Application of OsSPL7 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 OsSPL7 gene in regulation and control of nutritional indexes, cooking quality and taste quality of rice grains. According to the invention, the OsSPL7 gene for regulating and controlling the yield and quality of rice is cloned through reverse genetics, and the rice OsSPL7 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). Results of the specific embodiment of the invention show that the OsSPL7 gene is knocked out under the background of Nipponbare, compared with a wild type, the total starch content, the amylose content, the gel consistency and the hardness of the knocked-out strain are obviously lower than those of the wild type, and the alkali elimination value and the 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 OsSPL7 gene in regulating rice grain nutritional indexes, cooking quality and eating quality. Background Art

[0002] Rice (Oryza sativa L.) is an important food crop worldwide and one of the main foods in most Asian countries and some other regions. With the improvement of living standards and the gradual increase in consumers' requirements for rice quality, the taste and cooking quality of rice have become important considerations for consumers when purchasing. The taste and cooking quality directly affect the taste, stickiness, elasticity and softness of rice after cooking. These factors determine the market competitiveness of rice to a certain extent.

[0003] Traditional rice quality improvement focuses on increasing yield and improving disease resistance, while the improvement of taste and cooking quality is relatively lagging 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 taste, aroma, stickiness, and elasticity, while cooking quality involves the cooking time of rice, the stickiness after cooking, the softness and durability of rice (Cruz, CD, & Khush, GS Rice breeding: Current status and future prospects, Theoretical and Applied Genetics, 2000, 101 (1-2), 179-185.). Different varieties of rice have 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] At present, the improvement of rice taste and cooking quality mostly relies on traditional breeding methods, such as hybrid breeding and backcross breeding (Zhao et al., Traditional rice breeding and the need for quality improvement, Journal of Agricultural and Food Chemistry, 2019, 67 (23), 6385-6394.). Although these methods can improve the taste and cooking quality of rice to a certain extent, due to the long breeding cycle and large variability, it is difficult to meet the market 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 have gradually been 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 can cultivate rice varieties with excellent taste and cooking quality in a relatively short time by accurately identifying and regulating genes related to taste and cooking quality. However, although modern technology has made some progress in accelerating the improvement of rice taste and cooking quality, it still faces some challenges in practical application. First, improving rice taste and cooking quality requires the synergistic effect of multiple genes, and the genetic background is complex. The change of a single gene may not significantly improve the overall quality (Fujita, et al., Genetic basis of rice eating and cooking quality: From QTLs to genes. Rice, 2015, 8 (1), 12.). Secondly, environmental factors (such as climate, soil type, etc.) have an important influence on rice quality, and the universality and stability of the improvement scheme are limited (Egli et al., Effects of climate change on rice grain quality. Field Crops Research, 2016, 197, 151-161.). Therefore, digging out the key genes that control the taste quality of rice and developing a fast and efficient method for improving the taste and cooking quality of rice can significantly improve the eating experience and market competitiveness of rice without increasing production costs and complex operations, which has important theoretical significance and extensive 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 the present invention is to provide an application of the OsSPL7 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 OsSPL7 gene was knocked out in the Nipponbare background. Compared with the wild type, the total starch content, amylose content, gel consistency and hardness of the knockout strain 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 OsSPL7 gene, an OsSPL7 protein encoded by the OsSPL7 gene, or a biological material with the OsSPL7 gene knocked out in regulating nutritional indices, cooking quality, and taste 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 taste quality includes hardness and / or adhesion; 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, the method comprises inhibiting the expression of the OsSPL7 gene in rice, so as to reduce the total starch content, the amylose content, the gel consistency and the hardness, and improve the alkali elimination value and the adhesion.

[0009] Preferably, the biological material includes a recombinant vector and a recombinant bacterium.

[0010] The present invention provides the use of an OsSPL7 gene, an OsSPL7 protein encoded by the OsSPL7 gene, or a biological material with the OsSPL7 gene knocked out in cultivating transgenic rice of any of the following:

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

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

[0013] (3) Transgenic rice with high alkalinity value;

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

[0015] (5) Transgenic rice with low hardness;

[0016] (6) Highly adherent 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 comprises the step of inhibiting the expression of OsSPL7 gene in rice to obtain transgenic rice.

[0019] Preferably, the biological material includes a recombinant vector and a recombinant bacterium.

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

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

[0022] The present invention provides the use of the above-mentioned OsSPL7 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.

[0023] The present invention provides the use of the above-mentioned OsSPL7 gene mutant or the protein according to claim 8 in cultivating any of the following transgenic rice:

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

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

[0026] (3) Transgenic rice with high alkalinity value;

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

[0028] (5) Transgenic rice with low hardness;

[0029] (6) Transgenic rice with high adhesion.

[0030] As an additional solution, the present invention provides the use of the OsSPL7 gene, the OsSPL7 protein encoded by the OsSPL7 gene, or the biological material with the OsSPL7 gene knocked out in creating 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] The present invention provides the use of the above-mentioned OsSPL7 gene mutant or the above-mentioned protein in creating soft rice germplasm.

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

[0034] The present invention clones an OsSPL7 gene that regulates rice yield and quality through reverse genetics. The rice OsSPL7 gene provided by the present invention can be used as a gene related to improving rice quality, especially for regulating rice trait indicators, including important rice nutritional indicators: total starch content, amylose content; rice cooking quality: alkali digestion value and gel consistency; taste quality: hardness and adhesion. The results of the specific embodiments of the present invention show that compared with the wild type, the total starch content, amylose content, gel consistency and hardness of the knockout strain are significantly lower than those of the wild type, and the alkali digestion value and adhesion are significantly higher than those of the wild type. The OsSPL7 gene provided by the present invention provides an important gene resource and theoretical basis for the genetic improvement of nutrition and rice cooking and taste quality. The OsSPL7 gene and OsSPL7 gene mutant provided by the present invention provide a new direction for studying the molecular mechanism of rice endosperm development and quality formation, and at the same time expand the biological function of the rice SPL gene family in rice quality regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 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.

[0036] Figure 1 This is a diagram of tissue expression analysis for site prediction of the OsSPL7 gene;

[0037] Figure 2 This is the yeast single hybrid map of the interaction between OsSPL7 gene and granular starch synthase gene OsGBSSI;

[0038] Figure 3 This is a map of the CRISPR-Cas9 knockout vector containing the OsSPL7 gene target site sequence;

[0039] Figure 4 Schematic diagram of editing the OsSPL7 gene in the Nipponbare background;

[0040] Figure 5 : The phenotype and appearance trait analysis diagram of the polished rice of the wild type and the mutant osspl7; wherein, a is the appearance picture 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;

[0041] Figure 6 The figures are the analysis charts of the physical and chemical indexes of wild-type and mutant osspl7 rice; among them, a is the statistical bar graph of total starch content of different strains; b is the statistical bar graph of amylose content of different strains; c is the statistical bar graph of alkali consumption value of different strains; d is the statistical bar graph of gel consistency of different strains; e is the statistical bar graph of hardness of different strains; f is the statistical bar graph of adhesion of different strains. DETAILED DESCRIPTION

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

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

[0047] The sequence information of the OsSPL7 gene and the OsSPL7 protein encoded by the present 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] MEGNNGCGGSGATPRGVVGMHWAPVVTSPPSPQPPFLPPAPCRPDVQMQQQGGLTCLKLGKRPCFWGGDGAGQVAQGSGGGGGGGGGGSADQGKRKEKAATAVPVVPRCQVEGCDITLQGVKEYHRRHKVCEVHAKAPRVVVHGTEQRFCQQCSRFHVLAEFDDAKKSCRRRLAGHNERRRR SNASEAMARGSAHPGMPVLGHGFPPYGLPTSSAGALSLLSSARATGPWLMPTPDISARSSAALDELIAENRAALLSWQFFSDRQPPPAGRPTGRSPGSETAGGWHAHLQARPPPPGAGGQHENQSGHVTLDLMQATTAAGGSGAPFRPVPARPPKEGGDAGCTSDAWTPSPMEGARVV*.

[0052] Example 1 Analysis of predicted tissue expression patterns of rice OsSPL7 gene

[0053] The RAP-DB website (https: / / rapdb.dna.affrc.go.jp) was used to input the RAP_Locus number Os04g0551500 of the OsSPL7 gene for tissue expression pattern analysis. The prediction results showed that the OsSPL7 gene was highly expressed in rice embryo and endosperm ( Figure 1 ).

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

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

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

[0057] Table 1SD / - Trp / - Ura solid medium (containing X-Gal) components (1L)

[0058] Element Final concentration Dosage 40% Galactose 2％ 50mL 40% Persimmon Candy 1％ 25mL 10× Phosphate Buffer 1× 100mL X-Gal 80 μg / mL 4mL Agar 2％ 20g YNB 6.7g <![CDATA[SD / - Ura / - Trp]]> 0.72g

[0059] Note: After sterilization, cool the room temperature to below 60°C and add 1 mL K + , pour into a flat plate and set aside.

[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 50μg / mL kanamycin or ampicillin to inhibit bacteria.

[0064] 2. Preparation of competent yeast

[0065] (1) Use a sterilized pipette tip to pick up a small amount of yeast strain Y2H Gold, streak it on YPDA solid medium, and culture it upside down at 30°C 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 at 30°C and 250 r / min overnight;

[0066] (2) Inoculate the activated bacterial solution into 100 mL of fresh 1×YPDA liquid medium at a ratio of 1 / 1000 and culture at 30°C with shaking at 250 rpm for 3.5 h until the OD 600 Reach 0.5~0.6;

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

[0068] (4) Centrifuge at 5000 r / min for 3 min and discard the supernatant. Add 2 mL of Solution A to resuspend the precipitate. Aliquot 150 μL into each tube and store at -80°C for later use.

[0069] 3. Yeast single hybrid

[0070] - Trp / - Ura solid medium (without X-Gal) was cultured at 30°C for 2-3 days. Seven to eight monoclonal clones were picked and mixed in 100 μL sterile ddH2O. The concentrations were diluted to 10 -1 , 10 -2 and 10 -3 , respectively, pipette 10 μL of RBI onto SD / - Trp / - Ura solid medium (containing X-Gal) was cultured at 30°C and the color development results were observed. If the bacteria showed blue color, it indicated that there was a binding between the OsGBSSI promoter and the OsSPL7 gene ( Figure 2 ), take photos and record the results. Empty vectors pLacZi and pB42AD were used as negative controls.

[0071] (1) Carrier DNA was treated at 100°C 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 carrier DNA and transferred into freshly frozen and thawed yeast competent cells. The cells were cultured at 37°C and 200 rpm for 5 min.

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

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

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

[0075] (5) Add 100 μL Solution C to resuspend the precipitate and apply SD / - Trp / - Ura solid medium (containing X-Gal); the culture dish was sealed with sealing film and incubated upside down at 30°C for 3-5 days;

[0076] (6) Pick 7 to 8 monoclonal clones, mix them in 100 μL sterile ddH2O, and dilute them into 10 -1 , 10 -2 and 10 -3 Concentration gradient, 10 μL was pipetted and dotted on SD / - Trp / - The cells were cultured on Ura solid medium (containing X-Gal) at 30°C for 3-5 days, and the interactions were observed and photographed. Figure 2The results showed that the bacteria appeared blue, which means that there was 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 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 OsSPL7 gene. 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 of 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, Figure 3 As shown in the figure), 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 dimer: Take 1 μL of each of the 10 μM target site front and back primers (SEQ ID NO.4 and SEQ ID NO.5) and add 18 μL of Buffer Aneal, mix well, treat at 95°C for 3 min, and then slowly reduce the temperature to 20°C at about 0.2°C / second to obtain a double-stranded sequence containing the knockout target site;

[0084] 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;

[0085] 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 resuscitate for 1 h before applying to a plate coated with kanamycin resistance (Kana+);

[0086] 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.

[0087] 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 culture medium was added and revived in a constant temperature incubator at 28°C for 4 hours, and evenly spread on (kanamycin + rifampicin) K + / Rif resistant plates were plated at 28°C for 2-3 days, single clones were picked, positive clones were detected with hygromycin primers and expanded in 3 mL of liquid K+ / Rif medium, and positive K+ / Rif clones were further cultured. + / Rif bacterial liquid was sent to the company for genetic transformation of rice under the Nipponbare background, and a transgenic strain with OsSPL7 gene knockout was obtained.

[0088] Example 4 Obtaining a homozygous mutant of rice OsSPL7 gene

[0089] In order to identify the OsSPL7 knockout transgenic strain obtained above, the OsSPL7 knockout transgenic seedlings obtained were cultured in a normal temperature light incubator for about 1 week, and then the positive seedlings were identified. The specific steps are as follows:

[0090] Detection of transgenic seedlings with knockout of OsSPL7 gene: 20 T0 generation transgenic seedlings were obtained, and cultured in a normal temperature light incubator for about one week, and then DNA of the 20 seedlings was taken, and PCR amplified with OsSPL7cas9TF (5′-TGTGTGGCAG CCGCCGTTCCTCCCGC-3′, SEQ ID NO.6) and OsSPL7cas9TR (5′-AAACGCGGGAGGAAC GGCGGCTGCCA-3′, SEQ ID NO.7), and then sent to the company for sequencing, and the sequencing results were analyzed to obtain transgenic strains with knockout of OsSPL7 gene in which the translation of two proteins was terminated prematurely. The nucleotide sequence of the OsSPL7 gene mutant in the two homozygous knockout of OsSPL7 involved in the present invention is shown in SEQ ID NO.8 or SEQ ID NO.9, and the encoded protein is shown in SEQ ID NO.10 or SEQ ID NO.11.

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

[0092]

[0093] The nucleotide sequence of OsSPL7 gene mutant 2 (OsSPL7 gene deletion homozygous mutant 2) is as shown 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 the stable inherited T2 knockout strain, the target site was further sequenced to obtain the homozygous mutants osspl7-1 and osspl7-2 (such as Figure 4 ), and the mature rice seeds are harvested.

[0100] Example 5 Rice Quality Identification of OsSPL7 Gene Knockout Strains

[0101] The wild-type variety Nipponbare and the T2 generation homozygous mutant lines osspl7-1 and osspl7-2 with the OsSPL7 gene knocked out were cultivated in the field with conventional sunlight, water and fertilizer management, and mature seeds were harvested for the following experiments.

[0102] Rice appearance inspection: The wild type and knockout homozygous mutant brown rice were observed, and the chalky endosperm of the mutant was not significantly different from that of the wild type (such as Figure 5 100 mature seeds were randomly selected, and after removing the seed shells, the chalkiness and chalky grain rate were counted. The results are as follows Figure 5 The results showed that the chalkiness and chalky grain rate of the knockout strains were not significantly different from those of the wild-type Nipponbare.

[0103] Rice quality inspection:

[0104] 1. Determination of total starch content: The mature seeds of wild-type Nipponbare and knockout homozygous mutants osspl7-1 and osspl7-2 were shelled and ground into rice flour, and 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 min. 5 mL of 80% anhydrous ethanol was added again, and then centrifuged at a speed of 4000 g for 10 min. The supernatant was carefully sucked off with a gun, and 10 mL of 80% anhydrous ethanol was added to the precipitate, gently mixed, and further centrifuged at 4000 g for 10 min 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 in a boiling water bath for 30 min; cool to room temperature, add 4 mL of 2M KOH, and oscillate 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 in a 60°C water bath for 45 min, oscillating 2-3 times during the period, and finally adjust the volume to 100 mL and mix gently.

[0105] GOD-PAP was used to determine the glucose content. 1 mL of the above treated sample was taken into a 1.5 mL centrifuge tube, centrifuged at 4000 rpm for 10 min, 100 μL of the supernatant was taken into 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 3. All samples must be determined within 60 min.

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

[0107] 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

[0108] 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 6 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). 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.

[0110] 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 6 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.

[0111] 3. Determination of alkali elimination value: Select 6 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. Observe the decomposition of the rice endosperm and record the grade according to the national edible rice variety quality standard NT / T593-2021. The results are as follows: Figure 6As 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.

[0112] 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 6 As shown in d. 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 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 6 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.

[0114] 6. Adhesion determination: Weigh 3 g of wild-type and mutant rice flour respectively, add 25 mL of distilled water, and complete the adhesion determination of rice flour according to the operating procedures of TechMaster RVA rapid viscosity analyzer (Perten, Swiss), repeat 3 times, and the results are as follows: Figure 6 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.

[0115] 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 OsSPL7 gene, OsSPL7 protein encoded by OsSPL7 gene or biological material with knockout of OsSPL7 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 digestion value and / or gel consistency; the taste quality includes hardness and / or adhesion; 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.

2. The use according to claim 1, characterized in that: The method comprises inhibiting the expression of the OsSPL7 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. The use according to claim 1, characterized in that: The biological material includes a recombinant vector and a recombinant bacterium.

4. Use of the OsSPL7 gene, the OsSPL7 protein encoded by the OsSPL7 gene, or a biological material with the OsSPL7 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; 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.

5. The use according to claim 4, characterized in that: The method comprises the steps of inhibiting the expression of the OsSPL7 gene in rice to obtain transgenic rice.

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

7. An OsSPL7 gene mutant, characterized in that: The nucleotide sequence of the OsSPL7 gene mutant is shown in SEQ ID NO.8 or SEQ ID NO.

9.

8. The protein encoded by the OsSPL7 gene mutant according to claim 7, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.10 or SEQ ID NO.

11.

9. Use of the OsSPL7 gene mutant according to claim 7 or the protein according to claim 8 in regulating rice grain nutritional indexes, cooking quality and eating 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.

10. Use of the OsSPL7 gene mutant according to claim 7 or the protein according to claim 8 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) Transgenic rice with high adhesion.

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