Use of nog2 protein in regulating plant yield

Abstract

The application discloses application of NOG2 protein in regulating yield of plants. The application provides application of protein NOG2, a substance for regulating expression of a coding nucleic acid of the protein or a substance for regulating activity or content of the protein in any one of the following aspects: regulating yield, ear grain number and / or grain length of plants; preparing plants with high yield, increased ear grain number and / or increased grain length; and down-regulating or weakening or reducing expression of the coding nucleic acid of the protein NOG2 in plants, so as to improve yield, ear grain number and / or grain length of the plants, and the coding nucleic acid of the protein NOG2 is a high-quality gene for cultivating high-quality rice varieties and is used for cultivating high-yield rice.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of NOG2 protein in regulating plant yield. Background Technology

[0002] Rice is one of the world's most important food crops, with nearly half the world's population relying on it as a staple food and it providing the main source of energy for daily life. In recent years, with the continuous increase in global population and rapid urbanization, serious agricultural land occupation and degradation have occurred. Coupled with frequent outbreaks of pests and diseases and the continuous deterioration of the global climate, this poses a significant threat to global agricultural production and food security. After two major leaps in rice yield per unit area since the 1960s through dwarf breeding and the 1970s through hybrid breeding, a bottleneck has been encountered. The limited variety of rice germplasm resources and narrow genetic background are among the fundamental reasons for this phenomenon.

[0003] Asian cultivated rice ( Oryza sativa L.) is derived from common wild rice ( Oryzarufipogon Cultivated rice, derived from Griff., exhibits a more homogeneous genetic diversity compared to wild rice after long-term domestication. Wild rice, on the other hand, displays far more diverse genetic differentiation types, containing abundant genetic resources that can enhance rice yield. Therefore, the discovery and utilization of superior rice genetic resources has significant theoretical and practical value, and is also an important pathway to breaking through existing bottlenecks in high-yield breeding. Summary of the Invention

[0004] The technical problem solved by this invention is to provide a way to identify superior rice genes and obtain high-yielding rice with high panicle grain number and long grain.

[0005] To solve the above-mentioned technical problems, the first aspect of the present invention provides the use of protein NOG2, a substance that regulates the expression of the nucleic acid encoding the protein, or a substance that regulates the activity or content of the protein in any of the following:

[0006] A1) Regulating plant yield;

[0007] A2) Regulating the number of grains per spike;

[0008] A3) Regulates plant grain length;

[0009] A4) Preparation of high-yield plants;

[0010] A5) Prepare plants with increased grain number per spike;

[0011] A6) Prepare plants with increased grain length;

[0012] A7) Plant breeding;

[0013] The protein NOG2 is any one of a1)-a4):

[0014] a1) Proteins comprising the amino acid sequence shown in SEQ ID No. 2;

[0015] a2) Proteins derived from a1) having the same function by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2;

[0016] a3) is a protein that shares more than 80% identity with the protein shown in a1);

[0017] a4) Add a tag protein to the end of any of the proteins shown in a1)-a3) to obtain a fusion protein.

[0018] In the above text, plant breeding aims to increase plant yield, increase the number of grains per spike on the main stem, increase the grain length of plants, cultivate high-yielding plants, cultivate plants with increased number of grains per spike on the main stem, and / or cultivate plants with increased grain length.

[0019] In the above-described applications, the substance that regulates the expression of the protein-coding nucleic acid is any of the following biological materials:

[0020] B1) The nucleic acid molecule encoding the protein NOG2;

[0021] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0022] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0023] 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);

[0024] 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);

[0025] 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);

[0026] 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);

[0027] B8) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the nucleic acid encoding the protein NOG2, or inhibit, reduce, or downregulate the activity or content of the protein NOG2;

[0028] B9) expresses the gene encoding the nucleic acid molecule described in B8);

[0029] B10), an expression cassette containing the gene described in B9);

[0030] B11), a recombinant vector containing the gene described in B9), or a recombinant vector containing the expression cassette described in B10;

[0031] B12) recombinant microorganisms containing the gene described in B9), or recombinant microorganisms containing the expression cassette described in B10), or recombinant microorganisms containing the recombinant vector described in B11);

[0032] 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;

[0033] B14), transgenic plant tissue containing the 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;

[0034] B15), a transgenic plant organ containing the 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).

[0035] In the above-described applications, the nucleic acid molecule shown in B8 is encoded by its coding gene;

[0036] Or / and, the coding gene of the nucleic acid molecule shown in B8) includes: a sense segment, an antisense segment, and a spacer segment located between them;

[0037] The positive segment is the DNA molecule shown in SEQ ID No. 1 from the 5′ end, from the 956th to the 1298th segment;

[0038] The antonymous segment is the reverse complementary segment of the just segment.

[0039] In some embodiments, the coding gene of the nucleic acid molecule shown in B8) includes positions 212-1417 of SEQ ID No. 3.

[0040] In some embodiments, the coding gene of the nucleic acid molecule shown in B8) is positions 212-1417 of SEQ ID No. 3.

[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] The regulation is to increase, enhance, or raise, and / or to knock out, decrease, or reduce.

[0045] Upregulating or enhancing the expression of substances encoding the genes of said proteins, or the activity or content of said proteins, can reduce plant yield, number of grains per spike, and / or grain length. Downregulating or weakening the expression of substances encoding the genes of said proteins, or the activity or content of said proteins, can increase plant yield, number of grains per spike, and / or grain length.

[0046] The substance regulating the expression of the encoded nucleic acid 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; 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0047] In some implementations, the regulation of plant yield is to increase plant yield.

[0048] In some embodiments, the regulation of plant spikelet number is to increase the plant spikelet number.

[0049] In some embodiments, the regulation of plant grain length is to increase plant grain length.

[0050] In some embodiments, the substance that regulates the expression of the protein's encoding nucleic acid or the substance that regulates the activity or content of the protein is a substance that downregulates, weakens, or reduces the expression of the protein's encoding nucleic acid, or a substance that downregulates, weakens, or reduces the activity or content of the protein NOG2.

[0051] In some embodiments, the substance that downregulates or weakens or reduces the expression of the protein's encoding nucleic acid, or the substance that downregulates or weakens or reduces the activity or content of the protein NOG2, is the biological material described in B8)-B15).

[0052] In the above-described nucleic acid molecules (B1) or (B8), those skilled in the art can easily mutate the nucleotide sequence encoding the NOG2 protein of this 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 NOG2 protein isolated in this invention, as long as they encode and function as the NOG2 protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0053] 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.

[0054] 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.

[0055] 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 pWMB110 vector;

[0056] In the aforementioned biological materials, the expression cassettes described in B2) and B10) refer to DNA capable of expressing the gene in host cells. 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)); and promoters specific to seed storage proteins (e.g., beta-carotene, napin, etc.). The promoters of 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 Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627.

[0057] In B3) and 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 recombinant expression vectors using TaBON1, any type of 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 plant expression vectors using the genes 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 pCAMBIA1300, pTCK303 / JL1460, or pCAMBIA1301 vectors as expression vectors.

[0058] In the above applications, the nucleic acid molecule described in B1) is any one of the following DNA molecules: b1)-b3)

[0059] b1) The coding region includes the DNA molecule shown in SEQ ID No. 1;

[0060] b2) DNA molecules that hybridize with the DNA sequence defined in b1) under strict conditions and encode proteins with the same function;

[0061] DNA molecules that encode proteins with the same function and whose DNA sequences are defined in b3) and b1) have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology.

[0062] In some embodiments, the coding region of the nucleic acid molecule shown in b1) is the DNA molecule shown in SEQ ID No. 1.

[0063] In the above applications, the target gene of the nucleic acid molecule described in B9) is the gene encoding the protein shown in SEQ ID No. 2.

[0064] In the above text, the ORF of the gene encoding the protein shown in SEQ ID No. 2 may be as shown in SEQ ID No. 1.

[0065] In some embodiments, the nucleic acid molecule described in B9) is derived from the recombinant plasmid pRNAi- LEG2 The hairpin structure is expressed.

[0066] In some embodiments, the recombinant vector shown in B11) is a recombinant plasmid pRNAi- NOG2, The recombinant plasmid is derived from the vector pTCK303 / JL1460. Bam HI recognition sequence and Captain The small DNA fragment between the I-recognition sequences is replaced with the reverse complementary fragment of the DNA molecule shown in SEQ ID No. 1, from position 956 to 1298 starting from the open reading frame starter. Spe I identify sequences and Sac The DNA fragments between the I-identifying sequences are replaced with the nucleotide sequence shown in SEQ ID No. 1, which is the DNA molecule from position 956 to 1298 starting from the open reading frame.

[0067] Recombinant plasmid pRNAi- LEG2 To replace the vector pTCK303 / JL1460 with forward and reverse specific fragments respectively Spe I and Sac I identify sequences and Bam HI and Captain I identifies segments between sequences.

[0068] In some embodiments, B1)-B7) are substances that upregulate, enhance, or increase the expression of the gene encoding the protein or the activity or content of the protein. B8)-B15) are substances that knock out, reduce, or decrease the expression of the gene encoding the protein or the activity or content of the protein.

[0069] In a second aspect, the present invention provides a method for increasing plant yield, comprising the following steps: downregulating or weakening or reducing the expression of the nucleic acid encoding the protein NOG2 described in the first aspect in the plant, or downregulating or weakening or reducing the activity or content of the protein NOG2 described in the first aspect in the plant, thereby increasing plant yield.

[0070] Thirdly, the present invention provides a method for increasing the number of grains per ear in plants, comprising the following steps: downregulating or weakening or reducing the expression of the nucleic acid encoding the protein NOG2 described in the first aspect in plants, or downregulating or weakening or reducing the activity or content of the protein NOG2 described in the first aspect in plants, thereby increasing the number of grains per ear in plants.

[0071] Fourthly, the present invention provides a method for improving plant grain length, comprising the following steps: downregulating or weakening or reducing the expression of nucleic acid encoding the protein NOG2 described in the first aspect in the plant, or downregulating or weakening or reducing the activity or content of the protein NOG2 described in the first aspect in the plant, thereby improving plant grain length.

[0072] Fifthly, the present invention provides a method for cultivating high-yielding plants, comprising the following steps: downregulating, weakening, or reducing the expression of the nucleic acid encoding the protein NOG2 described in the first aspect in a recipient plant, or downregulating, weakening, or reducing the activity or content of the protein NOG2 described in the first aspect in the recipient plant, to obtain a target plant, which is a high-yielding plant. The high-yielding plant is defined as having an increased yield compared to the recipient plant.

[0073] Sixthly, the present invention provides a method for cultivating plants with increased grain number per ear, comprising the following steps: downregulating, weakening, or reducing the expression of the nucleic acid encoding the protein NOG2 described in the first aspect in a recipient plant, or downregulating, weakening, or reducing the activity or content of the protein NOG2 described in the first aspect in the recipient plant, to obtain a target plant, namely, a plant with increased grain number per ear. The plant with increased grain number per ear is defined as having an increased grain number per ear compared to the recipient plant.

[0074] In a seventh aspect, the present invention provides a method for cultivating plants with increased grain length, comprising the following steps: downregulating, weakening, or reducing the expression of the nucleic acid encoding the protein NOG2 described in the first aspect in a recipient plant, or downregulating, weakening, or reducing the activity or content of the protein NOG2 described in the first aspect in the recipient plant, to obtain a target plant, namely, a plant with increased grain length. The plant with increased grain length is defined as having increased grain length compared to the recipient plant.

[0075] The recipient plant contains nucleic acid encoding the protein NOG2 described in the first aspect.

[0076] The downregulation, weakening, or reduction of the expression of the gene encoding the aforementioned protein in the plant includes introducing the recombinant vector described in (B11) above into the target plant.

[0077] In an eighth aspect, the present invention provides the biomaterials described in B8)-B15) of the first aspect.

[0078] In the above text, the plant yield refers to the yield per plant;

[0079] And / or, the number of grains per spike is the number of grains per spike on the main stem of the plant;

[0080] And / or, the plant is any one of c1) to c5): c1) a dicotyledonous plant; c2) a monocotyledonous plant; c3) a grass; c4) rice; c5) indica rice.

[0081] In some embodiments, the rice variety is T5 or the Yunnan Yuanjiang common wild rice infiltration line TYIL17.

[0082] Experiments of this invention demonstrate that downregulating, weakening, or reducing the expression of the nucleic acid encoding the protein NOG2 in plants can increase plant yield, number of grains per panicle, and / or grain length. The nucleic acid encoding the protein NOG2 is a high-quality gene for cultivating high-yield rice varieties. Attached Figure Description

[0083] Figure 1 This study compares the main stem spike type, grain type, and yield per plant of T5 and TYIL17.

[0084] Figure 2 This study compares the main stem spike type, grain type, and yield per plant of T5 and complementary transgenic plants.

[0085] Figure 3 This study compares the main stem spike type, grain type, and yield per plant of TYIL17 with those of interference transgenic plants.

[0086] Figure 4 This study compares the main stem spike type, grain type, and yield per plant of T5 and overexpressing transgenic plants. Detailed Implementation

[0087] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. 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.

[0088] 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.

[0089] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0090] The vector pTCK303 / JL1460 is described in the following literature: Wang Z, Chen CG, Xu YY, Jiang RX, Han Y, Xu ZH, Chong K. A Practical Vector for Efficient Knockdown of GeneExpression in Rice ( Oryza sativa L.). Plant Mol Biol Rep, 2004, 22: 409-417, the vector is named pTCK303 in the article.

[0091] T5 is described in the following literature: Wu Sheng. Genes of rice plant height and yield. SD5 Cloning and functional analysis of the plant [D], the plant is named T5 in this paper. Beijing: China Agricultural University, 2021. It is available to the public from China Agricultural University. T5 belongs to indica rice.

[0092] The common wild rice of Yuanjiang, Yunnan, is described in the following literature: Tan L, Li X, Liu F, Sun X, Li C, Zhu Z, Fu Y, Cai H, Wang X, Xie D, Sun C. Control of a key transition from prostrate to erect growth in rice domestication. Nat Genet. 2008 Nov; 40(11):1360-4. The plant is named Yuanjiang common wild rice in the article; it is available to the public from China Agricultural University.

[0093] The Yunnan Yuanjiang common wild rice introgression line TYIL17 is a progeny obtained by multiple generations of backcrossing and continuous self-pollination between T5 and Yunnan Yuanjiang common wild rice. It is described in the following literature: Wu Sheng. Rice plant height and yield genes. SD5 Cloning and Functional Analysis of TYIL17 [D]. Beijing: China Agricultural University, 2021. Available to the public from China Agricultural University. TYIL17, the infiltrated line of common wild rice from Yuanjiang, Yunnan, is referred to as TYIL17 in this paper.

[0094] Agrobacterium tumefaciens strain EHA105 is described in the following literature: Ren Y, Wang Y, Liu F, Zhou K, Ding Y, Zhou F, Wang Y, Liu K, Gan L, Ma W, Han X, Zhang X, Guo X, Wu F, Cheng Z, Wang J, Lei C, Lin Q, Jiang L, Wu C, Bao Y, Wang H, Wan J. GLUTELIN PRECURSOR ACCUMULATION3 encodes a regulator of post-Golgivesicular traffic essential for vacuolar protein sorting in rice endosperm. Plant Cell, 2014, 26(1): 410-25. It is available to the public from China Agricultural University for replication of the experiments described in this application.

[0095] cDNA acquisition of T5 and TYIL17: Two-week-old seedlings of T5 and TYIL17 were used as experimental materials. Total RNA was extracted from the plants using the TIANGEN plant total RNA extraction kit, and cDNA was obtained by reverse transcription using the TIANGEN reverse transcription kit.

[0096] Example 1 LEG2 The discovery

[0097] Using common wild rice from Yuanjiang, Yunnan as the donor parent and the indica rice variety T5 as the recipient parent, an introgression line population (one of which was TYIL17) was constructed through multiple generations of backcrossing and continuous self-pollination. The panicle type, grain type, and yield per plant of T5 and TYIL17 were compared and statistically analyzed. The experiment was repeated three times, with 15 plants per replicate.

[0098] The experimental results are shown in Figure 1 af (a represents the main stem spike type, bar = 5 cm; b represents the grain type, bar = 1 cm; c represents the yield per plant, bar = 5 cm; df represents the statistical analysis of the number of grains per spike, grain length, and yield per plant). P <0.01 indicates a highly significant difference. The results showed that, compared with T5, TYIL17 had significantly lower main stem spike grain number, grain length, and yield per plant.

[0099] Map-based cloning and functional analysis were performed on the target gene regulating rice yield. The results showed that a QTL affecting rice yield was found on the long arm of chromosome 1, and it was named... LEG2Due to T5 leg2 A nonsense mutation occurred at exon 1 (+98-bp), causing premature termination of protein translation. Therefore, in T5... leg2 The open reading frame sequence is 99 bp in length, and the nog2 protein in T5 consists of 32 amino acid residues.

[0100] TYIL17 LEG2 The open reading frame of the gene is 1830 bp in length, and its nucleotide sequence is shown in SEQ ID No. 1. The protein encoded by this gene is named NOG2 protein, and its amino acid sequence is shown in SEQ ID No. 2. The NOG2 protein in TYIL17 consists of 609 amino acid residues.

[0101] Example 2 LEG2 Obtaining and phenotypically identifying complementary transgenic plants

[0102] I. Recombinant plasmid pCAMBIA1300- LEG2 Construction

[0103] Recombinant plasmid pCAMBIA1300- LEG2 The construction steps are as follows:

[0104] 1. Using two-week-old seedlings of TYIL17 as experimental material, genomic DNA was extracted and used as a template. Then, 90HB-F: 5'-AATTCGAGCTCGGTACCCGG GGATCC TGTCCTAACCCATCTCCCTC-3' (SEQ ID No. 4, underlined is restriction endonuclease) Bam HI recognition site) and 90HB-R: 5'-CCAGTGCCAAGCTTGCATGC CTGCAG AACGAGCTGAGTGTTGGAAA-3' (SEQ ID No. 5, underlined is restriction endonuclease) Psst Using the recognition site of I as primers, PCR amplification was performed and the DNA fragment was purified and recovered to obtain approximately 9.3 kb of DNA. LEG2 (The genome sequence of the gene).

[0105] 2. Using restriction endonucleases Bam HI and Psst I digested the plant expression vector pCAMBIA1300 with enzyme I and recovered approximately 9 kb of the vector backbone.

[0106] 3. The DNA fragment was ligated to the vector backbone to obtain the recombinant plasmid pCAMBIA1300- LEG2 .

[0107] Recombinant plasmid pCAMBIA1300- LEG2 To be LEG2 The genome sequence of the gene (NC 089035 and submission date CON10-JUL-2024) replaced the vector pCAMBIA1300. Bam HI and Psst The fragment between the I restriction sites yields the vector.

[0108] II. Obtaining Recombinant Agrobacterium

[0109] The recombinant plasmid pCAMBIA1300- LEG2 Recombinant Agrobacterium tumefaciens EHA105 was obtained by introducing it into Agrobacterium tumefaciens EHA105 / pCAMBIA1300- LEG2 .

[0110] III. T0 Generation LEG2 Obtaining complementary transgenic plants

[0111] The study employed Hiei et al. (Hiei Y, Ohta S, Komari T & Kumashiro T. Efficient transformation of rice) Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 1994, 6: 271-282) method, to recombinant Agrobacterium EHA105 / pCAMBIA1300- LEG2 Transplanted into T5 indica rice to obtain T0 generation. LEG2 Complementary transgenic plants.

[0112] IV. T0 generation LEG2 Detection of complementary transgenic plants by real-time quantitative PCR (RT-qPCR)

[0113] Randomly select 3 T0 generation LEG2 The complementary transgenic plants (named T0-CTP-1 to T0-CTP-3, respectively) were subjected to RT-qPCR detection. The specific steps are as follows:

[0114] 1. Represented by 3 T0 units respectively LEG2 Leaves of complementary transgenic plants were used as experimental materials. The total RNA extraction kit and reverse transcription kit of TIANGEN plant were used to obtain RNA from each T0 generation. LEG2 cDNA of complementary transgenic plants.

[0115] 2. RT-qPCR technology was used to detect the three T0 generations respectively. LEG2 In complementary transgenic plants LEG2 Relative expression levels (in terms of) KILL As an internal reference gene.

[0116] Detection LEG2 The forward primer 1D-15F: 5'-AAGGACCTTTGGCGTACTCA-3' (SEQ ID No. 6) and the reverse primer 1D-15R: 5'-TTGCTGGGAACGATTGACAA-3' (SEQ ID No. 7) were used for detection. KILL The forward primer UBI-F: 5'-CTGTCAACTGCCGCAAGAAG-3' (SEQ ID No. 8) and the reverse primer UBI-R: 5'-GGCGAGTGACGCTCTAGTTC-3' (SEQ ID No. 9).

[0117] Following the above method, T0 generation LEG2 The complementary transgenic plant was replaced with T5, while all other steps remained unchanged, to obtain T5. LEG2 The relative expression level.

[0118] In T5 LEG2 The relative expression level was used as 1 to statistically analyze the expression levels in complementary transgenic plants. LEG2 The relative expression levels of the three T0 generations were observed. The results showed that, compared to T5, the expression levels of the three T0 generations were significantly lower. LEG2 In complementary transgenic plants LEG2 The relative expression levels of all three increased significantly.

[0119] The above results indicate that T0-CTP-1, T0-CTP-2, and T0-CTP-3 are all T0 substitutions. LEG2 Genetically modified rice.

[0120] V. T2 generation homozygous LEG2 Obtaining complementary transgenic plants and detecting them by RT-qPCR

[0121] By self-crossing T0-CTP-1 to T0-CTP-3 for two consecutive generations, homozygous T2 generation was obtained. LEG2 The complementary transgenic plants were named CTP-1 to CTP-3, respectively.

[0122] Following the method in step four, RT-qPCR was performed on CTP-1 to CTP-3 and T5, respectively.

[0123] Test results are shown Figure 2 d ( P <0.01 indicates a highly significant difference. The results show that, compared to T5, CTP-1 to CTP-3... LEG2 The relative expression levels of all three increased significantly.

[0124] VI. Phenotypic Identification of T2 Generation Homozygous NOG2 Complementary Transgenic Plants

[0125] The seeds of the rice varieties to be tested (T5, CTP-1, CTP-2, and CTP-3) were planted in pots containing nutrient soil and vermiculite (volume ratio of nutrient soil to vermiculite was 1:1), and cultured at 28℃ with alternating light and dark conditions. During the growth and development process, the panicle type, grain type, and yield per plant of the rice varieties were compared and statistically analyzed. The experiment was repeated three times, with 15 plants per replicate.

[0126] Some experimental results can be found in Figure 2 The statistical analysis of ac and eg (a is the main stem spike type, bar = 5 cm; b is the grain type, bar = 1 cm; c is the yield per plant, bar = 5 cm; eg is the number of grains per spike, grain length and yield per plant) is performed. P <0.01 indicates a highly significant difference. The results showed that, compared with T5, the number of grains per spike, grain length, and yield per plant were all significantly reduced in CTP-1 to CTP-3.

[0127] Example 3 LEG2 Interference with the acquisition and phenotypic identification of transgenic plants

[0128] I. Recombinant plasmid pRNAi- LEG2 Construction

[0129] Recombinant plasmid pRNAi- LEG2 The construction steps are as follows:

[0130] 1. Synthetic primers

[0131] According to SEQ ID No. 1 LEG2 Based on the sequence, primers 90Ri-1F, 90Ri-1R, 90Ri-2F, and 90Ri-2R were designed and synthesized; the primer sequences are as follows:

[0132] 90Ri-1F: 5′-TTTCGAGATTTTCAATCGAT ACTAGT TCGGAAAACCTATTGCTGCA-3′ (SEQ ID No. 10, underlined is restriction endonuclease) Spe I's identification site);

[0133] 90Ri-1R: 5′-TTGAACGATCGGGGAAATTC GAGCTC TTGAGTATTGCATGGACCGC-3′ (SEQ ID No. 11, underlined is restriction endonuclease) Sac I. Identification site);

[0134] 90Ri-2F: 5′-TTCTGCAGGTCGACTCTAGA GGATCC TTGAGTATTGCATGGACCGC-3′ (SEQ ID No. 12, underlined is restriction endonuclease) Bam H I. Identification site);

[0135] 90Ri-2R: 5′-GCAGATCTGTCGACCTCGAG GGTACC TCGGAAAACCTATTGCTGCA-3′ (SEQ ID No. 13, underlined is restriction endonuclease) Captain I. Identification site).

[0136] 2. Using TYIL17 cDNA as a template and 90Ri-1F and 90Ri-1R as primers, PCR amplification and purification were performed to obtain DNA fragment A of approximately 340 bp.

[0137] 3. Using TYTIL17 cDNA as a template, and 90Ri-2F and 90Ri-2R as primers, PCR amplification and purification were performed to obtain DNA fragment B of approximately 340 bp.

[0138] 4. Using restriction endonucleases Spe I and Sac I digested the vector pTCK303 / JL1460 with enzyme I, and recovered approximately 14.6 kb of the vector backbone C.

[0139] 5. Ligate DNA fragment A with vector backbone C to obtain intermediate plasmid.

[0140] 6. Using restriction endonucleases Bam H I and Captain I enzyme digestion of the intermediate plasmid yielded approximately 14.9 kb of the vector backbone D.

[0141] 7. Ligate fragment B to vector backbone D to obtain recombinant plasmid pRNAi- LEG2 .

[0142] Based on the sequencing results, the recombinant plasmid pRNAi- LEG2 The structure is described as follows: the carrier pTCK303 / JL1460... Bam HI recognition sequence and Captain The I-recognition sequence is replaced by the reverse complementary fragment of the DNA molecule shown in SEQ ID No. 1, from position 956 to 1298 starting from the open reading frame. Spe I identify sequences and SacThe DNA fragments between the I-identifying sequences are replaced by nucleotide sequences as shown in the DNA molecule of SEQ ID No. 1, from position 956 to 1298 starting from the open reading frame.

[0143] Recombinant plasmid pRNAi- LEG2 To replace the vector pTCK303 / JL1460 with forward and reverse specific fragments respectively Spe I and Sac I identify sequences and Bam HI and Captain I identifies segments between sequences.

[0144] The sequence containing the specific fragment and part of the vector pTCK303 / JL1460 is SEQ ID No. 3. Among them, positions 212-554 of SEQ ID No. 3 are the reverse complementary sequence of the DNA molecule (antense fragment) shown in positions 956 to 1298 of SEQ ID No. 1 from the open reading frame starter; positions 1075-1417 of SEQ ID No. 3 are the DNA molecule sequence shown in positions 956 to 1298 of SEQ ID No. 1 from the open reading frame starter (sense fragment); positions 555-1074 of SEQ ID No. 3 is the spacer region; and the remaining part is the sequence of the vector pTCK303 / JL1460.

[0145] II. Obtaining Recombinant Agrobacterium

[0146] pRNAi- of recombinant plasmid LEG2 Recombinant Agrobacterium tumefaciens EHA105 was obtained by introducing it into Agrobacterium tumefaciens EHA105 / pRNAi- LEG2 .

[0147] III. T0 Generation LEG2 Interfering with the acquisition of transgenic plants

[0148] Recombinant Agrobacterium EHA105 / pRNAi- LEG2 Convert TYIL17 to obtain T0 generation LEG2 Interfering with transgenic plants.

[0149] IV. T0 generation LEG2 RT-qPCR detection of interference transgenic plants

[0150] Randomly select 3 T0 generation LEG2 The interference transgenic plants (named T0-RNAi-1 to T0-RNAi-3, respectively) were subjected to RT-qPCR detection. The specific steps are as follows:

[0151] 1. Represented by 3 T0 units respectively LEG2Using transgenic plants as experimental materials, the total RNA extraction kit and reverse transcription kit of TIANGEN plant were used to obtain the T0 generation of various transgenic plants. LEG2 Interfering with the cDNA of transgenic plants.

[0152] 2. RT-qPCR technology was used to detect the three T0 generations respectively. LEG2 Interference in transgenic plants LEG2 Relative expression level (detection) LEG2 The primers were 1D-15F and 1D-15R, with KILL As an internal reference gene, it is used for detection. KILL The primers are UBI-F and UBI-R.

[0153] Following the above method, T0 generation LEG2 The interference transgenic plant was replaced with TYIL17, with all other steps remaining unchanged, to obtain TYIL17. LEG2 The relative expression level.

[0154] In TYIL17 LEG2 The relative expression level was used as 1, and the expression levels in other rice plants were statistically analyzed. LEG2 The relative expression levels of the three T0 generations were as follows. The results showed that, compared to TYIL17, the expression levels of these three T0 generations were significantly lower. LEG2 Interference in transgenic plants LEG2 The relative expression levels of all three were significantly reduced.

[0155] The above results indicate that T0-RNAi-1 to T0-RNAi-3 are all T0 generation RNAi RNAi-1. LEG2 Interfering with transgenic plants.

[0156] V. T2 Generation LEG2 Obtaining and detecting interference transgenic plants by RT-qPCR

[0157] T0-RNAi-1 to T0-RNAi-2 were self-crossed for two consecutive generations to obtain the T2 generation homozygous strain. LEG2 The interfering transgenic plants were named RNAi-1 to RNAi-2, respectively.

[0158] Following the method in step four, RT-qPCR was performed on RNAi-1 to RNAi-2 and TYIL17, respectively.

[0159] Some test results can be found Figure 3 d ( P <0.01 indicates a highly significant difference. The results showed that, compared to TYIL17, RNAi-1 to RNAi-2... LEG2 The relative expression levels of all three were significantly reduced.

[0160] VI. Phenotypic identification of T2 generation homozygous silent strains

[0161] The seeds of the rice varieties to be tested (TYIL17, RNAi-1, and RNAi-2) were planted in pots containing nutrient soil and vermiculite (volume ratio of nutrient soil to vermiculite was 1:1), respectively, and cultured at 25℃ with alternating light and dark conditions. During the growth and development process, the panicle type, grain type, and yield per plant of the rice varieties were compared and statistically analyzed. The experiment was repeated three times, with 15 plants per replicate.

[0162] Some experimental results can be found in Figure 3 The statistical analysis of ac and eg (a is the main stem spike type, bar = 5 cm; b is the grain type, bar = 1 cm; c is the yield per plant, bar = 5 cm; eg is the number of grains per spike, grain length and yield per plant) is performed. P <0.01 indicates extremely significant difference). The results showed that, compared with TYIL17, RNAi-1 and RNAi-2 significantly increased the number of grains per spike, grain length, and yield per plant.

[0163] Example 4 LEG2 Obtaining and identifying the phenotypic characteristics of overexpressing transgenic plants

[0164] I. Recombinant plasmid pOE- LEG2 Construction

[0165] Recombinant plasmid pOE- LEG2 The construction steps are as follows:

[0166] 1. Synthetic primers

[0167] According to SEQ ID No. 1 LEG2 Based on the sequence, primers 90OE-F and 90OE-R were designed and synthesized; the primer sequences are as follows:

[0168] 90OE-F: 5′-TTCTGCAGGTCGACTCTAGA GGATCC ATGGAGAACCTCATCTCGCT-3′ (SEQ ID No. 14, underlined is restriction endonuclease) Bam H I's identification site);

[0169] 90OE-R: 5′-GAGCGGCCGCCACCGCGGTG GAGCTC CTATCTGGCCCACACAACCG-3′ (SEQ ID No. 15, underlined is restriction endonuclease) Sac I. Identification site).

[0170] 2. Using TYIL17 cDNA as a template and 90OE-F and 90OE-R as primers, PCR amplification and purification were performed to obtain a DNA fragment of approximately 1.8 kb.

[0171] 3. Using restriction endonucleases Bam H I and Sac I digested the vector pCAMBIA1301 with enzyme I, and recovered approximately 11.8 kb of the vector backbone.

[0172] 4. Ligate the DNA fragment to the vector backbone to obtain the recombinant plasmid pOE- LEG2 .

[0173] Recombinant plasmid pOE- LEG2 To transport the pCAMBIA1301 vector Bam H I and Sac The fragment between the I restriction sites is replaced with the one shown in SEQ ID No. 1. LEG2 Genes were obtained.

[0174] II. Obtaining Recombinant Agrobacterium

[0175] The recombinant plasmid pOE- LEG2 Recombinant Agrobacterium tumefaciens EHA105 was obtained by introducing it into Agrobacterium tumefaciens EHA105 / pOE- LEG2 .

[0176] III. T0 Generation LEG2 Obtaining transgenic plants by overexpression

[0177] Recombinant Agrobacterium EHA105 / pOE- LEG2 Transform T5 to obtain T0 generation LEG2 Overexpression of transgenic plants.

[0178] IV. T0 generation LEG2 RT-qPCR detection of overexpressing transgenic plants

[0179] Randomly select 3 T0 generation LEG2 The overexpressing transgenic plants (named T0-OE-1 to T0-OE-3, respectively) were subjected to RT-qPCR detection. The specific steps are as follows:

[0180] 1. Represented by 3 T0 units respectively LEG2 Using overexpressing transgenic plants as experimental materials, the total RNA extraction kit and reverse transcription kit of TIANGEN plant were used to obtain the T0 generation of transgenic plants. LEG2 Overexpression of cDNA in transgenic plants.

[0181] 2. RT-qPCR technology was used to detect the three T0 generations respectively. LEG2 In overexpressing transgenic plants LEG2 Relative expression level (detection) LEG2 The primers were 1D-15F and 1D-15R, with KILL As an internal reference gene, it is used for detection. KILL The primers are UBI-F and UBI-R.

[0182] Following the above method, T0 generation LEG2 The overexpressing transgenic plant was replaced with T5, and all other steps remained unchanged, resulting in T5. LEG2 The relative expression level.

[0183] In T5 LEG2 The relative expression level was used as 1, and the expression levels in other rice plants were statistically analyzed. LEG2 The relative expression levels of the three T0 generations were as follows. The results showed that, compared to T5, the expression levels of the three T0 generations were significantly lower. LEG2 In overexpressing transgenic plants LEG2 The relative expression levels of all of them increased significantly.

[0184] The above results indicate that T0-OE-1 to T0-OE-3 are all T0 generation. LEG2 Overexpression of transgenic plants.

[0185] V. T2 Generation LEG2 Obtaining overexpressing transgenic plants and detecting them by RT-qPCR

[0186] By self-crossing T0-OE-1 to T0-OE-2 for two consecutive generations, homozygous T2 generation was obtained. LEG2 The overexpressing transgenic plants were named OE-1 to OE-2, respectively.

[0187] Following the method in step four, RT-qPCR was performed on OE-1 to OE-2 and T5 respectively.

[0188] Some test results can be found Figure 4 d ( P <0.01 indicates a highly significant difference. The results show that, compared to T5, OE-1 to OE-2... LEG2 The relative expression levels of all of them increased significantly.

[0189] VI. Phenotypic identification of T2 generation homozygous silent strains

[0190] The seeds of the rice varieties to be tested (T5, OE-1, and OE-2) were planted in pots containing nutrient soil and vermiculite (volume ratio of nutrient soil to vermiculite was 1:1), respectively, and cultured at 25℃ with alternating light and dark conditions. During the growth and development process, the panicle type, grain type, and yield per plant of the rice varieties were compared and statistically analyzed. The experiment was repeated three times, with 15 plants per replicate.

[0191] Some experimental results can be found in Figure 4 The statistical analysis of ac and eg (a is the main stem spike type, bar = 5 cm; b is the grain type, bar = 1 cm; c is the yield per plant, bar = 5 cm; eg is the number of grains per spike, grain length and yield per plant) is performed. P <0.01 indicates a highly significant difference. The results showed that, compared with T5, the number of grains per spike, grain length, and yield per plant were significantly reduced in OE-1, OE-2, and OE-3.

[0192] The above results indicate that LEG2 It plays a very important role in regulating rice yield.

[0193] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Application of protein NOG2 in any of the following: A1) Regulating rice yield; A2) Regulating the number of grains per panicle in rice; A3) Regulate rice grain length; The protein NOG2 is any one of a1)-a2): a1) A protein with the amino acid sequence shown in SEQ ID No. 2; a2) Add a tag protein to the end of the protein shown in a1) to obtain the fusion protein.

2. The use of interfering RNA that downregulates, weakens, or reduces the expression of the nucleic acid encoding the NOG2 protein in any of the following: B1) Increase rice yield; B2) Increase the number of grains per panicle in rice; B3) Improve rice grain length; B4) Preparation of high-yield rice; B5) Prepare rice with increased grain number per panicle; B6) Prepare rice with increased grain length; The protein NOG2 is any one of a1)-a2): a1) A protein with the amino acid sequence shown in SEQ ID No. 2; a2) Add a tag protein to the end of the protein shown in a1) to obtain the fusion protein.

3. The application according to claim 2, characterized in that: The gene encoding the interfering RNA includes: a sense segment, an antisense segment, and a spacer segment between them; The positive segment is the DNA molecule shown in SEQ ID No. 1 from the 5′ end, from the 956th to the 1298th segment; The antonymous segment is the reverse complementary segment of the just segment.

4. A method for increasing rice yield, comprising the following steps: downregulating or weakening or reducing the expression of the nucleic acid encoding the protein NOG2 of claim 1 in rice, or downregulating or weakening or reducing the activity or content of the protein NOG2 of claim 1 in rice, thereby increasing rice yield.

5. A method for increasing the number of grains per panicle in rice, comprising the following steps: downregulating, weakening, or reducing the expression of the nucleic acid encoding the protein NOG2 of claim 1 in rice, or downregulating, weakening, or reducing the activity or content of the protein NOG2 of claim 1 in rice, thereby increasing the number of grains per panicle in rice.

6. A method for increasing rice grain length, comprising the following steps: downregulating or weakening or reducing the expression of the nucleic acid encoding the protein NOG2 of claim 1 in rice, or downregulating or weakening or reducing the activity or content of the protein NOG2 of claim 1 in rice, thereby increasing rice grain length.

7. A method for cultivating high-yield rice, comprising the following steps: downregulating or weakening or reducing the expression of the nucleic acid encoding the protein NOG2 of claim 1 in the recipient rice, or downregulating or weakening or reducing the activity or content of the protein NOG2 of claim 1 in the recipient rice, to obtain the target rice, which is high-yield rice.

8. A method for cultivating rice with increased grain number per panicle, comprising the following steps: downregulating or weakening or reducing the expression of the nucleic acid encoding the protein NOG2 of claim 1 in the recipient rice, or downregulating or weakening or reducing the activity or content of the protein NOG2 of claim 1 in the recipient rice, to obtain the target rice, namely, rice with increased grain number per panicle.

9. A method for cultivating rice with increased grain length, comprising the following steps: downregulating or weakening or reducing the expression of the nucleic acid encoding the protein NOG2 of claim 1 in the recipient rice, or downregulating or weakening or reducing the activity or content of the protein NOG2 of claim 1 in the recipient rice, to obtain the target rice, namely, rice with increased grain length.

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