Application of OsSPL7 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 an OsSPL7 gene in regulating nutritional index, cooking quality and eating quality of rice grains.The present application clones an OsSPL7 gene for regulating rice yield and quality through reverse genetics, and the rice OsSPL7 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 indexes (including important nutritional indexes of rice: total starch content, amylose content), cooking quality (alkali value and gel consistency) and eating quality (hardness and stickiness) of rice.The results of specific embodiments of the present application show that, in comparison with wild type, the total starch content, amylose content, gel consistency and hardness of knockout lines of the OsSPL7 gene in the Nipponbare background are significantly lower than those of the wild type, and the alkali value and stickiness are significantly higher than those of the wild type.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and plant genetic engineering, and in particular to the application of the OsSPL7 gene in regulating the nutritional indicators, cooking quality and palatability of rice grains. Background Technology

[0002] Rice (Oryza sativa L.) is an important food crop worldwide and a staple food in most Asian countries and some other regions. With rising living standards and increasingly higher consumer demands for rice quality, the taste and cooking quality of rice have become crucial factors for consumers when purchasing. Taste and cooking quality directly affect the texture, stickiness, elasticity, and softness of rice after cooking, and these factors, to a certain extent, determine the market competitiveness of rice.

[0003] Traditional rice quality improvement focuses primarily on increasing yield and enhancing disease resistance, while improvements in taste and cooking quality have lagged behind (Haug, W., & Lamberts, A. Rice breeding and quality improvement, Plant Breeding and Biotechnology, 2006, 4(3), 123-129). Taste quality mainly includes sensory indicators such as mouthfeel, aroma, stickiness, and elasticity, while cooking quality involves the cooking time, glutinousness, softness, and persistence of cooked rice (Cruz, CD, & Khush, GSRice breeding: Current status and future prospects, Theoretical and Applied Genetics, 2000, 101(1-2), 179-185). Different rice varieties exhibit significant differences in these quality indicators; therefore, improving rice taste and cooking quality has become an important research direction in rice breeding and processing technology.

[0004] Currently, the improvement of rice's eating taste and cooking quality largely relies on traditional breeding methods, such as hybridization breeding and backcrossing (Zhao et al., Traditional rice breeding and the need for quality improvement, Journal of Agricultural and Food Chemistry, 2019, 67(23), 6385-6394.). While these methods can improve rice's eating taste and cooking quality to some extent, their long breeding cycles and high variability make them difficult to meet the market's demand for rapid improvement. With the development of molecular biology and genomics, new breeding methods such as gene marker-assisted selection and gene editing technology are gradually being applied to rice quality improvement (Peng et al., Integration of genomic tools into rice breeding for quality improvement: New approaches and future directions. Plant Biotechnology Journal, 2022, 20(6), 991-1002.). These technologies, by accurately identifying and regulating genes related to eating taste and cooking quality, can cultivate rice varieties with excellent eating taste and cooking quality in a relatively short time. However, despite the progress made by modern technology in accelerating the improvement of rice eating and cooking quality, some challenges remain in practical application. First, improving rice eating and cooking quality requires the synergistic effect of multiple genes, resulting in a complex genetic background. Changes in a single gene may not significantly improve overall quality (Fujita, et al., Genetic basis of rice eating and cooking quality: From QTLs to genes. Rice, 2015, 8(1), 12.). Second, environmental factors (such as climate and soil type) have a significant impact on rice quality, limiting the universality and stability of improvement schemes (Egli et al., Effects of climate change on rice grain quality. Field Crops Research, 2016, 197, 151-161.). Therefore, identifying key genes controlling rice eating quality and developing rapid and efficient methods to improve rice eating and cooking quality can significantly enhance the eating experience and market competitiveness of rice without increasing production costs and complex operations, which has important theoretical significance and broad practical application value.This method can not only improve consumer acceptance of rice, but also provide strong support for the upgrading of the rice industry and food security. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the OsSPL7 gene in regulating the nutritional indicators, cooking quality, and eating quality of rice grains, thereby solving the problems existing in the prior art. Knocking out the OsSPL7 gene under Nipponbare background conditions, compared with the wild type, resulted in knockout lines with significantly lower total starch content, amylose content, gel consistency, and hardness, and significantly higher alkali spreading value and adhesiveness.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of the OsSPL7 gene, the OsSPL7 protein encoded by the OsSPL7 gene, or biomaterials with the OsSPL7 gene knocked out in regulating the nutritional indicators, cooking quality, and eating quality of rice grains. The nutritional indicators include total starch content and / or amylose content; the cooking quality includes alkali spreading value and / or gel consistency; the eating quality includes hardness and / or adhesiveness; the nucleotide sequence of the OsSPL7 gene is shown in SEQ ID NO.1; and the amino acid sequence of the OsSPL7 protein is shown in SEQ ID NO.2.

[0008] Preferably, this includes inhibiting the expression of the OsSPL7 gene in rice to reduce total starch content, amylose content, gel consistency and hardness, and improve alkali spreading value and adhesiveness.

[0009] Preferably, the biomaterial includes a recombinant vector and recombinant bacteria.

[0010] This invention provides the application of the OsSPL7 gene, the OsSPL7 protein encoded by the OsSPL7 gene, or biological material with the OsSPL7 gene knocked out in the cultivation of transgenic rice in any of the following:

[0011] (1) Genetically modified rice with low total starch content;

[0012] (2) Genetically modified rice with low amylose content;

[0013] (3) Genetically modified rice with high alkali digestibility;

[0014] (4) Genetically modified rice with low gel consistency;

[0015] (5) Low-hardness genetically modified rice;

[0016] (6) Highly adhesive transgenic rice;

[0017] The nucleotide sequence of the OsSPL7 gene is shown in SEQ ID NO.1; the amino acid sequence of the OsSPL7 protein is shown in SEQ ID NO.2.

[0018] Preferably, the method includes the step of inhibiting the expression of the OsSPL7 gene in rice to obtain transgenic rice.

[0019] Preferably, the biomaterial includes a recombinant vector and recombinant bacteria.

[0020] The present invention provides an OsSPL7 gene mutant, the nucleotide sequence of which is shown in SEQ ID NO. 8 or SEQ ID NO. 9.

[0021] The present invention provides a protein encoded by the above-mentioned OsSPL7 gene mutant, the amino acid sequence of which is shown in SEQ ID NO.10 or SEQ ID NO.11.

[0022] This invention provides the application of the above-mentioned OsSPL7 gene mutant or the above-mentioned protein in regulating the nutritional indicators, cooking quality and eating quality of rice grains. The nutritional indicators include total starch content and / or amylose content; the cooking quality includes alkali spreading value and / or gel consistency; and the eating quality includes hardness and / or adhesiveness.

[0023] This invention provides the use of the above-described OsSPL7 gene mutant or the protein of claim 8 in the cultivation of transgenic rice in any of the following:

[0024] (1) Genetically modified rice with low total starch content;

[0025] (2) Genetically modified rice with low amylose content;

[0026] (3) Genetically modified rice with high alkali digestibility;

[0027] (4) Genetically modified rice with low gel consistency;

[0028] (5) Low-hardness genetically modified rice;

[0029] (6) Highly adhesive transgenic rice.

[0030] As an additional option, the present invention provides the application of the OsSPL7 gene, the OsSPL7 protein encoded by the OsSPL7 gene, or biological materials with the OsSPL7 gene knocked out in the creation of soft rice germplasm.

[0031] The present invention also provides a method for creating soft rice germplasm, comprising the step of inhibiting the expression of the OsSPL7 gene in rice; the nucleotide sequence of the OsSPL7 gene is shown in SEQ ID NO.1.

[0032] This invention provides the application of the above-mentioned OsSPL7 gene mutant or the above-mentioned protein in the creation of soft rice germplasm.

[0033] The present invention discloses the following technical effects:

[0034] This invention cloned an OsSPL7 gene that regulates rice yield and quality using reverse genetics. The rice OsSPL7 gene provided by this invention can serve as a gene for improving rice quality, particularly for regulating rice trait indicators, including important nutritional indicators such as total starch content and amylose content; rice cooking quality such as alkali spreading value and gel consistency; and eating quality such as hardness and adhesiveness. Results from specific embodiments of this invention show that, compared to the wild type, the knockout lines had significantly lower total starch content, amylose content, gel consistency, and hardness, while significantly higher alkali spreading value and adhesiveness. The OsSPL7 gene provided by this invention provides important genetic resources and a theoretical basis for the genetic improvement of nutritional and cooking / eating quality of rice. The OsSPL7 gene and its mutants provide new directions for studying the molecular mechanisms of rice endosperm development and quality formation, while also expanding the biological functions of the rice SPL gene family in regulating rice quality. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is a plot showing the predicted tissue expression analysis of the OsSPL7 gene at different sites.

[0037] Figure 2 A yeast one-hybrid diagram showing the interaction between the OsSPL7 gene and the granular starch synthase gene OsGBSSI.

[0038] Figure 3 Map of CRISPR-Cas9 knockout vectors containing the OsSPL7 gene target site sequence;

[0039] Figure 4 A schematic diagram of OsSPL7 gene editing under a Nipponbare background;

[0040] Figure 5 The graphs show the phenotypic and appearance characteristics of polished rice from wild-type and mutant osspl7; where a is an image of the appearance of rice from different strains; b is a statistical bar chart of chalkiness from different strains; and c is a statistical bar chart of the grain percentage with chalkiness from different strains.

[0041] Figure 6 The graphs show the physicochemical properties of wild-type and mutant osspl7 rice. Among them, a is a statistical bar chart of total starch content of different strains; b is a statistical bar chart of amylose content of different strains; c is a statistical bar chart of alkali spreading value of different strains; d is a statistical bar chart of gel consistency of different strains; e is a statistical bar chart of hardness of different strains; and f is a statistical bar chart of adhesiveness of different strains. Detailed Implementation

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0045] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0046] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0047] The sequence information of the OsSPL7 gene and the OsSPL7 protein it encodes, provided by this invention, is as follows:

[0048] The nucleotide sequence of the OsSPL7 gene is shown in SEQ ID NO.1, specifically:

[0049]

[0050] The amino acid sequence of the OsSPL7 protein encoded by the OsSPL7 gene is shown in SEQ ID NO.2, specifically:

[0051] MEGNNGCGGSGATPRGVVGMHWAPVVTSPPSPQPPFLPPAPCRPDVQMQQQGGLTCLKLGKRPCFWGGDGAGQVAQGSGGGGGGGGGSADQGKRKEKAATAVPVVPRCQVEGCDITLQGVKEYHRRHKVCEVHAKAPRVVVHGTEQRFCQQCSRFHVLAEFDDAKKSCRRRLAGHNERRRR SNASEAMARGSAHPGMPVLGHGFPPYGLPTSSAGALSLLSSARATGPWLMPTPDISARSSAALDELIAENRAALLSWQFFSDRQPPPAGRPTGRSPGSETAGGWHAHLQARPPPPGAGGQHENQSGHVTLDLMQATTAAGGSGAPFRPVPARPPKEGGDAGCTSDAWTPSPMEGARVV*.

[0052] Example 1: Tissue Expression Pattern Analysis of Predicted OsSPL7 Gene in Rice

[0053] Tissue expression pattern analysis was performed using the RAP-DB website (https: / / rapdb.dna.affrc.go.jp) with the RAP_Locus number Os04g0551500 for the OsSPL7 gene. The prediction results showed that the OsSPL7 gene is highly expressed in rice embryos and endosperm. Figure 1 ).

[0054] Example 2: Yeast one-hybrid experiment on the interaction between the OsSPL7 gene and the granular starch synthase gene OsGBSSI.

[0055] 1. Preparation of yeast one-hybridization reagent

[0056] (1)SD / - Trp / - The components of Ura solid dual-deficiency culture medium (containing X-Gal) are shown in Table 1.

[0057] Table 1 SD / - Trp / - Components (1L) of Ura solid dual-deficiency culture medium (containing X-Gal)

[0058] Element Final concentration Dosage 40% galactose 2％ 50mL 40% persimmon sugar 1％ 25mL 10× phosphate buffer 1× 100mL X-Gal 80μg / mL 4mL Agar 2％ 20g YNB 6.7g <![CDATA[SD / - Ura / - Trp]]> 0.72g

[0059] Note: After sterilization, cool to below 60°C at room temperature, then add 1 mL of K. + Pour the flat plate into a container for later use.

[0060] (2) The components of Solutin A, Solutin B and Solutin C are shown in Table 2.

[0061] Table 2. Components of Solutin A, Solutin B, and Solutin C

[0062]

[0063] Note: When preparing various culture media, it is recommended to add 1 / 1000 of 50 μg / mL kanamycin or ampicillin to inhibit contaminating bacteria.

[0064] 2. Preparation of competent yeast cells

[0065] (1) Take a small amount of yeast strain Y2H Gold with the sterilization pipette tip, streak it on YPDA solid medium, and incubate it upside down at 30℃ for 3 days; pick a single clone the size of a match head and inoculate it into 5 mL of 1×YPDA liquid medium, and activate it overnight at 30℃ and 250 r / min.

[0066] (2) Inoculate the activated bacterial solution into 100 mL of fresh 1×YPDA liquid culture medium at a ratio of 1 / 1000, and incubate at 30℃ with shaking at 250 r / min for 3.5 h, until OD... 600 Reaching 0.5–0.6;

[0067] (3) Transfer the yeast culture into a 50 mL sterile centrifuge tube, centrifuge at 5000 r / min for 3 min to collect the cells, discard the supernatant, enrich twice; add 20 mL Solution A solution to resuspend the precipitate;

[0068] (4) Centrifuge at 5000 r / min for 3 min, discard the supernatant; add 2 mL Solution A solution to resuspend the precipitate; dispense 150 μL into each tube and store at -80℃ for later use.

[0069] 3. Yeast mono- or heterozygous

[0070] - Trp / - Ura solid dual-deficiency medium (excluding X-Gal), incubate at 30℃ for 2–3 days, pick 7–8 single colonies, mix them in 100 μL of sterile ddH2O, and then gradually dilute the concentration to 10⁻⁶. -1 10 -2 and 10 -3 10 μL of each sample was aspirated and dotted onto SD / - Trp / - Incubate on Ura solid dual-deficient medium (containing X-Gal) at 30°C and observe the color development results. If the bacterial cells turn blue, it indicates that there is a binding between the OsGBSSI promoter and the OsSPL7 gene. Figure 2 The results were recorded by taking photos. Empty vectors pLacZi and pB42AD were used as negative controls.

[0071] (1) The carrier DNA was treated at 100℃ for 5 min and placed on ice for 1 min; this was repeated once; 1 μg of plasmid (500 ng AD-plasmid + 500 ng BD-plasmid) was mixed with 5 μL of carrier DNA and transferred into freshly frozen and thawed yeast competent cells, and cultured at 37℃ in a shaker at 200 r / min for 5 min.

[0072] (2) Add 1 mL Solution B, mix gently, and incubate in a 30°C water bath for 60 min; invert the container several times at random times to mix.

[0073] (3) Centrifuge at 3000 r / min for 1 min and discard the supernatant;

[0074] (4) Add 800 μL Solution C to resuspend the precipitate, centrifuge at 3000 r / min for 1 min, and discard the supernatant;

[0075] (5) Resuspend the precipitate in 100 μL Solution C, and coat all solutions onto SD / - Trp / - Ura solid dual-deficiency medium (containing X-Gal); after sealing the petri dishes with sealing film, incubate upside down at 30°C for 3–5 days;

[0076] (6) Pick 7-8 single clones, mix them in 100 μL of sterile ddH2O, and dilute them sequentially to 10 μL. -1 10 -2 and 10 -3 Concentration gradient, 10 μL of each sample was spotted on SD / - Trp / - Incubate on Ura solid dual-deficiency medium (containing X-Gal) at 30℃ for 3–5 days, observe the interaction and take photographs. Results are as follows: Figure 2As shown in the image. The results show that the bacterial cells appear blue, indicating a binding between the OsGBSSI promoter and the OsSPL7 gene.

[0077] Example 3 Construction of OsSPL7 gene knockout vector in rice

[0078] Selection of gRNA target sequence: According to the CRISPR / Cas9 related experimental methods, a sequence containing NGG as the recognition site was selected on the exon of the OsSPL7 gene as the knockout target site. The sequence is shown in SEQ ID NO.3, specifically: 5′-GCAGCCGCCGTTCCTCCCGCCGG-3′, and the PAM sequence is CGG.

[0079] Design of upstream and downstream primers for gRNA oligonucleotide chain

[0080] The upstream primer was OsSPL7_gRNA_F: 5′-GCCCGCAGCCGCCGTTCCTCCCGC-3′, SEQ ID NO.4;

[0081] The downstream primer is OsSPL7_gRNA_F: 5′-GGCGCCGGCGGGAGGAACGGCGGCTGC-3′, SEQ ID NO. 5;

[0082] CRISPR / Cas9 vector construction: This example uses a plant Cas9 / gRNA plasmid construction kit (Catalog. No. BGK03, e.g.) Figure 3 As shown in the diagram, the target sequence is loaded to form a recombinant vector containing the OsSPL7 gene target site. The specific operation method is as follows:

[0083] Preparation of oligo dimers: Take 1 μL each of 10 μM target site primers (SEQ ID NO.4 and SEQ ID NO.5) and add 18 μL of Buffer Aneal. After mixing, treat at 95℃ for 3 min, and then slowly reduce to 20℃ at about 0.2℃ / second to obtain double-stranded sequences containing knockout target sites.

[0084] Oligo dimer construction into CRISPR / Cas9: Take 2 μL of Cas9 / gRNA vector, 1 μL of oligo dimer from step one, 1 μL of Enzyme Mix, add 6 μL of ddH2O, mix well, and react in a metal bath at 20℃ for 1 h.

[0085] E. coli transformation: Take 5-10 μL of the final product from step 2 and add it to 50 μL of freshly thawed DH5α competent cells. Gently mix, incubate on ice for 30 min, heat shock at 42℃ for 45 s, stand on ice for 2 min, then add 200 μL of antibiotic-free LB medium, place in a constant temperature shaker at 37℃ and 200 rpm, and after recovery for 1 h, plate on a kanamycin-resistant (Kana+) plate.

[0086] Bacterial PCR detection: The next day, single clones were picked and cultured in kanamycin-resistant liquid medium at 37°C in a shaker until the bacterial culture 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 plasmids of positive clones were extracted for later use.

[0087] Agrobacterium-mediated transformation and rice genetic transformation: The successfully constructed plasmid was transformed into Agrobacterium (EHA105): 1 μL of plasmid was injected into freeze-thawed Agrobacterium competent cells, then placed on ice for 5 min, in liquid nitrogen for 5 min, and in a 37°C water bath for 5 min. 300 μL of antibiotic-free LB culture was added, and the cells were incubated at 28°C for 4 h. The resulting solution was then evenly spread onto a substrate containing kanamycin and rifampin. + After incubating on Rif-resistant plates at 28°C for 2-3 days, single colonies were picked, and positive clones were obtained by detection with hygromycin primers. These positive clones were then expanded into 3 mL of liquid K+ / Rif medium for further K+ / Rif culture. + Rif bacterial culture was sent to the company for genetic transformation of rice under Nipponbare background, resulting in transgenic lines with the OsSPL7 gene knocked out.

[0088] Example 4: Obtaining homozygous mutants of the rice OsSPL7 gene

[0089] To identify the OsSPL7 gene knockout transgenic lines obtained above, the OsSPL7 gene knockout transgenic seedlings were cultured in a room temperature and light incubator for about one week, and then positive seedlings were identified. The specific steps are as follows:

[0090] Detection of OsSPL7 gene knockout transgenic seedlings: Twenty T0 generation transgenic seedlings were obtained and cultured in a room temperature light incubator for about one week. DNA from these 20 seedlings was then collected and amplified by PCR using OsSPL7cas9TF (5′-TGTGTGGCAG CCGCCGTTCCTCCCGC-3′, SEQ ID NO. 6) and OsSPL7cas9TR (5′-AAACGCGGGAGGAAC GGCGGCTGCCA-3′, SEQ ID NO. 7). The amplified DNA was then sent to a sequencing company for sequencing. Analysis of the sequencing results revealed two transgenic lines with premature translation termination of the OsSPL7 gene. The nucleotide sequences of the OsSPL7 gene mutants in the two homozygous knockout OsSPL7 mutants of this invention are shown in SEQ ID NO. 8 or SEQ ID NO. 9, and their encoded proteins are shown in SEQ ID NO. 10 or SEQ ID NO. 11.

[0091] The nucleotide sequence of OsSPL7 gene mutant 1 is as described in SEQ ID NO.8, specifically:

[0092]

[0093] The nucleotide sequence of OsSPL7 gene mutant 2 (OsSPL7 gene deletion homozygous mutant 2) is as described in SEQ ID NO.9, specifically:

[0094]

[0095] The amino acid sequence of the protein encoded by OsSPL7 gene mutant 1 (OsSPL7 gene deletion homozygous mutant 1) is shown in SEQ ID NO.10, specifically:

[0096] MEGNNGCGGSGATPRGVVGMHWAPVVTSPPSPQPPFLLAGAMQARRPDATARRADLP QAREAAVLLGWRRRRPGGAGERRRRRRRRWWFRGSGEEEGGDGGAGGAPLPGGGVR HYAPGSQGVPPAAQGVRGARQGPEGRRARHRAALLPAMQPVPRARGV*;

[0097] The amino acid sequence of the protein encoded by OsSPL7 gene mutant 2 (OsSPL7 gene deletion homozygous mutant 2) is shown in SEQ ID NO.11, specifically:

[0098] MEGNNGCGGSGATPRGVVGMHWAPVVTSPPSPQPPFLHAGAMQARRPDATARRADLP QAREAAVLLGWRRRRPGGAGERRRRRRRRWWFRGSGEEEGGDGGAGGAPLPGGGVR HYAPGSQGVPPAAQGVRGARQGPEGRRARHRAALLPAMQPVPRARGV*.

[0099] After obtaining stable T2 generation knockout lines, further sequencing of the target site yielded homozygous mutants osspl7-1 and osspl7-2 (e.g., Figure 4 As shown in the image, mature rice seeds were harvested.

[0100] Example 5: Rice quality identification of OsSPL7 gene knockout rice lines

[0101] Mature seeds were harvested from the field-grown wild-type cultivar Nipponbare and the T2 generation homozygous mutant lines osspl7-1 and osspl7-2 with the OsSPL7 gene knocked out, under conventional sunlight and water and fertilizer management, and used in the following experiments.

[0102] Rice appearance examination: Observation of brown rice from wild type and knockout homozygous mutants showed no significant difference in chalky endosperm between the mutant and wild type (e.g., Figure 5 (As shown in a). 100 mature seeds were randomly selected, and after removing the seed coat, the chalkiness and chalky seed percentage were statistically analyzed. The results are shown in Figure 1. Figure 5 See examples b and c. The results showed that the chalkiness and chalky grain rate of the knockout lines were not significantly different from those of the wild-type Nipponbare.

[0103] Rice quality assessment:

[0104] 1. Determination of total starch content: Mature seeds of wild-type Nipponbare and knockout homozygous mutants osspl7-1 and osspl7-2 were dehulled and ground into rice flour. The rice flour was carefully sieved through a 100-mesh sieve into clean resealable bags for later use. 50 mg of each sample was weighed, with three replicates, and placed in 50 mL centrifuge tubes. 5 mL of 80% anhydrous ethanol was gently added along the tube wall. The tubes were placed in an 85°C water bath for 5 min. Another 5 mL of 80% anhydrous ethanol was added, and the tubes were centrifuged at 4000 g for 10 min. The supernatant was carefully aspirated with a pipette, and 10 mL of 80% anhydrous ethanol was added to the precipitate. The mixture was gently mixed and centrifuged again at 4000 g for 10 min at room temperature. The centrifuged 50 mL centrifuge tubes were then quickly inverted onto filter paper and allowed to air dry to remove any remaining ethanol. Dissolve the prepared sample in 1 mL of ddH2O and boil in a water bath for 30 min. Cool to room temperature, add 4 mL of 2M KOH, and shake at room temperature for 30 min (to prevent clumping). Add 16 mL of sodium acetate (1.2 M, pH = 3.8) and 200 μL of amylase (3000 U / mL), and incubate in a water bath at 60°C for 45 min, shaking 2-3 times during the process. Finally, bring the volume to 100 mL and mix gently.

[0105] To determine glucose content using GOD-PAP, transfer 1 mL of the prepared sample to a 1.5 mL centrifuge tube and centrifuge at 4000 rpm for 10 min. Transfer 100 μL of the supernatant to a 10 mL test tube or centrifuge tube, add 3 mL of GOD-PAP (pre-treated by wrapping in aluminum foil and thawing on ice), and incubate at 37°C for 20 min. Invert the tube to mix thoroughly. Measure the absorbance at 510 nm using an ELISA reader (Infinite 200PRO, TECAN, Switzerland). Zero the sample using a blank prepared 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 solutions are shown in Table 3. All samples must be measured within 60 min.

[0106] Table 3 Gradient glucose solution standard sample reaction solution

[0107] Standard Sample 0 20μL 40μL 60μL 80μL 100μL 0.1M sodium acetate slow-release solution (pH = 4.75) 100μL 80μL 60μL 40μL 20μL 0 GOD-PAP 3mL 3mL 3mL 3mL 3mL 3mL

[0108] Based on the absorbance values ​​of the standard samples measured by the ELISA reader and their known concentrations, a standard curve and regression equation were established. Then, the absorbance value of each sample was substituted into the regression equation to obtain the corresponding glucose content, which was then converted into the total starch content (total starch content equals 0.9 times the glucose content, and the sample moisture content is calculated as 12%). Each sample was tested three times, and the average value was taken as the total starch content. The results are as follows: Figure 6 As described in section a. The results showed that the total starch content of the knockout homozygous mutant was significantly lower than that of the wild type.

[0109] 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). Remove the husks from the mature seeds of the material to be tested, grind them into rice flour, sieve through a 100-mesh sieve, and weigh 50mg of the rice flour and standard into a 50mL volumetric flask (3 replicates per group). Gently shake the volumetric flask to dissipate any rice flour adhering to the tube wall to the bottom.

[0110] Add 500 μL of anhydrous ethanol to a 50 mL volumetric flask and gently rotate the flask to ensure the rice flour is thoroughly mixed with the anhydrous ethanol. Then add 4.5 mL of 1M NaOH solution, rotating the flask while adding to flush any rice flour adhering to the inner wall to the bottom. Let the flask stand overnight at room temperature for 24 hours. Add ddH2O to bring the volume to 50 mL and gently shake to mix. Transfer 5 mL of ddH2O to a 10 mL test tube, then add 500 μL each of the blank, standard, and test sample (discard the first two pipette tips before pipetting to reduce experimental error). Add 100 μL of 1M acetic acid solution, 200 μL of KI-I2, and 4.2 mL of ddH2O to the test tube. Vortex the solution thoroughly and let it stand for 20 minutes. Measure the absorbance (OD) of each sample at 620 nm using an Infinite 200PRO microplate reader (TECAN, Switzerland). 620 Before use, zero the reaction mixture with a blank solution; plot a standard curve (standard concentrations of 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 6 As shown in b in the figure. The results showed that the amylose content of the knockout homozygous mutant was significantly lower than that of the wild type.

[0111] 3. Determination of Alkali Spreading Value: Select 6 plump whole rice grains and place them in a square box. Add 10 mL of 1.70% potassium hydroxide solution, cover the box, and place it in a constant temperature incubator at 30℃ for approximately 23 hours. Observe the decomposition of the rice endosperm and grade and record the results according to the national standard for edible rice varieties NT / T593-2021. The results are as follows: Figure 6As shown in c in the figure. The results showed that the basal extinction value of the knockout homozygous mutant was significantly higher than that of the wild type.

[0112] 4. Determination of gel consistency: The determination of gel consistency shall be carried out in accordance with GB / T 22294-2008 Grain and Oil Inspection: Gelatin Consistency of Rice. The specific steps are as follows: (1) Weigh 100mg of sieved (120 mesh) rice flour into a 10cm test tube; (2) Add 0.2mL of 95% ethanol, vortex and shake, then add 2mL of ethanol. (3) Place the test tube in a boiling water bath, cover the mouth of the test tube with a glass marble, and keep it gelatinized for 8 minutes. Adjust the depth of the test tube in the boiling water so that the elongation of the boiling rice flour does not exceed 2 / 3 of the length of the test tube. (4) After the rice flour gelatinization is complete, take out the test tube and cool it at room temperature for 5 minutes. (5) Keep the test tube in an ice water bath for 20 minutes, and then place it flat on the water platform of a 25℃ incubator for 1 hour. (6) Measure the length from the bottom of the test tube to the highest point of the rice glue with a ruler and record it as the consistency of the rice flour (mm). Set up 3 biological replicates for each sample and take the average value. The results are as follows. Figure 6 As shown in d in the figure. The results showed that the gel consistency of the knockout homozygous mutant was significantly lower than that of the wild type.

[0113] 5. Rice hardness determination: The hardness of wild-type and mutant rice was determined according to the operating procedure of the Shanghai Baosheng TA.XTC-18 texture analyzer. The results are as follows: Figure 6 As shown in e. The results showed that the rice hardness of the knockout homozygous mutant was significantly lower than that of the wild type.

[0114] 6. Adhesion Test: Weigh 3g of wild-type and mutant rice flour respectively, add 25mL of distilled water, and perform the adhesion test of the rice flour according to the operating procedure of the TechMaster RVA rapid viscosity analyzer (Perten, Swiss). Repeat the test three times. The results are as follows: Figure 6 As shown in f in the figure. The results showed that the rice flour adhesion of the knockout homozygous mutant was significantly higher than that of the wild type.

[0115] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of OsSPL7 gene knockout or biological materials used for OsSPL7 gene knockout in regulating total starch content, amylose content, cooking quality, and eating quality of rice grains, characterized in that, The cooking quality is defined as alkali spreading value and / or gel consistency; the eating quality is defined as hardness and / or adhesiveness; the nucleotide sequence of the OsSPL7 gene is shown in SEQ ID NO.1; The regulation is achieved by knocking out or inhibiting the expression of the OsSPL7 gene in rice, thereby reducing total starch content, amylose content, gel consistency and hardness, and improving alkali spreading value and adhesiveness.

2. The application according to claim 1, characterized in that, The biomaterials include recombinant vectors and recombinant bacteria.

3. The use of OsSPL7 gene knockout or biological materials used for OsSPL7 gene knockout in the cultivation of rice that falls into any of the following categories: (1) Rice with low total starch content in grains; (2) Rice with low amylose content in grains; (3) Rice with high alkali digestibility value; (4) Rice with low grain gel consistency; (5) Rice with low grain hardness; (6) Rice with high grain adhesion; The nucleotide sequence of the OsSPL7 gene is shown in SEQ ID NO.1; The cultivation method involves knocking out or inhibiting the expression of the OsSPL7 gene in rice to reduce the total starch content, amylose content, gel consistency, and hardness of rice grains, while improving alkali spreading value and adhesiveness.

4. The application according to claim 3, characterized in that, The biomaterials include recombinant vectors and recombinant bacteria.

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