Application of OsMYB1 gene in regulation and control of nutritional indexes and taste quality of rice

Abstract

The invention discloses application of an OsMYB1 gene in regulation and control of nutritional indexes and taste quality of rice, and belongs to the technical field of biotechnology and plant genetic engineering. The invention provides an application of an OsMYB1 gene, an OsMYB1 protein coded by the OsMYB1 gene or a biological material with the OsMYB1 gene knocked out in regulation and control of nutritional indexes and taste quality of rice grains. The nutritional indexes comprise the amylose content; the taste quality comprises hardness, chewiness and / or adhesiveness; the nucleotide sequence of the OsMYB1 gene is as shown in SEQ ID No. 1; the amino acid sequence of the OsMYB1 protein is as shown in SEQ ID No. 2. Gene resources are provided for rice breeding, and the OsMYB1 gene is applied to rice breeding and has important application value and practical significance.

Description

Technical Field

[0001] The invention relates to the technical fields of biotechnology and plant genetic engineering, and in particular to the application of OsMYB1 gene in regulating rice nutritional indexes and eating quality. Background Art

[0002] After the innovation of dwarf breeding, hybrid advantage utilization and green super rice, the yield of rice breeding has increased by leaps and bounds, and the rice market has also changed accordingly. Soft rice is favored by consumers due to its excellent taste. The main component of rice grains is starch, which is composed of two different polymers, amylose and amylopectin. Amylose is synthesized by GBSSI encoded by the waxy gene. If the amylose content is too low, that is, glutinous rice, although it tastes soft, it has too high viscosity and is not easy to digest. If the amylose content is too high, the taste is poor. Therefore, generally speaking, rice with a moderate amylose content (8% to 13%) usually tastes good and is a very popular soft rice in the domestic rice market.

[0003] Transcription factors are a class of proteins that have the ability to regulate gene expression. They recognize and regulate the expression of target genes by binding to specific sequences on DNA, and have important biological functions. MYB proteins are a class of transcription factors that are widely present in eukaryotes. Many MYB proteins have been reported in rice, involving various aspects of growth and development, but there are few reports on their involvement in regulating amylose synthesis. Therefore, in-depth exploration of genes related to amylose synthesis in rice and expansion of the biological functions of rice MYB protein family transcription factors are of great significance to rice breeding and improvement. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the OsMYB1 gene in regulating rice nutritional indicators and taste quality to solve the problems existing in the above-mentioned prior art. The present invention first reveals the function of the rice OsMYB1 gene in regulating rice amylose synthesis, and the experiments of the present invention prove that the rice gene OsMYB1 can regulate rice grain nutritional indicators and taste quality.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] Technical solution 1: Application of OsMYB1 gene, OsMYB1 protein encoded by OsMYB1 gene or biological material with OsMYB1 gene knocked out in regulating nutritional indicators and taste quality of rice grains, wherein the nutritional indicators include amylose content; the taste quality includes hardness, chewiness and / or adhesion; the nucleotide sequence of the OsMYB1 gene is shown in SEQ ID No.1; the amino acid sequence of the OsMYB1 protein is shown in SEQ ID No.2.

[0007] Furthermore, by knocking out or inhibiting the expression of the OsMYB1 gene, the amylose content, hardness and chewiness can be reduced, and the adhesion can be improved.

[0008] Furthermore, the biological material includes a recombinant vector containing the OsMYB1 gene.

[0009] Optionally, a host bacteria of the recombinant vector is included.

[0010] Technical solution 2: Use of OsMYB1 gene, OsMYB1 protein encoded by OsMYB1 gene or biological material with OsMYB1 gene knocked out in cultivating transgenic rice in any of the following items:

[0011] (1) Transgenic rice with low amylose content;

[0012] (2) Transgenic rice with low hardness;

[0013] (3) Transgenic rice with low chewiness;

[0014] (4) Highly adherent transgenic rice;

[0015] The nucleotide sequence of the OsMYB1 gene is shown in SEQ ID No.1; the amino acid sequence of the OsMYB1 protein is shown in SEQ ID No.2.

[0016] Furthermore, the biological material includes a recombinant vector or a recombinant bacterium.

[0017] Technical solution three: An OsMYB1 mutant gene, the amino acid sequence of the OsMYB1 mutant gene is shown as SEQ ID No.3 or SEQ ID No.4.

[0018] Technical solution 4: The protein encoded by the OsMYB1 mutant gene, the amino acid sequence of the protein is shown in SEQ ID No.5 or SEQ ID No.6.

[0019] Technical Solution 5: Application of the OsMYB1 mutant gene or the protein in regulating the nutritional indexes and taste quality of rice grains, wherein the nutritional indexes include the amylose content; and the taste quality includes hardness, chewiness and / or adhesion.

[0020] Technical solution six: Use of the OsMYB1 mutant gene or the protein in cultivating any of the following transgenic rice:

[0021] (1) Transgenic rice with low amylose content;

[0022] (2) Transgenic rice with low hardness;

[0023] (3) Transgenic rice with low chewiness;

[0024] (4) Highly adherent transgenic rice;

[0025] The nucleotide sequence of the OsMYB1 mutant gene is shown in SEQ ID No.3 or SEQ ID No.4; the amino acid sequence of the protein is shown in SEQ ID No.5 or SEQ ID No.6.

[0026] Furthermore, the biological material includes a recombinant vector containing the OsMYB1 mutant gene.

[0027] The present invention discloses the following technical effects:

[0028] The experiment of the present invention proves that the rice gene OsMYB1 can regulate the nutritional index and taste quality of rice grains, the nutritional index includes the content of amylose; the taste quality includes hardness, chewiness and / or adhesion; the nucleotide sequence of the OsMYB1 gene is shown in SEQ ID No.1; the amino acid sequence of the OsMYB1 protein is shown in SEQ ID No.2. The present invention provides gene resources for crop breeding, and the rice OsMYB1 gene can be applied to rice breeding. The present invention first discovered and cloned a new amylose-related gene OsMYB1, and obtained its functional loss mutant using gene editing technology. After the gene mutation, the amylose content of rice is downregulated and the hardness of rice is reduced. The gene and its encoded protein can be used for the molecular mechanism research of regulating the synthesis of amylose in rice and the genetic improvement of the taste quality of rice, which has important theoretical and practical significance for plant breeding and application. The present invention provides new gene resources for the molecular mechanism research of rice endosperm development and the creation of soft rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 This is a diagram of tissue expression analysis for site prediction of the OsMYB1 gene;

[0031] Figure 2 This is a map of the CRISPR-Cas9 knockout vector containing the OsMYB1 gene target site sequence;

[0032] Figure 3Schematic diagram of gene editing of OsMYB1 in the Nipponbare background;

[0033] Figure 4 Comparison of the plant types of the wild type Nipponbare and the mutants, including the plant morphology of the wild type (A) and the knockout mutants osmyb1-1 (B) and osmyb1-2 (C), as well as the comparison of plant height (D), effective tiller number (E), and 1000-grain weight (F) between the wild type and the knockout mutants osmyb1-1 and osmyb1-2;

[0034] Figure 5 The physical and chemical indexes of wild-type Nipponbare and mutant rice were measured, including observation of brown rice of wild type (A) and knockout mutants osmyb1-1 (B) and osmyb1-2 (C); comparison of chalky grain rate (D), chalkiness (E), amylose content (F), total starch content (G), total protein content (H), gel consistency (I), hardness (J), chewiness (K) and stickiness (L) of wild type and knockout mutants osmyb1-1 and osmyb1-2. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] OsMYB1 gene sequence:

[0041]

[0042] The protein sequence encoded by the OsMYB1 gene:

[0043] MASSSTTNTSDGAGKPASSSSSACPRGHWRPGEDEKLRQLVEKYGPQNWNSIAEKLEGRSGKSCRLRWFNQLDPRINKRPFTEEEEERLLAAHRHHGNKWALIARHFPGRTDNAVKNHWHVVRARRSRERSRLLARAAAAAAHPPPFSSYASQLDFSGGGASSGARNSSLCFGFGMINRSSSSSSSPAAAPFLIKSFNGTSYGTLLPATTSMAAAAQPVSTITFSSTPMRETLELMDAGGHENHGDVDGGGDKRKGVPYFDFLGVGV*(SEQ ID No.2);

[0044] The osmyb1-1 gene sequence:

[0045]

[0046] osmyb1-2 gene sequence:

[0047]

[0048] Protein sequence encoded by osmyb1-1 gene:

[0049] MASSSTTNTSDGAGKTRSSGSWRSTALRTGTPSPRSWRADQGRAVVSGGSTSWTRG STSGPSRRRRRSGCWRRTATTATSGPSSPATSPAAPTTPSRTTGTSSAPAAPASAPASSPAPPPP PPTRRRSPPTPASSTSAAAVRHQARAIARYASASA*(SEQ ID No. 5);

[0050] Protein sequence encoded by oscmyb1-2 gene:

[0051] MASSSTTNTSDGAGKPASSSSSASRGHWRPGEDEKLRQLVEKYGPQNWNSIAEKLEGRSGKSCRLRWFNQLDPRINKRPFTEEEEERLLAAHRHHGNKWALIARHFPGRTDNAVKNHWHVVRARRSRERSRLLAR AAAAAAHPPPFSSYASQLDFSGGGASSGARNSSLCFGFGMINRSSSSSSSSPAAAPFLIKSFNGTSYGTLLPATTSMAAAAQPVSTITFSSTPMRETLELMDAGGHENHGDVDGGGDKRKGVPYFDFLGVGV*(SEQ ID No.6);

[0052] Nucleotide sequence of OsMYB1cas9TF:

[0053] 5′-TCTCCCATTTCCCCTCTAATTCT-3′ (SEQ ID No. 7);

[0054] Nucleotide sequence of OsMYB1cas9TR:

[0055] 5'-TCCGATTCAAACCAAACAGGC-3' (SEQ ID No. 8).

[0056] Rice material: The original wild type material was the japonica rice variety Nipponbare (NIP).

[0057] Example 1 Analysis of the predicted tissue expression pattern of rice OsMYB1 gene

[0058] The RAP-DB website (https: / / rapdb.dna.affrc.go.jp) was used to input the RAP_Locus number of OsMYB1 and analyze the tissue expression pattern. The prediction results showed that OsMYB1 was expressed at the highest level in the embryo and endosperm of rice 3 days after flowering (e.g. Figure 1 shown).

[0059] Example 2 Construction of OsMYB1 gene knockout strains in rice

[0060] 1. Selection of gRNA target sequence

[0061] According to CRISPR / Cas9 related experimental methods, a 5′-TCGTCGTCGGCGTGCCCGAG-3′ (SEQ ID No. 9) sequence containing NGG as a recognition site was selected as a knockout target site on the OsMYB1 gene (the nucleotide sequence is shown in SEQ ID No. 1, and the protein sequence encoded by the OsMYB1 gene is shown in SEQ ID No. 2), and the PAM sequence is AGG;

[0062] 2. CRISPR / Cas9 vector construction

[0063] The plant Cas9 / gRNA plasmid construction kit (Catalog.No.VK005-01) was used to load the target sequence to form a recombinant vector containing the OsMYB1 gene target site (such as Figure 2 The specific operation method is as follows:

[0064] (1) Formation of oligo dimers

[0065] Take 5 μL of each 10 μM target site front and back primer and add 15 μL of ddH2O, mix well, treat at 95℃ for 3 minutes, slowly cool from 95℃ to 25℃, and treat at 16℃ for 5 minutes to obtain a double-stranded sequence containing the knockout target site;

[0066] (2) Insertion of oligo dimer into vector

[0067] Take 1 μL of Cas9 / gRNA vector, 1 μL of oligo dimer from step 1, 1 μL of buffer 1 and 2 respectively, add 6 μL of ddH2O, and react in a 16°C metal bath for 2 hours;

[0068] (3) E. coli transformation

[0069] Take 5-10 μL of the final product of step (2) and add it to 50 μL of DH5a competent cells that have just been thawed, flick to mix, place on ice for 30 minutes, heat shock at 42°C for 45 seconds, let stand on ice for 2 minutes, then add 200 μL of antibiotic-free LB, place in a 37°C constant temperature shaker at 200 rpm, and after one hour of recovery, place on a plate coated with kanamycin resistance (Kana+);

[0070] (4) Bacterial liquid PCR detection

[0071] The next day, a single clone was picked and placed in a kanamycin-resistant liquid culture medium, and cultured in a 37°C shaker until the bacterial solution became turbid. Sequencing was performed using the VK005 vector-specific sequencing primers provided in the kit; the sequencing results were analyzed using Snapgene software, and the plasmid of the positive clone bacterial solution was extracted for later use.

[0072] 3. Agrobacterium transformation and rice genetic transformation

[0073] The successfully constructed plasmid was transferred into Agrobacterium (EHA105): 1 μL of plasmid was injected into the frozen and thawed Agrobacterium competent cell on ice, and then placed on ice for 5 min, liquid nitrogen for 5 min, and 37°C for 5 min, and 300 μL of resistance-free LB was added to resuscitate at 28°C for 4 h, and evenly spread on a (kanamycin + rifampicin) K+ / Rif-resistant plate. After culturing at 28°C for 2-3 days, single clones were picked, and positive clones were obtained by detection with hygromycin primers and expanded in 3 mL of liquid K+ / Rif culture medium. The positive K+ / Rif bacterial solution was further sent to Wuhan Aidijing Biotechnology Co., Ltd. for rice genetic transformation under the Nipponbare background.

[0074] Example 3 Phenotypic analysis of rice OsMYB1 gene knockout strains

[0075] In order to identify the knockout transgenic strain obtained above, the transgenic seedlings obtained were cultured in a normal temperature light incubator for one week, and then the positive seedlings were identified. The specific steps are as follows:

[0076] 1. Knockout transgenic seedling detection

[0077] 25 T0 transgenic seedlings were obtained and cultured in a normal temperature light incubator for one week. DNA of the 25 seedlings was taken and amplified by PCR using OsMYB1cas9TF (nucleotide sequence as shown in SEQ ID No.7) and OsMYB1cas9TR (nucleotide sequence as shown in SEQ ID No.8) and then sent to the company for sequencing. The sequencing results were analyzed and the results showed that the translation of the two proteins was terminated prematurely (as shown in Figure 3The nucleotide sequences of the two homozygous mutants osmyb1-1 and osmyb1-2 obtained by knocking out the base deletion of the OsMYB1 gene of the present invention are shown as SEQ ID No.3 and SEQ ID No.4, respectively, and the encoded proteins are shown as SEQ ID No.5 and SEQ ID No.6, respectively.

[0078] 2. Phenotypic Identification

[0079] The T1 generation plants were sown in the natural field environment, the edge row effect was removed, the plants with normal morphology were selected, and 5-10 stable strains were selected. The important agronomic traits of different strains of knockout mutants were observed. It was found that there was no significant difference in plant morphology between the mutant and the wild type (such as Figure 4 After obtaining a stable genetic T2 knockout strain, the target site was further sequenced. After the target site was determined, mature rice seeds were harvested. The mature seeds were placed in a 65°C oven and dried to a constant weight. The mature seed shape was analyzed. Compared with the wild type, the thousand-grain weight of the mutant was slightly reduced (as shown in Figure 1). Figure 4 DF). Figure 5 Compared with the wild type, the chalky grain rate and chalkiness of the mutant were increased (as shown in Figure 5A). Figure 5 D and E in FIG.

[0080] Example 4 Detection method and analysis of rice taste quality of rice OsMYB1 gene knockout strain

[0081] Determination of amylose content: Grind the wild-type and OsMYB1 knockout mutant rice into rice flour, pass through a 100-mesh sieve, weigh 0.050 g of each, and place in a 50 mL volumetric flask. At the same time, prepare standard samples (0.4%, 10.6%, 16.2% and 26.5% amylose) for the same treatment. Slowly add 0.5 mL of 95% ethanol, gently shake the volumetric flask, then add 4.5 mL of 1 mol / L NaOH solution, and let stand at room temperature overnight; the next day, add deionized water, dilute to 50 mL, mix upside down, and let stand for 20 minutes. Accurately pipette 0.5mL of sample and standard into a 10mL test tube, add 5mL of ddH2O, 100μL of 1M acetic acid solution, 200μL of KI-I2 (2g I2+20g KI+1000mL ddH2O) solution and 4.2mL of ddH2O in sequence, mix well using an oscillator, and let stand at room temperature for 20min; pipette 0.2mL of the above mixture onto a transparent ELISA plate, and use an ELISA reader to measure the absorbance of the sample at a wavelength of 620nm. Calculate the amylose content of each sample (such as 4.2mL) based on the linear equation of the absorbance of the sample and the amylose content. Figure 5 ).

[0082] Total starch content determination: The total starch content in refined rice flour of wild-type NIP and OsMYB1 knockout mutant transgenic materials was determined using the Megazyme total starch assay kit K-TSTA (Megazyme, Ireland, UK). Figure 5 ).

[0083] Determination of total protein content: Weigh 0.1g refined rice flour and place it in a 100mL digestion tube, add 5mL concentrated sulfuric acid, place the digestion tube in a 290℃ digestion furnace, start timing, take it out and shake it every 15 minutes, generally shake it about 4 times, and time it for 60 minutes. Take out the digestion tube and cool it to room temperature. Add 0.75mL of hydrogen peroxide solution and put it into the 290℃ digestion furnace again to observe whether the solution in the digestion tube becomes clear. After the solution becomes clear, take it out, cool it to room temperature and make up the volume to 100mL. Use an enzyme marker to measure the absorbance of the sample and the standard sample at a wavelength of 280nm. Draw a linear equation based on the nitrogen content and absorbance OD value of the standard sample, calculate the nitrogen content in the sample based on the variance and convert it into protein content (such as Figure 5 ).

[0084] The gel consistency (GC) was determined according to the national edible rice variety quality standard NT / T593-2021. All data are based on three biological replicates, and the values ​​are the mean values. The error is the standard error of the mean (SEM). Different lowercase letters indicate significant differences (P<0.05). The statistical differences were tested by one-way ANOVA (such as Figure 5 1).

[0085] Determination of cooking taste quality: Wash the raw rice twice, soak it in water for 30 minutes, seal it and steam it at normal pressure for 30 minutes, then stir and simmer for 10 minutes. After simmering, cool it in a cooling box for 20 minutes, and finally cool it at room temperature 20-30℃ for 90 minutes. A rice taste meter (STA / A, Satake, Japan) is used to determine the score and comprehensive score of the hardness, viscosity and chewiness of the rice. After the test on 0d, the samples were placed in a 4℃ refrigerator for low temperature storage. The samples were taken out after 1d and 7d respectively, and the taste values ​​of the rice after different storage times (such as hardness, viscosity and chewiness) were tested. Figure 5 (as shown in JL in ).

[0086] In summary, except for a slight decrease in 1000-grain weight, the mutant after OsMYB1 gene knockout had no significant difference in plant morphology compared with the wild type; although OsMYB1 gene knockout increased the chalkiness and chalky grain rate of rice, it significantly reduced the amylose content of rice, and the hardness and chewiness of the mutant's mature endosperm were significantly reduced, while the stickiness was significantly increased, indicating that OsMYB1 gene knockout has better taste quality, and this gene has great application prospects in improving the taste quality of rice.

[0087] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of OsMYB1 gene, OsMYB1 protein encoded by OsMYB1 gene or biological material with knockout of OsMYB1 gene in regulating nutritional indexes and eating quality of rice grains, characterized in that: The nutritional index includes the content of amylose; the taste quality includes hardness, chewiness and / or adhesion; the nucleotide sequence of the OsMYB1 gene is shown in SEQ ID No.1; and the amino acid sequence of the OsMYB1 protein is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that: By knocking out or inhibiting the expression of the OsMYB1 gene, the amylose content, hardness and chewiness of rice grains can be reduced, and the adhesion can be improved.

3. The use according to claim 1, characterized in that: The biological material includes a recombinant vector or a recombinant bacterium.

4. Use of the OsMYB1 gene, the OsMYB1 protein encoded by the OsMYB1 gene, or a biological material with the OsMYB1 gene knocked out in cultivating any of the following transgenic rice: (1) Transgenic rice with low amylose content; (2) Transgenic rice with low hardness; (3) Transgenic rice with low chewiness; (4) Highly adherent transgenic rice; The nucleotide sequence of the OsMYB1 gene is shown in SEQ ID No.1; the amino acid sequence of the OsMYB1 protein is shown in SEQ ID No.

2.

5. The use according to claim 4, characterized in that: The biological material includes a recombinant vector or a recombinant bacterium.

6. An OsMYB1 mutant gene, characterized in that: The amino acid sequence of the OsMYB1 mutant gene is shown as SEQ ID No.3 or SEQ ID No.

4.

7. The protein encoded by the OsMYB1 mutant gene according to claim 6, characterized in that: The amino acid sequence of the protein is shown in SEQ ID No.5 or SEQ ID No.

6.

8. Use of the OsMYB1 mutant gene according to claim 6 or the protein according to claim 7 in regulating the nutritional indexes and taste quality of rice grains, characterized in that: The nutritional index includes the content of amylose; and the taste quality includes hardness, chewiness and / or adhesion.

9. Use of the OsMYB1 mutant gene according to claim 6 or the protein according to claim 7 in cultivating any of the following transgenic rice: (1) Transgenic rice with low amylose content; (2) Transgenic rice with low hardness; (3) Transgenic rice with low chewiness; (4) Transgenic rice with high adhesion.

10. The use according to claim 9, characterized in that: The biological material comprises a recombinant vector containing the OsMYB1 mutant gene.

Images