CN114989280A - A rice male fertility control gene STS1, its encoded protein and application - Google Patents

Abstract

The invention provides a rice male fertility control gene STS1, a protein encoded by the gene and application thereof, belonging to the field of plant biotechnology. The nucleotide sequence of the rice male fertility control gene STS1 has one of the following groups: 1) with the nucleotide sequence shown in SEQ ID NO.1; 2) with the nucleotide shown in SEQ ID NO.1 DNA sequences with sequences that are not less than 90% similar and have the same function; 3) DNA sequences that can hybridize to the DNA of 1) the sequence described under certain conditions; 4) Any of 1) to 3) DNA sequences The present invention also protects a method for affecting plant fertility by affecting the nucleotide sequence of STS1 or by regulating the transcription and expression of the STS1 gene, and using conventional genetic transformation methods to transform the STS1 gene into such as The male-sterile rice line obtained by the above-mentioned application enables the mutant to restore the wild-type phenotype.

Description

A rice male fertility control gene STS1, its encoded protein and application

technical field

The invention relates to the technical field of plant genes, in particular to a rice male fertility control gene STS1, a protein encoded by the gene, and applications.

Background technique

Rice is one of the most important crops for human nutrition and caloric intake, providing more than one-fifth of the calories consumed by all humans. The utilization of heterosis is the main method to cultivate new rice varieties, and it is also an important way to improve rice yield. Rice is a self-pollinating plant. The pistils and stamens are placed in the same small spikelets. The method of artificial emasculation is difficult to meet the large number of hybrid seeds required for production. Therefore, hybrid rice breeding relies on male sterile germplasm resources. Discover and utilize. That is to say, using this natural male-sterile rice as the female parent and artificially assisted pollination, a large number of hybrid seeds can be produced. However, for a long time, due to the overall limited exploration and utilization of male sterile germplasm resources, the currently widely used ones are limited to the two types of male sterility (three lines) and thermosensitive male sterility (two lines). Restricted by the relationship between restoration and protection, the latter is easily affected by the environment. More importantly, the actual sterile gene resources of these two types are limited, which limits the further improvement of the advantages of hybrid rice, and also has greater risks. With the development of current biotechnology, research in recent years has shown that the direct application of rice nuclear male sterile mutants in hybrid rice can be realized by making full use of a technical means called "Seed Production Technology (SPT)" Therefore, by isolating and cloning new types of nuclear male fertility control genes and creating new sterile materials, it is of great significance to expand the germplasm range of sterile lines urgently needed in hybrid rice

SUMMARY OF THE INVENTION

In view of the problems and defects in the prior art, the present invention provides a rice male fertility control gene STS1, a protein encoded by the gene, and applications thereof. The use of this gene and its encoded protein to regulate the male fertility of rice, to mutate the encoded gene through gene editing to obtain a new sterile line of rice, and to restore the mutant fertility through transgenic technology has important application value in agricultural production. The technical scheme of the present invention is:

In a first aspect, the present invention provides a rice male fertility control gene STS1, derived from rice, whose nucleotide sequence has one of the following groups: 1) has a nucleotide sequence as shown in SEQ ID NO.1; 2) A DNA sequence that is not less than 90% similar to the nucleotide sequence shown in SEQ ID NO. 1 and has the same function; DNA sequence; 4) a DNA sequence complementary to any of the sequences 1) to 3).

It is known in the art that the rice male fertility control gene of the present invention includes a highly homologous functional equivalent sequence that is highly homologous to the STS1 gene and has the same fertility control function. The highly homologous functional equivalent sequences include DNA sequences capable of hybridizing to the nucleotide sequence of the STS1 gene disclosed in the present invention under certain conditions. The "certain conditions" used in the present invention belong to common knowledge, including denaturation (for example, under the condition of 90-96°C, a certain pH, and in the presence of an organic solvent), annealing (at 40-65°C for renaturation), extension (under the action of polymerase at 68-75°C) and other means to carry out nucleic acid hybridization.

The functional equivalent sequence also includes at least 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity with the sequence shown in the STS1 gene disclosed in the present invention, and has fertility regulation function. DNA sequences can be isolated from any plant. Among them, the percentage of sequence similarity can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm (Bioinformatics, 4(1): 11-17, 1988), Needleman-Wunsch global alignment method (J.Mol. Biol., 48(3): 443-53, 1970), Smith-Waterman local alignment (J. Mol. Biol., 147: 195-197, 1981), Pearson and Lipman similarity search method (PNAS, 85 (8): 2444-2448, 1988), the algorithm of Karlin and Altschul (Altschul et al., J. Mol. Biol., 215(3): 403-410, 1990; PNAS, 90: 5873-5877, 1993). This is familiar to those skilled in the art.

"Can be isolated from any plant" as referred to above, including but not limited to Brassica, corn, wheat, sorghum, Camelina, mustard, castor bean, sesame, cottonseed, linseed, soybean, Arabidopsis, Bean, Peanut, Alfalfa, Oat, Rapeseed, Barley, Oat, Rye, Millet, Milo, Triticale, Einkorn, Spelt, Emmer , flax, Gramma grass (Gramma grass), rubgrass, false milo, fescue, perennial wheat grass, sugar cane, red raspberry moss, papaya, banana, safflower, oil palm, cantaloupe, apple, cucumber, dendrobium, Gladiolus, Chrysanthemum, Liliaceae, Cotton, Eucalyptus, Sunflower, Brassica, Beet, Coffee, Ornamental Plants and Pines, etc. Preferably, the plants include corn, soybean, safflower, mustard, wheat, barley, rye, rice, cotton and sorghum.

In a second aspect, the present invention also provides a biological material related to the rice male fertility control gene STS1, which is any one of the following 1) to 5):

1) the protein encoding the gene STS1;

2) an expression cassette containing the gene STS1;

3) a recombinant vector containing the gene STS1;

4) a recombinant microorganism containing the gene STS1;

5) A recombinant microorganism containing the recombinant vector of 3).

Further, the protein amino acid sequence of the coding gene STS1 has the sequence shown in SEQ ID NO. 2, or an amino acid sequence similar to the amino acid sequence shown in SEQ ID NO. 2 and having the same function.

Further, during the preparation of the gene-containing STS1 expression cassette, various DNA fragments can be manipulated to provide DNA sequences in proper orientations or in correct reading frames. For this purpose, adaptors or linkers can be used to join the DNA fragments, or other manipulations can be further included to provide convenient restriction enzyme sites and the like. In addition, the expression cassettes or recombinant vectors provided by the present invention can be inserted into plasmids, cosmids, yeast artificial chromosomes, bacterial artificial chromosomes or any other vector suitable for transformation into host cells. Preferred host cells are bacterial cells, especially for cloning or storage of polynucleotides, or for transforming plant cells, such as Escherichia coli, Agrobacterium tumefaciens and Agrobacterium rhizogenes. When the host cell is a plant cell, the expression cassette or recombinant vector can be inserted into the genome of the transformed plant cell. Insertions can be positioned or random. Preferably, insertion is effected, for example, by homologous recombination. Alternatively, the expression cassette or recombinant vector may remain extrachromosomal. The expression cassette or recombinant vector of the present invention may be present in the nucleus, chloroplast, mitochondria and/or plastid of a plant cell. Preferably, the expression cassette or recombinant vector of the present invention is inserted into the chromosomal DNA of the plant cell nucleus.

Further, the recombinant microorganism can be obtained by conventional methods or gene editing means, such as operably linking a promoter sequence with a reporter gene to form a transformable construct, and then transferring the construct into a plant; or The above constructs are subcloned for expression vectors used in transient expression experiments to test the function of the promoter or its regulatory region. The choice of an appropriate expression vector for testing the function of the promoter or regulatory region will depend on the host and the Methods for introducing expression vectors into hosts are well known to those of ordinary skill in the art. For eukaryotes, the regions in the vector include regions that control transcription initiation and regions that control processing. These regions are operably linked to reporter genes including YFP, UidA, GUS gene or luciferase. Expression vectors containing putative regulatory regions located in genomic fragments can be introduced into whole tissues, such as staged pollen, or into callus tissue.

In the third aspect, the application of the present invention is as follows: the present invention provides a method for affecting plant fertility by affecting the nucleotide sequence of STS1 or regulating the transcription and expression of the STS1 gene to obtain a new rice male sterile line, the Rice male sterile lines can be used to produce hybrid seeds.

The method for affecting plant fertility refers to changing the fertility of the plant by regulating the expression of the STS1 gene, such as causing male sterility of the plant. Specifically, depending on the specific application requirements, various methods can be used to influence the expression of the STS1 gene in plants, so as to achieve the effect of regulating the male fertility of plants. More specifically, regulating the expression of the STS1 gene can be performed using many tools available to those of ordinary skill in the art, for example, by mutation, mutagenesis, introduction of antisense genes, introduction of co-suppression or hairpin structures, and gene editing, etc. , can be used to disrupt the normal expression of the STS1 gene to obtain male sterile plants.

In addition, the present invention also provides a sterile mutant sequence of the STS1 gene and a male sterile mutant material thereof. More specifically, the male sterile mutant material is a process in which the plant body loses male fertility by mutating the endogenous STS1 gene in rice, or mutating the nucleotide sequence of a gene highly homologous thereto. The "mutation" includes but is not limited to the following methods, such as gene mutation caused by physical or chemical methods, chemical methods include mutagenesis caused by treatment with mutagen such as EMS, and the mutation can also be a point mutation, or It can be DNA deletion or insertion mutation, or it can be generated by means of gene silencing such as RNAi, site-directed mutagenesis, and gene editing.

Those skilled in the art should know that the male sterile material receptors created can include, but are not limited to, all conventional indica and japonica rice varieties currently known.

Specifically, the present invention adopts the above method to construct four rice male sterility mutants, and these four rice male sterility mutants all contain a mutated male sterility gene, and the nucleosides of the mutated male sterility gene The acid sequences are shown in SEQ ID NO.6 (sts1-1), SEQ ID NO.7 (sts1-2), SEQ ID NO.8 (sts1-3), and SEQ ID NO.9 (sts1-4). Compared with the wild type, among the four male sterile mutants, the sts1-1 mutant had a nucleotide substitution at a splicing recognition site in the coding region of the gene, resulting in three erroneous transcripts; The sts1-2 mutant has a nucleotide substitution in the 7th exon of the gene, which leads to the premature formation of the terminator; the sts1-3 mutant has a nucleotide insertion in the 2nd exon of the gene, This leads to the premature formation of the terminator; the sts1-4 mutant has a deletion of two bases in the 9th exon of the gene, which leads to the premature formation of the terminator. Mutations in this gene in all four mutants ultimately resulted in premature termination of protein translation.

In a fourth aspect, the present invention also relates to a method for restoring male sterility of a rice male sterile line, comprising the steps of: using conventional genetic transformation means to transform the STS1 gene into the rice obtained by the aforementioned application. Male sterile lines, which in turn allow mutants to restore the wild-type phenotype.

In a fifth aspect, the present invention also relates to the use of a sterile rice line in rice seed production, and cross-breeding is carried out with the male sterile mutant line of rice constructed and obtained as described above as the female parent. Specifically, in certain application embodiments, the STS1 gene provided by the present invention can be used to achieve the reproduction and maintenance of male sterile lines obtained by mutation of STS1 or other similar fertility-related genes.

More specifically, the breeding and maintenance of the above-mentioned male sterile line refers to using a homozygous recessive nuclear male sterile mutant as a transformation recipient material, and transforming the three closely linked target genes into the sterile mutant recipient plant. middle. The three target genes are fertility restoration gene, pollen inactivation gene and screening gene respectively. Among them, the fertility restorer gene can restore the sterile transformation by vegetative ability, and the pollen inactivation gene can inactivate the pollen containing the transformed exogenous gene, that is, lose the fertilization ability, and the screening gene can be used for transgenic seeds and non-transgenic seeds. The sorted non-transgenic seeds are used as sterile lines to produce hybrids, and the transgenic seeds are used as maintainer lines to continuously and stably produce sterile lines. The pollen-specific expression promoter provided by the present invention can be used for the specific expression of an exogenous gene in pollen, so as to avoid the adverse effects caused by the continuous expression of the exogenous gene in other plant tissues, and can also be used in plant pollen Functional analysis and identification of growth and development-related genes; it can be used for the creation of male sterile lines and restorer lines; it can be used in pollen abortion experiments to avoid biosafety problems caused by plant transgene drift or pollen escape. The creation of plant male sterile lines and restorer lines is of great significance.

Those skilled in the art should know that suitable screening genes include but are not limited to: chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, Glyphosate resistance gene, glyphosate resistance gene. The selectable marker gene can also be red fluorescent gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, anthocyanin p1 and other genes.

Furthermore, the transgenic plants of the present invention are prepared using transformation methods known to those skilled in the art of plant biotechnology. Any method can be used to transform the recombinant expression vector into plant cells to produce the transgenic plants of the present invention. Transformation methods can include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, and microinjection, among others. In a specific embodiment of the invention, the invention uses Agrobacterium-based transformation techniques.

Compared with the prior art, the present invention can obtain the following technical effects:

1) The present invention obtains mutants of rice male reproductive development by controlling the rice male fertility STS1 gene and its encoded protein, thereby realizing artificial control of the rice reproductive process.

2) The rice mutant obtained by the present invention has no obvious difference from the source parent in the vegetative period, and after entering the reproductive growth stage, the male reproductive development is abnormal, the pollen is aborted, and a sterile line with complete male sterility is obtained. The fertility of the sterile line Stable, unaffected by environmental conditions, capable of being restored by wild-type transgenes.

3) The gene and the sterile line produced by the mutation of the gene provide the necessary elements for the construction of the third-generation hybrid breeding system. The male sterile line produced by the gene mutation is used to produce hybrid seeds. "Three-line" and "two-line" hybrid technology is of great significance, and has very important application value in the construction of hybrid rice and agricultural production.

Of course, any product implementing the present invention does not necessarily need to achieve all the above-mentioned technical effects at the same time.

Description of drawings

Fig. 1 is the phenotype diagram of STS1 gene after mutation in Example 1 of the present invention; wherein A is wild type 9311 and mutant plant type; B is wild type and mutant panicle; C is wild type and mutant floret; D Anther morphology of wild-type and mutant; E is the pollen iodine staining of mutant and wild-type.

Fig. 2 is the clone of STS1 gene in Example 2 of the present invention; wherein A and B are the SNP index maps of mutants sts1-1 and sts1-2 respectively; C is the gene structure, mutation site and method of STS1 gene; D is Three erroneous transcripts produced by mutation in sts1-1; E is the co-segregation verification of mutant sts1-1 and sts1-2 mutation sites and phenotypes.

Figure 3 is a phenotype diagram of the mutant obtained by gene editing of the STS1 gene in Example 3 of the present invention; wherein A is the gene structure of the STS1 gene and the gene editing sites of the mutants sts1-3 and sts1-4 and the results after successful editing. Base changes; B is the plant type of wild-type Nipponbare and mutant sts1-3, sts1-4; C is anther morphology of wild-type Nipponbare and mutant sts1-3, sts1-4; D is wild-type Nipponbare and mutant sts1 -3. Pollen iodine staining of sts1-4.

Figure 4 is a phenotypic diagram of the sts1-1 mutant sports restorer line in Example 4 of the present invention; wherein A is the STS1 gene structure that restores fertility; B is the plant type of wild-type 9311, mutant and complementary lines; C is the floret of wild-type 9311, mutant and complementary lines; D is anther morphology of wild-type 9311, mutant and complementary lines; E is pollen iodine staining of wild-type 9311, mutant and complementary lines.

Detailed ways

In the description of the present invention, it should be noted that, if the specific conditions are not indicated in the examples, the conventional conditions or the conditions suggested by the manufacturer are carried out. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The inventor uses bioinformatics technology well known to those skilled in the art to determine a complete rice male fertility control gene STS1 from the rice genome, the nucleotide sequence is shown in SEQ ID NO. As shown in SEQ ID NO. 2; the full-length ORF of the STS1 gene is 1986 bp in length, encoding 661 amino acids.

The present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments, which are intended to explain rather than limit the present invention.

Example 1

Phenotype of STS1 Gene Mutation

Using the conventional gene mutation technology in the field, the conventional rice variety 9311 was mutagenized, and two male sterile mutants sts1-1 and sts1-2 were found in the obtained mutant library, and the nucleotide sequences are shown in SEQ ID NO. 6. As shown in SEQ ID NO.7, the two mutants and the wild type (WT) were stably inherited after multiple generations of crosses. The phenotypic analysis found that the mutant and the wild type showed similar vegetative growth, but could not produce mature pollen grains. For the powder-free male sterility (Figure 1). The same gene STS1 was further cloned from the two mutants by genome resequencing technology, see Example 2 for specific steps.

Example 2

Cloning of STS1 gene

1. Resequencing cloning of STS1 gene mutation

Two mutants, sts1-1 and sts1-2, were used as the female parent to cross with the wild-type body, and segregating populations were constructed respectively. The sequencing data was compared with the wild-type genome to obtain a map with a SNP index of 1 (as shown in Figures A and B in Figure 2). The mutation sites in the above map were further analyzed, and the individual plants and mutation sites isolated in the population were co-segregated and verified by PCR sequencing. The primers of the dots are sts1-1F (SEQ ID NO.10), sts1-1R (SEQ ID NO.11) and sts1-2F (SEQ ID NO.12), sts1-2R (SEQ ID NO.13), respectively. The gene where the dots are located is STS1. As shown in C, D, and E in Figure 2, the mutation pattern of sts1-1 and sts1-2, the three erroneous transcripts produced by mutation in sts1-1, and the co-segregation analysis of sts1-1 and sts1-2. Thus, it was confirmed that sts1-1 and sts1-2 were different variants of the same gene STS1. Among them, the nucleotide of a splicing recognition site on the intron of the coding region of the STS1 gene was replaced, resulting in the DNA sequence shown in SEQ ID NO. 6, resulting in the production of three misplaced transcripts, thereby obtaining sts1-1 Mutant; a nucleotide in the 7th exon of the coding region of the STS1 gene is replaced, resulting in a DNA sequence as shown in SEQ ID NO. line, the sts1-2 mutant.

The sequence of the wild-type STS1 gene is shown in SEQ ID NO.1, and the encoded protein sequence is shown in SEQ ID NO.2. The full-length ORF of the STS1 gene is 1986 bp in length and encodes 662 amino acids.

2. Full-length cDNA cloning of STS1 gene

Using the RNA of the normal wild-type rice Nipponbare variety as a template, the first-strand cDNA was synthesized, and the oligonucleotides at the 5' and 3' ends of the ORF sequence of the STS1 gene were used as PCR primers (SEQ ID NO.14 and SEQ ID NO.15). ), and amplified with high-fidelity Taq enzyme Primerstar to obtain a full-length cDNA amplification product of about 1.9K MFS1 gene. The amplified product was recombined into the corresponding restriction endonuclease multiple cloning site of the vector pEntry by EcoRI restriction enzyme digestion, and the recombinant was verified by sequencing. The recombinant transition plasmid vector was named pEntry-STS1.

The oligonucleotide sequence at the 5' end is (SEQ ID NO. 14):

5’-aaaaaagcaggctccgaattcatggccacgacgctgacg-3’

The oligonucleotide sequence at the 3' end is (SEQ ID NO. 15):

5’-caagaaagctgggtcgaattcttactgtgcaactgttccatctgtt-3’

Example 3

Using gene editing to create new STS1 mutant lines

The commercial CRSPR/Cas9 gene editing vector BGK030 (commercially available) was used as the vector for rice transgene. The vector encodes a bacterial origin of replication (ori), kanamycin resistance gene (Kan ^r ), hygromycin resistance gene ( ^Hygr ), gene editing gene (Cas9), 35S promoter, NOS gene termination signal Sequence and Multiple Cloning Site (MCS) for ligation of fragments of interest. Insert two guide sequences SG RNAs of STS1 gene into the restriction endonuclease sites respectively, the sequences are shown in SEQ ID NO.

4. STS1 gene transformation experiment in rice

The above BGK030-STS1-3 and BGK030-STS1-4 were introduced into Agrobacterium EHA105 by freeze-thaw method, and infected the callus of wild-type japonica variety Nipponbare, cultured and differentiated to obtain transgenic plants. The transformed plants were screened by hygromycin; the total DNA of leaves was extracted from the positive plants, and the transformed plants were further identified by PCR. Fertility phenotypes were investigated with transgenic T1 generation to verify STS1 gene function. As shown in Figure 3, plants with one base inserted and two bases deleted at the STS1 target site exhibited male sterility, and the obtained mutants were sts1-3 and sts1-4, respectively, and the nucleotide sequences were shown in SEQ ID NO.8, SEQ ID NO.9. That is, the sts1-3 mutant is a nucleotide insertion in the second exon of the coding region of the STS1 gene; the sts1-4 mutant is a deletion of two nucleotides in the 9th exon of the coding region of the STS1 gene. .

Example 4

Fertility Restoration of STS1 Gene Mutants

The STS1 gene complementary expression vector was constructed. In this example, pCMBIYA1300 was firstly digested with PstI and SacI restriction enzymes, and the wild-type STS1 complementary fragment containing the promoter amplified by SEQ ID NO.3 and SEQ ID NO.4 was approximately 6.3K, the sequence is shown in SEQ ID NO.5. Linked into the PstI and SacI sites of pCMBIYA1300, the recombinant was identified by restriction enzyme digestion and sequencing. Then, the mutant transgenic plants with complementary expression of rice STS1 were obtained by the transgenic method described in Example 3, and the transgenic T1 generation plants were used to observe them. of transgenic positive plants restored fertility.

To sum up, the present invention changes, increases or deletes individual nucleotide sequences of STS1 through cloning technology and gene editing method, resulting in loss of function of the encoded protein, thereby obtaining a male sterile line. The resulting male sterile line has complete fertility and is not affected by environmental conditions, and the transfer of the wild-type gene can restore the fertility. The male fertility control gene provided by the present invention and the sterile line generated based on the gene will provide the necessary sterile line germplasm resources for a new hybrid breeding system, and are of great significance to hybrid rice breeding.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the patent of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

sequence listing

<110> Sichuan Agricultural University

<120> A rice male fertility control gene STS1, its encoded protein and application

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

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Val Met Ser Leu Gly Asn Asp Ser Glu Val Gly Asn Thr Ile Lys Gly

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Gly Asp Gln Asp Ala Leu Pro Ser Ser Ser Gly Thr Asp Lys Ser Pro

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Asp Asp Ile Pro Ala Asn Asn His Arg Lys Met Ala Leu Leu Phe Ala

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Leu Leu Ser Ala Cys Val Ala Asp Lys Pro Val Ser Gln Glu Glu Glu

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Asp Arg Lys Ser Thr Arg Phe Arg Lys Gly Tyr Asp Ala Arg His Arg

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Val Ala Leu Arg Leu Leu Ser Thr Trp Leu Asp Val Lys Trp Ile Lys

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Met Glu Ala Ile Glu Val Met Val Ala Cys Ser Ala Met Ala Ala Ala

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Lys Glu Gln Glu Gln Ser Gln Glu Ser Ala Ser Pro Lys Ser Lys Trp

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Glu Lys Trp Lys Arg Gly Gly Ile Ile Gly Ala Ala Ala Leu Thr Gly

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Gly Ala Leu Leu Ala Ile Thr Gly Gly Leu Ala Ala Pro Ala Ile Ala

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Ala Gly Phe Gly Ala Leu Ala Pro Thr Leu Gly Thr Leu Val Pro Val

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Ile Gly Ala Ser Gly Phe Ala Ala Met Ala Thr Ala Ala Gly Ser Val

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Ala Gly Ser Val Ala Val Ala Ala Ser Phe Gly Ala Ala Gly Ala Gly

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Gly Ile Leu Ile Ser Gly Phe Ala Phe Asp Glu Asp Asp Phe Cys Arg

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Val Leu Ser Gly Leu Leu Ala Ala Phe Ala Trp Pro Ala Thr Leu Leu

465 470 475 480

Ala Ala Thr Asp Phe Ile Asp Ser Lys Trp Ser Val Ala Ile Asp Arg

485 490 495

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

500 505 510

Gln Gly Asn Arg Pro Val Thr Leu Ile Gly Phe Ser Leu Gly Ala Arg

515 520 525

Val Ile Phe Lys Cys Leu Gln Glu Leu Ala Leu Ser Ser Asp Asn Glu

530 535 540

Gly Leu Val Glu Arg Val Val Leu Leu Gly Ala Pro Val Ser Val Lys

545 550 555 560

Gly Glu Arg Trp Glu Ala Ala Arg Lys Met Val Ala Gly Arg Phe Val

565 570 575

Asn Val Tyr Ser Thr Asp Asp Trp Ile Leu Gly Val Thr Phe Arg Ala

580 585 590

Ser Leu Leu Thr Gln Gly Leu Ala Gly Ile Gln Ala Ile Asp Val Pro

595 600 605

Gly Val Glu Asn Val Asp Val Thr Glu Leu Val Asp Gly His Ser Ser

610 615 620

Tyr Leu Ser Ala Ala Gln Gln Ile Leu Glu His Leu Glu Leu Asn Thr

625 630 635 640

Tyr Tyr Pro Val Phe Val Pro Leu Ser Ala Ala Asn Glu Glu Thr Asp

645 650 655

Gly Thr Val Ala Gln

660

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 3

gctcatcttc gggctcacc 19

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 4

gacacggcca gaaacagcaa 20

<210> 5

<211> 6353

<212> DNA

<213> STS1 gene (Oryza sativa)

<400> 5

gctcatcttc gggctcacca aggacgtcat ctttcgcgcc gccttcggca cgcgcgacgg 60

cggcggccac ggcgagctgg aggttctcct gcaggagttc tccaagctgt tcggcgcgtt 120

caacgtcggg gacttcatcc cctggctcgc gtggctcgac ccgcacggca tcaaccgacg 180

gctccgcgcg gcgcgcgccg ccctcgacag cgtcatcgac aggatcatcg acgagcacgt 240

cagcaacccc gccggcgacg aggacgccga catggtggac gacatgctcg cgttcctcga 300

cgaggccgga cgagaccaaa ccggcggcgg cggcgagctg cagggcacgc tccggctgac 360

gcgcgacaac atcaaggcca tcataatggt acgtgccatt ctgacaccac tgccatggca 420

tgcacgtcgc gttgcttgat ggagttggcg acgacaagtg caggacttcg tcttcggcgg 480

gacggagacg gtggcgtcgg cgatcgagtg ggcgatggcg gagttgctgc acagccccgg 540

cgatctccgg cggctgcagg cggagctcgc cgacgtggtg gggctcgggc gcggcgtgga 600

ggagggcgac ctggagaagc tccccttcct caggtgcgtc gccatggaga cgctgcgtct 660

ccacccgccg atcccgctgc tcctccacga ggcggcggcg gactgcgtcg tcggcgggta 720

ctccgtgccg aggggcgccc gcgtggtggt caacgtgtgg agcgtcggcc gcgacgccgg 780

cgcgtggaag ggcgacgccg gcgcgttccg gccggcgagg ttcatggcgg gcggcgaggc 840

ggcggggatg gacctcaggg gcggatgctt cgagctcctg ccgttcgggt cggggcggag 900

ggcgtgcccc gccatcgtgc tcgggatgta cgagctggag ctcgtcgtcg cgcgcctcgt 960

ccacgcgttc gggtgggcgc cgccgggcgg cgtggcgccg gaggagctcg acatggcgga 1020

cggcttcggc ctcaccgcgc cgcgcgccgc gaggctccgc gccgtgccga cgccccggct 1080

cacctgcccg atgtgacgcg cgttctcgat ttggcgcgag agatgcgcaa cgcagctcga 1140

agtgtgactg tgacagtgtg agcgtgtaat ctggtgtgga catcgtatgc atcatgtgtg 1200

cttatggata attaaatata taaataatat ttggatgtgg atttcggctg aaattttctg 1260

tcatttcgaa aaaaattcgt cctagaattc aaatagaata ttttttttct cattattttt 1320

tatagttgca cattggctac attatctgta gaagcttctt gattcacgag atctagcggg 1380

cgtgacaata tggtcaaaga attctttacc ctagacatgg gcgtcgatat ggtcgtaaag 1440

ctgctcttca gcttagtagc cacatccata atttaatctt aggattattt ttacagttga 1500

ttccgatatt ttttcatcgt aaacatgggg ttttacatca ttaaaaaccc acatataaaa 1560

tcctttccct taaaagaatg atggtctttg aagcccttat ttcttttttt aatcttcgag 1620

tatgtatgaa tagtttgatt acgtgcattt taattatgta gagatttgga atgctattcg 1680

acaattatat tatcttaatg ttacattaag tgagatgctt ttattattta aaaatcgatt 1740

atattttaaa acaaaatctg aagcactccg tggaccgtgg ttacacgtgc caggtcgacg 1800

tgttcttctg gaagaaatct atgtctactt ttgaaatgga atatactacc tccgttaaaa 1860

ataagtacaa acgtaaataa ataagtacaa acgtaaatat ccgtatgaac gttcgtctta 1920

ttccaaaaat ttataaaaat aaattataaa aaaacagtca catataaaat attatttatg 1980

ttttataatc taatagtaac aaaaaaatat taatcataaa agataaagaa aaatttaaat 2040

aagataaacg ctgaatataa ataatatttt ataaagctgt acttatttta agacggaaag 2100

agtaaaaaat tttggaaaaa tttctagtgc agcatgctgc tcatttggga ggtgtgtgct 2160

gctgctctcc agtctccccc aaccaaaatt tgaccgagtt tggcacgaaa gaaaccgcct 2220

cgacgccgtc caacggaagc cagccgaggc gaccaaccca agggtaggcc taggcgtaaa 2280

ccctgcacct gaatcgatcg ccggcgatgg ccacgacgct gacgccgacg cagcgctacg 2340

cggcgggggc gctgctggcg ctcgcgctcc gccaggccca gatccaccag tccgtcctcc 2400

tcggcgccca ccaccaccac gacgacgacg acgaggaaca ggggcgcacc agcaccagca 2460

gcggcggcgg cggcggcagc tcctcctcct cctccaactc cggcgccggc gccgacgccg 2520

acctctggac acacgactcc cacggcctcc tccgccccgt cttcaggtgt tttctcgctc 2580

ctctccgctt gtaaatttgg ggggtttgag atgaatgctt ccgctttact tggcgatcgg 2640

ggggctctgc taggttcctg gagatcgatc ccaaggcgtg gtcggggctg gaggagacgg 2700

cggcgtcgtc cgaggccaag catcacatcg gcgcggtacg gcactcactg cattttctgc 2760

cctcgtattg taattggccg tgcgttttgg tcagtgttgc agtgatggtt tggggcttag 2820

gtgtttgatt atatgcctac aagactgttc aacttcaatt tccgcgtgtg cttgaccagt 2880

gaaatttgtg ttttcgtagc atctcatatc actctcattt tgtagtttct aaggataata 2940

ttcgaagaag atggcgaaag ctcctcagac agatccgttc aggagcttgc cttggcaaaa 3000

ggagttgatg tgatggtaat gagtttgggc aatgacagtg aagtgggtaa cacaattaaa 3060

ggtggggatc aagatgcttt gcctagcagc tctggaacag ataaatcccc aggggaaagc 3120

tctcatgatg accagttagg aatcaataaa ttgaccttag atgatattcc tgctaacaat 3180

caccggaaga tggcattgct gtttgctctt ctctcggctt gtgttgctga taaacctgta 3240

tcacaagagg aagaggacag aaaatcaacc cgcttcagaa agggttatga tgctcggcat 3300

cgtgttgctc ttcggctgct atctacatgg cttgatgtga agtggatcaa aatggtttgt 3360

acagactaaa ctttttttgga ttcagcagag taatcactat gggtaccaaa caatatccta 3420

aggcaattaa agaaaaaaga acccccaatg tttttcatgt ttcaccaaat ttgtcaccaa 3480

ccatgctttt tcgctgcagg aagcaattga ggttatggtt gcatgctctg ccatggcagc 3540

agcgaaagaa caagaacaat cacaagaaag cgcatcaccc aaaagtaaat gggaaaaatg 3600

gaagcgtgga ggaataattg gtgcagctgc tttgactgga ggagcactgt tggctattac 3660

tgggggtatg aaaaaacttc agaaattctg ctggacgctg aacatctgaa ctagtttctg 3720

tcgctcatat tactctaaac ggttgatatt ctcttccaac ttgacaggtt tagctgctcc 3780

agcaattgct gcagggtttg gtgctcttgc tccaaccctg ggcacacttg tccctgttat 3840

aggagctagc ggatttgctg ctatggctac cgctgcagga tctgttgctg gctctgtggc 3900

agttgctgca tcatttggag gtgtgtctac atgttcttca aaacccttct ggtgatgttg 3960

ttttgagctt tcgtttcgtc atgctacatt actatcagct gcagtctccc tttccttttg 4020

tcatgagggt tgcaatattg tttagtgaac ttacccaaga atatgcacac aacactttta 4080

ggggtgcatt ttacaattag ataatagttc gatggattac cagaagaagg ataagactta 4140

tacaatgtat gacccaaaat tgtgagaaac atatggatga aagcgtaatg agaatagttg 4200

tggtgtggaa attttctatt tattcatgct tatcctttga aatgatattt ttcaattagc 4260

tgctggagct ggcttgacgg ggagcaaaat ggccagaaga attgggagcg tgaaagaatt 4320

tgagttcaaa cctattggtg aaaaccataa tcagggtgtg agttcctttt ctctttctga 4380

tgtcacaaca aatttcattc tactagattt ttgttcaccc ttttagtagt caaactgatc 4440

tggtctggtt aaatgttctc aacactttca gcggcttgca gttggcatct taatttccgg 4500

atttgctttt gatgaggatg acttttgtag accttgggaa ggatggcagg ataacctaga 4560

gaggtataga gtggtttttt tgacatgttc tttttgctga tcactattct agtactttgt 4620

catgtgttac cagctaagag catccttaaa aatataattt caagagttga ataatgtctg 4680

gcaggtacat ccttcaatgg gagtctaagc atataattgc agtgagtaca gcaatacagg 4740

attggctgac atcaagtaat atttgtagtt ttgcccattt acactatcag caaaaacttc 4800

taatttacac tgttatttat gattcaggac ttgctatgga attgatgaag caaggtgcaa 4860

tgagaaccgt attaagtggt cttcttgcag catttgcatg gccagctaca ctgcttgcgg 4920

ctacagactt tattgatagt aaatggtctg ttgctattga caggtaattg caaactctcc 4980

catttctctt atgttgcttg cagagttgca gtgcacactg actatctaca gtattggtat 5040

tcttctgttg atcgacacat aagtttgtgt attcaaattt atgaagatta tgacgtaaca 5100

tttagacaaa cttttaatgt atatatccca cagtacatat attgcagtta aagacatcct 5160

agcaatttgt taacttagcc aatcaaaatc aactgtatct tctcagatca gataaggcag 5220

gaaaaatgct tgctgaagtg cttctcaagg gattgcaagg aaataggtaa ctccatatgc 5280

atttatttca ttagcgatgt tggccgctca gaaatatgcc cccctaatgc caattttttc 5340

ccaggcctgt gactctcatt gggttttcac taggtgcacg tgttatattt aagtgtttac 5400

aggaacttgc tttatcgagt gataatggta cctgttctgt aaccatctcc ttcatccata 5460

agattttcct ttcttggtgg caaattatca gttttaacca tctccatctc ttgtttgcaa 5520

ccagagggac ttgttgagag ggtggttctt cttggtgctc cagtttctgt gaaaggcgag 5580

cggtgggagg ctgcaaggaa ggtaatggcg atgaaatcat ttttttctct attatctctg 5640

taggaatggt gcaacgtaac aacaagaaac agtgaagttg ctgttcattt tatcccagct 5700

tcttccctcg gcgcttaatt cactcacttg tcctcatggc agatggttgc tggaagattt 5760

gtgaatgtgt actccacaga tgactggatt cttggtgtta cttttcgtgc gaggtaaaca 5820

gatttatgat ttcagagctc ctagaaagct catatcacca ttcccccatg tgagctatga 5880

cttaagtaat cttctcgtgc agtctgctaa cacaagggtt ggctggcatc caggccattg 5940

atgtccctgg tgttgaaaat gtaagcatct ggggtgccat tcttgtgttc ttacgtgatt 6000

ttcatctatc ttttacacgt ctttgtgtac aatgcaattt acaggtcgat gttacggagc 6060

ttgtcgatgg ccattcgtct tacctgtcgg cagcacaaca gatactggag catcttgaac 6120

taaataccta ctatccggtt tttgttcccc tgagcgcagc caacgaagaa acagatggaa 6180

cagttgcaca gtaacgtgtg ggcaggattc gatgaaaatc gtatgtagac aaccaaatgt 6240

aacagactat atatacaacc aaatgtaaca gactaactcg ttcaaactgt aaaccagtct 6300

cagcctgagg cgaatgtgtc tggttggtga ggcttgctgt ttctggccgt gtc 6353

<210> 6

<211> 4183

<212> DNA

<213> Rice male sterile mutant sts1-1 (Oryza sativa)

<400> 6

agtctccccc aaccaaaatt tgaccgagtt tggcacgaaa gaaaccgcct cgacgccgtc 60

caacggaagc cagccgaggc gaccaaccca agggtaggcc taggcgtaaa ccctgcacct 120

gaatcgatcg ccggcgatgg ccacgacgct gacgccgacg cagcgctacg cggcgggggc 180

gctgctggcg ctcgcgctcc gccaggccca gatccaccag tccgtcctcc tcggcgccca 240

ccaccaccac gacgacgacg acgaggaaca ggggcgcacc agcaccagca gcggcggcgg 300

cggcggcagc tcctcctcct cctccaactc cggcgccggc gccgacgccg acctctggac 360

acacgactcc cacggcctcc tccgccccgt cttcaggtgt tttctcgctc ctctccgctt 420

gtaaatttgg ggggtttgag atgaatgctt ccgctttact tggcgatcgg ggggctctgc 480

taggttcctg gagatcgatc ccaaggcgtg gtcggggctg gaggagacgg cggcgtcgtc 540

cgaggccaag catcacatcg gcgcggtacg gcactcactg cattttctgc cctcgtattg 600

taattggccg tgcgttttgg tcagtgttgc agtgatggtt tggggcttag gtgtttgatt 660

atatgcctac aagactgttc aacttcaatt tccgcgtgtg cttgaccagt gaaatttgtg 720

ttttcgtagc atctcatatc actctcattt tgtagtttct aaggataata ttcgaagaag 780

atggcgaaag ctcctcagac agatccgttc aggagcttgc cttggcaaaa ggagttgatg 840

tgatggtaat gagtttgggc aatgacagtg aagtgggtaa cacaattaaa ggtggggatc 900

aagatgcttt gcctagcagc tctggaacag ataaatcccc aggggaaagc tctcatgatg 960

accagttagg aatcaataaa ttgaccttag atgatattcc tgctaacaat caccggaaga 1020

tggcattgct gtttgctctt ctctcggctt gtgttgctga taaacctgta tcacaagagg 1080

aagaggacag aaaatcaacc cgcttcagaa agggttatga tgctcggcat cgtgttgctc 1140

ttcggctgct atctacatgg cttgatgtga agtggatcaa aatggtttgt acagactaaa 1200

ctttttttgga ttcagcagag taatcactat gggtaccaaa caatatccta aggcaattaa 1260

agaaaaaaga acccccaatg tttttcatgt ttcaccaaat ttgtcaccaa ccatgctttt 1320

tcgctgcagg aagcaattga ggttatggtt gcatgctctg ccatggcagc agcgaaagaa 1380

caagaacaat cacaagaaag cgcatcaccc aaaagtaaat gggaaaaatg gaagcgtgga 1440

ggaataattg gtgcagctgc tttgactgga ggagcactgt tggctattac tgggggtatg 1500

aaaaaacttc agaaattctg ctggacgctg aacatctgaa ctagtttctg tcgctcatat 1560

tactctaaac ggttgatatt ctcttccaac ttgacaggtt tagctgctcc agcaattgct 1620

gcagggtttg gtgctcttgc tccaaccctg ggcacacttg tccctgttat aggagctagc 1680

ggatttgctg ctatggctac cgctgcagga tctgttgctg gctctgtggc agttgctgca 1740

tcatttggag gtgtgtctac atgttcttca aaacccttct ggtgatgttg ttttgagctt 1800

tcgtttcgtc atgctacatt actatcagct gcagtctccc tttccttttg tcatgagggt 1860

tgcaatattg tttagtgaac ttacccaaga atatgcacac aacactttta ggggtgcatt 1920

ttacaattag ataatagttc gatggattac cagaagaagg ataagactta tacaatgtat 1980

gacccaaaat tgtgagaaac atatggatga aagcgtaatg agaatagttg tggtgtggaa 2040

attttctatt tattcatgct tatcctttga aatgatattt ttcaattagc tgctggagct 2100

ggcttgacgg ggagcaaaat ggccagaaga attgggagcg tgaaagaatt tgagttcaaa 2160

cctattggtg aaaaccataa tcagggtgtg agttcctttt ctctttctga tgtcacaaca 2220

aatttcattc tactagattt ttgttcaccc ttttagtagt caaactgatc tggtctggtt 2280

aaatgttctc aacactttca gcggcttgca gttggcatct taatttccgg atttgctttt 2340

gatgaggatg acttttgtag accttgggaa ggatggcagg ataacctaga gaggtataga 2400

gtggtttttt tgacatgttc tttttgctga tcactattct agtactttgt catgtgttac 2460

cagctaagag catccttaaa aatataattt caagagttga ataatgtctg gcaggtacat 2520

ccttcaatgg gagtctaagc atataattgc agtgagtaca gcaatacagg attggctgac 2580

atcaaataat atttgtagtt ttgcccattt acactatcag caaaaacttc taatttacac 2640

tgttatttat gattcaggac ttgctatgga attgatgaag caaggtgcaa tgagaaccgt 2700

attaagtggt cttcttgcag catttgcatg gccagctaca ctgcttgcgg ctacagactt 2760

tattgatagt aaatggtctg ttgctattga caggtaattg caaactctcc catttctctt 2820

atgttgcttg cagagttgca gtgcacactg actatctaca gtattggtat tcttctgttg 2880

atcgacacat aagtttgtgt attcaaattt atgaagatta tgacgtaaca tttagacaaa 2940

cttttaatgt atatatccca cagtacatat attgcagtta aagacatcct agcaatttgt 3000

taacttagcc aatcaaaatc aactgtatct tctcagatca gataaggcag gaaaaatgct 3060

tgctgaagtg cttctcaagg gattgcaagg aaataggtaa ctccatatgc atttatttca 3120

ttagcgatgt tggccgctca gaaatatgcc cccctaatgc caattttttc ccaggcctgt 3180

gactctcatt gggttttcac taggtgcacg tgttatattt aagtgtttac aggaacttgc 3240

tttatcgagt gataatggta cctgttctgt aaccatctcc ttcatccata agattttcct 3300

ttcttggtgg caaattatca gttttaacca tctccatctc ttgtttgcaa ccagagggac 3360

ttgttgagag ggtggttctt cttggtgctc cagtttctgt gaaaggcgag cggtgggagg 3420

ctgcaaggaa ggtaatggcg atgaaatcat ttttttctct attatctctg taggaatggt 3480

gcaacgtaac aacaagaaac agtgaagttg ctgttcattt tatcccagct tcttccctcg 3540

gcgcttaatt cactcacttg tcctcatggc agatggttgc tggaagattt gtgaatgtgt 3600

actccacaga tgactggatt cttggtgtta cttttcgtgc gaggtaaaca gatttatgat 3660

ttcagagctc ctagaaagct catatcacca ttcccccatg tgagctatga cttaagtaat 3720

cttctcgtgc agtctgctaa cacaagggtt ggctggcatc caggccattg atgtccctgg 3780

tgttgaaaat gtaagcatct ggggtgccat tcttgtgttc ttacgtgatt ttcatctatc 3840

ttttacacgt ctttgtgtac aatgcaattt acaggtcgat gttacggagc ttgtcgatgg 3900

ccattcgtct tacctgtcgg cagcacaaca gatactggag catcttgaac taaataccta 3960

ctatccggtt tttgttcccc tgagcgcagc caacgaagaa acagatggaa cagttgcaca 4020

gtaacgtgtg ggcaggattc gatgaaaatc gtatgtagac aaccaaatgt aacagactat 4080

atatacaacc aaatgtaaca gactaactcg ttcaaactgt aaaccagtct cagcctgagg 4140

cgaatgtgtc tggttggtga ggcttgctgt ttctggccgt gtc 4183

<210> 7

<211> 4183

<212> DNA

<213> Rice male sterile mutant sts1-2 (Oryza sativa)

<400> 7

agtctccccc aaccaaaatt tgaccgagtt tggcacgaaa gaaaccgcct cgacgccgtc 60

caacggaagc cagccgaggc gaccaaccca agggtaggcc taggcgtaaa ccctgcacct 120

gaatcgatcg ccggcgatgg ccacgacgct gacgccgacg cagcgctacg cggcgggggc 180

gctgctggcg ctcgcgctcc gccaggccca gatccaccag tccgtcctcc tcggcgccca 240

ccaccaccac gacgacgacg acgaggaaca ggggcgcacc agcaccagca gcggcggcgg 300

cggcggcagc tcctcctcct cctccaactc cggcgccggc gccgacgccg acctctggac 360

acacgactcc cacggcctcc tccgccccgt cttcaggtgt tttctcgctc ctctccgctt 420

gtaaatttgg ggggtttgag atgaatgctt ccgctttact tggcgatcgg ggggctctgc 480

taggttcctg gagatcgatc ccaaggcgtg gtcggggctg gaggagacgg cggcgtcgtc 540

cgaggccaag catcacatcg gcgcggtacg gcactcactg cattttctgc cctcgtattg 600

taattggccg tgcgttttgg tcagtgttgc agtgatggtt tggggcttag gtgtttgatt 660

atatgcctac aagactgttc aacttcaatt tccgcgtgtg cttgaccagt gaaatttgtg 720

ttttcgtagc atctcatatc actctcattt tgtagtttct aaggataata ttcgaagaag 780

atggcgaaag ctcctcagac agatccgttc aggagcttgc cttggcaaaa ggagttgatg 840

tgatggtaat gagtttgggc aatgacagtg aagtgggtaa cacaattaaa ggtggggatc 900

aagatgcttt gcctagcagc tctggaacag ataaatcccc aggggaaagc tctcatgatg 960

accagttagg aatcaataaa ttgaccttag atgatattcc tgctaacaat caccggaaga 1020

tggcattgct gtttgctctt ctctcggctt gtgttgctga taaacctgta tcacaagagg 1080

aagaggacag aaaatcaacc cgcttcagaa agggttatga tgctcggcat cgtgttgctc 1140

ttcggctgct atctacatgg cttgatgtga agtggatcaa aatggtttgt acagactaaa 1200

ctttttttgga ttcagcagag taatcactat gggtaccaaa caatatccta aggcaattaa 1260

agaaaaaaga acccccaatg tttttcatgt ttcaccaaat ttgtcaccaa ccatgctttt 1320

tcgctgcagg aagcaattga ggttatggtt gcatgctctg ccatggcagc agcgaaagaa 1380

caagaacaat cacaagaaag cgcatcaccc aaaagtaaat gggaaaaatg gaagcgtgga 1440

ggaataattg gtgcagctgc tttgactgga ggagcactgt tggctattac tgggggtatg 1500

aaaaaacttc agaaattctg ctggacgctg aacatctgaa ctagtttctg tcgctcatat 1560

tactctaaac ggttgatatt ctcttccaac ttgacaggtt tagctgctcc agcaattgct 1620

gcagggtttg gtgctcttgc tccaaccctg ggcacacttg tccctgttat aggagctagc 1680

ggatttgctg ctatggctac cgctgcagga tctgttgctg gctctgtggc agttgctgca 1740

tcatttggag gtgtgtctac atgttcttca aaacccttct ggtgatgttg ttttgagctt 1800

tcgtttcgtc atgctacatt actatcagct gcagtctccc tttccttttg tcatgagggt 1860

tgcaatattg tttagtgaac ttacccaaga atatgcacac aacactttta ggggtgcatt 1920

ttacaattag ataatagttc gatggattac cagaagaagg ataagactta tacaatgtat 1980

gacccaaaat tgtgagaaac atatggatga aagcgtaatg agaatagttg tggtgtggaa 2040

attttctatt tattcatgct tatcctttga aatgatattt ttcaattagc tgctggagct 2100

ggcttgacgg ggagcaaaat ggccagaaga attgggagcg tgaaagaatt tgagttcaaa 2160

cctattggtg aaaaccataa tcagggtgtg agttcctttt ctctttctga tgtcacaaca 2220

aatttcattc tactagattt ttgttcaccc ttttagtagt caaactgatc tggtctggtt 2280

aaatgttctc aacactttca gcggcttgca gttggcatct taatttccgg atttgctttt 2340

gatgaggatg acttttgtag accttgggaa ggatgacagg ataacctaga gaggtataga 2400

gtggtttttt tgacatgttc tttttgctga tcactattct agtactttgt catgtgttac 2460

cagctaagag catccttaaa aatataattt caagagttga ataatgtctg gcaggtacat 2520

ccttcaatgg gagtctaagc atataattgc agtgagtaca gcaatacagg attggctgac 2580

atcaagtaat atttgtagtt ttgcccattt acactatcag caaaaacttc taatttacac 2640

tgttatttat gattcaggac ttgctatgga attgatgaag caaggtgcaa tgagaaccgt 2700

attaagtggt cttcttgcag catttgcatg gccagctaca ctgcttgcgg ctacagactt 2760

tattgatagt aaatggtctg ttgctattga caggtaattg caaactctcc catttctctt 2820

atgttgcttg cagagttgca gtgcacactg actatctaca gtattggtat tcttctgttg 2880

atcgacacat aagtttgtgt attcaaattt atgaagatta tgacgtaaca tttagacaaa 2940

cttttaatgt atatatccca cagtacatat attgcagtta aagacatcct agcaatttgt 3000

taacttagcc aatcaaaatc aactgtatct tctcagatca gataaggcag gaaaaatgct 3060

tgctgaagtg cttctcaagg gattgcaagg aaataggtaa ctccatatgc atttatttca 3120

ttagcgatgt tggccgctca gaaatatgcc cccctaatgc caattttttc ccaggcctgt 3180

gactctcatt gggttttcac taggtgcacg tgttatattt aagtgtttac aggaacttgc 3240

tttatcgagt gataatggta cctgttctgt aaccatctcc ttcatccata agattttcct 3300

ttcttggtgg caaattatca gttttaacca tctccatctc ttgtttgcaa ccagagggac 3360

ttgttgagag ggtggttctt cttggtgctc cagtttctgt gaaaggcgag cggtgggagg 3420

ctgcaaggaa ggtaatggcg atgaaatcat ttttttctct attatctctg taggaatggt 3480

gcaacgtaac aacaagaaac agtgaagttg ctgttcattt tatcccagct tcttccctcg 3540

gcgcttaatt cactcacttg tcctcatggc agatggttgc tggaagattt gtgaatgtgt 3600

actccacaga tgactggatt cttggtgtta cttttcgtgc gaggtaaaca gatttatgat 3660

ttcagagctc ctagaaagct catatcacca ttcccccatg tgagctatga cttaagtaat 3720

cttctcgtgc agtctgctaa cacaagggtt ggctggcatc caggccattg atgtccctgg 3780

tgttgaaaat gtaagcatct ggggtgccat tcttgtgttc ttacgtgatt ttcatctatc 3840

ttttacacgt ctttgtgtac aatgcaattt acaggtcgat gttacggagc ttgtcgatgg 3900

ccattcgtct tacctgtcgg cagcacaaca gatactggag catcttgaac taaataccta 3960

ctatccggtt tttgttcccc tgagcgcagc caacgaagaa acagatggaa cagttgcaca 4020

gtaacgtgtg ggcaggattc gatgaaaatc gtatgtagac aaccaaatgt aacagactat 4080

atatacaacc aaatgtaaca gactaactcg ttcaaactgt aaaccagtct cagcctgagg 4140

cgaatgtgtc tggttggtga ggcttgctgt ttctggccgt gtc 4183

<210> 8

<211> 4184

<212> DNA

<213> Rice male sterile mutant sts1-3 (Oryza sativa)

<400> 8

agtctccccc aaccaaaatt tgaccgagtt tggcacgaaa gaaaccgcct cgacgccgtc 60

caacggaagc cagccgaggc gaccaaccca agggtaggcc taggcgtaaa ccctgcacct 120

gaatcgatcg ccggcgatgg ccacgacgct gacgccgacg cagcgctacg cggcgggggc 180

gctgctggcg ctcgcgctcc gccaggccca gatccaccag tccgtcctcc tcggcgccca 240

ccaccaccac gacgacgacg acgaggaaca ggggcgcacc agcaccagca gcggcggcgg 300

cggcggcagc tcctcctcct cctccaactc cggcgccggc gccgacgccg acctctggac 360

acacgactcc cacggcctcc tccgccccgt cttcaggtgt tttctcgctc ctctccgctt 420

gtaaatttgg ggggtttgag atgaatgctt ccgctttact tggcgatcgg ggggctctgc 480

taggttcctg gagatcgatc ccaaggcgtg gtcggggctg gaggagacgg cggcgtcgtc 540

cgaggccaag catcacatcg ggcgcggtac ggcactcact gcattttctg ccctcgtatt 600

gtaattggcc gtgcgttttg gtcagtgttg cagtgatggt ttggggctta ggtgtttgat 660

tatatgccta caagactgtt caacttcaat ttccgcgtgt gcttgaccag tgaaatttgt 720

gttttcgtag catctcatat cactctcatt ttgtagtttc taaggataat attcgaagaa 780

gatggcgaaa gctcctcaga cagatccgtt caggagcttg ccttggcaaa aggagttgat 840

gtgatggtaa tgagtttggg caatgacagt gaagtgggta acacaattaa aggtggggat 900

caagatgctt tgcctagcag ctctggaaca gataaatccc caggggaaag ctctcatgat 960

gaccagttag gaatcaataa attgacctta gatgatattc ctgctaacaa tcaccggaag 1020

atggcattgc tgtttgctct tctctcggct tgtgttgctg ataaacctgt atcacaagag 1080

gaagaggaca gaaaatcaac ccgcttcaga aagggttatg atgctcggca tcgtgttgct 1140

cttcggctgc tatctacatg gcttgatgtg aagtggatca aaatggtttg tacagactaa 1200

acttttttgg attcagcaga gtaatcacta tgggtaccaa acaatatcct aaggcaatta 1260

aagaaaaaag aacccccaat gtttttcatg tttcaccaaa tttgtcacca accatgcttt 1320

ttcgctgcag gaagcaattg aggttatggt tgcatgctct gccatggcag cagcgaaaga 1380

acaagaacaa tcacaagaaa gcgcatcacc caaaagtaaa tgggaaaaat ggaagcgtgg 1440

aggaataatt ggtgcagctg ctttgactgg aggagcactg ttggctatta ctgggggtat 1500

gaaaaaactt cagaaattct gctggacgct gaacatctga actagtttct gtcgctcata 1560

ttactctaaa cggttgatat tctcttccaa cttgacaggt ttagctgctc cagcaattgc 1620

tgcagggttt ggtgctcttg ctccaaccct gggcacactt gtccctgtta taggagctag 1680

cggatttgct gctatggcta ccgctgcagg atctgttgct ggctctgtgg cagttgctgc 1740

atcatttgga ggtgtgtcta catgttcttc aaaacccttc tggtgatgtt gttttgagct 1800

ttcgtttcgt catgctacat tactatcagc tgcagtctcc ctttcctttt gtcatgaggg 1860

ttgcaatatt gtttagtgaa cttacccaag aatatgcaca caacactttt aggggtgcat 1920

tttacaatta gataatagtt cgatggatta ccagaagaag gataagactt atacaatgta 1980

tgacccaaaa ttgtgagaaa catatggatg aaagcgtaat gagaatagtt gtggtgtgga 2040

aattttctat ttattcatgc ttatcctttg aaatgatatt tttcaattag ctgctggagc 2100

tggcttgacg gggagcaaaa tggccagaag aattgggagc gtgaaagaat ttgagttcaa 2160

acctattggt gaaaaccata atcagggtgt gagttccttt tctctttctg atgtcacaac 2220

aaatttcatt ctactagatt tttgttcacc cttttagtag tcaaactgat ctggtctggt 2280

taaatgttct caacactttc agcggcttgc agttggcatc ttaatttccg gatttgcttt 2340

tgatgaggat gacttttgta gaccttggga aggatggcag gataacctag agaggtatag 2400

agtggttttt ttgacatgtt ctttttgctg atcactattc tagtactttg tcatgtgtta 2460

ccagctaaga gcatccttaa aaatataatt tcaagagttg aataatgtct ggcaggtaca 2520

tccttcaatg ggagtctaag catataattg cagtgagtac agcaatacag gattggctga 2580

catcaagtaa tatttgtagt tttgcccatt tacactatca gcaaaaactt ctaatttaca 2640

ctgttattta tgattcagga cttgctatgg aattgatgaa gcaaggtgca atgagaaccg 2700

tattaagtgg tcttcttgca gcatttgcat ggccagctac actgcttgcg gctacagact 2760

ttattgatag taaatggtct gttgctattg acaggtaatt gcaaactctc ccatttctct 2820

tatgttgctt gcagagttgc agtgcacact gactatctac agtattggta ttcttctgtt 2880

gatcgacaca taagtttgtg tattcaaatt tatgaagatt atgacgtaac atttagacaa 2940

acttttaatg tatatatccc acagtacata tattgcagtt aaagacatcc tagcaatttg 3000

ttaacttagc caatcaaaat caactgtatc ttctcagatc agataaggca ggaaaaatgc 3060

ttgctgaagt gcttctcaag ggattgcaag gaaataggta actccatatg catttatttc 3120

attagcgatg ttggccgctc agaaatatgc ccccctaatg ccaatttttt cccaggcctg 3180

tgactctcat tgggttttca ctaggtgcac gtgttatatt taagtgttta caggaacttg 3240

ctttatcgag tgataatggt acctgttctg taaccatctc cttcatccat aagattttcc 3300

tttcttggtg gcaaattatc agttttaacc atctccatct cttgtttgca accagaggga 3360

cttgttgaga gggtggttct tcttggtgct ccagtttctg tgaaaggcga gcggtgggag 3420

gctgcaagga aggtaatggc gatgaaatca tttttttctc tattatctct gtaggaatgg 3480

tgcaacgtaa caacaagaaa cagtgaagtt gctgttcatt ttatcccagc ttcttccctc 3540

ggcgcttaat tcactcactt gtcctcatgg cagatggttg ctggaagatt tgtgaatgtg 3600

tactccacag atgactggat tcttggtgtt acttttcgtg cgaggtaaac agatttatga 3660

tttcagagct cctagaaagc tcatatcacc attcccccat gtgagctatg acttaagtaa 3720

tcttctcgtg cagtctgcta acacaagggt tggctggcat ccaggccatt gatgtccctg 3780

gtgttgaaaa tgtaagcatc tggggtgcca ttcttgtgtt cttacgtgat tttcatctat 3840

cttttacacg tctttgtgta caatgcaatt tacaggtcga tgttacggag cttgtcgatg 3900

gccattcgtc ttacctgtcg gcagcacaac agatactgga gcatcttgaa ctaaatacct 3960

actatccggt ttttgttccc ctgagcgcag ccaacgaaga aacagatgga acagttgcac 4020

agtaacgtgt gggcaggatt cgatgaaaat cgtatgtaga caaccaaatg taacagacta 4080

tatatacaac caaatgtaac agactaactc gttcaaactg taaaccagtc tcagcctgag 4140

gcgaatgtgt ctggttggtg aggcttgctg tttctggccg tgtc 4184

<210> 9

<211> 4122

<212> DNA

<213> Rice male sterile mutant sts1-4 (Oryza sativa)

<400> 9

agtctccccc aaccaaaatt tgaccgagtt tggcacgaaa gaaaccgcct cgacgccgtc 60

caacggaagc cagccgaggc gaccaaccca agggtaggcc taggcgtaaa ccctgcacct 120

gaatcgatcg ccggcgatgg ccacgacgct gacgccgacg cagcgctacg cggcgggggc 180

gctgctggcg ctcgcgctcc gccaggccca gatccaccag tccgtcctcc tcggcgccca 240

ccaccaccac gacgacgacg acgaggaaca ggggcgcacc agcaccagca gcggcggcgg 300

cggcggcagc tcctcctcct cctccaactc cggcgccggc gccgacgccg acctctggac 360

acacgactcc cacggcctcc tccgccccgt cttcaggtgt tttctcgctc ctctccgctt 420

gtaaatttgg ggggtttgag atgaatgctt ccgctttact tggcgatcgg ggggctctgc 480

taggttcctg gagatcgatc ccaaggcgtg gtcggggctg gaggagacgg cggcgtcgtc 540

cgaggccaag catcacatcg gcgcggtacg gcactcactg cattttctgc cctcgtattg 600

taattggccg tgcgttttgg tcagtgttgc agtgatggtt tggggcttag gtgtttgatt 660

atatgcctac aagactgttc aacttcaatt tccgcgtgtg cttgaccagt gaaatttgtg 720

ttttcgtagc atctcatatc actctcattt tgtagtttct aaggataata ttcgaagaag 780

atggcgaaag ctcctcagac agatccgttc aggagcttgc cttggcaaaa ggagttgatg 840

tgatggtaat gagtttgggc aatgacagtg aagtgggtaa cacaattaaa ggtggggatc 900

aagatgcttt gcctagcagc tctggaacag ataaatcccc aggggaaagc tctcatgatg 960

accagttagg aatcaataaa ttgaccttag atgatattcc tgctaacaat caccggaaga 1020

tggcattgct gtttgctctt ctctcggctt gtgttgctga taaacctgta tcacaagagg 1080

aagaggacag aaaatcaacc cgcttcagaa agggttatga tgctcggcat cgtgttgctc 1140

ttcggctgct atctacatgg cttgatgtga agtggatcaa aatggtttgt acagactaaa 1200

ctttttttgga ttcagcagag taatcactat gggtaccaaa caatatccta aggcaattaa 1260

agaaaaaaga acccccaatg tttttcatgt ttcaccaaat ttgtcaccaa ccatgctttt 1320

tcgctgcagg aagcaattga ggttatggtt gcatgctctg ccatggcagc agcgaaagaa 1380

caagaacaat cacaagaaag cgcatcaccc aaaagtaaat gggaaaaatg gaagcgtgga 1440

ggaataattg gtgcagctgc tttgactgga ggagcactgt tggctattac tgggggtatg 1500

aaaaaacttc agaaattctg ctggacgctg aacatctgaa ctagtttctg tcgctcatat 1560

tactctaaac ggttgatatt ctcttccaac ttgacaggtt tagctgctcc agcaattgct 1620

gcagggtttg gtgctcttgc tccaaccctg ggcacacttg tccctgttat aggagctagc 1680

ggatttgctg ctatggctac cgctgcagga tctgttgctg gctctgtggc agttgctgca 1740

tcatttggag gtgtgtctac atgttcttca aaacccttct ggtgatgttg ttttgagctt 1800

tcgtttcgtc atgctacatt actatcagct gcagtctccc tttccttttg tcatgagggt 1860

tgcaatattg tttagtgaac ttacccaaga atatgcacac aacactttta ggggtgcatt 1920

ttacaattag ataatagttc gatggattac cagaagaagg ataagactta tacaatgtat 1980

gacccaaaat tgtgagaaac atatggatga aagcgtaatg agaatagttg tggtgtggaa 2040

attttctatt tattcatgct tatcctttga aatgatattt ttcaattagc tgctggagct 2100

ggcttgacgg ggagcaaaat ggccagaaga attgggagcg tgaaagaatt tgagttcaaa 2160

cctattggtg aaaaccataa tcagggtgtg agttcctttt ctctttctga tgtcacaaca 2220

aatttcattc tactagattt ttgttcaccc ttttagtagt caaactgatc tggtctggtt 2280

aaatgttctc aacactttca gcggcttgca gttggcatct taatttccgg atttgctttt 2340

gatgaggatg acttttgtag accttgggaa ggatggcagg ataacctaga gaggtataga 2400

gtggtttttt tgacatgttc tttttgctga tcactattct agtactttgt catgtgttac 2460

cagctaagag catccttaaa aatataattt caagagttga ataatgtctg gcaggtacat 2520

ccttcaatgg gagtctaagc atataattgc agtgagtaca gcaatacagg attggctgac 2580

atcaagtaat atttgtagtt ttgcccattt acactatcag caaaaacttc taatttacac 2640

tgttatttat gattcaggac ttgctatgga attgatgaaa aggtgcaatg agaaccgtat 2700

taagtggtct tcttgcagca tttgcatggc cagctacact gcttgcggct acagacttta 2760

ttgatagtaa atggtctgtt gctattgaca ggtaattgca aactctccca tttctcttat 2820

gttgcttgca gagttgcagt gcacactgac tatctacagt attggtattc ttctgttgat 2880

cgacacataa gtttgtgtat tcaaatttat gaagattatg acgtaacatt tagacaaact 2940

tttaatgtat atatcccaca gtacatatat tgcagttaaa gacatcctag caatttgtta 3000

acttagccaa tcaaaatcaa ctgtatcttc tcagatcaga taaggcagga aaaatgcttg 3060

ctgaagtgct tctcaaggga ttgcaaggaa ataggtaact ccatatgcat ttatttcatt 3120

agcgatgttg gccgctcaga aatatgcccc cctaatgcca atttttttccc aggcctgtga 3180

ctctcattgg gttttcacta ggtgcacgtg ttatatttaa gtgtttacag gaacttgctt 3240

tatcgagtga taatggtacc tgttctgtaa ccatctcctt catccataag attttccttt 3300

cttggtggca aattatcagt tttaaccatc tccatctctt gtttgcaacc agagggactt 3360

gttgagaggg tggttcttct tggtgctcca gtttctgtga aaggcgagcg gtgggaggct 3420

gcaaggaagg taatggcgat gaaatcattt ttttctctat tatctctgta ggaatggtgc 3480

aacgtaacaa caagaaacag tgaagttgct gttcatttta tcccagcttc ttccctcggc 3540

gcttaattca ctcacttgtc ctcatggcag atggttgctg gaagatttgt gaatgtgtac 3600

tccacagatg actggattct tggtgttact tttcgtgcga ggtaaacaga tttatgattt 3660

cagagctcct agaaagctca tatcaccatt cccccatgtg agctatgact taagtaatct 3720

tctcgtgcag tctgctaaca caagggttgg ctggcatcca ggccattgat gtccctggtg 3780

ttgaaaatgt aagcatctgg ggtgccattc ttgtgttctt acgtgatttt catctatctt 3840

ttacacgtct ttgtgtacaa tgcaatttac aggtcgatgt tacggagctt gtcgatggcc 3900

attcgtctta cctgtcggca gcacaacaga tactggagca tcttgaacta aatacctact 3960

atccggtttt tgttcccctg agcgcagcca acgaagaaac agatggaaca gttgcacagt 4020

aacgtgtggg caggattcga tgaaaatcgt atgtagacaa ccaaatgtaa cagactatat 4080

atacaaccaa atgtaacaga ctaactcgtt caaactgtaa ac 4122

<210> 10

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 10

ctgtagccgc aagcag 16

<210> 11

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 11

ttgggagcgt gaaaga 16

<210> 12

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 12

caaatgctgc aagaagac 18

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 13

tgaaagcgta atgagaatag 20

<210> 14

<211> 39

<212> DNA

<213> STS1 gene (Oryza sativa)

<400> 14

aaaaaagcag gctccgaatt catggccacg acgctgacg 39

<210> 15

<211> 46

<212> DNA

<213> STS1 gene (Oryza sativa)

<400> 15

caagaaagct gggtcgaatt cttactgtgc aactgttcca tctgtt 46

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 16

ggccaagcat cacatcggcg cgg 23

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 17

gctatggaat tgatgaagca agg 23

Claims (9)

1. A rice male fertility control gene STS1, characterized in that: the STS1 gene is derived from rice, and its nucleotide sequence has one of the following groups: 1) has a nucleotide sequence as shown in SEQ ID NO.1 2) A DNA sequence that is not less than 90% similar to the nucleotide sequence shown in SEQ ID NO. 1 and has the same function; 3) Under certain conditions, it can hybridize with the DNA of the sequence described in 1) 4) a DNA sequence complementary to any of the sequences 1) to 3). 2. The biological material related to the rice male fertility control gene STS1 according to claim 1, wherein the biological material is any one of the following 1) to 5): 1) The protein encoding the gene STS1; 2) An expression cassette containing the gene STS1; 3) A recombinant vector containing the gene STS1; 4) Recombinant microorganisms containing the gene STS1; 5) A recombinant microorganism containing the recombinant vector described in 3). 3. The biological material according to claim 2, wherein the protein amino acid sequence of the coding gene STS1 has the sequence shown in SEQ ID NO.2. 4. The biological material according to claim 2, wherein the protein amino acid sequence of the coding gene STS1 is an amino acid sequence similar to the amino acid sequence shown in SEQ ID NO. 2 and having the same function. 5. The application of a rice male fertility control gene STS1 in the control of rice male fertility, characterized in that, by mutating the STS1 gene in the rice variety, or the mutation and the STS1 gene have a similarity of not less than 90% , and have the same functional DNA sequence to obtain male sterile material. 6. the application of a kind of rice male sterile material, it is characterized in that: the male sterile material obtained by the application described in claim 5 is carried out hybridization and backcross respectively with other materials, to obtain the stability of different backgrounds that cannot produce normal pollen Genetic male sterile lines. 7. An application of a rice male sterile material, characterized in that: the male sterile material obtained by the application according to claim 5 is further transformed by genetic engineering technology to form a derivative sterile line; "3G intelligent sterile line" created by "SPT" technology. 8. An application of a rice male sterile material, characterized in that: the male sterile material obtained by the application according to claim 5 or the derivative sterile line obtained by the application according to claim 8 is used as a hybrid rice sterile line The female parent is used in hybrid rice seed production. 9. a method for restoring the fertility of rice male sterile line is characterized in that: comprise the following steps: In step 1, DNA is extracted from a conventional rice variety with normal fertility as a template, and the wild-type STS1 genome sequence fragment of the gene is amplified by PCR technology. Sequences with at least 90% homology and the same function; Step 2, clone the genomic sequence fragment into a plant expression vector to construct an expression vector STS1-COM; In step 3, the expression vector STS1-COM is introduced into a male sterile rice line by conventional genetic transformation, and the obtained regenerated plants are identified by screening markers, and transformed plants with restored fertility are obtained after transplanting and surviving.

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