Application of BRW1 gene in rice in regulating grain weight traits

Abstract

The application belongs to the field of genetic engineering, and relates to a rice BRW1 gene and application thereof. A cDNA sequence of the rice BRW1 gene is shown as SEQ ID NO. 1, and an amino acid sequence of an RNA binding protein encoded by the rice BRW1 gene is shown as SEQ ID NO. 2. The application discloses a rice RNA binding protein gene BRW1 and a protein encoded by the rice RNA binding protein gene BRW1. The rice BRW1 gene controls grain weight, and the thousand-grain weight of a knockout mutant of the gene is significantly improved, and the grain length and the grain width are both increased. By mutating the BRW1 gene in the application, the grain weight of rice can be increased, and it is indicated that the gene is helpful to genetic improvement of the grain weight and the grain type of rice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to a rice RNA-binding protein encoding gene BRW1 and its application in regulating grain weight traits. Background Technology

[0002] Rice is one of the world's most important staple crops, and its yield is of great significance for ensuring food security and solving the problem of hunger. Grain weight is one of the three major factors determining rice yield, and it is also a complex agronomic trait, finely regulated by numerous genes. Although several rice grain weight regulation-related genes have been cloned, including GS3, GW2, GW8, GW6a, TGW6, TGW3 / GL6, TGW2, GSE5 / GW5, and GLW7, the molecular mechanisms and regulatory networks of grain weight still need to be further elucidated.

[0003] RNA-binding proteins (RBPs) are a class of proteins with RNA-binding domains that are widely involved in RNA splicing, transport, modification, and translation, playing important post-transcriptional regulatory roles. In recent years, multiple studies have found that RBPs also participate in the regulation of some important agronomic traits in crops. Currently, several rice RBP-encoding genes have been reported to affect grain weight, such as GL11, DGW1, and EOG1. Identifying and cloning more grain weight-regulating genes, including novel RBP-encoding genes, will not only provide further theoretical support for the genetic improvement of rice grain weight but also provide more genetic resources for the breeding of new rice varieties. Summary of the Invention

[0004] This invention reveals that the rice grain weight-related gene BRW1 encodes a novel RBP that negatively regulates grain weight. Knocking out BRW1 significantly increases rice grain weight, as well as grain length and width. Utilizing the function of BRW1 and applying it in plant genetic engineering is of great significance for improving rice grain weight.

[0005] The technical solution of this invention is as follows:

[0006] The first objective of this invention is to provide a rice BRW1 gene, the cDNA sequence of which is shown in SEQ ID NO. 1:

[0007]

[0008] A second objective of this invention is to provide an RNA-binding protein encoded by the aforementioned rice BRW1 gene, the amino acid sequence of which is shown in SEQ ID NO.2:

[0009] MGTKNQFDLLVDVDNDDPSHLIAAAEKKAAAAAASPKPAAQAKLPTKPPPPAQAVKES

[0010] RNYGAPAREGAGRNGPGRGSGGFGGGRIGQRRDFGEGDTNGVEGGYGASGFGDGIVRREE

[0011] GEHRPSERGHGPRQPYRGGGRRGGYTGGEAGDESGRAPHRAYERRSGTTGRGYGMKREGA

[0012] GRGNWGTVTDEALAQESGEAVSIEVSVTVTEENKQEDVPQSDEVEKHKEGESNEEEEKEPE

[0013] DKEMTLEEYEKVLEEKRKALLSLKVEERKVVVDKELQSMQQLSVKKDSDEVFIKLGSDKD

[0014] KKKENVERDERTRKSLSINEFLKPAEGERYYSPGGRGRGRGRGDRGGFRDGYSSRGPVAAPRIEDQAQFPGLAGRLVH*

[0015] A third objective of this invention is to provide a knockout mutant of the rice BRW1 gene.

[0016] The fourth objective of this invention is to provide the genetic engineering applications of the aforementioned rice BRW1 gene, the aforementioned RNA-binding protein, or the aforementioned rice BRW1 gene knockout mutant.

[0017] Furthermore, the application involves knocking out the rice BRW1 gene or reducing the expression level of RNA-binding protein to increase the weight, length, and width of hulled brown rice grains, or to increase the thousand-grain weight, length, width, and thickness of hulled rice grains.

[0018] Furthermore, the procedure includes the following steps:

[0019] 1) Transgenic construction of rice BRW1 gene mutants: Screening for knockout target sites of the rice BRW1 gene, designing BRW1 target site knockout primers based on the knockout target sites, obtaining the target sequence of the point mutation target fragment of the BRW1 gene, constructing the vector and transforming it into rice to obtain the CRISPR / Cas9 gene knockout mutant of rice BRW1.

[0020] 2) Screening and identification of rice mutants: The genome of young leaves of rice BRW1CRISPR / Cas9 gene knockout mutants was amplified using target detection primers, and the routine PCR products were sequenced to screen homozygous mutant plants, which are plants that increase the weight, length and width of rice grains.

[0021] Furthermore, 1) the knockout target sites of the rice BRW1 gene are knockout target site 1 and knockout target site 2; the nucleotide sequence of knockout target site 1 is shown in SEQ ID NO.3: ATGGTGCTCCAGCCCGTGA; the nucleotide sequence of knockout target site 2 is shown in SEQ ID NO.4: GAAGTCGAGAAACACAAGG.

[0022] Furthermore, 1) the BRW1 target site knockout primer sequences are shown in SEQ ID NO.5-SEQ ID NO.8:

[0023] SEQ ID NO.5: AATAATGGTCTCAGGCGATGGTGCTCCAGCCCGTGA;

[0024] SEQ ID NO.6:GATGGTGCTCCAGCCCGTGAGTTTTAGAGCTAGAAATAGC;

[0025] SEQ ID NO.7: CCTTGTGTTTCTCGACTTCCGCTTCTTGGTGCC;

[0026] SEQ ID NO. 8: ATTATTGGTCTCTAAACCTTGTGTTTCTCGACTTC.

[0027] Furthermore, 2) the target detection primers are shown in SEQ ID NO. 9 and SEQ ID NO. 10:

[0028] SEQ ID NO.9: GTATTCACCGCACATGGGGA;

[0029] SEQ ID NO. 10: ACTTGAAGATGGGCATGTGAGA.

[0030] Beneficial effects

[0031] 1. This invention discloses the rice BRW1 gene and the protein it encodes. The RBP protein gene BRW1 is reported for the first time in rice. By knocking out the rice BRW1 gene, its biological function is lost, and the grain weight, grain length, and grain width of rice grains are increased. It is expected to be used as a target gene for the genetic improvement and genetic engineering of rice.

[0032] 2. This invention provides theoretical and technical support for the breeding of high-yield rice varieties and is of great significance to rice production. Attached Figure Description

[0033] Figure 1 The mutation status of the rice BRW1 gene and its encoded protein, including:

[0034] (A) Gene sequence mutation status;

[0035] (B) Protein sequence mutation status; ZH11 in the figure is wild type.

[0036] Figure 2 Grain-related phenotypes of wild type and knockout mutant brw1, among which:

[0037] (A) Appearance of the grains;

[0038] (B) Thousand-grain weight, grain length, grain width and grain thickness; *p<0.05; **p<0.01.

[0039] Figure 3 Brown rice-related phenotypes of wild-type and knockout mutant brw1, among which:

[0040] (A) Appearance of brown rice;

[0041] (B) Thousand-grain weight, grain length, grain width and grain thickness of brown rice; **p<0.01; ns, no difference. Detailed Implementation

[0042] 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.

[0043] This invention is further illustrated by specific embodiments. Unless otherwise specified, all methods used in the embodiments are conventional methods. The primers and sequencing were performed by Nanjing Sipujin Biotechnology Co., Ltd. Various restriction endonucleases, ligases, DNA ladders, high-fidelity enzymes, vectors, etc. used in the experiments were purchased from Baoriyi Biotechnology (Beijing) Co., Ltd. RNA extraction kits were purchased from Beijing TransGen Biotechnology Co., Ltd. Reverse transcription kits were purchased from Novizan Biotechnology Co., Ltd. Plasmid extraction kits and gel extraction kits were purchased from Nanjing Maling Xuehai Biotechnology Co., Ltd. All methods were performed in accordance with the instructions.

[0044] Example 1: Gene Cloning

[0045] The BRW1 gene sequence was cloned using cDNA from germinating seeds of the japonica rice variety Zhonghua 11 as a template. The nucleotide and amino acid sequences of the rice BRW1 gene were obtained, and the nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO.2.

[0046] Example 2: Construction of BRW1 mutant plants

[0047] (I) Identification of mutation targets in the rice BRW1 gene:

[0048] Log in to the website http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html, screen for BRW1 gene targets, and select two 19bp target fragments specific targets in exon 2 and exon 3, corresponding to the two target sequences sgRNA1 and sgRNA2, as shown in SEQ ID NO.3 and SEQ ID NO.4;

[0049] Four BRW1 target mutation site-specific primers were designed for the specific target sites of the two selected target fragments. The knockout primer sequences are shown in SEQ ID NO.5-8.

[0050] Using the pCBC-MT1T2 vector diluted 100-fold as a template, PCR amplification was performed with four primers, followed by electrophoresis detection. The amplification products were then purified and recovered, and the specific targets of the BRW1 gene, sgRNA1 and sgRNA2, were cloned.

[0051] (II) Enzyme digestion-ligation system (15 μL):

[0052] PCR system: point mutation target DNA fragment, 2 μL; pBUE411 vector, 2 μL; 10xNEB T4 Buffer, 1.5 μL; 10xBSA, 1.5 μL; BsaI (NEB), 1 μL; T4Ligase (NEB) / high concentration, 1 μL; ddH2O, 6 μL;

[0053] Enzyme digestion-ligation PCR reaction program: 37℃, 5 hours; 50℃, 5 minutes; 80℃, 10 minutes.

[0054] (III) Obtaining mutant lines:

[0055] The specific target sgRNA of the constructed rice BRW1 gene was ligated into the pHUE411 vector through the BsaI restriction enzyme ligation system described in (II), and the resulting plasmid containing the pHUE411-Target vector was transformed into Agrobacterium. The Agrobacterium carrying the transformation plasmid was then transformed into the callus tissue of the wild-type japonica rice variety Zhonghua 11 using Agrobacterium-mediated rice transgenic technology.

[0056] (iv) Screening and Identification of Rice Mutants

[0057] Based on the physical locations of the target fragments at two specific target sites on exons 2 and 3 of the cloned BRW1 gene, PCR verification primers for mutant materials were designed. The upstream primer sequence for amplifying the mutant target sites is shown in SEQ ID NO. 9, and the downstream primer sequence is shown in SEQ ID NO. 10. Using the PCR verification primers for mutant materials, the CRISPR / Cas9 target sequence of rice BRW1 was amplified, and homozygous mutant plants were screened. Homozygous lines brw1-1, brw1-2, and brw1-3, which prematurely terminated translation in the mutants, were obtained. The mutation status of the BRW1 gene and its encoded amino acids in these lines is as described above. Figure 1 As shown.

[0058] Example 3: Phenotypic analysis of agronomic traits in mutant BRW1 plants

[0059] Compared to the wild-type Zhonghua 11, the rice mutants brw1-1, brw1-2, and brw1-3 showed significant increases in hulled grain weight, grain length, grain width, and grain thickness, as described above. Figure 2 As shown; however, the hulled brown rice exhibited significant increases in thousand-grain weight, grain length, and grain width, while grain thickness showed no significant difference, as described above. Figure 3 As shown.

[0060] 1,000-grain weight: After the seeds mature and are dried, three groups of plump rice seeds and brown rice are selected, with 100 grains in each group. The weight is then converted to statistical units after weighing using an electronic balance.

[0061] Grain length, grain width, and grain thickness: After the seeds mature and are dried, select plump rice seeds and brown rice, and use calipers to measure the grain length, grain width, and grain thickness.

[0062] It is evident that knocking out the BRW1 gene in rice can significantly increase the weight, length, and width of hulled brown rice grains, or increase the thousand-grain weight, length, width, and thickness of hulled rice grains.

[0063] 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. Rice BRW1 Genes or rice BRW1 The genetic engineering application of gene-encoded RNA-binding proteins, characterized by: The application is to knock out the rice. BRW1 The gene or the expression level of RNA-binding protein were reduced to increase the grain weight, grain length, and grain width of hulled brown rice, or the aforementioned rice was knocked out. BRW1 Genes or reduced expression of RNA-binding proteins can increase the thousand-grain weight, grain length, grain width, and grain thickness of rice grains with husks. The BRW1 The gene cDNA sequence is shown in SEQ ID NO. 1; The amino acid sequence of the RNA-binding protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, Includes the following steps: 1) Rice BRW1 Transgenic construction of gene mutants: screening rice BRW1 Gene knockout target sites, and design based on knockout target sites. BRW1 Target site knockout primers to obtain BRW1 The target sequence of the gene's point mutation is used to construct a vector, which is then transformed into rice to obtain rice. BRW1 CRISPR / Cas9 gene knockout mutant; 2) Screening and identification of rice mutants: Target detection primers were used to amplify rice mutants. BRW1 The genome of young leaves of CRISPR / Cas9 gene knockout mutants was sequenced using conventional PCR products, and homozygous mutant plants were screened. These plants were those with increased weight, length, and width of hulled brown rice grains, or those with increased thousand-grain weight, length, width, and thickness of hulled rice grains.

3. The application according to claim 2, characterized in that, The rice described in 1) BRW1 The gene knockout target sites are knockout target site 1 and knockout target site 2; the nucleotide sequence of knockout target site 1 is shown in SEQ ID NO.3: ATGGTGCTCCAGCCCGTGA; the nucleotide sequence of knockout target site 2 is shown in SEQ ID NO.4: GAAGTCGAGAAACACAAGG.

4. The application according to claim 2, characterized in that, As described in 1) BRW1 The target site knockout primer sequences are shown in SEQ ID NO.5-SEQ ID NO.8: SEQ ID NO.5: AATAATGGTCTCAGGCGATGGTGCTCCAGCCCGTGA; SEQ ID NO.6:GATGGTGCTCCAGCCCGTGAGTTTTAGAGCTAGAAATAGC; SEQ ID NO.7: CCTTGTGTTTCTCGACTTCCGCTTCTTGGTGCC; SEQ ID NO. 8: ATTATTGGTCTCTAAACCTTGTGTTTCTCGACTTC.

5. The application according to claim 2, characterized in that, The target detection primers described in section 2) are shown in SEQ ID NO. 9 and SEQ ID NO. 10: SEQ ID NO.9: GTATTCACCGCACATGGGGA; SEQ ID NO. 10: ACTTGAAGATGGGCATGTGAGA.

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