Application of rice BRW1 gene in regulation and control of grain weight character

Abstract

The invention belongs to the field of gene engineering, and relates to a rice BRW1 gene and application thereof. The cDNA (complementary deoxyribonucleic acid) sequence of the rice BRW1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the RNA binding protein coded by the rice BRW1 gene is shown as SEQ ID NO.2. The invention discloses the rice RNA binding protein gene BRW1 and the protein coded by the rice RNA binding protein gene BRW1. The rice BRW1 gene controls the grain weight of grains, the thousand grain weight of a knockout mutant of the gene is remarkably increased, and meanwhile the grain length and the grain width are increased. By mutating the BRW1 gene in the invention, the grain weight of rice grains can be increased, which indicates that the genetic improvement of the grain weight and grain shape of rice is facilitated by using the gene.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and particularly relates to a rice RNA binding protein coding gene BRW1 and its application in regulating grain weight traits. Background Art

[0002] Rice is one of the important staple food crops in the world, and its yield is of great significance for ensuring food security and solving the problem of hunger. Grain weight is one of the three main factors determining rice yield, and it is also a complex agronomic trait, which is finely regulated by numerous genes. Although multiple genes related to rice grain weight regulation have been cloned successively, including GS3, GW2, GW8, GW6a, TGW6, TGW3 / GL6, TGW2, GSE5 / GW5, GLW7, etc., the molecular mechanism and regulatory network of grain weight still need to be further analyzed.

[0003] RNA binding protein (RBP) is a class of proteins with RNA binding domains, which are widely involved in processes such as RNA splicing, transport, modification, and translation, and have important post-transcriptional regulatory functions. In recent years, many studies have found that RBP is also involved in the regulation of some important agronomic traits of crops. At present, multiple rice RBP coding genes have been reported in rice that can affect grain weight, such as GL11, DGW1, and EOG1. Identifying and cloning more genes related to grain weight regulation, including new RBP coding genes, can not only provide further theoretical basis for the genetic improvement of rice grain weight, but also provide more gene resources for the cultivation of new rice varieties. Summary of the Invention

[0004] The present invention has found through research that the rice grain weight-related gene BRW1 encodes a new RBP, which can negatively regulate grain weight; knocking out the BRW1 gene can significantly increase the grain weight of rice grains, and increase grain length and grain width. Utilizing the function of BRW1 and applying it in plant genetic engineering is of great significance for increasing rice grain weight.

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

[0006] The first object of the present invention is to provide the rice BRW1 gene, and the cDNA sequence of the BRW1 gene is shown as SEQ ID NO.1:

[0007]

[0008] The second object of the present invention is to provide the RNA-binding protein encoded by the aforementioned rice BRW1 gene, and the amino acid sequence of the RNA-binding protein is shown as SEQ ID NO.2:

[0009] MGTKNQFDLLVDVDNDDPSHLIAAAEKKAAAAAASPKPAAQAKLPTKPPPPAQAVKES

[0010] RNYGAPAREGAGRNGPGRGSGGFGGGRIGQRRDFGEGDTNGVEGGYGASGFGDGIVRREE

[0011] GEHRPSERGHGPRQPYRGGGRRGGYTGGEAGDESGRAPHRAYERRSGTGRGYGMKREGA

[0012] GRGNWGTVTDEALAQESGEAVSIEVSVTVTEENKQEDVPQSDEVEKHKEGESNEEEEKEPE

[0013] DKEMTLEEYEKVLEEKRKALLSLKVEERKVVVDKELQSMQQLSVKKDSDEVFIKLGSDKD

[0014] KKKENVERDERTRKSLSINEFLKPAEGERYYSPGGRGRGRGRGRGDRGGFRDGYSSRGPVAAPRIEDQAQFPGLAGRLVH*

[0015] The third object of the present invention is to provide a knockout mutant of the rice BRW1 gene.

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

[0017] Furthermore, the application is to knockout the rice BRW1 gene or reduce the expression level of the RNA-binding protein, so as to increase the brown rice grain weight, grain length and grain width of rice, or increase the 1000-grain weight, grain length, grain width and grain thickness of the hulled grains of rice.

[0018] Furthermore, it includes the following steps:

[0019] 1) Transgenic construction of rice BRW1 gene mutants: Screen the knockout target sites of the rice BRW1 gene. According to the knockout target sites, design BRW1 target site knockout primers to obtain the target sequence of the point mutation fragment of the BRW1 gene. After constructing the vector, transform rice to obtain the CRISPR / Cas9 gene knockout mutants of rice BRW1.

[0020] 2) Screening and identification of rice mutants: Use the target site detection primers to amplify the genome in the young leaves of rice BRW1 CRISPR / Cas9 gene knockout mutants, perform conventional PCR product sequencing, and screen for homozygous mutant plants, which are the plants with increased grain weight, grain length, and grain width of rice.

[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 sequences of the BRW1 target site knockout primers 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: ATTATTGGTCTCTAAACCCTTGTGTTTCTCGACTTC.

[0027] Furthermore, 2) the target site 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. The present invention discloses the rice BRW1 gene and the protein encoded thereby. The RBP protein gene BRW1 is reported for the first time in rice. By knocking out the rice BRW1 gene to make it lose its biological function, the grain weight, grain length, and grain width of rice grains are increased, and it is expected to be used as a target gene for the genetic improvement and genetic engineering of rice.

[0032] 2. The present invention provides theoretical and technical support for breeding high-yield rice varieties and is of great significance for rice production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 、Mutation situations of the rice BRW1 gene and the encoded protein, wherein:

[0034] (A) Mutation situation of the gene sequence;

[0035] (B) Mutation situation of the protein sequence; ZH11 in the figure is the wild type.

[0036] Figure 2 、Grain-related phenotypes of the wild type and the knockout mutant brw1, wherein:

[0037] (A) Appearance of grains;

[0038] (B) 1000-grain weight, grain length, grain width, and grain thickness; *p<0.05; **p<0.01.

[0039] Figure 3 、Brown rice-related phenotypes of the wild type and the knockout mutant brw1, wherein:

[0040] (A) Appearance of brown rice;

[0041] (B) 1000-grain weight, grain length, grain width, and grain thickness of brown rice; **p<0.01; ns, no difference. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] The following embodiments are used to further explain the present invention, but the embodiments do not limit the present invention in any form.

[0043] The present invention is further illustrated by specific embodiments. The methods used in the embodiments are all conventional methods unless otherwise specified. The primers and sequencing are completed by Nanjing Sipujin Biotechnology Co., Ltd.; various restriction endonucleases, ligases, DNA ladder, high-fidelity enzymes, vectors, etc. used in the experiments are purchased from Baoruiyi Biotechnology (Beijing) Co., Ltd.; the RNA extraction kit is purchased from Beijing TransGen Biotech Co., Ltd.; the reverse transcription kit is purchased from Novoprotein Scientific Inc.; the plasmid extraction kit and the gel extraction kit are purchased from Nanjing Merlin Xuehai Biotechnology Co., Ltd., and the methods are all carried out with reference to the instructions.

[0044] Example 1: Gene Cloning

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

[0046] Example 2: Construction of BRW1 Mutant Plants

[0047] (I) Determination of the gene mutation target site of rice BRW1:

[0048] Log in to the website http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html to screen for BRW1 gene target sites. Two specific target sites of 19bp target fragments were selected on the second exon and the third exon, and the corresponding two target sequences sgRNA1 and sgRNA2. The sequences are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0049] For the specific target sites of the two selected target fragments, 4 primers specific to the BRW1 target mutation site were designed. The knockout primer sequences are shown in SEQ ID NO.5-8;

[0050] Using the pCBC-MT1T2 vector diluted 100 times as a template for 4-primer PCR amplification, electrophoretic detection was performed, and the amplified product was purified and recovered to clone the specific target sites sgRNA1 and sgRNA2 of the BRW1 gene.

[0051] (II) Restriction Enzyme-Ligation System (15 μL):

[0052] PCR System: Point mutation target DNA fragment, 2 μL; pBUE411 vector, 2 μL; 10xNEB T 4 Buffer, 1.5 μL; 10xBSA, 1.5 μL; BsaI (NEB), 1 μL; T 4 Ligase (NEB) / High Concentration, 1 μL; ddH 2 O, 6 μL;

[0053] Restriction Enzyme-Ligation PCR Reaction Program: 37 °C, 5 hours; 50 °C, 5 minutes; 80 °C, 10 minutes.

[0054] (III) Obtaining of Mutant Lines:

[0055] The specific target sgRNA of the constructed rice BRW1 gene was ligated to the pHUE411 vector through the BsaI digestion and ligation system described in (2), and the plasmid containing the pHUE411-Target vector obtained was transformed into Agrobacterium tumefaciens; the Agrobacterium tumefaciens carrying the transformed plasmid was transformed into the callus of the wild-type japonica rice variety Zhonghua 11 through Agrobacterium-mediated rice transgenic technology.

[0056] (4) Screening and identification of rice mutants

[0057] According to the physical positions of the target fragments of the 2 specific targets on the 2nd exon and the 3rd exon of the cloned BRW1 gene, PCR verification primers for mutant materials were designed. The upstream primer sequence for amplifying the mutant target is shown in SEQ ID NO.9 in the sequence listing, and the downstream primer sequence is shown in SEQ ID NO.10 in the sequence listing. Using the PCR verification primers for mutant materials, the CRISPR / Cas9 target sequence of rice BRW1 was amplified to screen for homozygous mutant plants. Homozygous lines brw1-1, brw1-2, and brw1-3 with premature termination of translation of the mutant were obtained, and the mutation situations of the BRW1 gene and the encoded amino acids in these lines are as described Figure 1 as shown.

[0058] Example 3: Analysis of the agronomic trait phenotypes of mutant BRW1 plants

[0059] Compared with the wild-type Zhonghua 11, the hulled grains of the rice mutants brw1-1, brw1-2, and brw1-3 were significantly increased in 1,000-grain weight, grain length, grain width, and grain thickness, as described Figure 2 as shown; while their dehulled brown rice was significantly increased in 1,000-grain weight, grain length, and grain width, and there was no significant difference in grain thickness, as described Figure 3 as shown.

[0060] 1,000-grain weight: After the seeds were matured and dried, 3 groups of plump rice seeds and brown rice were selected, with 100 grains in each group. After weighing with an electronic balance, the statistical unit was converted.

[0061] Grain length, grain width, and grain thickness: After the seeds were matured and dried, plump rice seeds and brown rice were selected, and the grain length, grain width, and grain thickness were measured with a vernier caliper.

[0062] It can be seen that knocking out the rice BRW1 gene can significantly increase the 1,000-grain weight, grain length, and grain width of dehulled brown rice, or increase the 1,000-grain weight, grain length, grain width, and grain thickness of hulled grains.

[0063] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A rice BRW1 gene, characterized in that: The BRW1 gene cDNA sequence is shown as SEQ ID NO.

1.

2. The RNA binding protein encoded by the rice BRW1 gene according to claim 1, characterized in that: The amino acid sequence of the RNA binding protein is shown in SEQ ID NO.

2.

3. Knockout mutant of rice BRW1 gene.

4. Genetic engineering application of the rice BRW1 gene according to claim 1, the RNA binding protein according to claim 2, or the knockout mutant of the rice BRW1 gene according to claim 3.

5. The use according to claim 4, characterized in that: The application is to knock out the rice BRW1 gene or reduce the expression of RNA binding protein, thereby increasing the grain weight, grain length and grain width of hulled brown rice, or increasing the thousand-grain weight, grain length, grain width and grain thickness of hulled rice grains.

6. The use according to claim 5, characterized in that: The steps include: 1) Transgenic construction of rice BRW1 gene mutants: Screen the knockout target site of rice BRW1 gene, design BRW1 target site knockout primers according to the knockout target site, obtain the target sequence of the point mutation target fragment of BRW1 gene, construct the vector and transform rice to obtain the CRISPR / Cas9 gene knockout mutant of rice BRW1; 2) Screening and identification of rice mutants: The target detection primers were used to amplify the genome in the young leaves of the rice BRW1 CRISPR / Cas9 gene knockout mutant, and conventional PCR product sequencing was performed to screen homozygous mutant plants, that is, plants with increased rice grain weight, grain length, and grain width.

7. The use according to claim 6, characterized in that: 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.

8. The use according to claim 6, characterized in that: 1) The BRW1 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.

9. The use according to claim 6, characterized in that: 2) The target detection primers 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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