Genes regulating the formation of kelly belt and the balance of mineral nutrient elements and their application

Abstract

The application provides a gene for regulating the formation of a kelly band and the balance of mineral nutrient elements and application thereof. The gene for regulating the formation of a kelly band and the balance of mineral nutrient elements is named as OsSGN1. The application provides a new scheme for the development of a kelly band of a gramineous plant, the regulation of ion group homeostasis, the balance of mineral nutrient elements, and the improvement of plant height and yield traits.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, this invention relates to a gene that regulates the formation of Casparian strips and the balance of mineral nutrients and its application. Background Technology

[0002] Casparian strips have existed in the roots of ferns and angiosperms for approximately 400 million years. As an apoplast barrier surrounding the central vascular system in the root, the Casparian strip selectively absorbs mineral nutrients and plays a crucial role in regulating mineral transport and balance in Arabidopsis. However, the structure of the Casparian strip in rice differs significantly from that in Arabidopsis. First, previous studies suggested a two-layered Casparian strip structure in rice roots: one in the endodermis and another in the exodermal cells, where significant accumulation of lignin and suberin is observed in the cell walls. Second, previous research also reported differences in the structure of the rice endodermal Casparian strip compared to Arabidopsis. For example, in Arabidopsis, the Casparian strip on the endodermal cell wall is only distributed within a small area in the center of the cell wall, while in rice, it is distributed throughout the transverse and radial cell walls. These structural differences indicate a different Casparian strip development process in rice compared to Arabidopsis.

[0003] Previous studies have identified a gene called OsCASP1 in rice (Wang et al., 2019) and confirmed its involvement in the formation of the Casparian strip in the rice endodermis and the regulation of mineral homeostasis through subcellular localization observation, histochemical staining, and ionomics analysis. This was the first report of gene-level regulation of Casparian strip development and function in rice; however, the specific mechanism by which this gene functions remains unclear. Our latest research shows that a novel protein, GAPLESS, specifically located in the cell wall where the Casparian strip is located, can form a stable complex with OsCASP1 located at the CSD of the cell membrane. This complex binds the Casparian strip to the cell membrane, blocking the free diffusion of water and ions in the roots and further controlling nutrient transport and growth in rice.

[0004] However, there is still a need in this field to discover more regulatory factors that regulate the development or function of Casparian strips in plants, and thus regulate plant phenotypes, and to explore more clearly defined mechanisms of Casparian strip development. Summary of the Invention

[0005] The purpose of this invention is to provide a gene that regulates the formation of Casparian strips and the balance of mineral nutrients, and its application.

[0006] In a first aspect of the invention, there is provided an application of the OsSGN1 gene or the protein encoded thereon or the regulatory molecule thereof for: (i) regulating plant height; (ii) regulating Casparian strip development in plants; (iii) regulating the ionotype of plant leaves; and (iv) regulating yield traits in plants; wherein the plant is a grass.

[0007] In a preferred embodiment, the OsSGN1 gene or the protein it encodes includes its homologs.

[0008] In another preferred embodiment, the regulatory molecule is a downregulatory molecule that downregulates the expression or activity of the OsSGN1 gene or its encoded protein, wherein the downregulatory molecule of the OsSGN1 gene: (i) reduces plant height; (ii) increases Casparian strip holes, resulting in Casparian strip developmental defects; (iii) alters the ion composition of plant leaves, changes the levels of mineral nutrients, and causes them to exhibit low potassium and high calcium ion characteristics. Preferably, the alteration of the ion composition includes: reducing the ion levels of P, Mg, S, K, Cu, and Mo, and increasing the ion levels of Sr and Ca; (iv) reduces plant yield. Preferably, the reduction in yield includes: reducing the number of tillers, reducing the number of grains per spike, and reducing the weight of grains per spike.

[0009] In another preferred embodiment, the regulatory molecule is an upregulating molecule that upregulates the expression or activity of the OsSGN1 gene or its encoded protein (preferably, upregulation in plants with low / no OsSGN1 expression or low / no activity, such as increasing the expression or activity of OsSGN1 to normal or high levels), wherein the upregulating molecule of the OsSGN1 gene: (i) increases (including maintains) plant height; (ii) promotes (including maintains) the development of Casparian strips (normal development); (iii) maintains the mineral nutrient balance of plant leaves; and (iv) increases (including maintains) plant yield; preferably, increasing (including maintaining) yield includes: increasing (including maintaining) the number of tillers, increasing (including maintaining) the number of grains per spike, and increasing (including maintaining) the weight of grains per spike.

[0010] In another preferred embodiment, the downregulated molecule includes: a gene editing reagent that specifically edits the OsSGN1 gene, an interfering RNA molecule (such as siRNA, shRNA) or antisense nucleotide that specifically interferes with the expression of the OsSGN1 gene, and a reagent that knocks out the OsSGN1 gene through homologous recombination; preferably, the gene editing reagent that specifically edits the OsSGN1 gene is sgRNA, the nucleotide sequence of which is shown in SEQ ID NO:3.

[0011] In another preferred embodiment, the upregulated molecule includes: a polynucleotide (such as SEQ ID NO:1) or construct encoding OsSGN1, an upregulated molecule that promotes the promoter-driven ability of the OsSGN1 gene, and an upregulated molecule that interacts with the OsSGN1 protein to increase its expression or activity.

[0012] In another aspect of the present invention, a method for regulating plant traits is provided, comprising: regulating the expression or activity of the OsSGN1 gene or its encoded protein in a plant; wherein the plant traits include: (i) plant height; (ii) Casparian strip development; (iii) leaf ionome phenotype; (iv) yield; and the plant is a grass.

[0013] In a preferred embodiment, the method includes: downregulating the expression or activity of the OsSGN1 gene or its encoded protein, thereby: (i) reducing plant height; (ii) increasing Casparian strip pores, resulting in Casparian strip developmental defects; (iii) altering the ion composition of plant leaves, changing the levels of mineral nutrients, and giving them a low potassium and high calcium ion profile, preferably, the alteration of the ion composition includes: decreasing the ion levels of P, Mg, S, K, Cu, and Mo, and increasing the ion levels of Sr and Ca; (iv) reducing plant yield; preferably, reducing yield includes: reducing the number of tillers, reducing the number of grains per ear, and reducing the weight of grains per ear.

[0014] In another preferred embodiment, the expression or activity of the OsSGN1 gene or the protein it encodes is upregulated, thereby: (i) increasing (including maintaining) plant height; (ii) promoting (including maintaining) the development (normal development) of the Casparian strip; (iii) maintaining the ion profile of plant leaves including the normal potassium ion; and (iv) increasing (including maintaining) plant yield. Preferably, increasing (including maintaining) yield includes: increasing (including maintaining) the number of tillers, increasing (including maintaining) the number of grains per ear, and increasing (including maintaining) the weight of grains per ear.

[0015] In another preferred embodiment, the downregulation of the expression or activity of the OsSGN1 gene or its encoded protein includes: knocking out or silencing the OsSGN1-coding gene in a plant, or inhibiting the activity of the OsSGN1-coding protein; preferably, it includes: gene editing using a CRISPR system to knock out the OsSGN1-coding gene, knocking out OsSGN1 by homologous recombination, performing a loss-of-function mutation on OsSGN1 in a plant containing OsSGN1, or silencing OsSGN1 with an interfering molecule that specifically interferes with gene expression; preferably, the downregulation is performed using a gene editing reagent that specifically edits the OsSGN1 gene, the reagent comprising sgRNA, the nucleotide sequence of which is shown in SEQ ID NO:3.

[0016] In another preferred embodiment, the upregulation of the expression or activity of the OsSGN1 gene or the protein it encodes includes (but is not limited to): transferring the OsSGN1 gene or an expression construct or vector containing the gene into a plant; performing a gain-of-function mutation on OsSGN1; promoting OsSGN1 expression by expressing an enhancing promoter or a tissue-specific promoter; or promoting OsSGN1 expression by an enhancer.

[0017] In another preferred embodiment, the protein encoded by OsSGN1 (OsSGN1) comprises: (a) a protein with the amino acid sequence shown in SEQ ID NO:2; (b) a protein derived from (a) having the function of (a) formed by substituting, deleting, or adding one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the amino acid sequence shown in SEQ ID NO:2; (c) a protein having the function of (a) with an amino acid sequence that is 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more; e.g., 98% or more or 99% or more) identical to the amino acid sequence defined in (a); or (d) a protein formed by adding a tag or signal peptide to both ends of any of (a)-(c).

[0018] In another preferred embodiment, the plant includes the group below or the OsSGN1 is derived from the group below: cereal plants of the Poaceae family; preferably, the plant includes (but is not limited to): rice, wheat, millet, foxtail millet, corn, sorghum, millet, barley, rye, oats, and sedge.

[0019] In another aspect of the invention, an application of the plant OsSGN1 gene or the protein encoded therein is provided for use as a molecular marker for identifying plant traits; wherein the plant traits include: (i) plant height; (ii) Casparian strip development; (iii) leaf ionome phenotype; (iv) yield; and the plant is a grass.

[0020] In another aspect of the present invention, a method for analyzing plant traits is provided, the method comprising: analyzing the expression or activity of the OsSGN1 gene or its encoded protein in a test plant; if the expression or activity of the OsSGN1 gene or its encoded protein in the test plant is lower than (significantly lower, such as more than 10%, 20%, 30%, 50% or lower) the average value of the OsSGN1 gene or its encoded protein in this type of plant, then it is (i) low plant height (significantly low); (ii) exhibiting Casparian strip holes or Casparian strip developmental defects phenotype; (iii) the leaf ion profile exhibits low potassium and high calcium ion characteristics, preferably, the ion characteristics include: low (significantly low) ion levels of P, Mg, S, K, Cu, and Mo, and high (significantly high) ion levels of Sr and Ca. (iv) Plants with low (significantly low) yield; preferably, the low yield includes: low number of tillers, low number of grains per ear, and low grain weight per ear; if the expression or activity of the OsSGN1 gene or its encoded protein in the tested plant is higher than (significantly higher, such as 10%, 20%, 30%, 50% or more higher) the average value of the OsSGN1 gene or its encoded protein in this type of plant, then it is (i) a plant height phenotype (including normal); (ii) well-developed Casparian strips (including normal development); (iii) the leaf ion profile exhibits normal potassium ion ionic characteristics; (iv) Plants with high (including normal) yield; preferably, the high yield (including normal) includes: increased number of tillers (including normal), increased number of grains per ear (including normal), and increased grain weight per ear (including normal).

[0021] In a preferred embodiment, nucleic acid sequence analysis is performed using methods including sequencing, PCR amplification, restriction enzyme digestion, probe assay, hybridization, microarray analysis, and allele polymorphism analysis.

[0022] In another aspect of the present invention, a method for screening regulatory molecules that positively regulate plant traits is provided, wherein the plant is a grass, comprising: (1) adding a candidate substance to a system containing the OsSGN1 gene or its encoded protein; (2) detecting the expression or activity of the OsSGN1 gene or its encoded protein in the system of (1); if the candidate substance upregulates (significantly upregulates) the OsSGN1 gene or its encoded protein, then the candidate substance is a regulatory molecule that positively regulates plant traits; wherein the positively regulated plant traits include: (i) increasing (including maintaining) plant height; (ii) promoting (including maintaining) the development (normal development) of the Casparian strip; (iii) maintaining the normal potassium ion ion characteristics of the plant leaves; and (iv) increasing (including maintaining) plant yield.

[0023] In a preferred embodiment, a control group is also included to clearly distinguish the difference in OsSGN1 expression or activity between the test group and the control group.

[0024] In another preferred embodiment, the candidate substances include (but are not limited to): regulatory molecules designed to target OsSGN1 or its encoded proteins or their upstream or downstream proteins or genes (such as upregulatory molecules, small molecule compound gene editing constructs, etc.).

[0025] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0026] Figure 1 Comparison of the spatial structures of OsSGN1 protein in rice and proteins in Arabidopsis thaliana with less than 75% homology.

[0027] Figure 2 OsSGN1 is polarly located on the endodermal cell membrane facing the cortex in rice roots;

[0028] (A, B) Expression of OsSGN1 in the root of Zhonghua 11;

[0029] (C) Subcellular localization of OsSGN1 at different distances from the root tip in rice roots.

[0030] Figure 3 Phenotypic analysis of ossgn1 Casparian bands based on basic fuchsin staining;

[0031] (A, B) Casparian band phenotypic analysis of transgenic knockout materials of rice OsSGN1 from different perspectives;

[0032] (C) Statistical analysis of the number of pores on the radial wall per 100 μm of the Casparian strip at different locations from the root tip;

[0033] (D) Observation of the position of the Casparian strip between adjacent endothelial layers.

[0034] Figure 4 Phenotypic analysis of ossgn1;

[0035] (A) Phenotypic analysis of ossgn1 during the vegetative growth period;

[0036] (B) Phenotypic analysis of ossgn1 at the heading stage;

[0037] (C) Phenotypic analysis of ossgn1 during the grouting period;

[0038] (D) During the vegetative growth period, the new and old leaves of ossgn1 turn brown.

[0039] (E)ossgn1 grain number analysis;

[0040] (F) Plant height analysis of ossgn1 during the grain-filling stage;

[0041] (G) Analysis of the number of tillers in ossgn1 during the grouting period;

[0042] (H)ossgn1 ear grain weight analysis.

[0043] Figure 5 Phenotypic analysis of ossgn1 leaf ionome;

[0044] (A) PCA analysis of the ionome of wild-type ZH11 and ossgn1 leaves;

[0045] (B) The ionotype of ossgn1 leaves was determined using inductively coupled plasma mass spectrometry (ICP-MS).

[0046] In-situ qualitative analysis of mineral nutrients in leaves of (C)ossgn1 was performed using micro-area X-ray fluorescence spectrometry (XRF).

[0047] (D) ossgn1 leaf mineral nutrient element quantitative analysis was performed using inductively coupled plasma mass spectrometry (ICP-MS). Detailed Implementation

[0048] The inventors are dedicated to discovering more regulatory factors that control the development or function of Casparian strips in plants, and have revealed a gene that regulates Casparian strip formation and mineral nutrient balance, named OsSGN1. This invention proposes new solutions for the regulation of Casparian strip development, ionosphere homeostasis, and mineral nutrient balance in gramineous plants, as well as for improving plant height and yield traits.

[0049] the term

[0050] As used herein, "plant" includes grasses that express OsSGN1 or its homologs in the Poaceae family, or that contain OsSGN1 and the regulatory pathways it participates in. Based on knowledge in the art, plants expressing OsSGN1 or its homologs in the Poaceae family possess the mechanisms of action claimed in this invention and can achieve the technical effects claimed in this invention. In some embodiments, the plant is a crop, preferably a cereal crop, which is a crop with grains (ears). The "cereal crop" can be a Poaceae plant. In some preferred embodiments, the Poaceae plant includes: rice, barley, wheat, oats, rye, maize, sorghum, and Brachypodium distichum. The OsSGN1 includes its homologs (homologous genes and their encoded proteins).

[0051] As used in this invention, "grain" refers to the fruit or seed of a plant, and is also called ear grain in crops such as rice, corn, wheat, and barley.

[0052] Regarding "control plants," selecting appropriate control plants is a routine part of experimental design. These can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties of the same species or class as the plant being evaluated. Control plants can also be individuals from transgenic plants that have lost their transgenic components due to segregation. As used in this article, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0053] As used herein, the terms “enhance,” “upregulate,” “improve,” “promote,” or “enhance” are interchangeable and, in their application, should mean an increase of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25% or 30%, compared to the control plant as defined herein.

[0054] As used herein, the terms “reduction,” “downsizing,” or “reduction” are interchangeable and should be interpreted in the sense of a reduction of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25% or 30%, compared to the control plant as defined herein.

[0055] OsSGN1

[0056] This invention, through extensive systematic research and large-scale screening, identified the OsSGN1 gene, which regulates plant traits such as Casparian strip formation, leaf ionosphere homeostasis, and mineral nutrient balance. The spatial structure of the protein encoded by the OsSGN1 gene is shown below. Figure 1 The right image shows a protein in Arabidopsis thaliana that shares less than 75% homology with it. Figure 1 Compared to the left image, there are very significant differences in spatial structure.

[0057] In this invention, unless otherwise specified, the OsSGN1 protein includes its homologs (homologous proteins). The OsSGN1 can be a polypeptide (protein) having the amino acid sequence shown in SEQ ID NO:2, or it can include sequence variations having the same function as the OsSGN1 protein.

[0058] The variations include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5); and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the OsSGN1 protein (e.g., 80% or higher homology to the polypeptide sequence shown in SEQ ID NO:2; preferably 85% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and having the same function as the OsSGN1 protein is also included in this invention.

[0059] In this invention, the term "OsSGN1 protein" also includes its homologs. It should be understood that while this invention preferably studies OsSGN1 proteins derived from specific species, other polypeptides or genes derived from other species, particularly grasses, that are highly homologous to the OsSGN1 protein (e.g., having more than 80%, more particularly 85%, 90%, 95%, or even more than 98% sequence identity) are also within the scope of this invention.

[0060] In this invention, polypeptides derived from species other than rice that have high homology with the sequence of SEQ ID NO:2 or that play the same or similar role in the same or similar signaling pathways are also included.

[0061] The present invention also provides isolated proteins, which are fragments of the OsSGN1 protein or formed by adding other proteins or tags at both ends.

[0062] This invention also relates to a polynucleotide sequence encoding the OsSGN1 protein of this invention or a sequence variant thereof. The polynucleotide may be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. Its genomic sequence may be identical to the coding region sequence shown in SEQ ID NO:1 or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO:2, but differing from the sequence shown in SEQ ID NO:1 or its coding region sequence. This invention also relates to variants (variants) of the aforementioned polynucleotide encoding polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention.

[0063] The present invention also relates to a vector containing the aforementioned polynucleotide, and a host cell genetically engineered using the aforementioned vector or polypeptide to encode nucleic acids.

[0064] In this invention, the polynucleotide sequence encoding the polypeptide of this invention can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable in the host. An important characteristic of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements. Preferably, the expression vector may also selectively contain resistance elements, selection elements, or reporter gene elements, such as Bar or GUS.

[0065] When the aforementioned polynucleotide is expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. An enhancer is a cis-acting factor of DNA, typically consisting of approximately 10 to 300 base pairs, that acts on the promoter to enhance gene transcription.

[0066] Transforming host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. Plant transformation can be performed using methods such as Agrobacterium-mediated transformation or gene gun transformation, including spraying, leaf disc transformation, and rice embryo transformation.

[0067] Plant Transformation

[0068] The different growth environments and unique root structures of grasses dictate significant differences in the development and function of their Casparian strips compared to Arabidopsis thaliana, and the developmental process of the Casparian strip in grasses remains largely unknown. This invention reveals OsSGN1, specifically expressed in the endodermis and involved in the regulation of Casparian strip development, and utilizes CRISPR-Cas9 gene editing technology to knock out and obtain mutant materials with loss of related gene function. The inventors found that the OsSGN1 knockout mutant exhibited significant abnormal phenotypes in Casparian strip development and ionome analysis, suggesting that the rice Casparian strip does indeed play a crucial role in regulating mineral element homeostasis in rice. Further research revealed that OsSGN1 influences Casparian strip formation by facilitating the accumulation of OsCASP1 at the CSD site on the endodermal cell membrane and affecting the localization of OsCASP1.

[0069] Compared to Arabidopsis Casparian strip mutants, which exhibit high potassium and low calcium ion characteristics, rice Casparian strip mutants show increased calcium content but significantly decreased potassium content. This result indicates that, compared to Arabidopsis, the rice Casparian strip plays a unique role in regulating mineral element transport and could be an important potential target for improving the nutritional quality of gramineous plants. Research in this area is currently largely unexplored, leaving ample room for further investigation. The gene encoding the rice leaf ionome, as described in this invention, can provide genetic resources and technical support for improving mineral element-related traits in crops and creating nutritionally efficient crops.

[0070] Based on the inventor's new discovery, an application of OsSGN1 or its regulatory molecule is provided for: regulating plant height; regulating Casparian strip development in plants; regulating the ionotype of plant leaves; and regulating yield traits in plants.

[0071] It should be understood that, after learning about the regulatory role of OsSGN1 in grasses, various methods well known to those skilled in the art can be used to regulate the expression or activity of OsSGN1 as needed, and these methods are all included in this invention.

[0072] This invention provides a method for downregulating the OsSGN1 protein in plants, including targeted mutation, gene editing, or gene recombination of the OsSGN1 gene to achieve downregulation. As a more specific embodiment, any of the above methods transforms the OsSGN1 protein into its mutant form, thereby rendering it ineffective. As another more specific embodiment, a CRISPR / Cas9 system is used for gene editing. Suitable sgRNA target sites result in higher gene editing efficiency; therefore, suitable target sites can be designed and identified before gene editing. After designing specific target sites, in vitro cell activity screening is required to obtain effective target sites for subsequent experiments. Preferred gene editing reagents are provided in the embodiments of this invention.

[0073] The downregulating molecules of the OsSGN1 protein or its encoding gene refer to any substance that can reduce the activity of the OsSGN1 protein, reduce the stability of the OsSGN1 protein or its encoding gene, downregulate the expression of the OsSGN1 protein, reduce the effective duration of the OsSGN1 protein, inhibit the transcription and translation of the OsSGN1 gene, or reduce the phosphorylation / activation level of the protein. These substances can all be used in this invention as useful for downregulating the OsSGN1 protein. They can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. For example, the downregulating molecules are: gene editing reagents that specifically edit the OsSGN1 gene, interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of the OsSGN1 protein or other signaling pathway genes, etc.

[0074] As an alternative method, the method for downregulating the expression of OsSGN1 protein in plants may include: (1) transferring an interfering molecule that interferes with the expression of the OsSGN1 gene into plant cells, tissues, organs, or seeds to obtain plant cells, tissues, organs, or seeds transformed with the interfering molecule; (2) regenerating plants from the plant cells, tissues, organs, or seeds transformed with the interfering molecule obtained in step (1). Preferably, the method further includes: (3) selecting plant cells, tissues, or organs transformed with the vector.

[0075] In this embodiment of the invention, a CRISPR-CAS9 knockout expression vector was constructed using genetic engineering technology and transformed into wild-type Zhonghua 11 rice callus tissue via Agrobacterium tumefaciens invasion, resulting in the loss of expression in the wild-type rice. The plants exhibited disordered leaf ionome and reduced yield. To facilitate the identification and screening of transgenic plant cells or plants, the transformation vector contained antibiotic resistance markers (kanamycin, hygromycin), and the transformed plants were cultured into plants.

[0076] OsSGN1 activity can be upregulated by molecules that upregulate OsSGN1 expression or activity. These upregulating molecules include promoters, agonists, and activators. The terms "upregulation" and "promotion" include both upregulation and promotion of protein activity and protein expression. Any substance that can increase the activity of the OsSGN1 protein, improve the stability of the OsSGN1 gene or protein, upregulate the expression of the OsSGN1 gene, or increase the effective duration of action of the OsSGN1 protein can be used in this invention as a useful substance for upregulating the OsSGN1 gene or its encoded protein. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0077] As a preferred embodiment, a method for upregulating the expression or activity of OsSGN1 in plants is provided, the method comprising: transferring an expression construct or vector containing the OsSGN1 gene into plants.

[0078] Preferably, a method for preparing transgenic plants is provided, comprising: (1) transferring exogenous nucleic acid encoding OsSGN1 into a plant organ or tissue to obtain a plant tissue or organ transformed with said nucleic acid; and (2) regenerating a plant plant from the plant tissue or organ transformed with exogenous nucleic acid obtained in step (1).

[0079] As a preferred example, the method includes the steps of: (s1) providing Agrobacterium carrying an expression vector containing exogenous (recombinant) OsSGN1; (s2) contacting a plant tissue or organ with the Agrobacterium in step (s1) to transfer the OsSGN1 into and integrate it into the chromosome of the plant cell; and (s3) selecting plant cells, tissues or organs that have been transferred with the OsSGN1.

[0080] The present invention also includes plants obtained using any of the foregoing methods, said plants including transgenic plants introduced with said OsSGN1.

[0081] The method can be implemented using any appropriate conventional means, including reagents, temperature, pressure conditions, etc.

[0082] Applications as molecular markers

[0083] Based on the inventors' new findings, this invention also provides molecular markers suitable for analyzing plant traits, namely the OsSGN1 gene or its encoded protein; said plant traits include: (i) plant height; (ii) Casparian strip development; (iii) leaf ionome phenotype; and (iv) yield traits. This invention also relates to specific detection and analysis designed for the OsSGN1 gene or protein, and analytical strategies. Specifically, altering the leaf ionome phenotype further regulates the levels of mineral nutrients in the leaves, providing information on plants exhibiting changes in mineral nutrient levels.

[0084] As a preferred embodiment, the method for analyzing plant traits according to the present invention includes: analyzing the expression or activity of the OsSGN1 gene or its encoded protein in the test plant; if the expression or activity of the OsSGN1 gene or its encoded protein in the test plant is lower than the average value of the OsSGN1 gene or its encoded protein in this type of plant, then it is (i) low plant height; (ii) exhibiting Casparian strip holes or Casparian strip developmental defects phenotype; (iii) the leaf ion composition shows low potassium and high calcium ion characteristics; (iv) low yield plant. Preferably, the low yield includes: low number of tillers, low number of grains per ear, and low grain weight per ear; if the expression or activity of the OsSGN1 gene or its encoded protein in the test plant is higher than the average value of the OsSGN1 gene or its encoded protein in this type of plant, then it is a plant with (i) high plant height phenotype; (ii) well-developed Casparian strip; (iii) leaf ion profile exhibiting normal potassium ion ion characteristics; (iv) high yield; preferably, the high yield includes: increased number of tillers, increased number of grains per ear, and increased grain weight per ear.

[0085] Based on the novel findings of this invention, those skilled in the art can employ any of the various techniques known in the art or under development to analyze nucleic acid sequences, and these techniques are all included in this invention. The methods described include, but are not limited to: sequencing, PCR amplification, probe methods, hybridization, restriction enzyme digestion analysis, allele polymorphism analysis (such as melting curve analysis) for nucleic acid sequence identification, etc.

[0086] This invention has promising applications in molecular design breeding and crop variety improvement using genetic engineering technology.

[0087] After understanding the function of the OsSGN1 gene, it can be used as a molecular marker for targeted plant screening. This new discovery can also be used to screen for regulatory molecules (including potential substances) that positively regulate plant traits by modulating this mechanism.

[0088] This invention provides a method for screening regulatory molecules that positively regulate plant traits, wherein the plant is a grass, comprising: (1) adding a candidate substance to a system containing the OsSGN1 gene or its encoded protein; (2) detecting the expression or activity of the OsSGN1 gene or its encoded protein in the system of (1); if the candidate substance upregulates the OsSGN1 gene or its encoded protein, it indicates that the candidate substance is a regulatory molecule that positively regulates plant traits; wherein the positively regulated plant traits include: (i) increasing plant height; (ii) promoting the development of Casparian strips; (iii) maintaining the normal potassium ion characteristics of the ionosphere of plant leaves; and (iv) increasing plant yield.

[0089] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0090] The detection of protein-protein interactions and their strength can be achieved using a variety of techniques well-known to those skilled in the art, such as GST-Pull Down, bimolecular fluorescence complementation assays, yeast two-hybrid systems, or immunoprecipitation techniques.

[0091] Using the above methods, a class of substances that specifically act on the OsSGN1 protein or its encoding gene and have a regulatory effect on the improvement of traits in grass plants can be developed.

[0092] 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 that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0093] OsSGN1 genomic DNA sequence (SEQ ID NO:1)

[0094]

[0095] OsSGN1 protein sequence (SEQ ID NO:2)

[0096] MPPRQWRPMLASATKCCAAEDAVVAVDGSGGGGGLARCRPARSEFSRRLASFRKLSSMTNSPASSVAGAAEGGKDDGEEGGGGGGGVSGPLQLYSFSFSELRSITHDFSSSY LLGEGGFGAVHKGFVDAGMRPGLLPQPVAVKQLDIAGLQGHREWLAEVIFLGQFRHPHLVKLLGYCCEDEERLLVYEFMPRGSLENHLFKRISATVPWGTRLKIAIGAAKGL AFLHGASTPVIYRDFKASNILLDSEFTAKLSDFGLAKMGPEGSETHVTTRVMGTHGYAAPEYVMTGHLNIKSDVYSYGVVLLELLTGRRAMEHVRGRSLHADQVVKIVDWTR PYLGSSRRLRCIMDPRLAGHYSVKAARAVAHLAVQCTSPQPRDRPRMAAVVDALERLQGFKDMAVTVGLWPTNAPVAGRNAISAKIRAEVRGAGSGGGAASRRRSASAKLP*

[0097] CRISPR-Cas9 gene knockout sgRNA sequence (SEQ ID NO:3)

[0098] GATCTTCTTGGGGCAGTTC

[0099] Example 1: Expression characteristics of the OsSGN1 gene in rice

[0100] The following steps were taken to analyze the expression characteristics of the OsSGN1 gene in rice:

[0101] (1) Place the rice tissue material into a 2mL centrifuge tube, add 1.5mL of GUS staining buffer solution with 2mM X-Gluc substrate added in advance, cover the tube opening with perforated aluminum foil, and place it on ice to vacuum for 30min.

[0102] (2) Place the reaction in a 37°C oven and closely observe the staining of the tissue during the process to prevent over-staining. Stop the reaction once a blue signal is observed in the plant tissue with the naked eye.

[0103] (3) Rinse the plant material three times with ddH2O, and observe and photograph it under a regular optical microscope or observe it after preparing to embed and slice it.

[0104] Expression analysis results showed that the rice gene OsSGN1 was highly expressed in the root endodermis. Figure 2 A, B).

[0105] Example 2: Subcellular localization of OsSGN1 in rice roots

[0106] The subcellular localization of OsSGN1 in rice roots was analyzed using the following steps:

[0107] (1) Select the roots of the target rice area, fix them with 4% PFA, and vacuum for 30 min.

[0108] (2) After rinsing with PBS, incubate with 2 mg / ml Glycine at room temperature for 5 min, rinse with PBS again, then embed, trim, and section.

[0109] (3) Block with 2% BSA for 2-3 hours.

[0110] (4) The primary antibody dissolved in 2% BSA was incubated overnight on a shaker at 4°C at a ratio of 1:200.

[0111] (5) Shake and wash 3 times on a PBST shaker for 10 minutes each time.

[0112] (6) The fluorescent secondary antibody dissolved in 2% BSA was incubated at a ratio of 1:500 on a shaker at room temperature for 2 hours.

[0113] (7) Wash with PBST on a shaker for 10 minutes each time, 3 times.

[0114] (8) Dilute the Calcofluor White stock solution with PBST to a working solution of 0.1% and stain on a shaker at room temperature for 30 min.

[0115] (9) Wash the slides three times with PBST on a shaker for 10 minutes each time. Then replace the PBST with a mounting medium (glycerol:PBS = 1:1) and mount the slides. Store in the dark.

[0116] (10) Using a Leica SR5 or Leica SP8 laser confocal microscope, observe the fluorescence signal at the corresponding wavelength using the excitation light corresponding to the fluorescent secondary antibody.

[0117] Analysis revealed that the OsSGN1 protein is polarly localized on the cell membrane of the rice root endodermis layer towards the cortex. Figure 2 C).

[0118] Example 3: Analysis of Casparian strip development in rice

[0119] The steps for analyzing the lignin characteristics of rice roots are as follows:

[0120] (1) Dilute 5% (w / v) basic fuchsin ethanol staining solution with 75% lactic acid to make 0.2% (w / v) working solution, and add 3 mL to each well of a cleaned 6-well plate.

[0121] (2) Take a position about 3cm from the root tip of the rice root in a 6-well plate.

[0122] (3) Stain in the dark by vacuuming for 30 minutes, and after vacuuming, stain at room temperature in the dark for 1.5 hours.

[0123] (4) Rinse three times with ddH2O to remove the floating color on the slides. Add fresh 75% lactic acid to the 6-well plate. Decolorize by shaking on a horizontal shaker in the dark for at least 24 hours at room temperature, changing the 75% lactic acid at least three times during this period.

[0124] (5) Before observation, replace the 75% lactic acid solution with fresh one. Use a Leica SR5 or Leica SP8 laser confocal microscope with 561nm excitation light and observe the fluorescence signal at a wavelength of 600-650nm.

[0125] The observation results showed that the OsSGN1 gene loss-of-function mutant had an increased number of holes in the Casparian strip, indicating a developmental defect in the Casparian strip of the mutant. Figure 3 ).

[0126] Example 4: Field Phenotypic Analysis of Rice

[0127] 1. Preparation of ossgn1

[0128] The wild type of rice variety Zhonghua 11 (ZH11) was identified.

[0129] For Zhonghua 11, the OsSGN1 gene was knocked out using CRISPR-Cas9 technology to obtain the ossgn1 mutant. The sgRNA sequence is shown in SEQ ID NO:3.

[0130] Genotyping revealed different editing patterns in the resulting strains, three of which were named ossgn1-1, ossgn1-2, and ossgn1-3.

[0131] 2. Field phenotypic analysis of rice

[0132] (1) Field phenotypic observations and photographs were conducted on rice at different growth stages, namely the vegetative growth stage, the heading stage, and the grain-filling stage.

[0133] (2) During the vegetative growth period, observe and photograph the leaf color of wild-type and mutant rice to observe whether there is ion deficiency or excessive leaf color change.

[0134] (3) During the grain-filling stage, the plant height, number of tillers, number of grains per panicle and grain weight of wild-type and mutant rice were statistically analyzed.

[0135] Field growth phenotypic observations revealed that, compared to the wild type, both new and old leaves of ossgn1 developed brown spots, a typical symptom of potassium deficiency. Figure 4 ).

[0136] Statistical analysis of plant height and tiller number at the heading stage of different lines of the ossgn1 mutant revealed that the plant height and tiller number of ossgn1 were significantly reduced, and the yield phenotypes such as grain number and grain weight per ear were also significantly lower than those of the wild type. Figure 4 ).

[0137] The above results indicate that the normal development of the Casparian strip is crucial for maintaining normal growth and development of rice, absorption and transport of mineral elements, and yield.

[0138] Example 5: ICP-MS Ionome Analysis of Rice Leaves

[0139] The steps for ICP-MS ionome analysis of rice leaves are as follows:

[0140] (1) Weigh the cleaned special glass tubes using a micro balance and record the weight of 12 empty tubes.

[0141] (2) Cut the second leaf of the rice planted in hydroponics or picked in the field with ceramic scissors, hold it with plastic tweezers and wash it three times in 18MΩ ultrapure water, place it in a glass tube and dry it overnight in an oven at 70℃.

[0142] (3) After drying and cooling to room temperature, weigh the blades on a micro balance and calculate the dry weight of the blades in the corresponding empty tube.

[0143] (4) Add 1 mL of 70% nitric acid solution containing internal standard In to each glass tube in a fume hood and place it on a digestion furnace for digestion at 115°C for 4 hours. Avoid excessive airflow in the fume hood during digestion to prevent excessive nitric acid volatilization.

[0144] (5) After digestion is complete and the tube has cooled slightly, remove the glass tube to the tube rack, add 18.2 MΩ of ultrapure water to the fume hood and bring the volume up to 10 mL.

[0145] (6) Use a ceramic rod to thoroughly mix the solution in the tube, and use an electric pipette to transfer the solution in the glass tube to a clean 96-well sample plate, 1.7 mL per well.

[0146] (7) Take 850 μL of each sample into a clean pipette tip box, mix well, and then put it into a new 50 mL centrifuge tube as a mixed sample for instrument calibration.

[0147] (8) Ion group detection of the sample was performed by inductively coupled plasma mass spectrometry (ICP-MS), and the data were processed and analyzed using software Excel and R studio.

[0148] Analysis showed that the ossgn1 mutant leaf ionome underwent significant changes, particularly exhibiting low potassium and high calcium ion characteristics. Figure 5 Specifically, in the mutant, the ion levels of P, Mg, S, K, Cu, and Mo were significantly lower than those in the wild type, while the ion levels of Sr and Ca were significantly higher than those in the wild type.

[0149] Example 6: Screening of regulatory molecules

[0150] Test subject: Root endothelial cells, which normally express endogenous OsSGN1.

[0151] Test group: The above test subjects were treated with the candidate substances;

[0152] Control group: No candidate substance was introduced.

[0153] The expression and activity of OsSGN1 in the test group and control cells were detected and compared.

[0154] If the expression or activity of OsSGN1 in the test group is statistically higher (e.g., 50% or more higher) than that in the control group, it indicates that the candidate is a potential substance for increasing (including maintaining) plant height, promoting (including maintaining) the development of Casparian strips (normal development), maintaining the normal potassium ion ionic characteristics of plant leaves, and increasing (including maintaining) plant yield.

[0155] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of an OsSGN1 gene or its encoded protein or its regulatory molecule for: (i) regulating plant height; (ii) regulating Casparian strip development in plants; (iii) Regulating the ion composition of plant leaves; (iv) Regulating the yield traits of plants; the plants are grasses.

2. The application as described in claim 1, characterized in that, The regulatory molecule is a downregulatory molecule that downregulates the expression or activity of the OsSGN1 gene or its encoded protein, wherein the downregulatory molecule of the OsSGN1 gene: (i) reduces plant height; (ii) increases Casparian strip holes, resulting in Casparian strip developmental defects. (iii) Altering the ion composition of plant leaves and the levels of mineral nutrients to produce a low-potassium, high-calcium ion profile; preferably, altering the ion composition includes: decreasing the ion levels of P, Mg, S, K, Cu, and Mo, and increasing the ion levels of Sr and Ca; (iv) Reducing plant yield; preferably, reducing yield includes: reducing the number of tillers, reducing the number of grains per ear, and reducing the weight of grains per ear; or The regulatory molecule is an upregulatory molecule that upregulates the expression or activity of the OsSGN1 gene or its encoded protein, wherein the upregulatory molecule of the OsSGN1 gene: (i) increases plant height; (ii) promotes the development of Casparian strips; (iii) maintains the mineral nutrient balance of plant leaves; and (iv) increases plant yield. Preferably, increasing yield includes: increasing the number of tillers, increasing the number of grains per ear, and increasing the weight of grains per ear.

3. The application as described in claim 1, characterized in that, The downregulation molecules include: gene editing reagents that specifically edit the OsSGN1 gene, interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of the OsSGN1 gene, and reagents that knock out the OsSGN1 gene through homologous recombination; preferably, the gene editing reagent that specifically edits the OsSGN1 gene is sgRNA, the nucleotide sequence of which is shown in SEQ ID NO:3; or The upregulated molecules include: polynucleotides or constructs encoding OsSGN1, upregulated molecules that promote the promoter-driven ability of the OsSGN1 gene, and upregulated molecules that interact with the OsSGN1 protein to increase its expression or activity.

4. A method for regulating plant traits, comprising: Regulate the expression or activity of the OsSGN1 gene or its encoded protein in plants; wherein the plant traits include: (i) plant height; (ii) Casparian strip development; (iii) leaf ionome phenotype; (iv) yield; and the plant is a grass.

5. The method as described in claim 4, characterized in that, The method includes: downregulating the expression or activity of the OsSGN1 gene or its encoded protein, thereby: (i) reducing plant height; (ii) increasing Casparian strip holes, resulting in Casparian strip developmental defects; (iii) altering the ion composition of plant leaves, changing the levels of mineral nutrients, and causing them to exhibit low potassium and high calcium ion characteristics, preferably, the alteration of the ion composition includes: reducing the ion levels of P, Mg, S, K, Cu, and Mo, and increasing the ion levels of Sr and Ca; (iv) reducing plant yield; preferably, reducing yield includes: reducing the number of tillers, reducing the number of grains per ear, and reducing the weight of grains per ear; or Upregulating the expression or activity of the OsSGN1 gene or its encoded protein can thereby: (i) increase plant height; (ii) promote the development of Casparian strips; (iii) maintain the ion profile of plant leaves, including normal potassium ions; and (iv) increase plant yield. Preferably, increased yield includes: increasing the number of tillers, increasing the number of grains per spike, and increasing the weight of grains per spike.

6. The method as described in claim 5, characterized in that, The downregulation of the expression or activity of the OsSGN1 gene or its encoded protein includes: knocking out or silencing the OsSGN1-coding gene in plants, or inhibiting the activity of the OsSGN1-encoded protein; preferably, it includes: gene editing using a CRISPR system to knock out the OsSGN1-coding gene, knocking out OsSGN1 by homologous recombination, performing a loss-of-function mutation on OsSGN1 in plants containing OsSGN1, or silencing OsSGN1 with a specific gene-interfering molecule; preferably, the downregulation is performed using a gene-editing reagent specifically for editing the OsSGN1 gene, the reagent comprising sgRNA, the nucleotide sequence of which is shown in SEQ ID NO:3; or The upregulation of the expression or activity of the OsSGN1 gene or its encoded protein includes: transferring the OsSGN1 gene or an expression construct or vector containing the gene into a plant; performing a gain-of-function mutation on OsSGN1; promoting OsSGN1 expression by expressing an enhancing promoter or a tissue-specific promoter; or promoting OsSGN1 expression by an enhancer.

7. The application as described in any one of claims 1-3 or the method as described in any one of claims 4-6, characterized in that, The proteins encoded by OsSGN1 include: (a) proteins with the amino acid sequence shown in SEQ ID NO:2; (b) proteins derived from (a) that have the function of protein (a) formed by substitution, deletion or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:2; (c) proteins that have more than 80% identity with the amino acid sequence defined in (a) and have the function of protein (a); or (d) proteins formed by adding tags or signal peptides to both ends of any of the proteins (a)-(c).

8. The application as described in any one of claims 1-3 or the method as described in any one of claims 4-6, characterized in that, The plants include the following group or the OsSGN1 is derived from the following group: cereal plants of the Poaceae family; preferably, the plants include: rice, wheat, millet, foxtail millet, corn, sorghum, millet, barley, rye, oats, and short-stalked grass.

9. The application of a plant OsSGN1 gene or its encoded protein as a molecular marker for identifying plant traits; wherein, The plant traits include: (i) plant height; (ii) Casparian strip development; (iii) leaf ionotype; and (iv) yield. The plant is a grass.

10. A method for analyzing plant traits, the method comprising: Analyze the expression or activity of the OsSGN1 gene or its encoded protein in plants: If the expression or activity of the OsSGN1 gene or its encoded protein in the tested plant is lower than the average value of the OsSGN1 gene or its encoded protein in this type of plant, then it is (i) low plant height; (ii) exhibiting Casparian strip hole or Casparian strip developmental defect phenotype. (iii) The leaf ion profile exhibits low potassium and high calcium ion characteristics. Preferably, the ion characteristics include low levels of P, Mg, S, K, Cu, and Mo, and high levels of Sr and Ca ions. (iv) Low-yielding plants. Preferably, the low yield includes low number of tillers, low number of grains per ear, and low grain weight per ear. If the expression or activity of the OsSGN1 gene or its encoded protein in the tested plant is higher than the average value of the OsSGN1 gene or its encoded protein in this type of plant, then it is (i) a plant with a high plant height phenotype; (ii) well-developed Casparian strips; (iii) the leaf ion profile exhibits normal potassium ion characteristics; and (iv) a high-yielding plant. Preferably, the high yield includes: increased tiller number, increased grain number per ear, and increased grain weight per ear.

11. A method for screening regulatory molecules that positively regulate plant traits, wherein the plant is a grass (Poaceae family), comprising: (1) Add the candidate material to a system containing the OsSGN1 gene or its encoded protein; (2) Detect the expression or activity of the OsSGN1 gene or its encoded protein in the system of (1); if the candidate substance upregulates the OsSGN1 gene or its encoded protein, it indicates that the candidate substance is a regulatory molecule that positively regulates plant traits; wherein, the positively regulated plant traits include: (i) increasing plant height; (ii) promoting the development of Casparian strips; (iii) maintaining the normal potassium ion characteristics of the ionosphere of plant leaves; and (iv) increasing plant yield.

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