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

OsAPL protein and application of encoding gene thereof in regulation and control of plant yield Download PDF

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CN115247185B
CN115247185B CN202110453036.5A CN202110453036A CN115247185B CN 115247185 B CN115247185 B CN 115247185B CN 202110453036 A CN202110453036 A CN 202110453036A CN 115247185 B CN115247185 B CN 115247185B
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王柏臣
阎臻
晁青
李果
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Institute of Botany of CAS
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Abstract

The invention discloses an OsAPL protein and application of a coding gene thereof in regulating and controlling plant yield. The invention discloses an application of an OsAPL protein in any one of the following p 1) -p 4): p 1) regulating plant yield; p 2) regulating the size of plant seeds; p 3) growing transgenic plants with increased yield; p 4) cultivating transgenic plants with larger grains; the OsAPL protein is a 1) or a 2) or a 3) or a 4): a1 Amino acid sequence is a protein shown in sequence 1; a2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by the sequence 1; a3 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in the sequence 1 and is related to plant yield and/or grain size; a4 90% identical to the amino acid sequence shown in SEQ ID No. 1, derived from rice and related to plant yield and/or grain size.

Description

OsAPL protein and application of encoding gene thereof in regulation and control of plant yield
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an OsAPL protein and application of a coding gene thereof in regulating and controlling plant yield.
Background
Plants have evolved a vascular system throughout the plant body during adaptation to terrestrial life. The vascular system is responsible for the long distance transport of water, inorganic salts and photosynthetic products within the plant, and the rate of such transport determines the yield of the crop. Therefore, improving crop yield by regulating plant nutrient transport mechanisms has been one of the main objectives of agricultural science and technology research by breeders. Rice (Oryza sativa L.) is one of important grain crops, and the excavation of rice yield-related genes has important significance for cultivating high-yield rice varieties and improving rice yield.
Disclosure of Invention
In a first aspect, the present invention provides a novel use of an OsAPL protein.
The invention protects the application of the OsAPL protein in any one of the following p 1) -p 4):
p 1) regulating plant yield;
p 2) regulating the size of plant seeds;
p 3) growing transgenic plants with increased yield;
p 4) cultivating transgenic plants with larger grains;
the OsAPL protein is a 1) or a 2) or a 3) or a 4):
a1 Amino acid sequence is a protein shown in sequence 1;
a2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by the sequence 1;
a3 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in the sequence 1 and is related to plant yield and/or grain size;
a4 90% identical to the amino acid sequence shown in SEQ ID No. 1, derived from rice and related to plant yield and/or grain size.
Wherein sequence 1 consists of 355 amino acid residues.
The protein of a 2), wherein the tag refers to a polypeptide or protein which is fused and expressed together with the target protein by using a DNA in vitro recombination technology, so as 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.
The protein according to a 3) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.
In the protein described in the above a 4), the "identity" includes an amino acid sequence having 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown in the sequence 1 of the present invention.
The protein described in the above a 1), a 2), a 3) or a 4) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.
In a second aspect, the present invention provides novel uses of biological materials related to OsAPL proteins.
The invention protects the application of biological materials related to OsAPL proteins in any one of the following p 1) -p 4):
p 1) regulating plant yield;
p 2) regulating the size of plant seeds;
p 3) growing transgenic plants with increased yield;
p 4) cultivating transgenic plants with larger grains;
the biomaterial is any one of the following A1) to a 12):
a1 Nucleic acid molecules encoding osppl proteins;
a2 An expression cassette comprising A1) said nucleic acid molecule;
a3 A) a recombinant vector comprising the nucleic acid molecule of A1);
a4 A recombinant vector comprising the expression cassette of A2);
a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);
a6 A) a recombinant microorganism comprising the expression cassette of A2);
a7 A) a recombinant microorganism comprising the recombinant vector of A3);
a8 A) a recombinant microorganism comprising the recombinant vector of A4);
a9 A transgenic plant cell line comprising the nucleic acid molecule of A1);
a10 A transgenic plant cell line comprising the expression cassette of A2);
a11 A transgenic plant cell line comprising the recombinant vector of A3);
a12 A) a transgenic plant cell line comprising the recombinant vector of A4).
In the above applications, the nucleic acid molecule of A1) is a gene represented by the following B1) or B2) or B3) or B4):
b1 A genomic DNA molecule shown in sequence 2 or sequence 4;
b2 A cDNA molecule represented by SEQ ID No. 3;
b3 A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in B1) or B2) and encoding the above OsAPL protein;
b4 Under stringent conditions with a nucleotide sequence defined in B1) or B2) or B3), and a cDNA molecule or genomic DNA molecule encoding an OsAPL protein as described above.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
The nucleotide sequence encoding the OsAPL protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence encoding an OsAPL protein are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode an OsAPL protein and have the same function.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of a protein consisting of the amino acid sequence shown in the coding sequence 1 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.
In the above application, the stringent conditions are hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; alternatively, hybridization and washing of the membrane were performed at 65℃in a solution of 0.1 XSSPE (or 0.1 XSSC) and 0.1% SDS.
In the above applications, the expression cassette (OsAPL gene expression cassette) described in A2) containing a nucleic acid molecule encoding an OsAPL protein means a DNA capable of expressing an OsAPL gene in a host cell, and the DNA may include not only a promoter for initiating transcription of the OsAPL gene but also a terminator for terminating transcription of the OsAPL gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: a constitutive promoter; tissue, organ and development specific promoters and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter of cauliflower mosaic virus 35S: wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin, and soybean beta-cone (Beachy et al (1985) EMBO J. 4:3047-3053)). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), caMV 35S terminator, tml terminator, peaThe leguminous cS E9 terminator and the nopaline and octopine synthase terminator (see, e.g., odell et al (I) 985 ) Nature 313:810; rosenberg et al (1987) Gene,56:125; guerineau et al (1991) mol. Gen. Genet,262:141; proudroot (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).
The existing expression vector can be used for constructing a recombinant vector containing the OsAPL gene expression cassette. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.
In the above applications, the vector may be a plasmid, cosmid, phage or viral vector.
In the above application, the microorganism may be a yeast, a bacterium, an alga or a fungus, such as Agrobacterium.
In the application, the regulation and control are improved, and the regulation and control are specifically shown as follows: the higher the OsAPL protein content and/or activity in the plant or the higher the OsAPL gene expression level, the larger the plant seed and the higher the yield.
In a third aspect, the invention provides the use of a substance represented by m1 or m2 in any one of the following q 1) -q 4):
q 1) decreasing plant yield;
q 2) reducing plant kernel size;
q 3) growing transgenic plants with reduced yield;
q 4) cultivating transgenic plants with smaller kernels;
m1, an agent that inhibits or reduces the activity or content of an OsAPL protein in a plant;
m2, a substance that inhibits or interferes with expression of a nucleic acid encoding an OsAPL protein in a plant or a substance that knocks out a nucleic acid encoding an OsAPL protein in a plant.
In a fourth aspect, the invention provides a method of growing transgenic plants having increased yield and/or increased grain size.
The method for cultivating transgenic plants with improved yield and/or enlarged grains, which is protected by the invention, comprises the following steps: increasing the content and/or activity of OsAPL protein in the receptor plant to obtain a transgenic plant; the transgenic plant has a yield and/or grain that is greater than the recipient plant.
Further, the method for increasing the content and/or activity of the OsAPL protein in the receptor plant is to overexpress the OsAPL protein in the receptor plant; the over-expression method is to introduce the encoding gene of the OsAPL protein into a receptor plant.
The transgenic plant has a yield greater than that of the recipient plant, and is embodied by the transgenic plant seed having a hundred-grain weight greater than that of the recipient plant;
the transgenic plant has a grain that is larger than the recipient plant, and the transgenic plant has a grain that is wider and/or thicker than the recipient plant.
Furthermore, the encoding gene of the OsAPL protein is a DNA molecule shown in a sequence 3 in a sequence table.
In a fifth aspect, the invention provides a method of growing a transgenic plant having reduced yield and/or reduced grain size.
The method for cultivating transgenic plants with reduced yield and/or reduced grain size, which is protected by the invention, comprises the following steps: reducing the content and/or activity of OsAPL protein in the recipient plant to obtain a transgenic plant; the transgenic plant has a yield and/or grain that is smaller than the recipient plant.
Further, the method for reducing the content and/or activity of the OsAPL protein of claim 1 in a recipient plant is to introduce into the recipient plant a substance that interferes with the expression of the gene encoding the OsAPL protein.
The transgenic plant has a yield less than that of the recipient plant, and the transgenic plant has a seed weight less than that of the recipient plant;
the transgenic plant has a grain that is smaller than the recipient plant, and the transgenic plant has a grain that is smaller in length and/or width and/or thickness than the recipient plant.
Furthermore, the substance interfering with the expression of the encoding gene of the OsAPL protein is a DNA molecule shown in the 1 st to 323 rd positions of the sequence 5 or a vector containing the DNA molecule shown in the 1 st to 323 rd positions of the sequence 5.
In any one of the above applications or methods, the plant is a dicotyledonous plant or a monocotyledonous plant; further, the monocotyledonous plant is a plant of the Gramineae family; further, the gramineous plant is rice. In a specific embodiment of the invention, the rice variety is specifically japonica rice variety Kitaake (Oryza sativa L.cv.Kitaake).
The DNA molecules shown in the 1 st to 323 rd positions of the sequence 5 or the vector containing the DNA molecules shown in the 1 st to 323 rd positions of the sequence 5 also belong to the protection scope of the invention.
Experiments prove that the recombinant vector p3301UBI-R containing the OsAPL gene CDS sequence shown in the sequence 3 in the sequence table is transformed into rice to obtain T 3 The transgenic rice line has 9.1 percent of rice yield rise under normal conditions and is statistically significantly higher than wild rice. And transforming the recombinant vector pTCK303 containing partial CDS sequence shown in sequence 5 in the sequence table into rice to obtain T 3 The transgenic rice line has 43.5% lower rice yield under normal conditions and is statistically significantly lower than wild rice. The OsAPL protein has the function of regulating plant yield, and has important significance in molecular breeding and theoretical research for regulating crop yield.
Drawings
FIG. 1 shows the expression level of OsAPL gene in different rice lines after the p3301UBI-R vector is introduced in the present invention.
FIG. 2 shows the expression level of OsAPL gene in different rice lines after pTCK303-R vector is introduced into the present invention.
FIG. 3 shows the phenotype and statistical data of the rice line with the highest OsAPL gene expression level after the p3301UBI-R vector is introduced and the rice line with the lowest OsAPL gene expression level after the pTCK303-R vector is introduced.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
The biomaterial information in the following examples is as follows:
the p3301UBI-GFP/Flag vector is a vector obtained by inserting a GFP tag between BamHI and SacI cleavage sites of pCAMBIA3301 vector with UBI promoter and keeping other sequences of pCAMBIA3301 vector with UBI promoter unchanged. The pCAMBIA3301 vector with Ubi promoter is described in the literature "Mingda Luan, miaoyun Xu, yunming Lu, lanzhang, 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", which is available to the public from the national academy of sciences plant study. The biological material is only used for repeated experiments related to the invention and can not be used for other purposes.
pTCK303 vector is described in "Wang Z, chen CB, xu YY, jiang RX, han Y, xu ZH & Chong K. (2004) A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L.). Plant Molecular Biology Reporter 22:409-417." which is available to the public from the national academy of sciences plant study. The biological material is only used for repeated experiments related to the invention and can not be used for other purposes.
Agrobacterium tumefaciens EHA105 strain is described 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", and is available to the public from the national academy of sciences plant research. The biological material is only used for repeated experiments related to the invention and can not be used for other purposes.
The rice wild type material is japonica rice variety "Kitaake" (Oryza sativa L.cv.Kitaake) described 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 national academy of sciences plant research. The biological material is only used for repeated experiments related to the invention and can not be used for other purposes.
Example 1 acquisition of protein OsAPL and Gene encoding same
1. Soaking and swelling seed of rice Kitaake in water at room temperature for 48 hr, germinating in a 28 deg.C incubator for 24 hr, and transferring germinated rice seed into nutrient soil for two weeks. The whole plant is taken, quick frozen in liquid nitrogen, ground to extract total RNA, reverse transcribed and cDNA is obtained.
2. And (3) performing PCR amplification by taking the cDNA obtained in the step (1) as a template, 5'-GTAGACGCGTGGATCCATGTGCGTGCAGGGCGACT-3' as a forward primer and 5'-CCATGGTACCGGATCCCCCGTAGGATAGGTTCCTCGT-3' as a reverse primer to obtain a PCR product.
3. The amplified PCR product was subjected to agarose gel electrophoresis, and a DNA fragment of about 1.1kb in length was isolated and purified and sequenced. Sequencing results show that the nucleotide sequence of the DNA fragment is shown as the 1 st to 1068 th positions of the sequence 3 in the sequence table.
4. And (3) performing PCR amplification by taking the PCR product obtained in the step (2) as a template, 5'-GGGGTACCACTAGTAAGACCTCCACATGTACGGC-3' as a forward primer and 5'-CGGGATCCGAGCTCCTTCGTCTCGTAGACGTCGG-3' as a reverse primer.
5. The amplified PCR product was subjected to agarose gel electrophoresis, and a DNA fragment of about 300bp in length was isolated and purified and sequenced. Sequencing results show that the nucleotide sequence of the DNA fragment is shown as the 1 st-323 rd position of the sequence 5 in the sequence table.
The sequence 3 in the sequence table is the full-length coding region sequence of the protein OsAPL shown in the sequence 1 in the coding sequence table in the rice inbred line Kita. The gene encoding the protein OsAPL is named as gene OsAPL, and the 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
Cloning the DNA fragment shown in the 1 st to 1068 th positions of the sequence 3 in the sequence table between BamHI cleavage sites of the vector p3301UBI-GFP/Flag (the polyclonal cleavage site is positioned at the downstream of the corn Ubiquitin promoter), sequencing and comparing to obtain the recombinant overexpression vector p3301UBI-R.
The recombinant overexpression vector p3301UBI-R is obtained by inserting a DNA fragment shown in the 1 st to 1068 th positions of a sequence 3 in a sequence table between BamHI enzyme cutting sites of the vector p3301UBI-GFP/Flag, and keeping other sequences of the vector p3301UBI-GFP/Flag unchanged.
2. Acquisition of p3301UBI-R recombinant Agrobacterium tumefaciens
The recombinant overexpression vector p3301UBI-R is transformed into an agrobacterium tumefaciens EHA105 strain, and the recombinant agrobacterium tumefaciens R1 containing the recombinant vector p3301UBI-R is obtained after PCR detection.
2. Construction of recombinant interference vector pTCK303-R and recombinant Agrobacterium R2
1. Construction of pTCK303-R recombinant interference vector
The DNA fragment shown in the 1 st to 323 rd positions of the sequence 5 in the sequence table is digested twice and cloned between SacI and BamHI digestion sites of a vector pTCK303 (the multiple cloning site is positioned at the downstream of a 35S promoter), and then the recombinant interference vector pTCK303-R is obtained after sequencing and comparison.
The recombinant interference vector pTCK303-R is obtained by inserting a DNA fragment shown in the 1 st-323 rd position of the sequence 5 in the sequence table between SacI and BamHI cleavage sites of the vector pTCK303 and keeping other sequences of the vector pTCK303 unchanged.
2. Acquisition of pTCK303-R recombinant Agrobacterium
The recombinant vector pTCK303-R is transformed into an agrobacterium tumefaciens EHA105 strain, and the recombinant agrobacterium tumefaciens R2 containing the pTCK303-R recombinant vector is obtained after PCR detection.
3. Obtaining transgenic rice lines with different OsAPL gene expression levels
Transforming recombinant agrobacterium R1 and R2 into wild rice Kitaake by embryogenic callus infection method, and harvesting T 0 Harvesting T from generation rice lines 1 Seed generation; will each T 1 After germination of the seed, obtaining a resistant seedling for subsequent planting and seed collection to obtain T 2 Seed generation; will T 2 After germination of the seed, different transgenic lines are randomly selected for RT-PCR detection, and the rice lines which over-express the OsAPL and interfere with the OsAPL expression are determined. Transgenic rice lines with highest and lowest OsAPL gene expression level are selected for yield measurement and are respectively named as TRH and TRL.
T 0 Representing transformed contemporary plants, T 1 The generation represents T 0 Seed from generation selfing and plant grown from it, T 2 The generation is represented by T 1 Seed from the selfing generation and plants grown from it.
The specific operation steps of the agrobacterium-mediated rice transgenic process are as follows:
(1) Obtaining the rice mature embryo induced callus: taking the seeds of the ripe rice which are dried in the air, removing the shells, and placing the seeds in a sterile 100mL triangular flask. Adding 70% alcohol into the super clean bench, and sterilizing for 45 s. Then the seeds are transferred into a 2.5% sodium hypochlorite solution, a drop of Triton X-100 is added, the seeds are placed in a shaking table at 28 ℃ and are sterilized by shaking at 200rpm for 15min, and then the seeds are transferred into a 2.5% sodium hypochlorite solution and are sterilized by shaking at 200rpm for 15min in the shaking table at 28 ℃. After discarding the disinfectant, repeatedly washing the disinfectant with sterile distilled water for a plurality of times until the washed solution is clear and free of foreign matters. Pouring out the seeds, placing the seeds on a culture dish paved with sterile filter paper, and airing the seeds for 45min on an ultra-clean bench. The seeds are placed on an N6D solid culture medium in sequence and placed in a 28 ℃ incubator for 4 weeks. Picking tender yellow and smooth embryogenic callus to be transferred onto a new N6D solid culture medium for 5 days, and then using the culture medium for agrobacterium transformation.
(2) Culturing agrobacterium: the transformed Agrobacterium stored at-80℃was applied to YEB solid medium containing the corresponding antibiotic for streak activation, and then the Agrobacterium was cultured in a 28℃incubator for 3 days. Well-grown monoclonal strains were picked and streaked again on YEB solid medium containing the corresponding antibiotic. After 1 day of incubation at 28℃the matchhead-sized solid strain was scraped with a sterile spatula and suspended in 30mL of AAM (acetosyringone at a final concentration of 40 mg/L) liquid medium for transformation.
(3) Transformation and co-cultivation of Agrobacterium: small granular callus that grew vigorously after subculture was scraped from N6D medium and placed in a 100mL Erlenmeyer flask. The calli were suspended in AAM medium with Agrobacterium strain suspended, and after 20min of immersion, the AAM medium was discarded. The callus is placed on a culture dish paved with sterile filter paper, and is dried for 30min in an ultra-clean bench by blowing, and then is placed on a 2N6AS co-culture medium paved with a layer of filter paper. The calli were placed in a23℃incubator and dark cultured for 4 days.
(4) Cleaning and screening culture of agrobacterium: scraping the co-cultured callus into a sterile triangular flask by using a sterile medicine spoon, and flushing the callus with sterile distilled water for a plurality of times until the flushed water is clear. Adding a certain volume of sterile distilled water again, standing in an ultra-clean bench for 15min, and soaking twice with sterile distilled water containing carbenicillin with a final concentration of 400mg/L for 15min each time. Pouring the soaked callus into a culture dish paved with sterile filter paper, placing the culture dish in an ultra-clean bench, blowing for 3 hours, airing, and paving the callus on an N+ culture medium containing corresponding antibiotics (antibiotics on a culture medium for culturing the callus after R1 infection and dipropylamine phosphine with the concentration of 2.5mg/L, and R2 is 50mg/L hygromycin; the same applies below). Dark culture is carried out in an incubator at 30 ℃ once every two weeks for twice.
(5) Redifferentiation of resistant calli after infestation: and transferring the selected subcultured calli on a hypertonic culture medium according to different transgenic strains, and placing the calli in a 30 ℃ illumination incubator for illumination culture for 2 weeks until the calli are green. The green callus was again transferred to hypotonic medium until shoots developed. Sequentially transferring the tender buds to rooting culture mediums in triangular flasks according to different clones, and placing the rooting culture mediums in a light incubator at 30 ℃ for light culture for 2 weeks to obtain resistant seedlings.
(6) Water planting and transplanting of resistant seedlings: when the resistant young seedlings grow to the top of the triangular flask, the filter membrane at the top is removed, and the young seedlings are placed in the air. Meanwhile, sterile distilled water is added into the triangular flask, and domestication is carried out in an illumination incubator. After the leaves of the seedlings are straightened, the seedlings are pulled out of the triangular flask, the culture medium of the roots of the seedlings is washed off, and the seedlings are transplanted into a greenhouse for cultivation management. After plants are flowering and pollinated under normal greenhouse conditions, seeds are harvested.
The formula of the culture medium used in the agrobacterium-mediated rice callus transformation process comprises the following steps:
N6D solid Medium 1L: sucrose, 30g; NB base 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，278mg；Na 2 EDTA，373mg。
AA-5 100mL：NaH 2 PO4·2H 2 O，15g。
AA-6 100mL: nicotinic acid, 20mg; vitamin B1, 20mg; vitamin B6, 20mg; inositol, 2g.
AA-sol 100mL: arginine, 176.67mg; glycine, 75mg.
N6D screening Medium 1L: sucrose, 30g; NB base Medium,4.1g; casein,0.3g; L-Proline,2.875g;2,4-D,0.2g; gelrite,4g; ph=5.8; 50mg of hygromycin or 2mg of dipropylamine phosphine; 200mg of carboxin; 250mg of cephalosporin.
Hypertonic differentiation medium 1L: sucrose, 30g; 30g of sorbitol; MS Medium,4.43g; casein,0.5g; gelrite,4g; ph=5.8; hygromycin, 50mg or dipropionate phosphine, 2mg; carboxylic acid 200mg; cefuroxime, 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 dipropionate phosphine, 2mg; carboxylic acid 200mg; cefuroxime, 250mg; NAA,0.3mg;6-BA,3mg.
Rooting medium 1L: sucrose, 10g; MS Medium,2.215g; gelrite,4g; ph=5.8.
The method for detecting the transgenic rice by RT-PCR comprises the following steps: taking T 2 The rice seedlings 14 days after germination of the transgenic lines and the wild rice seedlings under the same germination conditions are used for extracting total RNA of rice leaves by using a plant RNA small extraction kit of Megan company, and DNA digestion is performed by using DNase I. After concentration was measured by NanoDrop2000 (Thermo Fisher, USA), 2ug was used to synthesize cDNA using Oligo d (T) as a primer using a reverse transcription kit from Invitrogen. cDNA of the OsAPL gene is amplified by specific quantitative detection primers QF and QR of the OsAPL gene, and an action 1 gene in rice is used as an internal reference, and the primers are AF and AR. And (3) analyzing the quantitative result to obtain the OsAPL gene expression levels in different transgenic rice lines.
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 results of the detection of the OsAPL gene expression levels in different transgenic rice lines are shown in FIG. 1 and FIG. 2. As a result, compared with the wild-type rice, the Line1 strain (OL 1) has the highest expression level of the OsAPL gene (figure 1), which is named OsAPL-TRH; the Line7 strain (TL 7) had the lowest expression level of the OsAPL gene (FIG. 2) and was designated TRL.
4. Phenotypic analysis of transgenic Rice
Respectively take T 3 The transgenic strain of the homozygous OsAPL-TRH rice (abbreviated as OE 1), the transgenic strain of the OsAPL-TRL rice (abbreviated as RNAi 7) and the wild rice Kitaake (WT) are cultivated in a greenhouse after seed germination. After 75 days of growth under normal conditions, T is selected 3 The mature seeds of the generation were subjected to statistical analysis of seed phenotype. Seeds (30 grains) with successful grouting of wild type and transgenic lines are randomly selected, and the grain size of the transgenic rice is measured by a micrometer.
The results are shown in FIG. 3 (FIG. 3A is wild-type mature seed morphology, FIG. 3B is RNAi7 mature seed morphology, FIG. 3C is OE1 mature seed morphology, FIG. 3D is hundred-grain weight detection result, FIG. 3E is seed length detection result, FIG. 3F is seed width detection result, FIG. 3G is seed thickness detection result). The results showed that the average lengths of seeds of the wild type rice Kitaake, osAPL-TRH rice transgenic line (OE 1) and the OsAPL-TRL rice transgenic line (RNAi 7) were 7.23mm, 7.09mm and 6.71mm, respectively, the average widths of seeds were 3.49mm, 3.54mm and 3.35mm, and the average thicknesses of seeds were 2.51mm, 2.64mm and 2.24mm, respectively. Compared with the wild type, the seed width of the OsAPL-TRH transgenic strain (OE 1) is increased by 1.4 percent, and the seed thickness is increased by 5.2 percent; compared with the wild type, the seed length of the OsAPL-TRL rice transgenic line (RNAi 7) is reduced by 7.2%, the width is reduced by 4%, and the thickness is reduced by 10.8%. More notably, the average seed hundred-grain weights of the wild type rice Kitaake, osAPL-TRH rice transgenic line (OE 1) and the OsAPL-TRL rice transgenic line (RNAi 7) were 2.09g, 2.28g and 1.18g, respectively. The mature seed hundred grain weight of the OsAPL-TRH transgenic line (OE 1) was increased by 9.1% compared to the wild-type, while that of the OsAPL-TRL rice transgenic line (RNAi 7) was decreased by 43.5% (FIG. 3). It is shown that rice seeds become smaller and the yield is reduced after the expression level of the OsAPL gene is reduced, and that excessive expression of the gene can lead to larger rice seeds and the yield to be increased. The result shows that the OsAPL protein and the encoding gene thereof have the function of regulating and controlling the rice yield, and the encoding gene of the OsAPL protein is overexpressed in rice, so that the rice yield can be improved.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
Sequence listing
<110> institute of plant Material at national academy of sciences
<120> OsAPL protein and application of encoding gene thereof 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 tgatgggagt gaagggcctc accctctacc acctcaagag ccatctccag 300
gtaacctagc tagctaacca caccttagct agctagggtt ttctcctagc ttagcttgtt 360
cctcgatctc tttctatcta tatgattcta tattcttatg 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 tcagttactc 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 ttacttatac 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 tttgtttatt tttataaggt 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 tgatgggagt gaagggcctc accctctacc acctcaagag ccatctccag 2700
gtaacctagc tagctaacca caccttagct agctagggtt ttctcctagc ttagcttgtt 2760
cctcgatctc tttctatcta tatgattcta tattcttatg 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 tcagttactc 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 ttacttatac 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. Use of an osapl protein in any one of the following p 1) -p 4):
    p 1) regulating plant yield;
    p 2) regulating the size of plant seeds;
    p 3) growing transgenic plants with increased yield;
    p 4) cultivating transgenic plants with larger grains;
    the OsAPL protein is a protein shown in the following a 1) or a 2):
    a1 Amino acid sequence is a protein shown in sequence 1;
    a2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by the sequence 1; the plant is rice.
  2. 2. Use of biological material related to OsAPL proteins in any of the following p 1) -p 4):
    p 1) regulating plant yield;
    p 2) regulating the size of plant seeds;
    p 3) growing transgenic plants with increased yield;
    p 4) cultivating transgenic plants with larger grains;
    the biomaterial is any one of the following A1) to a 12):
    a1 Nucleic acid molecules encoding osppl proteins;
    a2 An expression cassette comprising A1) said nucleic acid molecule;
    a3 A) a recombinant vector comprising the nucleic acid molecule of A1);
    a4 A recombinant vector comprising the expression cassette of A2);
    a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);
    a6 A) a recombinant microorganism comprising the expression cassette of A2);
    a7 A) a recombinant microorganism comprising the recombinant vector of A3);
    a8 A) a recombinant microorganism comprising the recombinant vector of A4);
    a9 A transgenic plant cell line comprising the nucleic acid molecule of A1);
    a10 A transgenic plant cell line comprising the expression cassette of A2);
    a11 A transgenic plant cell line comprising the recombinant vector of A3);
    a12 A transgenic plant cell line comprising the recombinant vector of A4);
    the OsAPL protein is a protein shown in the following a 1) or a 2):
    a1 Amino acid sequence is a protein shown in sequence 1;
    a2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by the sequence 1;
    the plant is rice.
  3. 3. The use 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 A genomic DNA molecule shown in sequence 2 or sequence 4;
    b2 A cDNA molecule represented by SEQ ID No. 3;
    b3 A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in B1) or B2) and encoding said OsAPL protein;
    b4 Under stringent conditions with a nucleotide sequence defined in B1) or B2) or B3), and a cDNA molecule or genomic DNA molecule encoding said OsAPL protein.
  4. 4. Use of a substance interfering with expression of a nucleic acid encoding an OsAPL protein in a plant in any one of the following q 1) -q 4):
    q 1) decreasing plant yield;
    q 2) reducing plant kernel size;
    q 3) growing transgenic plants with reduced yield;
    q 4) cultivating transgenic plants with smaller kernels;
    the substance interfering with the expression of the OsAPL protein coding nucleic acid in the plant is a DNA molecule shown in the 1 st to 323 rd positions of a sequence 5 or a vector containing the DNA molecule shown in the 1 st to 323 rd positions of the sequence 5;
    the plant is rice.
  5. 5. A method of growing a transgenic plant with increased yield and/or increased grain size comprising the steps of: increasing the content and/or activity of OsAPL protein in the receptor plant to obtain a transgenic plant; the transgenic plant has a yield and/or grain greater than the recipient plant; the plant is rice; the amino acid sequence of the OsAPL protein is shown as a sequence 1.
  6. 6. The method according to claim 5, wherein: the method for improving the content and/or activity of the OsAPL protein in the receptor plant is to overexpress the OsAPL protein in the receptor plant.
  7. 7. The method according to claim 6, wherein: the over-expression method is to introduce the encoding gene of the OsAPL protein into a receptor plant.
  8. 8. The method according to any one of claims 5-7, wherein: the encoding gene of the OsAPL protein is a DNA molecule shown in a sequence 3.
  9. 9. A method of growing a transgenic plant with reduced yield and/or reduced grain comprising the steps of: reducing the content and/or activity of OsAPL protein in the recipient plant to obtain a transgenic plant; the transgenic plant has a yield and/or grain that is smaller than the recipient plant; the plant is rice; the amino acid sequence of the OsAPL protein is shown as a sequence 1.
  10. 10. The method according to claim 9, wherein: the method for reducing the content and/or activity of the OsAPL protein in the receptor plant comprises the steps of introducing a substance interfering with the expression of an OsAPL protein coding gene into the receptor plant; the substance interfering the expression of the OsAPL protein coding gene is a DNA molecule shown in the 1 st-323 rd positions of the sequence 5 or a vector containing the DNA molecule shown in the 1 st-323 rd positions of the sequence 5.
  11. 11. DNA molecules shown in positions 1-323 of the sequence 5 or vectors containing the DNA molecules shown in positions 1-323 of the sequence 5.
CN202110453036.5A 2021-04-26 2021-04-26 OsAPL protein and application of encoding gene thereof in regulation and control of plant yield Active CN115247185B (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011239684A (en) * 2010-05-14 2011-12-01 National Institute Of Advanced Industrial Science & Technology Improvement of material productivity of plant using transcription factor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011239684A (en) * 2010-05-14 2011-12-01 National Institute Of Advanced Industrial Science & Technology Improvement of material productivity of plant using transcription factor

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
A rice mutant lacking a large subunit of ADP-glucose pyrophosphorylase has drastically reduced starch content in the culm but normal plant morphology and yield;Frederick R. Cook, et al.;Functional Plant Biology;第39卷;1068-1078 *
APL regulates vascular tissue identity in Arabidopsis;Martin Bonke, et al.;Nature;第426卷(第13期);181-186 *
Genbank accession number:BAD26189.1;Sasaki, T., et al.;Genbank;1-2 *
OsAPL controls the nutrient transport systems in the leaf of rice (Oryza sativa L.);Zhen Yan, et al.;Planta;第256卷;10-17 *
Starch reduction in rice stems due to a lack of OsAGPL1 or OsAPL3 decreases grain yield under low irradiance during ripening and modifies plant architecture;Masaki Okamura, et al.;Functional Plant Biology;第40卷;1137-1146 *

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