CN114921488A - Compound rice gene CS and application thereof - Google Patents

Abstract

The invention provides a double-grain rice gene CS and application thereof. The invention clones the double-grain rice gene CS for the first time, the gene codes a BR degrading enzyme CYP734A4, the CS promoter in the double-grain rice contains 34 AT copies of insertion relative to a reference genome, the promoter activity can be enhanced, and the CS gene in the double-grain rice is specifically up-regulated in the flower stalk and secondary branch meristem, thereby leading to the double-grain rice phenotype with shortened flower stalk and increased spike grain number, but the size and plant height of rice grains are not changed, the yield can be obviously increased by about 10 percent relative to non-double-grain rice under the same background, and a powerful means is provided for rice high yield breeding.

Description

Compound rice gene CS and application thereof

Technical Field

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

Background

The compound rice is a unique rice germplasm resource, and is characterized in that the pedicel is shortened, so that a plurality of grains cluster on one branch. At present, the multi-grain rice is mainly clustered by three grains. It should be noted that the double-grain rice is completely different from the reported three-flower mutant with development defects, the latter is that incomplete floral organs are formed on both sides of the middle complete grain due to the conversion of glume protection into the inner and outer palea, and the double-grain rice cannot develop into normal grains, and the double-grain rice has completely normal floral organs and grain development. Due to the dense grain resulting from clumping, some of the multiple grain rice behaves in the field as a spike like wheat and is therefore also called glume rice. This unique trait has attracted many scientists' efforts to clone the corresponding control genes over the past forty years. Almost all studies mapped candidate genes to chromosome 6 in the same interval by map-based cloning, but most did not clone candidate genes. Currently, only one report has been cloned into a particular gene, named SPED1, which was found to contain two point mutations that may be responsible for clustering. Surprisingly, another recent study has also mapped the gene of interest to this region using the clustered rice Nenjiang P164 material, but the sequencing finding did not contain the mutation site of SPED1, as described above, suggesting that this allele may only play a role in certain specific clustered rice. Considering that different researches utilize different materials, but target genes are positioned in the same interval, the true genetic mechanism of the cluster growth of the double-grain rice is still unsolved.

Brassinosteroids (BR) represent a class of steroid hormones and play an important role in the aspect of plant growth and development. In rice, BR can significantly regulate and control a plurality of important agronomic traits, such as plant height, leaf angle, grain size and the like, so that BR has important application potential. BR deletion mutants usually show phenotypes such as dwarfing, erect leaves and granule, and many of the different traits are negatively associated from the breeding point of view, so that genetic improvement of rice by using BR genes has certain difficulties. Solving this problem is of great significance to BR utilization. Interestingly, some studies found that the BR synthetic mutant d11 sometimes resulted in cluster growth of grains in addition to grain reduction, suggesting that BR may play a role in regulating the formation of double-grain rice.

In addition to the shortening of the pedicel, the increase of the number of grains per ear is possibly accompanied by the increase of the number of grains per ear, and although the yield increase potential attracts many breeders to try to introduce the trait to increase the rice yield, no scientific analysis report exists at present. Spike grain number is an important factor for determining rice yield, Japanese scientists clone a major quantitative trait locus gene Gn1a for controlling spike grain number, codes for CKX2 cytokinin oxidase, and the locus is widely existed in rice varieties, for example, one allele of Gn1a is contained in the southwest double-spike variety Shuhui R498, so that the spike grain number is increased, and the high yield is realized. Therefore, the panicle number gene has great application value in high-yield breeding of rice.

The double-grain rice as a special germplasm resource may have great application value in increasing the yield of rice, but the true genetic mechanism of clustering is still unsolved, and whether the grain number of ears can be increased or not is not determined. Whether the double-grain rice gene can be successfully cloned and utilized to increase the yield is a main problem to be solved urgently in the current double-grain rice utilization.

Disclosure of Invention

The invention aims to provide a double-grain 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 the number of grains per ear of rice and the trait of a clustering phenotype (without influencing other phenotypes), a rice gene CS is enhanced, and a rice plant with the expression of the gene CS being up-regulated is obtained.

The expression is upregulated in a manner 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 an enhancer.

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

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

(b) 1, protein which is derived from (a) and has the same function 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 double-stranded rice gene CS (derived from the double-stranded rice germplasm CS1) encoding a protein identical to that of rice gene CS; the promoter sequence is as follows:

a) 4, the nucleotide sequence shown as SEQ ID NO;

b) 4, nucleotide sequences with one or more nucleotides substituted, deleted and/or added and the same promoter activity;

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

d) A nucleotide sequence having 90% or more 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 containing the multiple grain rice gene CS, including but not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineered bacterium.

In a fourth aspect, the invention provides application of the compound rice gene CS or a biological material containing the same in promoting the traits of rice panicle number and clustering phenotype (without influencing other phenotypes).

In a fifth aspect, the invention provides an application of the compound rice gene CS or a biological material containing the same in preparing a transgenic plant.

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

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

1) making rice contain the compound rice gene CS; or

2) So that the rice overexpresses the compound rice gene CS.

Such methods include, but are not limited to, transgenics, crosses, backcrosses, selfs, or asexual propagation.

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

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

the double-grain rice gene CS of the invention codes a BR degrading enzyme CYP734A4, the CS promoter in the double-grain rice comprises an insertion of 34 AT copies relative to a Nipponbare reference genome, the promoter activity can be enhanced, and the CS gene in the double-grain rice is specifically up-regulated in the pedicel and secondary branch meristem, thereby leading to the phenotype of the double-grain rice with shortened pedicel and increased spike grain number, but the size and plant height of rice grains are not changed, and the yield can be remarkably increased by about 10 percent relative to non-double-grain rice under the same background, thereby providing a powerful means for high-yield breeding of rice.

Drawings

FIG. 1 is a chart of the cluster phenotype and statistics of the multi-grain rice in the preferred embodiment of the present invention. Wherein A is a phenotypic observation; b is a plant height statistical result, and the ordinate is the plant height (cm); c is the spike number statistical result, and the ordinate is the spike number; d is the statistical result of the spike length, and the ordinate is the spike length (cm); e is the statistical result of the first-level branch number, and the ordinate is the first-level branch number; f is the statistical result of the number of the secondary branches, and the ordinate is the number of the secondary branches; g is the grain number per spike statistical result, and the ordinate is the grain number per spike; h is a statistical result of the yield per mu, and the ordinate is the yield per mu (kg); i is the statistical result of grain length, 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 clusterin gene is not fully dominant in the preferred embodiment of the invention. Wherein NCS is not clustered; WCS is weak cluster: CS is clustering.

FIG. 3 is a diagram showing the disappearance of the clustered phenotype of the revertant mutants in the preferred embodiment of the present invention.

FIG. 4 is a graph of BRD3 modulating a multiple grain rice cluster phenotype in a preferred embodiment of the invention.

FIG. 5 shows that the expression level of BRD3 in CS1 young ear is significantly up-regulated in the preferred embodiment of the present invention.

FIG. 6 shows a promoter with significant enhancement of AT tandem repeat insertion in a preferred embodiment of the invention.

FIG. 7 shows that BRD3 is specifically expressed and upregulated in the meristems and pedicles of secondary shoot of double-grain rice in accordance with a preferred embodiment of the present invention.

Detailed Description

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

The invention adopts the following technical scheme:

the invention carries out phenotype observation and analysis on the compound rice, and proves that the compound rice promotes the development of secondary branches and stalks so as to promote the increase of spike grain number, but does not change the size and the plant height of rice grains, and the yield can be obviously increased by about 10 percent by field plot yield measurement.

In the invention, double-grain rice is subjected to large-scale sodium azide mutagenesis, two allelic phenotype reversion mutants are screened, a backcross separation population is constructed, and an extreme material mixed pool is subjected to repeated sequencing to finally clone a double-grain rice gene CS, so that a BR degrading enzyme is encoded, and the BR degrading enzyme is confirmed by gene editing and knockout.

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

The invention overcomes the problem of difficult cloning of the double-grain rice gene, identifies an excellent BR gene allele which promotes the number of grains per ear, and the insertion of a plurality of AT copies leads the BR degrading gene to be only expressed and up-regulated in the flower stalk and the secondary branch meristem, optimizes the space distribution of BR, thereby specifically promoting the number of grains per ear and simultaneously avoiding the possible negative effect caused by reduction of BR.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 phenotypic and genetic analysis of Multi-grain Rice

1. Near isogenic line construction

Wild type 9311 as donor parent, multiple grain rice germplasm CS1, namely rice (Oryza sativa) CS1 which is now deposited in China general microbiological culture Collection center, No. 3 Xilu-1-Chen-Yangxi district, Beijing, institute for microbiology, zip code 100101, preservation number CGMCC No.24373, preservation date 2022, 3 months and 1 day) as common microbiological center of China Committee for culture Collection of microorganismsRecurrent parent hybridization, progeny backcrossing for 5 generations and then selfing, screening individual plants with the plant phenotype similar to CS1 but not clustered in the selfing segregation population, and continuing selfing for at least 3 generations (BC) ₅ F ₃ ) Then, the non-clustered near isogenic line NCS1 was obtained.

2. Phenotypic observation and statistics of multiple-grain rice

And (3) the plant to be detected: the near isogenic line NCS1, multiple grain rice CS 1.

The plant height, the spike number, the spike length, the primary branch number, the secondary branch number, the spike grain number, the yield per mu, the grain length and the grain width of the plant to be detected in the mature period are counted, and the result is shown in figure 1. Results show that compared with NCS1, the end of the ear stem of the compound rice CS1 presents a cluster phenotype with 3 grains gathered together, the primary branch number has no obvious difference, the ear number is obviously increased due to the increase of the secondary branch number, but the plant height, the ear number, the ear length, the grain width and other important agronomic traits are not influenced, and the yield can be increased by about 10% in field plot measurement.

3. Genetic analysis of double-grain rice

Hybridizing the compound rice CS1 with wild ZH11 to obtain F ₁ Plant generation, F ₁ Inbreeding of the plant generations to obtain F ₂ Generation segregation population, pair F ₂ Counting the number of plants with different clustering characters of generation segregation population to obtain F ₂ The results are shown in FIG. 2.

The results show that hybridization F ₁ The generation exhibited a weakly clustered phenotype with only 2 kernels clustered at the end of the branch and stalk, F ₂ Three phenotypes were isolated by inbreeding, and the segregation ratios of the three phenotypes were not clustered: weak clustering: cluster growth is 65: 131: 45, approximately 1: 2: 1 (X) ² _0.05(2) 0.0951664 < 5.99), indicating that the clustering phenotype is regulated by a gene that is not fully dominant.

Example 2 cloning and verification of Multi-grain Rice Gene CS

1. Sodium azide mutagenesis compound grain rice germplasm CS1

Respectively preparing KH with concentration of 1M ₂ PO ₄ And 1M of H ₃ PO ₄ Solution of KH per 1000ml ₂ PO ₄ Solution with 120ml H ₃ PO ₄ The solutions were mixed well, the pH was adjusted to 3.0 with 1M NaOH solution to obtain phosphate buffer, and 1mM sodium azide solution was prepared with phosphate buffer. Weighing 500g of CS1 dry seeds, soaking the seeds in a sodium azide solution, placing the seeds in a fume hood at normal temperature for 6 hours, washing the seeds clean with clear water, replacing tap water to soak the seeds, placing the seeds in an incubator at 37 ℃ to accelerate germination, replacing water every 8 hours in the period, and sowing the seeds in the field after the seeds germinate.

2. Selection of revertant mutants

Sodium azide mutagenesis of M at CS1 ₀ At maturity of the generations, each individual was phenotypically identified in the field and revertants with the loss of clustered phenotype were selected among approximately 12000 individuals.

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

3. Sequencing and cloning cluster gene by BSA mixed pool

Will M ₁ Backcrossing the generation reversion mutants CS1-1 and CS1-2 with the compound rice CS1 to obtain F ₁ Generation of heterozygous plants, F ₁ Selfing to obtain F ₂ Generation segregating population at F ₂ And (3) performing phenotype identification in the filling stage of the generation segregation population, screening single plants with two extreme phenotypes of no clustering and clustering, mixing 30 fresh green leaves with the length of 2cm of the single plant for each extreme phenotype in a pool, storing at the temperature of-20 ℃, feeding samples, performing BSA (bovine serum albumin) re-sequencing, and cloning candidate genes.

BSA resequencing results show that only a small number of candidate genes are associated, and that the two groups of BSA resequencing results only have one candidate gene in common, namely the CS gene. The CS gene is a synonymous gene of BRD3 and codes for a BR degrading enzyme CYP734A4, which suggests that BR plays an important role in regulating the growth of the multi-grain rice cluster.

4. Cluster gene verification

Using double-grain rice CS1 as a receptor parent, knocking out BRD3 gene of CS1 by using CRISPR/Cas9 system, wherein the knocking-out target site is positioned on the first exon of BRD3 close to the initiation codon, and sgRNA5'-GTTCCTCGTCGGGTGCGTGAGGG-3', extracting T by CTAB method ₀ And (3) carrying out PCR amplification on total DNA of transgenic plant leaves by adopting a primer pair consisting of primers crBRD3-F and crBRD3-R, detecting a target site knockout sequence of an amplification 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 BRD3 gene of CS1 is knocked out and subjected to frame shift mutation, the clustering phenotype completely disappears, and the BRD3 is used for regulating the clustering phenotype of the double-grain rice.

Example 3 analysis of promoter variation and function of multiple Rice Gene

1. Analysis of BRD3 Gene sequence of double-grain Rice

Extracting total DNA of leaves of CS1 and NCS1 plants by a CTAB method, amplifying a coding region of BRD3 gene by adopting a primer pair consisting of primers BRD3-F and BRD3-R, amplifying a promoter region of BRD3 gene by adopting a primer pair consisting of primers pBRD3-F and pBRD3-R, and detecting the variation of the coding region and the promoter region of the BRD3 gene respectively by Sanger sequencing of an amplification product.

BRD3-F：5’-CGACCTGATCTCTCTGCGTTTG-3’，

BRD3-R：5’-TCACTATGCACACAACGGACAG-3’，

pBRD3-F：5’-AATAATGCCTAGCTTCTTTCCT-3’，

pBRD3-R：5’-CTCCTCCCCTGTTTCTTGAA-3’。

The results showed that the sequence of the coding region of the BRD3 gene of CS1, as shown in SEQ ID NO:2 (the amino acid sequence of the protein encoded by it is shown in SEQ ID NO: 1), did not undergo any non-synonymous variation, whereas the promoter sequence of the BRD3 gene of CS1, as shown in SEQ ID NO:4, contained an insertion of 34 AT tandem repeats, whereas this site of NCS1 contained only 6 AT repeats.

2. Analysis of BRD3 expression Pattern

Young ears of CS1 and NCS1 plants at the same period are taken at the booting stage of rice and immediately placed in liquid nitrogen for preservation at-80 ℃. After grinding by using 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 BRD3 gene in the young ears by using the cDNA as a template and adopting a qRT-PCR method (using the ubitin gene as an internal reference gene), detecting the expression of BRD3 gene by adopting a primer pair consisting of a primer qRT-BRD3-F and a primer qRT-BRD3-R, and detecting the expression of the Ubiquitin gene by adopting a primer pair consisting of a primer qRT-UBQ-F and a primer 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 mutation of CS1 in the BRD3 promoter region results in significantly higher expression level in young ears than NCS 1.

3. Activity analysis of BRD3 Gene promoter of double-grain Rice

1) Total DNA of CS1 seedlings of multi-grain rice was extracted.

2) And (2) carrying out PCR amplification by using 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 obtain a PCR amplification product.

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

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

Primer LUC-BRD3-F and primer LUC-BRD3-R are underlined as HindIII sites.

3) And (3) carrying out enzyme digestion on the PCR amplification product obtained in the step 2) by using restriction enzyme HindIII, and recovering the enzyme digestion product.

4) The LZ004-LUC vector was digested with restriction enzyme HindIII, and the vector backbone was recovered. LZ004-LUC vectors are offered by professor Yinyan, university of Iowa State university of Cell biology and genetics, USA, and for LZ004-LUC vectors, see Yanhai Yin, Dione Vafeados, Yi Tao, Shigeo Yoshiida, Tadao Asami and Joane Chory, Cell, Vol.120,249-259, DOI: 10.1016/j.cell.2004.11.044.

5) And (3) connecting the enzyme digestion product in the step 3) with the vector framework in the step 4) to obtain a recombinant vector pBRD3:: LUC. According to the sequencing results, the structure of the recombinant vector pBRD3:: LUC was described as follows: the fragment between the two HindIII restriction sites of the LUC vector was substituted into the DNA molecule shown as nucleotides 1 to 2199 from the 5' end of SEQ ID NO. 4.

6) Adopting the recombinant vector pBRD3 obtained in the step 5) to transform escherichia coli DH5 alpha by using LUC to obtain the recombinant strain.

7) The recombinant bacteria are plated on LB solid culture medium containing 50mg/ml spectinomycin, and are inversely cultured for 12 hours at 37 ℃.

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

9) 5-10. mu.g of plasmid was added to a 2mL centrifuge tube, and 100. mu.L of the prepared protoplast was added, and transformation was carried out by the method described in "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 high efficiency plasmid expression system for transformed gene expression and constructing light/chlorinated plasmid Process. plant houses 7: 30-43", and cultured at 28 ℃ for 12-16 hours.

10) After protoplast release, transcription activation experiments were performed with reference to the method in the literature "X Bai, Y Huang, Y Hu, H Liu, B Zhang, C Smaczniak, G Hu, Z Han, Y xing. duplication of an upstream plasmid of FZP innovations grain in yield in nature Plants, VOL 3, 885-.

The results are shown in FIG. 6. The result shows that the insertion of the AT tandem repeat sequence in the BRD3 gene promoter of the double-grain rice CS1 can obviously enhance the promoter activity, and the promoter activity is stronger when the number of AT copies is larger, so that the BRD3 expression is enhanced, the BR degradation is promoted, and the cluster phenotype is generated.

4. Analysis of spatial distribution of BRD3 Gene in Compound Rice

1) In the period of rice vegetative growth converted into reproductive growth, young ear meristems of CS1 and NCS1 plants are stripped and quickly placed into FAA fixing solution.

2) Vacuumizing in ice bath for 10 minutes, turning off a power supply for 10 minutes, and slowly discharging air; repeating for several times until the material sinks to the bottom of the tube, replacing the fixing solution once, and fixing for 4-16 hours in ice bath.

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

4) The mixture was treated with 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, and 90% xylene/10% chloroform at room temperature for 30 minutes.

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

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

7) placing the baked glass slide on a slide-spreading table, injecting sterilizing water between the glass plate and the slide-spreading table, placing the wax block on a slicing machine, slicing to a thickness of 8 μm, spreading on the slide-spreading table, and sucking off excessive water below the wax belt with clean filter paper.

8) The slide glass was dried in a 42 ℃ incubator for 36 hours, and the section was stored at 4 ℃.

9) Extracting total RNA of CS1 young ears, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification on an antisense probe template by taking the cDNA as a template and adopting a primer pair consisting of a primer anti-BRD3-F and a primer anti-BRD3-R, and carrying out PCR amplification on a sense probe template by adopting a primer pair consisting of a primer sense-BRD3-F and a primer sense-BRD3-R to obtain an amplification product.

In the primers anti-BRD3-F, anti-BRD3-R, sense-BRD3 and sense-BRD3-R, the protection bases are bolded, and recognition sites of SP6 and T7 transcriptases are underlined.

10) Connecting the amplified product to pGEM-T easy cloning vector, transforming Escherichia coli, identifying by PCR, selecting the target clone, adding the target clone into LB liquid culture medium containing 50mg/ml ampicillin at 200rpm, culturing for 12 hours, and extracting plasmid.

11) And (3) carrying out PCR amplification on the antisense probe template by taking the recombinant plasmid as a template and adopting a primer pair consisting of a primer anti-BRD3-F and a primer anti-BRD3-R, and carrying out PCR amplification on the sense probe template by adopting a primer pair consisting of a primer sense-BRD3-F and a primer sense-BRD3-R to obtain an amplification product, and recovering the product.

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

13) The probe was dissolved to prepare a hybridization solution, and the hybridization solution was prepared according to "YF Li, XQ Zeng, Y Li, L Wang, H Zhuang, Y Wang, J Tang, HL Wang, M Xiong, FY Yang. Multi-FLORET SPIKELET 2, a MYB Transcription Factor, determinines Spikeet Meristem face and Floral Organ Identity in Rice. plant Physiology, DOI: 10.1104/pp.20.00743' and the slide was photographed under a fluorescent microscope.

The results are shown in FIG. 7. The result shows that the insertion of AT tandem repeat sequence in BRD3 promoter of the double-grain rice CS1 leads to the up-regulation of gene expression in the flower stalk and secondary branch stalk meristem, optimizes the space distribution of BR AT the ear part, and leads to the double-grain rice phenotype of shortening the flower stalk and increasing the number of the ears.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> institute of crop science of Chinese academy of agricultural sciences

<120> multiple grain rice gene CS and application thereof

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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 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 (8)

1. A method for promoting the grain number of rice per ear and the phenotypic character of cluster growth is characterized in that a rice gene CS is enhanced to obtain a rice plant with the expression of the gene CS being up-regulated;

the expression is upregulated in a manner selected from the following 1) to 5), or optionally in 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 an enhancer;

wherein the rice gene CS is a gene encoding the following protein (a) or (b):

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

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

2. A multiple grain rice gene CS characterized in that the protein encoded by the multiple grain rice gene CS is the same as the protein encoded by the rice gene CS according to claim 1; the promoter sequence is as follows:

a) 4, the nucleotide sequence shown as SEQ ID NO;

b) 4, nucleotide sequences with one or more nucleotides substituted, deleted and/or added and the same promoter activity;

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

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

3. Biological material containing the gene of claim 2, said biological material comprising recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors or engineered bacteria.

4. Use of the gene of claim 2 or the biomaterial of claim 3 for promoting grain number per ear and clustering phenotype traits in rice.

5. Use of the gene of claim 2 or the biomaterial of claim 3 in the preparation of a transgenic plant.

6. Use of the gene of claim 2 or the biomaterial of claim 3 in plant breeding.

7. A method of promoting grain number per ear and a clustering phenotypic trait in rice, the method comprising:

1) allowing rice to comprise the gene of claim 2; or

2) Overexpresses in rice the gene of claim 2.

8. The method of claim 7, wherein the method comprises transgenesis, crossing, backcrossing, selfing, or asexual propagation.

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