CN115247185B - OsAPL protein and application of encoding gene thereof in regulation and control of plant yield - Google Patents

Abstract

The invention discloses the application of OsAPL protein and its encoding gene in regulating plant yield. The invention discloses the application of OsAPL protein in any of the following p1)-p4): p1) regulating plant yield; p2) regulating plant grain size; p3) cultivating transgenic plants with increased yield; p4) cultivating transgenic plants with enlarged grains Plant; the OsAPL protein is a1) or a2) or a3) or a4): a1) the amino acid sequence is the protein shown in sequence 1; a2) a tag is connected to the N-terminal or/and C-terminal of the protein shown in sequence 1 The obtained fusion protein; a3) a protein related to plant yield and/or grain size obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 1; a4) and The amino acid sequence shown in Sequence 1 is a protein with 90% identity, derived from rice, and related to plant yield and/or grain size.

Description

Application of OsAPL protein and its encoding gene in regulating plant yield

Technical field

The invention belongs to the field of biotechnology, and specifically relates to the application of OsAPL protein and its encoding gene in regulating plant yield.

Background technique

In the process of adapting to terrestrial life, plants evolved a vascular system that runs throughout the plant. The vascular system is responsible for the long-distance transport of water, inorganic salts and photosynthetic products in plants, and the rate of this transport determines crop yield. Therefore, improving crop yields by regulating plant nutrient transport mechanisms has always been one of the main goals of breeders' agricultural science and technology research. Rice (Oryza sativa L.) is one of the important food crops. The mining of rice yield-related genes is of great significance for cultivating high-yielding rice varieties and increasing rice yield.

Contents of the invention

In the first aspect, the present invention provides a new use for protecting OsAPL protein.

The present invention protects the application of OsAPL protein in any of the following p1)-p4):

p1) regulate plant yield;

p2) Regulate plant grain size;

p3) Cultivate transgenic plants with increased yield;

p4) Cultivate transgenic plants with enlarged grains;

The OsAPL protein is a1) or a2) or a3) or a4):

a1) The amino acid sequence is the protein shown in sequence 1;

a2) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of the protein shown in sequence 1;

a3) A protein related to plant yield and/or grain size obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 1;

a4) A protein with 90% identity to the amino acid sequence shown in Sequence 1, derived from rice, and related to plant yield and/or grain size.

Among them, sequence 1 consists of 355 amino acid residues.

In the protein described in a2) above, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.

In the protein described in a3) above, the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution, deletion and/or addition of no more than 10 amino acid residues.

In the protein described in a4) above, "identity" includes having 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher with the amino acid sequence shown in Sequence 1 of the present invention. Amino acid sequences with higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher homology.

The protein described in the above a1) or a2) or a3) or a4) can be artificially synthesized, or its encoding gene can be synthesized first and then biologically expressed.

In the second aspect, the present invention protects new uses of biological materials related to OsAPL protein.

The present invention protects the application of biological materials related to OsAPL protein in any of the following p1)-p4):

p1) regulate plant yield;

p2) Regulate plant grain size;

p3) Cultivate transgenic plants with increased yield;

p4) Cultivate transgenic plants with enlarged grains;

The biological material is any one of the following A1) to A12):

A1) Nucleic acid molecules encoding OsAPL protein;

A2) an expression cassette containing the nucleic acid molecule described in A1);

A3) A recombinant vector containing the nucleic acid molecule described in A1);

A4) A recombinant vector containing the expression cassette described in A2);

A5) Recombinant microorganism containing the nucleic acid molecule described in A1);

A6) A recombinant microorganism containing the expression cassette described in A2);

A7) A recombinant microorganism containing the recombinant vector described in A3);

A8) A recombinant microorganism containing the recombinant vector described in A4);

A9) A transgenic plant cell line containing the nucleic acid molecule described in A1);

A10) A transgenic plant cell line containing the expression cassette described in A2);

A11) A transgenic plant cell line containing the recombinant vector described in A3);

A12) A transgenic plant cell line containing the recombinant vector described in A4).

In the above application, the nucleic acid molecule described in A1) is the gene represented by the following B1) or B2) or B3) or B4):

B1) The genomic DNA molecule shown in sequence 2 or sequence 4;

B2) cDNA molecule shown in sequence 3;

B3) A cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined by B1) or B2) and encodes the above-mentioned OsAPL protein;

B4) A cDNA molecule or genomic DNA molecule that hybridizes to the nucleotide sequence defined by B1) or B2) or B3) under stringent conditions and encodes the above OsAPL protein.

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

Those of ordinary skill in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the OsAPL protein of the present invention. Those artificially modified nucleotides that have 75% or higher identity with the nucleotide sequence encoding the OsAPL protein, as long as they encode the OsAPL protein and have the same function, are derived from the nucleotide sequence of the present invention and are equivalent to Sequences of the invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 85% or higher, or 90% or higher, or 95% or Nucleotide sequences of higher identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

The above-mentioned 75% or above identity may be 80%, 85%, 90% or 95% or above identity.

In the above application, the stringent conditions are to hybridize and wash the membrane twice at 68°C for 5 minutes each time in a solution of 2×SSC and 0.1% SDS, and then in a solution of 0.5×SSC and 0.1% SDS at 68°C. Hybridize and wash the membrane twice at 68°C, 15 minutes each time; or hybridize and wash the membrane in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS at 65°C.

In the above application, the expression cassette containing the nucleic acid molecule encoding OsAPL protein (OsAPL gene expression cassette) described in A2) refers to the DNA that can express the OsAPL gene in the host cell. The DNA can not only include the promoter for initiating the transcription of the OsAPL gene. The terminator may also include a terminator that terminates the transcription of the OsAPL gene. Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters; tissue, organ and development specific promoters and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter 35S from cauliflower mosaic virus: wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter, pathogenesis-related 1 (PR1) from tobacco (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); tomatoes Protease inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (U.S. Patent 5,187,267); tetracycline inducible promoter (U.S. Patent 5,057,422) ; Seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (for example, promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J.4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminator (see, for example: Odell et al. ( ¹⁹⁸⁵ ) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) ) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

Existing expression vectors can be used to construct a recombinant vector containing the OsAPL gene expression cassette. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector may also contain the 3' untranslated region of the foreign gene, that is, containing the poly(A) signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal can guide poly(A) to be added to the 3′ end of the mRNA precursor, such as the Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant genes (such as soybean The untranslated regions transcribed at the 3' end of storage protein genes all have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but they must be the same as the coding The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The translation control signals and initiation codons come from a wide range of sources, and may be natural or synthetic. The translation initiation region can be derived from the transcription initiation region or from a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes encoding enzymes or luminescent compounds that can produce color changes (GUS genes, luciferase genes) that can be expressed in plants. genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to methotrexate, the EPSPS gene that confers resistance to glyphosate) or resistance to chemical reagent marker genes (such as herbicide resistance genes), mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene. Considering the safety of transgenic plants, the transformed plants can be directly screened by stress without adding any selective marker genes.

In the above applications, the vector can be a plasmid, cosmid, phage or viral vector.

In the above applications, the microorganism can be yeast, bacteria, algae or fungi, such as Agrobacterium.

In the above applications, the regulation is to increase, which is specifically reflected in the following: the higher the OsAPL protein content and/or activity in the plant or the higher the OsAPL gene expression, the larger the plant grains and the higher the yield.

In the third aspect, the present invention protects the application of the substance represented by m1 or m2 in any of the following q1)-q4):

q1) Reduce plant yield;

q2) Reduce plant grain size;

q3) Breed transgenic plants with reduced yield;

q4) Breed transgenic plants with smaller grains;

m1. Substances that inhibit or reduce the activity or content of OsAPL protein in plants;

m2, substances that inhibit or interfere with the expression of OsAPL protein-encoding nucleic acids in plants or substances that knock out OsAPL protein-encoding nucleic acids in plants.

In a fourth aspect, the present invention protects a method for cultivating transgenic plants with increased yield and/or larger grains.

The method for cultivating transgenic plants with increased yield and/or larger grains protected by the present invention includes the following steps: increasing the content and/or activity of OsAPL protein in recipient plants to obtain transgenic plants; the yield and/or grains of the transgenic plants larger than the recipient plant.

Further, the method for increasing the content and/or activity of OsAPL protein in the recipient plant is to overexpress the OsAPL protein in the recipient plant; the method for overexpression is to introduce the gene encoding the OsAPL protein into the recipient plant.

The yield of the transgenic plant is greater than that of the recipient plant, which is specifically reflected in the fact that the 100-seed weight of the seeds of the transgenic plant is greater than that of the recipient plant;

The grain of the transgenic plant is larger than that of the recipient plant, which specifically means that the width and/or thickness of the grain of the transgenic plant is larger than that of the recipient plant.

Furthermore, the encoding gene of the OsAPL protein is the DNA molecule shown in Sequence 3 in the sequence listing.

In a fifth aspect, the present invention protects a method for cultivating transgenic plants with reduced yield and/or smaller grains.

The method for cultivating transgenic plants with reduced yield and/or smaller grains protected by the present invention includes the following steps: reducing the content and/or activity of OsAPL protein in the recipient plant to obtain transgenic plants; the yield and/or grains of the transgenic plants smaller than the recipient plant.

Further, the method for reducing the content and/or activity of the OsAPL protein in claim 1 in a recipient plant is to introduce a substance that interferes with the expression of the gene encoding the OsAPL protein into the recipient plant.

The yield of the transgenic plant is less than that of the recipient plant, which specifically means that the 100-seed weight of the seeds of the transgenic plant is less than that of the recipient plant;

The grain of the transgenic plant is smaller than that of the recipient plant, which specifically means that the length and/or width and/or thickness of the grain of the transgenic plant is smaller than that of the recipient plant.

Furthermore, the substance that interferes with the expression of the gene encoding the OsAPL protein is a DNA molecule shown in positions 1-323 of Sequence 5 or a vector containing a DNA molecule shown in positions 1-323 of Sequence 5.

In any of the above applications or methods, the plant is a dicotyledonous plant or a monocotyledonous plant; further, the monocotyledonous plant is a gramineous plant; further, the gramineous plant is rice. In a specific embodiment of the present invention, the rice variety is specifically a japonica rice variety Kitaake (Oryza sativa L.cv. Kitaake).

The above-mentioned DNA molecules represented by positions 1-323 of Sequence 5 or vectors containing DNA molecules represented by positions 1-323 of Sequence 5 also belong to the protection scope of the present invention.

Experiments have shown that the recombinant vector p3301UBI-R containing the OsAPL gene CDS sequence shown in sequence 3 in the sequence list was transformed into rice, and the resulting T _3rd generation transgenic rice line increased rice yield by 9.1% under normal conditions, statistically speaking. The expression was extremely significantly higher than that of wild-type rice. The recombinant vector pTCK303 containing part of the CDS sequence shown in Sequence Listing 5 was transformed into rice, and the resulting T ₃ -generation transgenic rice line reduced rice yield by 43.5% under normal conditions, which was statistically significantly lower. in wild-type rice. This shows that OsAPL protein has the function of regulating plant yield and is of great significance in molecular breeding and theoretical research on regulating crop yield.

Description of the drawings

Figure 1 shows the expression levels of the OsAPL gene in different rice lines after the p3301UBI-R vector was introduced in the present invention.

Figure 2 shows the expression levels of OsAPL genes in different rice lines after the pTCK303-R vector was introduced in the present invention.

Figure 3 shows the yield phenotype and statistical data of the rice line with the highest OsAPL gene expression after the p3301UBI-R vector was introduced and the rice line with the lowest OsAPL gene expression after the pTCK303-R vector was introduced.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The test methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples were all purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following examples were repeated three times, and the results were averaged.

The biological material information in the following examples is as follows:

The p3301UBI-GFP/Flag vector is a vector obtained by inserting the GFP tag between the BamHI and SacI restriction sites of the pCAMBIA3301 vector with Ubi promoter, while keeping other sequences of the pCAMBIA3301 vector with Ubi promoter unchanged. The pCAMBIA3301 vector with Ubi promoter is documented in the literature "Mingda Luan, MiaoyunXu, Yunming Lu, Lan Zhang, Yunliu Fan, Lei Wang, Expression of zma-miR169 miRNAs and their target ZmNF-YA genes in response to abiotic stress in maize leaves, Gene , Volume 555, Issue 2, 2015, Pages 178-185, ISSN 0378-1119,” available to the public from the Institute of Botany, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

The pTCK303 vector is described in the literature "Wang Z, Chen CB, Xu YY, Jiang RX, Han Y, Xu ZH & ChongK. (2004) A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L.). Plant Molecular Biology Reporter 22 :409-417." available to the public from the Institute of Botany, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

Agrobacterium tumefaciens EHA105 strain is recorded in the literature "Qu LQ, ” is available to the public from the Institute of Botany, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

The wild-type rice material is the japonica rice variety "Kitaake" (Oryza sativa L.cv. Kitaake) and is recorded in the literature "Qu LQ, Xing YP, Liu WX, Xu XP, Song YR. (2008) Expression pattern and activity of six glutelin gene promoters in transgenic rice. Journal of Experimental Botany 59:2417–2424.”, available to the public from the Institute of Botany, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

Example 1. Obtaining protein OsAPL and its encoding gene

1. Take the seeds of rice Kitaake, soak them in water at room temperature for 48 hours, then place them in a 28°C incubator for 24 hours to germinate. Transfer the germinated rice seeds to nutrient soil and cultivate them for two weeks. The whole plant was snap-frozen in liquid nitrogen, ground and total RNA was extracted, reverse transcribed, and cDNA was obtained.

2. Use the cDNA obtained in step 1 as a template, use 5'-GTAGACGCGTGGATCCATGTGCGTGCAGGGCGACT-3' as the forward primer, and use 5'-CCATGGTACCGGATCCCCCGTAGGATAGGTTCCTCGT-3' as the reverse primer to perform PCR amplification to obtain a PCR product.

3. Perform agarose gel electrophoresis on the amplified PCR product, separate and purify the DNA fragment about 1.1kb in length, and sequence it. Sequencing results show that the nucleotide sequence of this DNA fragment is as shown in Sequence 3 No. 1-1068 in the sequence listing.

4. Use the PCR product obtained in step 2 as a template, use 5'-GGGGTACCACTAGTAAGACCTCCACATGTACGGC-3' as the forward primer, and use 5'-CGGGATCCGAGCTCCTTCGTCTCGTAGACGTCGG-3' as the reverse primer to perform PCR amplification.

5. Perform agarose gel electrophoresis on the amplified PCR product, separate and purify the DNA fragment of approximately 300 bp, and sequence it. The sequencing results showed that the nucleotide sequence of this DNA fragment is as shown in Sequence 5, No. 1-323 in the sequence listing.

Sequence 3 in the sequence listing is the full-length coding region sequence of the protein OsAPL shown in sequence 1 in the coding sequence listing of the rice inbred line Kita. The gene encoding the protein OsAPL is named gene OsAPL, and sequence 5 is the expression interference sequence of the OsAPL gene.

Example 2. Application of OsAPL protein in regulating rice yield

1. Construction of recombinant overexpression vector p3301UBI-R and recombinant Agrobacterium R1

1. Construction of p3301UBI-R recombinant overexpression vector

Clone the DNA fragment shown at positions 1-1068 of Sequence 3 in the sequence list into the vector p3301UBI-GFP/Flag between the BamHI restriction sites (the polyclonal restriction site is located downstream of the maize Ubiquitin promoter), and then After sequencing and comparison, the recombinant overexpression vector p3301UBI-R was obtained.

The recombinant overexpression vector p3301UBI-R is to insert the DNA fragment shown in the 1-1068th position of Sequence 3 in the sequence list between the BamHI restriction sites of the vector p3301UBI-GFP/Flag, and maintain the The vector obtained after keeping other sequences unchanged.

2. Obtaining p3301UBI-R recombinant Agrobacterium tumefaciens

The recombinant overexpression vector p3301UBI-R was transformed into Agrobacterium tumefaciens EHA105 strain, and after PCR detection, the recombinant Agrobacterium R1 containing the recombinant vector p3301UBI-R was obtained.

2. Construction of recombinant interference vector pTCK303-R and recombinant Agrobacterium R2

1. Construction of pTCK303-R recombination interference vector

The DNA fragment shown at positions 1-323 of Sequence 5 in the sequence listing was digested twice and cloned into the vector pTCK303 between the SacI and BamHI restriction sites (the multiple cloning site is located downstream of the 35S promoter), After sequencing and comparison, the recombinant interference vector pTCK303-R was obtained.

The recombinant interference vector pTCK303-R is obtained by inserting the DNA fragment shown in Sequence 5 No. 1-323 in the sequence list between the SacI and BamHI restriction sites of the vector pTCK303, while keeping other sequences of the vector pTCK303 unchanged. carrier.

2. Obtaining pTCK303-R recombinant Agrobacterium

The recombinant vector pTCK303-R was transformed into Agrobacterium tumefaciens EHA105 strain, and after PCR detection, the recombinant Agrobacterium R2 containing the pTCK303-R recombinant vector was obtained.

3. Obtaining transgenic rice lines with different OsAPL gene expression levels

Recombinant Agrobacterium R1 and R2 were transformed into wild-type rice Kitaake using the embryogenic callus infection method, and T ₀ generation rice lines were harvested and T ₁ generation seeds were harvested; after the respective T ₁ generation seeds were germinated, resistant seedlings were obtained Carry out subsequent planting and harvesting to obtain T ₂ -generation seeds; after germinating the T ₂ -generation seeds, randomly select different transgenic lines for RT-PCR testing to determine the rice lines that overexpress OsAPL and the rice lines that interfere with OsAPL expression. . The transgenic rice lines with the highest and lowest OsAPL gene expression were selected for yield measurement and named TRH and TRL respectively.

The T ₀ generation represents the transformed plant, the T ₁ generation represents the seeds produced by the T ₀ generation self-crossing and the plants grown from it, and the T ₂ generation represents the seeds produced by the T ₁ generation self-crossing and the plants grown from it.

The specific steps of the Agrobacterium-mediated rice transgenic process are as follows:

(1) Obtaining induced callus from mature embryos of rice: Take the dried seeds of mature rice, shell them and place them in a sterile 100mL Erlenmeyer flask. Add 70% alcohol to the ultra-clean bench and disinfect the surface for 45 seconds, while shaking the flask continuously. Then transfer the seeds to 2.5% sodium hypochlorite solution, add a drop of Triton Disinfect with shaking at 200rpm for 15min in a ℃ shaker. After discarding the disinfectant, rinse repeatedly with sterile distilled water several times until the rinsed solution is clear and free of foreign matter. Pour out the seeds and place them on a petri dish covered with sterile filter paper, and let them dry on a clean bench for 45 minutes. The seeds were placed on N6D solid medium in sequence, placed in a 28°C incubator, and cultured for 4 weeks. Pick bright yellow and smooth embryogenic callus and subculture them on the new N6D solid medium. After culturing for 5 days, they can be used for Agrobacterium transformation.

(2) Culture of Agrobacterium: Take the transformed Agrobacterium stored at -80°C, spread it on YEB solid medium containing the corresponding antibiotics for streak activation, and then place the Agrobacterium in an incubator at 28°C for cultivation 3 days. Pick monoclonal strains that grow well and streak them on YEB solid medium containing corresponding antibiotics for activation. After culturing for 1 day at 28°C, use a sterile spoon to scrape out the solid strain the size of a match head and suspend it in 30 mL AAM (the final concentration of acetosyringone is 40 mg/L) liquid culture medium in preparation for transformation.

(3) Agrobacterium transformation and co-culture: Scrape the small particles of calli that have grown vigorously after subculture from the N6D medium and place them in a 100 mL Erlenmeyer flask. Suspend the callus in AAM medium suspended with Agrobacterium strain. After soaking for 20 minutes, discard the AAM medium. Place the callus on a petri dish covered with sterile filter paper, air-dry it in a clean bench for 30 minutes, and then place it on a 2N6AS co-culture medium covered with a layer of filter paper. Place the callus in a 23°C incubator and cultivate it in the dark for 4 days.

(4) Agrobacterium removal and screening culture: Use a sterile spoon to scrape the callus after co-culture into a sterile Erlenmeyer flask, and rinse it with sterile distilled water several times until the rinsed water is clear. Add a certain volume of sterile distilled water again, let it stand in a clean bench for 15 minutes, and then soak twice in sterile distilled water containing carbenicillin with a final concentration of 400 mg/L, for 15 minutes each time. Pour the soaked callus into a petri dish covered with sterile filter paper, place it on a clean bench to air dry for 3 hours, and spread the callus on a culture medium containing the corresponding antibiotic (culture of R1-infected calli antibiotics and dipropylamine at a concentration of 2.5 mg/L, while R2 was 50 mg/L hygromycin; the same below) on the N+ medium. Place in a 30°C incubator for dark culture, subculture once every two weeks, and subculture twice.

(5) Redifferentiation of resistant calli after infection: The calli after screening were transferred to hypertonic culture medium in sequence according to different transgenic lines, placed in a 30°C light incubator, and illuminated Culture for 2 weeks until the callus turns green. The green calli were again transferred to hypotonic medium until shoots grew. The shoots were transferred to the rooting medium in Erlenmeyer bottles in sequence according to different clones, placed in a 30°C light incubator, and cultured under light for 2 weeks to obtain resistant seedlings.

(6) Hydroponics and transplanting of resistant seedlings: When the resistant seedlings grow to the top of the flask, remove the filter membrane at the top and place the seedlings in the air. At the same time, add sterile distilled water to the Erlenmeyer flask and acclimate in the light incubator. After the leaves of the seedlings stand straight, pull the seedlings out of the triangular flask, wash away the culture medium at the roots of the seedlings, and transplant them into a greenhouse for cultivation and management. Harvest the seeds after the plants have flowered and been pollinated under normal greenhouse conditions.

Medium formula used in Agrobacterium-mediated rice callus transformation process:

N6D solid medium 1L: sucrose, 30g; NB Basal Medium, 4.1g; Casein, 0.3g; L-Proline, 2.875g; 2,4-D, 0.2g; Gelrite, 4g; pH=5.8.

AAM Agrobacterium activation medium 1L: AA-1, 1mL; AA-2, 1mL; AA-3, 1mL; AA-4, 10mL; AA-5, 1mL; AA-6, 5mL; AA-Sol, 10mL; Casein, 0.5g; glucose, 36g; sucrose, 68.5g; aspartic acid, 0.3g; L-glutamine, 0.9g; inositol, 0.1g; KCl, 3g; acetosyringone, 40mg; pH=5.2 .

AA-1 100mL: MnSO ₄ ·6H ₂ O, 1g; H ₃ BO ₄ , 300mg; ZnSO ₄ ·7H ₂ O, 200mg; KI, 75mg; NaMoO ₄ ·2H ₂ O, 25mg; CuSO ₄ ·5H ₂ O, 2.5mg; CoCl ₂ ·6H ₂ O, 2.5mg.

AA-2 100mL: CaCl ₂ ·2H ₂ O, 15g.

AA-3 100mL: MgSO ₄ ·7H ₂ O, 25g.

AA-4 100mL: FeSO ₄ ·7H ₂ O, 278 mg; Na ₂ EDTA, 373 mg.

AA-5 100mL: NaH ₂ PO4·2H ₂ O, 15g.

AA-6 100mL: Niacin, 20mg; Vitamin B1, 20mg; Vitamin B6, 20mg; Inositol, 2g.

AA-sol 100mL: arginine, 176.67mg; glycine, 75mg.

N6D selection medium 1L: sucrose, 30g; NB Basal Medium, 4.1g; Casein, 0.3g; L-Proline, 2.875g; 2,4-D, 0.2g; Gelrite, 4g; pH=5.8; hygromycin 50mg Or dipropylamine 2mg; carbenzyl 200mg; cephalosporin 250mg.

Hypertonic differentiation medium 1L: sucrose, 30g; sorbitol, 30g; MS Medium, 4.43g; Casein, 0.5g; Gelrite, 4g; pH=5.8; hygromycin, 50mg or dipropylamine phosphine, 2mg; carbenzyl, 200mg; Cephalosporin, 250mg; NAA, 0.3mg; 6-BA, 3mg.

Hypotonic differentiation medium 1L: sucrose, 30g; MS Medium, 4.43g; Casein, 0.5g; Gelrite, 4g; pH=5.8; Hygromycin, 50mg or dipropylamine phosphonate, 2mg; Carbenzyl, 200mg; Cephalosporin, 250mg ; NAA, 0.3mg; 6-BA, 3mg.

Rooting medium 1L: sucrose, 10g; MS Medium, 2.215g; Gelrite, 4g; pH=5.8.

The above RT-PCR method for detecting transgenic rice is as follows: take rice seedlings 14 days after germination of the T ₂ generation transgenic line and wild-type rice seedlings under the same germination conditions, and use Megan's plant RNA mini-extraction kit to extract the rice leaves Total RNA was digested with DNase I. After measuring the concentration with NanoDrop2000 (Thermo Fisher, USA), 2ug was taken and used to synthesize cDNA using Invitrogen's reverse transcription kit using Oligo d(T) as a primer. The cDNA of the OsAPL gene was amplified with the specific quantitative detection primers QF and QR of the OsAPL gene, and the Actin1 gene in rice was used as an internal reference, and the primers were AF and AR. After analyzing the quantitative results, the OsAPL gene expression levels in different transgenic rice lines were obtained.

The sequences of the above primers are as follows:

QF:5'-CCGATCATGTCCGGCGACTC-3';

QR:5'-CATCACCGACGGGCTCCCCA-3';

AF:5'-ACCACAGGTATTGTGTTGGACTC-3';

AR:5'-AGAGCATATCCTTCATAGATGGG-3'.

The detection results of OsAPL gene expression in different transgenic rice lines are shown in Figures 1 and 2. The results show that compared with wild-type rice, the expression level of the OsAPL gene in the Line1 strain (OL1) is the highest (Figure 1), named OsAPL-TRH; the expression level of the OsAPL gene in the Line7 strain (TL7) is the lowest (Figure 2 ), named TRL.

4. Phenotypic analysis of transgenic rice

The T ₃ generation homozygous OsAPL-TRH rice transgenic line (OE1 for short), OsAPL-TRL rice transgenic line (RNAi7 for short) and wild-type rice Kitaake (WT) were taken respectively. After the seeds were germinated, they were cultured in the greenhouse. After 75 days of growth under normal conditions, mature seeds of the T ₃ generation were selected for statistical analysis of seed phenotypes. The seeds (30 seeds) that successfully filled the wild-type and transgenic lines were randomly selected, and the grain size of the transgenic rice was measured with a micrometer.

The results are shown in Figure 3 (Figure 3A shows the wild type mature seed morphology, Figure 3B shows the RNAi7 mature seed morphology, Figure 3C shows the OE1 mature seed morphology, Figure 3D shows the 100-seed weight detection results, Figure 3E shows the seed length detection results, Figure 3F is the seed width detection result, Figure 3G is the seed thickness detection result). The results showed that the average seed lengths of wild-type rice Kitaake, OsAPL-TRH rice transgenic line (OE1), and OsAPL-TRL rice transgenic line (RNAi7) were 7.23mm, 7.09mm, and 6.71mm respectively, and the average seed widths were 3.49. mm, 3.54mm, 3.35mm, and the average seed thicknesses are 2.51mm, 2.64mm, and 2.24mm respectively. Compared with the wild type, the seed width of the OsAPL-TRH transgenic line (OE1) increased by 1.4% and the seed thickness increased by 5.2%; while compared with the wild type, the seed length of the OsAPL-TRL rice transgenic line (RNAi7) It is reduced by 7.2%, the width is reduced by 4%, and the thickness is reduced by 10.8%. What is more noteworthy is that the average 100-seed weights of wild-type rice Kitaake, OsAPL-TRH rice transgenic line (OE1), and OsAPL-TRL rice transgenic line (RNAi7) are 2.09g, 2.28g, and 1.18g respectively. Compared with the wild type, the mature seed weight of the OsAPL-TRH transgenic line (OE1) increased by 9.1%, while that of the OsAPL-TRL rice transgenic line (RNAi7) decreased by 43.5% (Figure 3). This shows that after reducing the expression level of OsAPL gene, rice seeds become smaller and the yield is reduced, while overexpression of this gene will make the rice seeds become larger and the yield increase. The above results indicate that the OsAPL protein and its encoding gene have the function of regulating rice yield. Overexpression of the OsAPL protein encoding gene in rice can increase rice yield.

The above are only preferred embodiments of the present invention. It should be pointed out that those of ordinary skill in the art can also make several improvements and modifications without departing from the technical principles of the present invention. These improvements and modifications It should also be regarded as the protection scope of the present invention.

sequence list

<110> Institute of Botany, Chinese Academy of Sciences

<120> Application of OsAPL protein and its encoding gene in regulating plant yield

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 1

Met Cys Val Gln Gly Asp Ser Gly Leu Val Leu Thr Thr Asp Pro Lys

1 5 10 15

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

20 25 30

Val Thr Gln Leu Gly Gly Pro Asp Lys Ala Thr Pro Lys Thr Ile Met

35 40 45

Arg Val Met Gly Val Lys Gly Leu Thr Leu Tyr His Leu Lys Ser His

50 55 60

Leu Gln Lys Phe Arg Leu Gly Lys Gln Pro His Lys Glu Phe Ser Glu

65 70 75 80

His Ser Val Lys Glu Ala Ala Ala Met Glu Met Gln Arg Asn Ala Ala

85 90 95

Ser Ser Ser Gly Ile Met Gly Arg Ser Met Asn His Asp Arg Asn Val

100 105 110

Asn Asp Ala Ile Arg Met Gln Met Glu Val Gln Arg Arg Leu His Glu

115 120 125

Gln Leu Glu Val Gln Lys His Leu Gln Met Arg Ile Glu Ala Gln Gly

130 135 140

Lys Tyr Met Gln Ser Ile Leu Glu Lys Ala Tyr Gln Thr Leu Ala Ala

145 150 155 160

Gly Asp Val Ala Ala Ala Val Ala Cys Gly Pro Ala Gly Tyr Lys Ser

165 170 175

Leu Gly Asn His Gln Ala Ala Val Leu Asp Val Cys Ser Met Gly Phe

180 185 190

Pro Ser Leu Gln Asp Leu His Met Tyr Gly Gly Ala Gly Gly Gly His

195 200 205

Leu Asp Leu Gln Gln Gln Gln Pro Pro Ala Ser Thr Met Glu Ser Phe

210 215 220

Phe Ala Cys Gly Asp Gly Gly Gly Ser Leu Gly Lys Thr Ala Ala Lys

225 230 235 240

Thr Arg His Tyr Gly Gly Ala Gly Lys Ser Pro Met Met Trp Gly Val

245 250 255

Asp Asp Asp Asp Asp Asp Asp Asp Pro Ala Gly Lys Cys Gly Gly Gly

260 265 270

Gly His His Gln Leu Gln Met Ala Pro Pro Pro Met Met Asp Gly Gly

275 280 285

Ile Asp Val Met Asp Ser Leu Ala Ala Asp Val Tyr Glu Thr Lys Pro

290 295 300

Ile Met Ser Gly Asp Ser Thr Gly Ser Lys Gly Gly Gly Tyr Asp Val

305 310 315 320

Ala Ala Ala Ala Ser Lys Leu Glu Arg Pro Ser Pro Arg Arg Pro Pro

325 330 335

Gln Leu Gly Ser Pro Ser Val Met Ala Gly Ala Gln Thr Arg Asn Leu

340 345 350

Ser Tyr Gly

355

<210> 2

<211> 2027

<212> DNA

<213> Artificial Sequence

<400> 2

atgtgcgtgc agggcgactc cggcctggtc ctcaccaccg accccaagcc gcggctccgg 60

tggacggtgg agctccacga gcgcttcgtc gacgccgtca cccagctcgg cggccccgac 120

agtacgcatc atcaatctct caatcaattc tctccaaaaa aatcgaaaca agaattaatc 180

tcttctttct tacgatgaat tgatgatcgt cttgtcgtcg cagaggcgac gcccaagacg 240

atcatgaggg tgatggggagt gaagggcctc accctctacc acctcaagag ccatctccag 300

gtaacctagc tagctaacca caccttagct agctagggtt ttctcctagc ttagcttgtt 360

cctcgatctc tttctatcta tatgattcta tattctttatg ttggatgatt aactaaatct 420

gtttttggtt tcctgaatat atcatgaaca gaaattcagg ttagggaagc aaccgcacaa 480

ggagttcagc gagcactcag ttaaggaagg taatcaagat cttctagcat gcagtgtgat 540

tgatttcagt tttgaatttg aagttataac ttaatttcca ccaccagtgc aactgatagt 600

ctgaggcgac tgatttgttc ttgaaatttt ccacagccgc ggcaatggag atgcaaagaa 660

acgcagcatc ttcttcaggc ataatgggca gaagcatgaa ccatgagtaa gttcttttca 720

cagatcacct acactagtac actagtaaca gtacactagt actgtagaat taatccggtt 780

taagatttta atttctttct tagtccttgt tcagttatactc tttttttttg ttctttccta 840

actccatttt tgtgtttgct tgtgtgtgaa attctctctc tcagccgcaa cgtgaacgat 900

gccatcagaa tgcagatgga ggtgcaaaga aggctacatg agcaactaga ggtaattaac 960

aaaataaaag cgttgcatcc acatgcatat gcattcaaat taaattgcac caacaaattc 1020

acatgaagtt tcatcaagtt tctcctttca aataaagcac cagggaagtt tacctatcca 1080

cttttctgcc cttcattccc atgctgccat gcatacatac agccagctta gcttctcatc 1140

atgcatacat caacaactta aattacatac ctcaacattc tcatgaacct ttactttatac 1200

tacttaatta ttaccataac tacaagatta gttaattagg ctaataatta tgccctaatt 1260

agcaaagctc atcatcatgc atatatatgg acaatttgaa ttgtccaaca aattaaaatt 1320

gggcatgaat tctaattggt gacatgcagg tgcagaagca tctgcagatg aggatcgaag 1380

cgcaggggaa gtacatgcag tccatcctgg agaaagccta tcagacgctc gccgccggcg 1440

atgtcgcggc ggcggtggcg tgcggcccgg cggggtacaa atccctaggc aaccaccagg 1500

cggcggtgct cgacgtgtgc tccatgggct tcccttccct gcaagacctc cacatgtacg 1560

gcggcgccgg cggcggccac ctcgacctgc agcagcagca gccgccggcg tcgacgatgg 1620

agagcttctt cgcctgcggc gacggcggcg gctcgctggg gaagacggcg gcgaagacga 1680

ggcattacgg cggcgccggg aagagcccga tgatgtgggg cgtcgacgac gacgacgacg 1740

acgacgaccc ggccgggaag tgcggcggcg gcggccatca tcagctgcag atggcgccgc 1800

cgccgatgat ggacggcggc atcgacgtca tggactccct cgccgccgac gtctacgaga 1860

cgaagccgat catgtccggc gactcgacgg ggagcaaggg cggcggctac gacgtcgcgg 1920

cggcggcgtc gaagctggag aggccgtcgc cgcggcggcc gccgcagctg gggagcccgt 1980

cggtgatggc cggagctcag acgaggaacc tatcctacgg gtaatca 2027

<210> 3

<211> 1068

<212> DNA

<213> Artificial Sequence

<400> 3

atgtgcgtgc agggcgactc cggcctggtc ctcaccaccg accccaagcc gcggctccgg 60

tggacggtgg agctccacga gcgcttcgtc gacgccgtca cccagctcgg cggccccgac 120

aaggcgacgc ccaagacgat catgagggtg atgggagtga agggcctcac cctctaccac 180

ctcaagagcc atctccagaa attcaggtta gggaagcaac cgcacaagga gttcagcgag 240

cactcagtta aggaagccgc ggcaatggag atgcaaagaa acgcagcatc ttcttcaggc 300

ataatgggca gaagcatgaa ccatgaccgc aacgtgaacg atgccatcag aatgcagatg 360

gaggtgcaaa gaaggctaca tgagcaacta gaggtgcaga agcatctgca gatgaggatc 420

gaagcgcagg ggaagtacat gcagtccatc ctggagaaag cctatcagac gctcgccgcc 480

ggcgatgtcg cggcggcggt ggcgtgcggc ccggcggggt acaaatccct aggcaaccac 540

caggcggcgg tgctcgacgt gtgctccatg ggcttccctt ccctgcaaga cctccacatg 600

tacggcggcg ccggcggcgg ccacctcgac ctgcagcagc agcagccgcc ggcgtcgacg 660

atggagagct tcttcgcctg cggcgacggc ggcggctcgc tggggaagac ggcggcgaag 720

acgaggcatt acggcggcgc cgggaagagc ccgatgatgt ggggcgtcga cgacgacgac 780

gacgacgacg acccggccgg gaagtgcggc ggcggcggcc atcatcagct gcagatggcg 840

ccgccgccga tgatggacgg cggcatcgac gtcatggact ccctcgccgc cgacgtctac 900

gagacgaagc cgatcatgtc cggcgactcg acggggagca agggcggcgg ctacgacgtc 960

gcggcggcgg cgtcgaagct ggagaggccg tcgccgcggc ggccgccgca gctggggagc 1020

ccgtcggtga tggccggagc tcagacgagg aacctatcct acgggtaa 1068

<210> 4

<211> 4427

<212> DNA

<213> Artificial Sequence

<400> 4

aaatttgtat ggactttttt cattactcca ttccagaaaa aacgaattcc tagctacgaa 60

ggggtaggta gggttggtct tttttttaga cggaggggta ggtagttata agcctctcgc 120

cgtctaatta attattggct tgtcaaccaa aattaaaatt tggagttttt ttcatagagt 180

tttttcatca tattctattt tatagttttc cgctaacaat ataaatatta aagttttaca 240

tataaatttc ttttggttgt tttgtttatttttaaaggt ttatcagctc aaataatcaa 300

aggttaatta agtactccct ccggttccat ataaattgac gattttagac aaagttgagg 360

tcaaactttt ataattttga ccatcaataa ctttaaaaat atttagttta aagggactag 420

aacaacatat atagattttt ctttcaaagc actataataa aagtaaacat acatatattt 480

attatatgta ttataataaa aataatgtca aagatatatt ttgtagaccg tgtcattgtt 540

caaaacgtca attaaaatga aactggagtg agtaataaac actacccaca ttccttggta 600

gttgatgtag attttctgct ttaaatgtca ttttaggacg ctgtagaacg acagttaagt 660

cgtcgtgtgt tgaaataaca cgtactttca catctcattg tgctcttaat agtttttggt 720

caaataatga tcaaaagtat gtctagaacg acaaatctcg acaacaacat cgacaaatta 780

attaataaaa aaacaacaaa tttaatgtaa aaacaccgat gctccctggt caaaaaggga 840

cctggtatgc acacactata agtacaaaat aaaggtcagt acgtactagc ttacttggtt 900

tgttttctag tgccaaagag ggaaatataa aaagcagaga gagtatagat ccaatggtgt 960

gggggcacgc aaagacaaaa tcatgcatga tctgtttagt gccgagcaaa aggcacagtt 1020

taatccaaaa ctgatgctaa ttaactatat atgctttcat gcactaactg ttacatcctc 1080

tgattctgaa agagatgggt ggaagattgc aaagatcagg gggagaagat ccacataaat 1140

gtgttccaaa cacatgtcac atatatatgt acttaattac ttgtgtccca tccttaatga 1200

ctagttacat tatagtacat atactgacag aagcatatat aacagttgat ccaattttaa 1260

tatctgttta taggaatcct ttacgccaga cctaagaact tctttggatt aaggcatttc 1320

aacaaaatta ttttatgtgt aaaaagaatt ggaaagacta tgtaatgatc caaaaaacgg 1380

tgcaatgtcc ggtactgctt cacgtactat gggcctgttc agcaggccga ctgcgacggt 1440

tgcgactgcg cacagtgagt gtggcgctgt tcgtgccggc tgcggcagcc tcggcagcca 1500

aacaagccct atgagtaaat tatatctaga tatgtgttag ttactctatt tgcacctact 1560

atcatgataa ctattgaagt ttcgaaaccg taccttctca taattttttt acattttttt 1620

tttgcaacaa ccagtatttt acatagcgat tctaattatc catctaattt attttataac 1680

gttataaaat ttataaaaat ttatatgcta tataattagc tagtaataca tatatgtagc 1740

cctatatata ctaaccatgt tgtcgaaagg atattatatg agcacacact actcaattaa 1800

aaccaaaaga gagcccctct catcttttgg caaattaaag gaggggttga aggcatggag 1860

ttggggtcgg ccttggtggc tttttcccgg ccaggataga gaatatcacc cttgggcttt 1920

gtagcagaag actcctagct agctagctag ctagctagag agagaaacaa agaaagagaa 1980

agtttgtgtc acacacagac aaaaaaaaag gaagaagcag caaagccatc accccaagca 2040

aaagaggaga gagtgagaga gaaacccatc tagagagaga gagagactaa agagcatatg 2100

agcacaagct agagctacaa gtgtgatcaa gccatagata gagagagaga gagaggaaga 2160

gcgagcgaac cctattcctt gttcttgaat ctgtcgtctc caatcgggca ggatcaagga 2220

tcgacgaggt agagagagtt attattatta tagagagaga gaattaattc gagagagatc 2280

tagagagaga agaagaagag aggagatcat agaagaatgt tccctggctc gaagaagggc 2340

ggcggcggcg gcgccgcggt gagctcgggt gacggcggcg gcggcagggc ggcggcggcg 2400

atgtgcgtgc agggcgactc cggcctggtc ctcaccaccg accccaagcc gcggctccgg 2460

tggacggtgg agctccacga gcgcttcgtc gacgccgtca cccagctcgg cggccccgac 2520

agtacgcatc atcaatctct caatcaattc tctccaaaaa aatcgaaaca agaattaatc 2580

tcttctttct tacgatgaat tgatgatcgt cttgtcgtcg cagaggcgac gcccaagacg 2640

atcatgaggg tgatggggagt gaagggcctc accctctacc acctcaagag ccatctccag 2700

gtaacctagc tagctaacca caccttagct agctagggtt ttctcctagc ttagcttgtt 2760

cctcgatctc tttctatcta tatgattcta tattctttatg ttggatgatt aactaaatct 2820

gtttttggtt tcctgaatat atcatgaaca gaaattcagg ttagggaagc aaccgcacaa 2880

ggagttcagc gagcactcag ttaaggaagg taatcaagat cttctagcat gcagtgtgat 2940

tgatttcagt tttgaatttg aagttataac ttaatttcca ccaccagtgc aactgatagt 3000

ctgaggcgac tgatttgttc ttgaaatttt ccacagccgc ggcaatggag atgcaaagaa 3060

acgcagcatc ttcttcaggc ataatgggca gaagcatgaa ccatgagtaa gttcttttca 3120

cagatcacct acactagtac actagtaaca gtacactagt actgtagaat taatccggtt 3180

taagatttta atttctttct tagtccttgt tcagttatactc tttttttttg ttctttccta 3240

actccatttt tgtgtttgct tgtgtgtgaa attctctctc tcagccgcaa cgtgaacgat 3300

gccatcagaa tgcagatgga ggtgcaaaga aggctacatg agcaactaga ggtaattaac 3360

aaaataaaag cgttgcatcc acatgcatat gcattcaaat taaattgcac caacaaattc 3420

acatgaagtt tcatcaagtt tctcctttca aataaagcac cagggaagtt tacctatcca 3480

cttttctgcc cttcattccc atgctgccat gcatacatac agccagctta gcttctcatc 3540

atgcatacat caacaactta aattacatac ctcaacattc tcatgaacct ttactttatac 3600

tacttaatta ttaccataac tacaagatta gttaattagg ctaataatta tgccctaatt 3660

agcaaagctc atcatcatgc atatatatgg acaatttgaa ttgtccaaca aattaaaatt 3720

gggcatgaat tctaattggt gacatgcagg tgcagaagca tctgcagatg aggatcgaag 3780

cgcaggggaa gtacatgcag tccatcctgg agaaagccta tcagacgctc gccgccggcg 3840

atgtcgcggc ggcggtggcg tgcggcccgg cggggtacaa atccctaggc aaccaccagg 3900

cggcggtgct cgacgtgtgc tccatgggct tcccttccct gcaagacctc cacatgtacg 3960

gcggcgccgg cggcggccac ctcgacctgc agcagcagca gccgccggcg tcgacgatgg 4020

agagcttctt cgcctgcggc gacggcggcg gctcgctggg gaagacggcg gcgaagacga 4080

ggcattacgg cggcgccggg aagagcccga tgatgtgggg cgtcgacgac gacgacgacg 4140

acgacgaccc ggccgggaag tgcggcggcg gcggccatca tcagctgcag atggcgccgc 4200

cgccgatgat ggacggcggc atcgacgtca tggactccct cgccgccgac gtctacgaga 4260

cgaagccgat catgtccggc gactcgacgg ggagcaaggg cggcggctac gacgtcgcgg 4320

cggcggcgtc gaagctggag aggccgtcgc cgcggcggcc gccgcagctg gggagcccgt 4380

cggtgatggc cggagctcag acgaggaacc tatcctacgg gtaatca 4427

<210> 5

<211> 323

<212> DNA

<213> Artificial Sequence

<400> 5

aagacctcca catgtacggc ggcgccggcg gcggccacct cgacctgcag cagcagcagc 60

cgccggcgtc gacgatggag agcttcttcg cctgcggcga cggcggcggc tcgctgggga 120

agacggcggc gaagacgagg cattacggcg gcgccgggaa gagcccgatg atgtggggcg 180

tcgacgacga cgacgacgac gacgacccgg ccgggaagtg cggcggcggc ggccatcatc 240

agctgcagat ggcgccgccg ccgatgatgg acggcggcat cgacgtcatg gactccctcg 300

ccgccgacgt ctacgagacg aag 323

Claims (11)

1. Application of OsAPL protein in any of the following p1)-p4): p1) regulate plant yield; p2) Regulate plant grain size; p3) Cultivate transgenic plants with increased yield; p4) Cultivate transgenic plants with enlarged grains; The OsAPL protein is the protein shown in a1) or a2) below: a1) The amino acid sequence is the protein shown in sequence 1; a2) A fusion protein obtained by connecting a tag to the N-terminal or/and C-terminal of the protein shown in sequence 1; the plant is rice. 2. Application of biological materials related to OsAPL protein in any of the following p1)-p4): p1) regulate plant yield; p2) Regulate plant grain size; p3) Cultivate transgenic plants with increased yield; p4) Cultivate transgenic plants with enlarged grains; The biological material is any one of the following A1) to A12): A1) Nucleic acid molecules encoding OsAPL protein; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1); A4) A recombinant vector containing the expression cassette described in A2); A5) Recombinant microorganism containing the nucleic acid molecule described in A1); A6) A recombinant microorganism containing the expression cassette described in A2); A7) A recombinant microorganism containing the recombinant vector described in A3); A8) A recombinant microorganism containing the recombinant vector described in A4); A9) A transgenic plant cell line containing the nucleic acid molecule described in A1); A10) A transgenic plant cell line containing the expression cassette described in A2); A11) A transgenic plant cell line containing the recombinant vector described in A3); A12) A transgenic plant cell line containing the recombinant vector described in A4); The OsAPL protein is the protein shown in a1) or a2) below: a1) The amino acid sequence is the protein shown in sequence 1; a2) A fusion protein obtained by connecting a tag to the N-terminus or/and C-terminus of the protein shown in sequence 1; The plant is rice. 3. Application according to claim 2, characterized in that: A1) the nucleic acid molecule is a gene represented by the following B1) or B2) or B3) or B4): B1) The genomic DNA molecule shown in sequence 2 or sequence 4; B2) cDNA molecule shown in sequence 3; B3) A cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined by B1) or B2) and encodes the OsAPL protein; B4) A cDNA molecule or genomic DNA molecule that hybridizes to the nucleotide sequence defined by B1) or B2) or B3) under stringent conditions and encodes the OsAPL protein. 4. Use of substances that interfere with the expression of OsAPL protein-encoding nucleic acids in plants in any of the following q1)-q4): q1) Reduce plant yield; q2) Reduce plant grain size; q3) Breed transgenic plants with reduced yield; q4) Breed transgenic plants with smaller grains; The substance that interferes with the expression of OsAPL protein-encoding nucleic acid in plants is the DNA molecule shown in Sequence 5 No. 1-323 or a vector containing the DNA molecule shown in Sequence 5 No. 1-323; The plant is rice. 5. A method for cultivating transgenic plants with increased yield and/or larger grains, comprising the following steps: increasing the content and/or activity of OsAPL protein in recipient plants to obtain transgenic plants; the yield and/or The grain is larger than the recipient plant; the plant is rice; and the amino acid sequence of the OsAPL protein is shown in Sequence 1. 6. The method according to claim 5, characterized in that: the method for increasing the content and/or activity of OsAPL protein in the recipient plant is to overexpress the OsAPL protein in the recipient plant. 7. The method according to claim 6, characterized in that: the overexpression method is to introduce the gene encoding OsAPL protein into the recipient plant. 8. The method according to any one of claims 5 to 7, characterized in that: the encoding gene of the OsAPL protein is the DNA molecule shown in sequence 3. 9. A method for cultivating transgenic plants with reduced yield and/or smaller grains, comprising the following steps: reducing the content and/or activity of OsAPL protein in recipient plants to obtain transgenic plants; the yield and/or The grain is smaller than the recipient plant; the plant is rice; and the amino acid sequence of the OsAPL protein is shown in Sequence 1. 10. The method according to claim 9, characterized in that: the method for reducing the content and/or activity of OsAPL protein in the recipient plant is to introduce substances that interfere with the expression of OsAPL protein encoding genes into the recipient plant; The substance that interferes with the expression of the gene encoding the OsAPL protein is the DNA molecule shown in the 1-323rd position of Sequence 5 or the vector containing the DNA molecule shown in the 1st-323rd position of the Sequence 5. 11. The DNA molecule represented by Sequence 5 at positions 1-323 or a vector containing the DNA molecule represented by Sequence 5 at positions 1-323.