Application of OsC2H2 gene in regulating nutritional indexes and eating quality of rice

Abstract

The present invention discloses the use of the OsC2H2 gene in regulating rice nutritional indicators and taste quality, belonging to the fields of biotechnology and plant genetic engineering technology. The present invention provides the use of the OsC2H2 gene, the OsC2H2 protein encoded by the OsC2H2 gene, or a biological material with the OsC2H2 gene knocked out, in regulating rice grain nutritional indicators and taste quality. The nutritional indicators include amylose content; the taste quality includes hardness, chewiness, and / or adhesion. The nucleotide sequence of the OsC2H2 gene is shown in SEQ ID No. 1; the amino acid sequence of the OsC2H2 protein is shown in SEQ ID No. 2. The present invention provides a genetic resource for rice breeding, and the application of the OsC2H2 gene in rice breeding has important application value and practical significance.

Description

Technical Field

[0001] The present invention relates to the technical fields of biotechnology and plant genetic engineering, and in particular to the application of OsC2H2 gene in regulating rice nutritional indicators and taste quality. Background Art

[0002] Rice is one of the most important staple foods, serving as a staple food for more than half the world's population. Developing dwarf rice varieties, leveraging hybrid vigor, and developing green super rice have resulted in significant breakthroughs in rice production. However, with rapid social development and rising living standards, consumer demand for rice has shifted from simply "satisfying" to "eating well." Consequently, market demand for mid- to high-end rice is robust, with a particularly pressing need for high-quality, flavorful rice.

[0003] The primary component of rice grains is starch, which is composed of two distinct polymers: amylose and amylopectin. Amylose is synthesized by GBSSI, encoded by the Waxy gene, and its content is the primary factor influencing rice cooking and eating quality (ECQ). ECQ is the most critical quality trait of rice and can be quantitatively analyzed by measuring physical and chemical parameters such as amylose content (AC), hardness, and viscosity. Generally speaking, soft rice with a low AC content (8%-13%) generally has a better taste and is very popular in the domestic rice market.

[0004] Transcription factors are a class of proteins with the ability to regulate gene expression. They bind to specific sequences on DNA to recognize and regulate the expression of target genes, thus possessing important biological functions. Zinc finger proteins are a class of transcription factors that are widely present in eukaryotes. Many zinc finger proteins have been reported in rice, implicated in various aspects of growth and development, demonstrating their rich functionalities. However, few have been reported related to cooking and flavor quality. Therefore, further research into genes involved in rice starch synthesis and affecting rice cooking and flavor quality, and expanding the biological functions of the rice zinc finger protein family of transcription factors, is of great significance for rice breeding and improvement. Summary of the Invention

[0005] The present invention aims to address the aforementioned problems of the prior art by providing a method for using the OsC2H2 gene to regulate rice nutritional indicators and flavor quality. This invention, for the first time, reveals the function of the rice OsC2H2 gene in regulating rice amylose synthesis. Experiments conducted in this invention demonstrate that the rice gene OsC2H2 can regulate both nutritional indicators and flavor quality in rice grains.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical Solution 1: Application of the OsC2H2 gene, the OsC2H2 protein encoded by the OsC2H2 gene, or a biomaterial with the OsC2H2 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 OsC2H2 gene is shown in SEQ ID No. 1; and the amino acid sequence of the OsC2H2 protein is shown in SEQ ID No. 2.

[0008] Furthermore, by knocking out or inhibiting the expression of the OsC2H2 gene, the amylose content, hardness and chewiness of rice grains are reduced, and the adhesiveness is improved.

[0009] Furthermore, the biological material includes a recombinant vector containing the OsC2H2 gene.

[0010] Optionally, a host bacteria of the recombinant vector is included.

[0011] Technical Solution 2: Use of the OsC2H2 gene, the OsC2H2 protein encoded by the OsC2H2 gene, or a biomaterial with the OsC2H2 gene knocked out in cultivating any of the following transgenic rice:

[0012] (1) Transgenic rice with low amylose content;

[0013] (2) Transgenic rice with low hardness;

[0014] (3) Transgenic rice with low chewiness;

[0015] (4) Highly adhesive transgenic rice;

[0016] The nucleotide sequence of the OsC2H2 gene is shown in SEQ ID No. 1; the amino acid sequence of the OsC2H2 protein is shown in SEQ ID No. 2.

[0017] Furthermore, the biological material includes a recombinant vector or a recombinant bacterium.

[0018] Technical solution three: An OsC2H2 mutant gene, the amino acid sequence of the OsC2H2 mutant gene is shown as SEQ ID No. 3 or SEQ ID No. 4.

[0019] Technical Solution 4: The protein encoded by the OsC2H2 mutant gene, the amino acid sequence of the protein is shown in SEQ ID No. 5 or SEQ ID No. 6.

[0020] Technical Solution 5: Application of the OsC2H2 mutant gene or the protein in regulating the nutritional indicators and taste quality of rice grains, wherein the nutritional indicators include the amylose content; and the taste quality includes hardness, chewiness and / or adhesion.

[0021] Technical Solution 6: Use of the OsC2H2 mutant gene or the protein in cultivating any of the following transgenic rice:

[0022] (1) Transgenic rice with low amylose content;

[0023] (2) Transgenic rice with low hardness;

[0024] (3) Transgenic rice with low chewiness;

[0025] (4) Highly adhesive transgenic rice;

[0026] The nucleotide sequence of the OsC2H2 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.

[0027] Furthermore, the biological material includes a recombinant vector containing the OsC2H2 mutant gene.

[0028] The present invention discloses the following technical effects:

[0029] Experiments in this paper demonstrate that the rice gene OsC2H2 can regulate rice grain nutritional indicators and flavor quality. Nutritional indicators include amylose content, and flavor quality includes firmness, chewiness, and / or stickiness. The nucleotide sequence of the OsC2H2 gene is shown in SEQ ID No. 1, and the amino acid sequence of the OsC2H2 protein is shown in SEQ ID No. 2. This invention provides a genetic resource for crop breeding, enabling the application of the rice OsC2H2 gene in rice breeding. This invention, for the first time, discovered and cloned a novel amylose-related gene, OsC2H2, and generated its loss-of-function mutant using gene editing technology. Results showed that mutation of this gene resulted in a downregulation of rice amylose content and a significant improvement in the flavor and cooking quality of the rice. Therefore, the OsC2H2 gene and its encoded protein can be used to study the molecular mechanisms of rice amylose synthesis and genetically improve rice flavor and cooking quality, possessing important theoretical and practical significance for plant breeding and application. This invention provides a new genetic resource for studying the molecular mechanisms of rice endosperm development and for the development of soft rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0031] Figure 1 This is a map of the CRISPR-Cas9 knockout vector containing the OsC2H2 gene target site sequence;

[0032] Figure 2 Schematic diagram of gene editing of OsC2H2 in the Nipponbare background;

[0033] Figure 3 Comparison of plant types between the wild-type Nipponbare and mutants, including plant morphology of the wild type (A) and knockout mutants osc2h2-1 (B) and osc2h2-1 (C), as well as comparison of plant height (D), effective tiller number (E), and 1000-grain weight (F) between the wild type and knockout mutants osc2h2-1 and osc2h2-2;

[0034] Figure 4 The physical and chemical indicators of wild-type Nipponbare and mutant rice were measured, including observation of brown rice of wild type (A) and knockout mutants osc2h2-1 (B) and osc2h2-2 (C); and 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 osc2h2-1 and osc2h2-2. 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

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

[0040] OsC2H2 gene sequence:

[0041] ATGGGTGAGCAGATGGACAACGAGGAGCTCAACCTAAGTTTGTCGCTCCAGCCATCATATCCTTCAAGGTTTCAGACAGAGTTTTCGTGTTGCTACTGTCCTAAGAGGTTCCAGAGCTCTCAGGCATTAGGTGGCCACCAGAATGCACACAAGCTCCAGCGTAACCTTGCGAAGAGGAATCGAGAGGCTTTTCTATCAATAAGCCAAAGGAAGGGTGC AAATGCTGGAATAAAGGATGGAAGCTCTGCACTCTCTGCTGAGTCTATCTGCAAAATTTCAAGTGGAAAGAAGCATCATAAAGAGGCCTGGCAGGTGATGCAGGGATCATGTGGTTCATCATCTGGTACAGTGATGCAAAATCTATCGAACAGGATGTAGAAGACGAAGATCTATCAAATGGGACGATTGATCTGTCCTTAAAGTTATGA(SEQ ID No.1);

[0042] Protein sequence encoded by the OsC2H2 gene:

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

[0044] osc2h2-1 gene sequence:

[0045] ATGGGTGAGCAGATGGACAAACGAGGAGCTCAACCTAAGTTTGTCGCTCCAGCCAT CATATCCTTCAAGGTTTCAGACAGAGTTTTCGTGTTGCTACTGTCCTAA(SEQ ID No.3); osc2h2-2 gene sequence:

[0046] ATGGGTGAGCAGATGGACGAGGAGCTCAACCTAAGTTTGTCGCTCCAGCCATCATATCCTTCAAGGTTTCAGACAGAGTTTTCGTGTTGCTACTGTCCTAAGAGGTTCCAGAGCTCTCAGGCATTAGGTGGCCACCAGAATGCACACAAGCTCCAGCGTAACCTTGCGAAGAGGAATCGAGAGGCTTTTCTATCAATAAGCCAAAGGAAGGGTGCAAATGCTGGAATAAAGGATGGAAGCTCTGCACTCTCTGCTGAGTCTATCTGCAAAATTTCAAGTGGAAAGAAGCATCATAAAGAGGCCTGGCAGGTGATGCAGGGATCATGTGGTTCATCATCATCTGGTACAGTGATGCACAAATCTATCGAACAGGATGTAGAAGACGAAGATCTATCAAATGGGACGATTGATCTGTCCTTAAAGTTATGA(SEQ ID No.4);

[0047] Protein sequence encoded by osc2h2-1 gene:

[0048] MGEQMDKRGAQPKFVAPAIISFKVSDRVFVLLLS*(SEQ ID No.5);

[0049] Protein sequence encoded by osc2h2-2 gene:

[0050] MGEQMDEELNLSLSLQPSYPSRFQTEFSCCYCPKRFQSSQALGGHQNAHKLQRNLAK RNREAFLSISQRKGANAGIKDGSSALSAESICKISSGKKHHKEAWQVMQGSCGSSSSGTVM HKSIEQDVEDEDLSNGTIDLSLKL*(SEQ ID No.6);

[0051] Nucleotide sequence of OsC2H2cas9TF:

[0052] 5′-CCAGATGAGTCCAGCGAG-3′ (SEQ ID No. 7);

[0053] Nucleotide sequence of OsC2H2cas9TR:

[0054] 5′-CAGATCAATCGTCCATTTGATA-3′ (SEQ ID No. 8).

[0055] Rice material: The original wild type material was the japonica rice variety Nipponbare (NIP).

[0056] Example 1 Construction of OsC2H2 gene knockout strains in rice

[0057] 1. Selection of gRNA target sequence

[0058] According to CRISPR / Cas9 related experimental methods, the 5′-GGGTGAGCAGATGGACAACG-3′ (SEQ ID No. 9) sequence containing NGG as the recognition site was selected as the knockout target site on the OsC2H2 gene, and the PAM sequence was AGG;

[0059] 2. CRISPR / Cas9 vector construction

[0060] 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 OsC2H2 gene target site (such as Figure 1 The specific operation method is as follows:

[0061] (1) Formation of oligo dimers

[0062] Take 5 μL of each of the 10 μM target site front and back primers and add 15 μL of ddH2O. After mixing, 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.

[0063] (2) Insertion of oligo dimer into vector

[0064] Take 1 μL of Cas9 / gRNA vector, 1 μL of oligo dimer from step 1, 1 μL of buffer 1 and 2, add 6 μL of ddH2O, and react in a 16°C metal bath for 2 hours;

[0065] (3) Escherichia coli transformation

[0066] Take 5-10 μL of the final product of step 2 and add it to 50 μL of freshly thawed DH5a competent cells, flick to mix, incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, let it stand on ice for 2 minutes, then add 200 μL of antibiotic-free LB, place it in a 37°C constant temperature shaker at 200 rpm, and after one hour of recovery, add it to a plate coated with kanamycin resistance (Kana+);

[0067] (4) Bacterial liquid PCR detection

[0068] The next day, single clones were picked and placed in kanamycin-resistant liquid culture medium. The culture was cultured in a shaker at 37°C 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 plasmids of the positive clone bacterial solution were extracted and set aside.

[0069] 3. Agrobacterium transformation and rice genetic transformation

[0070] The successfully constructed plasmid was transformed into Agrobacterium (EHA105): 1 μL of plasmid was aspirated into the frozen-thawed Agrobacterium competent cell on ice, then placed on ice for 5 minutes, liquid nitrogen for 5 minutes, and 37°C for 5 minutes. 300 μL of antibiotic-free LB was added to resuscitate at 28°C for 4 hours, 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 into 3 mL of liquid K+ / Rif medium. The positive K+ / Rif bacterial solution was further sent to Wuhan Aidijing Biotechnology Co., Ltd. for rice genetic transformation under the Nipponbare background.

[0071] Example 2 Phenotypic Analysis of Rice OsC2H2 Gene Knockout Lines

[0072] In order to identify the knockout transgenic lines obtained above, the transgenic seedlings were cultured in a normal temperature light incubator for one week, and then the positive seedlings were identified. The specific steps are as follows:

[0073] 1. Knockout transgenic seedling detection

[0074] 25 T0 transgenic seedlings were obtained and cultured in a normal temperature light incubator for one week. DNA of the 25 seedlings was collected and amplified by PCR using OsC2H2cas9TF (nucleotide sequence shown in SEQ ID No. 7) and OsC2H2cas9TR (nucleotide sequence shown in SEQ ID No. 8). The DNA was 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 2). Figure 2 The nucleotide sequences of the two homozygous mutants of the OsC2H2 gene obtained by the present invention are shown in SEQ ID No.3 and SEQ ID No.4, and the encoded proteins are shown in SEQ ID No.5 and SEQ ID No.6.

[0075] 2. Phenotypic Identification

[0076] The T1 generation plants were sown in the natural environment of the field, 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 wild type (such as Figure 3 After obtaining a stable T2 knockout line, the target site was further sequenced. After the target site was confirmed, mature rice seeds were harvested. The mature seeds were dried in a 65°C oven to a constant weight. The grain shape of the mature seeds was analyzed. Compared with the wild type, the thousand-grain weight of the mutant was slightly reduced (as shown in Figure 4A). Figure 3 DF in the middle), observations on the brown rice of wild type and knockout mutants showed that compared with the wild type, the chalky grain rate and chalkiness of the mutants were increased (as shown in Figure 5). Figure 4 AC in Figure 1).

[0077] Example 3 Detection method and analysis of rice taste quality in rice OsC2H2 gene knockout strain.

[0078] Determination of Amylose Content: Grind wild-type and OsC2H2 knockout mutant rice into rice flour, pass through a 100-mesh sieve, and weigh 0.050 g of each into a 50 ml volumetric flask. Simultaneously prepare standard samples (0.4%, 10.6%, 16.2%, and 26.5% amylose) and perform the same procedure. Slowly add 0.5 ml of 95% ethanol, gently shake the flask, then add 4.5 ml of 1 mol / L NaOH solution. Let stand at room temperature overnight. The next day, add deionized water, bring the volume to 50 ml, mix thoroughly by inverting, and let stand for 20 minutes. Accurately pipette 0.5 ml of sample and standard into a 10 ml test tube, add 5 ml of ddH2O, 100 μl of 1 M acetic acid solution, 200 μl of KI-I2 (2 g I2 + 20 g KI + 1000 ml of ddH2O) solution and 4.2 ml of ddH2O in sequence, mix well using an oscillator, and let stand at room temperature for 20 minutes; pipette 0.2 ml of the above mixture onto a transparent ELISA plate, and use a microplate reader to measure the absorbance of the sample at a wavelength of 620 nm. Calculate the amylose content of each sample (such as ) based on the linear equation of the sample absorbance and amylose content. Figure 4 (as shown in F in the figure).

[0079] Total starch content determination: The total starch content in the refined rice flour of the wild-type NIP and OsC2H2 gene knockout mutant transgenic materials was determined using the Megazyme Total Starch Assay Kit K-TSTA (Megazyme, Ireland, UK). Figure 4 For specific steps, refer to the kit instructions.

[0080] 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, usually shake it 4 times, and time it for 60 minutes. Take out the digestion tube and cool it to room temperature. Add 0.75ml hydrogen peroxide solution and place it in a 290℃ digestion furnace again. Observe whether the solution in the digestion tube becomes clear. After the solution becomes clear, take it out, cool it to room temperature, and then adjust 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 4 (as shown in H in ).

[0081] The gel consistency (GC) was determined according to the National Standard for Quality of Edible Rice Varieties NT / T593-2021. All data are based on three biological replicates, and the values ​​are means with the standard error of the mean (SEM). Different lowercase letters indicate significant differences (P < 0.05). Statistical differences were tested by one-way ANOVA (e.g. Figure 4 1 in FIG).

[0082] 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 of 20-30℃ for 90 minutes. A rice taste meter (STA / A, Satake, Japan) is used to determine the hardness, viscosity, balance and comprehensive score of the rice. After the test on day 0, the sample was placed in a refrigerator at 4℃ for low temperature. The samples were taken out after 1 day and 7 days respectively, and the taste value of the rice after storage for different time periods (such as hardness, viscosity and chewiness) was tested. Figure 4 (as shown in JL in ).

[0083] In summary, after the OsC2H2 gene was knocked out, the mutant had no significant difference in plant morphology from the wild type except for a slight decrease in 1000-grain weight; although the knockout of the OsC2H2 gene increased the chalkiness and chalky grain rate of rice, it significantly reduced the amylose content of rice, and the hardness and chewiness of the mature endosperm of the mutant were significantly reduced, while the stickiness was significantly increased, indicating that the knockout of the OsC2H2 gene has better taste quality, and this gene has great application prospects in improving the taste quality of rice.

[0084] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of OsC2H2 gene or OsC2H2 gene knockout biomaterial in regulating rice grain nutritional indexes and taste quality, characterized in that: The nutritional indicator is amylose content; the taste quality is hardness, chewiness and / or adhesion; the nucleotide sequence of the OsC2H2 gene is shown in SEQ ID No. 1; The regulation is to achieve the effect of reducing the amylose content, hardness and chewiness of rice grains and improving adhesion by knocking out or inhibiting the expression of the OsC2H2 gene.

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

3. Use of the OsC2H2 gene or a biomaterial with the OsC2H2 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 adhesive transgenic rice; The nucleotide sequence of the OsC2H2 gene is shown in SEQ ID No. 1; the cultivation is to achieve the effect of reducing the amylose content, hardness and chewiness of rice grains and improving adhesion by knocking out or inhibiting the expression of the OsC2H2 gene.

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

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