Use of rice ALKBH8b gene or its encoded protein in regulating plant growth, yield or plant type

Abstract

The invention discloses rice ALKBH8b The invention relates to the use of a gene or its encoded protein in regulating plant growth, yield or plant type. ALKBH8b Gene mutation ALKBH8b Compared with wild-type rice, mutant rice showed increased plant height and leaf width; reduced leaf angle; increased rice panicle length, effective tiller number, and 100-grain weight, confirming ALKBH8b The gene or its protein has the function of regulating plant growth, yield or plant type. ALKBH8b The ALKBH8b protein was obtained by gene expression, and the enzyme activity experiment proved that the ALKBH8b protein is a m 6 A demethylase. The present invention has application prospects in regulating plant growth or plant type, increasing plant yield, etc.

Description

Technical Field

[0001] The present invention relates to a new use of genes isolated from crops, in particular to a new use of genes isolated from rice ( Oryza sativa ) ALKBH8 Use of a gene or its encoded protein in regulating plant growth, yield or plant type, belonging to ALKBH8 New areas of use for genes or their encoded proteins. Background Art

[0002] Epigenetic regulation plays a vital role in plant growth and development, yield, resistance to biotic and abiotic stresses, and response to external signals. In recent years, with the development of new technologies and the discovery of key mRNA methylation regulatory genes, a series of studies have shown that mRNA modification plays a key regulatory role in various biological processes in plants. 6 -methyladenine (m 6 A) is the most common mRNA methylation in plants and plays a key role in embryonic development, stem cell fate determination, epidermal hair bifurcation, leaf morphogenesis, flowering, fruit ripening, stress response and root development.

[0003] m 6 A is the methylation modification with the highest content and the most research in plants. In Arabidopsis, the methylation content changes dynamically in different tissues and developmental stages. 6 A is a reversible RNA modification, m 6 A methyltransferase promotes RNA methylation; 6 A demethylase removes part of the RNA that has been modified by methylation; m 6 A reader protein itself does not have the function of methylation or demethylation. The recognition protein plays its biological function in a reversible reaction by recruiting other proteins. 6 There are relatively many studies on genes related to A. However, the genes related to m 6 There are very few genes associated with A. Summary of the invention

[0004] The main purpose of the present invention is to ALKBH8 Gene, ALKBH8b protein, containing ALKBH8 Gene expression cassette or containing ALKBH8 Recombinant plant expression vectors of genes are used to regulate plant growth, yield or plant type.

[0005] To achieve the above purpose, the main technical solutions adopted by the present invention include:

[0006] One aspect of the present invention is to ALKBH8 Gene, ALKBH8b protein, containing ALKBH8Gene expression cassette or containing ALKBH8 Recombinant plant expression vectors of genes are used to regulate plant growth.

[0007] In a preferred embodiment of the present invention, the regulating plant growth is promoting plant growth; wherein the promoting plant growth includes increasing plant height or increasing leaf width.

[0008] Another aspect of the present invention is to ALKBH8 Gene, ALKBH8b protein, containing ALKBH8 Gene expression cassette or containing ALKBH8 Recombinant plant expression vectors of genes are used to regulate plant yield.

[0009] In a preferred specific embodiment of the present invention, the regulating plant yield is to increase plant yield; wherein, the increasing plant yield includes increasing rice panicle length, effective tiller number or 100-grain weight.

[0010] Another aspect of the present invention is to ALKBH8 Gene, ALKBH8b protein, containing ALKBH8 Gene expression cassette or containing ALKBH8 The recombinant plant expression vector of the gene is used to regulate the plant type; in a preferred embodiment of the present invention, the regulated plant type is a compact plant type, including reducing the leaf angle.

[0011] For reference, the present invention provides an embodiment. ALKBH8 Gene mutation ALKBH8 The gene expression level is reduced or the normal function of ALKBH8b protein is defective, thereby increasing plant height and leaf width; reducing leaf angle; increasing rice panicle length, effective tiller number, and 100-grain weight, thereby promoting plant growth or increasing plant yield.

[0012] The mutations include ALKBH8 One or more nucleotides are substituted, deleted and / or added to the nucleotide sequence of a gene or its promoter. Preferably, the mutation can be obtained by physical mutagenesis, chemical mutagenesis, or gene editing. Physical mutagenesis includes, but is not limited to, radiation mutagenesis, space breeding, etc.; chemical mutagenesis includes mutagenesis caused by treatment with mutagens such as EMS; the gene editing method includes, but is not limited to, ZFN, TALE and / or CRISPR / Cas, etc.

[0013] Those skilled in the art can use conventional methods such as conventional gene knockout or gene editing technology to modify the gene in plants. ALKBH8 Gene knockout mutation, such as constructing ALKBH8 Gene knockout vectors or constructed using gene editing technology ALKBH8CRISPR / Cas9 gene editing vectors, etc., can be used to edit genes in plants ALKBH8 Gene knockout or mutation are methods that are well known to those skilled in the art.

[0014] Those skilled in the art know that the main principle of the CRISPR / Cas gene editing system or gene editing method is to find the location of gene editing in the host genome through a nucleic acid fragment called guide RNA (gRNA), that is, the targeted DNA sequence, and then cut the DNA through the Cas protein. In this application, the Cas protein includes but is not limited to Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13 and / or Cas14.

[0015] The interference ALKBH8 The normal expression or normal function of a gene or promoter can be interfered with by RNA interference technology (RNAi) to interfere with the normal expression of the gene encoding the ALKBH8b protein or its promoter or to cause defects in its normal function. RNA interference technology is a conventional technology in the art. It specifically binds to the homologous region of the mRNA expressed by the target gene through a 21-23bp short-chain double-stranded RNA (siRNA) or a long-chain double-stranded RNA (dsRNA; double-strand RNA), thereby degrading the mRNA and achieving the effect of inhibiting gene expression.

[0016] A preferred specific embodiment of the present invention, a ALKBH8 The construction of gene editing vector includes: ALKBH8 The target sequence for introducing mutations into the gene and the fragments of the homology arms are connected to the vector backbone to obtain a gene editing vector.

[0017] The gene editing vector may also contain a selective marker gene for selecting transformed cells, for selecting transformed cells or tissues. The marker gene includes genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds. In addition, the marker gene also includes phenotypic markers, such as β-galactosidase and fluorescent protein.

[0018] The transformation protocol and the protocol for introducing the polynucleotide or polypeptide into the plant may vary depending on the type of plant or plant cell to be transformed. Suitable methods for introducing the polynucleotide into plant cells include microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-velocity ballistic bombardment. In certain embodiments, various transient transformation methods may be used to transform Medicago truncatula. ALKBH8Genes are provided to plants. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0019] A preferred embodiment of the present invention is a method for inhibiting plant growth, comprising: overexpressing in a plant ALKBH8 Gene, ALKBH8 The increased expression of the gene may enhance the function or activity of the ALKBH8b protein.

[0020] The overexpression can be achieved by constructing a plant recombinant expression vector, including: ALKBH8 The gene is connected to the expression control element to obtain a recombinant plant expression vector; the recombinant plant expression vector may be composed of a 5' non-coding region, the nucleotides shown in SEQ ID NO.1 and a 3' non-coding region; wherein the 5' non-coding region may include a promoter sequence, an enhancer sequence and / or a translation enhancement sequence; the promoter may be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter; the 3' non-coding region may include a terminator sequence, an mRNA cleavage sequence, etc. Suitable terminator sequences may be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase terminator regions.

[0021] In a preferred embodiment of the present invention, the plant is a crop; preferably, the crop is rice.

[0022] Another aspect of the present invention is to provide a rice-derived compound capable of regulating plant growth or yield. ALKBH8 gene or its encoded protein.

[0023] The present invention ALKBH8 The nucleotide sequence of the CDS of the gene is selected from any one of the nucleotide sequences described in (a) to (e) below:

[0024] (a) the polynucleotide sequence shown in SEQ ID No. 1;

[0025] (b) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No.3;

[0026] (c) a polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and the polynucleotide sequence still has the function of regulating plant growth, yield or plant type;

[0027] (d) a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence shown in any one of (a) to (c), and the polynucleotide sequence still has the function of regulating plant growth, yield or plant type;

[0028] (e) A polynucleotide sequence that is complementary to the polynucleotide sequence described in any one of (a) to (d), wherein the polynucleotide sequence still has the function of regulating plant growth, yield or plant type.

[0029] The percentage of sequence identity described in the present invention can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0030] Those skilled in the art can easily adopt known methods, such as directed evolution or point mutation, to ALKBH8 The nucleotide sequence of the gene is mutated. ALKBH8 Nucleotides with 75% or higher identity to the nucleotide sequence of a gene are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the encoded protein has the function of regulating plant growth or yield.

[0031] In addition, the nucleotide sequence described in the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; it can also be RNA, such as mRNA or hnRNA.

[0032] The present invention ALKBH8 The genomic nucleotide sequence of the gene is shown in SEQ ID No.2.

[0033] The ALKBH8b protein described in the present invention is a m 6 A demethylase, whose amino acid sequence is selected from the amino acid sequence shown in any one of the following (a)-(d):

[0034] (a) the amino acid sequence shown in SEQ ID NO.3;

[0035] (b) a protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.3, wherein the protein variant still has the function or activity of regulating plant growth, yield or plant type;

[0036] (c) a protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.3, wherein the protein variant still has the function or activity of regulating plant growth, yield or plant type;

[0037] (d) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 3, which protein still has the function or activity of regulating plant growth, yield or plant shape.

[0038] The present invention uses wild-type rice as experimental material and uses gene editing to modify the gene ALKBH8 The mutant strain obtained by mutation produced a defect in the normal function of ALKBH8b protein. Compared with the wild type, the plant height and leaf width of the mutant strain increased; the leaf angle decreased; the rice panicle length, effective tiller number, and 100-grain weight increased, that is, the growth performance and yield of the mutant strain were improved. Therefore, it can be known that the gene ALKBH8 The present invention further obtains ALKBH8b protein and proves through enzyme activity experiment that ALKBH8b protein is a m 6 A demethylase, and it is inferred that the m 6 A demethylase has the function of inhibiting plant growth. The present invention has application prospects in regulating plant growth and increasing plant yield.

[0039] Definitions of terms used in this invention

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods, devices and materials are now described.

[0041] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-strand or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences and explicitly specified sequences of conservative modifications thereof. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0042] "Stringent hybridization conditions" as used herein refer to conditions of low ionic strength and high temperature as known in the art. Typically, under stringent conditions, the detectable degree of hybridization of a probe to its target sequence is higher than the detectable degree of hybridization to other sequences (e.g., at least 2 times greater than background). Stringent hybridization conditions are sequence-dependent and will vary under different environmental conditions, with longer sequences specifically hybridizing at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the stringency of hybridization or washing conditions. For detailed guidance on nucleic acid hybridization, reference may be made to the literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid hybridization"). acidassays.1993). More specifically, the stringent conditions are usually selected to be about 5-10°C lower than the thermal melting point (Tm) of the specific sequence at a specified ionic strength pH. Tm is the temperature (under specified ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence in a balanced state (because the target sequence is present in excess, 50% of the probes are occupied in a balanced state at Tm). The stringent conditions may be the following conditions: wherein the salt concentration is lower than about 1.0 M sodium ion concentration at pH 7.0 to 8.3, usually about 0.01 to 1.0 M sodium ion concentration (or other salts), and the temperature is at least about 30°C for short probes (including but not limited to 10 to 50 nucleotides), and at least about 60°C for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least two times the background hybridization, optionally 10 times the background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42°C; or 5×SSC, 1% SDS, incubated at 65°C, washed in 0.2×SSC and washed in 0.1% SDS at 65°C. The wash can be performed for 5, 15, 30, 60, 120 minutes or longer.

[0043] The "plurality" mentioned in the present invention generally means 2-8, preferably 2-4; the "replacement" mentioned refers to the replacement of one or more nucleotide residues with different nucleotide residues; the "deletion" mentioned refers to the reduction in the number of nucleotide residues, that is, the lack of one or more nucleotide residues therein; the "insertion" mentioned refers to the change of the nucleotide residue sequence, which results in the addition of one or more nucleotide residues relative to the natural molecule.

[0044] The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

[0045] The term "operably linked" refers to a functional connection between two or more elements. The operably linked elements may be contiguous or non-contiguous.

[0046] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0047] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell.

[0048] The term "gene editing vector" refers to one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, together with vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selection markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 for ALKBH8 Flowchart for the construction of gene mutant strains;

[0050] Figure 2 For wild type and ALKBH8 Comparison of phenotypes of gene mutant strains at different stages; Figure 2 -A is wild type and ALKBH8 Comparison of phenotypes of gene mutants at 60 days; Figure 2 -B is wild type and ALKBH8 Comparison of phenotypes of gene mutants at 80 days;

[0051] Figure 3 For wild type and ALKBH8Statistical chart of the growth of gene mutant strains; Figure 3 -A is wild type and ALKBH8 Statistical graph of plant height of gene mutant strains; Figure 3 -B is wild type and ALKBH8 Leaf width statistics of gene mutant strains;

[0052] Figure 4 Wild type and ALKBH8 Statistical chart of the yield of gene mutant strains; among them, Figure 4 -A is wild type and ALKBH8 Statistical graph of leaf angles of gene mutant strains; Figure 4 -B is wild type and ALKBH8 Statistics of ear length of gene mutant strains; Figure 4 -C is wild type and ALKBH8 Statistical graph of effective tillering number of gene mutant strains; Figure 4 -D is wild type and ALKBH8 Statistics of 100-grain weight of gene mutant strains;

[0053] Figure 5 Construct a validation map for the pGEX 4T-1_GST_ALKBH8b protein expression vector; Figure 5 -A is ALKBH8 Figure 1. The cloning result of gene fragments. Figure 5 -B is ALKBH8 Figure 2. Cloning results of gene fragments and homology arms; Figure 5 -C is the result of linearization detection of pGEX4T-1 vector;

[0054] Figure 6 This is the electrophoresis result of ALKBH8b protein;

[0055] Figure 7 This is a diagram showing the enzyme activity test results of ALKBH8b protein. DETAILED DESCRIPTION

[0056] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0057] Test Example 1 ALKBH8 Gene mutation transformation, screening and functional identification experiments

[0058] 1 Construction of gene editing vector

[0059] according to ALKBH8The target sequence (ATGAGTCGCCGAATACCTCCCGG (SEQ ID No.4)) was designed based on the nucleotide sequence of the CDS of the gene (SEQ ID No.1), and the ALKBH8 The target sequence for introducing mutations in the gene and the target fragment of the homology arm were connected to the linearized vector backbone WMC025-pCBSG032. The connection system is shown in Table 1. After mixing on ice, the mixture was placed in a PCR instrument at 37°C for 30 min and then stored on ice or at 4°C.

[0060] ALKBH8The nucleotide sequence of the CDS of the gene is as follows: ATGGCGGCCGCCGCTGACGACGACGAGCTTCGCCGGCGGGCGGCGGCGCTCCGGGAGGTATTCGGCGACTCATCGGACAGCGAGGCCGACGACCTTCCCGTTGGCGGGGCGGGCCGGGAGCAGTGGAGGTGGGAGGCGGTGGAGGGGGTGAGGGGGCTATGGCTCTGCGCCGCCTTCCTCTCCGCCGACGAGCAGTCGCGCCTACTCACGGCGATCCGGCGAGAAGGATGGTTCAGCGATGCACGCAACCAAGCAATGAGATTTGGTGATCTTCCTTCATGGGCTGTTGAGCTTTCTGCGCTTATCCATGAGGCTATTTGTTTTGGTGATGTCCGTGTTGGTTGTGGTTTGGAGTTGAAAAATGAGGACGAAGATGCATGTCCTTTACCATCAGATTTGCTGTGGAGGAAACCGCTTTTTGACCAAATGATCGCAAACAGATACGAGCCAGGTGAGGGCATCTGTGCTCATGTTGATCTGATGCGCTTTGATGATGGAATCGCGATAGTCTCCCTTGAGTCACCATGCGTGATGCACTTCAGTCGGGCAGAACAAGAAGTTCCTATCTGTGAGACACTGGAGAGTGTGCATGCTGAACCTACAAAGATCCCTGTCTACCTGAACCCAGGGTCCCTTGTTCTCATGTCAGGCGACGCGAGATACTTATGGAAACATGAGATAAACCGCAAGCCAGGTGCTCAGCAATGGGGTGGCAGGGAGCTTGAGCAGCAGATAAGAACTTCAATTACACTGAGGAAGCTCCTTCCATCACCAAACTAA (SEQ ID No.1).

[0061] ALKBH8

[0062] The amino acid sequence of the ALKBH8b protein is as follows: MAAAADDDELRRRAAALREVFGDSSDSEADDLPVGGAGREQWRWEAVEGVRGLWLCAAFLSADEQSRLLTAIRREGWFSDARNQAMRFGDLPSWAVELSALIHEAICFGDVRVGCGLELKN EDEDACPLPSDLLWRKPLFDQMIANRYEPGEGICAHVDLMRFDDGIAIVSLESPCVMHFSRAEQEVPICETLESVHAEPTKIPVYLNPGSLVLMSGDARYLWKHEINRKPGAQQWGGRELEQQIRTSITLRKLLPSPN* (SEQ ID No. 3).

[0063] Table 1 Recombination connection system

[0064]

[0065] After taking the competent DH5α out of the refrigerator, quickly put it on ice. After 5 minutes, add 10 µL of the ligation product when the bacterial block is dissolved, let it stand on ice for 25 min, heat shock at 42°C for 45 s, let it stand on ice for 2 min (do not shake), add 100 µL of LB liquid culture medium without antibiotics, put it in 37°C, shake it at 200 rpm for 1 h, spread it with LB solid culture medium with kanamycin at a concentration of 50 μg / mL, and culture it at 37°C for one day.

[0066] 12 single clones were randomly picked from the cultured plates and placed in 2 mL sterile EP tubes. 500 µL of LB liquid culture medium was added to the sterile EP tubes in advance and the tubes were placed in a shaker at 37°C and 200 rpm for 6 h. 5 µL of bacterial solution was aspirated for PCR of each sample. The reaction system of bacterial solution PCR is shown in Table 2. Positive clones with the correct band size were selected by gel electrophoresis for sequencing.

[0067] The nucleotide sequences of primers QC-F / QC-R used in colony PCR are as follows:

[0068] QC-F: CTGGCGAAAGGGGGATGTGCTGCAA (SEQ ID No. 5);

[0069] QC-R: CTTAGACATGCAATGCTCAT (SEQ ID No. 6).

[0070] Table 2 Reaction system of bacterial liquid PCR

[0071]

[0072] The plasmid with the correct sequencing results was stored in a -20°C refrigerator to complete the construction of the gene editing vector.

[0073] 2 Genetic transformation of rice

[0074] Infection solution: 50 g sucrose, 2.2 g MS powder, 0.5 g MES, 500 μL Silwet L-77. First add 800 mL distilled water, adjust the pH value to 5.7 with 1 M KOH, add distilled water to make up to 1000 mL, and sterilize by high pressure.

[0075] Induction medium: N6 max Stock solution (10×) 100 mL, N6 min Stock solution (100×) 10 mL, Fe 2+ -EDTA stock solution (100×) 10 mL, vitamin stock solution (100×) 10 mL, 2, 4-D stock solution 2.5 mL, proline 0.6 g, CH 0.8 g, sucrose 30 g, plant gel 3 g, first add 900 mL of distilled water, adjust the pH value to 5.8 with 1 M KOH, add distilled water to make up to 1 L, then boil and dispense into 100 mL Erlenmeyer flasks, and sterilize by high pressure.

[0076] Solid co-culture medium: N6 max Stock solution (10×) 12.5 mL, N6 min Stock solution (100×) 1.25 mL, Fe 2+ -EDTA stock solution (100×) 1.25 mL, vitamin stock solution (100×) 2.5 mL, 2, 4-D stock solution 0.625 mL, proline 0.15 g, CH 0.2 g, sucrose 7.5 g, agar powder 2 g, first add 200 mL of distilled water, adjust the pH to 5.6 with 1 M KOH, add distilled water to 250 mL, and sterilize by high pressure. Before use, add 5 mL of 50% glucose and 250 μL of LAS stock solution.

[0077] Screening medium: N6 max Stock solution (10×) 25 mL, N6 min Stock solution (100×) 2.5 mL, Fe 2+-EDTA stock solution (100×) 2.5 mL, vitamin stock solution (100×) 2.5 mL, 2, 4-D stock solution 0.625 mL, proline 0.15 g, CH 0.2 g, sucrose 7.5 g, agar powder 2 g, first add 200 mL of distilled water, adjust the pH value to 6.0 with 1 M KOH, add distilled water to 250 mL, and sterilize by high pressure. When using, add 250 μL Hn (50 mg / ml) and 500 μL Cn (250 mg / mL), pour into a sterilized plate, blow dry on a clean bench for about 2 h before use.

[0078] Differentiation medium: MS max Stock solution (10×) 100 mL, MS min Stock solution (100×) 10 mL, Fe 2+ -EDTA stock solution (100×) 10 mL, vitamin stock solution (100×) 10 mL, KT stock solution 2.0 mL, NAA stock solution 0.2 mL, proline 0.6 g, CH 0.8 g, D-sorbitol 30 g, sucrose 30 g, plant gel 3.0 g, first add 900 mL of distilled water, adjust the pH value to 5.8 with 1 M KOH, add distilled water to make up to 1 L, then boil and dispense into 100 mL conical flasks, and sterilize by high pressure.

[0079] Rooting medium: MS max Stock solution (10×) 50 mL, MS min Stock solution (100×) 5 mL, Fe 2+ -EDTA stock solution (100×) 5 mL, vitamin stock solution (100×) 5 mL, sucrose 20 g, plant gel 3.0 g, first add 900 mL of distilled water, adjust the pH to 5.8 with 1 M KOH, add distilled water to 1 L, then boil and dispense into rooting tubes, and sterilize by high pressure.

[0080] Transform the gene editing vector into Agrobacterium: Place 1 µL of the gene editing vector into 20 µL of Agrobacterium culture medium (EHA105-WM), place in an ice bath for 5 min, freeze in liquid nitrogen for 5 min, place in a 37°C water bath for 5 min, and place in an ice bath for 5 min. Add 100 µL of antibiotic-free LB liquid medium, place in a 28°C, 200 rpm shaker for 2 h, directly apply to LB solid medium containing 50 μg / mL kanamycin + 50 μg / mL rifampicin, and culture at 28°C for two days.

[0081] Two days later, a single clone was picked and placed in a 5 mL sterile EP tube. 2 mL of LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampicin was added to the sterile EP tube in advance and cultured overnight. 400 μL of bacterial solution was placed in 100 μL 75% sterile glycerol, marked, and stored in a -80℃ refrigerator.

[0082] Select mature rice seeds, peel off the husk, pour into a 50 mL centrifuge tube, add 75% ethanol to disinfect for 1 min, pour out the ethanol, rinse with sterile water, pour out, add 30% sodium hypochlorite to disinfect for 20 min, pour out the sodium hypochlorite and rinse with sterile water 5-6 times. Use a pipette to absorb excess water and transfer the seeds to the induction medium, 20-25 seeds per dish. After the callus grows, the original embryo can be used for direct transformation.

[0083] Streak the Agrobacterium EHA105 containing the target gene vector on LB solid medium containing 50 μg / mL kanamycin and culture in the dark at 28°C for 2 days until a single colony appears. Prepare the infection solution and use a pipette to aspirate the infection solution to wash off the Agrobacterium on the plate to obtain the Agrobacterium suspension for co-cultivation of rice transformation.

[0084] Select a sufficient number of callus tissues (in good condition, bright yellow in color, round and hard in texture, and preferably with a particle diameter of about 3 mm) and place them in a 100 mL sterile conical flask, add an appropriate amount of Agrobacterium suspension (ensure that there is enough bacterial liquid in contact with the material), and place them at room temperature for infection for 20 minutes, shaking them from time to time. Pour out the bacterial liquid, place the callus tissue on a sterile filter paper to absorb the excess bacterial liquid, and then transfer it to a solid co-culture medium covered with a layer of sterile filter paper, and culture it in the dark at 26°C for 3 days.

[0085] ALKBH8 The flowchart of gene mutant construction is as follows Figure 1As shown in the figure, the specific steps are as follows. After 3 days of co-culture, the callus tissue was washed, and the callus on the solid co-culture medium was sown into a sterilized triangular flask with a 1 mL blue pipette tip, and sterile water was added to rinse both sides. The third time was rinsed with sterile water containing 500 μL / L carbenicillin. After the excess water was sucked off by the pipette, the callus was transferred to a sterile filter paper and the water on the callus was blown dry by the wind of the clean bench. The blowing time was controlled at about 30 min. After the callus was blown dry, it was transferred to the screening medium for screening culture. The culture conditions were 28-30℃ and dark culture. The screening time was 3-4 weeks. After one month of screening, bright yellow positive callus can be seen growing. At this time, the positive callus can be picked to the differentiation medium for differentiation and regeneration. 16 positive callus were placed on each differentiation dish and placed in a 28-30℃ greenhouse for light culture. Generally, green spots can emerge from the callus in about 10 days, and seedlings will differentiate after about 10 days. When the differentiated seedlings grow to about 2-3cm and have obvious root systems, they can be transferred to rooting medium to grow. The rooting medium should be poured into a relatively high bottle or tube so that the rooted seedlings have enough space to grow taller. The rooting culture conditions are 28-30℃ and sterile light culture.

[0086] After transplanting, the resistance gene and target site of T0 plants were detected by PCR. The reaction system and reaction procedure of PCR detection are shown in Tables 3 and 4. The primers for detecting resistance genes are HYG-F1 / HYG-R1, and the primers for detecting target sites are LOC_Os11g43610_J2281092-A1-F1 / R1. The nucleotide sequences of the two sets of primers are as follows:

[0087] HYG-F1: CAAAGATCGTTATGTTTATCGGCACT (SEQ ID No. 7);

[0088] HYG-R1: TTGGCGACCTCGTATTGGGAA (SEQ ID No. 8).

[0089] LOC_Os11g43610_J2281092-A1-F1: TATTACCGCCATTCCAAG (SEQ ID No. 9);

[0090] LOC_Os11g43610_J2281092-A1-R1:TCTATCTCAATCAACCCAC (SEQ ID No. 10).

[0091] Table 3 Reaction system for PCR detection

[0092]

[0093] Table 4 PCR test reaction procedure

[0094]

[0095] 3. Functional identification of mutant rice strains

[0096] 3.1 Results of growth performance evaluation of mutant rice

[0097] The rice seeds were spread flat on a petri dish lined with moist filter paper at 28°C for germination. After 3 days, the rice seeds were basically germinated and transferred to a rice culture box for culture at 28°C for about 30 days. They were then transferred to a rice field for continued growth. A measuring ruler was used to measure the distance from the root to the top of the rice 60 days after germination and the width of the widest part in the middle of the rice leaf.

[0098] The phenotypic results of rice at 60 and 80 days after germination are as follows Figure 2 -A. Figure 2 -B shows that compared with the wild type, the mutant strain showed a higher plant height; the statistical results of plant height are shown in Figure 3 -A shows that compared with the wild type, the mutant rice plant height increased significantly by 4.5%; the statistical results of leaf width are as follows Figure 3 As shown in Figure 2-B, the leaf width of the mutant rice was significantly increased by 4.0%. Therefore, the growth performance of the mutant was improved compared with the wild type.

[0099] 3.2 Yield identification results of mutant rice

[0100] The rice seeds were spread flat on a culture dish lined with moist filter paper at 28°C for germination. After 3 days, the rice seeds were basically germinated and transferred to a rice culture box for culture at 28°C for about 30 days. The seeds were transferred to a rice field and continued to grow until maturity. A protractor was used to measure the angle between the tangent at the base of the curved leaf and the main stem, a ruler was used to measure the distance from the base to the tip of the rice ear after mature harvest, a timer was used to count the number of tillers of a single rice plant, and an analytical balance was used to weigh the weight of 100 full rice seeds.

[0101] The yield of wild type and mutant rice is shown in Figure 4 As shown, Figure 4 -A is a statistical diagram of the leaf angles of wild-type and mutant rice strains; compared with the wild-type, the leaf angles of mutant rice strains are reduced, the plant shape is more compact, and it is more conducive to photosynthesis; Figure 4 -B is a statistical chart of wild-type and mutant rice panicle lengths. Compared with the wild-type, the mutant rice panicle length is significantly increased by 4.5%; Figure 4 -C is a statistical chart of the effective tiller numbers of wild-type and mutant rice. Compared with the wild-type, the effective tiller number of mutant rice was significantly increased by 14.2%; Figure 4-D is a statistical chart of the 100-grain weight of wild-type and mutant rice. Compared with the wild-type, the 100-grain weight of the mutant rice is significantly increased, by 3.7%. Therefore, compared with the wild-type, the yield of the mutant has been improved.

[0102] Experimental Example 2 ALKBH8b protein function identification test

[0103] 1 containing pGEX 4T-1_GST_ ALKBH8 Obtaining recombinant bacteria of protein expression vector

[0104] 1.1 Extraction of total RNA from wild-type rice

[0105] Take fully ground plant tissue powder, about 100 mg per tube, add 1 mL TRIzol, shake and mix for 1 min, and place on ice for 8 min; add 200 μL chloroform to extract, mix thoroughly and place on ice for 2 min; centrifuge at 13,523×g, 4℃ for 10 min; discard the supernatant, add 1 mL pre-cooled 70% ethanol, suspend the precipitate, and centrifuge at 13,523×g, 4℃ for 2 min; discard the supernatant as much as possible, place on ice, place in a clean bench and blow for 6 min, add 50 μL DEPC water to dissolve the precipitate; add DNase I to the mixed system according to Table 5 below, and enzymatically digest at 37℃ for 30 min; add 300 μL DEPC water, 200 μL water-saturated phenol and 200 μL chloroform, mix thoroughly; centrifuge at 13,523×g, 4℃ for 15 min; take 350 μL supernatant and place it in a new 1.5 mL RNase-free centrifuge tube, add 2 times the volume of pre-cooled anhydrous ethanol and 0.1 times the volume of 3 M NaAc, mix well, precipitate RNA at -80℃ for more than 2 h; centrifuge at 13,523×g, 4℃ for 10 min; discard the supernatant, add 1 mL of pre-cooled 70% ethanol, suspend the precipitate, and centrifuge at 13,523×g at 4℃ for 2 min (repeat this step once); discard the supernatant as much as possible, place on ice, place in a clean bench and blow for 6 min, dissolve RNA with 50 μL DEPC water; the obtained RNA is directly used for reverse transcription PCR or stored in a -80℃ refrigerator for later use.

[0106] Table 5 DNase I treatment system

[0107]

[0108] 1.2 cDNA synthesis

[0109] Collect mRNA from total RNA using oligo (dT) magnetic beads; reverse transcribe mRNA into cDNA using RT reverse transcriptase; determine RNA concentration of the sample and adjust the RNA concentration to be basically consistent with DEPC water; perform reverse transcription PCR according to the system and steps in Table 6; store the obtained cDNA in a -20°C refrigerator for later use.

[0110] Table 6 Reverse transcription system and steps

[0111]

[0112] 1.3 ALKBH8 Gene cloning

[0113] Using cDNA as a template, PCR reaction was performed using primers ALKBH8b_F / ALKBH8b_R. The PCR reaction system was shown in Table 7. The annealing temperature was 60°C, 35 cycles were performed, and the PCR product was amplified at 72°C for 1 min. The results of PCR amplification product detection by gel electrophoresis were as follows: Figure 5 -A, the correct positive clone was picked out according to its band size, and the recovered fragment was stored in a -20°C refrigerator for future use.

[0114] The nucleotide sequence of ALKBH8b_F / ALKBH8b_R is as follows:

[0115] ALKBH8b_F:ATGGCGGCCGCCGCTGA (SEQ ID No. 11);

[0116] ALKBH8b_R:TTAGTTTGGTGATGGAAGGAGCTTCCTCAGT (SEQ ID No. 12).

[0117] Table 7 ALKBH8 PCR reaction system for gene cloning

[0118]

[0119] 1.4 Construction of pGEX 4T-1_GST_ALKBH8b protein expression vector

[0120] by ALKBH8 The positive clone of the gene was used as a template, and the primers pGEX 4T-1_GST_ALKBH8b_F / pGEX4T-1_GST_ALKBH8b_R were used for PCR reaction. The PCR reaction system was shown in Table 8 below. The annealing temperature was 60°C, 35 cycles were performed, and the extension was 72°C for 10 min to amplify the PCR product with the homology arm of pGEX 4T-1. The results of gel electrophoresis detection of PCR amplification products were as follows: Figure 5-B, select the correct positive clone based on its band size, recover the fragment, and store it in a -20℃ refrigerator for future use. Bam HI and XOt I double enzyme digestion reaction, the specific reaction system is shown in Table 9 to obtain a linear vector, and the gel electrophoresis detection results are shown in Figure 5 -C. The linear vector was connected with the PCR product with homology arms using homologous recombination enzyme to obtain pGEX 4T-1_GST_ ALKBH8 The protein expression vector and homologous recombination reaction system are shown in Table 10.

[0121] The nucleotide sequence of pGEX 4T-1_GST_ALKBH8b_F / pGEX 4T-1_GST_ALKBH8b_R is as follows:

[0122] pGEX4T-1_GST_ALKBH8b_F:ATCTGGTTCCGCGTGGATCCATGGCGGCCGCCGCTGA (SEQ IDNo.13);

[0123] pGEX4T-1_GST_ALKBH8b_R:CACGATGCGGCCGCTCGAGTTAGTTTGGTGATGGAAGGAGCTTCCTCAGT (SEQ ID No. 14).

[0124] Table 8 ALKBH8 Gene and homology arm connection PCR reaction system

[0125]

[0126] Table 9 Carrier linearization reaction system

[0127]

[0128] Table 10 Homologous recombination reaction system

[0129]

[0130] 1.5 Conversion

[0131] The homologous recombination product was transferred into DH5a competent cells, placed on ice for 30 min, incubated at 42°C for 1 min, and on ice for 2 min. 700 μL of antibody-free LB was added, incubated at 37°C at 200 rpm for 1 h, and centrifuged at 5000 rpm for 1 min. The supernatant was discarded, and 200 μL of the precipitate was retained and coated with LB solid medium containing ampicillin antibiotic at a final concentration of 50 μg / mL.

[0132] 1.6 Identification of positive plasmids

[0133] Pick the larger monoclones cultured overnight and use primers ALKBH8b_F / ALKBH8b_R for colony PCR. The reaction system of colony PCR is shown in Table 11. The ones with the correct band size were selected for sequencing. Six monoclones with correct sequencing were selected and added to LB liquid culture medium containing ampicillin antibiotics. After culturing at 37°C and 250 rpm shaking for 6 hours, the plasmid was extracted. The plasmid was extracted using the TIANGEN rapid plasmid extraction kit. The specific steps refer to the instructions.

[0134] Table 11 Colony PCR reaction system

[0135]

[0136] 1.7 pGEX 4T-1_GST_ ALKBH8 Transformation of protein expression vector

[0137] 100 ng of plasmid was transferred into Rosetta competent cells, placed on ice for 30 min, heat-shocked at 42°C for 1 min, placed on ice for 2 min, 700 μL of antibiotic-free LB liquid medium was added, and cultured at 37°C, 200 rpm on a shaking incubator for 1 h. After that, the cells were centrifuged at 5000 rpm for 1 min, the supernatant was discarded, and 200 μL of the precipitate was retained and coated on LB solid medium containing ampicillin at a final concentration of 50 μg / mL.

[0138] 2. ALKBH8b protein induction expression and extraction

[0139] 2.1 Inducible expression of ALKBH8b protein

[0140] (1) Primary transfer: transfer 1% of the Rosetta bacteria containing the pGEX 4T-1_GST_ALKBH8b protein expression vector into LB liquid medium containing ampicillin at a final concentration of 50 μg / mL, shake the bacteria for 6 h, and the bacteria become turbid;

[0141] (2) Secondary transfer: transfer the newly shaken bacteria to a new LB liquid medium containing ampicillin at a final concentration of 50 μg / mL at a ratio of 2%, shake the bacteria for 2 h, and the absorbance OD value reaches about 0.55;

[0142] (3) Induce expression: Add IPTG solution to a concentration of 0.2 mM and induce overnight at 18°C ​​and 180 rpm.

[0143] 2.2 Magnetic bead method for extracting ALKBH8b protein

[0144] Centrifuge at 4℃, 8000 rpm for 5 min, collect Rosetta bacteria solution containing pGEX 4T-1_GST_ALKBH8b protein expression vector; resuspend with 50 mL of pre-cooled 1×Binding Buffer and place on ice; ultrasonic disruption at 50% power, with 5 s ultrasonic and 5 s cooling as one cycle, lasting about 20 min; centrifuge the disruption solution at 4℃, 10000 rpm for 20 min, and transfer the supernatant to a pre-cooled empty centrifuge tube; take 1 mL of magnetic beads and wash them twice with 1×Binding Buffer, then add 1 mL of 1×Binding Buffer to mix well and add to the supernatant disruption solution; put the flip instrument in the lower layer of a 4℃ refrigerator, select a suitable rack for flipping, and combine for 1 h; retain the binding solution, wash the magnetic beads 6-8 times with 1×Washing Buffer; use GSH competitive elution, and dilute GSH with 2×PBS to a final concentration of 10 mM, 20 mM, 30 mM, 40 mM, 50 200 μL of each mM was added and eluted in 200 μL order from low to high.

[0145] Prepare PAGE gel; pre-treat each sample by adding 10 μL 4×LDS Loading Buffer to 10 μL sample, 70°C, 10 min; use 1×MES Protein SDS running buffer for electrophoresis; after the electrophoresis, take out the gel and observe the gel. The electrophoresis results of ALKBH8b protein are shown in Figure 1. Figure 6 As shown, based on the different BSA concentrations in the electrophoresis diagram, the ALKBH8b protein concentration was calculated to be approximately 500 ng / μL.

[0146] 2.3 Verification of ALKBH8b protein enzyme activity

[0147] The enzyme activity function was verified using RNA probes. The specific reaction system is shown in Table 12. The reaction was carried out at 16°C for 12 h. The RNA probes were m 6 A modified RNA probe Primer_m 6 A_oryza and no m 6 A modified RNA probe Primer_CK_oryza, in which Primer_m 6 The nucleotide sequences of A_oryza and Primer_CK_oryza are as follows:

[0148] Primer_m 6 A_oryza: CTTACADDUAHTAAGCC (SEQ ID No. 15);

[0149] Primer_CK_oryza: CTTACADDUAHTAAGCC (SEQ ID No. 16);

[0150] Among them, D stands for A / G / T; H stands for A / C / T.

[0151] Table 12 Enzyme activity verification reaction system

[0152]

[0153] Dot-Blot verification: Take 1.5 μL of enzyme activity reaction product and spot on nylon membrane, let it stand and dry; crosslink at 254 nm for 24 s once, crosslink 4 times; use 10 mL of skim milk powder with a final concentration of 0.05 g / mL and 1×PBST (1×PBS+0.05%TWEEN20) to block for 30 min; discard the blocking solution, add primary antibody incubation (10 mL 1×PBST or ready-made primary antibody dilution) and proportionally diluted antibody, incubate for 2 h; wash with 1×PBST, 40 r / min, 15 min each time, repeat washing 3 times; discard the washing solution, add secondary antibody incubation (10 mL 1×PBST or ready-made secondary antibody dilution) and proportionally diluted antibody, incubate for 1.5 h; collect the secondary antibody incubation solution and place it at 4°C, wash with 1×PBST, 40 r / min, 15 min each time min, and repeat washing 3 times; use Bio-Tech's ultra-sensitive ECL chemiluminescence kit to soak the entire membrane and allow the antibodies on the membrane to glow in the chemiluminescence instrument, so that they can be detected; use a gel imaging instrument to take pictures and analyze the results. Figure 7 As shown, ALKBH8b protein is a m 6 A demethylase.

Claims

1. ALKBH8 The use of gene and ALKBH8b protein in regulating rice growth, yield or plant type, characterized in that: The rice ALKBH8 Gene mutation or silencing ALKBH8 The expression of the gene is reduced or the normal function of the ALKBH8b protein is defective; ALKBH8 The nucleotide sequence of the CDS of the gene is the nucleotide sequence shown in (a) or (b): (a) the polynucleotide sequence shown in SEQ ID No. 1; (b) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No.3; The amino acid sequence of the ALKBH8b protein is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that The regulating rice growth is to promote rice growth; the promoting rice growth is to increase plant height or increase leaf width.

3. The use according to claim 1, characterized in that The rice yield regulation is to increase the rice yield; the rice yield increase is to increase the rice panicle length, the number of effective tillers or the weight of 100 grains.

4. The use according to claim 1, characterized in that The method of regulating the rice plant type is to make the rice plant type compact and reduce the leaf angle.

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