Application of 830 protein and coding gene thereof in regulation and control of plant height

Abstract

The invention discloses an application of 830 protein and a coding gene thereof in regulation and control of plant height. The invention belongs to the technical field of biology, and particularly relates to an application of 830 protein and a coding gene thereof in regulation and control of plant height. The 830 protein is any one of the following proteins: A1) a protein with an amino acid sequence as shown in SEQ ID No.1; a2) a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues on the protein of A1), has 80% or more of identity with the protein of A1) and has the same function as the protein of A1); a3) a fusion protein obtained by connecting a protein label to the N terminal or / and the C terminal of A1) or A2). By inhibiting the expression of the 830 protein, the plant height of rice can be reduced, and the fusion protein has great application and popularization values for plant breeding, especially rice dwarf breeding.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the application of 830 protein and its encoding gene in regulating plant strain growth. Background Art

[0002] Rice is native to China and India. It is one of the world's major grain crops. In China, rice accounts for a quarter of the country's total grain crop area and over half of its production. Its cultivation history dates back 14,000 to 18,000 years. Rice is a key food crop. Besides being edible, its caryopsis can be used to make starch, wine, and vinegar. Rice bran can be used to make sugar, oil, and furfural for industrial and pharmaceutical applications. Rice stalks are a good feed, a raw material for papermaking, and textiles. Rice sprouts and roots are used medicinally.

[0003] In recent years, the rapid development of biotechnology has greatly promoted the innovation of plant breeding research methods and the continuous improvement of research levels. The formation of rice plant architecture is mainly determined by factors such as plant height, number of tillers, tiller angle, and panicle morphology. In rice breeding, improving plant architecture plays a vital role in increasing rice yield and has always been a key indicator for variety selection. Using biotechnology to introduce exogenous insecticide and herbicide resistance genes into plant genomes breaks the natural barriers that prevent hybridization between plant genera and even species, achieving the transfer of insect-resistant and herbicide-resistant genes. This allows plants to quickly and specifically achieve ideal plant architecture and mechanical weed control, while retaining their original favorable agronomic traits. Because each transgenic corn plant possesses a considerable degree of resistance, its insect and herbicide resistance is better and more stable than manual control, and it also saves manpower and material resources, effectively conserving social resources. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to regulate the plant height.

[0005] In order to solve the problems existing in the prior art, the present invention provides a protein.

[0006] The protein provided by the present invention may be any of the following proteins: A1) a protein with the amino acid sequence shown in SEQ ID No. 1; A2) a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein of A1) and having at least 75% identity with the protein of A1) and having the function of regulating plant height; For example, those skilled in the art can obtain a protein mutant having the same function as the amino acid sequence shown in SEQ ID No. 1 by replacing, deleting and / or adding one or more amino acids based on the amino acid sequence shown in SEQ ID No. 1 and conventional techniques in the art, without affecting its activity; A3) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0007] The name of the protein described in A1) above is 830.

[0008] In order to facilitate purification or detection of the protein in A1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing.

[0009] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0010] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0011] Those skilled in the art can readily mutate the nucleotide sequence encoding protein 830 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 protein 830 isolated from the present invention are derived from and are equivalent to the nucleotide sequence of the present invention, as long as they encode protein 830 and have the function of protein 830.

[0012] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0013] As used herein, identity refers to amino acid sequence or nucleotide sequence identity. Amino acid sequence or nucleotide sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, Advanced BLAST 2.1 can be used to calculate the identity of a pair of amino acid or nucleotide sequences by using blastp as the program, setting the Expect value to 10, 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. The identity value (%) can then be calculated.

[0014] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0015] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0016] In the above, the protein is derived from rice ( Oryza sativa L.).

[0017] The present invention also provides a biomaterial related to the above protein, which may be any of the following: B1) a nucleic acid molecule encoding the protein described above; 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) 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); 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); B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2); 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); C1) a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the protein mentioned above; C2) expressing a gene encoding the nucleic acid molecule described in C1); C3) an expression cassette containing the coding gene described in C2); C4) a recombinant vector containing the coding gene described in C2) or a recombinant vector containing the expression cassette described in C3); 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); 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); C7) a transgenic plant tissue containing the coding gene described in C2), or a transgenic plant tissue containing the expression cassette described in C3), or a transgenic plant tissue containing the recombinant vector described in C4); 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).

[0018] In the above biological materials, the nucleic acid molecule described in B1) may be the gene shown in E1) or E2) below: E1) a cDNA molecule or DNA molecule whose coding sequence is SEQ ID No. 2; E2) The nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No. 3.

[0019] The DNA molecule shown in SEQ ID No.2 (regulating plant height) 830 The gene) encodes protein 830 whose amino acid sequence is SEQ ID No.1.

[0020] The nucleotide sequence shown in SEQ ID No. 2 is the nucleotide sequence of the protein 830 encoding gene (CDS).

[0021] The present invention 830 The gene can be any nucleotide sequence that can encode protein 830. Considering the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons suitable for expression in a specific species as needed.

[0022] B1) The nucleic acid molecule may also include a nucleic acid molecule obtained by modifying the codon preference based on the nucleotide sequence shown in SEQ ID No. 3.

[0023] B1) The nucleic acid molecule may further include a nucleic acid molecule having a nucleotide sequence identity of more than 95% with the nucleotide sequence shown in SEQ ID No. 3.

[0024] The nucleic acid molecule described herein 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.

[0025] The vectors described herein are well known to those skilled in the art, and include, but are not limited to, plasmids, phages (such as lambda phage or M13 filamentous phage), cosmids (i.e., cosmids), Ti plasmids, or viral vectors. Specifically, the vector SG2027 may be used.

[0026] Existing plant expression vectors can be used to construct 830 Recombinant expression vectors for genes. Such plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors useful for plant microprojectile bombardment. Such plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. Examples include, but are not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (e.g., the rouge synthase Nos gene) and the 3' transcribed untranslated regions of plant genes (e.g., the soybean storage protein gene), all of which have similar functions.

[0027] use 830 When constructing a recombinant plant expression vector, any enhancing promoter or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to cauliflower mosaic virus (CAMV) 35S promoter and corn ubiquitin promoter, which can be used alone or in combination with other plant promoters. In addition, when using the gene construction plant expression vector of the present invention, 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 identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the start codon is extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

[0028] To facilitate identification and screening of transgenic plant cells or plants, the plant expression vectors used can be modified to include, but are not limited to, genes encoding enzymes or luminescent compounds that can be expressed in plants (such as the GUS gene or luciferase gene), antibiotic resistance markers (such as gentamicin and kanamycin), or chemical resistance marker genes (such as herbicide resistance genes). For safety reasons, it is possible to omit any selectable marker genes and directly screen for transformed plants using stress.

[0029] Utilize any vector that can guide the expression of foreign genes in plants to 830 By introducing genes or gene fragments into plant cells or recipient plants, transgenic cell lines and transgenic plants with altered plant height can be obtained. 830 Gene expression vectors can be transformed into plant cells or tissues through conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissues can be cultivated into plants.

[0030] As a specific embodiment, the recombinant vector may be SG2027-sgRNA. The structure of the recombinant vector SG2027-sgRNA is described as follows: The fragment between 5'-ctgtcgcttgtgtgagacc-3' and 5'-ctagctctaaaacagagacc-3' of the starting vector SG2027 is replaced with the fragment with the nucleotide sequence of SEQ ID No. 4, while the other sequences of the vector SG2027 remain unchanged. The recombinant plasmid SG2027-sgRNA can express the Cas9 protein and the specific sgRNA.

[0031] The microorganisms described herein may be yeast, bacteria, algae or fungi. Among them, the bacteria may be from the genus Escherichia ( Escherichia ), Erwinia ( Erwinia ), Agrobacterium tumefaciens ( Agrobacterium ), Flavobacterium ( Flavobacterium ), Alcaligenes spp. ( Alcaligenes ), Pseudomonas spp. ( Pseudomonas ), Bacillus spp. ( Bacillus ) etc. Specifically, it can be Agrobacterium tumefaciens EHA105.

[0032] In a specific embodiment, the recombinant microorganism can be recombinant Agrobacterium EHA105 / SG2027-sgRNA.

[0033] The recombinant Agrobacterium EHA105 / SG2027-sgRNA is a recombinant bacterium obtained by introducing the recombinant vector SG2027-sgRNA into Agrobacterium tumefaciens EHA105.

[0034] The present invention also provides the use of the aforementioned protein 830 or a substance regulating gene expression or a substance regulating the activity or content of the protein in any of the following applications: U1) 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; U2) Use of the protein or the substance regulating gene expression or the substance regulating the activity or content of the protein in the preparation of a product for regulating plant height; U3) Use of the protein or the substance regulating gene expression or the substance regulating the activity or content of the protein in cultivating plants with taller plants; U4) Use of the protein or the substance regulating gene expression or the substance regulating the activity or content of the protein in the preparation of products for cultivating plants with taller plants; U5) Use of the protein or the substance regulating gene expression or the substance regulating the activity or content of the protein in plant breeding.

[0035] 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 830 .

[0036] In the above application, the substance that regulates gene expression or the substance that regulates the activity or content of the protein may be a biological material related to the protein, and the biological material may be any one of the following: B1) a nucleic acid molecule encoding the protein described above; 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) 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); 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); B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2); 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); C1) a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the protein mentioned above; C2) expressing a gene encoding the nucleic acid molecule described in C1); C3) an expression cassette containing the coding gene described in C2); C4) a recombinant vector containing the coding gene described in C2) or a recombinant vector containing the expression cassette described in C3); 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); 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); C7) a transgenic plant tissue containing the coding gene described in C2), or a transgenic plant tissue containing the expression cassette described in C3), or a transgenic plant tissue containing the recombinant vector described in C4); 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).

[0037] 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 transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of the splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the 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).

[0038] The present invention also provides a method for regulating plant height, comprising regulating the activity and / or content of the aforementioned protein, or / and the expression level of the gene encoding the protein in the target plant, to regulate the plant height.

[0039] In the above method, regulating the activity and / or content of the protein 830 in the target plant, or / and the expression level of the gene encoding the protein, comprises introducing into the recipient plant a gene encoding the protein that inhibits, reduces or silences the protein. 830The substance is used to obtain the target plant with changed plant height; 830 The encoding gene encodes the protein 830.

[0040] The introduction refers to introduction by recombinant means, including but not limited to Agrobacterium-mediated transformation, biolistic methods, electroporation, in planta technology, and the like.

[0041] In the above applications and methods, the regulation may be improvement, enhancement or upregulation.

[0042] In the above applications and methods, the regulation may be inhibition, reduction or silencing.

[0043] To facilitate identification and screening of transgenic cells or plants, the recombinant expression vectors used can be modified, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants and produce color reactions, antibiotic resistance markers, or chemical resistance marker genes. Alternatively, the transformed plants can be directly screened for stress without adding any selectable marker genes.

[0044] The plants obtained by the above methods may be transgenic plants or plants obtained through conventional breeding techniques such as hybridization. In the above methods, the transgenic plants are understood to include not only first- and second-generation transgenic plants, but also their progeny. For transgenic plants, the gene can be propagated within the species or transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. Transgenic plants include seeds, callus tissue, whole plants, and cells.

[0045] The present invention also provides a method for cultivating plants with altered plant height, comprising: 1) increasing, enhancing and / or upregulating the expression level of the gene encoding the protein described above in the target plant, or / and increasing, enhancing and / or upregulating the activity and / or content of the gene encoding the protein described above, to obtain a plant with increased plant height; 2) Inhibiting, reducing or silencing the expression level of the gene encoding the aforementioned protein in the target plant, or / and inhibiting, reducing or silencing the activity and / or content of the gene encoding the aforementioned protein, to obtain a plant with reduced plant height.

[0046] As an embodiment of the present invention, the method for cultivating plants with reduced plant height comprises the following steps: (1) Constructing an expression vector containing SEQ ID No. 4; the expression vector is for inhibiting, reducing or silencing the 830 expression vectors for gene coding sequences; (2) introducing the expression vector constructed in step (1) into the plant; (3) Plants with reduced plant height were obtained through screening and identification.

[0047] Herein, the inhibition, reduction or silencing of the expression of the nucleic acid molecule encoding the 830 protein in the target plant can be achieved by introducing a gene editing vector targeting the nucleic acid molecule encoding the 830 protein into the target plant.

[0048] Specifically, the gene editing vector is based on Cas9 gene editing technology. Specifically, the gene editing vector expresses sgRNA and Cas9 protein. The sgRNA targets the nucleic acid molecule encoding the 830 protein. Specifically, the sgRNA targets the following sequence: 5'-AGGAGGCTGTCAAGATGTGCCGG-3'.

[0049] In the present invention, the purpose of plant breeding includes cultivating plants with increased / reduced plant height.

[0050] In the present invention, in a specific embodiment, in the genome of the plant with reduced plant height, "5'-AGGAGGCTGTCAAGATTGCCGGGACAGGAGGGACTT-3'" is used to replace the target plant's genomic DNA. 830 The "5'-AGGAGGCTGTCAAGATGTGCCGGGACAGGAGGGACTT-3'" sequence in the gene.

[0051] In the above application or method, the plant is any one of the following: N1) Monocots; N2) Gramineae; N3) Grasses; N4) Oryza plants; N5) Rice.

[0052] The present invention discloses the regulatory effect of 830 protein and its encoding gene on plant height. The present invention provides the application of 830 protein in regulating plant height. 830 Gene knocked out or 830 Gene expression is inhibited or 830 Gene editing can reduce plant height. 830 The gene-inhibiting substances and / or the substances that inhibit 830 protein can be applied in the field of plant breeding and can be used for dwarf plant breeding. They have great application and promotion value for plant breeding, especially rice breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram of the structure of the recombinant plasmid.

[0054] Figure 2 for830 Sequencing results of the plant mutation site and its surrounding nucleotides.

[0055] Figure 3 These are photos of plant phenotypes during the tillering stage.

[0056] Figure 4 This is the statistics of plant height during the tillering stage.

[0057] Figure 5 RNA m 5 Results of C modification level detection. DETAILED DESCRIPTION

[0058] 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.

[0059] 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.

[0060] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0061] The starting vector SG2027 in the following examples has been described in: Qu, R., Zhang, P., Liu, Q., Wang, Y., Guo, W., Du, Z., Li, X., Yang, L., Yan, S., & Gu, X. (2022). Genome-edited ATP BINDING CASSETTE B1 transporter SD8 knockouts show optimized rice architecture without yield penalty. Plant communications, 3(5), 100347. The public can obtain the biological material from the applicant for use only for repeating the experiments of the present invention and cannot be used for other purposes.

[0062] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.001 (***) indicated a highly significant difference.

[0063] Example 1. Obtaining 830 protein and its encoding gene The total RNA of rice variety Nipponbare was extracted using a plant total RNA extraction kit, and the first-strand cDNA was synthesized using the Quanshijin first-strand cDNA synthesis kit. Using rice cDNA as a template, PCR amplification was performed with specific primers. After gel recovery, ligation to the cloning vector, bacterial liquid PCR detection, and sequencing comparison, the correct 830 Gene.

[0064] Using the genomic DNA of rice Nipponbare as the reference genome, the gene encoding protein 830 is shown in SEQ ID No. 3 of the sequence listing, and the amino acid sequence of protein 830 is shown in SEQ ID No. 1 of the sequence listing. The coding sequence (CDS) encoding protein 830 is shown in SEQ ID No. 2 of the sequence listing.

[0065] Example 2: Preparation of gene-edited plants 1. sgRNA design and synthesis The target sequence binding region in sgRNA is: 5'-AGGAGGCTGTCAAGATGTGCCGG-3', and the target site is located at 830 The first exon of the gene, the target nucleotide sequence of the sgRNA is located at positions 32-54 of SEQ ID No.2 and positions 2066-2088 of SEQ ID No.3.

[0066] 2. Construction of recombinant plasmid Using vector SG2027 as the starting vector, a recombinant plasmid (circular plasmid) SG2027-sgRNA was prepared that can express the sgRNA in step 1. The recombinant vector SG2027-sgRNA was obtained by replacing the fragment between 5'-ctgtcgcttgtgtgagacc-3' and 5'-ctagctctaaaacagagacc-3' of the starting vector SG2027 with a fragment having the nucleotide sequence of SEQ ID No. 4, while keeping the other sequences of the vector SG2027 unchanged. The recombinant plasmid SG2027-sgRNA can express Cas9 protein and specific sgRNA. The schematic diagram of the recombinant plasmid structure is shown in Figure 1 .

[0067] 3. Genetic transformation and obtaining regenerated plants The recombinant plasmid SG2027-sgRNA prepared in step 2 was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / SG2027-sgRNA.

[0068] The embryonic callus of rice Nipponbare was genetically transformed with recombinant Agrobacterium using the Agrobacterium infection method, and then resistant callus was screened (resistance screening was performed using 100 mg / L hygromycin), followed by differentiation and regeneration culture, and then rooting culture was performed to obtain regenerated plants.

[0069] 4. Obtaining gene-edited plants and their offspring The regenerated plants obtained in step 3 were identified as follows: leaves were taken, genomic DNA was extracted, PCR amplification was performed using a primer pair consisting of primer F1 and primer R1, and the PCR amplification products were sequenced.

[0070] F1: 5'-CATCTTCTTGCTTGGAATGTG-3'; R1: 5'-CTTCTCATATGAACCATAAG-3'.

[0071] Through the above identification, a homozygous mutant plant (a homozygous mutant is a plant with the same mutation on a pair of homologous chromosomes) was screened from the regenerated plants obtained in step 3 and named 830 plant.

[0072] Sequencing showed that compared with the genomic DNA of rice Nipponbare (referred to as Nip), 830 The mutant plants differ only in that a single nucleotide is deleted in the gene encoding the 830 protein, namely the nucleotide "G" at position 2082 of SEQ ID No. 3 (corresponding to position 48 of SEQ ID No. 2). This nucleotide deletion causes a frameshift mutation, resulting in an amino acid change in the 830 protein and a loss of function of the 830 protein, thereby knocking out the 830 gene. The sequencing results of the mutation site and the surrounding nucleotides are shown in Figure 2 .

[0073] 830 The mutant plants are self-pollinated and seeds are harvested, which are then grown into plants, which are the T1 generation plants. The T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. 830 Mutant plants and their self-pollinated offspring are called 830 Mutant strains.

[0074] Example 3: Growth performance evaluation of gene-edited plants The rice seeds used for the test were: rice Nipponbare seeds (Nip), 830 T2 generation seeds of mutant lines.

[0075] Test plants were cultured under parallel conditions, specifically: test rice seeds were germinated and raised in a greenhouse (starting from the time of whitening, for a total of 3 weeks), 3-week seedlings were obtained and photographed; 3-week seedlings were transplanted to the fields in Langfang, Hebei and cultivated and managed normally.

[0076] See the photos of tillering plants cultivated and managed in the field. Figure 3 The statistical results of plant height at the booting stage are shown in Figure 4 (N=15). The results showed that: 830 The plant height of this strain is 83±6cm, which is significantly lower than that of rice Nipponbare.

[0077] Example 4, RNA m 5 C modification level The seeds used for the test are: rice Nipponbare seeds, 830 T2 generation seeds of the strain.

[0078] The test plants were cultured under parallel conditions, specifically: test rice seeds were taken, germinated and cultured in a greenhouse to the three-leaf stage, and the above-ground parts were taken to extract total RNA.

[0079] Total RNA was collected and RNA Dot-blot was performed. The specific antibody used was anti-m 5 C antibody (Diagenode, C15200081). The RNA amount was set at 100, 200, or 400 ng / μL.

[0080] The results are as follows Figure 5 As shown: Compared with Nipponbare, gene editing 830 RNA m 5 C modification levels decreased.

[0081] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A protein, wherein the protein is any of the following: A1) a protein with the amino acid sequence shown in SEQ ID No. 1; A2) a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein of A1) and having at least 80% identity with the protein of A1) and having the same function; A3) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of A1) or A2).

2. The protein according to claim 1, characterized in that: The protein is derived from rice.

3. A biomaterial related to the protein according to claim 1 or 2, wherein the biomaterial is any one of the following: B1) a nucleic acid molecule encoding the protein according to 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) 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); 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); B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2); 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); C1) a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the protein according to claim 1 or 2; C2) expressing a gene encoding the nucleic acid molecule described in C1); C3) an expression cassette containing the coding gene described in C2); C4) a recombinant vector containing the coding gene described in C2) or a recombinant vector containing the expression cassette described in C3); 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); 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); C7) a transgenic plant tissue containing the coding gene described in C2), or a transgenic plant tissue containing the expression cassette described in C3), or a transgenic plant tissue containing the recombinant vector described in C4); 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).

4. The biomaterial according to claim 3, characterized in that B1) The nucleic acid molecule is the gene shown in E1) or E2) below: E1) a cDNA molecule or DNA molecule whose coding sequence is SEQ ID No. 2; E2) The nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No.

3.

5. Application, characterized in that, The application is any of the following: U1) Use of the protein or gene expression regulating substance according to claim 1 or 2 or the substance regulating the activity or content of the protein in regulating plant growth; U2) Use of the protein or gene expression regulating substance according to claim 1 or 2, or a substance regulating the activity or content of the protein, in the preparation of a product for regulating plant height; U3) Use of the protein or gene expression regulating substance according to claim 1 or 2, or a substance regulating the activity or content of the protein, in cultivating plants with taller plants; U4) Use of the protein or gene expression regulating substance according to claim 1 or 2, or a substance regulating the activity or content of the protein, in the preparation of a product for cultivating plants with taller plants; U5) Use of the protein or gene expression regulating substance according to claim 1 or 2, or a substance regulating the activity or content of the protein in plant breeding.

6. The use according to claim 5, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein, and the biological material is any one of the following B1) to B7): B1) a nucleic acid molecule encoding the protein according to claim 1; 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) 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); 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); B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2); 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).

7. A method for regulating plant height, characterized in that: The method comprises regulating the activity and / or content of the protein of claim 1 or 2, or / and the expression level of the gene encoding the protein of claim 1 or 2 in the target plant to regulate the plant height.

8. The method according to claim 7, wherein: The regulating the activity and / or content of the protein according to claim 1 or 2, or / and the expression level of the gene encoding the protein according to claim 1 or 2 in the target plant includes introducing a substance that inhibits the expression of the gene encoding the protein into the recipient plant, thereby obtaining a target plant having a plant height lower than that of the recipient plant; the encoding gene encodes the protein according to claim 1 or 2.

9. A method for cultivating a plant with altered plant height, comprising: 1) increasing, enhancing and / or upregulating the expression level of the gene encoding the protein of claim 1 in the target plant, or / and increasing, enhancing and / or upregulating the activity and / or content of the gene encoding the protein of claim 1, to obtain a plant with increased plant height; 2) Inhibiting, reducing or silencing the expression level of the gene encoding the protein of claim 1 in the target plant, or / and inhibiting, reducing or silencing the activity and / or content of the gene encoding the protein of claim 1, to obtain a plant with reduced plant height.

10. The method according to any one of claims 7 to 9, characterized in that: The plant is any one of the following: N1) a monocotyledonous plant; N2) Gramineae; N3) Grasses; N4) Oryza spp.; N5) Rice.

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