Low absorbable protein and endosperm cell wall thickened rice and preparation method and application thereof

Abstract

The invention relates to a method for creating a rice material with low absorbable protein content and remarkably thickened endosperm cell walls and application of the rice material. Specifically, the invention provides a method for improving rice, and improved rice with unique characters is obtained by down-regulating the expression of glutelin genes and down-regulating the expression of globulin genes. In the improved rice, the content of absorbable protein is low, and endosperm cell walls are remarkably thickened. Besides, the improved rice is low in in-vitro digestion rate, has the potential postprandial blood sugar control effect and is suitable for preparing auxiliary food for treating chronic kidney disease patients, diabetic patients and the like.

Description

Technical Field

[0001] This invention belongs to the field of biological breeding. Specifically, this invention relates to rice with low absorbable protein and thickened endosperm cell walls, as well as its preparation method and application. Background Technology

[0002] Protein is the second most abundant nutrient in rice, and its content and composition have a significant impact on rice quality. The main storage proteins in rice include glutenins, globulins, and prolamins. Glutenins and globulins are stored in irregular type II protein bodies (PBII), which can be digested and absorbed by the human body, while prolamins are stored in globular type I protein bodies (PBI) and cannot be digested.

[0005] Rice is a typical high glycemic index (GI) food, generally with a GI value >70. Excessive consumption of rice can accelerate the development of diabetes. Existing research shows a negative correlation between the GI value of rice and its amylose content. With continuous improvements in rice quality, both indica and japonica rice have seen a decrease in amylose content, leading to a trend of increasing GI values. This is especially true for soft rice varieties from the south (amylose content around 10%) and glutinous rice varieties (amylose content <2%), whose GI values ​​often exceed 90 or even 100. This objectively contributes to the rapid increase in the number of diabetic patients. Therefore, developing new low-GI rice varieties specifically for patients with diabetes and obesity, and preventing and controlling the occurrence and development of diseases from the source through dietary adjustments (medicine and food sharing the same origin), is the most important and economical approach.

[0006] In conclusion, there is an urgent need in this field to develop rice varieties with low absorbable protein content and low glycemic index. Summary of the Invention

[0007] The purpose of this invention is to provide a rice variety with low absorbable protein content and a low glycemic index. This rice variety is suitable not only for the general population but also for patients or susceptible individuals with chronic kidney disease, diabetes, obesity, etc.

[0008] In a first aspect of the present invention, a method for improving rice varieties is provided, the method comprising the steps of:

[0009] (a) Provide a rice plant to be improved; and

[0010] (b) In the rice plants to be improved, the expression of glutenin gene and the expression of globulin gene are downregulated, thereby improving the rice variety.

[0011] In another preferred embodiment, the downregulation of glutenin gene expression is described as downregulation of GluB4 / B5 expression.

[0012] In another preferred embodiment, the downregulation of globulin gene expression is described as downregulation of Glb-1 expression.

[0013] In another preferred embodiment, the cell wall thickness of the mature endosperm is significantly increased in the improved rice variety.

[0014] In another preferred embodiment, the "significantly increased cell wall thickness" means that, compared with the thickness T0 of the mature endosperm cell wall of wild-type rice varieties (such as varieties with normal expression of glutenin family genes and Glb-1 genes, such as Ningjing 4), the thickness T1 of the mature endosperm cell wall of the improved rice variety satisfies T1 / T0≥400%, preferably≥600%, more preferably≥800%, such as 400%-850%.

[0015] In another preferred embodiment, the content of absorbable protein in the improved rice variety is significantly reduced.

[0016] In another preferred embodiment, the "significantly reduced absorbable protein content" means that, compared with the absorbable protein content C0 in the endosperm of wild-type rice varieties (such as varieties with normal expression of glutenin family genes and Glb-1 genes, such as Ningjing 4), the absorbable protein content C1 in the endosperm of the improved rice variety satisfies C1 / C0≤50%, preferably≤45%, more preferably≤40%, such as about 40%-50%.

[0017] In another preferred embodiment, the downregulation includes gene knockout or gene knockdown.

[0018] In another preferred embodiment, the downregulation refers to downregulating the expression of glutenin family genes and downregulating the expression of globulin genes.

[0019] In another preferred embodiment, downregulating the expression of F-glutenin family genes includes:

[0020] (A1) Expression of inactivated GluB4 / B5 gene;

[0021] (A2) Introduce deletion mutations into the GluB4 / B5 gene to form endogenous RNAi;

[0022] (A3) RNAi interference was used to target conserved regions of glutenin family genes, thereby downregulating the expression of glutenin family genes;

[0023] (A4) Introduce deletion or frameshift mutations into the genes of the glutenin family;

[0024] (A5) Any combination of A1 to A4 above.

[0025] In another preferred embodiment, the inactivated GluB4 / B5 gene includes LGC-1.

[0026] In another preferred embodiment, the sequence of LGC-1 has the nucleotide sequence shown in SEQ ID NO:24.

[0027] In another preferred embodiment, the "introduction of deletion mutations in the GluB4 / B5 gene" includes large deletions (such as...). Figure 6 (The large segment shown is missing).

[0028] In another preferred embodiment, the “conserved segment of the gluten family gene” is the 5' end conserved segment of GluA1.

[0029] In another preferred embodiment, the 5' conserved region of GluA1 has the nucleotide sequence shown in SEQ ID NO:26.

[0030] In another preferred embodiment, the "downregulation of gluten family gene expression" is achieved by interfering with all genes in the gluten family via RNAi.

[0031] In another preferred embodiment, the frameshift mutation results in a decrease or absence of glutenin content.

[0032] In another preferred embodiment, downregulating the expression of globulin family genes includes:

[0033] (B1) Expresses the inactivated Glb-1 gene;

[0034] (B2) Introduce deletion mutations or frameshift mutations into the Glb-1 gene;

[0035] (B3) Gene editing of the 5'-UTR downregulates the transcriptional level of Glb-1;

[0036] (B4) RNAi interference was performed on the Glb-1 gene to downregulate its expression;

[0037] (B5) Any combination of B1 to B4 above.

[0038] In another preferred embodiment, downregulating Glb-1 expression is achieved by gene editing to knock down or eliminate the Glb-1 gene.

[0039] In another preferred embodiment, the phrase "introducing a frameshift mutation into the Glb-1 gene" "includes a frameshift mutation involving a single base insertion (e.g.)" Figure 6 (The frameshift mutation shown).

[0040] In another preferred embodiment, the "RNAi interference of the Glb-1 gene" is achieved by constructing a transgenic interference vector and transforming rice to downregulate the expression of the Glb-1 gene.

[0041] In another preferred embodiment, the downregulation is performed by an operation selected from the group consisting of: hybridization breeding, gene editing, RNAi, or a combination thereof.

[0042] In another preferred embodiment, the gene editing includes gene editing based on gRNA and Cas9.

[0043] In another preferred embodiment, the gRNA includes: SEQ ID No:9 and SEQ ID No:10.

[0044] In another preferred embodiment, the RNAi includes RNAi performed using interfering primers.

[0045] In another preferred embodiment, the interfering primers include: SEQ ID No:19, SEQ ID No:20, SEQ ID No:21 and SEQ ID No:22.

[0046] In another preferred embodiment, step (b) further includes detecting the rice plant or cells, wherein the detection is selected from the group consisting of:

[0047] (1) Detect the genotype of rice and select plants that are homozygous for LGC-1 and homozygous for Glb-1 deletion, or homozygous for GluB4 / B5 deletion and homozygous for Glb-1 deletion.

[0048] (2) Detect the cell wall thickness of the mature endosperm of rice, and select plants with significantly increased cell wall thickness (e.g., plants with cell wall thickness approximately 8 times that of wild-type rice); and

[0049] (3) Detect the absorbable protein content in rice seeds and select plants with a significant decrease in absorbable protein.

[0050] In a second aspect of the invention, an improved rice is provided, said improved rice being obtained by the method described in the first aspect of the invention.

[0051] In another preferred embodiment, the improved rice has the following genotype: LGC-1 + / + and Glb-1 - / - A combination; or CRI-GluB4 / B5 - / - and CRI-Glb-1 - / - The combination of .

[0052] In a third aspect of the invention, a gene editing reagent is provided for downregulating the expression of absorbable protein-related polypeptides in rice cells or plants, wherein the absorbable protein-related polypeptides are a combination of glutenin family and globulin.

[0053] In another preferred embodiment, the gene editing reagent comprises:

[0054] (Z1) is a gene-editing reagent used to downregulate the glutenin family; and

[0055] (Z2) is a gene-editing reagent used to downregulate globulins.

[0056] In another preferred embodiment, the gene-editing reagent for downregulating the gluten family includes a gene-editing reagent for downregulating GluB4 / B5.

[0057] In another preferred embodiment, the gene-editing reagent for downregulating globulins includes a gene-editing reagent for downregulating Glb-1.

[0058] In another preferred embodiment, the GluB4 / B5 has a nucleotide sequence as shown in SEQ ID NO:23.

[0059] In another preferred embodiment, the Glb-1 has a nucleotide sequence as shown in SEQ ID NO:25.

[0060] In another preferred embodiment, the gene-editing reagent for downregulating GluB4 / B5 includes: SEQ ID NO.11 and SEQ ID NO.12.

[0061] In another preferred embodiment, the gene-editing reagent for downregulating Glb-1 includes: SEQ ID NO.13 and SEQ ID NO.14.

[0062] In another preferred embodiment, the gene-editing reagent is used to prepare a formulation for regulating the thickness of rice endosperm cell walls.

[0063] In another preferred embodiment, the formulation is used to improve rice, thereby obtaining rice varieties with a significant increase in the thickness of the rice endosperm cell walls.

[0064] In another preferred embodiment, the absorbable protein content in the rice variety is significantly reduced.

[0065] In a fourth aspect of the invention, a food product is provided, the food product containing or consisting of the endosperm of rice, wherein the rice is rice with glutenin and globulin gene knockout or knockdown.

[0066] In another preferred embodiment, the rice is rice with the glutenin gene and the Glb-1 gene knocked out or knocked down.

[0067] In another preferred embodiment, the rice is rice with GluA1 and Glb-1 genes knocked out or knocked down.

[0068] In another preferred embodiment, the rice is rice with GluB4 / B5 and Glb-1 genes knocked out or knocked down.

[0069] In another preferred embodiment, the cell wall thickness of the rice endosperm is significantly increased.

[0070] In another preferred embodiment, the food is used by the general population.

[0071] In another preferred embodiment, the food is used as food for patients with chronic kidney disease, diabetes, and / or obesity.

[0072] In a fifth aspect of the invention, there is provided the use of rice endosperm or a food containing said rice endosperm for preparing a low glycemic index food.

[0073] In a sixth aspect of the invention, there is provided an use of the food described in the fourth aspect of the invention for preparing a food that is administered to patients with chronic kidney disease, diabetes, and / or obesity.

[0074] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0075] Figure 1 This illustration shows the preparation process of the rice variety of the present invention in one embodiment. Taking W1721 and W0784 as examples, the aggregation process of the two genes LGC-1 and Glb-1 is shown. W1721 is a material carrying the low-glutenin gene LGC-1, and W0784 is a material carrying the globulin deletion gene Glb-1. After hybridization of the two materials, a new material containing both genes is bred using the pedigree method.

[0076] Figure 2 The SDS-PAGE electrophoresis patterns of total protein from seeds of Ningjing 4, W1721, W0784, and the new rice lines described in this invention are shown. The lanes are as follows: 1 is Ningjing 4, a conventional japonica rice variety used as a control; 2 is W1721, a rice variety containing only the low-glutenin gene (LGC-1); 3 is W0784, a rice material containing only the globulin deletion gene (Glb-1); 4 represents the new rice lines exhibiting both low-glutenin and globulin deletion traits.

[0077] Figure 3 This study demonstrates the detection of LGC-P3(A) and LGC-N(B) low-glutenin molecular markers in segregating generations of a population. The offspring of the segregating population were analyzed using LGC-P3 F / R primer pairs and LGC-N F / N primer pairs. If the LGC-P3 primer pair produced an amplification product while the LGC-N primer pair did not, the offspring carried the homozygous LGC-1 gene; if the LGC-P3 primer pair produced no amplification product while the LGC-N primer pair did, the offspring did not carry the LGC-1 gene; and if both primer pairs produced amplification products, the offspring carried the heterozygous LGC-1 gene. The lanes are shown below: M represents the DNA molecular weight standard, and 1-24 represent 24 individual plants from the segregating population.

[0078] Figure 4 This diagram illustrates the detection of the Glb-N1(A) and Glb-P1(B) globulin deletion molecular markers in segregating generations of a population. The offspring of the segregating population were analyzed using Glb-N1 F / R and Glb-P1 F / R primer pairs. If no amplification product was found with Glb-N1 F / R but with Glb-P1 F / R, the offspring were homozygous for globulin deletion. If amplification was found with Glb-N1 F / R but with no amplification product with Glb-P1 F / R, the offspring were not globulin-deficient. If both primer pairs produced amplification products, the offspring were heterozygous for the globulin deletion trait. The lanes are as follows: M represents the DNA molecular weight standard, and 1-24 represent the 24 individual plants in the segregating population.

[0079] Figure 5 The image shows the SDS-PAGE electrophoresis pattern of total protein from seeds of selected individual plants in the segregating population. Twelve seeds were randomly selected from each individual plant for total protein extraction and SDS-PAGE. As shown in the figure, lanes 1-12 represent 12 seeds from the same individual plant, and lanes 13-24 represent 12 seeds from another individual plant. The electrophoresis results were used to determine the presence of glutenin and globulin deficiency traits in the individual plants. Both individual plants were homozygous for glutenin and heterozygous for globulin. Blank lanes were used to distinguish multiple gels electrophoresed simultaneously; the second lane being empty indicates that it is the second gel from the same batch.

[0080] Figure 6The images show the locations of the GluB4 / B5 (A) and 26kDa (Glb-1) (B) globulin sgRNAs and the knockout effects. In Figure A, because GluB4 and GluB5 are inverted repeat sequences with 99.8% homology, a single sgRNA can cleave both sites simultaneously, resulting in a large deletion in the middle (CRI-GluB4 / 5). The post-transcriptional inverted complementary sequences form a hairpin structure, producing a small double-stranded RNA, thus interfering with the glutenin family and resulting in a hypoglutenin dominant trait. In Figure B, globulin knockout causes a 1bp base insertion (CRI-Glb-1), inducing a frameshift mutation that leads to premature translation termination. Arrows indicate the sgRNA locations.

[0081] Figure 7 The SDS-PAGE electrophoresis pattern of total protein from seeds of the homozygous line (CR-GluB4 / 5Glb1) with GluB4 / B5 and Glb1 knocked out is shown. NJ4 is Ningjing 4 (the transgenic recipient variety). In the homozygous line CR-GluB4 / 5Glb1, mature glutenin content decreased, the 26kDa globulin band was absent, and the content of absorbable protein was further significantly reduced.

[0082] Figure 8 This image shows the measurement of endosperm cell wall thickness in the new rice variety described in this invention. (AD) shows the cell wall morphology of the developing endosperm; (FI) shows the cell wall morphology of the mature seed endosperm. (E) and (J) show the statistical results of cell wall thickness in the developing endosperm (E) and mature endosperm (J), respectively. (B) and (D) are enlarged images within the red boxes in (A) and (C), respectively. NJ4, Ningjing 4; red arrows indicate cell walls, **** indicates P < 0.0001.

[0083] Figure 9 The cell wall thickness of the CR-GluB4 / 5Glb1 endosperm is shown. (AD) shows the cell wall morphology of the developing endosperm; (FI) shows the cell wall morphology of the mature seed endosperm. (E) and (J) show the statistical results of cell wall thickness in the developing endosperm (E) and mature endosperm (J), respectively. (B) and (D) are enlarged images within the red boxes in (A) and (C), respectively. The red arrows indicate the cell walls.

[0084] Figure 10 The SDS-PAGE electrophoresis patterns of seed proteins from strains obtained by reducing gluten content using RNA interference are shown. As can be seen from the figure, homozygous seeds exhibit a seed protein phenotype similar to LGC-1 mutants and CRI-GluB4 / B5 knockouts, with decreased gluten content and compensatory increases in globulin and prolamins. The lanes are as follows: 1 and 2 represent two homozygous single plants, and 3 represents Ningjing 4.

[0085] Figure 11The digestibility rates of different rice varieties are shown in Figure A. Figure A shows the digestibility rates of Ningjing 4 and the new rice line described in this invention; Figure B shows the digestibility rates of Ningjing 4 and CR-GluB4 / 5Glb1. The results indicate that when both genes are combined (GluB4 / B5 is knocked out or knocked down, and Glb-1 is knocked out or knocked down), the digestibility rate is significantly reduced. *, P < 0.05; **, P < 0.01. Detailed Implementation

[0086] Through extensive and in-depth research and long-term experimentation, the inventors have, for the first time, established a method for creating improved rice with low absorbable protein content and thickened endosperm cell walls. This method, by regulating the activity or expression of absorbable protein-related polypeptides and their encoding genes, has for the first time achieved a reduction in absorbable protein content in rice seeds and a thickening of the rice endosperm cell walls. Rice with low absorbable protein content and thickened endosperm cell walls is beneficial for the adjunctive treatment of chronic kidney disease and has the potential to control postprandial blood glucose. Based on this, the inventors completed this invention.

[0087] Specifically, the inventors utilized a low-gluten mutant W1721 (variety rights number CNA20070659.4), derived from W3660 (variety rights number CNA20020113.1), which carries the dominant low-gluten gene LGC-1, exhibiting a significant decrease in glutenin (absorbable) content and a significant increase in globulin (absorbable) and prolamins (non-absorbable). To further reduce the absorbable protein content in rice, a 26kDa globulin deletion mutant glb-1 was obtained through large-scale mutagenesis. The low-gluten gene LGC-1 and the 26kDa globulin deletion gene glb-1 were combined through hybridization breeding. New materials combining the two mutant genes were developed using pedigree hybridization breeding combined with molecular marker-assisted selection; or new materials combining the low-gluten deletion gene (e.g., CRI-GluB4 / B5) and the 26kDa globulin deletion gene CRI-Glb-1 were created through gene editing. By reducing the synthesis of rice storage proteins, especially absorbable proteins (mainly glutenin and globulin), through the above two methods, the synthesis of non-absorbable prolysin is promoted. At the same time, the cell wall is significantly thickened, which significantly reduces the digestion rate of starch. This creates a new functional rice material with low absorbable protein and thick cell wall, which has potential adjunctive therapeutic effects on chronic kidney disease, diabetes, obesity, etc.

[0088] The methods for reducing the content of absorbable proteins and increasing cell wall thickness are selected from one or more of the following methods in combination: (1) obtaining endogenous gene mutations through physical mutagenesis, chemical mutagenesis or natural mutation to reduce the content of absorbable proteins (glutenin and globulin); (2) knocking out or knocking down endogenous genes through gene editing technology or RNA interference technology to reduce the content of absorbable proteins (glutenin and globulin); (3) hybridization breeding and molecular marker-assisted selection breeding;

[0089] Therefore, the present invention provides a novel method for reducing starch digestion rate by increasing endosperm cell wall thickness by reducing absorbable storage proteins in rice. The method includes reducing the content of absorbable proteins in rice by aggregating the low-glutenin gene LGC-1 and the globule-deficient gene glb-1, or knocking out or knocking out the low-glutenin gene and the globule-deficient gene Glb-1, thereby increasing the synthesis of cell wall substances in rice and endowing rice with the function of lowering blood sugar.

[0090] the term

[0091] 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 invention pertains.

[0092] As used in this article, the terms "absorbable protein," "endogenously absorbable protein," "absorbable storage protein," and "endogenously absorbable storage protein" are used interchangeably and all refer to glutenin, 26kDa globulin, or a combination of glutenin and 26kDa globulin derived from rice. Because glutenin and globulin are stored in irregular type II protein bodies, they can be digested and absorbed by the human body, hence the term absorbable protein.

[0093] As used herein, "glutenin gene," "glutenin family gene," and "glutenin gene family" refer to genes that encode gluten, and the collection of gluten encoded by these genes constitutes the "glutenin family." Gluten is composed of acidic and basic subunits. In rice, gluten is encoded by multiple genes from four subfamilies: GluA, GluB, GluC, and GluD. These subfamilies are classified based on the similarity of the amino acid sequences encoded by their genes. In this invention, the glutenin genes used to obtain the rice varieties of this invention include, but are not limited to, the GluB4 / B5 gene of the GluB family and the GluA1 gene of the GluA family.

[0094] As used herein, "GluB4 / B5" refers to a sequence segment containing the GluB4 and GluB5 genes. The GluB4 and GluB5 genes are inverted repeat sequences, sharing 99.8% homology, and are closely adjacent. Both are involved in gluten synthesis, hence the collective term GluB4 / B5. The typical nucleotide sequence of GluB4 / B5 in this invention is shown in SEQ ID NO:23.

[0095] As used herein, "LGC-1" is a dominant low-glutenin gene; plants containing this gene exhibit significantly reduced glutenin content. LGC-1 is an inactivated gene formed by a large deletion of GluB4 / B5. This gene inhibits glutenin expression through RNA interference (RNAi) mechanism; normal expression or upregulation of its expression leads to a decrease in glutenin content. The typical nucleotide sequence of LGC-1 in this invention is shown in SEQ ID NO:24.

[0096] As used herein, "conserved region" and "conserved sequence" are used interchangeably and refer to similar or identical sequences in nucleic acid or protein sequences. Typically, conserved regions are crucial for the normal expression of genes or the normal function of proteins. Inactivation or deletion of conserved regions can lead to decreased or lost expression of related genes or function of proteins, thereby affecting related phenotypes. In this invention, the conserved regions of the glutenin family genes are identical or similar sequences present in most or all glutenin genes. Inactivation or deletion of these conserved regions can lead to decreased or abnormal expression of glutenin genes.

[0097] As used herein, the terms "globulin" and "26kDa globulin" are used interchangeably, both referring to 26kDa globulin derived from rice. The typical nucleotide sequence of the globulin gene in this invention is shown in SEQ ID NO:25.

[0098] As used herein, the term "inactivating variant" refers to a gene that has lost its original function due to the substitution, deletion, or insertion of one or more nucleotides, but can still be expressed normally. This invention relates to variants of genes encoding inactivating gluten or globulins, which can be naturally occurring or non-naturally occurring variants. These variants of gene encoding include substitution variants, deletion variants, and insertion variants.

[0099] This invention relates to rice and its preparation method.

[0100] As used herein, the terms "rice of the present invention," "double knockout rice of the present invention," "rice variety of the present invention," "new rice line of the present invention," and "glutB4 / B5 and Glb-1 double gene knockout rice of the present invention" refer to improved rice varieties in which the expression of glutB4 / B5 and Glb-1 is downregulated. Compared with wild-type rice varieties (such as varieties with normal expression of glutB4 / B5 and Glb-1 genes, such as Ningjing 4), the rice varieties of the present invention are characterized by low content of absorbable protein and significantly thickened endosperm cell walls.

[0101] In this invention, conventional hybridization breeding and / or genetic engineering methods can be used to manipulate wild-type rice varieties (i.e., varieties with normal expression of GluB4 / B5 and Glb-1 genes), rice varieties with GluB4 / B5 single gene knockout, or rice varieties with Glb-1 single gene knockout, thereby obtaining the rice varieties with GluB4 / B5 and Glb-1 double gene knockout of this invention.

[0102] As used in this article, the terms "hybrid breeding," "pedigree method," "generation increase," and "molecular marker-assisted breeding" refer to conventional breeding methods, techniques, or means. Hybrid breeding refers to the method of crossing parents to form hybrid offspring with genetic diversity, and then selecting new varieties that possess the superior traits of the parents without carrying undesirable traits through screening. The pedigree method is a commonly used selection method in hybrid breeding. Starting from the first segregating generation, individuals meeting the requirements are selected generation by generation according to the breeding objective until the required homozygosity is achieved, at which point the pedigrees are mixed and harvested as new varieties. Since most crops can only be planted once a year, developing new varieties through traditional breeding methods such as hybrid breeding is time-consuming. Because hybrid breeding is usually conducted at the individual biological level, manipulating the entire genome, it is impossible to accurately manipulate and select individual genes, resulting in poor predictability of offspring traits. Generation increase refers to accelerating the generation process in breeding, shortening the breeding cycle, and is used to address the problem of long breeding times in traditional methods. Molecular marker-assisted breeding utilizes the close linkage between molecular markers and target trait genes. By detecting molecular markers, the presence of the target gene can be detected, thus addressing the problem of poor predictability of traits in hybrid offspring.

[0103] In a preferred embodiment of this invention, the method employed is pedigree hybridization breeding and generational extension with molecular marker assistance.

[0104] In this invention, in a preferred embodiment (such as...) Figure 1The pedigree hybridization breeding and generation and molecular marker-assisted selection method used in the above-mentioned F1 generation hybrid seeds are as follows: (1) Using a low-gluten rice variety as parent A and a globulin deletion mutant as parent B, parent A and parent B are sexually hybridized to obtain F1 generation hybrid seeds; (2) F1 generation hybrid seeds are planted, and individual plant seeds are harvested. Individual plants are planted again to obtain F2 population; (3) Starting from the F2 generation population, individual plants with good agronomic traits are selected. Individual plants containing both the low-gluten gene (LGC-1) and the globulin deletion gene (glb-1) are selected from the above-mentioned individual plants using molecular marker-assisted selection. At maturity, the seeds of F2 individual plants are harvested (F3 generation). Then, the total protein of the 12 seeds of the selected individual plants is extracted individually and identified by SDS-PAGE electrophoresis; (4) For F2 generation hybrids with two genes, the total protein of the seeds is extracted individually and identified by SDS-PAGE electrophoresis. 3. Individual plants are further planted and identified, and individual plants with good agronomic traits and homozygous for both gene loci are selected for further planting. After maturity, F4 generation seeds are harvested; (5) From the F4 generation population, individual plants with stable agronomic traits and homozygous for both the low-glutenin gene (LGC-1) and the globule deletion gene (glb-1) are selected. The protein phenotype and agronomic traits are observed by adding generations. Individual plants with agronomic traits and both genes no longer segregate are selected and cultivated into new lines with aggregated low-glutenin and globule deletion traits; (6) Wherein, parent A is a rice material containing the rice low-glutenin gene (LGC-1); and parent B is a rice material containing the rice globule deletion gene (glb-1). Parent A or parent B can be obtained by physical or chemical mutagenesis, natural variation, gene editing technology, or RNA interference technology. This preferred example includes sexual hybridization between parent A and parent B. Both parent A and parent B can be used as the maternal or paternal parent.

[0105] In another preferred embodiment, wild-type rice varieties (i.e., varieties with normal expression of GluB4 / B5 and Glb-1 genes) are genetically engineered using gene editing techniques to obtain the rice variety with GluB4 / B5 and Glb-1 double gene knockout of the present invention.

[0106] As used in this article, the term "gene editing" refers to the process of modifying specific targets in the genome of an organism using gene editing technology, thereby efficiently and precisely inserting, deleting, or replacing genes to alter their genetic information and phenotypic characteristics. Commonly used gene editing technologies include zinc finger nuclease technology, transcription activator-like effector nuclease technology, and clustered regularly spaced short palindromic repeat-associated nuclease technology (CRISPR-Cas). Among them, CRISPR-Cas consists of a set of repetitive sequences, namely CRISPR sequences, and the nuclease Cas protein. The most common CRISPR-Cas system is the CRISPR-Cas9 (CRISPR / Cas9) system. CRISPR / Cas9 is mainly used for gene knockout and gene insertion or replacement. Gene knockout introduces double-strand breaks into the target gene, and the cell repairs the breaks through the non-homologous end joining mechanism. During this process, insertion or deletion mutations may be introduced, leading to the loss of function of the target gene. Gene insertion or replacement uses homologous DNA as a template, and the cell repairs double-strand breaks through the homologous recombination mechanism to introduce specific mutations or sequences into the target gene.

[0107] In this invention, the CRISPR / Cas9 system is used for gene knockout, or knockout. Specifically, CRISPR / Cas9 contains a single-stranded guide RNA (sgRNA) of the target gene and the Cas9 protein. The sgRNA consists of crRNA and tracrRNA, wherein the crRNA contains a 20 bp nucleotide sequence homologous to the target gene. Through complementary pairing with the target gene, the crRNA guides the Cas9 protein to the PAM sequence near the target gene, cleaving the PAM sequence 3-4 bases upstream, inducing double-strand breaks in the cell, and causing loss of function of the target gene. Those skilled in the art will understand that the term "sgRNA" as used herein refers to the 20 bp nucleotide sequence of the sgRNA determined by the target gene, including the sgRNA sequence itself or the nucleotide sequence encoding the sgRNA sequence. In a preferred embodiment, the sgRNA sequence is a sequence targeting GluB4 / B5 or a sequence targeting Glb-1.

[0108] The present invention also relates to a knockout vector of sgRNA containing a glutenin gene (GluB4 / B5) and a globulin gene (Glb-1), a host cell generated by genetic engineering using the knockout vector, and a method for knocking out the glutenin gene and the globulin gene using gene editing technology.

[0109] In this invention, CRISPR / Cas9-mediated gene knockout technology can target the genes using the sgRNAs of the glutenin and globulin genes, thereby reducing the expression of the genes. Generally, the steps are as follows: (1) transforming or transducing suitable host cells using the sgRNAs of the glutenin and globulin genes, or using a knockout vector containing the sgRNAs of the genes; (2) culturing the host cells under suitable culture conditions; and (3) generating CRISPR / Cas9 protein within the cells to knock out the genes.

[0110] As used herein, the terms "gene knockdown" and "knockdown" refer to the process of suppressing gene expression using inhibitors or repressive methods, without affecting the gene sequence itself. In this invention, the primary method of suppressing gene expression is RNA interference.

[0111] As used herein, the terms “RNA interference,” “RNA silencing,” “RNAi,” “RNAi interference,” and “gene interference” are used interchangeably and refer to the phenomenon of reduced or silenced gene expression caused by the degradation of homologous mRNA induced by double-stranded RNA (dsRNA). Specifically, long dsRNA is processed into small interfering RNA (siRNA) double strands by the endonuclease Dicer. Subsequently, the siRNA binds to the Ago protein, participating in the formation of the RNA-induced silencing complex (RISC). The siRNA is further divided into a guide strand and a delivery strand, both single strands. Finally, the delivery strand is degraded, and the guide strand-RISC complex binds to complementary target mRNA. Ago uses its endonuclease activity to cleave the target mRNA, and the resulting RNA fragments are easily degraded by exonucleases, thereby triggering posttranscriptional gene silencing.

[0112] In this invention, the LGC-1 gene is formed by the deletion of a large fragment of GluB4 / B5. It can be expressed normally and can form dsRNA to interfere with glutenin genes, thereby reducing glutenin gene expression. Furthermore, this invention also designs interfering sequences based on the conserved sequence of GluA1 to interfere with the glutenin gene family and reduce glutenin gene expression. The gene interference methods used to obtain the new rice lines of this invention include, but are not limited to, using RNAi to reduce glutenin expression.

[0113] In this invention, the sgRNA or interfering sequences of the glutenin and globulin genes described above can be inserted into a knockout vector or interfering vector. As used herein, the terms "knockout vector" and "interfering vector" refer to expression vectors containing foreign genes, including bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. Any plasmid and vector capable of replicating and remaining stable within a host cell can be used. An important characteristic of knockout or interfering vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements. Those skilled in the art can construct knockout or interfering vectors containing sgRNA or interfering sequences, as well as suitable transcriptional or translational control signals, using well-known techniques and methods. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The sgRNA or interfering sequence can be effectively linked to an appropriate promoter in the knockout or interfering vector to guide mRNA synthesis. The knockout or interfering vector should include a ribosome binding site for translation initiation and a transcription terminator. Typically, the knockout or interference vector should also include one or more selective marker genes that provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline, kanamycin, or ampicillin resistance for prokaryotic cell culture.

[0114] The knockout vector comprising the aforementioned suitable sgRNA sequence and a suitable promoter or control sequence, and the interference vector comprising the aforementioned suitable interference sequence and a suitable promoter or control sequence, as described in this invention, can be used to transform suitable host cells to enable them to express proteins. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples include: prokaryotic cells such as *Escherichia coli*, *Streptomyces*, and *Agrobacterium*; and fungal cells such as yeast and plant cells.

[0115] The knockout or interference vectors used in this invention can be used to transform host cells using conventional techniques well-known to those skilled in the art. When the host cell is a prokaryote such as *Escherichia coli*, the competent cell method can be used, i.e., the cells are treated with metal ions and collected after the exponential growth phase for absorption of the knockout vector; electroporation can also be used. When the host cell is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. For plant transformation, *Agrobacterium* transformation or gene gun transformation are preferred. In this invention, *Agrobacterium* transformation is used to transform plants. The transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain transgenic plants.

[0116] In a preferred embodiment of the present invention, by using gene editing technology to knock out or knock down rice storage protein genes, the content of endogenous absorbable storage proteins (glutenin and / or globulins) can be reduced, thereby reducing the total absorbable protein content in rice seeds; in addition, the synthesis of cell wall substances in seeds can be increased, thereby thickening the endosperm cell wall.

[0117] In a preferred embodiment, the representative method of the knockout includes the following steps: (1) designing specific CRISPR / Cas9 knockout primers based on rice storage protein genes; (2) constructing a CRISPR / Cas9 expression vector and using a 35S promoter to mediate the expression of CRISPR / Cas9; (3) using the constructed CRISPR / Cas9 vector to genetically transform rice callus tissue through Agrobacterium-mediated transformation, regenerating transgenic plants, and identifying transgenic plants with site-specific knockout of rice endogenous storage protein genes (glutenin and globulin) through specific PCR and subsequent sequencing technology; (4) obtaining genetically stable transgenic plants lacking low glutenin or prolysin through generation and molecular marker-assisted selection, and combining the low glutenin gene and the globulin deletion gene (glb-1) through sexual hybridization of single knockout plants; or obtaining double knockout plants by simultaneously knocking out the glutenin gene and the globulin gene (glb-1).

[0118] In a preferred embodiment, the representative method of knockdown includes the following steps: (1) designing specific RNAi interference primers based on rice storage protein genes; (2) constructing an interference vector and mediating the expression of the interference vector; (3) using the constructed interference vector to genetically transform rice callus tissue through Agrobacterium-mediated transformation, regenerating transgenic plants, and identifying transgenic plants with site-specific knockdown of rice endogenous storage protein genes (glutenin and globulin) through specific PCR and subsequent sequencing technology; (4) obtaining genetically stable transgenic plants with low glutenin through generation and molecular marker-assisted selection, and combining the low glutenin gene (GluA1-RNAi) and the globulin deletion gene (glb-1) through sexual hybridization; or obtaining double-knockdown plants by simultaneously knocking down the glutenin gene and the globulin gene.

[0119] As used herein, the term "transgenic plant" refers to a plant that has undergone gene knockout or knockdown. In this invention, a transgenic plant is a rice plant with double knockout of the GluB4 / B5 and Glb-1 genes, obtained through gene knockout (e.g., knocking out the GluB4 / B5 gene and / or knocking out the Glb-1 gene) or gene knockdown (e.g., knocking down the GluA1 gene and / or knocking down the Glb-1 gene), namely, CRI-GluB4 / B5. - / - and CRI-Glb-1 - / -The transgenic plants of this invention can be obtained by knocking out the glutenin gene and knocking down the Glb-1 gene, or by knocking down the glutenin gene and knocking out the Glb-1 gene.

[0120] As used herein, the term "homozygosity" refers to the presence of identical alleles at the same gene locus on homologous chromosomes. In this invention, LGC-1 homozygosity means that both alleles are LGC-1; Glb-1 deletion homozygosity means that both alleles are knocked out or inactivated Glb-1; and GluB4 / B5 deletion homozygosity means that both alleles are knocked out or inactivated GluB4 / B5.

[0121] As used herein, the terms "wild-type rice" or "conventional rice" refer to rice varieties that normally express the GluB4 / B5 and Glb-1 genes, such as Ningjing 4, or any rice variety with similar phenotypic characteristics to Ningjing 4. In this context, similar phenotypic characteristics mean that the SDS-PAGE electrophoresis pattern of seed proteins is the same or substantially the same as that of the wild type (e.g., Ningjing 4), and the endosperm cell wall thickness is the same or substantially the same as that of the wild type (e.g., Ningjing 4). Specifically, this means: (a) the absorbable protein content is the same or substantially the same as that of the wild type (e.g., Ningjing 4) (e.g., approximately 90–110%, or approximately 100%), and (b) the endosperm cell wall thickness is the same or substantially the same as that of the wild type (e.g., Ningjing 4) (a ratio of 80–120%).

[0122] As used herein, the term "glycemic index" refers to the relative rate at which blood glucose levels rise within two hours of consuming a food. The glycemic index of rice is negatively correlated with its amylose content; that is, the lower the amylose content, the higher the glycemic index. Consuming high-glycemic index foods (GI > 70) accelerates the development of diabetes, while consuming low-glycemic index foods can control the occurrence and development of related diseases. In this invention, the newly cultivated rice line is a low-glycemic index food, which helps in the prevention and control of related diseases.

[0123] The main advantages of this invention include:

[0124] (1) This invention utilizes hybridization breeding and gene editing methods to cultivate improved rice materials by regulating the activity or expression of endogenous absorbable protein-related polypeptides or their encoding genes in rice. Unexpectedly, it was found that the mature endosperm cell wall thickness of the improved rice materials was about 8 times greater than that of conventional rice.

[0125] (2) The absorbable protein content of the improved rice material described in this invention is significantly lower than that of conventional rice.

[0126] (3) The starch digestibility of the improved rice material described in this invention is significantly lower than that of conventional rice.

[0127] (4) The endosperm is the main component of rice. Therefore, rice products or foods based on the rice varieties of this invention not only have low absorbable protein (mainly glutenin and globulin), but also have a lower glycemic index due to a significant reduction in starch digestibility. Therefore, they are particularly suitable for patients or susceptible individuals with chronic kidney disease, diabetes, obesity, etc., which helps to prevent and control diseases such as diabetes and obesity from the source. They are also particularly suitable as food for patients with chronic kidney disease.

[0128] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0129] Material

[0130] W1721: A low-gluten rice line (CNA20070659.4), derived from the low-gluten rice line W3660 (CNA20020113.1). It exhibits reduced glutenin content but significantly increased globulin and prolamin content. W0784: A globulin-deficient line obtained through large-scale radiation mutagenesis combined with SDS-PAGE screening of Ningjing 4. Electrophoretic patterns show a lack of a 26kDa globulin band. Ningjing 4: A medium-maturing, medium-japonica conventional rice line W008 (CNA20080209.7), obtained through hybridization breeding.

[0131] method

[0132] SDS-PAGE electrophoresis is a method for identifying homozygous or heterozygous genes.

[0133] (1) Use the DYCZ-30 electrophoresis tank manufactured by Beijing Liuyi Instrument Factory. After the glass plates are assembled, seal the base of the glass plates with 1% agar solution that has been heated and melted.

[0134] (2) After the agar solidifies, pour in 15% separating gel solution (prepared according to Table 1) up to 1.5-2 cm from the top of the short plate, and then use a pipette to add 2 mL of 70% ethanol solution to seal the gel.

[0135] (3) After the concentrated gel solidifies, pour off 70% ethanol, pour in the prepared 7.5% concentrated gel solution (prepared according to Table 2), and insert 25-hole 1mm comb teeth (be careful to remove air bubbles under the comb teeth);

[0136] (4) After the concentrated gel solidifies, remove the comb teeth and add 1× electrode buffer (14.42g glycine, 3.03g Tris, 1g SDS, bring the volume to 1000mL).

[0137] (5) Spot 5 μL of protein extraction supernatant;

[0138] (6) During electrophoresis, first apply a constant voltage of 80V. After the indicator enters the separating gel, increase the voltage to 120V. Stop electrophoresis when the indicator is close to the bottom of the gel.

[0139] (7) Peel off the gel and immerse it in Coomassie Brilliant Blue staining solution (250 mL isopropanol, 100 mL acetic acid, 1 g Coomassie Brilliant Blue R250, and bring the volume to 1000 mL) for 1-2 hours.

[0140] (8) Discard the staining solution and add decolorizing solution (10% acetic acid) to decolorize. Change the solution several times in the middle until the bands are clear.

[0141] Table 1. SDS-PAGE Adhesive Preparation and Dosing Table

[0142]

[0143] Determination of mature endosperm cell wall thickness in rice materials

[0144] Measurement of endosperm cell wall thickness in materials containing two genes or transgenic knockout materials:

[0145] (1) Sampling and fixation: The endosperm developed 12 days after flowering was collected. The endosperm was cut into 1-2 mm thick slices using a double-edged blade and fixed in 2.5% glutaraldehyde solution at 4℃.

[0146] (2) Rinsing: Rinse the fixed sample three times with 0.1M phosphate buffer for 10 minutes each time;

[0147] (3) Dehydration: Fix the sections with 1% OsO4 solution for 1 h, then rinse with 0.1 M phosphate buffer 3 times, 10 min each time;

[0148] (4) Dehydration: Dehydrate using 50%-70%-80%-90% acetone in a gradient for 15 minutes each time, then dehydrate using 100% acetone 6 times for 30 minutes each time;

[0149] (5) Impregnation: Epon 812 resin and acetone are prepared in the proportions of 1:3 (V / V, 25%), 1:1 (50%), and 3:1 (75%). The resins are impregnated with 25%-50%-75%-100% concentration for 2h, 4h, 12h, and 12h respectively. Vacuum is applied during the impregnation process.

[0150] (6) Embedding: Fill the trapezoidal groove of the embedding plate with pure resin in advance, put it in the oven for pretreatment, and use toothpicks to transfer the impregnated sample into the embedding plate. Adjust the sample position as needed.

[0151] (7) Polymerization: The embedded plates were placed at 37℃, 45℃ and 60℃ for 48 hours each to allow for full polymerization;

[0152] (8) Trimming the block: Use a blade and sandpaper to trim the embedded block into a trapezoidal shape so that the slice area is less than 0.2mm × 0.2mm;

[0153] (9) Slicing: Use an ultramicrotome to cut slices 70-90nm thick and carefully transfer them to a copper grid.

[0154] (10) Staining: Stain with uranium oxide and lead acetate solutions for 5-15 min and 5-10 min respectively, then wash and dry in a 42℃ drying oven;

[0155] (11) Observation: The sample was observed and photographed using a Hitachi HT7800 transmission electron microscope;

[0156] (12) Statistics: The cell wall was measured and statistically analyzed using ImageJ software.

[0157] Determination of in vitro starch digestion rate in rice materials

[0158] (1) After removing the husks from the rice, the rice was milled to prepare finely milled rice that conforms to GB / T 5502-2018. Each rice grain was weighed and placed in a 2.0 mL centrifuge tube. Six replicates were set up for each family.

[0159] (2) Add water at a rice-to-water ratio of 1:1.2 (or according to the variety) to cook rice, and then add deionized water to 100μL;

[0160] (3) Grind and disperse separately, and add 400 μL of 0.05 M HCl solution containing 5 g / L trypsin and 3 3-4 mm glass beads. Shake and bathe in a water bath at 37 °C for 2 h.

[0161] (4) Add 200 μL NaAc (pH 5.2) to neutralize, add 200 μL enzyme mixture (containing 4 g / L trypsin and 12 g / L glucoamylase), shake in a water bath at 37°C, and start timing;

[0162] (5) Take 80 μL of the sample at 20 min, 2 h and 12 h respectively and put it into about 1 mL of alcohol to terminate the reaction, and then dry it;

[0163] (6) Add 1.6 mL of deionized water to each tube, vortex until fully dissolved, and then determine the glucose content using a high performance liquid chromatograph after passing through a 0.22 μm aqueous phase filter.

[0164] (7) Convert to starch content and calculate the percentage of digested starch in the rice grains relative to the sample weight.

[0165] Detection primer sequences for LGC-1 and glb-1 sites and gene editing primer sequences

[0166] The detection primer sequences and gene editing primer sequences designed and synthesized based on gene sequences are shown in Table 2.

[0167] Table 2. Primer sequences for detection of LGC-1 and glb-1 sites and gene editing primer sequences.

[0168]

[0169] Example 1: Using hybridization breeding and molecular markers to reduce the content of endogenous absorbable proteins and increase cell wall thickness

[0170] like Figure 1 As shown, in this embodiment, rice with dual gene mutations of LGC-1 and glb-1 was prepared using hybridization breeding and molecular marker methods. Specifically, in this embodiment, marker-assisted selection was performed using the detection primer pairs listed in Table 2. The method is as follows:

[0171] The rice variety W1721 (carrying the homozygous LGC-1 gene) was used as the female parent and crossed with the globulin-deficient variety W0784 (carrying the homozygous glb-1 gene) to obtain the F1 generation. The F2 generation was obtained by breeding in Hainan in winter and planting to form an F2 generation population of 1500 plants. 361 individual plants were selected through agronomic trait identification. 23 individual plants were retained after molecular marker-assisted selection and SDS-PAGE protein electrophoresis. F3 lines were planted, and 19 individual plants were selected through agronomic trait selection, molecular marker-assisted selection, and SDS-PAGE electrophoresis. F4 lines were planted, and 3 homozygous individual plants with both low glutenin and globulin deficiency traits and 4 heterozygous individual plants with both low glutenin and globulin deficiency traits were selected. F5 lines were planted, and 2 individual plants with both low glutenin and globulin deficiency and 1 heterozygous individual plant with both low glutenin and globulin deficiency were selected. F6 lines were planted, and 3 individual plants with low glutenin content and globulin deficiency were selected. F7 lines were planted, and 4 individual plants with low glutenin content and globulin deficiency were selected. F8 lines were also planted.

[0172] After screening and identification, four rice lines were obtained that were homozygous for both low glutenin and globulin deficiency traits. Among the four rice lines of this invention, LGC-1 and glb-1 were homozygous for both traits, and their traits were stable. Figure 1 The content of endogenous absorbable protein in this rice is reduced. Figure 2 ).

[0173] In the F2 generation, superior individual plants were selected based on agronomic traits. DNA was extracted from the leaves of these individual plants and analyzed by PCR. If both LGC-P3 F / R and LGC-N F / R produced amplification products, it indicated that the LGC-1 gene in this individual plant was heterozygous. If LGC-P3 F / R produced amplification products but LGC-N F / R did not, the LGC-1 locus was a homozygous genotype with low gluten content. If LGC-P3 F / R did not produce amplification products but LGC-N F / R did, the locus was a non-homozygous genotype with low gluten content. Figure 3 ).

[0174] Similarly, if Glb-P1 F / R and Glb-N1 F / R can amplify simultaneously, it indicates that the glb-1 deletion site of globulin is heterozygous; if Glb-P1 F / R amplifies a band while Glb-N1 F / R does not, the globulin deletion is homozygous; if Glb-P1 F / R does not amplify a band while Glb-N1 F / R amplifies a band, it indicates that the globulin gene at that site is normally homozygous. Figure 4 ).

[0175] After the F2 individual plants matured and produced seeds, the seeds were harvested and dried. Twelve seeds were randomly selected, and each seed was ground and added to 600 μL of protein extraction buffer (4% (w / v) SDS, 4M Urea, 5% (v / v) β-ME, and 0.125M Tris-HCl, pH 6.8, with a small amount of bromophenol blue added as an electrophoresis indicator). The mixture was incubated at 50℃ for 12 h (with frequent inversions to mix) to extract total protein. After centrifugation, 5 μL of the supernatant was collected for SDS-PAGE electrophoresis. The homozygosity and heterozygosity of the LGC-1 and glb-1 genes were determined based on the electrophoretic bands of each seed. Figure 5 ).

[0176] From the F3 population, we continued to select individual plants with excellent agronomic traits. We used a combination of molecular markers and SDS-PAGE to select individual plants until we obtained families with excellent agronomic traits and homozygous LGC-1 and glb-1 loci.

[0177] Cell wall thickness was measured in rice materials obtained through hybridization breeding.

[0178] The results show that:

[0179] For the developing endosperm (taking 12 days after fertilization (12DAF) as an example), the cell wall thickness of the developing endosperm of Ningjing 4 is 101.6 nm, while the cell wall thickness of the developing endosperm of the new rice line described in this invention is 238.2 nm, which is 2.34 times the cell wall thickness of the developing endosperm of Ningjing 4. Figure 8 AE; Table 3); For mature endosperm, the thickness of the mature endosperm cell wall of Ningjing 4 is 121.8 nm, while the thickness of the mature endosperm cell wall of the new rice line described in this invention is 1015 nm, which is 8.33 times the thickness of the mature endosperm cell wall of Ningjing 4 ( ). Figure 8 FJ; Table 3).

[0180] This indicates that, compared to wild-type rice varieties, rice varieties carrying the LGC-1 single mutation, or rice varieties carrying the glb-1 single mutation, the endosperm cell walls of the new rice lines of this invention exhibit an unexpectedly significant thickening. Compared to wild-type (such as Ningjing 4), the endosperm cell walls of the rice of this invention are more than 8 times thicker.

[0181] Increased cell walls in rice endosperm lead to a decrease in digestion rate, which in turn results in a decrease in the glycemic index (GI) of the corresponding rice products.

[0182] Example 2: Using CRISPR / Cas9 technology to reduce the content of endogenous absorbable proteins and increase cell wall thickness

[0183] In this embodiment, rice with double gene knockout of GluB4 / B5 and Glb-1 was prepared using gene editing methods. Specifically, in this embodiment, marker-assisted selection was performed using the detection primer pairs listed in Table 2.

[0184] The sgRNA sequences of GluB4 / B5 (SEQ ID NO. 9) and Glb-1 (SEQ ID NO. 10) were designed, and the CRI-GluB4 / B5-sgRNA and CRI-Glb1-sgRNA vectors were constructed and transformed into Agrobacterium EHA105, respectively. Details are as follows:

[0185] (1) Design of sgRNA sequence: Based on the gene sequences of GluB4 / B5 (NCBI Accession number: AB093593) and Glb-1 (LOC_Os05g41970), a highly specific sgRNA sequence with a length of 20bp was designed.

[0186] (2) Design of knockout primers: GGCA was added to the 5' end of the 20bp sequence of GluB4 / B5 sgRNA as the pre-knockout primer (SEQ ID NO.11). Similarly, the knockout primer for the Glb-1 gene was designed as the pre-knockout primer for Glb-1 (SEQ ID NO.13). After reverse complementation of the sgRNA sequence, AAAC was added to the 5' end to obtain the post-knockout primers (SEQ ID NO.12 for GluB4 / B5 sgRNA and SEQ ID NO.14 for Glb-1 gene). The primers were sent to a company for synthesis. Figure 6 ).

[0187] (3) Construction of the knockout vector: Add 1 μL of the left and right primer stock solution (100 μM) to a 200 μL PCR tube, then add 8 μL of ddH2O (annealed double-stranded primers); 95℃ for 5 min, then allow to cool to room temperature; prepare the following reaction system: ATP 1 μL, vector 1 μL, buffer 1 μL, oligo 0.2 μL, AarⅠ 0.2 μL, T4ase 0.2 μL, ddH2O 5.5 μL, and add 1 μL of annealed double-stranded primer mixture. Reaction program: 37℃ for 5 min; 20℃ for 5 min; 4℃ for 30 min (1×10 cycles).

[0188] (4) E. coli transformation: Take commercially available DH5α competent cells (Tiangen Biotech) and thaw on ice; add 10 μL of ligation vector, gently pipette to mix, and incubate on ice for 30 min; incubate in a 42℃ water bath for 45 s, then immediately incubate on ice for 2 min; add 700 μL of LB liquid medium (antibiotic-free), and incubate at 37℃ for 45 min on a shaker at 100 rpm; spread the bacterial culture evenly on LB medium (containing kanamycin antibiotic), place in a clean bench for about 30 min, and after the bacterial culture in the medium has completely dried, invert it in a 37℃ incubator for overnight incubation; after obtaining colonies, perform colony PCR detection. The primers used are sgRNA-F paired with its own right primer. Select 1-2 positive colonies for sequencing. After successful sequencing alignment, extract plasmids.

[0189] (5) Agrobacterium transformation: Agrobacterium EHA105 competent cells were placed on ice and thawed. 2-3 μL of plasmid was added and mixed with a pipette. After being rapidly frozen in liquid nitrogen for 5 min, the cells were immediately placed in a 37℃ water bath for 5 min of heat shock. After being removed and placed on ice for 2 min, 1 mL of antibiotic-free LB culture medium was added and the cells were placed in a 28℃ shaker for 3-4 h. The bacterial culture was then evenly spread on LB medium (containing kanamycin, hygromycin and rifampin), dried naturally in a clean bench, and then inverted in a 28℃ incubator for 2-3 days.

[0190] Transgenic knockout can be performed using any vector to obtain homozygous positive knockout plants, and then another vector can be used for knockout (sequential knockout); alternatively, simultaneous knockout can be performed by mixing two Agrobacterium tumefaciens bacterial solutions, or simultaneous knockout can be performed using a multi-gene knockout vector.

[0191] The specific steps for transforming Ningjing No. 4 rice using Agrobacterium-mediated transformation are as follows:

[0192] (1) First disinfection of seeds: First, remove the husks from the rice seeds, select clean and plump brown rice without mold spots, place them in 70% ethanol and shake to disinfect for several tens of seconds, discard the ethanol, and wash the seeds 3 to 4 times with sterile water.

[0193] (2) Second disinfection of seeds: Soak the washed seeds in 30% to 50% 84 disinfectant solution for 30 minutes;

[0194] (3) Callus differentiation: Discard the 84 disinfectant, wash the seeds again with sterile water 3-4 times, dry them, and then use sterile tweezers to pick them up and place them on N6 culture medium for culture. Callus will grow after about a month.

[0195] (4) Callus selection: Use sterile forceps to pick up the smaller but compact spherical callus particles differentiated from N6 medium and transfer them to a new N6 medium, and let them sit for a week before use;

[0196] (5) Preparation of Agrobacterium: Agrobacterium containing the correct recombinant plasmid is cultured in LB medium containing the corresponding antibiotics until OD600 = 0.6-0.8, and then placed in a refrigerator at 4℃ for later use;

[0197] (6) Callus infection: Take the above Agrobacterium suspension according to the amount of Agrobacterium required for infection, centrifuge, and resuspend in AS (acetylsuccine) liquid medium. Then soak the fresh callus from step 4 in this Agrobacterium resuspension for 30 min in the dark. Discard the infection solution and air dry the callus. Place a filter paper on the AS solid medium beforehand, place the dried callus on the filter paper, and incubate in the dark at 22°C for 3 days.

[0198] (7) Callus reculture: The above-cultured callus was washed with sterile water and then soaked in carbenicillin for about 20 minutes. The penicillin solution was then discarded. After the callus was dried, it was placed on the selection medium (containing carbenicillin and hygromycin) to continue growing.

[0199] (8) Callus differentiation: After new callus particles grow around the above-cultured callus, these new callus particles are placed in differentiation medium for further culture.

[0200] (9) Emergence: After tiny seedlings with roots have emerged on the differentiation medium, they are transferred to the rooting medium to continue growing until they form seedlings that are about 10 cm tall.

[0201] (10) Wash away the culture medium from the roots of the seedlings, add water to submerge the roots, and place them in a light incubator for about a week to harden them off before transplanting them to the field or greenhouse.

[0202] Identification of offspring of transgenic plants:

[0203] After the seeds of the T0 generation transgenic plants matured, SDS-PAGE was used to identify 12 randomly selected seeds from each plant (T1 generation). Plants with low glutenin trait (heterozygous or homozygous) or globulin deficiency trait (heterozygous or homozygous deficiency) were selected for further planting and identification. The same SDS-PAGE identification was performed on the T2 generation, selecting homozygous low glutenin plants or homozygous globulin deficiency plants. Figure 7 ).

[0204] SDS-PAGE electrophoresis revealed homozygous glutenin-containing monoclonal strains. PCR amplification of the GluB4 / B5 target site was performed using detection primers CRI-GluB4 / B5-F (SEQ ID NO. 15) and CRI-GluB4 / B5-R (SEQ ID NO. 16). The specific reaction system was: 10 μL of 2×Master mix, 2 μL each of GluB4 / B5-F / R (2 nmol), 1 μL of DNA, and water added to a final volume of 20 μL. The reaction program was: 95℃ for 3 min, (95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min) for 35 cycles, 72℃ for 3 min, and 10℃ forever. The amplified products were detected by 1.5% agarose gel electrophoresis. If no band was amplified, it indicated that the intermediate fragment had been knocked out.

[0205] PCR amplification of the Glb-1 target site was performed using CRI-Glb-F (SEQ ID NO.17) and CRI-Glb-R (SEQ ID NO.18). After amplification, 5 μL of the product was analyzed by 1.5% agarose gel electrophoresis, and then Sanger sequencing was performed using CRI-Glb-F (SEQ ID NO.15) to determine the knockout site sequence information. Figure 6 ).

[0206] Cell wall thickness was measured in double knockout rice materials.

[0207] The results show that:

[0208] For the developing endosperm (taking 12 days post-fertilization (12DAF) as an example), the cell wall thickness of the developing endosperm of Ningjing 4 was 101.6 nm, while the cell wall thickness of the developing endosperm of CR-GluB4 / 5Glb1 was 205.5 nm, which is 2.02 times the thickness of the cell wall of the developing endosperm of Ningjing 4. Figure 9AE; Table 3); For mature endosperm, the cell wall thickness of Ningjing 4 mature endosperm is 121.8 nm, while the cell wall thickness of CR-GluB4 / 5Glb1 mature endosperm is 927.6 nm, which is 7.62 times the thickness of Ningjing 4 mature endosperm cell wall ( ). Figure 9 FJ; Table 3).

[0209] This indicates that, compared to wild-type rice varieties, rice varieties with GluB4 / B5 knockout, or rice varieties with Glb-1 knockout, the endosperm cell walls of CR-GluB4 / 5Glb1 showed an unexpectedly significant thickening. Compared to wild-type varieties (such as Ningjing 4), the endosperm cell walls of the rice of this invention increased by approximately 7.6 times.

[0210] Increased cell walls in rice endosperm lead to a decrease in digestion rate, which in turn results in a decrease in the glycemic index (GI) of the corresponding rice products.

[0211] Table 3. Cell wall thickness of endosperm and mature endosperm from different materials

[0212]

[0213] Total protein was extracted from seeds of double knockout material and analyzed by SDS-PAGE. The results showed that the glutenin content was significantly reduced in the knockout family, while the 26kDa globulin content was missing. Figure 7 (Table 4) shows that the protein bands of the materials that were selected by hybridization and molecular marker-assisted selection to aggregate the LGC-1 gene and glb-1 gene in Example 1 are the same or basically the same.

[0214] Table 4. Proportions of different protein components in seeds of double knockout materials

[0215]

[0216] Example 3: Using RNA interference technology to reduce the content of endogenous absorbable proteins and increase cell wall thickness

[0217] In this embodiment, RNA interference technology was used to prepare rice with knockdown of both glutenin family genes and Glb-1.

[0218] Taking GluA1 (LOC_Os01g55690.1) as an example, interference segments were selected based on conserved sequences of the glutenin gene family. RNAi interference vector primers were designed, and the pUBI1390-GluA1 interference vector was constructed and transformed into Agrobacterium EHA105, as detailed below:

[0219] (1) Selection of interference segment: The conserved segments of the glutenin family were identified by comparing the glutenin gene sequence with DNAMAN software. The 61-973bp sequence conserved at the 5' end of GluA1 was selected as the interference segment.

[0220] (2) Design of interference vector primers: Homologous sequences of the two multiple cloning sites of the pUBI1390-RNAi interference vector were introduced into both ends of the interference primers to design two pairs of primers, GluA1-RNAi-F and GluA1-RNAi-R. The specific sequences are shown in Table 5.

[0221] Table 5 Primer sequences for GluA1-RNAi

[0222]

[0223] (3) Construction of the interference vector: The GluA1 gene fragment was amplified using two primer pairs, GluA1-RNAi-F and GluA1-RNAi-R, and the amplified positive and negative interference fragments were recovered. The pUBI1390-RNAi interference vector was digested with SacI restriction enzyme and the digested vector was recovered. The positive interference fragment was inserted into the SacI site of the interference vector by homologous recombination. Recombination reaction system: 50 μg each of digested vector and amplified fragment, 5 μL of recombinase, and ddH2O to make up to 10 μL; reaction program: 50℃ for 20 min, 4℃ for 5 min. The successfully constructed vector was digested again with SnaBI restriction enzyme and recovered. The negative interference fragment was inserted into the SnaBI site of the interference vector by homologous recombination again, completing the construction of the pUBI1390-RNAi-GluA1 vector.

[0224] (4) The methods for Escherichia coli transformation, Agrobacterium transformation and Agrobacterium-mediated transformation of rice are the same as in Example 2;

[0225] Identification of genetically modified offspring:

[0226] After the seeds of the T0 generation transgenic plants matured, SDS-PAGE was used to identify 12 randomly selected seeds from each plant (T1 generation). Those seeds with the low-gluten trait (either heterozygous or homozygous) were selected for further planting and identification. The same SDS-PAGE identification was performed on the T2 generation, selecting homozygous low-gluten plants. Figure 10 ).

[0227] Subsequently, by hybridizing the glb-1 gene deletion gene or further knocking out the Glb-1 gene, or by interfering with the Glb-1 gene via RNAi, rice lines with reduced glutenin content and glb-1 gene deletion or reduction were obtained. These rice lines had reduced absorbable protein content, significantly thickened cell walls, and reduced starch digestion rate.

[0228] Example 4: Determination of in vitro digestion rate

[0229] Increased cell wall size in rice endosperm leads to a decreased digestion rate, which in turn results in a lower glycemic index for the corresponding rice products. In this embodiment, the in vitro digestion rate of the double-knockout rice materials prepared in Examples 1 and 2 was determined, using the method described in "Determination of In Vitro Starch Digestion Rate of Rice Materials".

[0230] The test results showed that the digestion rate of the new rice line prepared by hybridization breeding and molecular marker methods was significantly lower than that of Ningjing 4 at all time points. Figure 11 A).

[0231] Similarly, the transgenic rice CR-GluB4 / 5Glb1 prepared based on gene editing of this invention showed significantly lower digestion rates at all time points than Ningjing 4 ( Figure 11 B).

[0232] This suggests that the rice of this invention is a low glycemic index food.

[0233] discuss

[0234] Currently, there are rice varieties on the market that control postprandial blood sugar, most of which are high-resistant starch (RS) rice. However, the inherent characteristics of high-RS rice result in poor appearance quality, characterized by opaque or powdery grains and poor taste. Some consumers report that it tastes "like chewing wax" and is "difficult to swallow," making it difficult for consumers to accept. Therefore, how to cultivate new low-GI rice varieties while maintaining rice quality is an urgent problem to be solved.

[0235] To address the issues of poor appearance and palatability in existing resistant starch rice varieties suitable for diabetics, this invention provides a new rice material that expands the applicable population, has better appearance and palatability, and also has the function of lowering postprandial blood glucose. The new rice line, synthesized by integrating the LGC-1 and glb-1 genes, demonstrates superior taste compared to the conventional japonica rice variety Ningjing 4, whether measured by machine or tasted manually.

[0236] Rice products or foods made from the rice varieties of this invention are not only low in absorbable protein (mainly gluten and globulin), but also have a lower glycemic index due to significantly reduced starch digestibility. Therefore, they are particularly suitable for patients or susceptible individuals with chronic kidney disease, diabetes, obesity, etc., and can help prevent and control diseases such as diabetes and obesity from the source. They are also particularly suitable as food for patients with chronic kidney disease.

[0237] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method of improving a rice variety, characterized by, The method comprises steps of: (a) providing a rice plant to be improved; and (b) down-regulating the expression of glutelin genes and down-regulating the expression of globulin genes in the rice plant to be improved, thereby improving the rice variety.

2. The method of claim 1, wherein, The improved rice variety has a significantly increased cell wall thickness of the mature endosperm.

3. The method of claim 1, wherein, The improved rice variety has a significantly decreased absorbable protein content.

4. The method of claim 1, wherein, The down-regulation comprises gene knockout or gene knockdown.

5. An improved rice plant, characterized by, The improved rice is obtained by the method of claim 1.

6. A gene editing reagent, characterized in that, The gene editing reagent is used to down-regulate the expression of absorbable protein related polypeptides in rice cells or plants, the absorbable protein related polypeptides being a combination of the glutelin family and globulin.

7. Use of a gene editing reagent according to claim 6, characterized in that, A preparation for regulating the cell wall thickness of rice endosperm.

8. A food product, characterized by, The food contains or consists of the endosperm of rice, wherein the rice is a rice with glutelin and globulin gene knockout or knockdown.

9. Use of endosperm of rice or a food containing the endosperm of rice, characterized in that, A food with a low glycemic index.

10. Use of a food product according to claim 8, characterized in that, A food for preparing a food to be administered to patients with chronic kidney disease, patients with diabetes, and / or patients with obesity.

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