Application of OsB3 gene in regulation and control of nutritional indexes, cooking quality and taste quality of rice grains

Abstract

The invention relates to the technical field of biotechnology and plant genetic engineering, in particular to application of an OsB3 gene in regulation and control of nutritional indexes, cooking quality and taste quality of rice grains. According to the invention, the OsB3 gene for regulating and controlling the yield and quality of rice is cloned through reverse genetics, and the rice OsB3 gene provided by the invention can be used as a related gene for creating soft rice; the method is especially used for regulating and controlling rice character indexes (including important nutritional indexes of rice: total starch content and amylose content), rice cooking quality (alkali elimination value and gel consistency) and taste quality (hardness and adhesion). The result of the specific embodiment of the invention shows that the chalkiness and chalkiness grain rate of the knockout strain are obviously lower than those of a wild type when the OsB3 gene is knocked out under the background of Nipponbare, the total starch, amylose, gel consistency and hardness are obviously lower than those of the wild type, and the alkali elimination value and adhesion are obviously higher than those of the wild type.

Description

Technical Field

[0001] The invention relates to the technical fields of biotechnology and plant genetic engineering, and in particular to the application of OsB3 gene in regulating rice grain nutritional indexes, cooking quality and taste quality. Background Art

[0002] Rice is an important food crop worldwide and one of the main sources of energy for humans. However, with the growth of population and the improvement of living standards, people have higher and higher requirements for rice quality. Although traditional rice breeding methods have achieved certain results in improving yield and stress resistance, they have certain limitations in rapidly improving rice quality.

[0003] Rice quality mainly includes appearance quality, taste quality and nutritional quality. Appearance quality such as grain shape, transparency, whiteness, etc. directly affects consumers' willingness to buy; taste quality such as taste, stickiness, elasticity, etc. determines the eating experience of rice; and nutritional quality such as protein content, vitamin content and trace element content are related to human health (Gong et al., Genetic improvements in rice grain quality: A review of elite genes and their applications in molecular breeding. Agronomy. 2023; 13 (5): 1375.). Soft rice refers to rice grains that can maintain good stickiness and soft taste during cooking, which is suitable for making rice balls, sushi and other foods (Marie et al., Rice quality: How is it defined by consumers, industry, food scientists, and geneticists? Trends in Food Science & Technology, 2019, 92: 122-137). Due to its unique taste and excellent processing characteristics, soft rice has a wide application market in the food industry. In recent years, with the increasing demand of consumers for diversified food taste, the demand for soft rice has gradually increased, so the development of new soft rice varieties has become an important research direction in rice breeding. Traditional soft rice varieties are mainly created through traditional hybrid breeding methods. Although this method can obtain some soft rice varieties, it usually has a long cycle and high cost, and may face the challenge of uncertainty in the hybrid breeding process. In addition, traditional breeding methods are often difficult to effectively improve rice yield and disease and pest resistance. Therefore, how to achieve efficient creation of soft rice without affecting other economic traits has become a difficult problem in rice breeding research.

[0004] In recent years, with the rapid development of genomics and molecular biology, gene editing technology has gradually become an important means of plant breeding. Therefore, it is of great theoretical significance and application value to study how to quickly create rice soft rice characteristics through rice genes and develop new varieties of soft rice with excellent quality, high yield and strong adaptability by combining modern gene editing technology. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the OsB3 gene in regulating rice grain nutritional indicators, cooking quality and taste quality, so as to solve the problems existing in the above-mentioned prior art. The OsB3 gene was knocked out in the Nipponbare background. Compared with the wild type, the chalkiness and chalky grain rate of the knockout strain were significantly reduced, the total starch, amylose, gel consistency and hardness were significantly lower than those of the wild type, and the alkali consumption value and adhesion were significantly higher than those of the wild type.

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

[0007] The present invention provides an application of an OsB3 gene, an OsB3 protein encoded by the OsB3 gene, or a biological material with an OsB3 gene knocked out in regulating nutritional indices, cooking quality, and eating quality of rice grains. The nutritional indices include total starch content and / or amylose content; the cooking quality includes alkali consumption value and / or gel consistency; the eating quality includes hardness and / or adhesion; the nucleotide sequence of the OsB3 gene is shown in SEQ ID NO.1; and the amino acid sequence of the OsB3 protein is shown in SEQ ID NO.2.

[0008] Preferably, the method comprises inhibiting the expression of the OsB3 gene in rice to reduce the total starch content, amylose content, gel consistency and hardness, and improve the alkali elimination value and adhesion.

[0009] Further preferably, the biological material includes a recombinant vector or a recombinant bacterium.

[0010] The present invention provides an application of an OsB3 gene, an OsB3 protein encoded by the OsB3 gene or a biological material with the OsB3 gene knocked out in regulating the chalkiness and chalky grain rate of rice. The nucleotide sequence of the OsB3 gene is shown in SEQ ID NO.1; the amino acid sequence of the OsB3 protein is shown in SEQ ID NO.2.

[0011] Preferably, the method comprises inhibiting the expression of the OsB3 gene in rice to achieve the effect of reducing the chalkiness and chalky grain rate of rice grains.

[0012] Further preferably, the biological material includes a recombinant vector or a recombinant bacterium.

[0013] The present invention provides the use of an OsB3 gene, an OsB3 protein encoded by the OsB3 gene, or a biological material with the OsB3 gene knocked out in cultivating transgenic rice of any of the following:

[0014] (1) Transgenic rice with low total starch content;

[0015] (2) Transgenic rice with low amylose content;

[0016] (3) Transgenic rice with high alkalinity value;

[0017] (4) Transgenic rice with low gel consistency;

[0018] (5) Transgenic rice with low hardness;

[0019] (6) Highly adherent transgenic rice;

[0020] (7) Transgenic rice with low chalkiness and chalky grain rate;

[0021] The nucleotide sequence of the OsB3 gene is shown in SEQ ID NO.1; the amino acid sequence of the OsB3 protein is shown in SEQ ID NO.2.

[0022] Further preferably, the biological material includes a recombinant vector or a recombinant bacterium.

[0023] The present invention provides an OsB3 gene mutant, the amino acid sequence of the OsB3 gene mutant is shown as SEQ ID NO.7 or SEQ ID NO.8.

[0024] The present invention provides a protein encoded by the above-mentioned OsB3 gene mutant, the amino acid sequence of the protein is shown in SEQ ID NO.9 or SEQ ID NO.10.

[0025] The present invention provides the use of the above-mentioned OsB3 gene mutant or the above-mentioned protein in regulating the nutritional indexes, cooking quality and taste quality of rice grains, wherein the nutritional indexes include total starch content and / or amylose content; the cooking quality includes alkali consumption value and / or gel consistency; and the taste quality includes hardness and / or adhesion.

[0026] The present invention provides the use of the above-mentioned OsB3 gene mutant or the above-mentioned protein in regulating the chalkiness and chalky grain rate of rice grains.

[0027] The present invention provides the use of the above-mentioned OsB3 gene mutant or the above-mentioned protein in cultivating any of the following transgenic rice:

[0028] (1) Transgenic rice with low total starch content;

[0029] (2) Transgenic rice with low amylose content;

[0030] (3) Transgenic rice with high alkalinity value;

[0031] (4) Transgenic rice with low gel consistency;

[0032] (5) Transgenic rice with low hardness;

[0033] (6) Highly adherent transgenic rice;

[0034] (7) Transgenic rice with low chalkiness and chalky grain rate.

[0035] As an additional solution, the present invention provides the use of OsB3 gene, OsB3 protein encoded by OsB3 gene or biological material with OsB3 gene knocked out in creating soft rice germplasm.

[0036] The invention provides a method for creating soft rice germplasm, comprising the step of inhibiting the expression of the OsB3 gene in rice; the nucleotide sequence of the OsB3 gene is shown as SEQ ID NO.1.

[0037] The present invention provides the use of the above-mentioned OsB3 gene mutant or the above-mentioned protein in creating soft rice germplasm.

[0038] The present invention discloses the following technical effects:

[0039] The present invention clones an OsB3 gene that regulates rice yield and quality through reverse genetics. The rice OsB3 gene provided by the present invention can be used as a related gene for creating soft rice, especially for regulating rice trait indicators (including important rice nutritional indicators: total starch content, amylose content), rice cooking quality (alkali consumption value and gel consistency) and taste quality (hardness and adhesion). The results of the specific embodiments of the present invention show that when the OsB3 gene is knocked out under the background of Nipponbare, compared with the wild type, the chalkiness and chalky grain rate of the knocked-out strain are significantly lower than those of the wild type, the total starch, amylose, gel consistency and hardness are significantly lower than those of the wild type, and the alkali consumption value and adhesion are significantly higher than those of the wild type. The present invention finds that after knocking out the OsB3 gene, under the premise of not changing the appearance quality, the hardness of rice is reduced, the adhesion is increased, and the rice cooking and taste quality is improved, which has important breeding utilization value. The rice OsB3 gene of the present invention provides an important gene resource and theoretical basis for the genetic improvement of nutrition and rice cooking and taste quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 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.

[0041] Figure 1 This is a diagram for site prediction and tissue expression analysis of the OsB3 gene;

[0042] Figure 2 This is a map of the CRISPR-Cas9 knockout vector containing the OsB3 gene target site sequence;

[0043] Figure 3 Schematic diagram of editing the OsB3 gene in the Nipponbare background;

[0044] Figure 4 The figures are the analysis diagrams of the phenotype and appearance characteristics of the polished rice of the wild type and the mutant osb3; wherein A is the appearance pictures of rice of different strains; B is the statistical bar graph of the chalkiness of different strains; C is the statistical bar graph of the chalkiness grain rate of different strains;

[0045] Figure 5 The figures are analysis charts of physical and chemical indexes of wild-type and mutant osb3 rice; A is a statistical bar graph of total starch content of different strains; B is a statistical bar graph of amylose content of different strains; C is a statistical bar graph of alkali consumption value of different strains; D is a statistical bar graph of gel consistency of different strains; E is a statistical bar graph of hardness of different strains; and F is a statistical bar graph of adhesion of different strains. DETAILED DESCRIPTION

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] The sequence information of the OsB3 gene and the protein encoded by the OsB3 gene provided by the present invention is as follows:

[0052] The nucleotide sequence of the OsB3 gene is shown in SEQ ID NO.1, specifically:

[0053] ;

[0054] The amino acid sequence of the protein encoded by the OsB3 gene (OsB3 protein) is shown in SEQ ID NO.2, specifically:

[0055] MELDTDPTKLKAKPIIKPKVEPCDDDDELPPPPPPASGSGEDWEATTPLAAGNPFFTALI AKSHLHPKFQMWIPPRFQHRLAEPEARTAAVLHSGGKSWATSYCGHLKMKKLDAGWSEFA VDNRLLVGDACVFELVAMGAAGGLEFQVQILRGGLPAEVVTSKGLTSDQPILIVD*.

[0056] Example 1 Analysis of predicted tissue expression patterns of rice OsB3 gene

[0057] The RAP-DB website (https: / / rapdb.dna.affrc.go.jp) was used to input the RAP_Locus number of the OsB3 gene, Os06g0112300, and the tissue expression pattern analysis was performed. The prediction results showed that the OsB3 gene was highly expressed 3-5 days after rice flowering ( Figure 1 ), indicating that the OsB3 gene may be closely related to rice seed development.

[0058] Example 2 Construction of OsB3 gene knockout vector in rice

[0059] Selection of gRNA target sequence: According to CRISPR / Cas9 related experimental methods, a sequence containing NGG as a recognition site was selected as the knockout target site on the exon of the OsB3 gene. The specific nucleotide sequence is shown in SEQ ID NO.11, specifically: 5′-ACTGATCCTACCAAGCTGAAGG-3′, and the PAM sequence is AGG.

[0060] Design of upstream and downstream primers of gRNA oligonucleotide chain

[0061] The upstream primer is OsB3_gRNA_F: 5′-GGACACTGATCCTACCAAGCTGA-3′, SEQ ID NO. 3;

[0062] The downstream primer is OsB3_gRNA_F: 5′-ACTGATCCTACCAAGCTGAAGGCCAAGC-3′, SEQ ID NO.4;

[0063] CRISPR / Cas9 vector construction: This example uses a plant Cas9 / gRNA plasmid construction kit (Catalog. No. BGK03, Figure 2 As shown), the target sequence is loaded to form a recombinant vector containing the OsB3 gene target site. The specific operation method is as follows:

[0064] Preparation of oligo dimers: Take 10 μM of the target site front and back primers (the upstream primer is OsB3_gRNA_F and the downstream primer is OsB3_gRNA_R) and add 18 μL of BufferAneal, mix well, treat at 95°C for 3 minutes, and then slowly reduce to 20°C at about 0.2°C / second to obtain a double-stranded sequence containing the knockout target site;

[0065] Construct oligo dimer into CRISPR / Cas9: Take 2 μL of Cas9 / gRNA vector, 1 μL of oligo dimer from step 1, 1 μL of Enzyme Mix, add 6 μL of ddH2O, mix well and react in a 20℃ metal bath for 1 hour;

[0066] E. coli transformation: 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 min, heat shock at 42°C for 45 s, let stand on ice for 2 min, then add 200 μL of antibiotic-free LB medium, place in a 37°C constant temperature shaker at 200 rpm, and recover for 1 h before applying kanamycin resistance (Kana + )

[0067] PCR detection of bacterial liquid: 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 liquid became turbid. Sequencing was performed using the BGK03 vector-specific sequencing primers provided in the kit; the sequencing results were analyzed using Snapgene software, and the plasmid of the positive clone bacterial liquid was extracted for later use.

[0068] Agrobacterium transformation and rice genetic transformation: The successfully constructed plasmid was transferred into Agrobacterium (EHA105): 1 μL of plasmid was injected into the frozen and thawed Agrobacterium competent cells on ice, and then placed on ice for 5 minutes, liquid nitrogen for 5 minutes, and a 37°C water bath for 5 minutes. 300 μL of antibiotic-free LB medium was added and revived at 28°C for 4 hours, and evenly applied to (kanamycin + rifampicin) K + / Rif-resistant plates were plated and cultured at 28°C for 2-3 days before picking single clones. Positive clones were detected with hygromycin primers and expanded with 3 mL of liquid K + / Rif medium, further positive K + / Rif bacterial liquid was sent to the company for genetic transformation of rice under the Nipponbare background, and a transgenic strain with OsB3 gene knocked out was obtained.

[0069] Example 3 Obtaining a homozygous mutant of rice OsB3 gene

[0070] In order to identify the transgenic strain with knockout of OsB3 gene obtained above, the transgenic seedlings with knockout of OsB3 gene were cultured in a normal temperature light incubator for about 1 week, and then positive seedlings were identified. The specific steps are as follows:

[0071] 20 T0 generation transgenic seedlings were obtained, placed in a normal temperature light incubator for culture for about one week, and then DNA of the 20 seedlings was taken, OsB3cas9TF (5′-TGTGTCACTGATCCTACCAAGCTGA-3′, SEQ ID NO.5) and OsB3cas9TR (5′-AAACTCAGCTTGGTAGGATCAGTGA-3′, SEQ ID NO.6) were used for PCR amplification and then sent to a company for sequencing, and the obtained sequencing results were analyzed to obtain transgenic plants with premature termination of two protein translations. The nucleotide sequence of the OsB3 gene mutant in the two homozygous knockouts of OsB3 is shown in SEQ ID NO.7 or SEQ ID NO.8, and the protein encoded by the mutant is shown in SEQ ID NO.9 or SEQ ID NO.10.

[0072] The nucleotide sequence of OsB3 gene mutant 1 is shown in SEQ ID NO.7, specifically:

[0073] ;

[0074] The nucleotide sequence of OsB3 gene mutant 2 is shown in SEQ ID NO.8, specifically:

[0075] ATGGAGCTGGACACTGATCCTGAAGGCCAAGCCCATCATCAAACCAAAAGTAGAACCCTGCGACGACGATGACGAGTTGCCGCCGCCGCCGCCGCCGGCTTCAGGATCCGGCGAGGATTGGGAGGCCACC ACCCCTCTCGCCGCCGGCAACCCCTTCTTCACCGCCCTCATCGCCAAGTCTCATCTCCACCCCAAGTTCCAGATGTGGATTCCACCTCGGTTCCAGCATCGGCTGGCGGAGCCGGAGGCGCGCACGGCGG CGGTGCTCCACTCCGGCGGCAAGTCGTGGGCGACGAGCTACTGCGGCCACCTCAAGATGAAGAAGCTGGACGCGGGATGGTCGGAGTTCGCGGTGGACAACCGGCTCCTGGTCGGGGACGCCTGCGTCTT CGAGCTCGTCGCCATGGGCGCCGCCGGAGGTCTGGAGTTCCAGGTGCAGATACTCCGCGGCGGCCTGCCGGCGGAGGTCGTCACCTCCAAGGGCCTCACCTCCGACCAACCCATCCTCATCGTCGACTAG;

[0076] The amino acid sequence of the protein encoded by the OsB3 gene mutant 1 is shown in SEQ ID NO.9, specifically:

[0077] MELDTDPTKREGQAHHQTKSRTLRRR*.

[0078] The amino acid sequence of the protein encoded by OsB3 gene mutant 2 is shown in SEQ ID NO.10, specifically:

[0079] MELDTDPEGQAHHQTKSRTLRRR*.

[0080] After obtaining the stable genetic knockout strain of OsB3 gene in the T2 generation, the target site was further sequenced to obtain the homozygous mutants osb3-1 and osbc-2 ( Figure 3 ), and harvest the mature rice seeds.

[0081] Example 4 Rice Quality Identification of OsB3 Gene Knockout Strains

[0082] Wild-type Nipponbare and T2 homozygous mutant lines osb3-1 and osb3-2 with OsB3 knockout were cultivated in the field with conventional sunlight, water and fertilizer management, and mature seeds were harvested for the following experiments.

[0083] Rice appearance inspection:

[0084] The brown rice of wild-type Nipponbare and knockout homozygous mutants (osb3-1 and osb3-2) was observed. 100 mature seeds were randomly selected, and the husks were removed. The chalkiness and chalky grain rate were counted. The results are as follows: Figure 4 The results showed that the chalkiness of the seeds and the chalky grain rate of the knockout homozygous mutant strain were significantly lower than those of the wild-type Nipponbare.

[0085] Rice quality inspection:

[0086] 1. Determination of total starch content: The mature seeds of wild-type Nipponbare and knockout homozygous mutants osb3-1 and osb3-2 were shelled and ground into rice flour. The rice flour was carefully sieved into a clean ziplock bag using a 100-mesh sieve for later use. 50 mg of each sample was weighed, three replicates were set, and placed in a 50 mL centrifuge tube. 5 mL of 80% anhydrous ethanol was gently added to the centrifuge tube along the tube wall, and it was placed in an 85°C water bath for 5 minutes. 5 mL of 80% anhydrous ethanol was added again, and then centrifuged at a speed of 4000g for 10 minutes. The supernatant was carefully sucked off with a gun, and 10 mL of 80% anhydrous ethanol was added to the precipitate. It was gently mixed, and further centrifuged at 4000g for 10 minutes at room temperature. Then, the centrifuged 50 mL centrifuge tube was quickly turned upside down on the filter paper to naturally air-dry the residual ethanol inside. Add 1 mL of ddH2O to the prepared sample and dissolve it, and boil it in a boiling water bath for 30 min; cool it to room temperature, add 4 mL of 2M KOH, and shake it at room temperature for 30 min (to prevent caking); add 16 mL of sodium acetate (1.2 M, pH = 3.8) and 200 μL of amyloglucosidase (3000 U / mL), and bathe it in a 60°C water bath for 45 min, shaking it 2 to 3 times during the period, and finally adjust the volume to 100 mL and mix gently.

[0087] GOD-PAP was used to determine the glucose content. 1 mL of the above treated sample was placed in a 1.5 mL centrifuge tube, centrifuged at 4000 rpm for 10 min, 100 μL of the supernatant was placed in a 10 mL test tube or a 10 mL centrifuge tube, 3 mL of GOD-PAP (taken out in advance, wrapped in tin foil, and placed on ice to melt) was added, and the tube was placed in a 37°C constant temperature water bath for 20 min, inverted, and fully mixed; the absorbance was measured at a wavelength of 510 nm using an ELISA reader (Infinite 200PRO, TECAN, Switzerland), and the blank was zeroed using the reaction solution treated with 100 μL of 0.1 M sodium acetate (pH 4.75) and 3 mL of GOD-PAP reagent under the same conditions. The remaining gradient glucose solution standard sample reaction solution is shown in Table 1. All samples must be determined within 60 min.

[0088] Table 1 Gradient glucose solution standard sample reaction solution

[0089] Standard samples 0 20μL 40μL 60μL 80μL 100μL 0.1M sodium acetate buffer solution (PH = 4.75) 100μL 80μL 60μL 40μL 20μL 0 GOD-PAP 3mL 3mL 3mL 3mL 3mL 3mL

[0090] According to the absorbance value of the standard sample measured in the microplate reader and the known concentration of the standard sample, a standard curve and a regression equation were established. Then, the absorbance value of each sample was substituted into the regression equation to obtain the corresponding glucose content, and finally converted into the total starch content (total starch content equals 0.9 times the glucose content, and the sample water content is calculated as 12%). Each sample was repeated three times, and the average value was finally used as the total starch content. The results are as follows: Figure 5 A in Figure 1. The results showed that the total starch content of the knockout homozygous mutant was significantly lower than that of the wild type.

[0091] 2. Determination of amylose content: The determination of amylose requires anhydrous ethanol, 1M NaOH, 1M acetic acid solution and KI-I2 (2g I2+20g KI+1000mL ddH2O). Shell the mature seeds of the material to be determined, grind them into rice flour, sieve them with a 100-mesh sieve, weigh 50mg of rice flour and standard sample into a 50mL volumetric flask (3 replicates per group), and gently shake the volumetric flask to shake the rice flour attached to the wall of the volumetric flask to the bottom of the flask.

[0092] Add 500 μL of anhydrous ethanol to a 50 mL volumetric flask, gently rotate the 50 mL volumetric flask so that the rice flour can be fully mixed with anhydrous ethanol; then add 4.5 mL of 1 M NaOH solution, rotate the bottle while adding, and wash the rice flour attached to the inner wall to the bottom of the bottle, and let it stand at room temperature for 24 hours overnight; add ddH2O to 50 mL, shake gently to mix; draw 5 mL of ddH2O into a 10 mL test tube, and then add 500 μL of blank, standard and sample to be tested (before drawing, do not use the first two shots, rinse the gun tip to reduce experimental errors), add 100 μL of 1 M acetic acid solution, 200 μL of KI-I2 and 4.2 mL of ddH2O to the test tube, use a vortex oscillator to shake the solution thoroughly, and let it stand for 20 minutes. Use an enzyme marker (Infinite 200PRO, TECAN, Switzerland) to measure the absorbance value OD of each sample at a wavelength of 620 nm. 620 Before use, adjust to zero with blank reaction solution; draw a standard curve (standard sample concentration 1.5%, 10.4%, 16.2% and 26.5%), and calculate the amylose content of each sample based on the prepared standard curve. The results are as follows: Figure 5 As shown in B. The results showed that the amylose content of the knockout homozygous mutant was significantly lower than that of the wild type.

[0093] 3. Determination of alkali elimination value: Select 10 full grains of polished rice and place them in a square box. Add 10 mL of 1.70% potassium hydroxide solution, cover the box, and place it in a 30°C constant temperature box for about 23 hours. Repeat 3 times. Observe the decomposition of the rice endosperm and record the classification according to the national edible rice variety quality standard NT / T593-2021. The results are as follows: Figure 5 As shown in C. The results showed that the alkali elimination value of the knockout homozygous mutant was significantly higher than that of the wild type.

[0094] 4. Determination of gel consistency: The determination of gel consistency was carried out according to GB / T 22294-2008 Grain and Oil Inspection Rice Gel Consistency, and the specific steps were as follows: (1) weigh 100 mg of sieved (120 mesh) refined rice flour into a 10 cm test tube; (2) add 0.2 mL of 95% ethanol, vortex and then add 2 mL 0.2mol / LKOH solution, vortex and oscillate again to mix; (3) Place the test tube in a boiling water bath, cover the mouth of the test tube with a glass marble, keep gelatinizing for 8 minutes, and adjust the depth of the test tube in the boiling water so that the elongation length of the boiling rice noodles does not exceed 2 / 3 of the length of the test tube; (4) After the gelatinization of the rice noodles is completed, take out the test tube and cool it at room temperature for 5 minutes; (5) After keeping the test tube in an ice water bath for 20 minutes, place it flat on a water platform in a 25℃ incubator and let it stand for 1 hour; (6) Use a ruler to measure the length from the bottom of the test tube to the highest point of the rice gelatin, which is recorded as the gelatin consistency of the rice noodles (mm). Set up 3 biological replicates for each sample and take the average value. The results are shown in the figure. Figure 5As shown in D. The results showed that the gel consistency of the knockout homozygous mutant was significantly lower than that of the wild type.

[0095] 5. Rice hardness test: Refer to the operation procedure of Shanghai Baosheng TA.XTC-18 texture analyzer to complete the hardness test of wild type and mutant. The results are as follows: Figure 5 As shown in E. The results showed that the hardness of the knockout homozygous mutant rice was significantly lower than that of the wild type.

[0096] 6. Adhesion determination: Weigh 3 g of rice flour of wild-type Nipponbare and knockout homozygous mutants (osb3-1 and osb3-2), add 25 mL of distilled water, and perform the adhesion determination of rice flour according to the operating procedures of TechMaster RVA rapid viscosity analyzer (Perten, Swiss). Repeat 3 times. The results are as follows: Figure 5 As shown in F. The results showed that the rice flour adhesion of the knockout homozygous mutant was significantly higher than that of the wild type.

[0097] 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 OsB3 gene, OsB3 protein encoded by OsB3 gene or biological material with knockout of OsB3 gene in regulating rice grain nutritional index, cooking quality and eating quality, characterized in that: The nutritional indicators include total starch content and / or amylose content; the cooking quality includes alkali consumption value and / or gel consistency; the taste quality includes hardness and / or adhesion; the nucleotide sequence of the OsB3 gene is shown in SEQ ID NO.1; and the amino acid sequence of the OsB3 protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The method comprises inhibiting the expression of the OsB3 gene in rice, thereby achieving the effects of reducing the total starch content, the straight-chain starch content, the gel consistency and the hardness, and improving the alkali elimination value and the adhesion.

3. Use of OsB3 gene, OsB3 protein encoded by OsB3 gene or biological material with knockout of OsB3 gene in regulating rice grain chalkiness and chalky grain rate, characterized in that: The nucleotide sequence of the OsB3 gene is shown in SEQ ID NO.1; the amino acid sequence of the OsB3 protein is shown in SEQ ID NO.

2.

4. The use according to claim 3, characterized in that: The method comprises inhibiting the expression of the OsB3 gene in rice to achieve the effect of reducing the chalkiness of rice grains and the chalky grain rate.

5. Use of OsB3 gene, OsB3 protein encoded by OsB3 gene or biological material with OsB3 gene knocked out in cultivating transgenic rice of any of the following: (1) Transgenic rice with low total starch content; (2) Transgenic rice with low amylose content; (3) Transgenic rice with high alkalinity value; (4) Transgenic rice with low gel consistency; (5) Transgenic rice with low hardness; (6) Highly adherent transgenic rice; (7) Transgenic rice with low chalkiness and chalky grain rate; The nucleotide sequence of the OsB3 gene is shown in SEQ ID NO.1; the amino acid sequence of the OsB3 protein is shown in SEQ ID NO.

2.

6. An OsB3 gene mutant, characterized in that: The amino acid sequence of the OsB3 gene mutant is shown in SEQ ID NO.7 or SEQ ID NO.

8.

7. The protein encoded by the OsB3 gene mutant according to claim 6, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.9 or SEQ ID NO.

10.

8. Use of the OsB3 gene mutant according to claim 6 or the protein according to claim 7 in regulating rice grain nutritional indexes, cooking quality and taste quality, characterized in that: The nutritional index includes total starch content and / or amylose content; the cooking quality includes alkali elimination value and / or gel consistency; and the taste quality includes hardness and / or adhesion.

9. Use of the OsB3 gene mutant according to claim 6 or the protein according to claim 7 in regulating the chalkiness and chalky grain rate of rice grains.

10. Use of the OsB3 gene mutant according to claim 6 or the protein according to claim 7 in cultivating any of the following transgenic rice: (1) Transgenic rice with low total starch content; (2) Transgenic rice with low amylose content; (3) Transgenic rice with high alkalinity value; (4) Transgenic rice with low gel consistency; (5) Transgenic rice with low hardness; (6) Highly adherent transgenic rice; (7) Transgenic rice with low chalkiness and chalky grain rate.

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