CN111333707B - Plant grain type related protein and coding gene and application thereof - Google Patents

Abstract

The invention discloses a plant grain type growth and development related protein OsSG2, and a coding gene and application thereof, belonging to the field of genetic engineering. The protein provided by the invention is named as OsSG2 protein and is the protein of the following (a) or (b): (a) a protein consisting of an amino acid sequence shown in SEQ ID No. 1; (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID NO.1, is related to the plant grain type and is derived from the SEQ ID NO. 1. The coding gene of the protein is introduced into a plant with abnormal grain type growth, so that a plant with normal grain type growth and development can be cultivated; the gene is knocked out in normal plants with reduced grain type. The protein and the coding gene thereof can be applied to the genetic improvement of rice grain types.

Description

Plant grain type related protein and coding gene and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and relates to a plant type growth and development related protein, and a coding gene and application thereof.

Background

Rice is an important food crop in the world, and as staple food, nearly half of the world population is cultivated. With the continuous increase of world population, the severe deterioration of natural environment and the continuous reduction of global cultivated land area, the continuous cultivation of high-yield, high-quality and good-adaptability rice has important significance for ensuring global grain safety and maintaining the peaceful and stable world. The yield of rice is mainly determined by factors such as spike grain number, spike per plant number, grain weight and the like, wherein the grain weight is mainly influenced by the seed grain type. Seed grain type is a complex trait in which grain length, grain width and grain thickness are important factors affecting grain weight. The size and shape of the seeds not only affect the appearance and quality of the seeds, but also have important influence on the quality and characters of the seeds, including cooking quality, processing quality and the like. Different countries and different regions of the same country which take the rice as food can select rice varieties with different grain types according to own preference and dietary habits. Such as the preference for long and thin rice in europe and most countries in asia, as well as in the south of china, but the preference for relatively short round rice in the north of china. Therefore, the molecular mechanism and the regulation network for controlling the rice grain type are further clarified, and important grain type genes are utilized for breeding application, so that the method has great significance for improving the yield and the quality of rice and ensuring the grain safety.

At present, a plurality of key genes for controlling grain size are cloned in rice, and mainly participate in ubiquitin proteasome signal pathways, G protein signal pathways, MAPK signals, plant hormone signals and other pathways to influence cell division or cell size to regulate and control seed size. Most of cloned genes are mainly used for detecting QTLs by constructing a genetic population, and are further cloned by constructing a backcross population, such as GW2, GS3 and the like; but also few genes are obtained by methods such as physical mutagenesis, chemical mutagenesis, T-DNA insertion and the like to obtain mutants with changed grain types, and genes are obtained by methods such as map-based cloning, insertion site detection or whole genome re-sequencing and the like. Although many grain type related genes are cloned, the number of genes actually used in production is small, so that further development of new grain type genes and thorough analysis of the molecular mechanism of regulation of grain types are important.

Disclosure of Invention

The invention aims to provide a plant grain type related protein, a coding gene and application thereof.

The rice grain type related protein OsSG2 provided by the invention is derived from a rice variety Ningjing No.1 and is a protein of the following (a) or (b):

(a) a protein consisting of an amino acid sequence shown by SEQ ID NO.1 in a sequence table;

(b) a protein which is derived from the SEQ ID NO.1 and is related to the particle type by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the SEQ ID NO. 1.

SEQ ID NO.1 of the sequence Listing consists of 810 amino acid residues.

The OsSG2 in the (b) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression. The gene encoding OsSG2 in (b) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in SEQ ID NO.2 or SEQ ID NO.3 of the sequence Listing, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 at its 5 'end and/or 3' end.

The gene (OsSG2) for coding the grain type related protein also belongs to the protection scope of the invention.

The gene OsSG2 can be a DNA molecule of the following 1) or 2) or 3) or 4):

1) DNA molecule shown as SEQ ID NO.2 in the sequence table;

2) a DNA molecule shown as SEQ ID NO.3 in the sequence table;

3) a DNA molecule which hybridizes with the DNA sequence defined in 1) or 2) under stringent conditions and encodes said protein;

4) DNA molecule which has more than 90% of homology with the DNA sequence limited by 1) or 2) or 3) and codes the related protein of the particle type.

SEQ ID NO.2 of the sequence Listing consists of 2433 nucleotides.

The stringent conditions may be in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, at 65. Hybridization and washing of membranes at C.

The recombinant expression vector containing any one of the genes also belongs to the protection scope of the invention.

The recombinant expression vector containing the gene can be constructed by using the existing plant expression vector.

The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal can direct polyadenylation to the 3 'end of the mRNA precursor, and untranslated regions transcribed from the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopalin synthase Nos), plant genes (e.g., soybean storage protein genes) all have similar functions.

When the gene is used for constructing a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added in front of transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV)35S promoter and maize Ubiquitin promoter (Ubiquitin), and the enhanced promoter or constitutive promoter can be used independently or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

The recombinant expression vector may be a recombinant plasmid obtained by recombinantly inserting the gene (OsSG2) between the multiple cloning sites HindIII and BamHI of pCUBi1390 vector. The recombinant plasmid can be pCUBi1390-OsSG 2; the pCUBi1390-OsSG2 was obtained by inserting the OsSG2 genomic coding sequence between the HindIII and BamHI multicloning sites of pCUBi1390 by recombinant techniques (Clontech, Infusion recombination kit).

pCUBi1390 containing OsSG2 was named pCUBi1390-OsSG 2.

The expression cassette, the transgenic cell line and the recombinant bacteria containing any one of the genes (OsSG2) belong to the protection scope of the invention.

Primer pairs for amplifying the full length or any fragment of the gene (OsSG2) also belong to the protection scope of the invention.

It is another object of the present invention to provide a method for breeding transgenic plants with altered grain type.

The method for cultivating the transgenic plant with the changed grain type, provided by the invention, is to introduce the gene into the plant with the shortened grain type length to obtain the transgenic plant with the normal grain type; knocking out the encoding gene of the protein in a normal plant to obtain a transgenic plant with a shortened grain type, and specifically, introducing the gene into the plant with the shortened grain type length through the recombinant expression vector; the grain-type length-shortened plant is sg2.

The protein, the gene, the recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant strain or the method can be applied to rice breeding.

The coding gene of the protein is introduced into a plant with abnormal grain type growth, so that the normal plant can be cultivated and developed through grain type growth; the gene is knocked out in normal plants with reduced grain type. The protein and the coding gene thereof can be applied to the genetic improvement of rice grain types.

Any vector capable of guiding the expression of the exogenous gene in the plant is utilized to introduce the gene for coding the protein into plant cells, so that a transgenic cell line and a transgenic plant can be obtained. The expression vector carrying the gene can transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and culture the transformed plant tissues into plants. The plant host to be transformed may be either a monocotyledonous or dicotyledonous plant, such as: tobacco, lotus roots, arabidopsis, rice, wheat, corn, cucumber, tomato, poplar, lawn grass, alfalfa and the like, and is particularly applied to rice.

The invention discovers, positions and clones a new particle type related protein gene OsSG2 for the first time. Introducing the coding gene of the protein into the plant with shortened grain type to obtain a transgenic plant with normal grain type development; the encoding gene of the protein is knocked out in normal plants, and transgenic plants with shortened grain types can be obtained. The protein and the coding gene thereof can be applied to the genetic improvement of crop grains.

Drawings

FIG. 1 is a table diagram of wild type Ningjing No.1 (WT) and mutant sg2 mature plant types (A wild type WT and mutant sg2 mature plant type comparison diagram; B wild type WT and mutant sg2 main stem internode and spike comparison diagram).

FIG. 2 is a phenotype analysis diagram of wild Ningjing No.1 (WT) and mutant sg2 grains (A comparison diagram of grain lengths of wild Ningjing No.1 (WT) and mutant sg 2; B comparison diagram of grain widths of wild Ningjing No.1 (WT) and mutant sg 2; C comparison diagram of grain widths of wild Ningjing No.1 (WT) and mutant sg 2; D average grain lengths of wild Ningjing No.1 (WT) and mutant sg 2; E average thousand-grain weight of wild Ningjing No.1 (WT) and mutant sg 2; and F average grain widths of wild Ningjing No.1 (WT) and mutant sg 2).

FIG. 3 is a scanning electron microscope observation of wild type Ningjing No.1 (WT) and mutant sg2 glume (A is a scanning electron microscope observation of wild type Ningjing No.1 (WT) and mutant sg2 exodermis cells; B is a scanning electron microscope observation of wild type Ningjing No.1 (WT) and mutant sg2 exodermis cells average length; C is a scanning electron microscope observation of wild type Ningjing No.1 (WT) and mutant sg2 exodermis cells average width; D is a scanning electron microscope observation of wild type Ningjing No.1 (WT) and mutant sg2 exodermis cells average length; E is a scanning electron microscope observation of wild type Ningjing No.1 (WT) and mutant sg2 exodermis cells average length).

FIG. 4 shows the initial mapping results of OsSG2 and the results of Mutmap association analysis (A is the initial mapping results of OsSG2 gene; B is the results of Mutmap association analysis of OsSG2 gene).

FIG. 5 shows the results of transgene knockout (plant type observation of A wild type Ningjing No.1 (WT), mutant sg2 and knockout plus line cr-1; sequencing identification and grain type observation of B wild type Ningjing No.1 (WT), mutant sg2 and knockout plus line cr-1).

FIG. 6 shows the result of transgene complementation.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1 discovery of a plant granule type-related protein and Gene encoding the same

First, obtaining rice grain type mutant

The Ningjing No.1 japonica rice variety is subjected to transgenic tissue culture to produce a mutant with small seed grains, which is named as sg2.

Compared with the wild type, the strain type of sg2 is compact after maturation, the strain height is obviously shortened, the length of the inverted internode is obviously shortened (figures 1A and B), the length of the seed grain is shortened, the width is narrowed, the thousand seed weight is obviously reduced (figures 2A-F), and the observation of the inner and outer epidermis of the seed glume shows that the length shortening and the width reduction of the sg2 seed grain are caused by the cell size (figures 3A-E).

Taken together, the phenotype of shortened grain length was caused by the altered cell size in mutant sg2.

Second, mutant Gene mapping

1. Preliminary mapping of mutant genes

Firstly, positive and negative crossing of wild Ningjing No.1 and a mutant sg2 are constructed, and the obtained seeds of a normal type and a mutant type in an inbred progeny of F1 meet the separation ratio of 3:1, so that the phenotype of grain reduction in sg2 is controlled by a single recessive nuclear gene.

The mutant sg2 is hybridized with indica rice variety 93-11 to construct a positioning population, extreme single plants with grain phenotypes very similar to mutant phenotypes are selected from an F2 segregation population (5000 strains) constructed by sg2 and 93-11, and DNA is extracted by using leaves of the extreme single plants. Firstly, carrying out polymorphism analysis on SSR primers between Ningjing No.1 and 93-11 by 565 covering the whole genome of rice, and selecting polymorphism markers uniformly distributed on 12 chromosomes for linkage analysis. The mutation sites are further positioned by using molecular markers on a public map and self-developed SSR and Indel molecular markers based on rice genome sequence data, and finally, the grain type key gene OsSG2 is initially positioned between two molecular markers, namely chromosome 2 markers DBB16 and HSP 38.

The method for the SSR marker analysis is as follows:

(1) the total DNA of the selected individual plant is extracted as a template, and the specific method is as follows:

firstly, taking about 0.2g of young and tender rice leaves, placing the young and tender rice leaves in an Eppendorf tube, placing a steel ball in the tube, freezing the Eppendorf tube filled with a sample in liquid nitrogen for 5min, and placing the tube on a 2000 model GENO/GRINDER instrument to crush the sample for 1 min.

② 660 mul of extract (solution containing 100mM Tris-HCl (pH 8.0), 20mM EDTA (pH 8.0), 1.4M NaCl,0.2g/ml CTAB) is added, mixed by intense vortex on a vortex machine, bathed in water at 65 ℃ for 30min, and shaken every 10 min.

③ adding 40 mu L of 20 percent SDS, carrying out warm bath at 65 ℃ for 10min, and slightly reversing and mixing the mixture up and down every two minutes.

Fourthly, 100 mu L of 5M NaCl is added and mixed gently.

Fifthly, 100 mu L of 10 xCTAB is added, the mixture is bathed for 10min at 65 ℃, and the mixture is mixed by intermittently and slightly reversing the upside down.

Sixthly, adding 900 mu L of chloroform, fully and uniformly mixing, and centrifuging at 12000rpm for 3 min.

Seventhly, transferring the supernatant to a 1.5mL Eppendorf tube, adding 600 mu L of isopropanol, uniformly mixing, and centrifuging at 12000rpm for 5 min.

Eighthly, discarding the supernatant, rinsing the precipitate once by using 70 percent (volume percentage) of ethanol, and drying at room temperature.

Ninthly, adding 100. mu.L of 1 XTE (a solution obtained by dissolving 121 g of Tris in 1 liter of water and adjusting pH to 8.0 with hydrochloric acid) to dissolve the DNA.

Sample of red (2 μ L) was electrophoresed to detect DNA mass and its concentration was determined by DU800 spectrophotometer (Bechman Instrument Inc.U.S.A.).

(2) Diluting the extracted DNA to about 20 ng/. mu.L, and performing PCR amplification as a template;

PCR reaction (10. mu.L): 1. mu.L of DNA (20 ng/. mu.L), 1. mu.L of upstream primer (2 pmol/. mu.L), 1. mu.L of downstream primer (2 pmol/. mu.L), 10xBuffer (MgCl)₂ free)1μL，dNTP(10mM)0.2μL，MgCl₂(25mM)0.6μL，rTaq(5u/μL)0.1μL，ddH₂O5.1. mu.L, 10. mu.L total.

PCR reaction procedure: denaturation at 94.0 deg.C for 5 min; denaturation at 94.0 deg.C for 30s, annealing at 55 deg.C for 30s, and extension at 72 deg.C for 1min, and circulating for 35 times; extending for 7min at 72 ℃; storing at 10 deg.C. The PCR reaction was performed in an MJ Research PTC-225 thermal cycler.

(3) SSR-tagged PCR product detection

The amplification products were analyzed by 8% native polyacrylamide gel electrophoresis. The molecular weight of the amplified product is compared by taking 50bp DNA Ladder as a control, and silver staining is performed for color development.

The SSR and Indel markers described above were developed as follows:

(1) integrating SSR markers of a public map with a rice genome sequence, and downloading BAC/PAC clone sequences near mutation sites. Searching potential SSR sequences (the repetition times are more than or equal to 6) in the clone by SSR Hunter (Liqiang et al, heredity, 2005, 27(5): 808-; comparing the SSRs and sequences adjacent to 400-500 bp thereof with corresponding indica rice sequences on line at NCBI through a BLAST program, and preliminarily deducing that the PCR product of the SSR primer has polymorphism between indica rice and japonica rice if the SSR repetition times of the SSRs and the sequences are different; then, the SSR primers are designed by using Primer Premier 5.0 software and synthesized by Shanghai Invitrogen/Nanjing Kingsrei Biotech Co. The self-designed SSR paired primers are mixed in equal proportion, the polymorphism between Ningjing No.1 and 93-11 is detected, and the molecular marker with polymorphism is used as a molecular marker for positioning the OsSG2 gene. The molecular markers used for localization are shown in table 1.

(2) Development of Indel markers

Design of Indel primers: downloading clone sequences of Nipponbare near a positioning interval, comparing the clone sequences with indica rice variety 93-11 sequences in a database, searching positions where 3 or more than 3 bases are deleted between the clone sequences and the indica rice variety, and designing a Primer containing a deletion site by using Primer Premier 5.0 software so as to obtain the Primer with polymorphism shown in Table 1.

PCR reaction system for InDel marker analysis: 2ul of DNA (20ng/ul), 2ul of Primer1(10pmol/ul), 2ul of Primer2(10pmol/ul), 2ul of 10xBuffer (MgCl2free)2ul, 0.4ul of dNTP (10mM), 1.2ul of MgCl2(25mM), 0.4ul of rTaq (5u/ul), 10ul of ddH2O 10ul, and 20ul of total volume. The amplification reaction was performed on a PTC-200(MJ Research Inc.) PCR instrument: 3min at 94 ℃; 94 ℃ for 30sec, 55 ℃ (primer varied, adjusted) for 45sec, 72 ℃ for 2.5min, 35 cycles; 5min at 72 ℃.

The PCR product was purified and recovered according to the procedure of the kit (Beijing Tiangen Co.). After digestion overnight, the PCR product was separated by electrophoresis on 1-4% agarose gel, stained with EB, and photographed under an ultraviolet lamp. dCAPS was separated on 8% native PAGE gel and silver stained.

TABLE 1 molecular markers for Primary localization

2. Mutmap analysis of mutant genes

(1) Association analysis of mutant genes

The genetic analysis result shows that the phenotype of grain reduction in the sg2 mutant is controlled by a single recessive nuclear gene, and the initial positioning result shows that the positioning interval of sg2 spans the silk grain and cannot be further finely positioned. Further cloning was therefore performed using the method of Mutmap as follows:

firstly, screening 30 single plants with the same grain phenotype as the wild type from an F2 group of sg2/WT to respectively and equivalently mix DNA to form a wild type mixed pool, respectively and equivalently mixing 30 single plants with the same grain phenotype as the mutant to form a mutant mixed pool, then sequencing four samples of wild type Ningjing No.1, mutant sg2, the wild type mixed pool and the mutant mixed pool by using a HiSeq2500 sequencing platform, and then performing correlation analysis by using a Mutmap method to obtain a result as shown in figure 4.

(2) Obtaining of mutant Gene

As a result of primary localization and a result of Mutmap analysis, it was found that a deletion of 8 bases was present in the OsSG2 gene in the primary localization region (FIG. 5B)

Primers were designed based on the mapped sites and the sequences were as follows:

primer1：5'-GAGAATTCCACGCCGCTACG-3'(SEQ ID NO.4)

primer2：5'-GAAGAAATTTCTAGGAGGCA-3'(SEQ ID NO.5)

PCR amplification is carried out by taking primer1 and primer2 as primers and taking Ningjing No.1 cDNA as a template to obtain the target gene.

The amplification reaction was performed on a PTC-200(MJ Research Inc.) PCR instrument: 3min at 94 ℃; 30sec at 94 ℃, 45sec at 60 ℃, 3min at 72 ℃ and 35 cycles; 5min at 72 ℃. The PCR product was recovered and purified, cloned into a vector pEASY (Beijing Quanji Co., Ltd.), transformed into E.coli DH 5. alpha. competent cells (Beijing Tiangen Co., Ltd., CB101), and positive clones were selected and sequenced. The sequencing result shows that the fragment obtained by PCR reaction has the nucleotide sequence shown as SEQ ID NO.2 in the sequence table and encodes a protein (from ATG to TGA) consisting of 810 amino acid residues (see SEQ ID NO.1 in the sequence table). The protein shown in SEQ ID No.1 is named as OsSG2 (namely the OsSG2 gene in the gene mapping), and the coding gene of the protein shown in SEQ ID No.1 is named as OsSG2.

Example 2 obtaining and identifying transgenic plants

Construction of recombinant expression vector

Taking cDNA of Ningjing No.1 as a template, carrying out PCR amplification to obtain a coding sequence of the OsSG2 gene, wherein the PCR primer sequence is as follows:

primer3 (sequence HindIII restriction site underlined):

5'-CCGGCGCGCCAAGCTTATTAATCACTATGCCTACTTCATAGTATATTAT-3'(SEQ ID NO.6)

primer4 (underlined sequence is the BamHI site):

5'-GAATTCCCGGGGATCCTGTCTTAACACCCCTCACAGCATCATATTCCCA-3'(SEQ ID NO.7)

the above-mentioned primers are positioned in promoter region and coding region of gene shown by SEQ ID No.3, and the PCR product can be recovered and purified. The PCR product was cloned into the vector pCAMBIA1390 using the HD Cloning Kit recombination Kit (Takara Co.).

The primers are respectively positioned in the sequences shown in SEQ ID NO.3, the amplification product comprises all the coding region parts of the gene, and the PCR product is recovered and purified. Cloning the PCR product into a vector pCUBi1390 by using an Infusion recombination kit (Clontech) to construct pCUBi1390-OsSG 2; recombination reaction system (10.0 μ L): PCR product 5.4. mu.L (50-100ng), pCUBi1390 vector 1.6. mu.L (30-50ng), 5. mu.L of Infusion buffer, and 1. mu.L of Infusion enzyme mix. After brief centrifugation, the mixed system was subjected to water bath at 37 ℃ for 0.5 hour or more, and 2.5. mu.L of the reaction system was used to transform E.coli DH 5. alpha. competent cells (Beijing Tiangen Co.; CB101) by heat shock method. All the transformed cells were spread evenly on LB solid medium containing 50mg/L kanamycin.

After culturing at 37 ℃ for 16h, clone-positive clones were picked and sequenced. As a result of sequencing, a recombinant expression vector containing the gene shown in SEQ ID NO.3 was obtained, pCUBi1390 containing SG2 was named pCUBi1390-OsSG2, and the OsSG2 gene was inserted between the multiple cloning sites HindIII and BamHI.

According to the sequence of SEQ ID NO.3, the site-directed knockout is carried out by using CRISPR technology, and the primers are as follows:

Primer5：

5'-GGCAGCGGTACGAGGGGCTCCTCA-3'(SEQ ID NO.8)

Primer6：

5'-AAACTGAGGAGCCCCTCGTACCGC-3'(SEQ ID NO.9)

the two primers are fused into a PCAMBIA1305.1 vector, and the gene is subjected to site-directed knockout and is named as PCAMBIA1305.1-CRISPR-OsSG2.

II, obtaining recombinant agrobacterium

The pCAMBIA1390-OsSG2 and the PCAMBIA1305.1-CRISPR-OsSG2 are respectively transformed into Agrobacterium EHA105 strain (purchased from Invitrogen corporation of America) by a heat shock method to obtain recombinant strains, and plasmids are extracted for PCR and enzyme digestion identification. The recombinant strains which are identified correctly by PCR and enzyme digestion are respectively named as EH-pCAMBIA1390-OsSG2 and EH-PCAMBIA1305.1-CRISPR-OsSG 2.

Thirdly, obtaining of transgenic plants

The method for transforming the EH-p CAMBIA1390-OsSG2 into the rice mutant sg2 and transforming the EH-PCAMBIA1305.1-CRISPR-OsSG2 into Ningjing No.1 rice comprises the following steps:

(1) culturing EH-p CAMBIA1390-OsSG2 and EH-PCAMBIA1305.1-CRISPR-OsSG for 216 hours at 28 ℃, collecting thalli, diluting the thalli into N6 liquid culture medium (Sigma company, C1416) containing 100 mu mol/L until the concentration is OD600 approximately equal to 0.5, and obtaining a bacterial liquid;

(2) mixing the mutant sg2 and the mature embryo embryonic callus of Ningjing No.1 rice cultured for one month with the bacterial liquid obtained in the step (1) for infection for 30min, sucking the bacterial liquid by filter paper, transferring the bacterial liquid into a co-culture medium (N6 solid co-culture medium, Sigma company), and co-culturing for 3 days at 24 ℃;

(3) inoculating the callus of step (2) on N6 solid selection medium containing 100mg/L paromomycin (Phyto Technology Laboratories, Inc.) for the first selection (16 days);

(4) selecting healthy callus, transferring the healthy callus to an N6 solid screening culture medium containing 100mg/L paromomycin for secondary screening, and subculturing once every 15 days;

(5) selecting healthy callus, transferring the healthy callus to an N6 solid screening culture medium containing 50mg/L paromomycin for third screening, and subculturing once every 15 days;

(6) selecting the resistant callus to transfer to a differentiation culture medium for differentiation;

obtaining positive plants of T0 generation which are differentiated into seedlings. Mutant sg2 and Ningjing No.1 were used as negative controls.

Fourth, identification of transgenic plants

1. PCR molecular characterization

Extracting genome DNA from the T1 generation complementary positive plants obtained in the third step, and amplifying by using the genome DNA as a template and using primers Primer7 and Primer8 as a Primer pair (Primer 7: 5 '-GCTCCACCATGTTATCACATCAAT-3' (SEQ ID NO.10) and Primer 8: 5 '-CACAGAAGTGATACGAGTGC-3' (SEQ ID NO. 11)). Extracting genome DNA from the T1 generation site-directed knockout positive plant obtained in the third step, and amplifying by using the genome DNA as a template and the primers Primer9 and Primer10 as a Primer pair (Primer 9: 5 '-TTGAGAATTCCACGCCGCTA-3' (SEQ ID NO.12) and Primer 10: 5 '-CGGGGATGAGCACCGGCTCC-3' (SEQ ID NO. 13)). And (3) PCR reaction system: 2ul of DNA (20ng/ul), 2ul of Primer5(10pmol/ul), 2ul of Primer6(10pmol/ul), 2ul of 10xBuffer (MgCl2free)2ul, 0.4ul of dNTP (10mM), 1.2ul of MgCl2(25mM), 0.4ul of rTaq (5u/ul), 10ul of ddH2O 10ul, and 20ul of total volume. The amplification reaction was performed on a PTC-200(MJ Research Inc.) PCR instrument: 3min at 94 ℃; 30sec at 94 ℃, 45sec at 55 ℃, 1min at 72 ℃ and 35 cycles; 5min at 72 ℃.

The PCR product was purified and recovered by using a kit (Beijing Tiangen Co.). The PCR product was detected by electrophoresis in 1% agarose.

2. Phenotypic identification

Transgenic plants of T1 generation transformed PCUBi1390-OsSG2 and PCAMBIA1305.1-CRISPR-OsSG2, wild Ningjing No.1 and mutant sg2 are planted in the field of the soil bridge experimental base of Nanjing agriculture university. The transgenic complementation family is identified to recover the wild phenotype, and the transgenic site-directed knockout family is highly identical to the mutant phenotype. It was thus confirmed that the phenotype of the granule type abnormality was controlled by the OsSG2 gene, i.e., the OsSG2 gene was a granule type-associated gene (FIGS. 5-6).

Sequence listing

<110> Nanjing university of agriculture

<120> plant grain type associated protein, coding gene and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 810

<212> PRT

<213> Ningjing rice variety Ningjing No.1 (Ningjing 1)

<400> 1

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Ala Met Gly Gly Glu Glu Ala Ala Ala Arg Ala Ala Gln Lys Arg Tyr

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

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Trp Tyr Trp Ala His Leu Glu Pro Val Leu Ile Pro Ala Ala Asp Thr

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Gly Met Pro Pro Lys Ala Val Lys Leu Arg Cys Gly Leu Cys Ser Ala

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Val Phe Ser Ala Ser Asn Pro Ser Arg Thr Ala Ser Glu His Leu Lys

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Arg Gly Thr Cys Pro Asn Phe Ser Ala Pro Pro Pro Gly Ala Ala Ala

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Ala Ser Gly Ser Ser Gly Ser Gln His Gln Gln Gln Thr Pro Gln Ala

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Ala Leu Gln Ala Leu Pro Pro Pro Asn Ser Thr Ala Ser Ser Pro Ile

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Pro Ile Ser Ser Ile Ala Pro Ser Ser Pro Arg His Pro His His His

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Ser Gln Pro Gln Gln Pro Gln Ser His His His His His His His Ser

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Gly Ser Arg Lys Arg His Ser Met Pro Pro Ala Tyr Thr Ala Ala Glu

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Pro Val Ser His His His His Leu Val Val Val Asp Pro Ser Thr Val

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Tyr Ser Pro Pro Leu Pro Ala Leu Pro Pro Pro Pro Pro Gln Gln Pro

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Gln Ser Ala Leu Val Leu Ser Gly Gly Lys Glu Asp Leu Gly Ala Leu

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

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Pro Gly Ala Met Leu Pro Lys Pro Gln Ala Asp Ala Ala Leu Ala Leu

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Leu Ala Glu Trp Phe Leu Glu Ser Ser Gly Gly Val Ser Leu Ser Ala

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Val Ala Asn Pro Lys Leu Arg Ser Phe Leu Arg His Val Gly Leu Pro

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

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Ala Glu Ala Arg Ala Asp Ala Thr Ala Arg Val Arg Asp Ala Leu Phe

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Phe Gln Leu Ala Ala Asp Gly Trp Arg Glu Gln Val Val Thr Leu Cys

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Val Asn Leu Pro Asn Gly Thr Ser Val Phe His Arg Gly Val Pro Val

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Pro Ala Pro Ala Pro Ser Asp Tyr Ala Glu Glu Val Leu Leu Asp Ala

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Val Ala Ser Val Ser Ala Ser Gly Ser Ser Asn Asp Leu His His Cys

385 390 395 400

Ala Gly Ile Val Ala Asp Arg Phe Lys Ser Lys Ala Leu Arg Asp Leu

405 410 415

Glu Asn Lys His His Trp Met Val Asn Leu Ser Cys Gln Ile His Gly

420 425 430

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

435 440 445

Ser Ala Ala Ala Lys Ser Ala Lys Leu Ala Ala Tyr Phe Asn Ala Lys

450 455 460

Pro Thr Val Arg Ser Leu Leu His Lys His Gln Ile Gln Glu Leu Gly

465 470 475 480

His Ala Ser Leu Leu Arg Val Ala His Val Pro Phe Asn Ser Ser Gly

485 490 495

Ser Asp Tyr Arg Ala Ala Phe Glu Met Leu Glu Asp Val Leu Thr Ser

500 505 510

Ala Arg Pro Leu Gln Leu Ala Val Leu Glu Glu Ser Tyr Lys Leu Val

515 520 525

Cys Ile Asp Asp Ser Ala Ala Arg Glu Met Ala Asp Met Leu Gln Asp

530 535 540

Gly Ser Phe Trp Ser Glu Val Glu Ala Val His Leu Leu Val Lys Leu

545 550 555 560

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

565 570 575

Gln Cys Leu Pro Leu Trp Glu Asp Leu Arg Gly Lys Val Arg Asp Trp

580 585 590

Cys Asp Lys Phe Asn Thr Asp Glu Gly Ala Ala Leu Asn Val Val Glu

595 600 605

Lys Arg Phe Arg Lys Asn Tyr His Pro Ala Trp Ser Ala Ala Phe Ile

610 615 620

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

625 630 635 640

Pro Phe Lys Phe Leu Thr Pro Asp Gln Glu Lys Asp Val Asp Met Leu

645 650 655

Ile Thr Arg Met Val Ser Arg Glu Glu Ala His Ile Ala Val Met Glu

660 665 670

Leu Met Lys Trp Arg Thr Glu Gly Leu Asp Pro Leu Tyr Ala Gln Ala

675 680 685

Val Gln Val Arg Gln Pro Asp Pro Ser Thr Gly Lys Met Lys Val Ala

690 695 700

Asn Lys Gln Ser Ser Arg Leu Val Trp Glu Thr Cys Leu Ser Glu Leu

705 710 715 720

Lys Ser Leu Gly Lys Val Ala Val Arg Leu Ile Phe Leu His Ala Thr

725 730 735

Ala Arg Gly Phe Arg Cys Ser Pro Ser Met Leu Arg Trp Leu Ser Ala

740 745 750

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

755 760 765

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

770 775 780

Asp Lys Asp Ala Glu Leu Leu Thr Glu Gly Asp Asp Asp Val Leu Asn

785 790 795 800

Glu Pro Gly Ser Leu Glu Arg Ser Ser Val

805 810

<210> 2

<211> 2433

<212> DNA

<213> Ningjing rice variety Ningjing No.1 (Ningjing 1)

<400> 2

atgtcgacgg ccgccaagga ggaggcggca gcgtcggctc cggcgccggc aatgggcggc 60

gaggaggcgg cggcgcgggc ggcgcagaag cggtacgagg ggctcctcac ggtgcgggcc 120

aaggcggtca agggcaaggg cgcctggtac tgggcgcacc tggagccggt gctcatcccc 180

gccgccgaca ccgggatgcc gcccaaggcc gtcaagctgc gctgcggcct ctgctccgcc 240

gtcttctccg cctccaaccc atcgcgcacg gcttcggagc acctcaagcg gggcacctgc 300

cccaacttct ccgcgccacc gccgggcgcc gccgcggcat ccggttcgtc cggttcgcag 360

catcagcagc agacgccaca ggcggcgctg caggcgttgc cgccgcccaa ctcgacggca 420

tcgtccccta tcccaatctc gtccattgcg ccctcgtcgc cgcgccaccc gcaccaccac 480

tcgcagcccc aacagccgca gtcgcatcac caccatcacc atcactccgg cagccggaag 540

cgccactcga tgcctccggc gtacacggcc gcggagcccg tgtcccacca ccaccatctc 600

gtcgtcgtcg acccgtcgac ggtttactcc cctccgctgc ccgccctgcc gccgccacca 660

ccgcaacaac cgcagtcggc gctcgtgctc tccggcggca aggaggatct cggcgcgttg 720

gccatgctgg aggacagcgt gaagcggctc aagtcgccca aggcgtcgcc gggggcgatg 780

ctgccgaagc cgcaggccga cgcggcgctc gccctgctcg ccgaatggtt tctcgagtcg 840

tcgggcggcg tgtcgctgtc ggccgtggcg aacccgaaac tccggtcctt cctccgccac 900

gtcggcctgc cggagctgca gcggacagac ctggcggggg ctcggctgga tgcgcggttc 960

gccgaggccc gcgccgacgc caccgcgcgc gtccgcgacg cgctcttttt ccagctcgcg 1020

gcggacgggt ggcgggagca ggtggtaact ctgtgtgtca acctcccaaa tggaacatcc 1080

gtgttccacc gcggcgtgcc ggtccccgcc ccggcgccct cggactacgc cgaggaggtg 1140

ttgctggacg cggtggcgtc cgtgtccgcg tctggctcct ccaacgacct gcaccattgc 1200

gcgggcatcg tggctgaccg cttcaagtca aaggcccttc gtgatctgga gaacaagcac 1260

cattggatgg tgaacctgtc ctgccaaatc catggcttca cccggctggt tcgggacttc 1320

gcgcgggagc tccctctgtt ccgctccgct gcggctaaat ccgccaagct ggccgcctac 1380

ttcaatgcca agccgacggt gcggtccctg ctgcacaagc accaaatcca ggagctcggg 1440

catgcgtcgc ttctccgtgt ggcgcatgtg ccattcaata gcagcggcag cgactaccgt 1500

gcggccttcg agatgctgga ggacgtgttg acctctgctc gcccgctcca gctcgccgtc 1560

ttggaagagt cctataagct ggtctgcatc gacgattcgg ccgccaggga gatggcggac 1620

atgttgcagg atgggtcatt ctggagtgag gttgaggccg tgcatctgct cgtgaagctg 1680

atcatggaca tggtgaaaga gatggagact gacaggcctc tggttggcca atgcctgcct 1740

ctctgggagg acctacgtgg caaggttagg gattggtgcg ataaattcaa cactgatgag 1800

ggcgctgccc tgaatgtggt ggagaaaagg ttcaggaaga actaccatcc agcctggtca 1860

gcggctttca tattggatcc tctctaccta atcaaggatg ccagtgggcg ctacctcccg 1920

cccttcaagt tcttgactcc tgatcaagag aaggatgtgg acatgctcat caccaggatg 1980

gtgtcgcggg aggaggcgca cattgcggtc atggagctca tgaaatggcg gacggaaggg 2040

ctggacccgt tgtatgcgca ggcggtgcag gtccggcagc ctgacccgtc caccgggaag 2100

atgaaggtcg caaacaagca gagcagccgc cttgtctggg agacgtgcct gagcgagctc 2160

aagtccctcg ggaaggtggc tgtacggctc atcttcctcc atgccactgc tagggggttc 2220

aggtgctcac cctccatgct gcgttggctc tctgctcctg gcagtttagc cggtggcatt 2280

gatcgtgcgc accggctagt gttcgttgct gcaaattcca agctagagag gagggatttc 2340

tcaagtgatg aagacaagga tgctgagtta ctcactgaag gggacgatga tgtgctaaac 2400

gagcctggca gtttggagcg ctcctcagtg taa 2433

<210> 3

<211> 8494

<212> DNA

<213> Ningjing rice variety Ningjing No.1 (Ningjing 1)

<400> 3

attaatcact atgcctactt catagtatat tattgttcat tgccttccgg atgtcttttc 60

aacaccacat gtttactgag gcttctaatt tctattgtgg tgcatacttt aaatgtataa 120

tttgtgaaaa ttgacatatt cttttggcaa ggagtttgta ccacgtgcat ttctaagtac 180

aatattcaat cactaatttt agagtaaatt tcataaaact acaactattt agacaaaact 240

atattttgct gtaacttcat atgaacattt ctaaaggctg caactatatt gcatgcgagc 300

gattttataa aaaataaaac tacaacttaa aagtgatatg agtgtttgac atgtggttct 360

ttgacaaagt tgcatttttc tcataaattg tcaactatgg ttgtatgacc aatcaaagtt 420

gtagcaaatt aattgttttg acatatagtt ttctgaaatt atcataaatc taaattatca 480

tatagttttc tgaaattact tatttggttt gtagcatatt tgttcacaca cacacacaca 540

catatatcta tatatattcg aaagatctta agttttatta ggccagccac cgagttataa 600

tgtttattgt cctatattag ttcttgaagc acaatcaaca acttcagaag gtctacatat 660

atagccagat tggaaagctc atcggtgtat tcactattgg tactcattca ctattggtac 720

tgtcagcacc atttgttgtg gtctgtaaat ccatgttcat gagaataccg tgtctaagca 780

ctcgtatcac ttctgtgata tgagtgataa atttttcaaa cacagtacag ttttgcacta 840

tttatgtcca acgtttcacc gttcgtttta tttgaaaact ttttcaaata agatggacgg 900

tcaaacatta gacacggatt ttcacagctg tacttatttt tagaacggag gtattaacat 960

ttcttttatc tagcgcatat attttaatag accaggcaag aaaaggaaaa atgataggta 1020

gagtgtcgat agactgtata gtacgtacag ttgccaacag tccccgtttc ttcagttata 1080

gacttttata tggctactgg ttacaatatc tttatctgac gtccaactct tttcatatta 1140

ctacagtgga ggaaaaacag aaaaaaaaag gagaactatt ttttttaatc taacctgcct 1200

aatccaaaat atatccaaac cttattaatt aattacagct gtgatggtat atacatacac 1260

acacaattat tttcttgtgc atgctaatct actatataca aatggctctg tttgagtaca 1320

aatgcaaata attgtggttg aatggactta attttttggt cctgtgtgtt atccagcgga 1380

gttacgttac agctataccg cattgatgaa aattaatgtt ttacatcaac cccgctatag 1440

aaaagtctct ctctccggaa tgatattgat atatatggtg ctgcatgcat gcatgttctt 1500

gcgcaatata ttacggtgat ttttattctt ctctgatcag tgatcactcc atacattgcg 1560

gggtactagt aattaatcaa ccgtctggct gttgcagcta gcaacgcatg caaatcttac 1620

tgtgtgtttt gtgatgctca aatctaactg ttacagtcgt acttaccgtt gcgccagaca 1680

agcacaagcc acagaaaagg tcatacgcag tacagtacta tatacagtaa ccgttccatg 1740

catgatgcta attatctgca tgtactttta cttggctaat gactccagat taatttgtgt 1800

ataccacatg catgtatacc agcgtacgta ttttcctgca gtatatacta ctactagtag 1860

ttaggtacgt acgtacgtat gtatagccac tagccagatt ggaaagctag ctgcagcctg 1920

caggtgtagc gtatgagagc atgtgaaatt cctaaataat tgagggtaca cagccttttg 1980

ctgtcctaga aacagtcgag aaattcaacc aaacttgcca gctttttcaa acgcttattt 2040

atcataatgt ggatgggtta gctctagctt gcttgcttac tcgctttctc cccagtggag 2100

gagggaagta ttgtacgccg cccttaattg tctgtctagc ttgcctgcaa atactcacta 2160

ctgctacgtg tataatgcgt gcagttcata cgctgtccgg cgcctcacca aaactggtac 2220

cccaccccac ccttctatat atgtttagcc acgcacgcca ttgacaacta ctacgtcctt 2280

tttttcccca tatagacgcc acagcaaaat actcaatgct actaagtata cgcgattgat 2340

aactattagt atgtttggta aaatgccact gtcacacggt taataatgac gatgaatttg 2400

gcttgatgct aaaaaatgcc ctagatttta ctgactttgt cagagagatt tgtagctttt 2460

cagtcgtaac catttgtttt tatgactttt gaattaactg atcatctgga cttgagattt 2520

tctttctcca cccacaggaa aaaaatgaat aattctatgt taaataagcg atgtgctgtt 2580

actattgcgg aggtacacgt atcatgggag caggtcaggt agccgaaatg gaggttttgt 2640

aaggtgttta tacggcagcg gcgcggcggc aaatttgcgc tgccgccggc cggggggaac 2700

gaaaagggag cgagaggtag gaaaggctaa acaaggggga gggcgtgggc tctcacgcca 2760

cggcctctag ggccaccaac tattccaccg caataattgt gggagagagg agaggagaag 2820

agaggtgtgt ggtggtggtc atgcatagta gccgaacaca atacacttgt acaatcctcc 2880

cccttatttt acatataggc ctttgtacca taaagaatag actaggcatt ataccgagag 2940

gcattcctct tgtgggagtg ggcagtcaac cctgccccaa agaccagacc aaaaaaaatt 3000

aaagaaaaga aaaagggtgt agagaagtag tctggataga gagagagatg gtggctctca 3060

ggctctcaag cttgctcctc ctcttcttct tccataaatt cctaggttgc ccctctcctg 3120

ctttctcttc ttcttcaatc tccatcccca tcatctatct atccatcatc catcagacca 3180

gacccttcct cctctcccct gcctaccggc agcagcaccc agcagtaagc ccccaagcag 3240

taagctatag ctgcctgtgt gaggccccaa aatctctcta ctctcccttt ctccatcccc 3300

acacacattc ctccgatatc tagtatatct ccctactaca ccatcatcca ggctacatta 3360

gtttggtctc tttttttttt cttttataag ttttgggttt ttcctcacca ccaccacaag 3420

cgaggagttg cacaaccatc caaagggagg ggagtgcaag aattgtgagc tgccacaagt 3480

gatatttttc caattttgtt tttgggcaag tgtagtgcac tcactcacac actaggcagg 3540

ggcaggggca gggcggccta taaataaaaa gaaaaaaaaa gagagagaag atatattact 3600

agtagtattt gctattggaa gaagccaaag gcaggggagg aggaagagtc ttcttctgct 3660

gctctctctc ttctcttctc aattcatcct ccttttgtcc ctatcaaacc ctttttttct 3720

gtctctctct ttctaccttg gcattgtttt ctcgcaaatc ttgctttcta aacccccaaa 3780

acctcccgga aaacaaccca cccaccccgc gcgtggatgg acggcccgcg gccgccgccg 3840

ccgcccaatg cgctgcgccg atcgcggttg tttccgacga ccacgccgcc gccgtccgcc 3900

accgggggtc tccggcatta gaacccgggc agttgagaat tccacgccgc tacggcggga 3960

ttggattagc gggcgggcgg cggtcaggga agaggaggtt ctctcttgtg gtcgttgccg 4020

ctggaggtga tcgatcgatc ggtcggtcgt ctcgcggggc gatcggtcgg catgtcgacg 4080

gccgccaagg aggaggcggc agcgtcggct ccggcgccgg caatgggcgg cgaggaggcg 4140

gcggcgcggg cggcgcagaa gcggtacgag gggctcctca cggtgcgggc caaggcggtc 4200

aagggcaagg gcgcctggta ctgggcgcac ctggagccgg tgctcatccc cgccgccgac 4260

accgggatgc cgcccaaggc cgtcaagctg cgctgcggcc tctgctccgc cgtcttctcc 4320

gcctccaacc catcgcgcac ggcttcggag cacctcaagc ggggcacctg ccccaacttc 4380

tccgcgccac cgccgggcgc cgccgcggca tccggttcgt ccggttcgca gcatcagcag 4440

cagacgccac aggcggcgct gcaggcgttg ccgccgccca actcgacggc atcgtcccct 4500

atcccaatct cgtccattgc gccctcgtcg ccgcgccacc cgcaccacca ctcgcagccc 4560

caacagccgc agtcgcatca ccaccatcac catcactccg gcagccggaa gcgccactcg 4620

atgcctccgg cgtacacggc cgcggagccc gtgtcccacc accaccatct cgtcgtcgtc 4680

gacccgtcga cggtttactc ccctccgctg cccgccctgc cgccgccacc accgcaacaa 4740

ccgcagtcgg cgctcgtgct ctccggcggc aaggaggatc tcggcgcgtt ggccatgctg 4800

gaggacagcg tgaagcggct caagtcgccc aaggcgtcgc cgggggcgat gctgccgaag 4860

ccgcaggccg acgcggcgct cgccctgctc gccgaatggt ttctcgagtc gtcgggcggc 4920

gtgtcgctgt cggccgtggc gaacccgaaa ctccggtcct tcctccgcca cgtcggcctg 4980

ccggagctgc agcggacaga cctggcgggg gctcggctgg atgcgcggtt cgccgaggcc 5040

cgcgccgacg ccaccgcgcg cgtccgcgac gcgctctttt tccagctcgc ggcggacggg 5100

tggcgggagc aggtggtaac tctgtgtgtc aacctcccaa atggaacatc cgtgttccac 5160

cgcggcgtgc cggtccccgc cccggcgccc tcggactacg ccgaggaggt gttgctggac 5220

gcggtggcgt ccgtgtccgc gtctggctcc tccaacgacc tgcaccattg cgcgggcatc 5280

gtggctgacc gcttcaagtc aaaggccctt cgtgatctgg agaacaagca ccattggatg 5340

gtgaacctgt cctgccaaat ccatggcttc acccggctgg ttcgggactt cgcgcgggag 5400

ctccctctgt tccgctccgc tgcggctaaa tccgccaagc tggccgccta cttcaatgcc 5460

aagccgacgg tgcggtccct gctgcacaag caccaaatcc aggagctcgg gcatgcgtcg 5520

cttctccgtg tggcgcatgt gccattcaat agcagcggca gcgactaccg tgcggccttc 5580

gagatgctgg aggacgtgtt gacctctgct cgcccgctcc agctcgccgt cttggaagag 5640

tcctataagc tggtctgcat cgacgattcg gccgccaggg agatggcgga catgttgcag 5700

gatgggtcat tctggagtga ggttgaggcc gtgcatctgc tcgtgaagct gatcatggac 5760

atggtgaaag agatggagac tgacaggcct ctggttggcc aatgcctgcc tctctgggag 5820

gacctacgtg gcaaggttag ggattggtgc gataaattca acactgatga gggcgctgcc 5880

ctgaatgtgg tggagaaaag gttcaggaag aactaccatc cagcctggtc agcggctttc 5940

atattggatc ctctctacct aatcaaggat gccagtgggc gctacctccc gcccttcaag 6000

ttcttgactc ctgatcaaga gaaggatgtg gacatgctca tcaccaggat ggtgtcgcgg 6060

gaggaggcgc acattgcggt catggagctc atgaaatggc ggacggaagg gctggacccg 6120

ttgtatgcgc aggcggtgca ggtccggcag cctgacccgt ccaccgggaa gatgaaggtc 6180

gcaaacaagc agagcagccg ccttgtctgg gagacgtgcc tgagcgagct caagtccctc 6240

gggaaggtgg ctgtacggct catcttcctc catgccactg ctagggggtt caggtgctca 6300

ccctccatgc tgcgttggct ctctgctcct ggcagtttag ccggtggcat tgatcgtgcg 6360

caccggctag tgttcgttgc tgcaaattcc aagctagaga ggagggattt ctcaagtgat 6420

gaagacaagg atgctgagtt actcactgaa ggggacgatg atgtgctaaa cgagcctggc 6480

agtttggagc gctcctcagt gtaacaccct tatttaattg ttcgtttctt tttctctatt 6540

agcctttttt agaagttaat ccctaggaat ttgctagaat ttccaccccc tacctatttt 6600

tttgcctcct agaaatttct tcctcgtgcc catgctgtta attcttactt cctcactcta 6660

gattttggtg gggtggggag tggggcatga gatggagata tgcatccttt gttcatcaag 6720

tttggttaag aatgactgtc tcttgccata tatgattcca tggggccatt gaacttgcag 6780

agattaatgg aagtctttct gtcaatatgt tcatagtcgg tggtacaccg aaagcgggaa 6840

gaatgtgtaa ccattttttt ttgtgaaaat taataatatt gatatgcaat tgaatgcaat 6900

ctgactgccg tctatatatc tattattgct ttcttcaaat tgcactgtta cttcttttac 6960

attgttcttg aacgtgtggt gctctgttcc tagttaatac taccatacat cactccactc 7020

aacagtccac actgagcagg cttcgaacaa agagtaataa catcccttta ttgttttgac 7080

cgtgattaac tcaacaccag gaactcttgc tactctgaaa gagaaatgct caccggatgc 7140

taattaataa tatccttgtg tggtttcttc taacgcgcct ggccttgttt ctggaggcat 7200

tgtgcatcat tggagcttgg cagcttctgt gatcctgtca tgactcatga aggcgaggct 7260

cactttgcaa cagcatcagc caagaaggta catatctcat cttctaggat gttctttcgg 7320

ttctactcca tggcatttgg cttttcagct ggacctgcta tgctggaagt ttaaacagta 7380

gcagcagcag aagcagctcc agctcggttt attacgaggt gagcggtgcc aactttcaca 7440

ttttgccttt tctcagtgtt actcgtagca tttactgttt ttctccagag taggcacaac 7500

ggcaatgccg caatgggttt ttctgcattg tgtgatcatg tgagattatc atgagcacgg 7560

tcgttgtcgt cgtcgtcatc ttcttcttcg tgctccttgc cttggtcgtc gcccaaccgg 7620

gcgatgattg cgccggttcg catggcttca tctcgacggt atcggaaagg ctccggtccg 7680

gagagggatg aaaagccgga gttgaaggtg cagacacggt aggctttttg ctcctccccc 7740

atccccatgc ccatcaccac ggccagggcc tttgacaatc atgccttgca tcaaaacatg 7800

tccttttagt ttgcaggggg tacaagcttg ccaaggcagt gatcgattcc ctttcctatc 7860

attgggatgg gcacctcggt tgctgcctac cggagagctc gccgtgcttt cgatccaatc 7920

tgccatgaga gctcctctcc ttgcccttct ggtcttatgg tttgatggag aaaagagcgt 7980

ttgattggag cggcagggat gtgttcttgg acaggagaag aggaaagaaa agcctgtgta 8040

atacaggctg ttcctgcatc cgtaccaatt ggggtttttc ttttttcgct cggcttctct 8100

gttgctcctg gctcctgctg ctctctcctc ccctcagctg tccaagcaca tgctttacct 8160

gactaatcat taccatttga gctttattag gcgtcgtcgc agagaacgga ggagatgatt 8220

attggacata atgcagtcag tattcagcaa gtgatggtga gtatcgttgg tgctggcaca 8280

tctccttgtt tttccttaat tttcctcctg tgtcagtact gctggggctg tgtagttgtt 8340

gctaaagtag tagtagcagc agcagcagta caaactattg ctggaatggg tgtattctgc 8400

tttttctcag tcgcttttgc aacttgatgt actatgtttt gtacacatgt tgtgtagtag 8460

ttgggaatat gatgctgtga ggggtgttaa gaca 8494

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gagaattcca cgccgctacg 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaagaaattt ctaggaggca 20

<210> 6

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccggcgcgcc aagcttatta atcactatgc ctacttcata gtatattat 49

<210> 7

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gaattcccgg ggatcctgtc ttaacacccc tcacagcatc atattccca 49

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggcagcggta cgaggggctc ctca 24

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aaactgagga gcccctcgta ccgc 24

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gctccaccat gttatcacat caat 24

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cacagaagtg atacgagtgc 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttgagaattc cacgccgcta 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cggggatgag caccggctcc 20

Claims (4)

1. The gene shown in SEQ ID NO.3, a recombinant expression vector containing the gene shown in SEQ ID NO.3, an expression cassette or a recombinant bacterium are applied to rice breeding with changed grain size.
2. 2. The use according to claim 1, wherein the gene shown in SEQ ID No.3 is introduced into rice with abnormal grain growth to breed normal rice; the gene is knocked out from normal rice with a reduced grain type to obtain rice with a reduced grain type.
3. 3, the gene shown in SEQ ID NO.3, a recombinant expression vector containing the gene shown in SEQ ID NO.3, an expression cassette or a recombinant bacterium are applied to culturing transgenic rice with normal grain size; the transgenic rice with normal grain type is the transgenic rice with normal grain and thousand grain weight.
4. 4. A method for cultivating transgenic rice with normal grain type growth and development is to introduce a gene shown in SEQ ID NO.3 into grain type abnormal rice to obtain the transgenic rice with normal grain type development; the rice with abnormal grain type development is rice with reduced grain length and thousand seed weight; the transgenic rice with normal grain type is the transgenic rice with normal grain and thousand grain weight.

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