Application of rice OsGSK3 gene in regulation and control of grain size
By using CRISPR/Cas9 gene editing technology on the OsGSK3 gene, rice grain size was regulated, solving the problem of insufficient rice grain regulation gene resources and realizing the regulation of rice grain size and yield improvement.
Patent Information
- Application Number
- CN202411056590.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-02
- Publication Date
- 2026-02-03
AI Technical Summary
In existing technologies, the genetic resources for regulating rice grain size are limited, the advantages of traditional hybrid breeding are weakened, and it is difficult to increase rice yield with limited germplasm resources.
Rice grain size can be regulated by CRISPR/Cas9 gene editing technology on the OsGSK3 gene. The CRISPR/Cas9 nuclease is used to specifically cut the GSK3 genomic DNA of the rice recipient plant, thereby changing its expression and regulating grain size.
It has enabled the regulation of rice grain size, provided new genetic resources, improved rice yield and quality, and enhanced breeding potential.
Smart Images
Figure CN121449705A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant genetic engineering and genetic breeding, and relates to a gene related to the regulation of rice grain size, its encoding and application. Background Technology
[0002] Rice is one of the most important food crops, a staple food for more than half of the world's population, and also an important model plant for functional gene research. With decreasing arable land and rapid population growth, current rice yield increases still rely on limited rice germplasm resources. The advantages of traditional hybrid breeding are gradually weakening, while transgenic rice technology has the potential to unlock further yield increases.
[0003] Rice grain weight is determined by grain length, width, and thickness, and is a complex quantitative trait regulated by multiple genes. In recent years, some key genes regulating rice grain size have been reported, but only the molecular regulatory mechanisms of some genes have been elucidated. Therefore, identifying more key genes regulating rice grain size and elucidating their molecular mechanisms is of great significance for improving rice yield and breeding high-yielding and high-quality rice varieties.
[0004] OsGSK3 is a conserved serine / threonine protein kinase in eukaryotes, which is widely present and highly conserved in the eukaryotic world. Summary of the Invention
[0005] The purpose of this invention is to provide a protein related to the regulation of rice grain size, its encoding gene, and its applications.
[0006] The protein provided by this invention, named OsGSK3, is derived from rice (Oryza sativa L.) and is as follows (a) or (b):
[0007] (a) A protein consisting of the amino acid sequence shown in Sequence 1 of the sequence listing;
[0008] (b) A protein derived from sequence 1 with substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of sequence 1, and which is related to the regulation of rice grain quality.
[0009] To facilitate the purification of the protein shown in (a) above, a tag as shown in the table below can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid residue sequence of sequence 1 in the sequence listing.
[0010] Table: Sequence of Labels
[0011] Label residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL
[0012] The protein in (b) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically. The encoding gene of the protein in (b) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in Sequence 2 of the sequence listing, and / or by performing a missense mutation of one or more base pairs.
[0013] The nucleic acid molecules encoding the protein are also within the scope of protection of this invention.
[0014] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA, hnRNA, or tRNA.
[0015] In one embodiment of the present invention, the nucleic acid molecule is specifically a gene encoding the protein (named OsGSK3), and the gene may be any one of the following DNA molecules 1)-3):
[0016] The gene is any one of the following DNA molecules: 1)-3)
[0017] 1) The DNA molecule shown in sequence 2 of the sequence listing;
[0018] 2) The DNA molecule shown in sequence 3 of the sequence listing;
[0019] 3) A DNA molecule that hybridizes under strict conditions to any of the DNA molecules specified in 1)-2) and encodes a protein derived from sequence 1 that is related to the regulation of rice grain size;
[0020] 4) A DNA molecule that has more than 90% identity with any of the defined DNA sequences in 1)-3) and encodes a protein derived from sequence 1 that is related to the regulation of rice grains.
[0021] Sequence 2 is the cDNA sequence of the OsGSK3 gene, and sequence 3 is the sequence of the OsGSK3 gene in the rice genome.
[0022] Recombinant vectors, expression cassettes, transgenic cell lines, or recombinant microorganisms containing the aforementioned nucleic acid molecules are also within the scope of protection of this invention. The recombinant vector may be a recombinant expression vector or a recombinant cloning vector.
[0023] The recombinant expression vector can be constructed using existing plant expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN, or other derived plant expression vectors. The plant expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylated nucleotide signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated nucleotide signal can guide the addition of polyadenylated nucleotides to the 3' end of the mRNA precursor. When constructing a recombinant expression vector using the gene described above, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide, such as the CaMV35S promoter of cauliflower mosaic virus, the Ubiquitin gene promoter (pUbi), the stress-inducible promoter rd29A, etc., which can be used alone or in combination with other plant promoters. Furthermore, when constructing a recombinant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The translation control signals and start codons are widely available and can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vector can be processed, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants, antibiotic resistance markers, or chemical reagent resistance marker genes. Alternatively, without adding any selective marker genes, plants can be directly screened for transformation under stress.
[0024] In this invention, the recombinant expression vector is a recombinant plasmid obtained by inserting the OsGSK3 gene into the multiple cloning sites (such as Sac I and Hind III) of the pMDC99 vector. More specifically, it is a recombinant plasmid (named pMDC99-OsGSK3) obtained by replacing the small fragment between the SacI and Hind III restriction sites of the pMDC99 vector with the DNA fragment shown in sequence 2 of the sequence listing.
[0025] The expression cassette consists of a promoter capable of initiating the expression of the gene, the gene, and a transcription termination sequence.
[0026] The transgenic cell line is a non-reproductive material into which the gene has been transferred.
[0027] The use of the protein, nucleic acid molecule, recombinant vector, expression cassette, transgenic cell line, or recombinant microorganism in any of the following is also within the scope of protection of this invention:
[0028] (a) Plant breeding and / or seed production;
[0029] (b) Regulation of plant seed size.
[0030] This invention also provides a method for cultivating transgenic plants.
[0031] The method for cultivating transgenic plants provided by this invention may be either A or B as follows:
[0032] A. A method for cultivating transgenic plants with reduced seed length includes the following steps:
[0033] (a1) The gene encoding the GSK3 protein is introduced into a recipient plant with the genotype gsk3 / gsk3 to obtain a transgenic plant expressing the gene; the seed size of the recipient plant is due to the enhanced ability of the recipient plant to express the functional GSK3 protein;
[0034] (a2) Obtain transgenic plants with smaller seeds from the transgenic plants obtained in step (a1); the encoding gene can be introduced into the recipient plant through the above recombinant expression vector;
[0035] B. Methods for cultivating transgenic plants with increased seed size include the following steps:
[0036] (b1) By suppressing the expression of GSK3 protein in plants with the genotype GSK3 / GSK3 recipient, transgenic plants were obtained;
[0037] (b2) Obtain transgenic plants with larger seeds from the transgenic plants obtained in step (b1). The increase in seed size of the transgenic plants is due to the loss of function of the GSK3 protein in the recipient plant.
[0038] In step (b1) of the method, the expression of the encoding gene in the recipient plant is inhibited by specifically cutting the genomic DNA sequence encoding the GSK3 protein in the recipient plant using a CRISPR / Cas9 nuclease, thereby causing the recipient plant to lose the ability to express the functional GSK3 protein.
[0039] Specifically, the target fragment for the CRISPR / Cas9 nuclease to specifically cleave the genomic DNA sequence encoding the GSK3 protein in the recipient plant is a fragment within the genomic DNA sequence encoding the GSK3 protein in the recipient plant that conforms to the sequence arrangement rule of 5'-NX-NGG-3' or 5'-CCN-NX-3'; N represents any one of A, G, C, and T, 14 ≤ X ≤ 30, and X is an integer (e.g., X is 20), and NX represents X consecutive deoxyribonucleotides. More specifically, the target fragment is "5'-GAAGGGAGTGATCCTGTCAC-3' (i.e., positions 138-158 of sequence 2)" in the genomic DNA sequence encoding the GSK3 protein in the recipient plant.
[0040] In this invention, the plant can be either a monocotyledonous plant or a dicotyledonous plant. The monocotyledonous plant includes plants of the Poaceae family, specifically rice.
[0041] In embodiments of the present invention, the recipient plant used in cultivating transgenic plants with reduced grain size is specifically the rice mutant gsk3, and the recipient plant used in cultivating transgenic plants with increased grain size is specifically the wild-type rice material ZH11.
[0042] The GSK3 gene of this invention can control grain size in rice. That is, homozygous mutation or deletion of this gene can significantly increase the size of rice seeds, and normal expression of the GSK3 gene in materials with gene mutation or deletion can restore the seed phenotype.
[0043] This invention provides new genetic resources for the research and application of important yield traits of rice grain size, such as grain length, grain width, and thousand-grain weight. Attached Figure Description
[0044] Figure 1 Creation and identification of rice osgsk3 mutants (A) GSK3 gene structure, with the target site located in the second exon; (B) Sequencing results of the target site; (C) Full-length OsGSK3 protein and osgsk3-1 / 2 mutant protein.
[0045] Figure 2 Phenotypic observation of rice osgsk3 mutants: (AB) Seeds of ZH11 and osgsk3-1 at maturity; (A) scale bar is 2 mm, (B) scale bar is 1 mm; (CD) Grain length (C) and grain width (D) of ZH11 and osgsk3-1 at maturity, n=50; (H) 100-grain weight of ZH11 and osgsk3-1 at maturity, n=3. Values in the figures are mean ± standard deviation. Detailed Implementation
[0046] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in at least three replicates, and the results were averaged.
[0047] The wild-type rice varieties ZH11 and osgsk3 were collected and preserved in our laboratory. The genotype of ZH11 is GSK3 / GSK3, and the genotype of the mutant is gsk3 / gsk3.
[0048] Rice genome sequencing information was obtained from the National Rice Research Center database, which can be found at the following link: http: / / www.ricedata.cn / .
[0049] Example 1: Creation of the gsk3-CRISPR mutant
[0050] The inventors of this invention designed an experiment to edit the GSK3 gene in the wild-type rice material ZH11. Specifically, they used the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats Associated 9) gene editing system to perform site-specific editing of the GSK3 gene genome sequence in the recipient rice plant ZH11. CRISPR-Cas9 technology can cut DNA at specific sites on the genome. Taking advantage of the fact that an organism's DNA strand repair cannot always be 100% accurate, the reconnected DNA strand will have a sequence difference compared to before the cut, thus changing the gene sequence and the encoded protein accordingly.
[0051] In this specific experiment, based on the characteristics of the CRISPR-Cas9 system, a specific sequence on the second exon of the GSK3 gene was selected as the sgRNA (single guide RNA) target sequence 5'-GAAGGGAGTGATCCTGTCAC-3' (i.e., positions 138-158 of sequence 2), and ligated into the pC1300-Cas9 vector to construct the GSK3 gene knockout vector pC1300-Cas9-GSK3. Agrobacterium-mediated transformation was used to transform the rice recipient plant ZH11. The resulting transgenic plants were examined, and homozygous T-insertion mutants were obtained in the ZH11 background. This mutation causes OsGSK3 to prematurely terminate after encoding some incorrect proteins, thus missing a key kinase domain.
[0052] Example 2: Observation of the seed phenotype of gsk3-CRISPR mutant
[0053] The gsk3-CRISPR transgenic plants obtained in Example 1 and the wild-type rice material ZH11, used as the transgenic recipient, were grown under natural field conditions. After maturity, the grains were harvested, and the seeds were scanned using a ScanMarker i560 scanner. Grain length and width were measured using SC-G software, with at least 40 seeds from each material measured. The weight of 100 seeds from each material was calculated using an analytical balance (Mettler, AL104) and converted to a thousand-grain weight. Representative photographs of the gsk3-CRISPR transgenic plants and wild-type rice ZH11 seeds, along with the statistical results, are shown below. Figure 2 As shown.
[0054] Based on the research results of the above embodiments, it is evident that the OsGSK3 gene of this invention is a gene related to the regulation of rice grain size; that is, homozygous mutations in this gene lead to larger rice seeds and increased 100-grain weight. This invention provides a new gene resource for the study of yield traits in plants, especially rice. Selecting rice materials with superior GSK3 alleles for genetic improvement of rice will play an important role in the field of rice breeding.
Claims
1. Protein is either (a) or (b) as follows: (a) A protein consisting of the amino acid sequence shown in Sequence 1 of the sequence listing; (b) A protein derived from Sequence 1 with substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of Sequence 1, and which is related to the regulation of seed size in plants.
2. A nucleic acid molecule encoding the protein of claim 1.
3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is a gene encoding the protein of claim 1, and the gene is any one of the following DNA molecules 1)-4): 1) The DNA molecule shown in sequence 2 of the sequence listing; 2) The DNA molecule shown in sequence 3 of the sequence listing; 3) A DNA molecule that hybridizes under strict conditions with any of the DNA molecules specified in 1)-2) and encodes a protein derived from sequence 1 that is related to the regulation of plant seed size; 4) A DNA molecule that has more than 90% identity with any of the defined DNA sequences in 1)-3) and encodes a protein derived from sequence 1 that is related to the regulation of plant seed size.
4. A recombinant vector, expression cassette, transgenic cell line, or recombinant microorganism containing the nucleic acid molecule described in claim 2 or 3.
5. The recombinant vector according to claim 4, characterized in that: The recombinant vector is a recombinant expression vector or a recombinant cloning vector.
6. The use of the protein of claim 1, the nucleic acid molecule of claim 2 or 3, or the recombinant vector, expression cassette, transgenic cell line, or recombinant microorganism of claim 4 or 5 in any of the following: (a) Plant breeding and / or seed production; (b) Regulating plant seed size.
7. The methods for cultivating transgenic plants are as follows: The method for cultivating transgenic plants provided by this invention may be either A or B as follows: A. A method for cultivating transgenic plants with reduced seed length includes the following steps: (a1) The gene encoding the GSK3 protein is introduced into a recipient plant with the genotype gsk3 / gsk3 to obtain a transgenic plant expressing the gene; the seed size of the recipient plant is due to the enhanced ability of the recipient plant to express the functional GSK3 protein; (a2) Obtain transgenic plants with smaller seeds from the transgenic plants obtained in step (a1); the encoding gene can be introduced into the recipient plant through the above recombinant expression vector; B. A method for cultivating transgenic plants with increased seed size, comprising the following steps: (b1) By suppressing the expression of GSK3 protein in plants with the genotype GSK3 / GSK3 recipient, transgenic plants were obtained; (b2) Obtain transgenic plants with larger seeds from the transgenic plants obtained in step (b1). The increase in seed size of the transgenic plants is due to the loss of function of the GSK3 protein in the recipient plant.
8. The method according to claim 7, characterized in that: In step (a), the encoding gene is introduced into the recipient plant via the recombinant expression vector of claim 5.
9. The application or method according to claim 7 or 8, characterized in that: The plant is a monocotyledonous plant or a dicotyledonous plant.
10. The application or method according to any one of claims 7-9, characterized in that: The monocotyledonous plants are grasses (Poaceae). The grass species mentioned are specifically rice.