with interfering molecules that specifically interfere with the expression of the gene coding for NAL8, gene editing with the CRISPR system to knock out the gene coding for NAL8, knocking out the gene coding for NAL8 by homologous recombination, or loss-of-function mutation of NAL8 in plants containing NAL8 (e.g.mutation of the amino acid corresponding to position 228 of the sequence SEQ ID NO:3, such as Thr).

In another aspect of the present invention, there is provided the use of NAL8 or a modulator thereof for modulating plant type traits, yield traits, organelle development, hormone signalling or cell cycle; the regulator comprises an up regulator or a down regulator.

In a preferred embodiment, the plant type trait comprises: plant height, leaf width, leaf length.

In another preferred embodiment, said yield traits comprise: seed number of each ear, seed width, seed weight and seed setting number.

In another preferred embodiment, the organelle comprises: chloroplasts, mitochondria; preferably, said NAL8 or its modulator modulates chloroplast or mitochondria stability, alignment, development, photosynthetic product and lipid product levels.

In another preferred embodiment, the hormone signal comprises: auxin or cytokinin signals.

In another preferred embodiment, said modulating the plant cell cycle comprises: regulating the number of cells in division phase and S phase, and regulating the expression of cell cycle related gene.

In another preferred embodiment, the NAL8 up-regulation includes (but is not limited to): exogenous NAL8 encoding gene (gDNA or cDNA, also containing promoter) or expression construct or vector containing the encoding gene; or a reagent that functionally gains point mutation in NAL 8; preferably, the mutant is reverted to the NAL8 wild-type in plants in which the NAL8 mutation (e.g.the mutation corresponding to amino acid 228 of the sequence of SEQ ID NO: 3) has occurred (e.g.gene editing reagents, gene recombination reagents, site-directed mutagenesis reagents).

In another preferred embodiment, said NAL8 down-regulator includes (but is not limited to): agents that knock out or silence NAL8, agents that inhibit NAL8 activity; preferably, the method comprises the following steps: an interfering molecule that specifically interferes with the expression of the gene encoding NAL8, a CRISPR gene editing agent, a homologous recombination agent, or a site-directed mutagenesis agent directed to NAL8 that functionally null mutates NAL 8.

In another aspect of the invention, there is provided the use of plant NAL8 (including polypeptides and genes) as a molecular marker for identifying plant type traits, yield traits, organelle development, hormone signalling or cell cycle.

In a preferred embodiment, the plant type trait comprises: plant height, leaf width, leaf length.

In another preferred embodiment, said yield traits comprise: seed number of each ear, seed width, seed weight and seed setting number.

In another preferred embodiment, the organelle comprises: chloroplasts, mitochondria; preferably, said NAL8 or its modulator modulates chloroplast or mitochondria stability, alignment, development, photosynthetic product and lipid product levels.

In another preferred embodiment, said modulating the plant cell cycle comprises: regulating the number of cells in division phase and S phase, and regulating the expression of cell cycle related gene.

In another aspect of the present invention, there is provided a method for targeted selection or identification of plants, the method comprising: identification of NAL8 expression or sequence characteristics in test plants: if the NAL8 of the test plant is highly expressed or normally expressed, the test plant is a plant with normal plant type character or yield character such as plant height, leaf width, normal or improved seed number or seed weight (such as thousand seed weight) per ear, or a plant with stable mitochondria and chloroplasts and normal development, or a plant with low auxin content or high cytokinin content, or a plant with stable cell cycle; if the NAL8 of the test plant is low or not expressed, or the NAL8 polypeptide in the test plant has mutation at the 228 th amino acid corresponding to the SEQ ID NO. 3 sequence, the plant is characterized by plant type character or yield character, such as plant height reduction, leaf width reduction, leaf length increase, seed number reduction per ear, seed weight reduction, seed width reduction, seed set number reduction or yield reduction, or is a chloroplast or mitochondria unstable plant, or is a plant with high auxin content or low cytokinin content, or is a plant with changed cell cycle (such as G) ₁Increased stage cells, decreased cells in the division stage and the S stage, and decreased expression of cell cycle related genes).

As a preferred mode of the aforementioned aspects of the invention, the polypeptide of NAL8 is: (a) 3, as shown in SEQ ID NO; or (b) a polypeptide derived from (a) and having the function of (a) a polypeptide formed by substituting, deleting or adding one or more (e.g., 1 to 20; preferably 1 to 10; more preferably 1 to 5) amino acid residues to the amino acid sequence shown in SEQ ID NO:3, and the amino acid at the position corresponding to position 228 of the sequence shown in SEQ ID NO:3 is Ala; or (c) a polypeptide having an amino acid sequence which is 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more; e.g., 98% or more or 99% or more) identical to the amino acid sequence defined in (a) and having the function of the polypeptide of (a), and the amino acid at the position corresponding to position 228 in the sequence of SEQ ID No. 3 is Ala; or (d) a fragment of SEQ ID NO. 3 (e.g., 100 to 227aa in length, more specifically 120aa, 150aa, 160aa, 180aa, 200aa, 210aa) having (a) a polypeptide function, but with the presence of the amino acid corresponding to position 228 of the sequence of SEQ ID NO. 3, and with the amino acid being Ala.

As a preferred mode of the aforementioned aspects of the present invention, the plant includes (but is not limited to): gramineae, leguminous plants or cruciferae plants.

In another aspect of the invention, an isolated polypeptide (NAL 8) is provided^A228TA protein) which is: (a') a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 5; or (b ') a polypeptide derived from (a ') and having the function of the polypeptide of (a ') in which the amino acid sequence shown in SEQ ID NO:5 is substituted, deleted or added by one or more (e.g., 1 to 20; preferably 1 to 10; more preferably 1 to 5) amino acid residues, and the amino acid at the position corresponding to position 228 of the sequence shown in SEQ ID NO:5 is Thr; or (c ') a polypeptide which has an amino acid sequence that is 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more; e.g., 98% or more or 99% or more) identical to the amino acid sequence defined in (a ') and which has the function of the polypeptide (a '), and the amino acid at the position corresponding to position 228 of the sequence of SEQ ID NO. 5 is Thr; or (d ') a fragment of SEQ ID NO. 5 (e.g., 100 to 227aa in length, more specifically 120aa, 150aa, 160aa, 180aa, 200aa, 210aa) having the polypeptide function of (a'), but with the presence of the amino acid corresponding to position 228 of the sequence of SEQ ID NO. 5, and the amino acid is Thr.

In another aspect of the invention, there is provided an isolated polynucleotide encoding the isolated polypeptide.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, phenotypic characteristics of the nal8 mutant.

(A) Plant types of wild type TQ and nal 8; (B) leaf shapes of TQ and nal 8; (C) spike type of TQ and nal 8; (D-I) phenotypic comparison statistics of TQ and nal8, including plant height (D), sword leaf width (E), panicle number (F), yield per plant (G), thousand kernel weight (H) and seed set rate (I).

FIG. 2, positional cloning and evolutionary analysis of the NAL8 gene.

(A) Bit clone of NAL 8; (B) NAL8 protein trimer and NAL8^A228TPredicting the structure of the protein trimer; (C) aligning NAL8 protein conserved region sequences in different species; (D) genetic distance and genetic relationship analysis of NAL8 protein in different species.

Fig. 3, NAL8 gene complementation experimental validation and phenotype statistics.

(A-B) TQ, NAL8 mutant and two independent complementation plants NAL8^comThe plant type phenotype (A) and the flag leaf type (B); (C) TQ, NAL8 mutant and two independent complementary plants NAL8^comCounting the width of the sword leaf; (D-E) TQ, NAL8 mutant and two independent complementation plants NAL8^comEar type (D) and granule type (E); (F-K) TQ, NAL8 mutant and two independent complementation plants NAL8^comThe seed width (F), the seed length (G), the thousand seed weight (H), the plant height (I), the spike grain number (J) and the sword leaf length (K).

FIG. 4, NAL8 knockout plants show leaf thinning and reduction in panicle number.

(A) ZH11 and two independent NAL8^CRISPRKnocking out the plant type phenotype of the transgenic plant; (B) ZH11 and two independent NAL8^CRISPRKnocking out the sword leaf type of the transgenic plant; (C) ZH11 and two independent NAL8^CRISPRKnocking out the panicle phenotype of the transgenic plant; (D-L) ZH11 and NAL8^CRISPRThe width (D) of folium xiphocae, the plant height (E), the grain number (F) per ear, the thousand-grain weight (G), the grain width (H), the grain length (I), the length (J) of folium xiphocae, the tillering number (K) and the editing site (L).

FIG. 5, phenotype showing leaf thinning and reduction in panicle number by suppressing NAL8 expression.

(A) TQ and two independent NAL8^RNAiThe plant type phenotype of the transgenic plant; (B) TQ and two independent NAL8^RNAiTransgenic plant sword leaf type; (C) TQ and two independent NAL8^RNAiA transgenic plant panicle phenotype; (D-K) TQ and two independent NAL8^RNAiThe relative expression level (J) of RNA in the leaves of these samples of the transgenic plants, namely, the width of the leaf of flag (D), the height of the transgenic plants (E), the number of grains per ear (F), the width of the grains (G), the length of the grains (H), the length of the leaf of flag (I) and NAL 8.

Fig. 6, overexpressing NAL8, did not show a phenotype that is significantly different from ZH 11.

(A) ZH11 and two independent NAL8^OEThe plant type phenotype of the transgenic plant; (B) ZH11 and two independent NAL8^OETransgenic plant sword leaf type; (C) ZH11 and two independent NAL8 ^OETransgenic plant panicle phenotype; (D-K) ZH11 and two independent NAL8^OEThe relative expression level (J) of RNA in the leaves of these samples of the transgenic plants, i.e., the leaf width (D), plant height (E), number of grains per ear (F), grain width (G), grain length (H), leaf length (I) and NAL8, was determined.

Fig. 7, nal8 mutant showed thylakoid dysplasia, reduced photosynthetic and lipid production.

(A-D) TQ leaf chloroplast ultrastructure; (E-H) nal8 mutant leaf chloroplast ultrastructure; (I-J) the mitochondrial ultrastructure of the TQ leaf; (K-L) nal8 mutant leaf mitochondrial ultrastructure.

FIG. 8, TQ and nal8 mutant virtualization, section cell ploidy analysis, and cell cycle gene expression level detection.

(A-D) TQ and nal8 mutant seedling leaf cross-section virtual slice picture; (E-H) number of leaf vascular bundles (E), leaf thickness (F), number of xylem cells (G) and length of xylem cells (H) of TQ and nal8 mutants; (I) detecting cell ploidy results of TQ and nal8 mutant seedling root tip cell flow cytometry; (J) TQ and nal8 mutant seedling root tip cell cycle distribution statistical chart; (K) relative expression level of TQ and nal8 mutant seedling leaf cell cycle related gene RNA.

FIG. 9, TQ and nal8 mutant endogenous hormone levels.

(A-H) auxin (IAA) content (A), auxin methyl ester (Me-IAA) content (B), N of TQ and nal8 mutant⁶- (delta 2-isopentenyl) adenine (iP) content (C), trans-zeatin (tZ) content (D), Jasmonic Acid (JA) content (E), Salicylic Acid (SA) content (F), indolebutyric acid (ICA) content (G) and cis-zeatin (cZ) content (H).

FIG. 10, TQ and nal8 mutant transcriptome sequencing differential gene KEGG analysis and GO classification analysis.

(A) Sequencing differential gene KEGG enrichment map of TQ and nal8 mutant transcriptome; (B) TQ and nal8 mutant transcriptome sequencing GO taxonomic enrichment maps.

FIG. 11, TQ and nal8 mutant proteome analysis and Nitric Oxide (NO) staining results.

(A-B) TQ and nal8 mutant leaf proteome analysis differential protein clustering plots, wherein A plot is protein clustering analysis of up-regulated expression compared with TQ, and B plot is protein clustering analysis of down-regulated expression compared with TQ; (C) analysis of differential protein mitochondrial key protein fold difference heatmap analysis of TQ and nal8 mutant leaf proteome; (D) analyzing a difference protein quantity wain graph for the proteome of the TQ and nal8 mutant leaf; (E) analyzing protein groups of the mutant TQ and nal8 leaves to obtain differential protein volcano maps; (F) and (4) carrying out statistics on detection results of NO stains at root tips of TQ and nal8 mutants and fluorescence signals.

Fig. 12, wild type NAL8 gene and mutated gene and protein sequences, boxes indicate the positions of mutated nucleotides and amino acids.

Detailed Description

The invention researches and reveals a novel gene for regulating plant characters for the first time, which is named NAL8(Narrow Leaf 8), encodes a polypeptide with important biological functions, can regulate plant type characters and yield characters of plants, and is a genetic improvement gene with potential balance of rice Leaf width and panicle number. The NAL8 gene also has the ability to modulate organelle development in plant cells and to modulate the cell cycle. In addition, the invention also discloses application of the NAL8 as a plant trait regulation target.

NAL8

In the present invention, unless otherwise specified, NAL8 refers to a polypeptide having the sequence of SEQ ID NO. 3 or a gene encoding the same, and also includes a variant of the sequence having the same function as NAL8 polypeptide. The coding gene may be gDNA or cDNA, and may contain a promoter. For example, the gDNA has a nucleotide sequence shown by SEQ ID NO. 1, the cDNA has a nucleotide sequence shown by SEQ ID NO. 2, and the promoter has a nucleotide sequence shown by SEQ ID NO. 6. The sequences encoding the genes also include sequences that are degenerate to the sequences provided herein.

Variants of the NAL8 polypeptide include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. Any protein having high homology (such as 70% or more homology with the polypeptide sequence shown in SEQ ID NO: 3; preferably 80% or more homology; more preferably 90% or more homology, such as 95%, 98% or 99% homology) with the NAL8 polypeptide and having the same function as the NAL8 polypeptide is also included in the present invention.

Polypeptides derived from other species than rice, which have a high homology with the polypeptide sequence of SEQ ID NO. 3 or which exert the same or similar effects in the same or similar regulatory pathways, are also included in the present invention.

In the present invention, "NAL 8" also includes the homologue thereof. It is understood that while NAL8 from rice of a particular species is preferably studied in the present invention, other polypeptides or genes from other species that are highly homologous (e.g., have greater than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) to said NAL8 are also contemplated by the present invention.

The invention also includes a mutant form of a NAL8 polypeptide mutant which has been mutated to occur at amino acid 228 of the sequence corresponding to SEQ ID No. 3. The mutant does not have the function of the wild NAL8 polypeptide, and the plant type character, the yield character, the organelle or the cell cycle of the plant with the mutant are obviously changed.

The invention also includes the polynucleotide for coding the polypeptide mutant, which has the sequence shown in SEQ ID NO. 4. Degenerate sequences are also included.

Method for improving plants

As used herein, a "plant (crop)" is a plant comprising/expressing "NAL 8" or a homologue thereof; preferably, the "plant" is a plant suitable for transgenic manipulation. The plant may be a dicot, monocot or gymnosperm; may include crops, floral or forestry plants, etc. Preferably, the "plant" includes (but is not limited to): gramineae (e.g., plants of the genus oryza, triticum, zea); cruciferous plants (such as arabidopsis, brassica); leguminous plants. More specifically, the plants include (but are not limited to): wheat, barley, rye, rice, corn, sorghum, sugar beet, apple, pear, plum, peach, apricot, cherry, strawberry, raspberry, blackberry, bean, lentil, pea, soybean, canola, mustard, poppy, sweet wormwood, olea, sunflower, coconut, castor oil plant, cocoa bean, peanut, gourd, tobacco, oil palm, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, grapefruit, spinach, garland, asparagus, cabbage, chinese cabbage, pakchoi, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nut, coffee, eggplant, sugarcane, tea, pepper, grape tree, oyster hemp, banana, poplar, willow, pine, fir, eucalyptus, euphorbia, lucerne, coconut, sago, natural rubber tree, ornamental plants, and the like.

Based on the new findings of the present inventors, the present invention provides a method for improving a plant, the method comprising: regulating and controlling the expression or activity of NAL8 in plant body, and further regulating and controlling plant type character, yield character, organelle development, hormone character or cell cycle of plant, wherein the plant type character or yield character specifically includes plant height, leaf width, leaf length, seed number of each ear, seed width, seed weight and seed setting number.

In one aspect, the present invention provides a method for conferring plant type or yield traits in plants, particularly in plants that express low (including no) NAL8, as increased plant height, increased leaf width, increased number of seeds per ear or increased seed grain weight (e.g., thousand kernel weight), comprising: up-regulates the expression or activity of NAL 8.

In another aspect, the present invention provides a method for expressing plant type traits or yield traits of a plant as reduced plant height, reduced leaf width, increased leaf length, reduced number of seeds per ear, reduced seed grain weight, reduced seed grain width, reduced number of seeds set or reduced yield, comprising: downregulating expression or activity of NAL 8.

In another aspect, the present invention provides a method for stabilizing mitochondrial, intracytoplasmic thylakoids, developing normally or maintaining stable cell cycle in plants, particularly in plants that have low (including no) NAL8 expression, comprising: up-regulates the expression or activity of NAL 8.

In another aspect, the invention provides a method of destabilizing thylakoids or mitochondria in the chloroplast of a plant or modulating the cell cycle, comprising: downregulating the expression or activity of NAL 8. The modulated cell cycle comprises; increase at G₁Cells in stage, decreased cells in the division stage, S stage, or down-regulated cell cycle-associated gene expression.

It is understood that, knowing the function of the NAL8, various methods known to those skilled in the art can be used to modulate the expression or activity of NAL 8. For example, NAL8 may be overexpressed or NAL8 expression may be reduced or absent using a variety of methods well known to those skilled in the art.

In the invention, the up-regulation agent of the NAL8 protein or the coding gene thereof comprises an accelerant, an agonist and an activator. The terms "up-regulation" and "promotion" include "up-regulation", "promotion" of protein activity or "up-regulation", "promotion" of protein expression. Any substance that increases the activity of NAL8, increases the stability of NAL8, upregulates the expression of NAL8, increases the effective duration of NAL8 can be used in the present invention as a useful substance for upregulating NAL 8. They may be chemical compounds, chemical small molecules, biological molecules. The biomolecule may be at the nucleic acid level (including DNA, RNA) or at the protein level. The NAL8 protein or the up-regulation agent thereof are particularly suitable for being applied to a class of plants, the expression of the NAL8 of the NAL8 protein or the up-regulation agent thereof is lower than the average value of the class of plants or the NAL8 of the NAL8 of the NAL8 protein is not expressed; thus, the application of the NAL8 protein or its up-regulator can restore the wild-type phenotype or better phenotype to the plants.

In the present invention, the NAL8 protein or its coding gene down-regulator refers to any substance that can reduce the activity of NAL8 protein, reduce the stability of NAL8 protein or its coding gene, down-regulate the expression of NAL8 protein, reduce the effective acting time of NAL8 protein, or inhibit the transcription and translation of NAL8 gene, and these substances can be used in the present invention as useful substances for down-regulating NAL 8. They may be chemical compounds, small chemical molecules, biomolecules. The biomolecule may be at the nucleic acid level (including DNA, RNA) or at the protein level. For example, the down-regulating agent is: an interfering RNA molecule or antisense nucleotide that specifically interferes with NAL8 gene expression; or a gene editing reagent that specifically edits NAL8, and the like.

The invention also provides a method for up-regulating NAL8 expression in a plant, which comprises the following steps: the gene coding for NAL8 or an expression construct or vector containing said coding gene is transferred into plants.

The invention also provides a method for down-regulating the expression of NAL8 in plants, which comprises the step of carrying out targeted mutation, gene editing or gene recombination on NAL8 so as to realize down-regulation. As a more specific example, the NAL8 can be converted into a mutant thereof (e.g., a mutation corresponding to amino acid 228 of SEQ ID NO: 3) by any of the methods described above, thereby altering the plant characteristics. As a more specific example, gene editing is performed using CRISPR/Cas9 system to knock out or down regulate a target gene. An appropriate sgRNA target site will lead to higher gene editing efficiency, so that an appropriate target site can be designed and found before gene editing is initiated. After designing a specific target site, in vitro cell activity screening is also required to obtain an effective target site for subsequent experiments.

As another embodiment of the present invention, there is provided a method of down-regulating the expression of NAL8 in a plant, comprising: (1) transferring an interference molecule interfering with NAL8 gene expression into a plant cell, tissue, organ or seed to obtain the plant cell, tissue, organ or seed transformed with the interference molecule; (2) regenerating the plant cell, tissue, organ or seed transformed with the interfering molecule obtained in step (1) into a plant. Preferably, the method further comprises: (3) selecting a plant cell, tissue or organ into which said vector has been transferred; and (4) regenerating the plant cell, tissue or organ of step (3) into a plant.

In the present invention, the differences between NAL8 and its mutant (mutated at amino acid 228 in the sequence corresponding to SEQ ID NO: 3) are more clear by comparison. Thus, as a preferred mode of the present invention, the regulation is carried out by mutating the 228 th amino acid corresponding to the sequence of SEQ ID NO. 3 or by disrupting the domain in which the amino acid is located.

The technical scheme of the invention can be applied to molecular design breeding in various ways.

Plant directional screening or targeted screening regulatory molecules

After the function of NAL8 is known, it can be used as a molecular marker to perform the directional screening of plants. Substances or potential substances that can directionally regulate plant type traits, yield traits, organelles or cell cycle of plants by modulating this mechanism can also be screened based on this new finding.

Accordingly, the present invention provides a method for targeted selection or identification of plants, the method comprising: identification of NAL8 expression in test plants: if the NAL8 of the test plant is highly expressed or normally expressed, the plant is a plant with normal plant height, leaf width, normal or improved plant type character or yield character, normal or improved seed number or seed weight (such as thousand seed weight) of each ear, or a plant with stable thylakoid in mitochondria and chloroplasts and normal development, or a plant with stable cell cycle; if NAL8 of the test plant is low or not expressed, or the test plant is a transgenic plantThe NAL8 polypeptide in the test plant has mutation at the 228 th amino acid corresponding to the SEQ ID NO. 3 sequence, and is a plant with plant type character or yield character which is shown as plant height reduction, leaf width reduction, leaf length increase, seed number reduction per spike, seed weight reduction, seed width reduction, seed number reduction or yield reduction, or is a plant with unstable thylakoid or mitochondria in chloroplast, or is a plant with changed cell cycle (such as G)₁Increased stage cells, decreased cells in the division stage and the S stage, and decreased expression of cell cycle related genes).

The invention provides a method for screening and regulating plant type characters, yield characters, organelles or cell cycles of plants, which comprises the following steps: adding a candidate substance to a system containing or expressing NAL 8; detecting the expression or activity of NAL8 in said system; if the candidate substance up-regulates the expression or activity of NAL8, the candidate substance is a regulator which can lead the plant type character or the yield character of the plant to be expressed as plant height, leaf width, normal or improved number of seeds per spike or seed weight (such as thousand seed weight), or stable thylakoid, normal development or stable cell cycle in mitochondria and chloroplasts; if the candidate substance down-regulates the expression or activity of NAL8, the candidate substance is indicated to be a regulator which can make the plant type character or yield character of the plant show that the plant height is reduced, the leaf width is reduced, the leaf length is increased, the number of seeds per spike is reduced, the seed weight is reduced, the seed width is reduced, the seed setting number is reduced or the yield is reduced, or thylakoid or mitochondria in chloroplast are unstable, or the cell cycle is changed.

Methods for targeting a protein or gene or a specific region thereof to screen for substances that act on the target are well known to those skilled in the art and all of these methods can be used in the present invention. The candidate substance may be selected from: peptides, polymeric peptides, peptidomimetics, non-peptidic compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, nucleic acid sequences, and the like. Depending on the kind of substance to be screened, it is clear to the skilled person how to select a suitable screening method.

Through large-scale screening, a kind of potential substances which specifically act on NAL8 and have the regulation and control effects on plant type traits, yield traits, organelles or cell cycles can be obtained.

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. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBrook et al, molecular cloning, A laboratory Manual, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Materials and methods

1. Map bit cloning

EMS chemical mutagenesis is carried out by utilizing indica rice variety extra green (TQ), leaf type and spike type mutant nal8 is screened, and then the mutant and the TQ are backcrossed to purify background. Generation of F by selfing after hybridization with japonica rice variety Jiahua No. 1₂Isolating the population. Statistical segregation population phenotypes were found to contain a ratio of normal leaf-type panicle type to mutant phenotype close to 3: 1, according with Mendelian genetic rules. First, the present inventors performed BSA pool-mixing method using 772 strains of F2 rice, and found that the candidate gene was located at the end of the short arm of chromosome seven between molecular markers 07-30610 and 07-52402. Then, the present inventors shortened the targeting interval to about 72kb between the molecular markers NAL8-Q and NAL8-C1 using 10447 rice. Since the number of crossovers is small due to the proximity to the centromere, 12 candidate genes were predicted by the co-discovery in the 72kb range, and most of them were transposon genes. The inventor designs a primer to sequence a gene for coding a protein, and discovers that the 682 th base of a coding region of a candidate gene LOC _ Os07G15880 is mutated from G to A, so that the 228 th alanine is mutated into threonine. Therefore, the inventors hypothesized that the candidate gene LOC _ Os07g15880 might be responsible for the phenotype of the nal8 mutant.

TABLE 1 primer sequences

2. Transgene complementation verification, overexpression, RNA interference and establishment of CRISPR/Cas9 gene editing plant material

To further confirm that LOC _ Os07g15880 is a phenotype causing a reduction in the number of fine leaves and panicles of the nal8 mutant, the present inventors constructed a DNA fragment of LOC _ Os07g15880 derived from wild type Teqing including a promoter region of 2.5kb, a total length of gDNA of about 5.3kb and a downstream 3' UTR region of about 500bp to about 8kb on a pCAMBIA1300 complementation vector, genetically transformed by a rice seed-induced callus transformation method mediated by Agrobacterium tumefaciens EHA105 strain, and screened for transgenic positive strains by hygromycin.

The full-length cds of the NAL8 gene from the wild type is amplified by PCR and cloned into an overexpression vector containing the promoter drive of CaMV 35S.

An artificial small molecule interference fragment is designed and constructed to be constructed on an overexpression vector driven by a CaMV35S promoter to strongly inhibit the expression of a NAL8 gene.

Two target points of NAL8 gene are designed by using CRISPR/Cas9 gene editing technology to construct a gene knockout carrier for rice genetic transformation.

By genetic transformation of transgenic positive lines, stable T is obtained₂Transgenic plants are generated and are used for next physiological and biochemical experiments and phenotype investigation. The influence of NAL8 gene on the growth and development of rice is explored genetically.

TABLE 2 primer sequences

3. Histological staining and subcellular localization of NAL8 gene

In order to study the distribution of the NAL8 gene in plant tissues, including the range of plant subcellular organelles affected by the gene and the acting sites, the inventor utilizes a GUS gene reporter system and a qRT-PCR method to study the distribution pattern of the NAL8 gene in different tissues of rice.

NAL8_ProGUS 5' end oligonucleotide primer sequence is (SEQ ID NO: 37):

5’-AAAACTGCAGGGAGAGGAGAGAGGAGGGGAGTGGTGGGTG-3’

the 3' end primer sequence is (SEQ ID NO: 38):

5’-GGCGGGTACCTTTATTGCGATAATAGTGGTGCCATTGCGC-3’

the 5' end oligonucleotide primer sequence of qRT-NAL8 is (SEQ ID NO: 39):

5’-CCCATGCCAGAGAAGCTACCAAC-3’

the 3' end primer sequence is (SEQ ID NO: 40):

5’-ATTCCTGGCCCTCTCAGTCAG-3’

the result shows that NAL8 has expression in all tissues of rice and belongs to a ubiquitous expression distribution mode. GUS staining and qRT-PCR results can both prove.

Cytological assays

The nal8 mutant showed significant leaf narrowing and spike grain reduction compared to the TQ wild type. The present inventors observed changes in the state of cell elongation or division using a flow cytometer and a virtual slice; the FAA fixed sample was dehydrated and observed by X-ray microscopy.

As a result, the number of vascular bundles of the nal8 mutant was increased, and the leaf thickness, the number of xylem cells and the length were significantly decreased, as compared to the wild-type leaf.

The present inventors performed cell ploidy analysis on both TQ and nal8 mutants. In general, the number of tetraploid cells is relatively increased in the cells in the vigorous division phase, and the cell cycle distribution is also changed. Selecting young rice root tip cells, extracting cell nuclei, staining with a nuclear dye DAPI, analyzing the cell ploidy of each sample, comparing the relative cell division rates of TQ and nal8 mutant cells, and assisting qRT-PCR analysis to obtain the expression difference of cell cycle related genes.

5. Organelle morphology detection

The inventors obtained young leaves of TQ and nal8 mutants and prepared ultrathin sections, and observed the chloroplast and mitochondrial ultrastructure with an electron microscope.

As a result, it was found that the nal8 mutant exhibited a reduced number of chloroplast stackings, a discontinuous distribution of thylakoids, and reduced photosynthetic and lipid production compared to wild type TQ.

6. Transcriptome sequencing and proteome analysis of TQ and nal8 mutants

To further investigate the effect of NAL8 mutations on various metabolic synthesis reactions occurring in the mitochondria and chloroplasts of the NAL8 mutant, the inventors performed transcriptome and proteome analyses using TQ and NAL8 mutant leaf material. The transcriptome mainly detects the difference of the two different mRNAs in the transcription level, and the proteome directly reflects the difference of the expression levels of the two proteins.

As a result, the key gene signal pathways of the difference between the two genes are similar, and the transcription level and the protein translation level of a large number of chloroplast photomorphogenesis, mitochondrial carbon metabolism and TCA circulating genes are obviously influenced. Ultimately morphologically affecting plant growth and development.

Example 1 obtaining of mutants with reduced grain count and narrowed leaf Width and determination of phenotypic control genes

The inventor carries out EMS mutagenesis on indica rice variety Terqing (TQ) to obtain a mutant nal8 with reduced grain number per ear and narrowed leaf width.

The phenotype of the wild type and the nal8 mutant was observed (FIGS. 1A to I), and it was found that the nal8 mutant exhibited phenotypes such as a significant reduction in leaf width (FIGS. 1B and E), a reduction in the number of grains per ear (FIGS. 1C and F), a reduction in thousand kernel weight (FIG. 1H), a partial reduction in grain width, a reduction in the number of seeds (FIG. 1I), a reduction in plant height (FIG. 1D), and a reduction in yield (FIG. 1G).

F utilizing nal8 mutant and japonica rice variety Jiahua No. 1₂The segregation population judges that the phenotype of the segregation population, such as leaf width, spike grain number and the like, is a single-gene recessive mutation, and successfully locates and clones the candidate gene NAL8 by a map-based cloning method and the like. The inventors found that the candidate gene NAL8 cloned from the NAL8 mutant had a mutation from G to A at position 682 of CDS, This results in the mutation of alanine to threonine at position 228 of the amino acid sequence (FIG. 2A).

The genome sequence of the wild type NAL8 gene is shown as SEQ ID NO. 1, the coding region sequence is shown as SEQ ID NO. 2, the protein sequence is shown as SEQ ID NO. 3, and the promoter sequence is shown as SEQ ID NO. 6.

The coding region sequence of NAL8 gene of mutant NAL8 is shown in SEQ ID NO. 4, and the protein sequence is shown in SEQ ID NO. 5. The 682 nucleotide of the coding region is mutated G → A relative to the wild type, and the 228 th alanine → serine mutation in the encoded protein occurs. The amino acid positions where the mutations occurred are shown in FIG. 12.

Sequence conservation analysis shows that the NAL8 gene is highly conserved in animals and plants, and the 228 th amino acid is a highly conserved site (FIG. 2C, D). Meanwhile, the structural finding that NAL8 forms homotrimers was predicted, and the mutation site caused partial changes in the trimer structure (fig. 2B). Accordingly, the inventors speculate that this mutation may cause loss of function of NAL8 protein, resulting in the NAL8 phenotype.

The inventor verifies that the gene is the cause of the nal8 mutant phenotype by constructing a full-length gDNA complementary vector transgene, the complementary plant can return to the phenotype close to the wild type, and the plant height (figure 3A, I), the leaf shape of the sword leaf (figure 3B), the leaf width (figure 3C), the spike shape and the spike grain (figure 3D, E), the grain width (figure 3F), the thousand grain weight (figure 3H) and the spike grain number (figure 3J) are all recovered. That is, the plants with low NAL8 expression were returned to wild type phenotype, plant height was increased, leaf width was widened, grain number per ear was increased, and thousand kernel weight was increased.

Therefore, NAL8 gene is a genetic improvement gene with potential to balance leaf width and panicle number of rice.

Example 2, NAL8 transgenic plant phenotype showed a phenotype consistent with NAL8

The inventor prepares NAL8 knockout transgenic plants through a CRISPR/Cas9 gene editing system, and observes the phenotypic change of the NAL8 knockout transgenic plants. The positions targeted by CRISPR/Cas9 gene editing are shown in figure 4L.

As a result, the knockout plant was found to exhibit phenotypes such as a significant decrease in plant height (fig. 4A, E), a thinning of leaf blade (fig. 4B), a decrease in the number of grains per ear (fig. 4C, F), a narrowing of leaf width (fig. 4D), an increase in leaf length (fig. 4J), a decrease in thousand kernel weight (fig. 4G), a decrease in grain width (fig. 4H), an increase in grain length (fig. 4I), and a decrease in tiller number (fig. 4K).

Similar to the knockout plants, the RNAi-interfered transgenic plants also showed significant reduction in plant height (fig. 5A, E), thinning of leaves (fig. 5B), reduction in grain number per ear (fig. 5C, F), narrowing of leaf width (fig. 5D), and reduction in grain width (fig. 5G).

Whereas overexpression of NAL8 showed an increase in the number of particles per ear (fig. 6C, F), with insignificant leaf width variation (fig. 6D).

Therefore, the inventor believes that NAL8 has significant effects on phenotype such as grain per ear and leaf width of plants.

Example 3 NAL8 correlates with mitochondrial and chloroplast stability, number and arrangement of leaf vascular bundle cells

The inventor discovers that the chloroplast of the nal8 mutant is obviously changed, the stacking structure is thinned and discontinuous, thylakoid development is abnormal, and the quantity of photosynthetic products and lipid products is reduced through transmission electron microscope observation of TQ and nal8 mutants (figures 7A-L).

Not only the organelle level changes, but at the tissue level, the inventors observed virtual sections of leaf cross-sections at TQ and nal8 seedling stage using X-ray microscopy (FIGS. 8A-D). Compared to TQ, the nal8 mutant increased leaf vascular bundle number (fig. 8E), decreased leaf thickness (fig. 8F), decreased xylem cell number (fig. 8G), and decreased xylem cell length (fig. 8H). The leaf parenchyma cell number was reduced (FIG. 8G), the vascular bundle arrangement was sparse, and the like, suggesting that the cell division process of the nal8 mutant was affected.

Therefore, NAL8 encodes a subunit of the inhibin complex, affecting the number and arrangement of leaf vascular bundle cells by affecting mitochondrial and chloroplast stability.

Example 4 NAL8 influencing cell division and cell size by regulating the expression of cell cycle-related genes

The present inventors examined the ploidy of TQ and nal8 cells by flow cytometry, and as a result, found that the nal8 mutant was present in G₁Cells in stage significantly more than wild type TQ, and in division stage S significantly less than TQ (FIG. 8J). These results indicate that NAL8 is involved in the cell division process.

The expression level of most cell division positive regulatory factor genes was found to be significantly down-regulated in the nal8 mutant by qRT-PCR detection of the expression level of cell cycle-related genes (FIG. 8K).

These data indicate that amino acid mutations in NAL8 protein down-regulate cell cycle-associated gene expression in the NAL8 mutant and further influence cell division and elongation to alter leaf width and spike architecture.

Example 5 involvement of NAL8 in auxin signalling pathway and cytokinin signalling pathway response Process

Since the nal8 mutant showed various plant growth phenotype, the present inventors speculated that some hormones affecting plant growth and development and rice yield might play a role. Thus, the present inventors determined endogenous rice hormone content, including auxin, cytokinin, jasmonic acid, salicylic acid, etc., using LC-MS analysis.

By measuring endogenous hormones of the TQ and nal8 mutants, the content of auxin IAA was increased in the nal8 mutant (fig. 9A) and the content of methyl ester IAA was decreased in the nal8 mutant (fig. 9B) compared to TQ; whereas the cytokinin content decreased in the nal8 mutant (FIGS. 9C and D); jasmonic (fig. 9E), salicylic (fig. 9F), indolecarboxylic acid ICA (fig. 9G) and trans-zeatin (fig. 9H) levels were not much changed.

The present inventors further determined the difference in total RNA expression levels between TQ and nal8 mutants using RNA-seq. The results showed that the expression levels of the signaling pathway genes of various hormones including auxin, cytokinin and brassinolide were changed, and the expression levels of genes involved in the important synthesis and metabolism of mitochondria and chloroplasts were also significantly changed (FIGS. 10A-B). Especially, most important chloroplast genes are obviously down-regulated in the nal8 mutant, which is consistent with the obvious structural change of nal8 chloroplast.

Example 6 NAL8 influences chloroplast and mitochondrial protein and Nitric Oxide (NO) profiles

The inventors of the present invention performed a microscopic staining observation of the root tips of TQ and nal8 mutants with Nitric Oxide (NO) indicator DAF-FM DA, and found that NO was significantly decreased in the nal8 mutant distribution, indicating that inhibin plays an important role in NO transport metabolism (fig. 11F).

The present inventors have also studied using proteomics and the like, and found that not only the transcription level but also the TQ and nal8 mutants were greatly different at the translation level. The present inventors identified 570 different proteins in total, and most of the proteins with a large fold difference were distributed in pathways such as ribosome, carbon metabolism, TCA cycle, and pyruvate metabolism (FIGS. 11A to E).

Taken together, the present inventors believe that NAL8, as a molecular chaperone, affects a variety of processes closely related to cell division by affecting mitochondrial and chloroplast stability, ultimately affecting rice leaf morphology and panicle number.

Discussion of the preferred embodiments

According to the above, the mutant of rice variety Terqing (TQ) is obtained by EMS chemical mutagenesis, the number of seeds per ear (number of grains per ear) is reduced, and the Leaf width is narrowed, and the mutant is named as nal8(Narrow Leaf 8). The inventor hybridizes a nal8 mutant with a japonica rice variety Jiahua No. 1 and separates the obtained F₂The isolated population is consistent with the ratio of normal leaf width to narrow leaf being 3: 1, it was revealed that the gene controlling the nal8 mutant was a monogenic recessive mutation. The present inventors succeeded in mapping and cloning the NAL8 gene which controls the phenotype of NAL8 by map-based cloning. After sequencing, the 228 th amino acid of the NAL8 gene in the NAL8 mutant is mutated from alanine to threonine. The inventor confirms NAL8 by using a transgenic genetic complementation experiment^A228TThe mutation caused the phenotype of nal 8. Both the inhibition of NAL8 expression and the knockout of NAL8 can cause the phenotype of leaf width narrowing and spike grain number reduction in rice. The NAL8 gene encodes a molecular chaperone of inhibin, and transmission electron microscopy shows that compared with the wild type, the NAL8 mutant shows phenotypes of reduced chloroplast stacking number, irregular shape, obviously reduced quantity of photosynthetic carbon products and lipid products, and the like, and the results show that the mutation of NAL8 can cause instability of chloroplast and mitochondrial states. In addition, the inventors found a large amount of photosynthesis, carbon metabolism, TCA cycle and the like through RNA-Seq experiments and proteome experiments The key gene of the important biochemical reaction pathway is severely inhibited in the nal8 mutant. Endogenous hormone measurements also revealed that mutant auxin was significantly upregulated compared to wild type and cytokinin was significantly decreased. At the same time, the cell division rate of the nal8 mutant was significantly reduced compared to the wild type as measured by flow cytometry. These results indicate that NAL8 is a novel chaperone that regulates the balance of the rice source pool by maintaining the structural stability of mitochondria and chloroplasts.

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 or 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 appended claims of the present application.

Sequence listing

<110> Shanghai Life science research institute of Chinese academy of sciences

<120> novel gene NAL8 for controlling plant type, yield and other traits of plants and application thereof

<130> 194440

<160> 40

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4720

<212> DNA

<213> Rice (Oryza sativa L)

<400> 1

acctttattg cgataatagt ggtgccattg cgcaagccaa ggagcctaga tcgcatcaaa 60

agtccaaaca cattttacgg cggtatcacc tcattcgcga gatagtgggt agaggagatg 120

tgaagatatg caaaatacat acggatttga atgttgcaga tctgttaaca aagccactcc 180

ctcagcctaa gcatgaggca cacactaggg caatgggtat aagatacgtt tatgattgac 240

tctagtgcaa gtgggagact attagtgata tgccctagag acaatcatag atgattgtat 300

cacatactat gtttacatat ttatctgaca ttgttccttg aaatagataa ctccttattg 360

gttaatgaat atgtgattct ttcatgagac tctttatgtt gtttgttact atttctaaag 420

gatccttgat caaatatcat atgtggaaca aatatgttta tatgatcagc acatgtatta 480

attgatgatc atgtctcatg gatcatagta tagagatacc aaattaataa tgtggacaca 540

tgttggtgaa catgttgttg gatagaccca acatgagaca ctgcaagagc catatgtgtt 600

gtgtcatcag tgatctcatt tagtgttggt gttgaatcct tagacctgag attatcatgg 660

ttctcaacat gtgtagcagc ttacttaggg actgctaaac gctactccgt aaatgggtag 720

ctataaaagt agttttcagg tatgctatga aacatgtagt gggatatgaa taaccaagat 780

gggatttgct cctcctgtgg agagatatct ctgggcccct cgatgttgta gattatgaaa 840

gtgcatggcc atgccaaagg tgattgagga gtcaatcaca agttatataa tctatcaaca 900

ggttcgagtg aatgattgag ctattagagg atggcacata tctagccttg agcttaatcg 960

atatcgtgag gcaaaggggt tcatacaagt atacactaga ggttcagccg atatgatctt 1020

tatgtatgcc tggtgggtca atacgttctg ctaagggccg ctgttgacgc gtggaccgaa 1080

aaggagtttt cgggttacag ccgagtatat atgaacctat agggtcgcac gtttaatggg 1140

ccggaataag gggattggat agagatccaa tatgagctta attcggatag tgatccgaat 1200

aggagtccta cgggtcttgg aggcttgagt gatggattct atatattcgt gaggggcgtg 1260

accggcagag gcattgtatc acgtgagaaa ccctagccgc cacttccctc cccgagcaaa 1320

accctagcca cgcgcgggtg ctagcacatc tgcgcgtggc gttccgtccc tgtacgtgtg 1380

gataccggta gaggcgccgc tggtttgcga tgctgatcgg cgtgggagta cggcgaggag 1440

aacgcatgag gaggagaagg tcgagccggt gtgatcaact acttcctcta catcgacgcg 1500

cgctacttcg cgagagttcc ttcgacttct cagcgttttc ttccgctgca cagcgcgtcg 1560

agtggtaacg atctatgatc taataattgt atggttcttg gtttacgcga tagaaaattt 1620

tgatttatgc tatcgtagcc tacgcgtatc ccaacacgtc ggtccagctc ctcgcctcct 1680

cggtggatgc cacacgcggt agatgggagg gagtaaatga ggggaagaga gagagggggg 1740

agaagagagg ggtagtatag taaaatgtgc tttttttggt agggatactc attgggctac 1800

taggggtatc ctacattgtg acttgtgagg ggagtttcct agagagtact ctcagttagt 1860

gagggcacaa atagagtatc cctgattggt gatgccctta gtactaagca gagcccatgt 1920

tatttaaaaa acaagtaccg tatccccact ctcccatgta tataagtacc gcatcctctc 1980

tctcatatat actccagccg tttcacaatg taaatcattc tagcatttcc cacattcata 2040

ttgatgttaa gggtatccat aatgtagtga agaagtgggt attagggatt aaaatattct 2100

tactactagg taagatacat tgtggaaata gtaaatagct attaccaagc cataagctta 2160

ctattaggtt atagcacttt gtggtgggtc ctacctattt tttaattgtg gcatggctca 2220

tgtttggggt tatttttcag atgggtccta ccttattacc cactctacac atatatacat 2280

tgtgatgcat cttcaatcta ctattaccat cttacatgga cccaccttac tacctacttt 2340

tgcaacatac attgaggatg ccctaatgaa tctagataga tactagaatg acttatattg 2400

tgaaacagag gaagtatatc attttcaaaa gaaagcacca tgcagtcata ccccattgca 2460

ctggtatttt tttaataaaa ccacttatat tttttaaatc gtatgttatt ttaaagattt 2520

gtttaaacca ttttactcca ctcaaaacat gtaaaaaaac aagtctaata ttgaatatat 2580

tttgaatagc aatatagtta ctttaaatac catcaagagt tagtaccgtg aagagtacga 2640

tggctcaaac ggaatctaaa aataatacat ggtttaaaat atgttatatt tggtacatct 2700

agatttatta atattaatat tattgtggga aattttagaa tgacttaaag tataaaacgg 2760

agggagtatt ataaaagaac atgtctactt caccgagaga aaaaaacaac acgagaaaaa 2820

cctggcgagc acaccaccac cgcatcaccg gtccaccgct cttgccttcc cctttcttgc 2880

agtgcaggaa agaaccccac gccctcatcc aatcccagcc gtccatccac catccaacgc 2940

ctctcaccgc gccgctcacc tcctcaagac caccacccac cactcccctc ctctctcctc 3000

tccattcctc ctcgcttcga caaacaaaac cccccaaaac cctagcctcc gccgcccgcc 3060

gccgccgccg ccgcgcaacc cccgagctcg agcggcgcga tgaacatcaa gggcggaggc 3120

cgggtcccgg tgcccccggc cggcgcgggc acgctcgtca agctggtggt gctcggcggc 3180

acggccgtgt acgccgccgt caacagcctc tacaacgtcg agggtggcca ccgcgccatc 3240

gtcttcaacc gcatccaggg aatcaaggac aaggtaaccg gacacgccca tccccccgcc 3300

ccttccctgt tccatttcgt tacttggatg tgccgcgtgg tttggttgcg gcgttaacga 3360

ttcggtcgtt taggggagag attggttgga aataaatatt tggtctcgcg cgctatctta 3420

tgttaggttg cgtggtaaat tgtgttttga agcaaatcta tttgggttta taggtgctat 3480

ctggtaaatc tgaactaacc aggtggtctt catgatgtgt gagatacctg cctatcttta 3540

gataacttgt ttagctgcag cgatttatca ggctgaaaac atgacaagtg ttccctttct 3600

tgtgggtgat atcatttgtt gaggtttgag gattaaaaag gtcaaccatt gtgtgctgga 3660

actgattcca agttcttggt aaaaaatcca atttgtagta tgattgactg ctgattccag 3720

taccttatgc cttgaaaaga gaattacctc tgaaacaaca cgatcgctct gtaccaatgt 3780

gtgtggtttc cagtctggct tgcactgagg ttatgaaatt ttcctgcgct agatacctgt 3840

aattggttct agaaagcagc tgctttgtct tatttattgg cctgcggcag gtccacgagt 3900

accatgccgt ggtgtagttc agaacagata gtcacatgat gtttctacct tcatttttta 3960

tgttgtttgt tgaaatgatc tgaatgtggt aaacacttat tgaaattaaa ttgtaaatga 4020

acacgactac ttttgtttta tcattcctta aaaaccattt gaaaatatgg taacattcat 4080

cacagtccag tcgatttaat gcacacgaga ccactgctgg ttggaagatg gtgccaagat 4140

caactgaaaa ccattgactt ctttcagtgt ttgccgcatt gtggtttttt tataccaaat 4200

ttaccatgga caagtgcaat cgcattattt gattttagtc tggcagtctg gctgtgacca 4260

ttatatatgg ctcgccatca gtacaatcag ggcatattga agttgtttgt gttcacaaaa 4320

aacacacatt cttttctgta ctcacaatga actgcacatt tagttctgtc acacatgcac 4380

ctttttcttg taatttcttt aaatgtggtt catgggagta acacatttcc ttttaaaata 4440

tgtatcttta ttttatcact atgtggtttc ttatgctctt tctttctctc ttttttcctt 4500

ttcaaaaagt aataaaatgt attgttatta atggcattca attgtggttc tctgtttcat 4560

aatgcaactt aaatagcatt aaatttgtta gatttgttga aaatatctgt tatcatatat 4620

cttctgctct aactatctta atcgacctta gatggccggt tcctagcaat atactcagga 4680

acagcctaga taacgacaga ggctgctttc accttcttgc 4720

<210> 2

<211> 870

<212> DNA

<213> Rice (Oryza sativa L)

<400> 2

atgaacatca agggcggagg ccgggtcccg gtgcccccgg ccggcgcggg cacgctcgtc 60

aagctggtgg tgctcggcgg cacggccgtg tacgccgccg tcaacagcct ctacaacgtc 120

gagggtggcc accgcgccat cgtcttcaac cgcatccagg gaatcaagga caaggtatac 180

cctgagggga ctcactttat gattccatgg tttgagaggc caatcattta tgatgtccgt 240

gctcgtccca atcttgtgga gagtacttct ggaagcaggg atctccagat ggtgaaaatt 300

ggtctccgtg tccttacaag gcccatgcca gagaagctac caactatcta caggactctg 360

ggggagaact tcaatgagag agttttgcct tcaattatcc atgaaacact taaagctgtt 420

gtcgcacaat acaatgcgag tcagctaatc acacagagag agaccgtgag tagggagata 480

agaaagatac tgactgagag ggccaggaat tttaatattg cccttgatga tgtgtccatc 540

acaagcctga gcttcggaaa ggagttcact catgctattg aagccaaaca ggttgctgca 600

caagaagctg agcgtgctaa gttcattgtt gagaaagctg agcaagacaa gaggagtgcg 660

attatcaggg cacagggtga agctaaaagt gctgagctga ttggtcaagc cattgcaaac 720

aaccctgctt tccttgccct caggcagatc gaagctgcca gggagatctc tcatacgatg 780

tcatcttcag ctaacaaggt gttcctggac tccaatgatc tcttgctcaa tctccagcag 840

ctcaccgtgg caaacaagtc gaagaaatga 870

<210> 3

<211> 289

<212> PRT

<213> Rice (Oryza sativa L)

<400> 3

Met Asn Ile Lys Gly Gly Gly Arg Val Pro Val Pro Pro Ala Gly Ala

1 5 10 15

Gly Thr Leu Val Lys Leu Val Val Leu Gly Gly Thr Ala Val Tyr Ala

20 25 30

Ala Val Asn Ser Leu Tyr Asn Val Glu Gly Gly His Arg Ala Ile Val

35 40 45

Phe Asn Arg Ile Gln Gly Ile Lys Asp Lys Val Tyr Pro Glu Gly Thr

50 55 60

His Phe Met Ile Pro Trp Phe Glu Arg Pro Ile Ile Tyr Asp Val Arg

65 70 75 80

Ala Arg Pro Asn Leu Val Glu Ser Thr Ser Gly Ser Arg Asp Leu Gln

85 90 95

Met Val Lys Ile Gly Leu Arg Val Leu Thr Arg Pro Met Pro Glu Lys

100 105 110

Leu Pro Thr Ile Tyr Arg Thr Leu Gly Glu Asn Phe Asn Glu Arg Val

115 120 125

Leu Pro Ser Ile Ile His Glu Thr Leu Lys Ala Val Val Ala Gln Tyr

130 135 140

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

145 150 155 160

Arg Lys Ile Leu Thr Glu Arg Ala Arg Asn Phe Asn Ile Ala Leu Asp

165 170 175

Asp Val Ser Ile Thr Ser Leu Ser Phe Gly Lys Glu Phe Thr His Ala

180 185 190

Ile Glu Ala Lys Gln Val Ala Ala Gln Glu Ala Glu Arg Ala Lys Phe

195 200 205

Ile Val Glu Lys Ala Glu Gln Asp Lys Arg Ser Ala Ile Ile Arg Ala

210 215 220

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

225 230 235 240

Asn Pro Ala Phe Leu Ala Leu Arg Gln Ile Glu Ala Ala Arg Glu Ile

245 250 255

Ser His Thr Met Ser Ser Ser Ala Asn Lys Val Phe Leu Asp Ser Asn

260 265 270

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

275 280 285

Lys

<210> 4

<211> 870

<212> DNA

<213> Rice (Oryza sativa L)

<400> 4

atgaacatca agggcggagg ccgggtcccg gtgcccccgg ccggcgcggg cacgctcgtc 60

aagctggtgg tgctcggcgg cacggccgtg tacgccgccg tcaacagcct ctacaacgtc 120

gagggtggcc accgcgccat cgtcttcaac cgcatccagg gaatcaagga caaggtatac 180

cctgagggga ctcactttat gattccatgg tttgagaggc caatcattta tgatgtccgt 240

gctcgtccca atcttgtgga gagtacttct ggaagcaggg atctccagat ggtgaaaatt 300

ggtctccgtg tccttacaag gcccatgcca gagaagctac caactatcta caggactctg 360

ggggagaact tcaatgagag agttttgcct tcaattatcc atgaaacact taaagctgtt 420

gtcgcacaat acaatgcgag tcagctaatc acacagagag agaccgtgag tagggagata 480

agaaagatac tgactgagag ggccaggaat tttaatattg cccttgatga tgtgtccatc 540

acaagcctga gcttcggaaa ggagttcact catgctattg aagccaaaca ggttgctgca 600

caagaagctg agcgtgctaa gttcattgtt gagaaagctg agcaagacaa gaggagtgcg 660

attatcaggg cacagggtga aactaaaagt gctgagctga ttggtcaagc cattgcaaac 720

aaccctgctt tccttgccct caggcagatc gaagctgcca gggagatctc tcatacgatg 780

tcatcttcag ctaacaaggt gttcctggac tccaatgatc tcttgctcaa tctccagcag 840

ctcaccgtgg caaacaagtc gaagaaatga 870

<210> 5

<211> 289

<212> PRT

<213> Rice (Oryza sativa L)

<400> 5

Met Asn Ile Lys Gly Gly Gly Arg Val Pro Val Pro Pro Ala Gly Ala

1 5 10 15

Gly Thr Leu Val Lys Leu Val Val Leu Gly Gly Thr Ala Val Tyr Ala

20 25 30

Ala Val Asn Ser Leu Tyr Asn Val Glu Gly Gly His Arg Ala Ile Val

35 40 45

Phe Asn Arg Ile Gln Gly Ile Lys Asp Lys Val Tyr Pro Glu Gly Thr

50 55 60

His Phe Met Ile Pro Trp Phe Glu Arg Pro Ile Ile Tyr Asp Val Arg

65 70 75 80

Ala Arg Pro Asn Leu Val Glu Ser Thr Ser Gly Ser Arg Asp Leu Gln

85 90 95

Met Val Lys Ile Gly Leu Arg Val Leu Thr Arg Pro Met Pro Glu Lys

100 105 110

Leu Pro Thr Ile Tyr Arg Thr Leu Gly Glu Asn Phe Asn Glu Arg Val

115 120 125

Leu Pro Ser Ile Ile His Glu Thr Leu Lys Ala Val Val Ala Gln Tyr

130 135 140

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

145 150 155 160

Arg Lys Ile Leu Thr Glu Arg Ala Arg Asn Phe Asn Ile Ala Leu Asp

165 170 175

Asp Val Ser Ile Thr Ser Leu Ser Phe Gly Lys Glu Phe Thr His Ala

180 185 190

Ile Glu Ala Lys Gln Val Ala Ala Gln Glu Ala Glu Arg Ala Lys Phe

195 200 205

Ile Val Glu Lys Ala Glu Gln Asp Lys Arg Ser Ala Ile Ile Arg Ala

210 215 220

Gln Gly Glu Thr Lys Ser Ala Glu Leu Ile Gly Gln Ala Ile Ala Asn

225 230 235 240

Asn Pro Ala Phe Leu Ala Leu Arg Gln Ile Glu Ala Ala Arg Glu Ile

245 250 255

Ser His Thr Met Ser Ser Ser Ala Asn Lys Val Phe Leu Asp Ser Asn

260 265 270

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

275 280 285

Lys

<210> 6

<211> 3099

<212> DNA

<213> Rice (Oryza sativa L)

<400> 6

acctttattg cgataatagt ggtgccattg cgcaagccaa ggagcctaga tcgcatcaaa 60

agtccaaaca cattttacgg cggtatcacc tcattcgcga gatagtgggt agaggagatg 120

tgaagatatg caaaatacat acggatttga atgttgcaga tctgttaaca aagccactcc 180

ctcagcctaa gcatgaggca cacactaggg caatgggtat aagatacgtt tatgattgac 240

tctagtgcaa gtgggagact attagtgata tgccctagag acaatcatag atgattgtat 300

cacatactat gtttacatat ttatctgaca ttgttccttg aaatagataa ctccttattg 360

gttaatgaat atgtgattct ttcatgagac tctttatgtt gtttgttact atttctaaag 420

gatccttgat caaatatcat atgtggaaca aatatgttta tatgatcagc acatgtatta 480

attgatgatc atgtctcatg gatcatagta tagagatacc aaattaataa tgtggacaca 540

tgttggtgaa catgttgttg gatagaccca acatgagaca ctgcaagagc catatgtgtt 600

gtgtcatcag tgatctcatt tagtgttggt gttgaatcct tagacctgag attatcatgg 660

ttctcaacat gtgtagcagc ttacttaggg actgctaaac gctactccgt aaatgggtag 720

ctataaaagt agttttcagg tatgctatga aacatgtagt gggatatgaa taaccaagat 780

gggatttgct cctcctgtgg agagatatct ctgggcccct cgatgttgta gattatgaaa 840

gtgcatggcc atgccaaagg tgattgagga gtcaatcaca agttatataa tctatcaaca 900

ggttcgagtg aatgattgag ctattagagg atggcacata tctagccttg agcttaatcg 960

atatcgtgag gcaaaggggt tcatacaagt atacactaga ggttcagccg atatgatctt 1020

tatgtatgcc tggtgggtca atacgttctg ctaagggccg ctgttgacgc gtggaccgaa 1080

aaggagtttt cgggttacag ccgagtatat atgaacctat agggtcgcac gtttaatggg 1140

ccggaataag gggattggat agagatccaa tatgagctta attcggatag tgatccgaat 1200

aggagtccta cgggtcttgg aggcttgagt gatggattct atatattcgt gaggggcgtg 1260

accggcagag gcattgtatc acgtgagaaa ccctagccgc cacttccctc cccgagcaaa 1320

accctagcca cgcgcgggtg ctagcacatc tgcgcgtggc gttccgtccc tgtacgtgtg 1380

gataccggta gaggcgccgc tggtttgcga tgctgatcgg cgtgggagta cggcgaggag 1440

aacgcatgag gaggagaagg tcgagccggt gtgatcaact acttcctcta catcgacgcg 1500

cgctacttcg cgagagttcc ttcgacttct cagcgttttc ttccgctgca cagcgcgtcg 1560

agtggtaacg atctatgatc taataattgt atggttcttg gtttacgcga tagaaaattt 1620

tgatttatgc tatcgtagcc tacgcgtatc ccaacacgtc ggtccagctc ctcgcctcct 1680

cggtggatgc cacacgcggt agatgggagg gagtaaatga ggggaagaga gagagggggg 1740

agaagagagg ggtagtatag taaaatgtgc tttttttggt agggatactc attgggctac 1800

taggggtatc ctacattgtg acttgtgagg ggagtttcct agagagtact ctcagttagt 1860

gagggcacaa atagagtatc cctgattggt gatgccctta gtactaagca gagcccatgt 1920

tatttaaaaa acaagtaccg tatccccact ctcccatgta tataagtacc gcatcctctc 1980

tctcatatat actccagccg tttcacaatg taaatcattc tagcatttcc cacattcata 2040

ttgatgttaa gggtatccat aatgtagtga agaagtgggt attagggatt aaaatattct 2100

tactactagg taagatacat tgtggaaata gtaaatagct attaccaagc cataagctta 2160

ctattaggtt atagcacttt gtggtgggtc ctacctattt tttaattgtg gcatggctca 2220

tgtttggggt tatttttcag atgggtccta ccttattacc cactctacac atatatacat 2280

tgtgatgcat cttcaatcta ctattaccat cttacatgga cccaccttac tacctacttt 2340

tgcaacatac attgaggatg ccctaatgaa tctagataga tactagaatg acttatattg 2400

tgaaacagag gaagtatatc attttcaaaa gaaagcacca tgcagtcata ccccattgca 2460

ctggtatttt tttaataaaa ccacttatat tttttaaatc gtatgttatt ttaaagattt 2520

gtttaaacca ttttactcca ctcaaaacat gtaaaaaaac aagtctaata ttgaatatat 2580

tttgaatagc aatatagtta ctttaaatac catcaagagt tagtaccgtg aagagtacga 2640

tggctcaaac ggaatctaaa aataatacat ggtttaaaat atgttatatt tggtacatct 2700

agatttatta atattaatat tattgtggga aattttagaa tgacttaaag tataaaacgg 2760

agggagtatt ataaaagaac atgtctactt caccgagaga aaaaaacaac acgagaaaaa 2820

cctggcgagc acaccaccac cgcatcaccg gtccaccgct cttgccttcc cctttcttgc 2880

agtgcaggaa agaaccccac gccctcatcc aatcccagcc gtccatccac catccaacgc 2940

ctctcaccgc gccgctcacc tcctcaagac caccacccac cactcccctc ctctctcctc 3000

tccattcctc ctcgcttcga caaacaaaac cccccaaaac cctagcctcc gccgcccgcc 3060

gccgccgccg ccgcgcaacc cccgagctcg agcggcgcg 3099

<210> 7

<211> 21

<212> DNA

<213> primers (Primer)

<400> 7

acacaagtat caggtagtcc c 21

<210> 8

<211> 22

<212> DNA

<213> primers (Primer)

<400> 8

cgggtaaaac aatgacatca gc 22

<210> 9

<211> 20

<212> DNA

<213> primers (Primer)

<400> 9

caatcccaga attcaagagc 20

<210> 10

<211> 21

<212> DNA

<213> primers (Primer)

<400> 10

gaaatacatc acagagcaac g 21

<210> 11

<211> 20

<212> DNA

<213> primers (Primer)

<400> 11

aatgattgtt tgattgagcc 20

<210> 12

<211> 23

<212> DNA

<213> primers (Primer)

<400> 12

cctagctatg aaattcttac gga 23

<210> 13

<211> 23

<212> DNA

<213> primers (Primer)

<400> 13

attattactt ctcaatttgc aga 23

<210> 14

<211> 20

<212> DNA

<213> primers (Primer)

<400> 14

cccttccaaa ttgtggggat 20

<210> 15

<211> 21

<212> DNA

<213> primers (Primer)

<400> 15

gtattgcaat ccaatatgct t 21

<210> 16

<211> 23

<212> DNA

<213> primers (Primer)

<400> 16

tgacttccgg attaatttta ggg 23

<210> 17

<211> 21

<212> DNA

<213> primers (Primer)

<400> 17

taatgcaaga gagacaacat c 21

<210> 18

<211> 22

<212> DNA

<213> primers (Primer)

<400> 18

tctactcata aattaagagg tg 22

<210> 19

<211> 22

<212> DNA

<213> primers (Primer)

<400> 19

ggacgaaagg gtatttgatt gg 22

<210> 20

<211> 22

<212> DNA

<213> primers (Primer)

<400> 20

cctcaaggtg gtctccttct cc 22

<210> 21

<211> 21

<212> DNA

<213> primers (Primer)

<400> 21

cccgcataaa tggataagct g 21

<210> 22

<211> 22

<212> DNA

<213> primers (Primer)

<400> 22

tatgcctaca acaaattggt ga 22

<210> 23

<211> 20

<212> DNA

<213> primers (Primer)

<400> 23

acccaccttt tgtcctctcc 20

<210> 24

<211> 23

<212> DNA

<213> primers (Primer)

<400> 24

tgactctagt gcaagtggga gac 23

<210> 25

<211> 20

<212> DNA

<213> primers (Primer)

<400> 25

attgaagcca aacaggttgc 20

<210> 26

<211> 20

<212> DNA

<213> primers (Primer)

<400> 26

ttgaccaatc agctcagcac 20

<210> 27

<211> 23

<212> DNA

<213> primers (Primer)

<400> 27

ggcacatcaa gggcggaggc cgg 23

<210> 28

<211> 23

<212> DNA

<213> primers (Primer)

<400> 28

aaacccggcc tccgcccttg atg 23

<210> 29

<211> 23

<212> DNA

<213> primers (Primer)

<400> 29

gccgtcaagc tggtggtgct cgg 23

<210> 30

<211> 23

<212> DNA

<213> primers (Primer)

<400> 30

aaacccgagc accaccagct tga 23

<210> 31

<211> 31

<212> DNA

<213> primers (Primer)

<400> 31

aaaactgcag atgaacatca agggcggagg c 31

<210> 32

<211> 35

<212> DNA

<213> primers (Primer)

<400> 32

tttttgagct ccatttcttc gacttgtttg ccacg 35

<210> 33

<211> 40

<212> DNA

<213> primers (Primer)

<400> 33

agttattaaa attcctggcc ctacaggaga ttcagtttga 40

<210> 34

<211> 40

<212> DNA

<213> primers (Primer)

<400> 34

tgtagggcca ggaattttaa taactgctgc tgctacagcc 40

<210> 35

<211> 40

<212> DNA

<213> primers (Primer)

<400> 35

cttaggggca gcaattttaa taattcctgc tgctaggctg 40

<210> 36

<211> 40

<212> DNA

<213> primers (Primer)

<400> 36

aattattaaa attgctgccc ctaagagagg caaaagtgaa 40

<210> 37

<211> 40

<212> DNA

<213> primers (Primer)

<400> 37

aaaactgcag ggagaggaga gaggagggga gtggtgggtg 40

<210> 38

<211> 40

<212> DNA

<213> primers (Primer)

<400> 38

ggcgggtacc tttattgcga taatagtggt gccattgcgc 40

<210> 39

<211> 23

<212> DNA

<213> primers (Primer)

<400> 39

cccatgccag agaagctacc aac 23

<210> 40

<211> 21

<212> DNA

<213> primers (Primer)

<400> 40

attcctggcc ctctcagtca g 21

Claims (20)

1. A method for modulating plant type traits or yield traits in a plant comprising: modulating expression or activity of NAL8 in a plant, thereby modulating plant type traits or yield traits in the plant; the polypeptide of the NAL8 is a polypeptide of an amino acid sequence shown in SEQ ID NO. 3; the plant is a gramineous plant;

Wherein (a) the expression or activity of NAL8 is up-regulated, so that the plant type character or yield character of the plant is represented as: plant height is increased, the number of seeds per spike is increased or the seed grain weight is increased, and the up-regulation is as follows: transferring a coding gene of NAL8 or an expression construct or a vector containing the coding gene into a plant, or returning a mutant to a NAL8 wild type in a plant in which a NAL8 228 th mutation occurs; or

(b) The expression or activity of NAL8 is down-regulated, so that the plant type character or yield character of the plant is represented as: the plant height is reduced, the leaf width is reduced, the leaf length is increased or the yield is reduced, and the down regulation is as follows: knocking out or silencing a gene encoding NAL8 or inhibiting the activity of NAL8 in a plant.

2. The method of claim 1, wherein in (b), the NAL8 is silenced with an interfering molecule that specifically interferes with the expression of a gene encoding NAL8, the gene editing is performed with the CRISPR system to knock out the gene encoding NAL8, and the gene encoding NAL8 is knocked out by homologous recombination.

3. The method of claim 1, wherein in (a), the expression or activity of NAL8 is up-regulated in plants that have low NAL8 expression, such that the plant type or yield traits of the plants are as follows: the plant height is increased, the number of seeds per spike is increased or the seed grain weight is increased.

4. The method of claim 1, wherein the yield reduction is a reduction in the number of seeds per ear, a reduction in seed weight, a reduction in seed width, or a reduction in seed set.

5. A method of modulating development of a plant organelle comprising: (i) up-regulating the expression or activity of NAL8 in a plant which expresses NAL8 to stabilize and develop normally mitochondria and chloroplasts of the plant, wherein the up-regulation is as follows: transferring the coding gene of NAL8 or an expression construct or a vector containing the coding gene into a plant, or returning the mutant to a NAL8 wild type in a plant in which the 228 th mutation of NAL8 occurs; or

(ii) Down-regulating the expression or activity of plant NAL8, thereby destabilizing the chloroplast or mitochondria of the plant, by: knocking out or silencing a gene encoding NAL8 or inhibiting NAL8 activity in a plant;

the polypeptide of the NAL8 is a polypeptide of an amino acid sequence shown in SEQ ID NO. 3; the plant is a gramineous plant.

6. The method of claim 5, wherein in (ii), NAL8 is silenced with an interfering molecule that specifically interferes with the expression of a gene encoding NAL8, gene editing is performed with CRISPR system to knock out the gene encoding NAL8, and the gene encoding NAL8 is knocked out by homologous recombination.

7. The method of claim 5, wherein in (i), when the plant's mitochondria, chloroplasts are stable and normally developed, the plant's organelles are represented by: the stack structure of chloroplast, the maintenance of chloroplast quantity, the maintenance of continuous distribution of thylakoids in chloroplast or the maintenance of photosynthetic product and lipid product quantity.

8. The method of claim 5, wherein in (ii), when the chloroplast or mitochondria of the plant are unstable, the organelles of the plant are represented by: the stack structure of chloroplast is thinned, the stack number of chloroplast is reduced, the stack structure of chloroplast is discontinuous, the chloroplast is irregular in shape, thylakoid bodies in chloroplast are discontinuously distributed, and the quantity of photosynthetic products and lipid products is reduced.

9. A method of modulating the cell cycle of a plant comprising:

(A) up-regulating NAL8 expression or activity in plants that express NAL8 low, maintaining cell cycle stability, said up-regulation being: transferring the coding gene of NAL8 or an expression construct or a vector containing the coding gene into a plant, or returning the mutant to a NAL8 wild type in a plant in which the 228 th mutation of NAL8 occurs; or

(B) Downregulating NAL8 expression or activity, thereby modulating cell cycle and increasing the expression of NAL8 at G ₁A cell in stage (iii) that reduces cells in division stage, S stage, or down-regulates cell cycle-associated gene expression, the down-regulated NAL8 expression or activity being a knock-out or silencing of a gene encoding NAL8 or inhibiting NAL8 activity in a plant; the cell cycle related gene is selected from CyCD4, CDKA1, CycA2, 1, CycA2, 2, CycA2, 3, CycB2, 1, CycB2, 2, CDC20 or CyCD4 and 1;

the polypeptide of NAL8 is a polypeptide of an amino acid sequence shown in SEQ ID NO. 3; the plant is a gramineous plant.

10. The method of claim 9, wherein in (B), NAL8 is silenced with an interfering molecule that specifically interferes with the expression of a gene encoding NAL8, gene editing is performed with the CRISPR system to knock out the gene encoding NAL8, and the gene encoding NAL8 is knocked out by homologous recombination.

11. A method of modulating plant hormone signaling, comprising:

(1) up-regulating NAL8 expression or activity in plants that express NAL8, thereby reducing auxin content or increasing cytokinin content in the plant; the up-regulation is as follows: transferring the coding gene of NAL8 or an expression construct or a vector containing the coding gene into a plant, or returning the mutant to a NAL8 wild type in a plant in which the 228 th mutation of NAL8 occurs; or

(2) Downregulating the expression or activity of NAL8, thereby increasing the auxin content or cytokinin content of the plant; the downregulating expression or activity of NAL8 is knocking out or silencing a gene encoding NAL8 or inhibiting activity of NAL8 in a plant;

the polypeptide of the NAL8 is a polypeptide of an amino acid sequence shown in SEQ ID NO. 3; the plant is a gramineous plant.

12. Use of NAL8 or a modulator thereof for modulating plant type traits, yield traits, organelle development, hormone signalling or cell cycle in a plant; the regulator comprises an up regulator or a down regulator; the polypeptide of the NAL8 is a polypeptide of an amino acid sequence shown in SEQ ID NO. 3; the plant is a gramineous plant;

the NAL8 up-regulator is: exogenous NAL8 coding gene or expression construct or vector containing the coding gene, or an agent for reverting the mutant to NAL8 wild type in plants where the NAL8 position 228 mutation occurs;

the NAL8 down-regulator is an agent for knocking out or silencing NAL8 or an agent for inhibiting NAL8 activity, and comprises: interfering molecules specifically interfering with NAL8 coding gene expression, CRISPR gene editing agents or homologous recombination agents or site-directed mutation agents against NAL 8.

13. The use according to claim 12, wherein the plant-type trait comprises: plant height, leaf width, leaf length.

14. Use according to claim 12, wherein said yield traits comprise: seed number of each ear, seed width, seed weight and seed setting number.

15. The use according to claim 12, wherein said organelles comprise: chloroplasts, mitochondria; the NAL8 or its regulator regulates stability, arrangement structure, development, photosynthetic product and lipid product amount of chloroplast or mitochondria.

16. The use according to claim 12, for modulating the cell cycle of a plant comprising: regulating the number of cells in the division stage and the S stage, and regulating the expression of cell cycle related genes; the cell cycle related gene is selected from CyCD4, CDKA1, CycA2, 1, CycA2, 2, CycA2, 3, CycB2, 1, CycB2, 2, CDC20 or CyCD4 and 1.

17. Use of a plant NAL8 as a molecular marker for identifying plant type traits, yield traits, organelle development, hormone signaling, or cell cycle; the polypeptide of the NAL8 is a polypeptide of an amino acid sequence shown in SEQ ID NO. 3; the plant is a gramineous plant.

18. The use according to claim 17, wherein the plant-type trait comprises: plant height, leaf width and leaf length; or

Said yield traits comprise: seed number of each ear, seed width, seed weight and seed setting number; or

The organelle comprises: chloroplasts, mitochondria; the NAL8 or its regulator can regulate stability, arrangement structure, development, photosynthetic product and lipid product amount of chloroplast or mitochondria; or

The hormone signals include: auxin or cytokinin signals; or

The method for regulating the cell cycle of the plant comprises the following steps: regulating the number of cells in the division stage and the S stage, and regulating the expression of cell cycle related genes; the cell cycle related gene is selected from CyCD4, CDKA1, CycA2, 1, CycA2, 2, CycA2, 3, CycB2, 1, CycB2, 2, CDC20 or CyCD4 and 1.

19. A method for targeted selection or identification of plants, comprising: identification of NAL8 expression or sequence characteristics in test plants:

if the NAL8 of the test plant is highly expressed or normally expressed, the test plant is a plant with normal plant height, leaf width, normal or improved plant type character or yield character, or a plant with stable mitochondria and chloroplasts and normal development, or a plant with low auxin content or high cytokinin content, or a plant with stable cell cycle;

If the NAL8 of the test plant is low expressed or not expressed, or the NAL8 polypeptide in the test plant is mutated into Thr at the 228 th amino acid corresponding to the SEQ ID NO. 3 sequence, the test plant is a plant with plant type character or yield character of reduced plant height, reduced leaf width, increased leaf length or reduced yield, or a plant with unstable chloroplast or mitochondria, or a plant with high auxin content or low cytokinin content, or a plant with changed cell cycle;

the polypeptide of NAL8 is a polypeptide of an amino acid sequence shown in SEQ ID NO. 3; the plant is a gramineous plant.

20. The method of claim 19, wherein the yield reduction is a reduction in the number of seeds per ear, a reduction in seed weight, a reduction in seed width, or a reduction in seed set.

Images