Application of rice GW7.2 gene in regulation and control of grain weight

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a rice GW7.2 gene and application thereof in regulation and control of rice grain weight and grain length. The invention discloses a rice GW7.2 gene and a protein coded by the rice GW7.2 gene, the genome nucleotide sequence of the GW7.2 gene is shown as SEQ ID NO.1, the nucleotide sequence of a coding region of the GW7.2 gene is shown as SEQ ID NO.2, and the amino acid sequence of the coded protein is shown as SEQ ID NO.3. The invention further discloses a preparation method of the rice GW7.2 gene. The invention provides a technology for increasing the grain weight and the grain length of rice by using a rice gene GW7.2, and finds that the GW7.2 gene controls the grain weight and the grain length of the rice, and knockout of the GW7.2 gene enables the grain weight of the rice to be remarkably increased and the grain length to be remarkably increased, so that the GW7.2 gene has potential utilization value in genetic improvement and molecular breeding of the grain weight and the grain shape of the rice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and relates to a rice F-box protein-coding gene. GW7.2 And its application in regulating rice grain weight and grain length. Background Technology

[0002] Rice yield is determined by three factors: the number of effective panicles, the number of grains per panicle, and grain weight. Grain weight is influenced by grain shape (grain length, grain width, and grain thickness). Increasing grain weight or enlarging grain shape can significantly increase rice yield. Currently, several genes affecting rice grain weight or grain shape have been cloned, for example... GS3 , GW5 , TGW6 , GS5 , GW6 wait.

[0003] Since rice grain weight and grain shape are complex agronomic traits regulated by multiple genes, our understanding of their molecular regulatory networks is still very limited. Therefore, discovering more genes that affect rice grain weight and grain shape is of great significance for the genetic improvement of rice grain traits and high-yield molecular breeding. Summary of the Invention

[0004] This invention has identified genes related to the regulation of rice grain weight and grain length. GW7.2 Encoding a novel F-box protein that negatively regulates grain weight and length. This is achieved by knocking out... GW7.2 Genes can significantly increase grain length, thereby increasing rice grain weight, indicating that... GW7.2 Genes can serve as important target genes for molecular breeding of rice grain weight and grain length, and have potential value for genetic breeding and genetic engineering improvement of high-yield rice.

[0005] This invention discloses rice GW7.2 The gene and the sequence of its encoded F-box protein, the GW7.2 The nucleotide sequence of the gene is shown in SEQ ID NO.1. GW7.2 The gene open reading frame sequence is shown in SEQ ID NO.2. GW7.2 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.3.

[0006] The main technical problem solved by this invention is to utilize GW7.2 As a preferred embodiment of this invention, genes that enhance rice grain weight and length can be constructed using CRISPR / Cas9 technology. GW7.2 Gene knockout mutants are used to increase the weight and length of rice grains.

[0007] The objective of this invention can be achieved through the following technical solutions: The first object of the present invention is to provide a rice GW7.2 protein, the amino acid sequence of which is shown in SEQ ID NO.3: MANLQFLLKRLAKLRCFRRRRRRAAKPNAATPSPAARPAPTPTPTPPPPLAVLSDDVLREIFVRVPSPADLARAATACAGFRRVITEPSFLRRFRAADNQPALLGFIDVGGG FVPAEPPHPSAAAAAGAAARDVVDFACPFLPSSPDPWRRRDVLDGRVLFSRGAVGGGGEVDGQDDDPGFMDLAVCDPLSRRYVLLPAVPADLAASAQLHNLLDLQPFFAPP RDDDDDGGGGTSFRVMYMARCQSKLVVFTFSSDTQQWSSTSYDGWGILVAATPSQETALTQRHHAHGCIFWFLRWAKKLLVLDTFTMELSTINLPSSELIEIQQVAIVESA RGGIGMFAMVDEILDSTFDMFYVVWDPEGANKWPLERLMKLPVEFRYNLVGAAGGYLLVQGISVQGPVQDQVCFTVELKTFKVEMFCETRRTLIGADLFAGFAPSLSPSV.

[0008] A second objective of this invention is to provide rice that encodes the aforementioned rice GW7.2 protein. GW7.2 Gene.

[0009] Furthermore, the rice GW7.2 The nucleotide sequence of the gene is selected from either a) or b): a) The genome sequence is shown in SEQ ID NO.1:

[0010] b) The coding region sequence is shown in SEQ ID NO.2:

[0011] The third objective of this invention is to provide rice GW7.2 The gene knockout vector is a CRISPR / Cas9 vector, and the knockout target site sequence is shown in SEQ ID NO.4 and / or SEQ ID NO.5.

[0012] A fourth objective of this invention is to provide the aforementioned rice GW7.2 protein or the aforementioned rice GW7.2 Application of genes or the aforementioned knockout vectors in regulating rice yield traits.

[0013] Furthermore, reducing the expression level of GW7.2 protein in rice or reducing the expression level of GW7.2 protein in rice GW7.2 Increasing gene expression levels or introducing knockout vectors into rice can improve rice yield.

[0014] Furthermore, the improved yield traits include increasing rice grain length and / or increasing rice grain weight.

[0015] In a particular embodiment, the rice of the present invention GW7.2 Genetic engineering applications of genes include: 1) Rice GW7.2 CRISPR / Cas9 vector construction for gene screening in rice GW7.2 Gene knockout targets, and design based on knockout targets. GW7.2 Dual-target CRISPR / Cas9 knockout primers were obtained GW7.2 The CRISPR / Cas9 mutation target sequence of the gene is used to construct... GW7.2 CRISPR / Cas9 gene knockout vector.

[0016] 1) The above GW7.2 The gene knockout target sequence is shown in SEQ ID NO.4 and / or SEQ ID NO.5.

[0017] SEQ ID NO.4: AAAGCGGCTGGCCAAACTG; SEQ ID NO. 5: CGCGCCGCCGACAACCAGC.

[0018] Furthermore, 1) the aforementioned GW7.2 The target knockout primer sequences are shown in SEQ ID NO.6 - SEQ ID NO.9: SEQ ID NO.6: AATAATGGTCTCAGGCGAAAGCGGCTGGCCAAACTG; SEQ ID NO.7: GAAAGCGGCTGGCCAAACTGGTTTTAGAGCTAGAAATAGC; SEQ ID NO.8: GCTGGTTGTCGGCGGCGCCGCTTCTTGGTGCC; SEQ ID NO.9: ATTATTGGTCTCTAAACGCTGGTTGTCGGCGGCGCG.

[0019] 2) Rice GW7.2 Construction and identification of gene knockout mutants: After constructing the vector, rice was transformed using Agrobacterium-mediated transformation to obtain rice. GW7.2 CRISPR / Cas9 gene knockout mutants of the gene. Target detection primers were used to amplify rice. GW7.2 The genome sequence of the young leaves of the gene knockout mutant was sequenced using routine PCR products for screening. GW7.2 Homozygous mutant plants of the gene are rice plants with increased grain weight and grain length.

[0020] Furthermore, 2) the target detection primers are shown in SEQ ID NO.10 and SEQ ID NO.11: SEQ ID NO.10: AAATTCATATCCTCAAGTTGAAGAGA; SEQ ID NO. 11: GAAGAGGACGCGGCCGTCGAGGACGT. Beneficial effects

[0021] This invention discloses rice for the first time GW7.2 Genes and their encoded protein sequences, and GW�.2 Application of genes in regulating rice grain weight and length. This invention involves knocking out rice... GW7.2 This gene can significantly increase rice grain weight and length, and it holds promise as a target gene for genetic improvement and genetic engineering of rice grain weight and shape. Therefore, this invention provides a simple and effective method for breeding high-yielding rice varieties. Attached Figure Description

[0022] Figure 1 : GW7.2 Mutation details of gene knockout mutants (A), grain appearance (B), grain shape and weight (C); scale bar is 5 mm, ** indicates P <0.01, WT is wild type.

[0023] Figure 2 : GW7.2 Appearance (A), grain shape, and grain weight (B) of brown rice from gene knockout mutants; scale bar is 5 mm, ** indicatesP <0.01, WT is wild type. Detailed Implementation

[0024] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0025] The following description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0026] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and raw materials involved are all commercially available conventional products; unless otherwise specified, the test methods involved are all conventional methods.

[0027] Example 1: GW7.2 Construction and identification of knockout mutant plants 1) GW7.2 Gene knockout vector construction Obtain rice from the NCBI database (https: / / www.ncbi.nlm.nih.gov). GW7.2 The genome sequence and coding region sequence of the gene and the amino acid sequence of the encoded protein, the genome nucleotide sequence is shown in SEQ ID NO.1, the coding region nucleotide sequence is shown in SEQ ID NO.2, and the corresponding amino acid sequence is shown in SEQ ID NO.3.

[0028] Using CRISPR / Cas9 target screening tools, such as the CRISPR-PLANT online tool, in GW7.2 Two distinct knockout targets were identified in the gene exon region, and the target sequences are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0029] SEQ ID NO.4: AAAGCGGCTGGCCAAACTG; SEQ ID NO. 5: CGCGCCGCCGACAACCAGC.

[0030] Target-specific primers for constructing CRISPR / Cas9 gene knockout vectors were designed based on the target sequence. The specific primer sequences are shown in SEQ ID NO.6 - SEQ ID NO.9: SEQ ID NO.6: AATAATGGTCTCAGGCGAAAGCGGCTGGCCAAACTG; SEQ ID NO.7: GAAAGCGGCTGGCCAAACTGGTTTTAGAGCTAGAAATAGC; SEQ ID NO.8: GCTGGTTGTCGGCGGCGCCGCTTCTTGGTGCC; SEQ ID NO.9: ATTATTGGTCTCTAAACGCTGGTTGTCGGCGGCGCG.

[0031] Using the pCBC-MT1T2 vector and target-specific primers, construct GW7.2 The sgRNA expression cassette was knocked out at the target site. Subsequently, the sgRNA expression cassette was ligated into the binary vector pHUE411 using the restriction enzymes Bsa1 and T4 ligase, completing the process. GW7.2 Construction of CRISPR / Cas9 gene knockout vectors.

[0032] 2) GW7.2 Construction and screening of knockout mutants The constructed CRISPR / Cas9 gene knockout vector was transformed into Agrobacterium tumefaciens. Using Agrobacterium-mediated rice transgenic technology, the Agrobacterium carrying the CRISPR / Cas9 gene knockout vector plasmid was transformed into callus tissue of the japonica rice variety Nipponbare. Transgenic plants were obtained through differentiation and culture. Based on the cloned... GW7.2 The nucleotide sequences of two specific target regions of the gene were used to design PCR primers for verifying transgenic mutants. The upstream and downstream primer sequences for the mutant target verification PCR are shown in SEQ ID NO.10 and SEQ ID NO.11, respectively. SEQ ID NO.10: AAATTCATATCCTCAAGTTGAAGAGA; SEQ ID NO. 11: GAAGAGGACGCGGCCGTCGAGGACGT.

[0033] Genomic DNA was extracted from leaves of transgenic mutant materials and amplified using PCR. GW7.2 The sequences of the two target regions of the gene were obtained, and the PCR products were sequenced to determine the mutation status of the two target sites. Homozygous mutant plants were then screened. Ultimately, a homozygous mutant line exhibiting premature termination of translation in GW7.2 was obtained. gw7.2-1 and gw7.2-2 Among these strains GW7.2 The gene mutation situation is as described above. Figure 1 As shown in Figure A.

[0034] Example 2: GW7.2 Phenotypic analysis of knockout transgenic plants For mutantsgw7.2-1 and gw7.2-2 The grain shape and weight of the wild type were also examined.

[0035] A mutant equivalent to wild-type Nipponbare was discovered. gw7.2-1 and gw7.2-2 The length and thousand-kernel weight of the hulled seeds both increased significantly, as described above. Figure 1 As shown in B and 1C.

[0036] Meanwhile, mutant gw7.2-1 and gw7.2-2 The grain length and thousand-grain weight of the hulled brown rice also increased significantly, as described above. Figure 2 As shown; however, there were no significant changes in the grain width and thickness of the mutant's hulled and unhulled grains.

[0037] It can be seen that rice GW7.2 The gene knockout can significantly increase the grain length and weight of both hulled and dehulled rice grains.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A rice GW7.2 protein, characterized in that, The amino acid sequence of the GW7.2 protein is shown as SEQ ID NO.

3.

2. Rice gene encoding the rice GW7.2 protein of claim 1. GW7.2 gene.

3. The rice plant of claim 2 GW7.2 gene characterized in that, The rice GW7.2 The nucleotide sequence of the gene is selected from a) or b): a) the genomic sequence is shown as SEQ ID NO. 1; b) the coding region sequence is shown as SEQ ID NO.

2.

4. Oryza sativa GW7.2 A knock-out vector of a gene characterized in that, The knockout vector is a CRISPR / Cas9 vector, and the knockout target site sequence is shown as SEQ ID NO. 4 and / or SEQ ID NO.

5.

5. The rice GW7.2 protein of claim 1 or the rice GW7.2 gene of claim 2 or 3 or the knockout vector of claim 4 for use in modulating yield traits in rice.

6. Use according to claim 5, characterized in that, reducing the expression level of GW7.2 protein in rice or reducing the expression level of GW7.2 protein in rice GW7.2 Gene expression or knock-out vector into rice can increase the yield of rice.

7. Use according to claim 5 or 6, characterized in that, The yield-increasing traits include increasing rice grain length and / or increasing rice grain weight.

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