Protein OsD6p1 Related to Rice Growth and Development, Its Encoding Gene and Application

Abstract

The present invention discloses a rice growth and development-related protein OsD6p1, its encoding gene, and applications. The present invention belongs to the field of biotechnology, and specifically relates to a rice growth and development-related protein OsD6p1, its encoding gene, and applications. The protein provided by the present invention is the OsD6p1 protein, which may be as follows: A1) a protein with an amino acid sequence of SEQ ID No. 1; A2) a protein obtained by substituting and / or deleting and / or adding amino acid residues to the protein of A1), having more than 80% identity with the protein of A1) and having the function of regulating plant stress resistance; A3) a fusion protein obtained by connecting a protein tag to one end of A1) or A2). By regulating the expression level of the gene encoding the OsD6p1 protein, the growth and development performance of rice can be changed, providing new materials for the breeding of superior rice varieties and playing a positive role in accelerating the improvement of rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a rice growth and development-related protein OsD6p1, a coding gene thereof and an application thereof. Background Art

[0002] Frequent global extreme heat waves have caused rice yield reductions, posing a serious threat to food security. Studying the mechanisms by which rice responds to heat stress and cultivating heat-tolerant varieties are key challenges in rice breeding applications. DNA N6-adenine methylation (6mA) is a novel epigenetic mark in eukaryotes that plays a key role in regulating gene expression, DNA damage repair, and development. Currently, research on DNA 6mA regulation of eukaryotic development and environmental stress responses has primarily focused on green algae, nematodes, fruit flies, mammals, and the model plant Arabidopsis thaliana. However, the function and mechanism of DNA 6mA in regulating rice growth, development, and stress tolerance remain unclear. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to regulate the growth and development performance of rice.

[0004] In order to solve the above technical problems, the present invention provides a protein.

[0005] The protein provided by the present invention may be any of the following proteins:

[0006] A1) a protein having an amino acid sequence of SEQ ID No. 1;

[0007] A2) a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein of A1) and having an identity of 80% or more to the protein of A1) and having a function of regulating plant growth and development;

[0008] A3) a fusion protein obtained by connecting a protein tag to one end of A1) or A2);

[0009] The protein may be named OsD6p1 protein.

[0010] Among the above proteins, the protein is derived from rice (Oryza sativa).

[0011] In the above-mentioned proteins, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, blastp can be used as the program, with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained.

[0012] In the above proteins, the above 80% or greater identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0013] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0014] In the above protein, sequence 1 (SEQ ID No. 1) consists of 812 amino acid residues.

[0015] In order to solve the above technical problems, the present invention also provides biological materials related to the protein.

[0016] The biomaterial provided by the present invention can be any of the following:

[0017] B1) a nucleic acid molecule encoding the protein;

[0018] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0019] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0020] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0021] 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);

[0022] B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2);

[0023] B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2);

[0024] C1) a nucleic acid molecule that inhibits, reduces or silences the expression of a gene encoding the protein or a nucleic acid molecule that inhibits, reduces or silences the activity or content of the protein;

[0025] C2) expressing the gene encoding the nucleic acid molecule described in C1);

[0026] C3) an expression cassette containing the coding gene described in C2);

[0027] C4) a recombinant vector containing the coding gene described in C2), or a recombinant vector containing the expression cassette described in C3);

[0028] C5) a recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4);

[0029] C6) a transgenic plant cell line containing the encoding gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4);

[0030] C7) transgenic plant tissue containing the encoding gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4);

[0031] C8) A transgenic plant organ containing the encoding gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

[0032] In the above-mentioned biological materials, the nucleic acid molecule in B1) can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0033] B1) Within the nucleic acid molecule, those skilled in the art can readily mutate the nucleotide sequence encoding the OsD6p1 protein of the present invention using known methods, such as directed evolution or point mutagenesis. Artificially modified nucleotide sequences that are 75% or more identical to the nucleotide sequence of the isolated OsD6p1 protein of the present invention are derived from and equivalent to the nucleotide sequence of the present invention, as long as they encode the OsD6p1 protein and possess the function of the OsD6p1 protein.

[0034] The aforementioned 75% or greater identity may be 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0035] In the above biological material, the nucleic acid molecule in B1) may be the protein encoding gene OsD6p1.

[0036] B1) The nucleic acid molecule may be the following DNA molecule (a1), (a2), or (a3):

[0037] (a1) a DNA molecule whose coding region is shown in Sequence 2 in the Sequence Listing;

[0038] (a2) the DNA molecule shown in Sequence 2 in the Sequence Listing;

[0039] (a3) a DNA molecule that hybridizes to a defined DNA molecule under stringent conditions and encodes the protein;

[0040] (a4) A DNA molecule derived from rice that is 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% identical to the defined DNA molecule and encodes the protein.

[0041] B1) The nucleic acid molecule may specifically be a DNA molecule whose coding sequence of the coding strand is shown in Sequence 2.

[0042] Herein, the vector is well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage), cosmid (i.e., cosmid), Ti plasmid or viral vector. Specifically, it can be vector SG2027.

[0043] In the above-mentioned biological materials, the expression cassettes described in B2) and C3) refer to DNA capable of expressing the gene in the host cell. This DNA may include not only a promoter to initiate gene transcription but also a terminator to terminate gene transcription. Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus, a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); the tomato proteinase inhibitor II promoter (PIN2) or the LAP promoter (both inducible by methyl jasmonate); a heat shock promoter (U.S. Pat. No. 5,187,267); a tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); a seed-specific promoter, such as the millet seed-specific promoter pF128 (CN101063139B (China Patent No. 20071106)). 0099169.7)), seed storage protein-specific promoters (e.g., phaseolin, napin, oleosin, and soybean beta-conglycin promoters (Beachy et al. (1985) EMBO J. 4: 3047-3053). These can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, e.g., 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).

[0044] In B3) above, a plant expression vector can be used to construct a recombinant expression vector containing the gene expression cassette. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, for example. When using OsD6p1 to construct a recombinant expression vector, 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 or the ubiqutin gene promoter (pUbi). These promoters can be used alone or in combination with other plant promoters. In addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, but must be in frame with the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are diverse and can be natural or synthetic. The translation initiation region can be derived from the transcription initiation region or a structural gene.

[0045] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as by adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.), or chemical resistance marker genes (such as herbicide resistance genes).

[0046] The present invention also provides the use of the above-mentioned protein or a substance regulating the expression of the gene or a substance regulating the activity or content of the protein in regulating plant growth and development. The gene encodes the above-mentioned protein, and the application can be any of the following:

[0047] P1) Use of the protein or the substance regulating gene expression or the substance regulating the activity or content of the protein in regulating plant growth and development,

[0048] P2) Use of the protein or a substance regulating gene expression or a substance regulating the activity or content of the protein in the preparation of a product for cultivating plants with advantageous growth and development,

[0049] P3) Use of the protein or the substance regulating gene expression or the substance regulating the activity or content of the protein in improving plant germplasm resources.

[0050] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the protein OsD6p1.

[0051] The present invention also provides a method for regulating plant growth and development, which comprises regulating plant growth and development by regulating the expression of a gene encoding the protein OsD6p1 or regulating the activity or content of the protein.

[0052] In the above, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the cell nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0053] Herein, the regulation may be up-regulation, enhancement, or increase, or down-regulation, inhibition, or reduction.

[0054] Herein, the regulating plant growth and development may be up-regulating, enhancing or improving plant growth and development performance, or may be down-regulating, inhibiting or reducing plant growth and development performance.

[0055] Herein, the regulation of the expression of the gene encoding the protein may be inhibition, reduction or down-regulation of the expression of the gene encoding the protein. Inhibition, reduction or down-regulation of the expression of the gene encoding the protein may be achieved by gene knockout or gene silencing.

[0056] Gene knockout refers to the phenomenon of inactivating a specific target gene through gene editing technology. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.

[0057] The gene silencing refers to the phenomenon of not expressing or underexpressing a gene without damaging the original DNA. Gene silencing is based on the premise that the DNA sequence is not changed, so that the gene is not expressed or underexpressed. Gene silencing can occur at two levels. One is gene silencing at the transcriptional level due to DNA methylation, heterochromatinization and position effects, and the other is post-transcriptional gene silencing, that is, gene inactivation by specifically inhibiting the target RNA at the level after gene transcription, including antisense RNA, co-suppression, gene repression (quelling), RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.

[0058] The present invention also provides a method for up-regulating, enhancing or improving the growth and development performance of rice.

[0059] The method for upregulating, enhancing or improving the growth and development performance of rice provided by the present invention comprises the following steps: upregulating, enhancing or improving the expression of the gene encoding the protein in rice, so that the growth and development performance of rice is higher than that of recipient rice.

[0060] The present invention provides a method for down-regulating, inhibiting or reducing plant growth and development performance, comprising the following steps: down-regulating, inhibiting or reducing the expression of the protein encoding gene in recipient rice, so that the growth and development performance of the rice is lower than that of the recipient rice.

[0061] In the above method, the down-regulation, inhibition or reduction of the expression of the gene encoding the protein in rice is to knock out the gene encoding the protein in target rice.

[0062] In the above method, the knockout is achieved through the CRISPR / Cas9 system.

[0063] In the above method, the target of gene editing by the CRISPR / Cas9 system is positions 2941-2960 of sequence 3 or positions 270-289 of the corresponding sequence 2.

[0064] The substances for gene editing the gene encoding the protein can specifically be sgRNA and Cas9 protein.

[0065] The substance for gene editing the gene encoding the protein can specifically be a DNA molecule encoding sgRNA and a DNA molecule encoding Cas9 protein.

[0066] The substance for gene editing the gene encoding the protein can specifically be an expression vector having a DNA molecule encoding sgRNA and an expression vector having a DNA molecule encoding Cas9 protein.

[0067] The substance for gene editing the gene encoding the protein can specifically be an expression vector having a DNA molecule encoding sgRNA and a DNA molecule encoding Cas9 protein.

[0068] The substance for gene editing the gene encoding the protein is specifically the recombinant plasmid pSG2027-OsD6p1.

[0069] In the above method, the knockout of the protein encoding gene in the target rice may be performed by performing at least one of the following mutations on the protein encoding gene shown in Sequence 2 in the rice genome:

[0070] 1) replacing 5'-GAAGTTGGCTAAGGAGGCCCTGGCAGAGAATAAG-3' in the protein encoding gene in rice genomic DNA with 5'-GAAG-3', thereby knocking out the gene encoding OsD6p1 protein;

[0071] 2) The 5'-GAAGTTGGCTAAGGAGGCCC-3' in the gene encoding the protein in rice genomic DNA was replaced with 5'-GAAGTTGGCTAAGGAGGCCC-3', thereby knocking out the gene encoding the OsD6p1 protein.

[0072] The present invention also provides a method for cultivating rice with improved growth and development performance.

[0073] The method for cultivating rice provided by the present invention comprises the following steps: up-regulating, enhancing or improving the expression of the coding gene of the rice protein to obtain target rice, wherein the growth and development performance of the target rice is higher than that of the rice.

[0074] In the above applications and methods, the plant may be m1) or m2) or m3) or m4):

[0075] m1) monocotyledonous or dicotyledonous plants;

[0076] m2) Grasses;

[0077] m3) Oryza plants;

[0078] m4) Rice.

[0079] In the above applications and methods, regulating the growth and development of the plant can be embodied in any of the following ways:

[0080] 1) Plant height;

[0081] 2) internode length of plants;

[0082] 3) plant spike length;

[0083] 4) Number of first (second) level branches of the plant.

[0084] The present invention uses CRISPR / Cas9-mediated gene editing technology to perform site-directed mutagenesis or knockout of the gene encoding the rice growth and development-related protein OsD6p1, providing new materials for rice variety breeding and playing a positive role in accelerating the improvement of rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 Schematic diagram of the structure of the recombinant plasmid pSG2027-OsD6P1.

[0086] Figure 2 The nucleotide sequence of the mutation site and its surrounding nucleotides is shown in Figure 2.

[0087] Figure 3 The phenotype and statistical chart of rice plant height.

[0088] Figure 4 The phenotype and statistical results of rice panicle type.

[0089] Figure 5 The results show the abundance of DNA 6mA modification detected by UHPLC-MS / MS. DETAILED DESCRIPTION

[0090] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0091] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0092] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0093] The pSG2027 vector in the following examples is described in: Zhang Q, Liang Z, Cui X, et al. N6-methyladenine DNA methylation in Japonica and Indica rice genomes and its association with gene expression, plant development, and stress responses. Molecular Plant, 2018, 11(12): 1492-1508. The public can obtain it from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0094] EXCEL software was used for statistical analysis of the data, and SPASS software was used for multiple comparisons of the data.

[0095] Example 1: Obtaining the Rice OsD6P1 Gene

[0096] DNA was extracted from the leaves of rice variety Nipponbare and used as template to generate the PCR product using primers OsD6P1-F: 5'-ATGGACATGAAAGCCAGGG-3'; OsD6P1-R: 5'-GTCATCAATATCTTGGACC-3'. MaxSuper-Fidelity DNA Polymerase (Cat. No. P505-d1, Vazyme) was used for PCR amplification to obtain the amplified product (i.e., the coding region of the OsD6P1 gene). The coding sequence (CDS) of the OsD6P1 gene in the rice variety Nipponbare is SEQ ID No. 2, and the encoded amino acid sequence is SEQ ID No. 1 for the OsD6P1 protein. The genomic gene encoding the OsD6P1 protein in the genomic DNA of the rice variety Nipponbare is shown in the sequence listing as SEQ ID As shown in No. 3, positions 2001-2017 are the first exon, positions 2238-2319 are the second exon, positions 2556-2678 are the third exon, positions 2894-3037 are the fourth exon, positions 3147-3249 are the fifth exon, positions 3607-3735 are the sixth exon, positions 5982-6052 are the seventh exon, positions 6500-6669 are the eighth exon, positions 6754-6847 are the ninth exon, positions 7615-7712 are the tenth exon, positions 7792-7927 are the eleventh exon, positions 8020-8126 are the twelfth exon, and positions 8722-88 Position 17 is the 13th exon, positions 8891-8978 are the 14th exon, positions 9066-9183 are the 15th exon, positions 9274-9385 are the 16th exon, positions 9455-9578 are the 17th exon, positions 9661-9738 are the 18th exon, positions 9880-9998 are the 19th exon, positions 10461-10514 are the 20th exon, positions 10691-10793 are the 21st exon, positions 15928-16056 are the 22nd exon, positions 16766-16877 are the 23rd exon, and positions 17297-17328 are the 24th exon.

[0097] Example 2: Construction of recombinant plasmid pSG2027-OsD6P1 for knocking out the OsD6P1 gene

[0098] The schematic diagram of the structure of the recombinant plasmid pSG2027-OsD6P1 is shown in Figure 1The nucleotide sequence of the recombinant plasmid pSG2027-OsD6P1 is Sequence 4 in the sequence listing. pSG2027-OsD6P1 expresses an sgRNA targeting the OsD6P1 gene. The target site of the sgRNA is located in the fourth exon of the OsD6P1 gene. The nucleotide sequence of the target site of the sgRNA is positions 2941-2960 of SEQ ID No. 3. pSG2027-OsD6P1 contains an sgRNA gene expression cassette with a nucleotide sequence of positions 2697-5135. The sgRNA gene is shown in nucleotides 519-538 of Sequence 4 in the sequence listing. Nucleotides 59-268 are a promoter that initiates transcription of the sgRNA gene, and nucleotides 7052-7247 are a terminator that terminates transcription of the sgRNA gene.

[0099] pSG2027-OsD6P1 contains a Cas9 protein gene expression cassette with a nucleotide sequence of positions 2725-6988, nucleotides 5136-9399 in sequence 4 encode the Cas9 protein, nucleotides 2650-2859 are a promoter that initiates transcription of the Cas9 protein gene, and nucleotides 9643-9838 are a terminator that terminates transcription of the Cas9 protein gene.

[0100] Example 3: Obtaining and Identifying OsD6P1 Gene Knockout Rice

[0101] 1. Obtaining OsD6P1 gene knockout rice

[0102] The recombinant plasmid pSG2027-OsD6P1 obtained in step 2 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium. Embryogenic calli of rice Nipponbare were genetically transformed with the recombinant Agrobacterium using the Agrobacterium infection method. Resistant calli were then selected (resistance screening was performed using 100 mg / L hygromycin), followed by differentiation and regeneration culture, and then rooting culture to obtain regenerated plants.

[0103] The specific steps are as follows:

[0104] (1) Take out the mature seeds of the plant, remove the shells, and select the plump, smooth and sterile seeds for disinfection.

[0105] (2) Inoculate the sterilized seeds onto the induction medium and culture them in the dark at 28°C for about 14 days. Select the callus with good appearance and good growth ability.

[0106] (3) The recombinant vector pSG2027-OsD6P1 constructed in Example 2 was introduced into Agrobacterium tumefaciens EHA105 to obtain a recombinant bacterium named EHA105 / pSG2027-OsD6P1.

[0107] (4) Take the recombinant bacteria obtained in step (3) and resuspend the bacteria in infection medium to obtain EHA105 / pSG2027-OsD6P1 bacterial suspension.

[0108] (5) Soak the Nipponbare callus tissue from step (2) in the EHA105 / pSG2027-OsD6P1 bacterial suspension prepared in step (4) for 20 min. After infection, discard the bacterial suspension, remove the callus tissue, dry it with sterile filter paper, and then place it on a co-cultivation medium containing acetosyringone and glucose and incubate it in the dark at 28°C for 50-55 h.

[0109] (6) After completing step (5), callus tissues without obvious Agrobacterium on the surface were selected and transferred to an antibacterial medium containing cephalosporin, and cultured in the dark at 28°C for 3-4 days.

[0110] (7) The cultured callus tissue was transferred to a screening medium supplemented with hygromycin and cephalosporin and cultured in the dark at 28°C for 30 days, with subculture every 10 days.

[0111] (8) After completing step (7), fresh hygromycin-resistant callus tissue was taken and inoculated into a pre-regeneration medium. The cells were cultured in the dark at 28°C for 7 days, and then placed in a light culture room (12 h light / 12 h dark) for another 7 days. The cells were then transferred to a regeneration medium and cultured in the light until regenerated plants were grown to obtain candidate OsD6P1 gene knockout plants.

[0112] The rice induction and differentiation medium were both MS medium.

[0113] 2. Identification of OsD6P1 gene knockout rice

[0114] Plants to be tested: Nipponbare (CK) and the candidate OsD6P1 gene knockout plants obtained in Example 3.

[0115] Genomic DNA was extracted from leaves of the test plants. PCR amplification was performed using the primer pair consisting of OsD6P1-F1 and OsD6P1-R1, using the genomic DNA as a template. The pSG2027-OsD6P1 plasmid served as a positive control (V), and the recipient variety Nipponbare served as a negative control (CK). The resulting product was then sequenced. Plants with overlapping peaks near the target site, as identified above, were heterozygous for the edited gene and designated T0-generation transgenic OsD6P1 knockout rice.

[0116] OsD6P1-F1: 5'-GCATATTCAAAAGTTGACAAG-3';

[0117] OsD6P1-R1: 5'-TCCATTTCAACACCCTGGTTC-3'.

[0118] The resulting T0-generation transgenic OsD6P1 knockout rice was cultured to the T2 generation. Each generation of cultured rice was self-pollinated and then identified by PCR to screen for homozygous lines. Two homozygous mutant plants (i.e., with identical mutations on both homologous chromosomes) were obtained and designated osd6p1#1 and osd6p1#2.

[0119] Sequencing revealed that, compared to the genomic DNA of rice Nipponbare (denoted by Nip), the gene encoding the OsD6p1 protein in both homologous chromosomes of the osd6p1#1 plant (denoted by osd6p1#1) underwent the following mutation: "5'-GAAGTTGGCTAAGGAGGCCCTGGCAGAGAATAAG-3' (corresponding to positions 6845-6875 of SEQ ID No. 3 and positions 270-300 of SEQ ID No. 2)" to "5'-GAAG-3'", resulting in a translational frameshift of the amino acids following the editing site, thereby knocking out the gene encoding the OsD6p1 protein. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 2. Figure 2 .

[0120] Sequencing revealed that, compared with the genomic DNA of rice Nipponbare (denoted by Nip), the gene encoding the OsD6p1 protein in both homologous chromosomes of the osd6p1#2 plant (denoted by osd6p1#2) underwent the following mutation: "5'-GAAGTTGGCTAAGGAGGCCC-3' (corresponding to positions 6845-6864 of SEQ ID No. 3 and positions 270-289 of SEQ ID No. 2)" mutated to "5'-GAAGTTGGCTAAGGAGCCC-3'", resulting in a frameshift in the translation of the amino acids following the editing site and premature termination of the protein sequence, thereby knocking out the gene encoding the OsD6p1 protein. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 2. Figure 2 .

[0121] The T2 generation homozygous mutant rice plants of the OsD6p1 gene were further cultivated to obtain T3 generation OsD6p1 plants without transgenic elements, and the phenotypic characteristics were identified.

[0122] The osd6p1#1 plant is self-pollinated and seeds are harvested. These seeds are then grown into plants, which are known as the T1 generation. The T1 generation plants are self-pollinated and seeds are harvested, which are known as the T2 generation. The osd6p1#1 plant and its self-pollinated offspring are referred to as the osd6p1#1 line.

[0123] The osd6p1#2 plant is self-pollinated and seeds are harvested. These seeds are then grown into plants, which are the T1 generation. The T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation. The osd6p1#2 plant and its self-pollinated offspring are referred to as the osd6p1#2 line.

[0124] Example 4: Comparison of rice production traits

[0125] The plants to be tested are: T3 generation homozygous lines of rice Nip, mutant osd6p1#1 and osd6p1#2.

[0126] The seeds of each test strain were germinated and raised in a greenhouse (starting from the time of dew, for a total of 3 weeks) to obtain 3-week seedlings; the 3-week seedlings were transplanted to the field in Langfang, Hebei and cultivated and managed normally, and the plant height, internode length, number of first (second) level branches, and ear length of the test strains were measured. At least 30 individual plant data were counted for each material.

[0127] The growth of the plants to be tested is shown in Figure 3 Compared with Nip, the plant height and internode length of osd6p1#1 and osd6p1#2 were significantly reduced ( Figure 3 ); spike length and number of first (second) level branches were significantly decreased ( Figure 4 ).

[0128] Example 5: DNA 6mA methylation modification level

[0129] The plants to be tested were homozygous T3 plants of the rice Nip, osd6p1#1, and osd6p1#2 lines. Seeds of each test line were germinated and cultured in a greenhouse until the three-leaf stage. Genomic DNA was extracted, and the abundance of DNA 6mA modification was detected using ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). An Agilent 6400 triple quadrupole liquid chromatography-mass spectrometry instrument was used, with Watsons distilled water (0.1% formic acid) and acetonitrile (0.1% formic acid) as the mobile phases. A GOLDaQ column (100 mm x 2.1 mm) with a pore size of 1.9 μm was used, and ion-pair injection detection was set. DNA 6mA methylation modification levels were measured.

[0130] The results are as follows Figure 5 As shown, the DNA 6mA modification levels were increased in osd6p1#1 and osd6p1#2 mutant plants compared with Nip.

[0131] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of a substance that reduces the expression of a protein-coding gene or a substance that reduces the content of said protein in any of the following: P1) Use in reducing the plant height of rice or / and the internode length or / and the panicle length or / and the number of primary and secondary branches; P2) Use in the preparation of a rice product with reduced plant height or / and internode length or / and panicle length and the number of primary and secondary branches of rice; The protein has an amino acid sequence shown in SEQ ID No.1; The substance that reduces the expression of the protein-coding gene or reduces the content of said protein is any one of the following C1)-C5) substances: C1) A nucleic acid molecule that inhibits or reduces or silences the expression of the coding gene of said protein; C2) The coding gene expressing the nucleic acid molecule of C1); C3) An expression cassette containing the coding gene of C2); C4) A recombinant vector containing the coding gene of C2), or a recombinant vector containing the expression cassette of C3); C5) A recombinant microorganism containing the coding gene of C2), or a recombinant microorganism containing the expression cassette of C3), or a recombinant microorganism containing the recombinant vector of C4).

2. The application according to claim 1, wherein: c1) The nucleic acid molecule is a DNA molecule expressing an sgRNA targeting the protein-coding gene described in claim 1 or an sgRNA targeting the protein-coding gene described in claim 1 and a DNA molecule encoding the Cas9 protein; The target sequence of the sgRNA corresponds to positions 2941-2960 of sequence 3 in the sequence listing or positions 270-289 of corresponding sequence 2.

3. The application according to claim 1, wherein: c4) The recombinant vector is recombinant plasmid pSG2027- OsD6p1 , and the nucleotide sequence of the pSG2027- OsD6p1 is Sequence 4 in the Sequence Listing.

4. A method for down-regulating, inhibiting or reducing the growth and development performance of rice, characterized in that: The method includes downregulating or inhibiting or reducing the expression level of the coding gene of the protein described in claim 1 in the recipient rice to reduce the plant height of rice and / or reduce the internode length of rice and / or reduce the panicle length of rice and / or reduce the number of primary and secondary branches of rice.

5. The method according to claim 4, wherein: The method includes introducing into the rice a substance that downregulates or inhibits or reduces the expression level of the coding gene of the protein in the recipient rice; the substance that downregulates or inhibits or reduces the expression level of the coding gene of the protein in the recipient rice is any one of the following C1)-C5) substances: C1) A nucleic acid molecule that inhibits or reduces or silences the expression of the coding gene of said protein; C2) The coding gene expressing the nucleic acid molecule of C1); C3) An expression cassette containing the coding gene of C2); C4) A recombinant vector containing the coding gene of C2), or a recombinant vector containing the expression cassette of C3); C5) A recombinant microorganism containing the coding gene of C2), or a recombinant microorganism containing the expression cassette of C3), or a recombinant microorganism containing the recombinant vector of C4).

6. The method according to claim 4 or 5, characterized in that: The downregulation or inhibition or reduction of the expression of the coding gene of the protein in rice is the knockout of the coding gene of the protein in the target rice; The knockout is achieved through the CRISPR / Cas9 system; The gene editing target of the CRISPR / Cas9 system is positions 2941-2960 of sequence 3 or positions 270-289 of corresponding sequence 2.

7. The method according to claim 6, wherein: The recombinant vector for gene editing by the CRISPR / Cas9 system is the recombinant plasmid pSG2027- OsD6p1 , and the nucleotide sequence of the pSG2027- OsD6p1 is Sequence 4 in the sequence listing.

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