Application of OsB3 gene in regulating rice grain nutrition index, cooking quality and eating quality

Abstract

The present application relates to the field of biotechnology and plant genetic engineering technology, and particularly relates to application of OsB3 gene in regulating rice grain nutrition index, cooking quality and eating quality. The present application clones an OsB3 gene for regulating rice yield and quality through reverse genetics, and the rice OsB3 gene provided by the present application can be used as a related gene for creating soft rice, and is particularly used for regulating rice trait index (including important rice nutrition index: total starch content, amylose content), rice cooking quality (alkali-soluble value and gel consistency) and eating quality (hardness and stickiness). The results of specific embodiments of the present application show that, in the Nipponbare background, the chalkiness and chalky grain rate of the knockout strain of the OsB3 gene 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-soluble value and stickiness are significantly higher than those of the wild type.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and plant genetic engineering technology, in particular to the application of OsB3 gene in regulating rice grain nutritional indicators, cooking quality and eating quality. BACKGROUND

[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 improvement of living standards, people have increasingly high demands for rice quality. Although traditional rice breeding methods have achieved certain success in improving yield and stress resistance, they have certain limitations in rapidly improving rice quality.

[0003] Rice quality mainly includes appearance quality, eating quality and nutritional quality. Appearance quality such as grain shape, transparency and whiteness directly affects consumers' willingness to purchase; eating quality such as taste, stickiness and elasticity determines the eating experience of rice; and nutritional quality such as protein content, vitamin content and trace element content is 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, 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 taste of food, the demand for soft rice has gradually increased, so developing new soft rice varieties has become an important research direction in rice breeding. Traditional soft rice varieties are mainly created by traditional hybrid breeding methods. Although this method can obtain some soft rice varieties, it usually has a long cycle, high cost and may face the challenge of uncertainty in the hybrid breeding process. In addition, traditional breeding methods are also often difficult to effectively improve the yield and disease resistance of rice, 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 technology, gene editing technology has gradually become an important means of plant breeding. Therefore, it has important theoretical significance and application value to study how to realize the rapid creation of soft rice characteristics of rice genes, and to develop new soft rice varieties with excellent quality, high yield and strong adaptability by combining modern gene editing technology. SUMMARY

[0005] The purpose of the present application is to provide the application of OsB3 gene in regulating the nutritional index, cooking quality and eating quality of rice grains, in order to solve the problems existing in the prior art. Compared with the wild type, the chalkiness and chalky grain rate of the knockout strain are significantly reduced, the total starch, amylose, gel consistency and hardness are significantly lower than the wild type, and the alkali value and adhesion are significantly higher than the wild type.

[0006] In order to achieve the above purpose, the present application provides the following scheme:

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

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

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

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

[0011] Preferably, the expression of the OsB3 gene is inhibited in rice to reduce 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 application provides application of an OsB3 gene, an OsB3 protein coded by the OsB3 gene or a biological material knocking out the OsB3 gene in cultivating transgenic rice in any one of the following aspects:

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

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

[0016] (3) transgenic rice with high alkali value;

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

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

[0019] (6) transgenic rice with high adhesiveness;

[0020] (7) transgenic rice with low chalkiness and chalky kernel rate;

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

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

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

[0024] The application provides a protein coded by the OsB3 gene mutant, and the amino acid sequence of the protein is shown as SEQ ID NO. 9 or SEQ ID NO. 10.

[0025] The application provides application of the OsB3 gene mutant or the protein in regulating nutritional indexes, cooking quality and eating quality of rice grains, wherein the nutritional indexes include total starch content and / or amylose content; the cooking quality includes alkali value and / or gel consistency; and the eating quality includes hardness and / or adhesiveness.

[0026] The application provides application of the OsB3 gene mutant or the protein in regulating chalkiness and chalky kernel rate of rice grains.

[0027] The application provides application of the OsB3 gene mutant or the protein in cultivating transgenic rice in any one of the following aspects:

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

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

[0030] (3) transgenic rice with high alkali value;

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

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

[0033] (6) transgenic rice with high adhesiveness;

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

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

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

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

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

[0039] The present application clones an OsB3 gene for regulating rice yield and quality through reverse genetics, and the rice OsB3 gene provided by the present application can be used as a related gene for creating soft rice, and is especially used for regulating rice trait indexes (including important nutritional indexes of rice: total starch content, amylose content), rice cooking quality (alkali value and gel consistency), and eating quality (hardness and adhesiveness). The results of the specific embodiments of the present application show that, in the Nipponbare background, the chalkiness and chalky grain rate of the knockout strain of the OsB3 gene 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 value and adhesiveness are significantly higher than those of the wild type. The present application finds that, after knocking out the OsB3 gene, the hardness of rice is reduced, the adhesiveness is increased, and the cooking and eating quality of rice is improved without changing the appearance quality, which has important breeding utilization value. The rice OsB3 gene of the present application provides important gene resources and theoretical basis for genetic improvement of nutrition and rice cooking and eating quality. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below only illustrate some of the embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative effort.

[0041] Figure 1 Figure for predicting the expression of OsB3 gene website organization analysis;

[0042] Figure 2 Figure for CRISPR-Cas9 knockout vector containing OsB3 gene target site sequence;

[0043] Figure 3 Figure for editing OsB3 gene in Nipponbare background;

[0044] Figure 4 Figure for wild type and mutant osb3 milled rice phenotype and appearance trait analysis; wherein, A is the appearance picture of rice in different strains; B is the statistical column chart of chalkiness degree in different strains; C is the statistical column chart of chalkiness degree in different strains;

[0045] Figure 5 Figure for wild type and mutant osb3 rice physicochemical index determination analysis; wherein, A is the statistical column chart of total starch content in different strains; B is the statistical column chart of amylose content in different strains; C is the statistical column chart of alkali solubility value in different strains; D is the statistical column chart of gel consistency in different strains; E is the statistical column chart of hardness in different strains; F is the statistical column chart of adhesiveness in different strains. DETAILED DESCRIPTION

[0046] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0047] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range, and any other stated value or intermediate value in the range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict between the content of the specification and that of any incorporated literature, the content of the specification controls.

[0049] Many modifications and variations of this application of the application described herein will be apparent to those of ordinary skill in the art without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0050] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation. Accordingly, the use of "including" and "comprising" and variations thereof herein is intended to be broad and encompass the terms "consisting of" and "consisting essentially of" and variations thereof, subject to any limitations specifically indicated.

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

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

[0053] ATGGAGCTGGACACTGATCCTACCAAGCTGAAGGCCAAGCCCATCATCAAACCAAAAGTAGAACCCTGCGACGACGATGACGAGTTGCCGCCGCCGCCGCCGCCGGCTTCAGGATCCGGCGAGGATTGGGAGGCCACCACCCCTCTCGCCGCCGGCAACCCCTTCTTCACCGCCCTCATCGCCAAGTCTCATCTCCACCCCAAGTTCCAGATGTGGATTCCACCTCGGTTCCAGCATCGGCTGGCGGAGCCGGAGGCGCGCACGGCGGCGGTGCTCCACTCCGGCGGCAAGTCGTGGGCGACGAGCTACTGCGGCCACCTCAAGATGAAGAAGCTGGACGCGGGATGGTCGGAGTTCGCGGTGGACAACCGGCTCCTGGTCGGGGACGCCTGCGTCTTCGAGCTCGTCGCCATGGGCGCCGCCGGAGGTCTGGAGTTCCAGGTGCAGATACTCCGCGGCGGCCTGCCGGCGGAGGTCGTCACCTCCAAGGGCCTCACCTCCGACCAACCCATCCTCATCGTCGACTAG;

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

[0055] MELDTDPTKLKAKPIIKPKVEPCDDDDELPPPPPPASGSGEDWEATTPLAAGNPFFTALIAKSHLHPKFQMWIPPRFQHRLAEPEARTAAVLHSGGKSWATSYCGHLKMKKLDAGWSEFAVDNRLLVGDACVFELVAMGAAGGLEFQVQILRGGLPAEVVTSKGLTSDQPILIVD*.

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

[0057] Using the RAP-DB website (https: / / rapdb.dna.affrc.go.jp), the RAP_Locus number Os06g0112300 of the OsB3 gene was inputted to perform tissue expression pattern analysis. The prediction result showed that the OsB3 gene was highly expressed in rice 3-5 days after floweringFigure 1 ), indicating that the OsB3 gene is closely related to the development of rice seeds.

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

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

[0060] Design of upstream and downstream primers on 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] Construction of CRISPR / Cas9 vector: In this embodiment, a plant Cas9 / gRNA plasmid construction kit (Catalog. No. BGK03, as shown) was used. After loading the target sequence, a recombinant vector containing the target site of OsB3 gene was formed. The specific operation method is as follows: Figure 2

[0064] Preparation of oligo dimer: Take 10 μM target site forward and reverse primers (upstream primer is OsB3_gRNA_F and downstream primer is OsB3_gRNA_R) each 1 μL and add 18 μL Buffer Aneal, mix well, then treat at 95℃ for 3 min, then slowly reduce to 20℃ at about 0.2℃ / sec, to obtain a double-stranded sequence containing the knockout target site;

[0065] Construction of oligo dimer into CRISPR / Cas9: Take Cas9 / gRNA vector 2 μL, oligo dimer 1 μL of step one, Enzyme Mix 1 μL, add 6 μL ddH2O, mix well and react at 20℃ metal bath for 1 h;

[0066] ​E. coli transformation: Take the final product of step two 5-10 μL into just thawed 50 μL of DH5a competent cells, mix gently, ice bath for 30 min, 42℃ heat shock for 45 s, ice for 2 min, then add 200 μL of LB medium without antibiotics, placed in a constant temperature shaker at 37℃, 200 rpm, recover for 1 h, then plate on kanamycin resistant (Kana + ) plate;

[0067] Bacterial liquid PCR detection: The next day, single colonies were picked in kanamycin resistant liquid medium, cultured at 37℃ in a shaker until the bacterial liquid became turbid, and sequencing was performed using the BGK03 carrier specific sequencing primer provided in the kit; the sequencing results were analyzed using Snapgene software, and the plasmid of the positive clone bacterial liquid was extracted for standby.

[0068] Agrobacterium transformation and rice genetic transformation: The successfully constructed plasmid was transferred into Agrobacterium (EHA105): 1 μL of the plasmid was taken and added to ice-frozen Agrobacterium competent cells, then placed on ice for 5 min, liquid nitrogen for 5 min, 37℃ water bath for 5 min, and 300 μL of LB medium without antibiotics was added and incubated at 28℃ for 4 h, then evenly coated on (kanamycin + rifampicin) K + / Rif resistant plate, cultured at 28℃ for 2-3 days, then single colonies were picked and detected using the hygromycin primer to obtain positive clones and expand 3 mL of liquid K + / Rif medium, and the positive K + / Rif bacterial liquid was sent to the company for rice genetic transformation in the Nipponbare background, and transgenic lines with OsB3 gene knockout were obtained.

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

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

[0071] Obtain 20 strains of T0 generation transgenic seedlings, place in normal temperature light incubator for about 1 week, take 20 strains of seedlings DNA, use OsB3cas9TF(5'-TGTGTCACTGATCCTACCAAGCTGA-3', SEQ ID NO.5) and OsB3 cas9TR(5'-AAACTCAGCTTGGTAGGATCAGTGA-3', SEQ ID NO.6) for PCR amplification, then send company sequencing, the sequencing results obtained are analyzed, two kinds of protein translation termination are obtained Transgenic plants, the nucleotide sequence of the OsB3 gene mutant in the two homozygotes of the present application is shown in SEQ ID NO.7 or SEQ ID NO.8, and the encoded protein 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] ATGGAGCTGGACACTGATCCTACCAAGCGTGAAGGCCAAGCCCATCATCAAACCAAAAGTAGAACCCTGCGACGACGATGACGAGTTGCCGCCGCCGCCGCCGCCGGCTTCAGGATCCGGCGAGGATTGGGAGGCCACCACCCCTCTCGCCGCCGGCAACCCCTTCTTCACCGCCCTCATCGCCAAGTCTCATCTCCACCCCAAGTTCCAGATGTGGATTCCACCTCGGTTCCAGCATCGGCTGGCGGAGCCGGAGGCGCGCACGGCGGCGGTGCTCCACTCCGGCGGCAAGTCGTGGGCGACGAGCTACTGCGGCCACCTCAAGATGAAGAAGCTGGACGCGGGATGGTCGGAGTTCGCGGTGGACAACCGGCTCCTGGTCGGGGACGCCTGCGTCTTCGAGCTCGTCGCCATGGGCGCCGCCGGAGGTCTGGAGTTCCAGGTGCAGATACTCCGCGGCGGCCTGCCGGCGGAGGTCGTCACCTCCAAGGGCCTCACCTCCGACCAACCCATCCTCATCGTCGACTAG;

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

[0075] ATGGAGCTGGACACTGATCCTGAAGGCCAAGCCCATCATCAAACCAAAAGTAGAACCCTGCGACGACGATGACGAGTTGCCGCCGCCGCCGCCGCCGGCTTCAGGATCCGGCGAGGATTGGGAGGCCACCACCCCTCTCGCCGCCGGCAACCCCTTCTTCACCGCCCTCATCGCCAAGTCTCATCTCCACCCCAAGTTCCAGATGTGGATTCCACCTCGGTTCCAGCATCGGCTGGCGGAGCCGGAGGCGCGCACGGCGGCGGTGCTCCACTCCGGCGGCAAGTCGTGGGCGACGAGCTACTGCGGCCACCTCAAGATGAAGAAGCTGGACGCGGGATGGTCGGAGTTCGCGGTGGACAACCGGCTCCTGGTCGGGGACGCCTGCGTCTTCGAGCTCGTCGCCATGGGCGCCGCCGGAGGTCTGGAGTTCCAGGTGCAGATACTCCGCGGCGGCCTGCCGGCGGAGGTCGTCACCTCCAAGGGCCTCACCTCCGACCAACCCATCCTCATCGTCGACTAG;

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

[0077] MELDTDPTKREGQAHHQTKSRTLRRR*.

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

[0079] MELDTDPEGQAHHQTKSRTLRRR*.

[0080] After obtaining the stable hereditary knockout OsB3 gene T2 generation knockout strain, the target site is further sequenced to obtain homozygous mutant osb3-1 and osbc-2( Figure 3 ), and mature rice seeds are obtained.

[0081] Example 4: Rice quality identification of rice OsB3 gene knockout strain

[0082] Wild type Nipponbare and knockout homozygous mutant lines osb3-1 and osb3-2 were cultivated in the field, and the mature seeds were collected for the following experiments.

[0083] Rice appearance investigation:

[0084] The appearance of the wild type Nipponbare and knockout homozygous mutants (osb3-1 and osb3-2) was observed. 100 mature seeds were randomly selected, and the chalkiness and chalky grain rate were calculated after removing the seed coat. The results are shown in Table 1. Figure 4 The results show that the chalkiness and chalky grain rate of the knockout homozygous mutant lines are significantly lower than those of the wild type Nipponbare.

[0085] Rice quality investigation:

[0086] 1. Total starch content determination: The mature seeds of wild type Nipponbare and knockout homozygous mutants osb3-1 and osb3-2 were dehulled and ground into rice powder. The rice powder was carefully sieved through a 100-mesh screen into a clean ziplock bag for use. 50 mg of each sample was weighed and set up in triplicate in a 50-mL centrifuge tube. 5 mL of 80% anhydrous ethanol was added to the centrifuge tube along the wall, and it was placed in a 85°C water bath for 5 min. Another 5 mL of 80% anhydrous ethanol was added, and then centrifuged at 4000 g for 10 min. The supernatant was carefully aspirated with a gun, and 10 mL of 80% anhydrous ethanol was added to the precipitate and mixed gently. Further centrifugation at 4000 g for 10 min was performed, and then the 50-mL centrifuge tube was inverted on filter paper, and the residual ethanol was naturally air-dried. The prepared sample was dissolved in 1 mL of ddH2O and boiled in a water bath for 30 min. After cooling to room temperature, 4 mL of 2M KOH was added, and the mixture was shaken at room temperature for 30 min (to prevent clumping). 16 mL of sodium acetate (1.2M, pH=3.8) and 200 μL of starch glucosidase (3000 U / mL) were added, and the mixture was water-bathed at 60°C for 45 min, with shaking 2-3 times during the process. Finally, the volume was adjusted to 100 mL, and the mixture was mixed gently.

[0087] GOD-PAP to determine the glucose content, take 1 mL of the above-mentioned sample to 1.5 mL centrifuge tube, 4000 rpm centrifugation for 10 min, take 100 μL supernatant to 10 mL test tube or 10 mL centrifuge tube, add 3 mL GOD-PAP (take out in advance, wrapped with tin foil, placed on ice to melt), 37℃ constant temperature water bath for 20 min, upside down, fully mixed; using enzyme label instrument (Infinite 200PRO, TECAN, Switzerland) at 510 nm wavelength to determine the absorbance value, with 100 μL of 0.1M sodium acetate (pH4.75) and 3mL GOD-PAP reagent under the same conditions to process blank zero, the remaining gradient glucose solution standard sample reaction liquid is shown in Table 1. All samples must be determined within 60 min.

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

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

[0090] According to the standard sample absorbance value measured in the enzyme label instrument and the known concentration of the standard sample, a standard curve and a regression equation are established, then the absorbance value of each sample is substituted into the regression equation to obtain the corresponding glucose content, and finally the total starch content is converted (the total starch content is equal to 0.9 times the glucose content, the sample moisture content is calculated as 12%), each sample is repeated three times, and finally the average value is taken as the total starch content, the results are shown in Figure 5 The results show that the total starch content of the knockout homozygous mutant is significantly lower than that of the wild type.

[0091] 2, Amylose content determination: The determination of amylose requires anhydrous ethanol, 1M NaOH, 1M acetic acid solution and KI-I2 (2g I2+20g KI+1000mL ddH2O). The mature seeds of the material to be determined are shelled, ground into rice powder, sieved with a 100 mesh sieve, 50 mg of the rice powder and standard sample are weighed into a 50 mL volumetric flask (3 replicates per group), the volumetric flask is gently shaken, and the rice powder adhered to the wall of the volumetric flask is shaken to the bottom of the flask.

[0092] Into a 50 mL volumetric flask, 500 μL of absolute ethanol was added, and the 50 mL volumetric flask was gently rotated to facilitate the uniform mixing of the rice powder in the absolute ethanol; 4.5 mL of 1 M NaOH solution was then added while rotating the flask to wash the rice powder adhering to the inner wall into the bottom of the flask, and the flask was left to stand at room temperature overnight for 24 h; ddH2O was added to make up to 50 mL, and the mixture was gently shaken to mix; 5 mL of ddH2O was then taken into a 10 mL test tube, and 500 μL of the blank, standard sample, and sample to be tested (before taking, the first two were not used, and the gun head was rinsed to reduce experimental error) were added; 100 μL of 1 M acetic acid solution, 200 μL of KI-I2, and 4.2 mL of ddH2O were added to the test tube, and the solution was shaken well using a vortex shaker, and left to stand for 20 min. The absorbance OD of each sample was measured at a wavelength of 620 nm using an enzyme marker (Infinite 200PRO, TECAN, Switzerland) 620 , and the blank reaction solution was used to zero before use; a standard curve was drawn (standard sample concentrations of 1.5%, 10.4%, 16.2%, and 26.5%), and the amylose content of each sample was calculated according to the prepared standard curve, and the results are shown as B in Figure 5 . The results showed that the amylose content of the knockout homozygous mutant was significantly lower than that of the wild type.

[0093] 3. Alkali digestion value determination: 10 full and whole milled rice grains were selected and placed in a square box, 10 mL of 1.70% potassium hydroxide solution was added, the lid was covered, and it was placed in a 30°C constant temperature box for about 23 h, with 3 repeats. The decomposition of the endosperm of the rice grains was observed, and the classification record was made according to the national edible rice variety quality NT / T593-2021 standard, and the results are shown as C in Figure 5 . The results showed that the alkali digestion value of the knockout homozygous mutant was significantly higher than that of the wild type.

[0094] 4. Gel consistency determination: The determination of gel consistency was carried out according to GB / T 22294-2008 Grain and Oil Testing Rice Gel Consistency, and the specific steps were as follows: (1) 100 mg of milled rice powder that had been sieved (120 mesh) was weighed into a 10 cm test tube; (2) 0.2 mL of 95% ethanol was added, and after vortex shaking, 2 mL of 0.2 mol / L KOH solution was added, and the mixture was vortexed again; (3) the test tube was placed in a boiling water bath, a glass marble was added to the mouth of the test tube, and the gelatinization was maintained for 8 min, and the depth of the test tube in the boiling water was adjusted so that the elongation length of the boiling rice powder did not exceed 2 / 3 of the length of the test tube; (4) after the gelatinization of the rice powder was completed, the test tube was removed and cooled for 5 min at room temperature; (5) after 20 min in an ice water bath, the test tube was placed flat on the water platform of a 25°C incubator and left to stand for 1 h; (6) the length from the bottom of the test tube to the highest point of the rice gel was measured with a ruler, and recorded as the gel consistency of the rice powder (mm), and 3 biological repeats were set for each sample, and the average value was taken, and the results are shown as Figure 5The results show that the consistency of knock-out homozygous mutant is significantly lower than that of wild type.

[0095] 5. Rice hardness determination: The hardness determination of wild type and mutant was completed according to the operation procedure of Shanghai Baosheng TA.XTC-18 texture meter, and the results are shown in Fig. 5. Figure 5 The results show that the hardness of knock-out homozygous mutant is significantly lower than that of wild type.

[0096] 6. Adhesiveness determination: 3 g of wild type Nipponbare and knock-out homozygous mutant (osb3-1 and osb3-2) rice powder were weighed respectively, 25 mL of distilled water was added, and the adhesiveness determination of rice powder was completed according to the operation procedure of TechMaster RVA rapid viscosity analyzer (Perten, Swiss), 3 times of repetition, and the results are shown in Fig. 6. Figure 5 The results show that the adhesiveness of knock-out homozygous mutant rice powder is significantly higher than that of wild type.

[0097] The above-described embodiments are only to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. The application of biological material knocking out OsB3 gene in regulating the nutritional index, cooking quality and eating quality of rice grain, characterized in that, The nutrition index is total starch content and amylose content; the cooking quality is alkali-soluble value and gel consistency; the eating quality is hardness and adhesiveness; the nucleotide sequence of the OsB3 gene is shown as SEQ ID NO. 1; the amino acid sequence encoded by the OsB3 gene is shown as SEQ ID NO. 2; by knocking out the OsB3 gene in rice, the effects of reducing total starch content, amylose content, gel consistency and hardness, and improving alkali-soluble value and adhesiveness are achieved.

2. Application of biological material knocking out OsB3 gene in regulating the chalkiness and chalky grain rate of rice grains, characterized in that, The nucleotide sequence of the OsB3 gene is shown as SEQ ID NO. 1; the amino acid sequence encoded by the OsB3 gene is shown as SEQ ID NO. 2; by knocking out the OsB3 gene in rice, the effects of reducing the chalkiness of rice grains and the chalky kernel rate are achieved.

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