Application of rice GIP1 gene in regulation and control of grain length and thousand grain weight
By overexpressing or knocking out the GIP1 gene in rice, grain size can be regulated, solving the problem of the lack of genes regulating rice grain size in existing technologies, thereby increasing grain length and thousand-grain weight and improving rice yield.
Patent Information
- Application Number
- CN202511498592.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-20
- Publication Date
- 2025-12-30
AI Technical Summary
In existing technologies, there are relatively few key genes that regulate rice grain size, making it difficult to effectively increase rice yield through gene modification.
This study provides the rice GIP1 gene and its encoded protein, and describes how to regulate grain size by overexpressing or knocking out this gene in rice. Specific expression methods, including application in rice, can be used to regulate grain size by introducing new expression vectors and promoter systems.
By regulating rice grain size, grain length and thousand-grain weight can be significantly increased, thereby improving rice yield and providing new genetic resources for rice breeding.
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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 length and thousand-grain weight, its encoding and application. Background Technology
[0002] With the continuous increase in the world's population, the demand for food crops is also growing. Rice is one of the most important food crops, and with the available arable land area constantly decreasing, how to increase rice yield per unit area to meet the demand is a serious problem currently facing rice breeding research.
[0003] Rice yield per plant is determined by the number of effective panicles, the number of grains per panicle, and the thousand-grain weight. Grain size is closely related to thousand-grain weight and is one of the important factors affecting yield. In recent years, some key genes regulating rice grain size have been cloned, but only a few can be truly applied in production. Therefore, identifying more key genes regulating grain size and elucidating their molecular mechanisms is of great value for improving rice yield and breeding high-yielding and high-quality rice varieties.
[0004] GIP1 encodes Di19-1 (drought-induced19), a member of the rice transcription factor family containing a zinc finger structure. The Di19 transcription factor family has seven members in rice, and previous analyses of their functions have mainly focused on stress-related factors; how Di19-1 regulates rice grain size remains unclear. Our study found that GIP1 can regulate rice grain length and thousand-grain weight, which is of great significance for improving rice yield and breeding high-yielding and high-quality rice varieties. 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 GIP1, 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 3 of the sequence listing;
[0008] (b) A protein derived from sequence 3 with substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of sequence 3, and which is related to the regulation of rice grain size.
[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 3 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-tagII 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 GIP1), 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 1 of the sequence listing;
[0019] 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 3 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 3 that is related to the regulation of rice grain size.
[0021] Sequence 2 is the cDNA sequence of the GIP1 gene, and sequence 1 is the sequence of the GIP1 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 GIP1 gene into the multiple cloning site of the pCambia2300-35S-eGFP vector. More specifically, it is a recombinant plasmid (named p35S::GIP1-eGFP) obtained by replacing the small fragment between the BamHI and SalI restriction sites of the pCambia2300-35S-eGFP 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 provides the GIP1 gene for improving rice grain size, wherein the size traits include grain length and thousand-grain weight.
[0031] In a preferred embodiment, the GIP1 gene is used to increase rice grain size, including overexpression of the GIP1 gene, which can increase grain length and thousand-grain weight. Therefore, this invention discloses a novel gene regulating rice grain size.
[0032] The GIP1 gene of this invention can regulate grain size in rice, that is, enhanced function of this gene can increase the size of rice grains.
[0033] 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.
[0034] In an embodiment of the present invention, the recipient plant used in cultivating transgenic plants with increased grain size is specifically the wild-type rice material Zhonghua 11.
[0035] This invention provides new genetic resources for the research and application of rice seed size traits. Attached Figure Description
[0036] Figure 1 、 A. Seed size phenotype of GIP1 overexpression material; B. Seed length and thousand-seed weight measurement results.
[0037] Figure 2 A. Grain phenotype of GIP1 knockout mutant; B. Grain length and thousand-grain weight measurement results. Detailed Implementation
[0038] 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.
[0039] The wild-type rice variety Zhonghua 11 and p35S::GIP1-eGFP were created and preserved in our laboratory. The genotype of Zhonghua 11 is GIP1 / GIP1, and the overexpression material was constructed as p35S::GIP1-eGFP.
[0040] 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 / .
[0041] Example 1 Creation of GIP1 overexpression materials and observation of seed size
[0042] This study overexpressed GIP1 in the background of the japonica rice variety Zhonghua 11. Primers were designed as GIP1-F-BamHI: gtacccggggatccATGGACTCGGAGCACTGGATC; GIP1-R-SalI: gtactagtgtcgacATCTCCAAATAAAGTAGTGAGCAGC to amplify the target fragment, which was then ligated into the pCambia2300-35S-eGFP vector digested with BamHI and SalI to construct p35S::GIP1-eGFP. This p35S::GIP1-eGFP was further genetically transformed into Zhonghua 11 materials to obtain GIP1 overexpression material. The obtained GIP1 overexpression material was screened, and seed phenotypes were measured. The results showed that the grain length and thousand-grain weight of the p35S::GIP1-eGFP overexpression material were significantly increased compared to Zhonghua 11.
[0043] Based on the above research results, it is evident that the GIP1 gene provided by this invention is a gene related to the regulation of rice grain size; that is, overexpression of this gene leads to longer and heavier rice grains. This invention provides a new gene resource for the study of grain size traits in plants, especially rice. Selecting rice materials with superior GIP1 alleles for genetic improvement of rice grain size will play an important role in the field of rice breeding.
Claims
1. A protein, which is either (a) or (b) : (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in the Sequence Listing; (b) a protein derived from SEQ ID NO: 3 by substitution and / or deletion and / or addition of one or several amino acid residues, and which is associated with seed quality in plants.
2. A nucleic acid molecule encoding the protein of claim 1. The nucleic acid molecule is a gene encoding the protein of claim 1, which is any one of the following 1) to 4) : 1) a DNA molecule shown in SEQ ID NO: 2 in the Sequence Listing; 2) a DNA molecule shown in SEQ ID NO: 1 in the Sequence Listing; 3) a DNA molecule hybridizing under stringent conditions with any one of the DNA molecules defined in 1) to 2) and encoding a protein derived from SEQ ID NO: 3 which is associated with regulation of grain length and thousand seed weight in plants; 4) a DNA molecule having more than 90% identity with any one of the DNA sequences defined in 1) to 3) and encoding a protein derived from SEQ ID NO: 3 which is associated with regulation of grain length and thousand seed weight in plants.
4. A recombinant vector, an expression cassette, a transgenic cell line or a recombinant microorganism containing the nucleic acid molecule of claim 2 or 3.
3. The nucleic acid molecule of claim 2, wherein: The recombinant vector is a recombinant expression vector or a recombinant cloning vector.
6. Use of the protein of claim 1 or the nucleic acid molecule of claim 2 or 3 or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant microorganism of claim 4 or 5 in any one of the following: (a) plant breeding and / or seed production; (b) regulation of grain length and thousand seed weight in plants.
7. A method for breeding a transgenic plant, which is as follows: The method for breeding a transgenic plant provided by the present application can be as follows A: A. A method for breeding a transgenic plant having increased grain length and thousand seed weight, comprising the following steps: (a1) increasing the expression of GIP1 protein in a receptor plant having genotype GIP1 / GIP1 to obtain a transgenic plant; (a2) obtaining a transgenic plant having increased grain length and thousand seed weight from the transgenic plant obtained in step (a1), the increase in grain length and thousand seed weight of the obtained transgenic plant being caused by overexpression of the GIP1 protein in the receptor plant.
5. The recombinant vector of claim 4, wherein: In step (a), the coding gene is introduced into the receptor plant by the recombinant expression vector of claim 5. The plant is a monocotyledonous plant or a dicotyledonous plant. The monocotyledonous plant is a plant of the family Poaceae. The plant of the family Poaceae is specifically rice. 8. The method of claim 7, wherein: 9. Use or method according to claim 7 or 8, characterized in that: 10. Use or method according to any one of claims 7 to 9, characterized in that: