CN114921488B - Composite grain rice gene CS and application thereof - Google Patents

Abstract

The invention provides a compound rice gene BRD3 and application thereof. The invention clones the BRD3 gene of the compound grain rice for the first time, the gene codes a BR degrading enzyme CYP734A4, the BRD3 promoter in the compound grain rice comprises 34 AT copies inserted relative to a reference genome, the activity of the promoter can be enhanced, and the BRD3 gene in the compound grain rice is specifically expressed and upregulated in the pedicel and secondary branch meristem, thereby leading to the phenotype of the compound grain rice with shortened pedicel and increased spike grain number, but not changing the grain size and plant height of the rice, and obviously increasing the yield by about 10 percent relative to the non-compound grain rice under the same background, and providing a powerful means for high-yield breeding of the rice.

Description

Composite grain rice gene CS and application thereof

Technical Field

The invention relates to the field of biotechnology and plant genetic breeding, in particular to a compound grain rice gene CS and application thereof.

Background

Multiple grain rice is a unique rice germplasm resource and is characterized in that the pedicel is shortened so that multiple grains are clustered on one branch. Most of the existing compound rice mainly comprises three clusters. It should be noted that the multiple rice is completely different from the reported three-flower mutant with developmental defects, which is caused by transformation of glume into palea, resulting in incomplete flower organs formed on both sides of the middle intact seed, and unable to develop into normal seed, whereas the multiple rice has completely normal flower organs and seed development. Because of the grain density caused by clustering, part of the multi-grain rice appears in the field as ears like wheat, and is therefore also called glume rice. This unique trait has attracted many scientists to strive to clone the corresponding control gene over the last forty years. By map-based cloning, almost all studies mapped candidate genes to the same region of chromosome 6, but most did not clone to candidate genes. At present, only one example of a reported clone is named SPED1, and the inclusion of two point mutations is found to be probably responsible for clustering. Surprisingly, another recent study using P164 in oryza sativa as a material also mapped the gene of interest to this region, but sequencing found that it did not contain the above-described mutation site of SPED1, suggesting that this allele may only play a role in some specific oryza sativa. Considering that different studies utilize different materials, but the target genes are all located in the same interval, the true genetic mechanism of the cluster of the multi-grain rice is still an undeveloped puzzle.

Brassinosteroids (BR) represent a class of sterols hormones and play an important role in the aspect of plant growth and development. In rice, BR significantly regulates a plurality of important agronomic traits, such as plant height, leaf angle, grain size and the like, and therefore has important application potential. BR deletion mutants often exhibit dwarf, erect leaves, small grains, etc. phenotypes, many of which are negatively associated from a breeding perspective, making genetic improvement of rice using BR genes difficult. Solving this problem has important significance for BR utilization. Interestingly, some studies found that BR synthetic mutant d11 sometimes resulted in clumping of the grain in addition to grain size reduction, suggesting that BR may have a role in regulating the formation of multiple grain rice.

In addition to the shortened pedicel, multiple grain rice may be accompanied by an increase in spike grain number, although this yield-increasing potential has attracted many breeders to try to introduce this trait to increase rice yield, no scientific analysis has been reported at present. The number of grains per ear is an important factor for determining the yield of rice, and Japanese scientists cloned the major quantitative trait locus gene Gn1a for controlling the number of grains per ear, encoding CKX2 cytokinin oxidase, which is widely present in rice varieties, for example, in the southwest heavy spike variety Shuhui R498, which contains one allele of Gn1a, resulting in an increase in the number of grains per ear and thus high yield. Therefore, the spike number gene has great application value in rice high-yield breeding.

The compound grain rice is used as a special germplasm resource, which may have great application value in rice yield increase, but because the true genetic mechanism of cluster generation is still an undeveloped puzzle, whether the number of spike grains can be increased is not clear. Whether the rice gene can be cloned successfully and the yield of the compound rice is increased is a main problem to be solved in the current compound rice utilization.

Disclosure of Invention

The invention aims to provide a compound rice gene CS and application thereof.

In order to achieve the purpose of the invention, in a first aspect, the invention provides a method for promoting rice spike grain number and cluster phenotype character (without affecting other phenotypes), enhancing rice gene CS and obtaining a rice plant with up-regulated gene CS expression.

The means for up-regulating expression is selected from the following 1) to 5), or an optional combination:

1) By introducing a plasmid having the gene;

2) By increasing the copy number of the gene on the plant chromosome;

3) By altering the promoter sequence of said gene on the plant chromosome;

4) By operably linking a strong promoter to the gene;

5) By introducing enhancers.

In the present invention, the rice gene CS is a gene encoding the following protein (a) or (b):

(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1;

(b) And (b) a protein which is derived from (a) and has equivalent functions and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1.

The CDS sequence of the rice gene CS is shown as SEQ ID NO.2, and the genome sequence is shown as SEQ ID NO. 3.

In a second aspect, the present invention provides a rice-plural-grain gene CS (derived from rice-plural-grain germplasm CS 1), the protein encoded by the rice-plural-grain gene CS being the same as the protein encoded by the rice gene CS; the promoter sequence is as follows:

a) A nucleotide sequence shown as SEQ ID NO. 4;

b) The nucleotide sequence shown in SEQ ID NO. 4 is a nucleotide sequence with the same promoter activity by substituting, deleting and/or adding one or more nucleotides;

c) A nucleotide sequence which hybridizes to the sequence shown in SEQ ID No. 4 and expresses the same functional protein under stringent conditions, i.e., in a 0.1 XSSPE solution containing 0.1% SDS or in a 0.1 XSSC solution containing 0.1% SDS, at 65℃and washing the membrane with the solution; or (b)

d) Nucleotide sequence having more than 90% homology with the nucleotide sequence of a), b) or c) and having the same promoter activity.

In a third aspect, the present invention provides a biological material comprising the multiple grain rice gene CS, including but not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineering bacteria.

In a fourth aspect, the invention provides the use of the multiple grain rice gene CS or a biological material containing the gene in promoting rice grain number and cluster phenotype (without affecting other phenotypes) traits.

In a fifth aspect, the present invention provides the use of the multiple rice gene CS or a biological material comprising the gene in the preparation of transgenic plants.

In a sixth aspect, the present invention provides the use of the multiple rice gene CS or a biological material containing the gene in plant breeding.

In a seventh aspect, the invention provides a method of promoting rice grain number and cluster phenotype (without affecting other phenotypes) traits comprising:

1) Allowing rice to contain the compound rice gene CS; or (b)

2) And (3) enabling the rice to overexpress the compound rice gene CS.

Such methods include, but are not limited to, transgenesis, crosses, backcrosses, selfing, or asexual propagation.

In an eighth aspect, the invention provides a CRISPR/Cas9 system targeting the multiple grain rice gene CS, having an sgRNA sequence of 5'-GTTCCTCGTCGGGTGCGTGAGGG-3'.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the CS gene of the compound rice codes a BR degrading enzyme CYP734A4, the CS promoter in the compound rice comprises 34 AT copies inserted relative to a Japanese reference genome, the activity of the promoter can be enhanced, and the CS gene in the compound rice is specifically and upwardly regulated in the pedicel and secondary branch meristems, thereby leading to the phenotype of the compound rice with shortened pedicel and increased spike number, but not changing the size and plant height of rice seeds, and obviously increasing the yield by about 10 percent relative to non-compound rice under the same background, and providing a powerful means for high-yield breeding of rice.

Drawings

FIG. 1 shows the phenotype and statistics of multiple grain rice cluster in a preferred embodiment of the invention. Wherein A is a phenotypic observation; b is a plant height statistical result, and the ordinate is plant height (cm); c is the spike count statistical result, and the ordinate is the spike count; d is the spike length statistical result, and the ordinate is the spike length (cm); e is the first-level branch number statistical result, and the ordinate is the first-level branch number; f is the statistical result of the secondary branch number, and the ordinate is the secondary branch number; g is the spike number statistical result, and the ordinate is the spike number; h is a mu yield statistical result, and the ordinate is mu yield (kg); i is grain length statistics result, and the ordinate is grain length (mm); j is the grain width statistics and the ordinate is the grain width (mm).

FIG. 2 shows that the clustering gene is not fully dominant in the preferred embodiment of the invention. Wherein NCS is a non-cluster; WCS is a weak cluster: CS is tufting.

FIG. 3 shows the disappearance of the cluster phenotype of revertants in a preferred embodiment of the invention.

FIG. 4 is a schematic representation of BRD3 regulated multiple grain rice clustering phenotype in accordance with a preferred embodiment of the present invention.

FIG. 5 shows that BRD3 expression levels in CS1 young ears are significantly up-regulated in the preferred embodiment of the invention.

FIG. 6 shows that AT tandem repeat insertion significantly enhances the promoter in the preferred embodiment of the present invention.

FIG. 7 shows that BRD3 is specifically expressed and up-regulated in secondary branch meristems and pedicles of oryza sativa in a preferred embodiment of the invention.

Detailed Description

The invention clones the compound grain rice gene CS for the first time, reveals the genetic variation of the gene, deeply clarifies the action mechanism of the gene, discovers that the gene can obviously promote the grain number of rice ears and utilizes the gene to increase the yield.

The invention adopts the following technical scheme:

the phenotype observation analysis is carried out on the compound rice, and the compound rice is proved to promote the development of secondary branches so as to promote the increase of the spike grain number, but the grain size and the plant height of the rice are not changed, and the yield can be obviously increased by about 10 percent in the field district.

The invention screens phenotype revertants of two alleles by carrying out large-scale sodium azide mutagenesis on the compound rice, finally clones the compound rice gene CS by constructing a backcross segregation population and sequencing by an extreme material mixed pool, encodes a BR degrading enzyme and confirms the gene editing knockout.

Sequencing analysis finds that the CS promoter in the multi-grain rice contains 34 AT copies inserted relative to a reference genome, so that promoters with different AT numbers are constructed, the activity of the promoters is gradually enhanced along with the increase of AT copies, and in-situ hybridization combined with quantitative gene expression analysis finds that the insertion of a plurality of ATs leads to the up-regulation of gene specific expression in pedicel and secondary branch meristems, thereby leading to the phenotype of the multi-grain rice with shortened pedicel and increased spike number.

The invention overcomes the problem of difficult cloning of the compound grain rice gene, identifies an excellent BR gene allele for promoting the grain number of the spike, and the BR degradation gene is only up-regulated in the pedicel and secondary branch meristem by inserting a plurality of AT copies, thereby optimizing the spatial distribution of BR, specifically promoting the grain number of the spike and simultaneously avoiding the possible negative effect caused by BR reduction.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

Example 1 phenotype and genetic analysis of multiple grain Rice

1. Construction of near isogenic lines

Wild type 9311 is used as a donor parent, a compound rice germplasm CS1, namely rice (Oryza sativa) CS1 is preserved in China general microbiological culture Collection center, north Chen Xway No. 1 and No. 3 of the Korean area of Beijing, china academy of sciences microbiological study, post code 100101, preservation number CGMCC No.24373, and preservation date 2022, 3 months and 1 day) is used as a recurrent parent for hybridization, after 5 generations of back cross, selfing is carried out, single plants with plant phenotypes similar to CS1 but not clustered are selected in a selfing segregation population, and the selfing is continued for at least 3 generations (BC) ₅ F ₃ ) Thereafter, a near isogenic NCS1 was obtained which did not cluster.

2. Phenotype observation and statistics of compound rice

And (3) measuring plants: near isogenic line NCS1, composite grain rice CS1.

The plant height, the spike number, the spike length, the first-stage branch number, the second-stage branch number, the spike grain number, the mu yield, the grain length and the grain width of the plant to be tested are counted, and the result is shown in figure 1. The result shows that compared with NCS1, the end of the ear branch of the compound grain rice CS1 presents a cluster generation phenotype that 3 grains are gathered together, the number of the first branch is not obviously different, the number of the ear branch is obviously increased due to the increase of the number of the second branch, but the plant height, the ear number, the ear length, the grain width and other important agronomic characters are not influenced, and the yield can be increased by about 10 percent in the field district.

3. Genetic analysis of multiple grain rice

Hybridization of composite grain rice CS1 with wild ZH11 to obtain F ₁ Plants of the generation F ₁ F is obtained after the selfing of the generation plants ₂ Generation of segregating population, pair F ₂ Counting the plant numbers of different cluster growth traits of the generation separation population to obtain F ₂ The separation ratio was substituted and the results are shown in FIG. 2.

The results indicate that hybridization F ₁ The generation exhibits a weak cluster phenotype with only 2 clusters clustered at the end of the shoot, F ₂ Three phenotypes were isolated by generation selfing, and the isolation ratio of the three phenotypes was no cluster: weak clusters: tufting = 65:131:45, approaching 1:2:1 (χ) ² _0.05(2) = 0.0951664 < 5.99), indicating that the clustering phenotype is regulated by an incompletely dominant gene.

Example 2 cloning and validation of the multiple Rice Gene CS

1. Sodium azide mutagenesis multiplex rice germplasm CS1

KH with concentration of 1M is respectively arranged ₂ PO ₄ And H of 1M ₃ PO ₄ Solution, KH per 1000ml ₂ PO ₄ Solution and 120ml H ₃ PO ₄ The solution is mixed evenly, the pH value is regulated to 3.0 by 1M NaOH solution, thus obtaining phosphate buffer solution, and the sodium azide solution with the concentration of 1mM is prepared by the phosphate buffer solution. 500g of CS1 dry seeds are weighed and soaked in sodium azide solution, the seeds are washed clean by clean water after being soaked in a fume hood for 6 hours at normal temperature, the seeds are soaked in tap water, the seeds are placed in a 37 ℃ incubator for germination acceleration, water is changed every 8 hours until the seeds germinate, and then field sowing is carried out.

2. Revertant screening

Sodium azide mutagenesis M at CS1 ₀ The mature period of the generation was phenotypically identified for each individual in the field, and revertants with the loss of the clusterin phenotype were selected from about 12000 individual.

The results are shown in FIG. 3. The results show that 2 revertant individuals with the cluster phenotype disappeared after CS1 is subjected to sodium azide mutagenesis are screened, and are named as CS1-1 and CS1-2 respectively, and other phenotypes of the plants are not changed obviously.

3. Sequencing clone clustering gene of BSA mixed pool

Will M ₁ The generation revertant mutants CS1-1 and CS1-2 are backcrossed with the compound grain rice CS1 to obtain F ₁ Hybrid plants are replaced, F ₁ F is obtained after the generation is selfed ₂ Segregating populations of generations, at F ₂ And (3) carrying out phenotype identification in the grouting period of the generation separation population, screening single plants which are not clustered and have two extreme phenotypes, respectively taking 30 fresh green leaves with the single plant length of 2cm for each extreme phenotype, carrying out pool mixing, preserving at-20 ℃, carrying out BSA (bovine serum albumin) resequencing, and cloning candidate genes.

BSA resequencing results showed that only a few candidate genes were associated and that the two sets of BSA resequencing results only had one common candidate gene, the CS gene. The CS gene is a synonymous gene of BRD3 and codes for a BR degrading enzyme CYP734A4, suggesting that BR plays an important role in regulating cluster growth of multiple grain rice.

4. Clustering gene validation

Taking compound rice CS1 as a receptor parent, knocking out BRD3 gene of CS1 by using a CRISPR/Cas9 system, wherein a knocking-out target site is positioned on a first exon of BRD3 close to a start codon, the sgRNA sequence is 5'-GTTCCTCGTCGGGTGCGTGAGGG-3', and extracting T by using a CTAB method ₀ And (3) carrying out PCR amplification on total DNA of leaves of the transgenic plant by adopting a primer pair consisting of the primers crBRD3-F and crBRD3-R, detecting a target site knockout sequence of an amplified product by Sanger sequencing, and screening a T0 generation homozygous knockout mutant.

crBRD3-F：5’-CATCCTCGCCTTCCCATTT-3’，

crBRD3-R：5’-CATCCGTCCTCAACATACCG-3’。

The results are shown in FIG. 4. The result shows that after the frame shift mutation occurs in the BRD3 gene knockout of CS1, the cluster phenotype completely disappears, and the BRD 3-regulated cluster phenotype of the multi-grain rice is verified.

Example 3 variation of Gene promoter and functional analysis of multiple grain Rice

1. BRD3 gene sequence analysis of multiple grain rice

Extracting total DNA of leaves of CS1 and NCS1 plants by using a CTAB method, amplifying a BRD3 gene coding region by using a primer pair consisting of primers BRD3-F and BRD3-R, amplifying a BRD3 gene promoter region by using a primer pair consisting of primers pBRD3-F and pBRD3-R, and detecting mutation of the coding region and the promoter region of the BRD3 gene by using Sanger sequencing of amplified products.

BRD3-F：5’-CGACCTGATCTCTCTGCGTTTG-3’，

BRD3-R：5’-TCACTATGCACACAACGGACAG-3’，

pBRD3-F：5’-AATAATGCCTAGCTTCTTTCCT-3’，

pBRD3-R：5’-CTCCTCCCCTGTTTCTTGAA-3’。

The results show that the sequence of the BRD3 gene coding region of CS1 is shown as SEQ ID NO.2 (the amino acid sequence of the coding protein is shown as SEQ ID NO. 1), NO nonsensical mutation occurs, the sequence of the BRD3 gene promoter of CS1 is shown as SEQ ID NO. 4, 34 AT series repeats are inserted, and the NCS1 site only contains 6 AT repeats.

2. BRD3 expression Pattern analysis

Young ears of CS1 and NCS1 plants are stripped at the same period in the booting stage of rice, and are immediately placed in liquid nitrogen for preservation at-80 ℃. Grinding with a mortar, extracting total RNA of young ears by using a Tirzol method, carrying out reverse transcription to obtain cDNA, detecting the expression quantity of a BRD3 gene in young ears by using a qRT-PCR method by using the cDNA as a template (using a Ubiquitin gene as an internal reference gene), detecting the expression of the BRD3 gene by using a primer pair consisting of primers qRT-BRD3-F and qRT-BRD3-R, and detecting the expression of the Ubiquitin gene by using a primer pair consisting of primers qRT-UBQ-F and qRT-UBQ-R.

qRT-BRD3-F：5’-CAGCCGTCCAATTAGCTACTAT-3’，

qRT-BRD3-R：5’-ACTATGCACACAACGGACA-3’，

qRT-UBQ-F：5’-GCCCAAGAAGAAGATCAAGAAC-3’，

qRT-UBQ-R：5’-CATATACCACGACCGTCAAAAC-3’。

The results are shown in FIG. 5. The results show that the variation of CS1 in the BRD3 promoter region leads to significantly higher expression levels in young ears than NCS1.

3. Analysis of BRD3 Gene promoter Activity of multiple grain Rice

1) Extracting total DNA of CS1 seedlings of the compound rice.

2) And (2) taking the DNA obtained in the step (1) as a template, and adopting a primer pair consisting of a primer LUC-BRD3-F and a primer LUC-BRD3-R to carry out PCR amplification to obtain a PCR amplification product.

LUC-BRD3-F:5’-AAGCTTAATAATGCCTAGCTTCTTTCCT-3’,

LUC-BRD3-R:5’-AAGCTTCTCCTCCCCTGTTTCTTGAA-3’。

In the primers LUC-BRD3-F and LUC-BRD3-R, hindIII cleavage sites are underlined.

3) And (3) cutting the PCR amplification product obtained in the step (2) by using restriction enzyme HindIII, and recovering the cut product.

4) The LZ004-LUC vector is digested with restriction enzyme HindIII, and the vector skeleton is recovered. LZ004-LUC vectors are given by the university of Aiwa State biology and genetic college Yin Yanhai in the United states, and for relevant LZ004-LUC vectors see Yanhai Yin, dione Vafeados, yi Tao, shigeo Yoshida, tadao Asami and Joanne Chory, cell, vol.120,249-259.DOI:10.1016/j.cell.2004.11.044.

5) And (3) connecting the enzyme digestion product of the step (3) with the vector skeleton of the step (4) to obtain a recombinant vector pBRD 3:LUC. Based on the sequencing results, the structure of the recombinant vector pBRD 3:LUC is described as follows: the fragment between the two HindIII cleavage sites of the LUC vector was replaced by a DNA molecule shown as nucleotide 1-2199 of SEQ ID NO. 4 from the 5' end.

6) The recombinant vector pBRD3 obtained in the step 5) is adopted to transform escherichia coli DH5 alpha to obtain recombinant bacteria.

7) Recombinant strain coated plates containing 50mg/ml spectinomycin in LB solid medium were cultured for 12 hours at 37℃in an inverted manner.

8) And (3) inoculating the monoclonal strain obtained in the step (7) into an LB liquid medium containing 50mg/ml spectinomycin, culturing AT 200rpm for 12 hours to obtain recombinant bacterial suspension, sequencing after extracting plasmids greatly, and screening recombinant plasmids containing different AT copy numbers.

9) Into a 2mL centrifuge tube, 5-10. Mu.g of plasmid was added, and 100. Mu.L of the prepared protoplast was added, and the protoplast transformation was performed by the method described in the references "Zhang Y, su J, duan S, ao Y, dai J, liu J, wang P, li Y, liu B, feng D, wang J, wang H.A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chlorinated processes.plant Methods 7:30-43", and the mixture was incubated at 28℃for 12-16 hours.

10 After protoplast dissociation was completed, transcriptional activation experiments were performed as described in the references "X Bai, Y Huang, Y Hu, H Liu, B Zhang, C Smaczniak, G Hu, Z Han, Y Xing. Multiplexing of an upstream silencer of FZP increases grain yield in ri.

The results are shown in FIG. 6. The result shows that the AT series repetitive sequence insertion in the BRD3 gene promoter of the compound rice CS1 can obviously enhance the promoter activity, and the promoter activity is stronger when the AT copy number is larger, so that the BRD3 expression is enhanced, the BR degradation is promoted, and the generation of the cluster phenotype is caused.

4. BRD3 gene spatial distribution analysis of compound rice

1) In the period of rice nutrition growth to reproductive growth, young ear meristems of CS1 and NCS1 plants are peeled off and quickly put into FAA fixing solution.

2) Vacuumizing in ice bath for 10 min, turning off power supply for 10 min, and slowly deflating; repeating for several times until the material is sunk to the bottom of the tube, changing the fixing liquid once, and fixing in an ice bath for 4 to 16 hours.

3) The fixative was decanted and dehydrated with 50%, 70%, 85%, 95%, 100% gradient ethanol.

4) 100% ethanol at room temperature for 2 hours, 50% ethanol/50% xylene at room temperature for 1 hour, 90% xylene/10% chloroform at room temperature for 30 minutes.

5) The material was transferred to a glass petri dish and 100% paraffin embedding material was changed.

6) The embedded material is trimmed into regular trapezoid blocks by a scalpel,

7) Placing the baked glass slide on a spreading table, injecting sterilizing water between the glass plate and the spreading table, placing the wax block on a slicing machine, flattening the slice with the thickness of 8 μm on the spreading table, and sucking the excessive water under the wax band with clean filter paper.

8) The slides were dried in a 42℃incubator for 36 hours and stored at 4℃for slicing.

9) Extracting CS1 young ear total RNA, reversely transcribing into cDNA, using the cDNA as a template, adopting a primer pair consisting of a primer anti-BRD3-F and a primer anti-BRD3-R to carry out PCR amplification on an antisense probe template, and adopting a primer pair consisting of a primer sense-BRD3-F and a primer sense-BRD3-R to carry out PCR amplification on the sense probe template, thus obtaining an amplification product.

The primers anti-BRD3-F, anti-BRD3-R, sense-BRD3 and sense-BRD3-R are bolded as protecting bases and underlined as SP6 and T7 transcriptase recognition sites.

10 The amplified product was ligated to pGEM-T easy cloning vector, E.coli was transformed, and after PCR identification, the objective clone was selected, added to LB liquid medium containing 50mg/ml ampicillin at 200rpm, and cultured for 12 hours, to extract the plasmid.

11 The recombinant plasmid is used as a template, a primer pair consisting of a primer anti-BRD3-F and a primer anti-BRD3-R is used for carrying out PCR amplification on the antisense probe template, a primer pair consisting of a primer sense-BRD3-F and a primer sense-BRD3-R is used for carrying out PCR amplification on the sense probe template, an amplification product is obtained, and the product is recovered.

12 2. Mu.g of the recovered product was used as a transcription template, anti-sense and sense probes were transcribed with T7 transcriptase, the probes were precipitated, collected, resuspended in 50. Mu.l DEPC water and stored at-70 ℃.

13 Dissolving probe, and preparing hybridization solution, see the references YF Li, XQ Zeng, Y Li, L Wang, H Zhuang, Y Wang, J Tang, HL Wang, M Xiong, FY Yang. MULTI-FLORET spike 2,a MYB Transcription Factor,Determines Spikelet Meristem Fate and Floral Organ Identity in Rice.Plant Physiology,DOI:10.1104/pp.20.00743", the slides were photographed under a fluorescent microscope.

The results are shown in FIG. 7. The result shows that the AT tandem repeat sequence insertion in the BRD3 promoter of the compound rice CS1 leads to the up-regulation of the gene specificity in the flower stalk and secondary branch stalk meristems, optimizes the spatial distribution of BR in the ear part and leads to the phenotype of compound rice with shortened flower stalk and increased ear grain number.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

<110> institute of crop science at national academy of agricultural sciences

<120> composite grain rice gene CS and application thereof

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ctcgcgttct accactactg gaggaagatc tacgggccga cgttcttgat ttggttcggg 300

ccgacgccgc ggctcacggt ggcggagccg gagatggtgc gggagatctt cctcacgcgc 360

gccgaggcgt tcgaccgcta cgaggcgcac cccgtggtcc ggcagctgga gggcgacggg 420

ctcgtcagcc tccacggcga caagtgggct caccaccgcc gcgtcctcac ccccggcttc 480

taccccgaca acctcaaccg gctggtgccg cacgtcggca ggtcggtggc ggcgctggcg 540

gagaggtggc gcgccatggc gtgcgccggc ggcggcgagg tggaggtgga cgtggcggag 600

tggttccagg cggtggcgga ggaggccatc acgcgcgcca cgttcggccg cagctacgac 660

tccggccgcg tcgtgttccg cttgcaggcc cgcctcatgg cgttcgcctc cgaggccttc 720

cgcaaggtgc tcgtcccggg atacaggttc ctgccgacca agaagaacag gatgtcgtgg 780

ggcctggaca gggagatcag gcgcggcctg gtccggctca tcggccggcg cagtggcggc 840

gacggcggcg aggaagacga gaccaccacc gagctcaaag acaagcagga cagcggcttc 900

aacgacttgc tggggctcat gatcaatgcc ggcgtggaca ggacgatgcc ggtggaggac 960

atggtggagg agtgcaagac cttcttcttc gccggcaagc agacgaccac caacctgctc 1020

acctgggcca ccgtgctgct cgccatgcac ccggactggc aggaccgcgc ccgccgcgag 1080

gtcctcgccg tctgcggcga tgccgccggc gagctcccca ccaaggacca cctccccaag 1140

ctcaagacgc tcgggatgat cctcaacgag acgctgcgcc tgtacccgcc ggcggtggcc 1200

accatccgcc gcgccaagtt cgacgtcacc ctcggcggcg gtggcgacgg cgacgccgga 1260

ggcatccata tcccgcgcga cacggagctg ctcgtcccga tcatggcgat ccaccacgac 1320

gcccggttgt gggggcccga cgcggcccag ttcaacccgg cgaggttcgc cagcggcgcg 1380

gcgcgcgcgg cgaagcaccc gctcgccttc atcccgttcg ggctgggctc ccgcatgtgc 1440

atcggccaga gcctcgccat cctcgaggcc aagctcacca tggccgtcct cctccagcgc 1500

ttcgacctcg cgctctcgcc cacctacgtg cacgccccca ccgtgctgat gctgctccac 1560

ccgcagtacg gcgcgccgtt gatcttccgg ccgcgccaat ctcagccgtc caattag 1617

<210> 3

<211> 2001

<212> DNA

<213> Rice (Oryza sativa)

<400> 3

atgatggagg cggtggccgt ggcggcggcg gtgctgctgc tgctgcacgt ggcggcgagg 60

gtggcggacg cggtgtggtg gcggccgagg cggctggagg cgcacttcgc ggggcagggg 120

gtgcgcggcc cgccgtaccg gttcctcgtc gggtgcgtga gggagatggt ggcgctcatg 180

gcggaggcca ccgcgaagcc catgccgccc gccgcgccgc acaacgcgct ccccagggtg 240

ctcgcgttct accactactg gaggaagatc tacggtatgt tgaggacgga tgaattttgt 300

gtgcttgctc gtgtctgatc agatggatta atggcggtcg tcgcggttgc agggccgacg 360

ttcttgattt ggttcgggcc gacgccgcgg ctcacggtgg cggagccgga gatggtgcgg 420

gagatcttcc tcacgcgcgc cgaggcgttc gaccgctacg aggcgcaccc cgtggtccgg 480

cagctggagg gcgacgggct cgtcagcctc cacggcgaca agtgggctca ccaccgccgc 540

gtcctcaccc ccggcttcta ccccgacaac ctcaacgtga gtctctcctc tgtttcttca 600

tcctccgatc gatcggggcg cacccgcgat gacgacgacg acgatggctg acacgtgctc 660

tgtctctgtc tctctcttgc agcggctggt gccgcacgtc ggcaggtcgg tggcggcgct 720

ggcggagagg tggcgcgcca tggcgtgcgc cggcggcggc gaggtggagg tggacgtggc 780

ggagtggttc caggcggtgg cggaggaggc catcacgcgc gccacgttcg gccgcagcta 840

cgactccggc cgcgtcgtgt tccgcttgca ggcccgcctc atggcgttcg cctccgaggc 900

cttccgcaag gtgctcgtcc cgggatacag gtacggtaca cgccacgaac aacccaaaaa 960

actcccaaac gattcgccat tctcgccgaa attgggctca cggttggcgc cggaattcga 1020

tcgaacaggt tcctgccgac caagaagaac aggatgtcgt ggggcctgga cagggagatc 1080

aggcgcggcc tggtccggct catcggccgg cgcagtggcg gcgacggcgg cgaggaagac 1140

gagaccacca ccgagctcaa agacaagcag gacagcggct tcaacgactt gctggggctc 1200

atgatcaatg ccggcgtgga caggacgatg ccggtggagg acatggtgga ggagtgcaag 1260

accttcttct tcgccggcaa gcagacgacc accaacctgc tcacctgggc caccgtgctg 1320

ctcgccatgc acccggactg gcaggaccgc gcccgccgcg aggtcctcgc cgtctgcggc 1380

gatgccgccg gcgagctccc caccaaggac cacctcccca agctcaagac ggtacgcaca 1440

ccaaaccata tccatggccc agtgggggta ttcccgtaat tccacgccga caaaagtctc 1500

accttgttgg ttctcgctgt catcttcttc cagctcggga tgatcctcaa cgagacgctg 1560

cgcctgtacc cgccggcggt ggccaccatc cgccgcgcca agttcgacgt caccctcggc 1620

ggcggtggcg acggcgacgc cggaggcatc catatcccgc gcgacacgga gctgctcgtc 1680

ccgatcatgg cgatccacca cgacgcccgg ttgtgggggc ccgacgcggc ccagttcaac 1740

ccggcgaggt tcgccagcgg cgcggcgcgc gcggcgaagc acccgctcgc cttcatcccg 1800

ttcgggctgg gctcccgcat gtgcatcggc cagagcctcg ccatcctcga ggccaagctc 1860

accatggccg tcctcctcca gcgcttcgac ctcgcgctct cgcccaccta cgtgcacgcc 1920

cccaccgtgc tgatgctgct ccacccgcag tacggcgcgc cgttgatctt ccggccgcgc 1980

caatctcagc cgtccaatta g 2001

<210> 4

<211> 2199

<212> DNA

<213> Rice (Oryza sativa)

<400> 4

aataatgcct agcttctttc ctacaaaaaa gtttgaaagc tattttttga cgtagattta 60

attttgatcg ataaaaataa agctggacgt actcgcacaa aaattaaatg agtgctgcac 120

gtcacattta tattgggtca ctctttgttg agagctcctt gtccttgcat gcaggtcact 180

ttgcaaagta ctagcacaac aattagttga ttaaatgcac aagcgaaaag aaaatgcata 240

ctagtagtat cagacagttc ctacttatca tttgaatttt gaaaccttgt tttttttcag 300

ataatttaat ataagtataa actttgtgtc tttgcgctaa ttaggaatga acacaaccaa 360

tttgttgtct ttaatatttt tagaacctgt cgagtgaaat catacacata cgactctacc 420

aaatctgcgc atacgctcag ttatatgagc tcatatgaat caatttacca tagatgttga 480

ccaaactggg acaatgttta gaatttaatt agcacctgag atttttttct aataatggaa 540

gtgtgatgcc taatcactgg actcggtaca gcgctatcca taatttaaat aagcaaacga 600

tttattcgat cgggtgtttt caatcaacac acaacttcaa agagttaata attattatta 660

gcaggtgaag aatcaatcat tgggcatctt gaagaaacca catcatatca ggacatgtag 720

gtaggaggaa gacatcccaa attaagcggc ctgctcaaac cgtgttctga cctgcgtaaa 780

caggcaggga caagctgggt catactaagt tttgtggtct ggtctgaact accctaccac 840

attaacatcc aaaacaattt ccaatatata tatatatata tatatatata tatatatata 900

tatatatata tatatatata tatatatata tatatatata tattcaagca ctaaacagtg 960

gaacgtgttt ctcatggtta cagtgaaaac cgtattaaca ccattcctaa ttgtagctga 1020

ttagtctctc tttgtatagc tctggtgcag tagttttatt ttttacttac taaggaagca 1080

gctgcttttt caatagaaat tgagagacga gaggtgctga ccttcattta tttagcagat 1140

gatagaagat gtatacgcgg gtgtgtgcga atagcgatgt atgtattctt tcacataata 1200

aaaaaaaaac ctcatccggt ttttaatatt tgaagatgtt gactgttggc attaaaaaat 1260

atctatttat cttattaaaa aattatgtaa tatgcaaaag tatagatctt attaaaataa 1320

atcacaacaa aaataaataa taatcacgta attttttcaa tataatgaat agtcaataaa 1380

caatgttaaa cattaaaaac cagataaacc agtttaaatt ttagaatata attttttttg 1440

cactaaatac tataaagctg acataagtac atagtagaca cataatataa taagtacttc 1500

ctccgtccca aaataagcgc agccatgagt tttttatcca actttaatcg ttcgtcttat 1560

ttaaattttt cttttgaaaa cactaaaaaa tataatcacg tataaaatgt tatttatatt 1620

ttataatcta atagcaataa aaaatattat tataaaaaaa ttaaataaaa cagatgatca 1680

aagttgaaaa aaaaacttat gcctgcgttt atttgggttt atttgggact gagagagtac 1740

atcacaaact ttcaatcact caagagtaag cagcacacgc aagcaaaagc cttgccttca 1800

ctcccctcct ctcatttttc ccaactgcaa cgaatgaaaa tgttgttcta caaaaaagag 1860

aatgaaaatg ccacgtcagc acccccaagc tcggtcagct gaactgaagg cggttttctc 1920

acacttcacc tacctcgttg acgcctcccc cctttctctc tccatcccat ctgaatttac 1980

caagcagcca ccaccaacag atcatcgtgc tgggccggtg ggccccacct ccgccgcccc 2040

gcttcgctat ataacccccg cctcctcccg ccatcctcgc cttcccattt cgaatccaaa 2100

cccccaaccc aaccgccgcc accactcacc ggcgcaacca ccggcggcga cctgatctct 2160

ctgcgtttgt gtgctctgtt tcaagaaaca ggggaggag 2199

Claims (4)

1. A method for promoting rice spike number and cluster phenotype character, which is characterized in that the rice gene BRD3 is enhanced to obtain a rice plant with up-regulated gene BRD3 expression;

the means for up-regulating expression is selected from the following 1) to 4), or an optional combination:

1) By introducing a plasmid having the gene;

2) By increasing the copy number of the gene on the plant chromosome;

3) By altering the promoter sequence of said gene on the plant chromosome;

4) Operably linking the strong promoter shown in SEQ ID NO. 4 to said gene;

wherein, the rice gene BRD3 is a gene encoding the following proteins:

a protein consisting of the amino acid sequence shown in SEQ ID NO. 1.

2. Use of the rice gene BRD3 of claim 1 for promoting rice grain number and clusterin phenotype traits.

3. A method for promoting rice grain number and cluster phenotype, the method comprising: the rice gene BRD3 of claim 1 is overexpressed by rice.

4. A method according to claim 3, wherein the method comprises transgenesis, crossing, backcrossing, selfing or asexual reproduction.