Corn small grain and grain weight related protein RPABC2a and coding gene and application thereof

Abstract

The application discloses corn small kernel and kernel weight related protein RPABC2a and a coding gene and application thereof, belongs to the technical field of biology, and specifically discloses the application of a protein, a substance for regulating the expression of a coding gene of the protein, or a substance for regulating the activity or content of the protein in any one of the following applications: A1) reducing the seed volume of a plant in the family Poaceae and / or preparing a product for reducing the seed volume of a plant in the family Poaceae; A2) reducing the kernel weight of a plant in the family Poaceae and / or preparing a product for reducing the kernel weight of a plant in the family Poaceae.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the corn kernel and kernel weight-related protein RPABC2a, its encoding gene, and its applications. Background Technology

[0002] Corn is my country's largest grain crop, with a stable planting area of ​​over 600 million mu (approximately 40 million hectares) annually. It spans three core industrial chains: food grain, feed grain, and deep-processing raw materials. It holds an irreplaceable strategic position in consolidating the foundation of national food security, ensuring a stable supply of feed grain, and supporting the development of the bio-manufacturing industry.

[0003] my country's maize production has achieved 100% commercial supply of hybrid seeds, with an annual demand of 1.2 billion kilograms. This demand continues to rise slightly with increased planting density and the promotion of new planting models such as strip intercropping. Under this scale effect, even slight fluctuations in seed utilization efficiency per unit area will translate into tens of thousands of tons of seed demand or loss nationwide, significantly impacting seed production cost control and the overall efficiency of the seed industry.

[0004] Currently, my country's maize production has fully entered the mechanization stage, with the adoption rate of single-seed precision planting technology exceeding 85%. The commercial model of pricing and packaging based on the number of seeds has become the industry mainstream, and seed companies have an increasingly urgent need for maize varieties with suitable kernel size. Maize varieties with suitable kernel size are compatible with mainstream precision planters, reducing seed packaging, storage, and transportation costs, and improving seedling uniformity and overall yield. Therefore, researching technologies for regulating maize kernel size is of great guiding significance for breeding maize varieties with suitable kernel size that meet the requirements of single-seed precision planting and for promoting cost reduction and efficiency improvement in the maize seed industry. Summary of the Invention

[0005] The technical problem solved by this invention is to reduce the volume and weight of plant seeds, especially corn seeds.

[0006] To address the above problems, the present invention provides a method for reducing the volume and weight of plant seeds.

[0007] The method includes reducing plant seed volume and grain weight by knocking out or reducing or decreasing the expression of protein-coding genes in plants, and / or the activity and / or content of said proteins.

[0008] The protein is any of the following proteins:

[0009] B1) Proteins with amino acid sequences as shown in SEQ ID NO:2;

[0010] B2) A protein having more than 80% identity and the same function as the protein shown in B1) obtained by substituting and / or deleting and / or adding amino acid residues.

[0011] B3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0012] In this application, the plant may be corn.

[0013] To address the aforementioned problems, the present invention also provides a method for cultivating seeds with reduced volume and weight.

[0014] The method includes knocking out or reducing or decreasing the expression level of the gene encoding the protein mentioned above in the target plant, and / or the activity and / or content of the protein to obtain a plant with reduced seed volume and grain weight, wherein the seed volume and / or grain weight of the plant with reduced seed volume and grain weight is lower than that of the target plant.

[0015] In the above method, knocking out or reducing or decreasing the expression of the gene encoding the protein mentioned above in the plant includes introducing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3) into the target plant.

[0016] In the above text, the nucleic acid molecule may be the nucleic acid molecule described in SEQ ID NO:1.

[0017] To address the above problems, the present invention also provides the following applications.

[0018] The use of a protein, a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the activity or content of the protein in any of the following;

[0019] A1) Application in reducing the seed volume of grasses and / or application in the preparation of products that reduce the seed volume of grasses;

[0020] A2) Applications and / or preparations for reducing grain weight in gramineous plants;

[0021] The protein is any of the following proteins:

[0022] F1) The amino acid sequence is that of the protein shown in SEQ ID NO:2;

[0023] F2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in F1) that has more than 80% identity with and has the same function as the protein shown in F1).

[0024] F3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of F1) or F2);

[0025] The substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein is any one of the following:

[0026] B1) Nucleic acid molecules that encode the above proteins;

[0027] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0028] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0029] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0030] B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0031] B6) Transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0032] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2), or transgenic plant organs containing the recombinant vector described in B3);

[0033] B8) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of genes encoding the aforementioned proteins, or inhibit, reduce, or downregulate the activity or content of the aforementioned proteins;

[0034] B9) The gene encoding the nucleic acid molecule described in B8);

[0035] B10), an expression cassette containing the gene encoding described in B9);

[0036] B11), a recombinant vector containing the encoding gene described in B9), or a recombinant vector containing the expression cassette described in B10;

[0037] B12) recombinant microorganisms containing the encoding gene described in B9), or recombinant microorganisms containing the expression cassette described in B10), or recombinant microorganisms containing the recombinant vector described in B11);

[0038] B13), a transgenic plant cell line containing the gene described in B9), or a transgenic plant cell line containing the expression cassette described in B10), or a transgenic plant cell line containing the recombinant vector described in B11;

[0039] B14), transgenic plant tissue containing the encoding gene described in B9), or transgenic plant tissue containing the expression cassette described in B10), or transgenic plant tissue containing the recombinant vector described in B11;

[0040] B15), a transgenic plant organ containing the encoding gene described in B9), or a transgenic plant organ containing the expression cassette described in B10), or a transgenic plant organ containing the recombinant vector described in B11).

[0041] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0042] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0043] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0044] Of the proteins described above, SEQ ID No:2 consists of 142 amino acid residues. It is named RPABC2a protein, and its encoding gene is the Rpabc2a1 gene.

[0045] In this application, the regulation may be knocking out, reducing, or decreasing.

[0046] In this application, the knockout, reduction, or decrease of the expression of the gene encoding the protein, or the activity or content of the protein, can reduce the seed volume and / or grain weight of grass plants.

[0047] In the above applications, the protein is derived from corn.

[0048] In the above text, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0049] In the nucleic acid molecules described in B1) or B8), those skilled in the art can easily mutate the nucleotide sequence encoding the protein RPABC2a of the present invention using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides that have 80% or more identity with the nucleotide sequence of the protein RPABC2a isolated in the present invention, as long as they encode and function the protein RPABC2a, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0050] The aforementioned 80% or higher degree of identity can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sameness.

[0051] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0052] In this document, the vectors described are known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the pROKII vector;

[0053] In the aforementioned biological materials, the expression cassette described in B2) or B9) refers to DNA capable of expressing the gene in a host cell. This DNA may include not only promoters that initiate gene transcription but also terminators that terminate gene transcription. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this 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 of cauliflower mosaic virus; wound-inducible promoters from tomatoes, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically induced promoters from tobacco, pathogenesis-related (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both induced by jasmonic acid methyl ester); heat shock promoters (US Patent 5,187,267); tetracycline-inducible promoters (US Patent 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 2007 1)). 0099169.7), seed storage protein-specific promoters (e.g., promoters of beta-conglycin, 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 full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (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 AcidsRes. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0054] In B3) or B11) above, the recombinant vector can be a recombinant expression vector containing the gene expression cassette constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, pMDC85, or pCAMBIA1391-Xb. When constructing a recombinant expression vector using MOG1, any enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters. Furthermore, when constructing a plant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. As a specific embodiment, this application uses the pbinGFP4 vector as the expression vector.

[0055] As a specific embodiment, the microbial strain in the recombinant microorganism may be Agrobacterium EHA105.

[0056] In the above applications, the nucleic acid molecule described in B1) is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:1.

[0057] In some embodiments, the present invention relates to sequence information as follows:

[0058] SEQ ID No:1 (Sequence 1) is as follows:

[0059] ATGGCGGACGACGATTACAACGAAATTGACATGGGGTATGAGGATGAGCCCCCAGAACCGGAGATTGAGGAAGGAGCTGAAGAAGAGCCTGAGAACAACGAAGATGCCCCTGATGACGTCATAGGTGGTGAGGGTGAAGATAAGGAACAGGAAAAGACCAAGCGTGAACGCAAAACAACAAAATACATGACGAAGTACGAACGTGCGCGCATCCTGGGTACACGAGCCTTGCAGATAAGCATGAATGCTCCAGTCATGGTTGAGCTTGAGGGTGAAACTGACCCTCTTGAGATTGCAATGAAGGAGCTGAGAGCGCGCAAGATACCCTTCACGATCAGACGGTACTTACCTGATGGAAGCTATGAAGACTGGGGAGTAGACGAGCTCATTGTGGAGGACTCCTGGAAACGCCAGGTTGGTGGAGACTGA。

[0060] SEQ ID No:2 (Sequence 2) is as follows:

[0061] MADDDYNEIDMGYEDEPPEPEIEEGAEEEPENNEDAPDDVIGGEGEDKEQEKTKRERKTTKYMTKYERARILGTRALQISMNAPVMVELEGETDPLEIAMKELRARKIPFTIRRYLPDGSYEDWGVDELIVEDSWKRQVGGD*。

[0062] SEQ ID No:3 (Sequence 3) is as follows:

[0063]

[0064] SEQ ID No:4 (Sequence 4) is as follows:

[0065] 5'-AAGAGAAAAGGCCGGTAGGC-3';

[0066] SEQ ID No:5 (Sequence 5) is as follows:

[0067] 5'-TGCGTGATACCGAAACCACA-3';

[0068] SEQ ID No:6 (Sequence 6) is as follows:

[0069] 5'-GCTCTTCKTCYATAATGRCAATT-3';

[0070] SEQ ID No:7 (Sequence 7) is as follows:

[0071] 5'-ATGGCGGACGACGATTACAACG-3';

[0072] SEQ ID No:8 (Sequence 8) is as follows:

[0073] 5'-TCAGTCTCCACCAACCTGGCGTT-3';

[0074] SEQ ID No:7 (Sequence 9) is as follows:

[0075] 5'-ACTTACCTGATGGAAGCTATGAA-3';

[0076] SEQ ID No:8 (Sequence 10) is as follows:

[0077] 5'-CACTGCACCACATACCCTTT-3';

[0078] SEQ ID No:9 (Sequence 11) is as follows:

[0079] 5'-AGGATATCAAGAAAGCTATTAAGGC-3';

[0080] SEQ ID No:10 (Sequence 12) is as follows:

[0081] 5'-GTAGCCCCACTCGTTGTCG-3';

[0082] SEQ ID No:11 (Sequence 13) is as follows:

[0083] 5'-TATCTCTAGAGGATCCATGGCGGACGACGATTACAAC-3';

[0084] SEQ ID No:12 (Sequence 14) is as follows:

[0085] 5'-TGCTCACCATGGATCCGTCTCCACCAACCTGGCG-3'.

[0086] To address the above problems, the present invention provides a method for cultivating plants with low seed volume and grain weight.

[0087] The method includes knocking out or reducing or decreasing the expression level of the gene encoding the protein mentioned above in the target plant, and / or the activity and / or content of the protein to obtain plants with low seed volume and grain weight, wherein the seed volume and / or grain weight of the low seed volume plants are lower than those of the target plant.

[0088] In any of the methods or applications described above, the plant is any of the following:

[0089] J1) Grasses (Poaceae family);

[0090] J2) Plants of the genus *Zea*;

[0091] J3) Corn.

[0092] The aforementioned proteins or nucleic acid molecules.

[0093] This invention has discovered a gene associated with plant seed volume and grain weight, which has been named Rpabc2a1. This gene can be used to improve the seed volume and grain weight of maize, which is of great significance for breeding new maize varieties. Attached Figure Description

[0094] Figure 1 The phenotype and molecular identification of the maize Rpabc2a1 gene mutant rpabc2a1-1 are shown in Figure (A). The differential loci of the Rpabc2a1 gene in wild-type and rpabc2a1-1 are shown in Figure (B). The molecular identification of the rpabc2a1-1 mutant is shown in Figures (CD). The phenotypic identification of rpabc2a1-1 and wild-type seeds is shown in Figure (E). The 100-seed weight of mature seeds of rpabc2a1-1 and wild-type seeds is shown in Figure (F). The seed length of mature seeds of rpabc2a1-1 and wild-type seeds is shown in Figure (G). The seed width of mature seeds of rpabc2a1-1 and wild-type seeds is shown in Figure (H). The seed thickness of mature seeds of rpabc2a1-1 and wild-type seeds is shown in Figure (I).

[0095] Figure 2The phenotypic and molecular identification of the maize Rpabc2a1 gene mutant rpabc2a1-2 are shown in Figure (A). The differentially expressed sites of the Rpabc2a1 gene in wild-type and rpabc2a1-2 are shown in Figure (B). The molecular identification of the rpabc2a1-2 mutant is shown in Figures (CD). The phenotypic identification of rpabc2a1-2 and wild-type seeds is shown in Figure (E). The 100-seed weight of mature seeds of rpabc2a1-2 and wild-type seeds is shown in Figure (F). The seed length of mature seeds of rpabc2a1-2 and wild-type seeds is shown in Figure (G). The seed width of mature seeds of rpabc2a1-2 and wild-type seeds is shown in Figure (H). The seed thickness of mature seeds of rpabc2a1-2 and wild-type seeds is shown in Figure (I).

[0096] Figure 3 (A) The expression of Rpabc2a1 in various tissues of maize: Root, Stem, Leaf, Husk, Silk, Tassel, Endosperm, and Embryo; (B) Subcellular localization of RPABC2a in tobacco; In each figure, * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001. Detailed Implementation

[0097] This invention provides the maize kernel and kernel weight-related protein RPABC2a, its encoding gene, and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0098] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0099] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0100] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0101] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0102] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0103] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative and not intended to limit the scope of the invention. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0104] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0105] Example 1: Phenotypic and molecular identification of the maize Rpabc2a1 gene mutant rpabc2a1-1

[0106] I. Molecular identification of the rpabc2a1-1 mutant

[0107] This invention targets the Rpabc2a1 gene and screens for Rpabc2a1 mutant materials from a maize Mu transposon insertion mutant library. It was found that the Mu transposon is inserted into the 5'UTR region of the Rpabc2a1 gene. Figure 1 A), and named this material rpabc2a1-1.

[0108] The material was molecularly identified using PCR. Based on the genomic sequences flanking the insertion site and the Mu transposon-specific sequence, a specific primer PCR genotyping system was designed: forward primer F1 5'-AAGAGAAAAGGCCGGTAGGC-3' (SEQ ID NO: 4), reverse primer R1 5'-TGCGTGATACCGAAACCACA-3' (SEQ ID NO: 5), and Mu transposon-specific primer MuTIR5 5'-GCTCTTCKTCYATAATGRCAATT-3' (SEQ ID NO: 6). The PCR reaction system consisted of 20 μL: 10 μL of 2×Rapid Taq Master Mix, 0.5 μL each of forward and reverse primers (10 μmol / L), 1 μL of genomic DNA (100 ng / μL), and 8 μL of ddH2O. The reaction program was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 60 °C annealing for 30 s, 72 °C extension for 1 min, 34 cycles; 72 °C final extension for 5 min. The homozygous mutant amplified only a 500 bp Mu insertion-specific band, the wild-type mutant amplified only a 1000 bp wild-type gene band, and the heterozygous mutant amplified both bands simultaneously.

[0109] Genotyping of single-plant leaf DNA was performed using agarose gel electrophoresis, and the results are shown below. Figure 1 (B) confirms that the Mu transposon of rpabc2a1-1 was not lost and can be stably inherited.

[0110] II. Phenotypic Identification of rpabc2a1-1

[0111] Using rpabc2a1-1 as the male parent and maize inbred line B73 as the female parent, a hybridization was conducted to harvest F1 generation seeds. F1 seed plants were individually planted in the field, bagged, and self-pollinated to harvest F2 generation segregating ears and seeds. All materials were planted in the same experimental field under the same cultivation and management measures to ensure consistent growth conditions.

[0112] See results Figure 1 As shown in (CH), the 100-grain weight, grain length, grain width, and grain thickness of rpabc2a1-1 were all significantly reduced.

[0113] Using cDNA from maize variety B73 as a template, and primers 5'-ATGGCGGACGACGATTACAACG-3' (SEQ ID No:7) and 5'-TCAGTCTCCACCAACCTGGCGTT-3' (SEQ ID No:8), the Rpabc2a1 gene was amplified by PCR.

[0114] Sequencing revealed that the CDS sequence of the Rpabc2a1 gene is SEQ ID No:1, the nucleotide sequence is SEQ ID No:3, and the amino acid sequence of the encoded protein is SEQ ID No:2. This protein was named RPABC2a.

[0115] SEQ ID No:1 (Sequence 1) is as follows:

[0116] ATGGCGGACGACGATTACAACGAAATTGACATGGGGTATGAGGATGAGCCCCCAGAACCGGAGATTGAGGAAGGAGCTGAAGAAGAGCCTGAGAACAACGAAGATGCCCCTGATGACGTCATAGGTGGTGAGGGTGAAGATAAGGAACAGGAAAAGACCAAGCGTGAACGCAAAACAACAAAATACATGACGAAGTACGAACGTGCGCGCATCCT GGGTACACGAGCCTTGCAGATAAGCATGAATGCTCCAGTCATGGTTGAGCTTGAGGGTGAAACTGACCCTCTTGAGATTGCAATGAAGGAGCTGAGAGCGCGCAAGATACCCTTCACGATCAGACGGTACTTACCTGATGGAAGCTATGAAGACTGGGGAGTAGACGAGCTCATTGTGGAGGACTCCTGGAAACGCCAGGTTGGTGGAGACTGA.

[0117] SEQ ID No:2 (Sequence 2) is as follows:

[0118] MADDDYNEIDMGYEDEPPEPEIEEGAEEEPENNEDAPDDVIGGEGEDKEQEKTKRERKTTKYMTKYERARILGTRALQISMNAPVMVELEGETDPLEIAMKELRARKIPFTIRRYLPDGSYEDWGVDELIVEDSWKRQVGGD*.

[0119] SEQ ID No:3 (Sequence 3) is as follows:

[0120]

[0121] III. Detection of Rpabc2a1 expression level

[0122] Seed samples of rpabc2a1-1 and wild type were collected separately and stored in liquid nitrogen to obtain the corresponding test samples. Total RNA was extracted from the test samples using the Trizo1 method, and then the first-strand cDNA was reverse transcribed using a reverse transcription kit (Vazyme). The cDNA was diluted 50 times with sterile water and used as a template. The expression level of the Rpabc2a1 gene in seeds was detected by real-time quantitative PCR (qRT-PCR) (Gapdh gene Zm00001eb173410 was used as an internal reference gene).

[0123] The primers for detecting the Rpabc2a1 gene are 9520-F: 5'-ACTTACCTGATGGAAGCTATGAA-3' (SEQ ID No: 9) and 9520-R: 5'-CACTGCACCACATACCCTTT-3' (SEQ ID No: 10).

[0124] The primers for detecting the Gapdh gene are GAPDH-F: 5'-AGGATATCAAGAAAGCTATTAAGGC-3' (SEQ ID No: 11) and GAPDH-R: 5'-GTAGCCCCACTCGTTGTCG-3' (SEQ ID No: 12).

[0125] Real-time quantitative PCR was performed on an Applied Biosystems 7500 Real Time PCR system (ABI, USA), with each experiment repeated in triplicate. The method reported by Livak KJ and Schmittgen TD (2001) was used, i.e., 2... -∆∆CT Calculate the relative expression level.

[0126] ΔΔCT=(CT. Target-CT. Ubiquitin)Time x-(CT. Target-CT. Ubiquitin)Time 0

[0127] Time x represents any time point, and Time 0 represents the target gene expression at 1-fold after Ubiquitin correction.

[0128] Test results are shown Figure 1(I). Compared with the wild type, the expression level of the Rpabc2a1 gene in rpabc2a1-1 seeds was significantly reduced, indicating that the Mu transposon insertion significantly inhibited the transcriptional expression of the Rpabc2a1 gene, thereby leading to a decrease in seed volume and weight.

[0129] Example 2: Phenotypic and molecular identification of the maize Rpabc2a1 gene allelic mutant rpabc2a1-2

[0130] I. Molecular identification of the allelic mutant rpabc2a1-2

[0131] This invention continues to screen for different types of Rpabc2a1 allelic mutant materials from the maize Mu transposon insertion mutant library, naming this material rpabc2a1-2, whose Mu transposon is precisely inserted into the first intron region of the maize Rpabc2a1 gene ( Figure 2 A).

[0132] Using the same experimental methods and primer sequences as in Example 1, genotyping of single-plant leaf DNA was performed by agarose gel electrophoresis. The homozygous mutant amplified only a 250 bp Mu insertion-specific band, the wild-type amplified only a 1000 bp wild-type gene band, and the heterozygous mutant amplified both bands. The results are as follows: Figure 2 As shown in (B), the Mu transposon insertion of rpabc2a1-2 was not lost and could be stably inherited.

[0133] II. Phenotypic Identification of rpabc2a1-2

[0134] Using rpabc2a1-2 as the male parent and maize inbred line B73 as the female parent, a hybridization was performed to harvest F1 generation seeds. F1 seed plants were individually planted in the field, bagged, and self-pollinated to harvest F2 generation segregating ears and seeds. All materials were planted in the same experimental field under the same cultivation and management measures to ensure consistent growth conditions.

[0135] The rpabc2a1-2 seeds have a similar phenotype to the rpabc2a1-1 seeds in Example 1, but the 100-seed weight, seed length, seed width, and seed thickness of the rpabc2a1-2 seeds are significantly reduced. The results are as follows: Figure 2 As shown in (CH).

[0136] When rpabc2a1-1 and rpabc2a1-2 materials were crossed, the offspring seeds also showed a phenotype of reduced volume and grain weight.

[0137] III. Detection of Rpabc2a1 expression level in allelic mutant rpabc2a1-2

[0138] Seed samples from the rpabc2a1-2 allelic mutant and wild type were collected and stored in liquid nitrogen to obtain the corresponding test samples. Total RNA was extracted from the test samples using the Trizo1 method, and then the first-strand cDNA was reverse transcribed using a reverse transcription kit (Vazyme). The cDNA was diluted 50 times with sterile water and used as a template. The relative expression level of the Rpabc2a1 gene was detected by real-time quantitative PCR (Gapdh gene Zm00001eb173410 was used as an internal reference gene).

[0139] The same experimental methods, statistical methods, and primer sequences as in Example 1 were used for detection. The detection results are shown below. Figure 2 (I). The results showed that the expression level of the Rpabc2a1 gene in the seeds of the rpabc2a1-2 allelic mutant was also significantly reduced compared with the wild type, indicating that the insertion of the Mu transposon into the intron region also affected the transcriptional expression of the Rpabc2a1 gene, which in turn led to a decrease in seed volume and grain weight.

[0140] Example 3: Expression and subcellular localization analysis of RPABC2a in various maize tissues.

[0141] I. Real-time quantitative PCR

[0142] Total RNA was extracted from various tissues of the maize inbred line B73, and the first strand of cDNA was synthesized by reverse transcription using reverse transcriptase. The first strand of cDNA was used as a template, and the Gapdh gene Zm00001eb173410 was used as an internal reference gene for real-time quantitative analysis.

[0143] The primers for detecting the Rpabc2a1 gene are 9520-F: 5'-ACTTACCTGATGGAAGCTATGAA-3' (SEQ ID No: 9) and 9520-R: 5'-CACTGCACCACATACCCTTT-3' (SEQ ID No: 10).

[0144] The primers for detecting the Gapdh gene are GAPDH-F: 5'-AGGATATCAAGAAAGCTATTAAGGC-3' (SEQ ID No: 11) and GAPDH-R: 5'-GTAGCCCCACTCGTTGTCG-3' (SEQ ID No: 12).

[0145] Test results as follows Figure 3 (A) shows that the Rpabc2a1 gene is expressed in all tissues of B73, with the highest expression level in the endosperm.

[0146] II. Subcellular localization of RPABC2a

[0147] 1. Construction of the RPABC2a-pbinGFP4 vector

[0148] To investigate the subcellular localization of RPABC2a, this study constructed a vector for the RPABC2a-pbinGFP4 fusion protein. The CDS of Rpabc2a1 without a stop codon was ligated into the plant expression vector pbinGFP4 fusion plant expression vector. Specific amplification primers for the N-terminal and C-terminal fusion of the pbinGFP4 tag were designed for the target gene coding region sequence: For the C-terminal fusion vector (35S:Gene:GFP), the upstream primer had a CACC adapter sequence added to its 5' end, and the downstream primer had a TGCTCCTGCTCC adapter sequence added to its 5' end; for the N-terminal fusion vector (35S:GFP:Gene), the upstream primer had a CACCTGCTCCTGCTCC adapter sequence added to its 5' end. After sequencing verification, the protein was named RPABC2a-GFP. Using B73 cDNA as a template, the primers used are shown below:

[0149] Nb.GFP-F:

[0150] 5'-TATCTCTAGAGGATCCATGGCGGACGACGATTACAAC-3' (SEQ ID No: 13)

[0151] Nb.GFP-R:

[0152] 5'-TGCTCACCATGGATCCGTCTCCACCAACCTGGCG-3' (SEQ ID No: 14)

[0153] 2. Transient expression in tobacco leaves

[0154] (1) Transient transformation and culture of tobacco: Select healthy tobacco plants with 6-7 fully expanded true leaves that have grown for 4-6 weeks. Use a 1 mL syringe with the needle removed to inject the Agrobacterium tumefaciens solution into the mesophyll cells from the back of the leaf, so that the solution can soak the entire leaf. After injection, the plants are first cultured in a completely dark environment at 28℃ for 12-16 h, and then cultured under normal light conditions. Samples are taken after 3 days for fluorescence observation.

[0155] (2) Laser confocal microscopy observation: Take tobacco leaves from the injection area to prepare temporary slides, use laser confocal scanning microscope to observe the subcellular distribution of GFP fluorescence signal, collect images and complete the subcellular localization analysis of the target gene.

[0156] The results are as follows Figure 3 As shown in (B), RPABC2a is a protein that is mainly located in the cell nucleus.

[0157] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for reducing the volume and / or weight of plant seeds, characterized in that, This includes reducing plant seed volume and / or seed weight by knocking out or reducing or decreasing the expression of protein-coding genes in plants, and / or the activity and / or content of said proteins; The protein is any of the following proteins: B1) The amino acid sequence is that of the protein shown in SEQ ID NO:2; B2) A protein having more than 80% identity and the same function as the protein shown in B1) obtained by substituting and / or deleting and / or adding amino acid residues. B3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

2. A method for cultivating plants with reduced seed volume and / or reduced seed weight, characterized in that, This includes knocking out or reducing or decreasing the expression level of the gene encoding the protein of claim 1 in the target plant, and / or the activity and / or content of the protein to obtain plants with reduced seed volume and / or reduced grain weight, wherein the seed volume of the plant is reduced and / or the grain weight is less than that of the target plant.

3. The method as described in claim 1 or 2, characterized in that, The knockout, reduction, or decrease of expression of the gene encoding the protein of claim 1 or 2 in the plant comprises introducing into the target plant the nucleic acid molecule of claim 4B1), the expression cassette of claim 4B2), or the recombinant vector of claim 4B3).

4. The use of a protein, a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the activity or content of the protein in any of the following: A1) Application in reducing the seed volume of grasses and / or application in the preparation of products that reduce the seed volume of grasses; A2) Application in reducing the grain weight of grasses and / or application in the preparation of products that reduce the grain weight of grasses; The protein is any of the following proteins: F1) The amino acid sequence is that of the protein shown in SEQ ID NO:2; F2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in F1) that has more than 80% identity with and has the same function as the protein shown in F1). F3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of F1) or F2); The substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein is any one of the following: B1) A nucleic acid molecule encoding the protein of claim 1 or 2; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2), or transgenic plant organs containing the recombinant vector described in B3); B8) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein of claim 1 or 2, or inhibit, reduce, or downregulate the activity or content of the protein; B9) The gene encoding the nucleic acid molecule described in B8); B10), an expression cassette containing the gene encoding described in B9); B11), a recombinant vector containing the encoding gene described in B9), or a recombinant vector containing the expression cassette described in B10; B12) recombinant microorganisms containing the encoding gene described in B9), or recombinant microorganisms containing the expression cassette described in B10), or recombinant microorganisms containing the recombinant vector described in B11); B13), a transgenic plant cell line containing the encoding gene described in B9), or a transgenic plant cell line containing the expression cassette described in B10), or a transgenic plant cell line containing the recombinant vector described in B11; B14), transgenic plant tissue containing the encoding gene described in B9), or transgenic plant tissue containing the expression cassette described in B10), or transgenic plant tissue containing the recombinant vector described in B11; B15), a transgenic plant organ containing the encoding gene described in B9), or a transgenic plant organ containing the expression cassette described in B10), or a transgenic plant organ containing the recombinant vector described in B11).

5. The application according to claim 4, characterized in that, The protein is derived from corn.

6. The application according to claim 4 or 5, characterized in that, B1) The nucleic acid molecule described is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:

1.

7. A method for cultivating seeds with reduced volume and / or reduced weight, characterized in that, This includes downregulating or weakening or reducing the expression level of the gene encoding the protein of claim 1 in the target plant, and / or reducing the activity and / or content of the protein to obtain a reduction in seed volume and / or grain weight, wherein the seed volume and / or grain weight of the plant is lower than that of the target plant.

8. The method as described in any one of claims 1-3 or 7, or the application as described in any one of claims 4-6, characterized in that, The plant is any one of the following: J1) Grasses (Poaceae family); J2) Plants of the genus *Zea*; J3) Corn.

9. The protein or nucleic acid molecule as described in claim 4.

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