CN109112157B

CN109112157B - Silencer CNV-18bp of rice panicle development gene and application thereof in rice yield improvement

Info

Publication number: CN109112157B
Application number: CN201710483025.5A
Authority: CN
Inventors: 邢永忠; 白旭峰; 黄勇
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2017-06-22
Filing date: 2017-06-22
Publication date: 2020-12-04
Anticipated expiration: 2037-06-22
Also published as: CN109112157A

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a silencer CNV-18bp of a rice panicle development gene and application thereof in rice yield improvement. The silencer is named CNV-18bp and is positioned at 5309bp upstream of the initiation codon ATG of the SGDP7 gene, and the sequence of the silencer is shown as SEQ ID NO. 1. The sequence has the characters of controlling the number of glumes per spike, grain shape/grain weight of rice, and the characters are related to the yield characters of rice. The invention also clones the allele of SGDP7 gene, which can control the number of glume flowers of rice per spike, thousand grain weight, grain length and yield, and the nucleotide sequence of the allele is shown as SEQ ID NO: 2, respectively. The invention also comprises biological function verification of the pleiotropic gene SGDP7 and alleles thereof, preparation of molecular markers and application thereof in rice yield improvement.

Description

Silencer CNV-18bp of rice panicle development gene and application thereof in rice yield improvement

Technical Field

The present invention belongs to the field of plant gene engineering technology. In particular to a silencer CNV-18bp of a rice panicle development gene and application thereof in rice yield improvement. The silencer is named CNV-18bp and is positioned at 5309bp upstream of the initiation codon ATG of the SGDP7 gene, and the sequence of the silencer is shown as SEQ ID NO. 1. The sequence has the characteristics of controlling the number of glumes per spike, the grain shape and the grain weight of rice, and the characteristics are related to the yield characteristics of the rice. The invention also clones the allele of SGDP7 gene, which can control the number of glume flowers of rice per spike, thousand grain weight, grain length and yield, and the nucleotide sequence of the allele is shown as SEQ ID NO: 2, respectively. The invention also comprises biological function verification of the pleiotropic gene SGDP7 and alleles thereof, preparation of molecular markers and application thereof in rice yield improvement.

Background

World food safety is one of the problems of close attention of people, and how to further improve the rice yield has important significance on food safety of China and even the world population. The rice yield mainly comprises three main components of tillering number, glume flower number per spike and thousand-grain weight; the number of glumes of each spike of rice is mainly related to the number of secondary branches of each spike of rice. The influence effect on the yield is more prominent than the grain shape and the thousand grain weight.

The number of glume flowers of each ear is controlled by a plurality of quantitative character sites and is divided into a major gene and a minor gene. They can be obtained by constructing the near isogenic line F₂It is isolated and cloned using the map-based cloning technique. In 1986, Alan Coulson, Cambridge university, first proposed map-based cloning (Coulson et al, 1986) by constructing F₂Separating the population, and gradually reducing the candidate segments into small segments or even single genes according to the recombinant individual by utilizing the linkage relation between the molecular marker and the gene locus. Further, the present invention applies a similar technical route (as shown in FIG. 1) by complementation verification through genetic transformation. In recent years, a plurality of topic groups in different countries successfully separate the number of grains per ear and the yield major gene NAL1/LSCHL4/qTSN4 on the rice chromosome 4 by using a map-based cloning strategy (Fujita et al 2013; Zhang et al 2014; Taguchi-Shiobara et al 2015; Fabre et al 2016). In addition, in 2015, Chinese scientists identified the expression of a copy number variation site regulation grain shape gene GL7 through a map-based cloning strategy, thereby realizing the regulation of rice grain shape variation (Wang et al 2015).

The invention utilizes a recombinant inbred line F derived from rice varieties Nanyang occupation (large-grain ear) and Sichuan 7 (small-grain ear)₇In generations, the RI68 and RI76 families have the separation of secondary branch number, spikelet number, grain length and grain weight, specifically, the number of large-grain flower plants with few glumes and the number of small-grain flower plants with many glumesAnd (3) nearly: 1, separating. The segregation population is taken as a near isogenic line (Bai et al, BMC Genetics2010,11:16), and the pleiotropic QTL-SGDP7 clone which simultaneously controls the number of glumes of each spike, the grain weight, the grain length and the yield of rice is finally cloned by utilizing a map-based cloning strategy. In an RI 68-derived family, the number of flowers per spike (239) and the yield per plant (22.4g) of an allelic type of 'Chuan 7' from a rice variety are respectively and obviously greater than the number of flowers per spike (119) and the yield per plant (15.0g) of the allelic type of Nanyang, the thousand seed weight (17.0g) of the 'Chuan 7' allelic type is slightly less than the thousand seed weight (20.6g) of the allelic type of the 'Nanyang', and the yield per plant is increased by 49.3%; in the RI 76-derived near isogenic line, the number of spikelets per ear (162) and yield per plant (17.4g) from the "chuan 7" allele were significantly greater than the number of spikelets per ear (118) and yield per plant (14.8g), respectively, of the "nanyang occupancy" genotype. The thousand seed weight (16.4g) of the 'Chuan 7' allele type is slightly less than the thousand seed weight (19.5g) of the 'Nanyang' genotype, and the yield of a single plant is increased by 17.5 percent. Its clone can be used for increasing yield and breeding of rice. Through further research, we found that an 18bp repetitive sequence (which inhibits the expression of downstream genes and is therefore defined as the silencer CNV-18bp) is inserted into the gene type "Chuan 7" at 5309bp upstream of the initiation codon ATG of the SGDP7 gene. In addition, the 18bp insertion fragment is proved to increase the rice yield by more than 17.5 percent through research. Meanwhile, a molecular marker (InDel712) is designed aiming at the insertion and deletion site, and can be directly used for molecular marker assisted breeding.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, separates and clones a pleiotropic gene SGDP7 and an allele for controlling the rice yield such as the number of glumes per ear, the grain shape, the grain weight and the yield, and also relates to the application of SGDP7 gene and an allele molecular marker. The insertion of the 18bp sequence at 5309bp upstream of the SGDP7 gene results in the reduction of the expression level of the gene, so that the grain shape and the grain weight are slightly reduced, but the number of secondary branches is increased to form multiparticulates and increase the yield. The applicants have named this pleiotropic gene cloned as SGDP7 and identified an 18bp insert upstream of its start codon ATG. Meanwhile, a molecular marker InDel712 is designed aiming at the insertion/deletion.

The invention is realized by the following technical scheme:

the invention clones a pleiotropic gene SGDP7 for controlling the number of flowers per ear, grain shape, grain weight and yield of rice from a rice variety 'Chuan 7' by a plant genetic engineering method and a transgenic method, and inserts an 18bp repetitive fragment (the nucleotide sequence of which is shown as SEQ ID NO:1) at 5309bp upstream of an SGDP7 gene initiation codon ATG in the rice variety 'Chuan 7' compared with a reference genome (Nipponbare genome) and a rice variety 'Nanyang occupation'. The nucleotide sequence (Chuan 7 allele sequence) of the whole gene (comprising 6489bp upstream sequence and 957bp CDS) is shown as SEQ ID NO: 2, SEQ ID NO: 3 (note: the SGDP7 Nanyang allele has the same CDS as the Sichuan 7 allele), and the sequence of the encoded protein is shown as "SEQ ID NO: 4 "(note: SGDP7 Nanyang occupied allele encodes the same amino acid as the Sichuan 7 allele). The allele of SGDP7 gene is cloned from rice variety "Nanyang zhan", and the nucleotide sequence (also genetic complementary transformation sequence) of the allele is shown as SEQ ID NO: 5, respectively.

The applicant transforms the pleiotropic gene SGDP7 (i.e. the allele cloned from Nanyang variety) into the allele of the near isogenic line 'Chuan 7' (the near isogenic line is derived from the recombinant inbred line F derived from the rice varieties 'Nanyang variety' and 'Chuan 7') by transgenic method₇The technical route is shown in figure 1), the number of secondary branches and the number of glumes per ear of a transgenic positive single plant are reduced, the grain weight and the grain length are increased, and the transgenic positive single plant conforms to the expected phenotype.

The invention has the advantages that:

(1) based on QTL positioning results, the invention separates and clones a pleiotropic gene SGDP7 for regulating and controlling the number of glumes per ear, the grain shape, the grain weight and the yield of rice. The gene regulates the rice yield and lays a further foundation for improving the rice yield.

(2) The invention detects that an 18bp insertion/deletion polymorphic site exists at 5309bp upstream of the ATG start codon of the SGDP7 gene. The site is a functional site of SGDP7 gene, and can negatively regulate the expression of SGDP7, thereby regulating the yield of rice.

(3) The invention designs a specific molecular marker InDel712 aiming at the 18bp insertion/deletion fragment, and the molecular marker can be used for rice molecular marker assisted breeding.

The more detailed technical scheme is shown in the detailed description.

Drawings

Sequence listing SEQ ID NO:1 is the nucleotide sequence of an 18bp fragment inserted at 5309bp upstream of the ATG initiation codon of the pleiotropic gene SGDP 7.

Sequence listing SEQ ID NO: 2 is the complete sequence of the rice Chuan 7 allele of the pleiotropic gene SGDP7 (wherein the ATG upstream is 6489 bp; the CDS region sequence is 957bp, 318 amino acid sequences are coded).

Sequence listing SEQ ID NO: 3 is the CDS sequence of the pleiotropic gene SGDP7 (note: the SGDP7 Nanyang allele has the same CDS as the SGDP7 Sichuan 7 allele).

Sequence listing SEQ ID NO: 4 is the protein sequence encoded by the pleiotropic gene SGDP7 (note: the Nanyang dominant allele SGDP7 and the Sichuan 7 allele SGDP7 encode the same amino acids).

Sequence listing SEQ ID NO: 5 is the nucleotide sequence of the Nanogan allele SGDP7 of the pleiotropic gene SGDP7 (also a genetically complementary transforming sequence).

Fig. 1 is a general technical roadmap of the present invention.

FIG. 2 is a schematic diagram showing the fine localization of SGDP7 gene and the prediction of candidate gene in the present invention. Description of reference numerals: FIG. 2, panel a, is a fine localization candidate interval of SGDP7 on rice chromosome 7; panel b in figure 2 is a diagram of all variant sites (including SNP and InDel) of the fine localization candidate interval; panel c in FIG. 2 is a drawing showing the SGDP7 candidate gene and its upstream interval; the d diagram in FIG. 2 is a display diagram of the complementary fragment size and relative position of genetic transformation, and the polymorphic site SNP4 base variation and InDel712 insertion sequence.

FIG. 3 differential expression analysis of SGDP7 in young ears of two genotypes of the near isogenic line (Nanyang occupation, NIL-NN and Chuan 7, NIL-CC). FIG. 4 maps of the original plasmid pCAMBIA1301 used for cloning the SGDP7 transformation sequence and the transformation plasmids pCAMBIA1301-SGDP7 constructed according to the present invention. Description of reference numerals: FIG. 4 is a drawing a showing the map of the original plasmid pCAMBIA1301 and the genetic complementation of the transformation fragment of SGDP 7; panel b in FIG. 4 is a map of the transformation plasmid pCAMBIA1301-SGDP 7.

FIG. 5 is a graph showing spike type and granule phenotype of SGDP7 near isogenic lines. Description of reference numerals: NIL-NN is shown on the left of FIG. 5, and NIL-CC is shown on the right of FIG. 5.

FIG. 6 is a phenotypic comparison analysis of the yield and yield-related traits of two genotypes of the near isogenic line (Nanyang occupation, NIL-NN and Chuan 7, NIL-CC). Description of reference numerals: "Panel number per plant" of panel a in FIG. 6 is the effective ear number, which is not significantly different between the two genotypes NIL-NN and NIL-CC; "No. of primary branches" of the b-diagram in FIG. 6 is the number of branches, which is not significantly different between the two genotypes of NIL-NN and NIL-CC; in the c diagram of FIG. 6, "No. of secondary branches" indicates the number of secondary branches, and the number of secondary branches of NIL-CC is significantly greater than that of NIL-NN; "Ratio of NSB/NPB" of the d diagram in FIG. 6 is the Ratio of the number of secondary branches to the number of primary branches, and the Ratio of the number of secondary branches to the number of primary branches of NIL-CC is significantly greater than the Ratio of the number of secondary branches to the number of primary branches of NIL-NN; "Spikeets per panicle" of the e plot in FIG. 6 is the number of flowers per ear, with the number of flowers per ear for NIL-CC being significantly greater than the number of flowers per ear for NIL-NN; "1000-grain weight" of plot f in FIG. 6 is the thousand seed weight, which is significantly greater for NIL-NN than for NIL-CC; the "gain yield per plant" of the g diagram in FIG. 6 is the individual yield, and the individual yield of NIL-CC is significantly higher than that of NIL-NN.

FIG. 7 is a 3% agarose gel electrophoresis image of the genotype of different rice varieties detected by PCR amplification using InDel712 as a marker. Description of reference numerals: in the figure: "NYZ" indicates the Nanyang band-occupied type, "C7" phenotype Chuan 7 band type; the lane 1 is a DNA marker band, the lane 2-18 is the same as the band 7, and the lane 19-31 is the same as the band occupied by the south ocean.

Detailed Description

According to the technical scheme described in FIG. 1, the applicant utilized the rice varieties "Nanyang zhan" and "Chuan 7" (present by Roli military researcher, agricultural Gene center, Shanghai)Derived recombinant inbred line F₇The generation population constructs genetic linkage maps and collects phenotypes, and after quantitative trait loci analysis, the quantitative trait loci (Bai et al, BMC Genetics2010,11:16) of the number of glumes, thousand grain weight and grain length of each ear are located at the lower end of the long arm of the 7 th chromosome (RM22065-RM 5720). 185 recombinant inbred lines were screened and RI68 was found to be heterozygous with RI76 line in the interval "RM 22065-RM 5720". Harvesting the two lines of seeds for planting to obtain F₂Isolating the population. This segregating population is the near isogenic line for SGDP 7.

Near isogenic line F for planting SGDP7₂The large population (6890 strains) was screened for recombinant individuals using SSR-related common markers and newly developed InDel and SNP markers (molecular marker primer sequences are shown in Table 2), and SGDP7 was finally mapped to 17.8-kb. In addition to 6 SNP differences, an 18bp insertion/deletion polymorphic site was found upstream of LOC _ Os07g47330 within the localization interval; for this purpose, "LOC _ Os07g 47330" was used as a candidate gene for SGDP7, which initiated a 6489bp segment (containing the 18bp insert) upstream of the codon "ATG" as the gene regulatory segment (FIG. 2; see SEQ ID NO:1 for sequence). Furthermore, expression analysis revealed that "LOC _ Os07g 47330" was significantly higher in NIL-NN than in NIL-CC (see FIG. 3).

Through comparative sequencing analysis, the sequence of the allele of the two candidate genes is basically the same as that of the allele of the Nipponbare. Therefore, we utilized the BAC clone A0044I19 of "Nipponbare". An 8.2-kb DNA fragment (i.e., DF1, see FIG. 2) was obtained by using PstI and SpeI separation, and the "Nipponbare" allelic base "C" of SNP2 was replaced with "Nanyang occupied" allelic base "T" by PCR amplification and fragment recombination; the above-mentioned isolated and recombinant fragment was then cloned into a complementary vector pCAMBIA1301(pCAMBIA1301 is a publicly reported and widely used vector: http:// www.cambia.org/day/cambia/585, see FIG. 4a) to obtain a transformation plasmid pCAMBIA1301-SGDP7, see FIG. 4b), and the transformation plasmid pCAMBIA1301-SGDP7 was transformed into rice of the receptor SGDP7 near isogenic line Chuan 7 genotype. Through phenotype investigation and statistical analysis of transgenic positive plants, the following results are found: the fragments may complement the phenotype. "LOC _ Os07g 47330" (and containing an upstream fragment of 6489 bp) was confirmed to be the SGDP7 gene.

The following examples further define the invention and describe the isolated cloning of the SGDP7 gene and the application of the process in the development of functional markers.

Example 1: SGDP7 near isogenic line construction and utility evaluation

185 recombinant inbred lines F constructed by planting rice varieties Nanyang in and Chuan 7 in a rice experimental base of agricultural university in Wuhan Huazhong (a gift from Roli army researchers in Shanghai city agricultural biological Gene center)₇Pedigrees (Bai et al, BMC Genetics2010,11:16) and QTL mapping analysis using molecular markers covering almost the entire genome to construct genetic linkage maps and then combining phenotypes found: a pleiotropic QTL for respectively controlling thousand-grain weight, grain length and number of glumes per ear exists in the region of 'RM 22065-RM 5720' at the tail end of the long arm of the 7 th chromosome of rice. In view of this result, the present inventors examined all recombinant inbred lines for lines that were heterozygous in the segment "RM 22065-RM 5720" and homozygous at other genomic positions, and found that "RI 68" and "RI 76" (Bai et al, BMC Genetics2010,11:16) lines belong to this case. Therefore, the progeny of this RI68 and RI76 line were harvested and planted, and it was found that segregation of thousand kernel weight, grain length and number of glume flowers per ear occurred in the progeny, with a segregation ratio close to 3: 1; their progeny population is therefore referred to as the SGDP7 near isogenic line (Bai et al, BMC Genetics2010,11: 16). The different phenotypic values for each genotype in the two near isogenic populations are summarized in Table 1 and FIGS. 5-6.

TABLE 1 phenotypic comparison analysis of near isogenic lines of SGDP7 (RI68)

In table 1, "transit" represents a phenotypic Trait; "NIL-NN" represents the Nanyang account genotype of the near isogenic line; "NIL-CC" represents the gene type of Chuan 7 of the near isogenic line; "PN" represents the effective spike number of a single plant; "NPB" represents the number of branches at a time; "NSB" represents the number of secondary branches; "NSB/NPB" represents the ratio of the number of secondary branches to the number of primary branches; "SPP" represents the number of glume flowers per ear; "GL" represents grain length; "TGW" represents thousand kernel weight; the phenotypic values are mean ± standard deviation P values are obtained from the t test.

Example 2: fine localization and candidate Gene determination of SGDP7

1. Recombinant Individual screening and Fine localization of SGDP7

6890 SGDP7 isogenic line segregating populations were grown in Wuhan Hubei, China in the growing season of 2009 (5 months to 10 months). The SSR molecular markers "RM 22065", "RM 5720", "RM 6389" and "RM 22115" (see http:// www.gramene.org/markers /) and the self-created polymorphism markers "InDel 76", "InDel 712" and "SNP 1-SNP 7" (primer sequences are shown in Table 2) of the gene localization segment are used to screen recombinant individuals and finally localize the gene into the segment of "SNP 4-SNP 8" (17.8kb) (FIG. 2).

2. Identification of candidate genes

An 18bp insertion/deletion polymorphic site was identified from the above candidate segment, and the site was located upstream of LOC _ Os07g47330, and for this purpose, "LOC _ Os07g 47330" was used as a candidate gene for SGDP7 in the present invention. The coding region of the candidate gene has no mutation before the two parents of Nanyang and Chuan 7. On the other hand, there was a polymorphic site of insertion/deletion (18 bp fragment inserted into "Chuan 7") at 5309bp upstream of the ATG initiation codon, and 2 SNP polymorphic sites (SNP2 and SNP4) were present, while SNP2 was not in the candidate region (see FIG. 2). In addition, differential expression of LOC _ Os07g47330 in young ears of two near isogenic lines (NIL-NN and NIL-CC) was detected by the Real-Time PCR method (see Bai et al, Scientific Reports,2016,6:19022), indicating that LOC _ Os07g47330 is significantly higher expressed in NIL-NN than in NIL-CC (see FIG. 3).

TABLE 2 related primer sequences designed for SGDP7 localization, cloning and sequencing

Note: the sequences in table 2 are all primer sequences for molecular markers. Wherein, InDel712 is a functional marker, and the rest markers are markers in the positioning process.

Example 3: transgenic complementation assay for SGDP7

Through comparative sequencing analysis, the southern ocean occupational allele sequences of the two candidate genes are basically identical with the allele sequences of Nipponbare. Thus, the BAC clone "A0044I 19" from Nipponbare was used. The BAC clone A0044I19 was digested with both PstI and SpeI to obtain an 8.2-kb DNA fragment (DF1, see FIG. 2), and the SNP2 of this fragment was replaced by the Nanyang allele (T); the isolated fragment and the empty vector pCAMBIA1301 (see a in FIG. 4) obtained by double digestion of PstI and SpeI were then purified and recovered (Fermentas Genomic DNA Purification Kit, Thermo Fisher Scientific Inc) and then used with T₄DNA ligase (Promega) ligation cloning of the transformation fragments into the "pCAMBIA 1301" expression vector (see FIG. 4, panel b), respectively; the candidate gene LOC _ Os07g47330 carried by the transformation vector is transformed into the rice with gene type Chuan 7 in the SGDP7 near isogenic line by utilizing an agrobacterium-mediated genetic transformation method. Among them, the resulting "LOC _ Os07g 47330" transgenic positive individual (T)₁Passage) phenotype very similar to the phenotype of the individual southern ocean occupational genotype of the SGDP7 near isogenic line (table 3). LOC _ Os07g47330 was therefore considered a candidate gene. SGDP7 was successfully cloned.

TABLE 3 phenotype of the complementation of transgenic Individual plants transformed with SGDP7

Description of table 3: "Genotype" represents Genotype; "NPB" represents the number of branches at a time; "NSB" represents the number of secondary branches; "NSB/NPB"

Representing the ratio of the number of secondary branches to the number of primary branches; "SPP" is the number of glume flowers per spike; "GL" is the grain length; "TGW" is the thousand grain weight

Example 4: detection of SGDP7 genotypes of different rice varieties by using molecular marker InDel712

A molecular marker primer InDel712 (see Table 2) is designed aiming at the 18bp insertion/deletion polymorphic site. The PCR amplification system is as follows: 2 XGC buffer (http:// www.takarabiomed.com.cn /) 8. mu.l, dNTP 4. mu.l, InDel712 primer (forward + reverse) 2. mu.l (2mM),

DNA template 2. mu.l, r-Taq (http:// www.takarabiomed.com.cn /) 0.3. mu.l, ddH₂The volume of O is filled to 25 μ l. The PCR reaction program is: PCR amplification was performed at 95 ℃ for 5 minutes, 95 ℃ for 30 seconds, 64 ℃ for 30 seconds, 72 ℃ for 30 seconds, and 35 cycles at 72 ℃ for 5 minutes and 25 ℃ for 1 minute. Adding bromophenol blue into the PCR amplification product, loading the PCR amplification product into a 3% agarose gel for electrophoresis (100 watts) for 15 minutes, and then performing gel photography on an imaging system. The detection results are shown in FIG. 7. The rice variety with 18bp insertion is of the same band type as Chuan 7(C7), while the rice variety without insertion is of the same band type as Nanyang occupation (NYZ).

The transgene of the invention comprises the following specific steps:

the correctly cloned plasmid is introduced into the Sichuan 7 genotype of the SGDP7 near isogenic line through the agrobacterium-mediated rice genetic transformation system

Inducing callus, subculturing, pre-culturing, infecting, co-culturing, screening the callus with hygromycin resistance, differentiating, rooting, training seedlings and transplanting to obtain transgenic plants. Agrobacterium-mediated rice (japonica rice subspecies) genetic transformation system was optimized based on the method reported by Hiei et al (see: Efficient transformation of rice, Oryza sativa L., mediated by Agrobacterium and sequence analysis of the bases of the T-DNA, 1994, Plant Journal 6: 271-282).

The main steps of genetic transformation, culture medium and the method for preparing the same are as follows:

(1) reagent and solution abbreviations

The abbreviations for the phytohormones used in the medium of the present invention are as follows: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin ); KT (Kinetin ); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indoleacetic acid); 2,4-D (2, 4-dichlorphenoxyacetic acid, 2,4-Dichlorophenoxyacetic acid); AS (acetosyringone); CH (Casein enzymic Hydrolysate, hydrolyzed Casein); HN (Hygromycin B, Hygromycin); DMSO (Dimethyl Sulfoxide); n6max (N6 macronutrient solution); n6mix (N6 trace element composition solution); MSmax (MS macronutrient component solution); MSmix (MS microelement component solution)

(2) Main solution formulation

1) N6 medium macroelement mother liquor (prepared as 10-fold concentrated solution (10 ×)):

the reagents are dissolved one by one, and then the volume is adjusted to 1000 ml by distilled water at room temperature.

2) Preparing N6 culture medium microelement mother liquor (according to 100 times of concentrated solution (100X))

The above reagents were dissolved at room temperature and made up to 1000 ml with distilled water.

3) Ferric salt (Fe2EDTA) stock solution (prepared as 100X concentrate);

3.73 g of disodium ethylene diamine tetraacetate (Na2 EDTA.2H2O) and 2.78 g of FeSO 4.7H2O are respectively dissolved, mixed and added with distilled water to be constant volume to 1000 ml, and the mixture is heated and bathed for 2 hours at 70 ℃ and stored for standby at 4 ℃.

4) Vitamin stock solution (prepared according to 100X concentrated solution)

Adding distilled water to a constant volume of 1000 ml, and storing at 4 ℃ for later use.

5) MS culture medium macroelement mother liquor (MSmax mother liquor) (prepared according to 10X concentrated solution)

6) MS culture medium microelement mother liquor (MSmin mother liquor) (prepared according to 100X concentrated solution)

7) Preparation of 2,4-D stock solution (1 mg/ml):

weighing 100 mg of 2,4-D, dissolving with 1 ml of 1N potassium hydroxide for 5 minutes, adding 10 ml of distilled water to dissolve completely, then fixing the volume to 100 ml, and storing at room temperature.

8) Preparation of 6-benzylaminopurine (6-BA) stock solution (1 mg/ml):

weighing 100 mg of 6-BA, dissolving for 5 minutes by using 1 ml of 1N potassium hydroxide, adding 10 ml of distilled water to dissolve completely, then fixing the volume to 100 ml, and storing at room temperature.

9) Formulation of stock solutions of naphthaleneacetic acid (NAA) (1 mg/ml):

weighing 100 mg of NAA, dissolving with 1 ml of 1N potassium hydroxide for 5 minutes, adding 10 ml of distilled water to dissolve completely, fixing the volume to 100 ml, and storing at 4 ℃ for later use.

10) Formulation of Indole Acetic Acid (IAA) stock solution (1 mg/ml):

weighing 100 mg of IAA, dissolving with 1 ml of 1N potassium hydroxide for 5 minutes, adding 10 ml of distilled water to dissolve completely, fixing the volume to 100 ml, and storing at 4 ℃ for later use.

11) Preparation of glucose stock solution (0.5 g/ml):

weighing 125 g of glucose, dissolving with distilled water to a constant volume of 250 ml, sterilizing and storing at 4 ℃ for later use.

12) Preparation of AS stock solution:

weighing 0.392 g of AS, adding 10 ml of DMSO for dissolving, subpackaging into 1.5 ml of centrifuge tubes, and storing at 4 ℃ for later use.

13)1N potassium hydroxide stock solution

Weighing 5.6 g of potassium hydroxide, dissolving with distilled water to a constant volume of 100 ml, and storing at room temperature for later use.

(3) Culture medium formula for rice genetic transformation

1) Induction medium

Adding distilled water to 900 ml, adjusting pH to 5.9 with 1N potassium hydroxide, boiling, diluting to 1000 ml, packaging into 50 ml triangular flask (25 ml/bottle), sealing, and sterilizing at 121 deg.C for 25 min;

2) subculture medium

Adding distilled water to 900 ml, adjusting pH to 5.9 with 1N potassium hydroxide, boiling, diluting to 1000 ml, packaging into 50 ml triangular flask (25 ml/bottle), sealing, and sterilizing.

3) Pre-culture medium

Adding distilled water to 250 ml, adjusting pH to 5.6 with 1N potassium hydroxide, sealing, and sterilizing as above.

The medium was dissolved by heating and 5 ml of glucose stock solution and 250. mu.l of AS stock solution were added before use and dispensed into petri dishes (25 ml/dish).

4) Co-culture medium

Adding distilled water to 250 ml, adjusting pH to 5.6 with 1N potassium hydroxide, sealing, and sterilizing by conventional method.

5) Suspension culture medium

Adding distilled water to 100 ml, adjusting pH to 5.4, subpackaging into two 100 ml triangular bottles, sealing, and sterilizing according to the above method.

1 ml of sterile glucose stock solution and 100. mu.l of AS stock solution were added before use.

6) Selection medium

Adding distilled water to 250 ml, adjusting pH to 6.0, sealing, and sterilizing as above.

The medium was dissolved before use and added to 250. mu.l of HN (50 mg/ml) and 400. mu.l of CN (250 mg/ml) and dispensed into petri dishes (25 ml/dish). (Note: the concentration of carbenicillin in the first selection medium was 400 mg/L, and the concentration of carbenicillin in the second and subsequent selection media was 250 mg/L).

7) Pre-differentiation culture medium

Adding distilled water to 250 ml, adjusting pH to 5.9 with 1N potassium hydroxide, sealing, and sterilizing as above.

The medium was dissolved before use, 250. mu.l of HN (50 mg/ml) 250. mu.l of CN (250 mg/ml) and dispensed into petri dishes (25 ml/dish).

8) Differentiation medium

Distilled water was added to 900 ml, and the pH of the medium was adjusted to 6.0 with 1N potassium hydroxide.

Boiling, adding distilled water to 1000 ml, packaging into 50 ml triangular flask (50 ml/bottle), sealing, and sterilizing.

9) Rooting culture medium

Distilled water was added to 900 ml, and the pH of the medium was adjusted to 5.8 with 1N potassium hydroxide.

Boiling, adding distilled water to 1000 ml, packaging into raw tube (25 ml/tube), sealing, and sterilizing.

(4) Agrobacterium-mediated genetic transformation procedure

Callus induction

Shelling mature SGDP7 near isogenic line Chuan 7 genotype seeds, then sequentially treating the seeds with 70% ethanol for 1 minute, and disinfecting the surfaces of 0.15% mercuric chloride (HgCl2) seeds for 15 minutes;

washing the seeds with sterilized water for 4-5 times;

placing the seeds on an induction medium;

the inoculated culture medium is placed in a dark place for culturing for 4 weeks at the temperature of 25 +/-1 ℃.

3.2 callus subculture

The bright yellow, compact and relatively dry embryogenic calli were selected and placed on subculture medium for 2 weeks in the dark at 25 + -1 deg.C.

3.3 preculture

Compact and relatively dry embryogenic calli were selected and placed on pre-culture medium for 2 weeks in the dark at 25 + -1 deg.C.

3.4 Agrobacterium culture

1) Agrobacterium EHA105 (a strain from an Agrobacterium strain publicly used by CAMBIA) was pre-cultured for two days at 28 ℃ in LA medium with a corresponding resistance selection (see: preparation of LA medium J. SammBruke et al, molecular cloning instructions, third edition, King Dong Yan et al (translation), scientific Press, 2002, Beijing);

the Agrobacterium is transferred to a suspension medium and cultured on a shaker at 28 ℃ for 2-3 hours.

3.5 Agrobacterium infection

1) Transferring the pre-cultured callus to a sterilized bottle;

adjusting the suspension of Agrobacterium to OD 6000.8-1.0;

soaking the callus in agrobacterium tumefaciens suspension for 30 minutes;

transferring the callus to sterilized filter paper and sucking to dry; then placed on a co-culture medium to be cultured for 3 days at a temperature of 19-20 ℃.

3.6 callus wash and selection culture

1) Washing the callus with sterilized water until no agrobacterium is visible;

soaking in sterilized water containing 400 mg/L Carbenicillin (CN) for 30 min;

transferring the callus to sterilized filter paper and sucking to dry;

transferring the callus to selective medium for selective culture for 2-3 times, each time for 2 weeks.

3.7 differentiation

1) Transferring the resistant callus to a pre-differentiation culture medium and culturing for 5-7 days in a dark place;

transferring the pre-differentiation cultured callus to a differentiation culture medium, and culturing under illumination at 26 ℃.

3.8 taking root

1) Cutting off roots generated during differentiation;

then transferred to rooting medium and cultured for 2-3 weeks under illumination at 26 ℃.

3.9 transplantation

Washing off residual culture medium on the roots, transferring the seedlings with good root systems to a field isolation environment, and managing the field as the common field.

Through the transgenic method, three transgenic rice positive plants are successfully obtained, and further investigation on the transgenic positive plants shows that the glume number of each ear of the transgenic plants obtained by the invention is obviously reduced, and the thousand grain weight and the grain length are obviously increased (see table 3, figure 4 and figure 5).

Reference to the literature

1.Alan Coulson,John Sulston,Sydney Brenner,Jonathan Karn.(1986)Toward a physical map of the genome of the nematode Caenorhabditis elegans.Proc Natl Acad Sci U S A,83:7821-7825.

2.Daisuke Fujita,Kurniawan Rudi Trijatmiko,Analiza Grubanzo Tagle,Maria Veronica Sapasap,Yohei Koide,Kazuhiro Sasaki,Nikolaos Tsakirpaloglou,Ritchel Bueno Gannaban,Takeshi Nishimura,Seiji Yanagihara,Yoshimichi Fukuta,Tomokazu Koshiba,Inez Hortense Slamet-Loedin,Tsutomu Ishimaru,Nobuya Kobayashi*.(2013)NAL1allele from a rice landrace greatly increases yield in modern indica cultivars.Proc Natl Acad Sci U S A,2013,110:20431-20436.

3.Denis Fabre*,Dewi E.Adriani,Michael Dingkuhn,Tsutomu Ishimaru,Bermenito Punzalan,Tanguy Lafarge,Anne Clement-Vidal,Delphine Luquet.(2016)The qTSN4Effect on Flag Leaf Size,Photosynthesis and Panicle Size,Benefits to Plant Grain Production in Rice,Depending on Light Availability.Front Plant Sci,7,Article 623.

4.Fumio Taguchi-Shiobara*,Tatsuya Ota,Kaworu Ebana,Taiichiro Ookawa,Masanori Yamasaki,Takanari Tanabata,Utako Yamanouchi,Jianzhong Wu,Nozomi Ono,Yasunori Nonoue,Kazufumi Nagata,Shuichi Fukuoka,Hideyuki Hirabayashi,Toshio Yamamoto,Masahiro Yano.(2015)Natural Variation in the Flag Leaf Morphology of Rice Due to a Mutation of the NARROW LEAF 1Gene in Oryza sativa L.Genetics,201,795-808.

5.Guang-Heng Zhang,Shu-Yu Li,Li Wang,Wei-Jun Ye,Da-Li Zeng,Yu-Chun Rao,You-Lin Peng,Jiang Hu,Yao-Long Yang,Jie Xu,De-Yong Ren,Zhen-Yu Gao,Li Zhu,Guo-Jun Dong,Xing-Ming Hu,Mei-Xian Yan,Long-Biao Guo,Chuan-You Li,Qian Qian*.(2014).Lschl4from japonica cultivar,which is allelic to nal1,increases yield of indica super rice 93-11.Mol Plant,7,1350-1364

6.Xufeng Bai,Lijun Luo,Wenhao Yan,Mallikarjuna Rao Kovi,Wei Zhan and Yongzhong Xing*.Genetic dissection of rice grain shape using a recombinant inbred line population derived from two contrasting parents and fine mapping a pleiotropic quantitative trait locus qGL7.BMC Genet,2010,11:16.

7.Xufeng Bai,Yong Huang,Donghai Mao,Mi Wen,Li Zhang,Yongzhong Xing*.(2016)Regulatory role of FZP in the determination of panicle branching and spikelet formation in rice.Sci Rep-UK,6:19022.

8.Yuexing Wang,Guosheng Xiong,Jiang Hu,Liang Jiang,Hong Yu,Jie Xu,Yunxia Fang,Longjun Zeng,Erbo Xu,Jing Xu,Weijun Ye,Xiangbing Meng,Ruifang Liu,Hongqi Chen,Yanhui Jing,Yonghong Wang,Xudong Zhu*,Jiayang Li*,Qian Qian*.Copy number variation at the GL7locus contributes to grain size diversity in rice.Nat Genet,2015,47:944-948。

SEQUENCE LISTING

<110> university of agriculture in Huazhong

<120> silencer CNV-18bp of rice panicle development gene and application thereof in rice yield improvement

<130>

<141> 2017-05-10

<160> 5

<170> PatentIn version 3.1

<210> 1

<211> 18

<212> DNA

<213> Rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(18)

<223>

<400> 1

gcacgcacgc acggacgc 18

<210> 2

<211> 8262

<212> DNA

<213> Rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(8262)

<223>

<400> 2

tctgcagaac aagtgaagtt agcctctctc tctctctctc tcgtctacga ccacaccatg 60

catcatcatc atcatcagca atgtgcttac gtgaacagtg ctctactcct acacatatca 120

tcctatcctt ctacccccgt tccatccatc tgtactgtgt acatcatctc gactcttctt 180

atcctctaca cgcacgacac ttggactgca tgcccggtcg gtcaagagct gagctagcta 240

tacccatcac atgtatacat ttgacagcta tatgtatata gactagtgtg agtgtgtgac 300

acactggtag tcgtctacta gtgagtagtg actcgagctg cttgctatgc tattgctatc 360

tagctacctg tgtgtcatgc ctttcactcg ctcatggccg ggatttacct tttcgttttg 420

gaaataaaaa aacccttctc accggtttac ccaaaaactg ataaaatcgg ggattttcaa 480

tcaatgtact acggaaacta gcctaaaact gatatgtaac cctaaattga ccgtgggcag 540

tttcacgaaa accaagcggt tctcgtgaaa accgatcgtt ttttgaaacc tgctcatagc 600

tgtatcttta acacatgaaa aatggtcaga gacattatga aaatgattat tgaccggtca 660

aaattttttc atatttacca atttaactat ttattaacga ctgcaaaatg tcgttggaaa 720

agaaaaacaa aaatgctata tgggagaaat ggaaaaggtc aaaaacgata tgaaaacaat 780

gctcagttgg ccgataattt ctccttttta gaacaagaat ttctagctat caccatttcc 840

attccttatt attgatgctt tgtatggtaa caagagagaa aattaaaaaa tgttgtagct 900

agggttgtta aatgttgtgt aaaaaagaaa gaaaagaaaa caggtactcc tacgtagtaa 960

gacaccagaa gcaagtcgta gttgagtaca aagcgagagg cgcgccgggg gagagtatgg 1020

cgccggccgg tcgcgcgcgg cggtgtgcat tgcgccggtg gtgcatacat agcccgcgcg 1080

cggcgtgcgt gcgcagggca gctaccacag ctagctagcc gaacgatcga tcgatcggcg 1140

cggcgcgcgc gcagacgccg tcacgcacgc acgcacggac gcgcacgcac gcacggacgc 1200

gcacgcgcac gcgcgcgccc acgtcctggg agcggccggc gcggcgcggc agcccagagc 1260

gcgcgctata gtagctagcg ttgtcggcgc cgtagccggt gtacaagtct ctcgtgcgcg 1320

ccgcggccgt atggctcggc tctgtggctg tggctggctg gcgtcccgcg gggcaatgcg 1380

gcggcgacac gtcgtcccac ggggaggcca ctctcactcg cacgacctac tgttcccttg 1440

tacacggcgt cggcgtagcc gtctccattc ttttttactg cctctacctt atcatctcac 1500

caatcccaat ggcatgcata tacttttttt ttcttttacg aaaccatatg ttttatgtta 1560

cagtgtaagt gtttcaatgt tttctaaacg atcatatttc ccgtccttaa gaatctacgg 1620

ggaacaatgt aacacgaggt atcaatataa tataaccgtt ggtttagcag gggtatgatc 1680

gggctaacga aaatgagata ccgctgctaa tcactgccca ctcagccaat agcgacatcc 1740

tctgtgccac tctcttctcc tctgttccat ctaccgcatc aggtcgtccg tgccggccgt 1800

ctgcgccgcg ccgccatcgc acatcgaggt caccacacta cagtcgcatc gaggctacca 1860

ccgggaatgt ccccatctag gattcgtgac cttcatgccc gtgctctcgc caccatctct 1920

cgttgccctt gcctctcgcc accccagccc tgcgatttag ggctagtttc tctcactgct 1980

cttccccggc gatttttttc ctatcgtgaa agagagaaga aagcgaaaca cgaaagaaag 2040

tgatgccgca cgtgaaagta cagacgtacc ctctcaccca cgataccgct tcaatgaggt 2100

acggacgatt tttgaccgtt ggatgatgct agatcaacgg tcacgatttg ccatttggta 2160

ctgcacaacc ttaagaacag taaaatacct cttttaaaac tagttaagtt aaaccaacaa 2220

cattagttaa gaaatcaaca aataaaaatc catacaagag tccttaaact caaatcctta 2280

gaaaacacat accaaattgt ttagcaactg attaagcttc taatcttagc agtcggttag 2340

cagtttggaa aacatgctaa aaggaaaaca ttgccactac tagctcacta ccgaacgggg 2400

ctttataata gaactccaag actctgatct ctaacataca ttttttgtca ccatcattct 2460

ccttaagcaa caacgaccaa caccgatgtc ccctgaaaat cactcatggc atacacttct 2520

accatgtatc ttacttaaat ccaaaaaaac atatatatct attatatagt atgtagatat 2580

ctaccacaca tcatatctcc attcttctac ctacatagcc ttgcccaggc tgctgctgca 2640

gggtccaggg agtggttgct gcgtgctctg cctctgcctc tgcatccatg atcatccttg 2700

taggcaaagc tcgcacaagt ggcctagtag ctgggactgg gagagacgaa gatgcatttc 2760

atctctgtgt gtataattgc atgtgtagac agtagtagtg tgtagagtgt agagccagcc 2820

tatagcaatg tatggtcagg agcccccttt tgcaaggagc tgtagtctcg tacttgtcag 2880

ttttagctcc aacacataca ctcctctgta ctagcttctt cttcttctac tactactctc 2940

agtttagata tcatatgctg cttcactgat catcacctgc cacctgccaa tcccaagtaa 3000

acaaaagagt taacaacaag aaaccatgca tgcttatgtt aatcattgtt cagtcaaatg 3060

caggacaatg ccattgggga tgatcagacc aatcaacatg gatcatagga acttgaagag 3120

tgggaacaat ctttcattcg ttagatcagt gccaaatcat catcttcagt tgcgtaattg 3180

cttgtcattt ttggtggtcg atataaaatg ggacaagtgt gctgtagtgc actctcctct 3240

tggtacttgg tcgcttgttt aattaataac ttagtaatca atcacataga caacagcaac 3300

actaacactg tgagatcgaa ctgtttgacc agtacaataa ccttgtccaa tctgcgagct 3360

gaagatttga acttgatgca gtactaatta attgccatca attatctgaa aactagaaac 3420

agagttttcg tagcaagcac tgacaattgt taaggtacca atacaattgg aatgatatcc 3480

accaaaatat ctaatatatg gaagtttttt ggaattagca tagaatcaac aattgaaaat 3540

cagagttcaa attcgaaatg attaaagtaa gcgttctatg tggagctttt gttctgtgaa 3600

ttgtgagtgc accttttcac tactaaactc tgtctgatct ccttttattc aaaccacaac 3660

ttggtgagac aaatgtgcat gccccaagtg gtctttttct acctttccct tgattatgtt 3720

tagtgaactg ccttgcatac ttctacaatt gcacatacac acacacacac acaaacaccc 3780

acacccacac tgtcctagca tacatattat ttttttgggg tgtgtgtctt ttccacaaaa 3840

cttgttggtt ctataaggga ggtctaaagt caaagtagtg caaaggccac ccccaaaacc 3900

cgggtgtgac cctaaggaat agacaagaac atgtgtcttg tgtgctgtgc tcgtttccat 3960

cataattata attatcagtt taatctggtc tctctgcaaa ttaactaact tatgcatttc 4020

tagtacatct tgtcaaggcc attgtgtgga gcctccttgc ccttagattt ctcctttttt 4080

ctccccatct ccagttgaac accaccttct cctcctcaca ccttttcaga tgtctagcta 4140

gggcttgacc tgaccccatc ttgcaatgca atattttcac aggccaacac atatatgcca 4200

aattcacaca caagagaggg gtgtagtagg ggaagtcaag gtcatcagat aagatctctc 4260

tctctgcatg cactctacac atgtagctct cctctcattg tactatactt gcatttgtag 4320

gaagctaagg cattagatgc aagaattgcc ccaaatgcag ctctccataa gaatccaaat 4380

tctcattgtt ctcatgaggg atggaagtca aggctttgta cactccaaga aaagcctttt 4440

ctagatgctc caaagttaaa caaacaagag gaattaacag tgctaatttc actctgcatt 4500

atatatgctt aattaatcaa ttgcaaacat ttttttcata agaaataaaa aaatcagcat 4560

atccactaga ttgatacttc atccgtttca ggttctaaga cctatatgaa tgtgtgcaat 4620

gctaaaatgt cttataatta acttgaaacg aagatagtat agattatgtg atgttctcta 4680

tttgaagcct tttacaagat gctattacac aacagctaat taattaatgt ggaggagaaa 4740

ttagacaggt ggccggtgaa aagtagtatc cacctcagtt gttgttttgc ttaaacctca 4800

catggcacac acttaacagt cggtcgagtt gatgttgcta tatacattgg atgcattacc 4860

acctcgacga cgtcttcaat tcgtctctct ccggtttggc tatagctcta gctctctctc 4920

tttctttttg actccaaaaa ggcatctatt aggtttatta aaatggctct agagagacat 4980

gaaagggata gttctcttgt tttgctgatt taatatcact cctttcgatt tgttctcatt 5040

tgaacctaac accggcacgc acatgcgcac taatctatag aatgtccatt gtgattaaat 5100

tatcggcaag aattcaatgg gaattgtgag ggataatgat cgttacaagc attttatttt 5160

aattaatctg tctagctagc tcatgcaaat gaacaaagca aagcttcact agagaaaagg 5220

agtcaaagaa ggggagaccc actggaccct cacctcatgt gagtgtcact caaggttgca 5280

tgaatggaac caaaagaaat ataccaaaaa aattacacag ggaatgccat gagaaagagt 5340

gcccttctac acatttcaca ctgttgttca tctagttttg agggcaagct aaccactcca 5400

tttgaaagct tcttaagtgc ttatgcaatg aggcaaatat aagtatagct ttaaggtttt 5460

tttttgtttg atctttttaa cttgcatgca tgcatgtatt gcgcaagcaa gcacatggat 5520

acgaaggaaa gcaaagccat atggcctgta aaaaatgggc attattgtgt gtagtagcat 5580

tttgacatgt atcataatat atagctatag aagtagtatc ttggagcaaa gatttcattt 5640

tttaaaattt attggagcac acaaattaca attgttaaaa aggtggatga ctaggttaca 5700

tatgttctct tctttccctg atccttttcc taacccagtg gcaggcatct tttcattatc 5760

tgattagttt gtaatactgt ctaactcagg agtgaatcat gactccatgc aagcttaagg 5820

cagaaattat tagaactagc ttaattaaac cacaattaga atatatgtag gaccagcaaa 5880

atttaaagag ttgctttggc aggggcttct tggtcactag cttgattaat gtggcacttt 5940

cagatgctgc ctggtgttgg actgtatatg ttgtatgcca tacccccata ttatacctat 6000

aggatgataa ctttgcatac atgtgtggta gagagctagc atgcattgtc atcataaatg 6060

aagtgtatga aaactaaggg gagaacattc caaccattct taattttgaa gcaacacact 6120

cacacacaaa cattagtagc acacaacatt cccctctact ggtcctaagg ttaaattagg 6180

gcacacaaat ccaaacctca cattggaaaa caagatgaat acttacaaac taaccttgat 6240

atgatgcagc tataggtcta gctaacacaa gtgcagtgca tgcatgccac ccctctctgc 6300

catctcatcc acagtgacct ctctctccaa gacccattta aataccaccc ttgaatccct 6360

cccatttgag cactacacac aagctaagct tagctcgagc tcattcatat accttagctt 6420

agctaaagcc tagctcactc tcactctcac tcttgcattg cattgcaata ttgcaatatt 6480

gcattggtca tgaacactcg aggcagcggc agtagcagca gcagcagcag cagccaggcc 6540

agcctgatgg cgttctcgga gccgccgaag ccggcgagcc agccgtcgcc gccgtcgtcg 6600

ccgatgagcg agcggccgcc gtcggggcgc agcaggcggc gcgcgcagga gccagggagg 6660

ttcctcggcg tgcggcggcg gccgtggggg aggtacgctg ccgagatacg cgacccgacc 6720

accaaggagc ggcactggct gggcacgttc gacacggcgc aggaggcggc gctggcgtac 6780

gaccgcgccg cgctgtccat gaagggcgcc caggcgcgca ccaacttcgt ctacacccac 6840

gccgcctaca actacccgcc gttcctcgcg ccgttccacg cgccgcagta cgccgctgcc 6900

gccgccgcgc cgtcctcggt gcagtacggc ggcggcgtgg gcgcggcgcc gcacattggc 6960

tcgtacggtc accaccacca ccaccaccac caccacggac atggcgcggc gtcgggcgcg 7020

tcgtcggtgg gcgagtgctc gacgatgccg gtgatggtcc cggttgatcc ccaccgctcc 7080

agcatgtcgt cgtcgctgct ggacatggac aggaacggcc acgacttcct cttctccggc 7140

gccgacgaca actccgggta cctgagcagc gtggtgccgg agagctgcct ccggccgagg 7200

ggcggcggcg ccgcggccga tcatcaggac atgcggcgct actccgacgc cgacgcctac 7260

ggcatgatgg ggctccggga ggacgtcgac gacctcgcgc agatggtcgc cggcttctgg 7320

ggcggcggcg acgcggcgga ccagctcggc gcctgcgggt tcccggcgag cggcggcgcc 7380

gccgacatgg tcgcctcgtc gcagggctcc gactcctact ctccattcag cttcctctcc 7440

cattgatcga gttgcaccgg gcattgcatg gctaatcacg cactttgcct ctgagcgatc 7500

atgcatatat catttcctgt ctaccatgat cagatcgagt tgatcttgat caatctgcgt 7560

ttgatcgatc gagcaagaga tcgagtagta tagcagcagc agcagcagca gctagcagta 7620

gatcgaacaa gaagagtggc ttggcgccat ttctagttaa ttaagtagag cttcttcaaa 7680

tatgtactta ggcagatgat ctctctctct ctctctctct ctctctctct ctctctctct 7740

cttctatatt agcattagta cctacttagc catcatattc atgcatgcat gcatgatata 7800

ttgcatggca cagcttgtta atttcttcta gcatcttaat caattaatcc atgacctggt 7860

tcaaaatgtc caattcgatt ttacatttta caactaacct gcaattagat gacaagaatg 7920

gcgccgcgag aagtacacac atatgtagta ctgacaatgg attgagaaat ggagaagaaa 7980

attgaagaag atgaggaact gaacgacatg ggcaacaagt acagaaaagc tgggaaaaag 8040

gagggaagca attgacagtt caacggaaag ctcacactgg acaagtgtca gccaagcaac 8100

catttcgtca ggtacttgat gctgattctt gcgtacgtca ggttcagaat ttcagaaatt 8160

cagatgatga ctgacagtgt ccgaaatgct ctctacgcaa gtgcaggggt tgatcaaatc 8220

cctgtagctt gttttgatgt tggatcgatc catcactagt ac 8262

<210> 3

<211> 957

<212> DNA

<213> Rice (Oryza sativa)

<220>

<221> CDS

<222> (1)..(957)

<223>

<400> 3

atg aac act cga ggc agc ggc agt agc agc agc agc agc agc agc cag 48

Met Asn Thr Arg Gly Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Gln

1 5 10 15

gcc agc ctg atg gcg ttc tcg gag ccg ccg aag ccg gcg agc cag ccg 96

Ala Ser Leu Met Ala Phe Ser Glu Pro Pro Lys Pro Ala Ser Gln Pro

20 25 30

tcg ccg ccg tcg tcg ccg atg agc gag cgg ccg ccg tcg ggg cgc agc 144

Ser Pro Pro Ser Ser Pro Met Ser Glu Arg Pro Pro Ser Gly Arg Ser

35 40 45

agg cgg cgc gcg cag gag cca ggg agg ttc ctc ggc gtg cgg cgg cgg 192

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

50 55 60

ccg tgg ggg agg tac gct gcc gag ata cgc gac ccg acc acc aag gag 240

Pro Trp Gly Arg Tyr Ala Ala Glu Ile Arg Asp Pro Thr Thr Lys Glu

65 70 75 80

cgg cac tgg ctg ggc acg ttc gac acg gcg cag gag gcg gcg ctg gcg 288

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

85 90 95

tac gac cgc gcc gcg ctg tcc atg aag ggc gcc cag gcg cgc acc aac 336

Tyr Asp Arg Ala Ala Leu Ser Met Lys Gly Ala Gln Ala Arg Thr Asn

100 105 110

ttc gtc tac acc cac gcc gcc tac aac tac ccg ccg ttc ctc gcg ccg 384

Phe Val Tyr Thr His Ala Ala Tyr Asn Tyr Pro Pro Phe Leu Ala Pro

115 120 125

ttc cac gcg ccg cag tac gcc gct gcc gcc gcc gcg ccg tcc tcg gtg 432

Phe His Ala Pro Gln Tyr Ala Ala Ala Ala Ala Ala Pro Ser Ser Val

130 135 140

cag tac ggc ggc ggc gtg ggc gcg gcg ccg cac att ggc tcg tac ggt 480

Gln Tyr Gly Gly Gly Val Gly Ala Ala Pro His Ile Gly Ser Tyr Gly

145 150 155 160

cac cac cac cac cac cac cac cac cac gga cat ggc gcg gcg tcg ggc 528

His His His His His His His His His Gly His Gly Ala Ala Ser Gly

165 170 175

gcg tcg tcg gtg ggc gag tgc tcg acg atg ccg gtg atg gtc ccg gtt 576

Ala Ser Ser Val Gly Glu Cys Ser Thr Met Pro Val Met Val Pro Val

180 185 190

gat ccc cac cgc tcc agc atg tcg tcg tcg ctg ctg gac atg gac agg 624

Asp Pro His Arg Ser Ser Met Ser Ser Ser Leu Leu Asp Met Asp Arg

195 200 205

aac ggc cac gac ttc ctc ttc tcc ggc gcc gac gac aac tcc ggg tac 672

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

210 215 220

ctg agc agc gtg gtg ccg gag agc tgc ctc cgg ccg agg ggc ggc ggc 720

Leu Ser Ser Val Val Pro Glu Ser Cys Leu Arg Pro Arg Gly Gly Gly

225 230 235 240

gcc gcg gcc gat cat cag gac atg cgg cgc tac tcc gac gcc gac gcc 768

Ala Ala Ala Asp His Gln Asp Met Arg Arg Tyr Ser Asp Ala Asp Ala

245 250 255

tac ggc atg atg ggg ctc cgg gag gac gtc gac gac ctc gcg cag atg 816

Tyr Gly Met Met Gly Leu Arg Glu Asp Val Asp Asp Leu Ala Gln Met

260 265 270

gtc gcc ggc ttc tgg ggc ggc ggc gac gcg gcg gac cag ctc ggc gcc 864

Val Ala Gly Phe Trp Gly Gly Gly Asp Ala Ala Asp Gln Leu Gly Ala

275 280 285

tgc ggg ttc ccg gcg agc ggc ggc gcc gcc gac atg gtc gcc tcg tcg 912

Cys Gly Phe Pro Ala Ser Gly Gly Ala Ala Asp Met Val Ala Ser Ser

290 295 300

cag ggc tcc gac tcc tac tct cca ttc agc ttc ctc tcc cat tga 957

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

305 310 315

<210> 4

<211> 318

<212> PRT

<213> Rice (Oryza sativa)

<400> 4

Met Asn Thr Arg Gly Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Gln

1 5 10 15

Ala Ser Leu Met Ala Phe Ser Glu Pro Pro Lys Pro Ala Ser Gln Pro

20 25 30

Ser Pro Pro Ser Ser Pro Met Ser Glu Arg Pro Pro Ser Gly Arg Ser

35 40 45

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

50 55 60

Pro Trp Gly Arg Tyr Ala Ala Glu Ile Arg Asp Pro Thr Thr Lys Glu

65 70 75 80

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

85 90 95

Tyr Asp Arg Ala Ala Leu Ser Met Lys Gly Ala Gln Ala Arg Thr Asn

100 105 110

Phe Val Tyr Thr His Ala Ala Tyr Asn Tyr Pro Pro Phe Leu Ala Pro

115 120 125

Phe His Ala Pro Gln Tyr Ala Ala Ala Ala Ala Ala Pro Ser Ser Val

130 135 140

Gln Tyr Gly Gly Gly Val Gly Ala Ala Pro His Ile Gly Ser Tyr Gly

145 150 155 160

His His His His His His His His His Gly His Gly Ala Ala Ser Gly

165 170 175

Ala Ser Ser Val Gly Glu Cys Ser Thr Met Pro Val Met Val Pro Val

180 185 190

Asp Pro His Arg Ser Ser Met Ser Ser Ser Leu Leu Asp Met Asp Arg

195 200 205

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

210 215 220

Leu Ser Ser Val Val Pro Glu Ser Cys Leu Arg Pro Arg Gly Gly Gly

225 230 235 240

Ala Ala Ala Asp His Gln Asp Met Arg Arg Tyr Ser Asp Ala Asp Ala

245 250 255

Tyr Gly Met Met Gly Leu Arg Glu Asp Val Asp Asp Leu Ala Gln Met

260 265 270

Val Ala Gly Phe Trp Gly Gly Gly Asp Ala Ala Asp Gln Leu Gly Ala

275 280 285

Cys Gly Phe Pro Ala Ser Gly Gly Ala Ala Asp Met Val Ala Ser Ser

290 295 300

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

305 310 315

<210> 5

<211> 8244

<212> DNA

<213> Rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(8244)

<223>

<400> 5

tctgcagaac aagtgaagtt agcctctctc tctctctctc tcgtctacga ccacaccatg 60

catcatcatc atcatcagca atgtgcttac gtgaacagtg ctctactcct acacatatca 120

tcctatcctt ctacccccgt tccatccatc tgtactgtgt acatcatctc gactcttctt 180

atcctctaca cgcacgacac ttggactgca tgcccggtcg gtcaagagct gagctagcta 240

tacccatcac atgtatacat ttgacagcta tatgtatata gactagtgtg agtgtgtgac 300

acactggtag tcgtctacta gtgagtagtg actcgagctg cttgctatgc tattgctatc 360

tagctacctg tgtgtcatgc ctttcactcg ctcatggccg ggatttacct tttcgttttg 420

gaaataaaaa aacccttctc accggtttac ccaaaaactg ataaaatcgg ggattttcaa 480

tcaatgtact acggaaacta gcctaaaact gatatgtaac cctaaattga ccgtgggcag 540

tttcacgaaa accaagcggt tctcgtgaaa accgatcgtt ttttgaaacc tgctcatagc 600

tgtatcttta acacatgaaa aatggtcaga gacattatga aaatgattat tgaccggtca 660

aaattttttc atatttacca atttaactat ttattaacga ctgcaaaatg tcgttggaaa 720

agaaaaacaa aaatgctata tgggagaaat ggaaaaggtc aaaaacgata tgaaaacaat 780

gctcagttgg ccgataattt ctccttttta gaacaagaat ttctagctat caccatttcc 840

attccttatt attgatgctt tgtatggtaa caagagagaa aattaaaaaa tgttgtagct 900

agggttgtta aatgttgtgt aaaaaagaaa gaaaagaaaa caggtactcc tacgtagtaa 960

gacaccagaa gcaagtcgta gttgagtaca aagcgagagg cgcgccgggg gagagtatgg 1020

cgccggccgg tcgcgcgcgg cggtgtgcat tgcgccggtg gtgcatacat agcccgcgcg 1080

cggcgtgcgt gcgcagggca gctaccacag ctagctagcc gaacgatcga tcgatcggcg 1140

cggcgcgcgc gcagacgccg tcacgcacgc acgcacggac gcgcacgcgc acgcgcgcgc 1200

ccacgtcctg ggagcggccg gcgcggcgcg gcagcccaga gcgcgcgcta tagtagctag 1260

cgttgtcggc gccgtagccg gtgtacaagt ctctcgtgcg cgccgcggcc gtatggctcg 1320

gctctgtggc tgtggctggc tggcgtcccg cggggcaatg cggcggcgac acgtcgtccc 1380

acggggaggc cactctcact cgcacgacct actgttccct tgtacacggc gtcggcgtag 1440

ccgtctccat tcttttttac tgcctctacc ttatcatctc accaatccca atggcatgca 1500

tatacttttt ttttctttta cgaaaccata tgttttatgt tacagtgtaa gtgtttcaat 1560

gttttctaaa cgatcatatt tcccgtcctt aagaatctac ggggaacaat gtaacacgag 1620

gtatcaatat aatataaccg ttggtttagc aggggtatga tcgggctaac gaaaatgaga 1680

taccgctgct aatcactgcc cactcagcca atagcgacat cctctgtgcc actctcttct 1740

cctctgttcc atctaccgca tcaggtcgtc cgtgccggcc gtctgcgccg cgccgccatc 1800

gcacatcgag gtcaccacac tacagtcgca tcgaggctac caccgggaat gtccccatct 1860

aggattcgtg accttcatgc ccgtgctctc gccaccatct ctcgttgccc ttgcctctcg 1920

ccaccccagc cctgcgattt agggctagtt tctctcactg ctcttccccg gcgatttttt 1980

tcctatcgtg aaagagagaa gaaagcgaaa cacgaaagaa agtgatgccg cacgtgaaag 2040

tacagacgta ccctctcacc cacgataccg cttcaatgag gtacggacga tttttgaccg 2100

ttggatgatg ctagatcaac ggtcacgatt tgccatttgg tactgcacaa ccttaagaac 2160

agtaaaatac ctcttttaaa actagttaag ttaaaccaac aacattagtt aagaaatcaa 2220

caaataaaaa tccatacaag agtccttaaa ctcaaatcct tagaaaacac ataccaaatt 2280

gtttagcaac tgattaagct tctaatctta gcagtcggtt agcagtttgg aaaacatgct 2340

aaaaggaaaa cattgccact actagctcac taccgaacgg ggctttataa tagaactcca 2400

agactctgat ctctaacata cattttttgt caccatcatt ctccttaagc aacaacgacc 2460

aacaccgatg tcccctgaaa atcactcatg gcatacactt ctaccatgta tcttacttaa 2520

atccaaaaaa acatatatat ctattatata gtatgtagat atctaccaca catcatatct 2580

ccattcttct acctacatag ccttgcccag gctgctgctg cagggtccag ggagtggttg 2640

ctgcgtgctc tgcctctgcc tctgcatcca tgatcatcct tgtaggcaaa gctcgcacaa 2700

gtggcctagt agctgggact gggagagacg aagatgcatt tcatctctgt gtgtataatt 2760

gcatgtgtag acagtagtag tgtgtagagt gtagagccag cctatagcaa tgtatggtca 2820

ggagccccct tttgcaagga gctgtagtct cgtacttgtc agttttagct ccaacacata 2880

cactcctctg tactagcttc ttcttcttct actactactc tcagtttaga tatcatatgc 2940

tgcttcactg atcatcacct gccacctgcc aatcccaagt aaacaaaaga gttaacaaca 3000

agaaaccatg catgcttatg ttaatcattg ttcagtcaaa tgcaggacaa tgccattggg 3060

gatgatcaga ccaatcaaca tggatcatag gaacttgaag agtgggaaca atctttcatt 3120

cgttagatca gtgccaaatc atcatcttca gttgcgtaat tgcttgtcat ttttggtggt 3180

cgatataaaa tgggacaagt gtgctgtagt gcactctcct cttggtactt ggtcgcttgt 3240

ttaattaata acttagtaat caatcacata gacaacagca acactaacac tgtgagatcg 3300

aactgtttga ccagtacaat aaccttgtcc aatctgcgag ctgaagattt gaacttgatg 3360

cagtactaat taattgccat caattatctg aaaactagaa acagagtttt cgtagcaagc 3420

actgacaatt gttaaggtac caatacaatt ggaatgatat ccaccaaaat atctaatata 3480

tggaagtttt ttggaattag catagaatca acaattgaaa atcagagttc aaattcgaaa 3540

tgattaaagt aagcgttcta tgtggagctt ttgttctgtg aattgtgagt gcaccttttc 3600

actactaaac tctgtctgat ctccttttat tcaaaccaca acttggtgag acaaatgtgc 3660

atgccccaag tggtcttttt ctacctttcc cttgattatg tttagtgaac tgccttgcat 3720

acttctacaa ttgcacatac acacacacac acacaaacac ccacacccac actgtcctag 3780

catacatatt atttttttgg ggtgtgtgtc ttttccacaa aacttgttgg ttctataagg 3840

gaggtctaaa gtcaaagtag tgcaaaggcc acccccaaaa cccgggtgtg accctaagga 3900

atagacaaga acatgtgtct tgtgtgctgt gctcgtttcc atcataatta taattatcag 3960

tttaatctgg tctctctgca aattaactaa cttatgcatt tctagtacat cttgtcaagg 4020

ccattgtgtg gagcctcctt gcccttagat ttctcctttt ttctccccat ctccagttga 4080

acaccacctt ctcctcctca caccttttca gatgtctagc tagggcttga cctgacccca 4140

tcttgcaatg caatattttc acaggccaac acatatatgc caaattcaca cacaagagag 4200

gggtgtagta ggggaagtca aggtcatcag ataagatctc tctctctgca tgcactctac 4260

acatgtagct ctcctctcat tgtactatac ttgcatttgt aggaagctaa ggcattagat 4320

gcaagaattg ccccaaatgc agctctccat aagaatccaa attctcattg ttctcatgag 4380

ggatggaagt caaggctttg tacactccaa gaaaagcctt ttctagatgc tccaaagtta 4440

aacaaacaag aggaattaac agtgctaatt tcactctgca ttatatatgc ttaattaatc 4500

aattgcaaac atttttttca taagaaataa aaaaatcagc atatccacta gattgatact 4560

tcatccgttt caggttctaa gacctatatg aatgtgtgca atgctaaaat gtcttataat 4620

taacttgaaa cgaagatagt atagattatg tgatgttctc tatttgaagc cttttacaag 4680

atgctattac acaacagcta attaattaat gtggaggaga aattagacag gtggccggtg 4740

aaaagtagta tccacctcag ttgttgtttt gcttaaacct cacatggcac acacttaaca 4800

gtcggtcgag ttgatgttgc tatatacatt ggatgcatta ccacctcgac gacgtcttca 4860

attcgtctct ctccggtttg gctatagctc tagctctctc tctttctttt tgactccaaa 4920

aaggcatcta ttaggtttat taaaatggct ctagagagac atgaaaggga tagttctctt 4980

gttttgctga tttaatatca ctcctttcga tttgttctca tttgaaccta acaccggcac 5040

gcacatgcgc actaatctat agaatgtcca ttgtgattaa attatcggca agaattcaat 5100

gggaattgtg agggataatg atcgttacaa gcattttatt ttaattaatc tgtctagcta 5160

gctcatgcaa atgaacaaag caaagcttca ctagagaaaa ggagtcaaaa aaggggagac 5220

ccactggacc ctcacctcat gtgagtgtca ctcaaggttg catgaatgga accaaaagaa 5280

atataccaaa aaaattacac agggaatgcc atgagaaaga gtgcccttct acacatttca 5340

cactgttgtt catctagttt tgagggcaag ctaaccactc catttgaaag cttcttaagt 5400

gcttatgcaa tgaggcaaat ataagtatag ctttaaggtt tttttttgtt tgatcttttt 5460

aacttgcatg catgcatgta ttgcgcaagc aagcacatgg atacgaagga aagcaaagcc 5520

atatggcctg taaaaaatgg gcattattgt gtgtagtagc attttgacat gtatcataat 5580

atatagctat agaagtagta tcttggagca aagatttcat tttttaaaat ttattggagc 5640

acacaaatta caattgttaa aaaggtggat gactaggtta catatgttct cttctttccc 5700

tgatcctttt cctaacccag tggcaggcat cttttcatta tctgattagt ttgtaatact 5760

gtctaactca ggagtgaatc atgactccat gcaagcttaa ggcagaaatt attagaacta 5820

gcttaattaa accacaatta gaatatatgt aggaccagca aaatttaaag agttgctttg 5880

gcaggggctt cttggtcact agcttgatta atgtggcact ttcagatgct gcctggtgtt 5940

ggactgtata tgttgtatgc cataccccca tattatacct ataggatgat aactttgcat 6000

acatgtgtgg tagagagcta gcatgcattg tcatcataaa tgaagtgtat gaaaactaag 6060

gggagaacat tccaaccatt cttaattttg aagcaacaca ctcacacaca aacattagta 6120

gcacacaaca ttcccctcta ctggtcctaa ggttaaatta gggcacacaa atccaaacct 6180

cacattggaa aacaagatga atacttacaa actaaccttg atatgatgca gctataggtc 6240

tagctaacac aagtgcagtg catgcatgcc acccctctct gccatctcat ccacagtgac 6300

ctctctctcc aagacccatt taaataccac ccttgaatcc ctcccatttg agcactacac 6360

acaagctaag cttagctcga gctcattcat ataccttagc ttagctaaag cctagctcac 6420

tctcactctc actcttgcat tgcattgcaa tattgcaata ttgcattggt catgaacact 6480

cgaggcagcg gcagtagcag cagcagcagc agcagccagg ccagcctgat ggcgttctcg 6540

gagccgccga agccggcgag ccagccgtcg ccgccgtcgt cgccgatgag cgagcggccg 6600

ccgtcggggc gcagcaggcg gcgcgcgcag gagccaggga ggttcctcgg cgtgcggcgg 6660

cggccgtggg ggaggtacgc tgccgagata cgcgacccga ccaccaagga gcggcactgg 6720

ctgggcacgt tcgacacggc gcaggaggcg gcgctggcgt acgaccgcgc cgcgctgtcc 6780

atgaagggcg cccaggcgcg caccaacttc gtctacaccc acgccgccta caactacccg 6840

ccgttcctcg cgccgttcca cgcgccgcag tacgccgctg ccgccgccgc gccgtcctcg 6900

gtgcagtacg gcggcggcgt gggcgcggcg ccgcacattg gctcgtacgg tcaccaccac 6960

caccaccacc accaccacgg acatggcgcg gcgtcgggcg cgtcgtcggt gggcgagtgc 7020

tcgacgatgc cggtgatggt cccggttgat ccccaccgct ccagcatgtc gtcgtcgctg 7080

ctggacatgg acaggaacgg ccacgacttc ctcttctccg gcgccgacga caactccggg 7140

tacctgagca gcgtggtgcc ggagagctgc ctccggccga ggggcggcgg cgccgcggcc 7200

gatcatcagg acatgcggcg ctactccgac gccgacgcct acggcatgat ggggctccgg 7260

gaggacgtcg acgacctcgc gcagatggtc gccggcttct ggggcggcgg cgacgcggcg 7320

gaccagctcg gcgcctgcgg gttcccggcg agcggcggcg ccgccgacat ggtcgcctcg 7380

tcgcagggct ccgactccta ctctccattc agcttcctct cccattgatc gagttgcacc 7440

gggcattgca tggctaatca cgcactttgc ctctgagcga tcatgcatat atcatttcct 7500

gtctaccatg atcagatcga gttgatcttg atcaatctgc gtttgatcga tcgagcaaga 7560

gatcgagtag tatagcagca gcagcagcag cagctagcag tagatcgaac aagaagagtg 7620

gcttggcgcc atttctagtt aattaagtag agcttcttca aatatgtact taggcagatg 7680

atctctctct ctctctctct ctctctctct ctctctctct ctcttctata ttagcattag 7740

tacctactta gccatcatat tcatgcatgc atgcatgata tattgcatgg cacagcttgt 7800

taatttcttc tagcatctta atcaattaat ccatgacctg gttcaaaatg tccaattcga 7860

ttttacattt tacaactaac ctgcaattag atgacaagaa tggcgccgcg agaagtacac 7920

acatatgtag tactgacaat ggattgagaa atggagaaga aaattgaaga agatgaggaa 7980

ctgaacgaca tgggcaacaa gtacagaaaa gctgggaaaa aggagggaag caattgacag 8040

ttcaacggaa agctcacact ggacaagtgt cagccaagca accatttcgt caggtacttg 8100

atgctgattc ttgcgtacgt caggttcaga atttcagaaa ttcagatgat gactgacagt 8160

gtccgaaatg ctctctacgc aagtgcaggg gttgatcaaa tccctgtagc ttgttttgat 8220

gttggatcga tccatcacta gtac 8244

Claims

1. The nucleotide sequence is shown in a sequence table SEQ ID NO: 2 in the application of the silencer CNV-18bp regulating and controlling sequence of the SGDP7 gene in controlling the number of glumes per ear, thousand grain weight, grain length and yield of rice, and is characterized in that the nucleotide sequence of the silencer CNV-18bp regulating and controlling sequence is shown as SEQ ID NO:1, the silencer CNV-18bp is located 5309bp upstream of the initiation codon "ATG" of SGDP7 gene.

The application of SGDP7 gene in controlling the number of glumes per ear, thousand grain weight, grain length and yield of rice is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO: 2, respectively.