Plant type or flowering phase regulation gene and application thereof

Abstract

The invention provides a plant type or flowering phase regulation gene and application thereof. The regulatory gene is LC4 and comes from gramineous plant rice. By optimizing the expression level of the LC4 (expression is reduced or is not expressed), the plant type or flowering phase phenotype can be regulated and controlled, so that the purposes of guiding agricultural production, promoting lodging resistance and close planting, promoting high yield in unit area, advancing the time of grain marketing and improving economic benefits are achieved. The invention provides a new way for variety improvement of gramineous plants.

Description

Technical Field

[0001] The present invention belongs to the field of plant biology; more specifically, the present invention relates to a plant type or flowering period regulating gene and its application. Background Art

[0002] Gramineous plants, such as rice, wheat, corn, sorghum, etc., are very important crops in human production and life. Regarding the factors regulating the close planting of gramineous plants, little is known in this field.

[0003] The research related to plant close planting includes the following aspects: (1) Stomatal genes and photosynthesis efficiency: Gramineous plants and broad-leaved plants use the same genes to produce stomata, that is, the openings on the leaves that allow water and gas to pass through. The opening and closing of these stomata are controlled by specific genes, which affect the photosynthesis efficiency and gas exchange of plants. For example, dumbbell-shaped guard cells are more efficient in photosynthesis than kidney-shaped guard cells because they require less solute and water to achieve the same pore size. However, these genes mainly affect the gas exchange and photosynthesis efficiency of plants, rather than directly regulating the close planting of plants. (2) Genome and evolutionary research: The genome research of gramineous plants reveals their early evolutionary history and genetic mechanisms. For example, the genome sequencing and evolutionary genomics research of Pharus reveals the relationship between the whole genome duplication (WGD) event and the genes related to spikelet development. These studies provide an in-depth understanding of the genetic diversity and adaptability of gramineous plants, but do not directly involve the regulatory genes of close planting traits. (3) Agricultural practices related to close planting: In agricultural practices, close planting is usually determined according to environmental factors such as soil fertility status and rainfall conditions, rather than being completely regulated by genes. However, it may be possible to breed gramineous plant varieties more suitable for close planting through genetic improvement, but this requires further genetic research and breeding practices.

[0004] The genes that have been studied include: (1) The "brake gene" DPY1 of brassinolide signal, DPY1 regulates the molecular mechanism of leaf drooping and erectness, affects the plant type of gramineous crops, and may thus affect the planting density and yield of crops. (2) The BTA2 gene, which is a gene suitable for close planting of rice. This gene regulates the tillering angle of rice by controlling the content and distribution of auxin. Overexpression of this gene can increase the yield under moderate close planting. (3) The ZmYUC2 and ZmYUC4 genes: These two genes specifically regulate the root architecture of maize and have an important impact on the traits of maize resistance to close planting and lodging, and thus have application potential under close planting conditions. The discovery and research of these genes provide an important theoretical basis and molecular targets for the molecular breeding of high-yield close planting of gramineous plants based on plant type improvement.

[0005] At present, the methods for regulating the flowering period of grasses mainly include two categories: gene regulation and non-gene regulation. In terms of gene regulation, researchers have discovered some genes that can regulate the flowering period of grass crops. For example, researchers have discovered a gene VRN11 that can prevent grass crops from entering the flowering period before the arrival of the appropriate season. The HSP101 gene was also discovered, which affects the expression of genes in multiple flowering time regulatory pathways under normal growth conditions. In addition, as in rice, the OsTPR075-OsFTIP1 / 9-RFT1 / Hd3a molecular module was revealed as a new mechanism for regulating the heading period (flowering period) of rice.

[0006] In terms of non-genetic regulation, one is light regulation: using the light requirements of flower bud differentiation of different plants, artificially supplementing light or shading, so that the plants can bloom earlier or later. This includes long-day treatment and short-day treatment, which are suitable for different plants and seasons. Another is temperature regulation: according to the temperature requirements of plant flower bud differentiation, artificially increasing or decreasing the temperature can also make the plants bloom earlier or later. The warming treatment method and the cooling treatment method are respectively suitable for plants that require high and low temperatures to bloom. In addition, it also includes chemical regulation: the application of plant growth regulators is also a method of regulating the flowering period of plants, which can make plants bloom earlier or later.

[0007] In summary, there are still few genes that have been clearly identified in the field that can directly regulate the density or flowering period of grasses. This field also needs to further analyze the genetic mechanism of density traits or flowering period traits through multi-omics methods such as genomics, transcriptomics and phenomics, combined with field trials and breeding practices, to explore valuable and suitable targets for regulation. Summary of the invention

[0008] The purpose of the present invention is to provide a plant type or flowering period regulating gene and application thereof.

[0009] In a first aspect of the present invention, an application of a down-regulating molecule of LC4 is provided for optimizing the phenotype of Gramineae plants; wherein the optimization of the phenotype of Gramineae plants includes: increasing the number of tillers, reducing plant height, promoting compact plant type, and promoting early flowering.

[0010] In one or more embodiments, the down-regulating molecule down-regulates the expression or activity of LC4, including: an agent that causes a loss-of-function mutation in LC4, an agent that knocks out or silences LC4, and an agent that inhibits the activity of LC4.

[0011] In one or more embodiments, the down-regulating molecule includes: a CRISPR gene editing reagent, a homologous recombination reagent, a site-directed mutagenesis reagent, or an interfering molecule that specifically interferes with the expression of the gene encoding LC4.

[0012] In one or more embodiments, the downregulating molecule is a CRISPR gene editing reagent; preferably a CRISPR / Cas12 reagent.

[0013] In one or more embodiments, the CRISPR / Cas12 reagent comprises a gRNA, and the gRNA comprises: TTTGAGCATGGTTCAATCAAAGATGAT.

[0014] In one or more embodiments, the gramineous plants with optimized phenotypes have one or more of the following properties: the number of plants planted per unit area increases (can be densely planted), has lodging resistance, the yield per unit area increases, and the growth cycle is short (early maturity, early listing time of new rice).

[0015] In one or more embodiments, the gramineous plants are water-saving and drought-resistant plants.

[0016] In another aspect of the present invention, there is provided an application of the endogenous LC4 of a gramineous plant, which is used as a target gene for identifying the phenotype of a gramineous plant or as a target gene for directional screening of plants; wherein, the phenotype includes: tiller number, plant height, plant type compactness, flowering period.

[0017] In one or more embodiments, the expression or sequence characteristics of the LC4 protein or its gene in a test plant are identified; if the LC4 protein or its gene in the test plant has low expression (including no expression) or low activity (including no activity), then it is a plant with a high tiller number, low plant height, compact plant type, and early flowering (compared with the average level of this type of plant).

[0018] In another aspect of the present invention, there is provided a method for optimizing the phenotype of a gramineous plant, comprising: reducing the expression or activity of LC4 in the gramineous plant; wherein, optimizing the phenotype of the gramineous plant includes: increasing the tiller number, reducing the plant height, promoting plant type compactness, and promoting early flowering.

[0019] In one or more embodiments, the method includes (but is not limited to): performing a loss-of-function mutation on LC4 in a plant expressing LC4, knocking out or silencing the coding gene of LC4, or inhibiting the activity of LC4.

[0020] In one or more embodiments, the performing a loss-of-function mutation on LC4 includes: performing targeted modification on the coding gene of LC4 to reduce its function / activity; preferably, causing a frameshift in its coding gene so that translation terminates prematurely.

[0021] In one or more embodiments, causing a frameshift in its coding gene so that translation terminates prematurely (for example, inserting a foreign gene fragment into the LC4 gene, disrupting its coding sequence to downregulate LC4 or inactivate it).

[0022] In one or more embodiments, the method includes: performing gene editing with a CRISPR system to knockout the coding gene of LC4; silencing LC4 with an interfering molecule that specifically interferes with the expression of the coding gene of LC4; knocking out the coding gene of LC4 by homologous recombination; or, for the purpose of downregulating LC4 in a plant, constructing a plant screening library formed by gene insertion-deletion, mutation-deletion, or deletion-deletion, and screening for plants (mutants) in which LC4 is downregulated from the library.

[0023] In one or more embodiments, the method is implemented with a CRISPR gene editing reagent; preferably, the CRISPR gene editing reagent is a CRISPR / Cas12 reagent; more preferably, the CRISPR / Cas12 reagent includes a gRNA, and the gRNA includes: TTTGAGCATGGTTCAATCAAAGATGAT.

[0024] In one or more embodiments, after preparing a gramineous plant with an altered phenotype by the method, it further includes: hybridizing a plant in which the expression or activity of LC4 has been regulated with a plant into which the protein of LC4 or its coding gene has not been introduced to obtain hybrid offspring.

[0025] In another aspect of the present invention, there is provided a gramineous plant cell, tissue, or organ, which contains an exogenous LC4 downregulating molecule; preferably, the downregulating molecule is as defined above.

[0026] In one or more embodiments, the LC4 is derived from or the gramineous plant includes (but is not limited to): rice, wheat, panicum miliaceum, Setaria italica, maize, sorghum, millet, barley, rye, oats, Brachypodium distachyon.

[0027] In one or more embodiments, the gramineous plant is rice.

[0028] In one or more embodiments, the rice includes water-saving and drought-resistant varieties.

[0029] In one or more embodiments, the LC4 is the LC4 derived from a gramineous plant.

[0030] In one or more embodiments, the LC4 is the LC4 derived from rice.

[0031] In one or more embodiments, the LC4 includes its homologs.

[0032] In one or more embodiments, the amino acid sequence of the protein of LC4 is selected from the following group:

[0033] (i) A protein having the amino acid sequence shown in SEQ ID NO: 2;

[0034] (ii) A protein having a homology of ≥ 80% (preferably ≥ 85%, ≥ 90%, ≥ 95% or ≥ 98%) with the amino acid sequence shown in SEQ ID NO: 2 and having the function of regulating traits;

[0035] (iii) A protein formed by adding a tag sequence or a cleavage site sequence to the N- or C-terminus of the protein having the amino acid sequence shown in SEQ ID NO: 2, or adding a signal peptide sequence to its N-terminus.

[0036] In one or more embodiments, the LC4 gene includes a cDNA sequence, a genomic sequence (gDNA), or a sequence artificially optimized or modified based on them.

[0037] In one or more embodiments, the terms down-regulation, inhibition, reduction or decrease mean significant down-regulation, inhibition, reduction or decrease, such as down-regulation, inhibition, reduction or decrease by 5%, 10%, 20%, 40%, 60%, 80%, 100%, 200%, 300%, 500%, 800% or higher.

[0038] In one or more embodiments, the terms increase, promotion, improvement or up-regulation mean significant increase, promotion, improvement or up-regulation, such as increase, promotion, improvement or up-regulation by 5%, 10%, 20%, 40%, 60%, 80%, 100%, 200%, 300%, 500%, 800% or higher.

[0039] In one or more embodiments, the down-regulated expression includes absent expression.

[0040] In one or more embodiments, the terms high expression or high activity refer to a statistically significant increase in expression or activity compared to the average value of the expression or activity of the same or similar plants.

[0041] In one or more embodiments, the terms low expression or low activity refer to a statistically significant decrease in expression or activity compared to the average value of the expression or activity of the control plants (including the same or similar plants).

[0042] Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Gene editing materials of lc4-cas9 (lc4-cr1) with ZH11 as the transformation background and their phenotypic analysis.

[0044] Figure 2, Gene editing lines and editing sequence analysis of lc4-cas12 (lc4-cr2) under the background of ZH11.

[0045] Figure 3 , Phenotypic analysis of gene editing lines of lc4-cas12 (lc4-cr2) under the background of ZH11.

[0046] Figure 4 , Sequence and phenotypic analysis of lc4-cas12 gene editing materials under the background of HuHan 1505.

[0047] Figure 5 , Sequence and phenotypic analysis of lc4-cas12 gene editing materials under the background of HuHan 106.

[0048] Figure 6 , Sequence and phenotypic analysis of lc4-cas12 gene editing materials under the background of HuHan 1516.

[0049] Figure 7 , Sequence and phenotypic analysis of lc4-cas12 gene editing materials under the background of Daohuaxiang 2.

[0050] Figure 8 , Sequence and phenotypic analysis of lc4-cas12 gene editing materials under the background of Daohuaxiang Yangzhou.

[0051] Figure 9 , Sequence and phenotypic analysis of lc4-cas12 gene editing materials under the background of Songgeng 6. Detailed implementation manners

[0052] Through in-depth research, the present inventors have revealed a novel target for regulating the plant type or flowering period phenotype of gramineous plants. Through in-depth research, screening and experiments, the inventors have isolated a gene involved in the regulation of plant type or flowering period phenotype from the gramineous plant rice, named LC4. By optimizing the expression level of LC4 (reducing expression or making it non-expressed), the plant type or flowering period phenotype can be regulated, so as to guide agricultural production, promote lodging resistance and close planting, promote high yield per unit area, advance the grain market time, and improve economic benefits. The present invention provides a new way for the variety improvement of gramineous plants.

[0053] In the present invention, unless otherwise specified, a "water-saving and drought-resistant" plant refers to a new type of gramineous variety that has both high-yield and high-quality characteristics and water-saving and drought-resistant characteristics. During the breeding process of water-saving and drought-resistant plants, some varieties are discarded due to unfavorable traits such as insufficiently compact plant type, too high plant height, overly vigorous vegetative growth, and late flowering, while the present invention can effectively change this situation.

[0054] In the present invention, the "optimized trait" or "improved trait" refers to improving the characteristics of gramineous plants, which may include, for example, regulating the phenotype of gramineous plants.

[0055] In the present invention, the "gramineous plants" include plants expressing LC4. According to the knowledge in the art, for plants in which LC4 or its homologs exist, the mechanism as claimed in the present invention inherently exists, so it can be expected that the technical effects as in the present invention can also be achieved.

[0056] "Control plants" are applied in the present invention. Selecting appropriate "control plants" is a routine part of experimental design, which may include corresponding wild-type plants or corresponding transgenic plants without the target gene. Control plants are generally the same plant species or even varieties that are the same as or belong to the same category as the plants to be evaluated. Control plants can also be individuals in which the transgenic plants are lost due to segregation. As used herein, control plants refer not only to whole plants but also to plant parts, including seeds and seed parts.

[0057] In the present invention, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a target gene sequence is not normally naturally occurring, then the promoter is exogenous to the target gene. A specific sequence is "exogenous" to the cell into which it is inserted.

[0058] In the present invention, "introducing", "importing" or "transforming" includes transferring an exogenous polynucleotide into a host cell, and there is no particular limitation on the methods of "introducing", "importing" or "transforming" in the present invention.

[0059] LC4 gene

[0060] In the present invention, the LC4 refers to the protein having the sequence of SEQ ID NO:2 or its encoding gene (LC4 gene), and also includes sequence variant forms having the same function as the LC4 protein. The encoding gene can be gDNA or cDNA, and can also include a promoter. For example, the cDNA has the nucleotide sequence shown in SEQ ID NO:1. The sequence of the encoding gene also includes sequences degenerate with the sequences provided in the present invention.

[0061] Variant forms of the LC4 protein include (but are not limited to): deletions, insertions and / or substitutions of several (e.g., 1 - 40, preferably 1 - 30, more preferably 1 - 20, most preferably 1 - 10, even more preferably 1 - 8, 1 - 5) amino acids, as well as addition or deletion of one or several (e.g., within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. Any protein with high homology to the described LC4 protein (such as having a homology of 80% or higher with the protein 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 function of regulating the phenotype of gramineous plants should also be included in the present invention.

[0062] In addition, proteins derived from other gramineous plants other than rice, which have relatively high homology with the protein sequence shown in SEQ ID NO:2 or play the same or similar roles in the same or similar regulatory pathways, are also included in the present invention.

[0063] The present invention also includes mutant forms of LC4 or protein truncations (fragments), as long as they can retain the activity of the full-length protein in regulating the phenotype of gramineous plants.

[0064] In the present invention, the described LC4 also includes its homologs. That is, other proteins or genes obtained from other species and highly homologous to the LC4 (such as having a sequence identity of more than 60%, such as 70%, 80%, 85%, 90%, 95%, even 98%) are also within the scope considered in the present invention.

[0065] The polynucleotide (gene) encoding the LC4 protein can be a natural gene from gramineous plants or their degenerate sequences, which can be understood by those of ordinary skill in the art.

[0066] Vectors containing the described coding sequence, as well as host cells genetically engineered with the vector or protein coding sequence, are also included in the present invention. Methods well-known to those skilled in the art can be used to construct appropriate expression vectors.

[0067] The host cell can be a plant cell. Agrobacterium-mediated transformation or particle bombardment, such as the leaf disc method, immature embryo transformation method, etc., can generally be used for plant transformation; preferably, the Agrobacterium method. Transformed plant cells, tissues or organs can be regenerated into plants by certain methods, thereby obtaining plants with altered traits relative to the wild type. However, the present invention also encompasses cells, tissues or organs that cannot be directly regenerated into plant varieties.

[0068] Application

[0069] Based on the inventors' new discoveries, the present invention provides a method for regulating the phenotype of gramineous plants or preparing gramineous plants with phenotypic changes, including: regulating the expression or activity of LC4 in gramineous plants.

[0070] On the one hand, by downregulating the expression or activity of LC4 in plants, the number of tillers is increased, the plant height is reduced, the plant type is promoted to be compact, and early flowering is promoted. The regulation enables the gramineous plants to have one or more of the following properties: the number of plants planted per unit area is increased (capable of being densely planted), having lodging resistance, the yield per unit area is increased, and the growth cycle is short (early maturity, early listing time of new rice). Thus, water-saving and drought-resistant plants are obtained.

[0071] On the other hand, if necessary, the expression or activity of LC4 in plants can also be upregulated, or the expression or activity of LC4 can be increased in plants with low expression of LC4, thereby reducing the number of tillers, increasing the plant height, promoting the expansion of the plant type, and promoting late flowering.

[0072] In the present invention, the downregulating molecule of the LC4 protein or its coding gene refers to any substance that can reduce the activity of the LC4 protein, reduce the stability of the LC4 protein or its coding gene, downregulate the expression of the LC4 protein, reduce the effective action time of the LC4 protein, inhibit the transcription and translation of the LC4 gene, or reduce the phosphorylation / activation level of the protein. These substances can all be used in the present invention as substances useful for downregulating LC4. They can be biomolecules, compounds, small chemical molecules, etc. The biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level. For example, the downregulating molecule is: an interfering RNA molecule or an antisense nucleotide that specifically interferes with the expression of the LC4 gene; or a gene editing reagent that specifically edits LC4, etc.

[0073] The present invention also provides a method for downregulating LC4 in plants, including: performing a loss-of-function mutation on LC4 in plants containing LC4, knocking out or silencing the coding gene of LC4, or inhibiting the activity of LC4; preferably, the loss-of-function mutation of LC4 includes: performing targeted modification on the coding gene of LC4 to reduce its function / activity; preferably, causing a frameshift in its coding gene so that translation terminates prematurely.

[0074] As a particularly preferred embodiment, a system based on CRISPR / Cas12 or CRISPR / Cas9 (the former is more preferred) can be used for gene editing to knock out or downregulate the target gene. Appropriate sgRNA target sites will bring higher gene editing efficiency, so before starting gene editing, appropriate target sites can be designed and found. After designing specific target sites, in vitro cell activity screening is also required to obtain effective target sites for subsequent experiments.

[0075] As another alternative embodiment, the downregulator may be an interfering RNA molecule specific for the LC4 gene (such as siRNA, shRNA, miRNA, etc.). The interfering RNA can be delivered into cells by using an appropriate transfection reagent, or can also be delivered into cells by using a variety of techniques known in the art. In some embodiments, RNAi is used to inhibit the LC4 gene. RNAi is an evolutionarily conserved cellular defense mechanism for controlling the expression of foreign genes in most eukaryotes including humans. RNAi is usually triggered by double-stranded RNA (dsRNA) and causes sequence-specific mRNA degradation of single-stranded target RNA. The mediator of mRNA degradation is small interfering RNA duplexes (siRNAs), which are usually generated by the intracellular cleavage of long dsRNA. The length of siRNAs is usually about 21 nucleotides (for example, 21-23 nucleotides). After the small RNA or RNAi is introduced into cells, it is believed that the sequence is delivered to an enzyme complex called RISC (RNA-induced silencing complex). RISC recognizes the target and cleaves it with an endonuclease. It is worth noting that if a larger RNA sequence is delivered into cells, the RNase III enzyme (Dicer) will convert the longer dsRNA into 21-23 nt ds-siRNA fragments. In some embodiments, the interfering effect is carried out by using the shRNA technique. shRNA is an RNA sequence that can make a tight hairpin turn and can be used to silence gene expression through RNA interference. shRNA uses a vector introduced into cells and utilizes a promoter (such as U6) to ensure that shRNA is always expressed. This vector is usually passed on to daughter cells, enabling the inheritance of gene silencing. The shRNA hairpin structure is cleaved into siRNA by the cellular machinery and then binds to the RNA-induced silencing complex (RISC). This complex binds and cleaves the mRNAs that match the siRNA it binds to. shRNA is transcribed by RNA polymerase III.

[0076] As an alternative embodiment, an antisense compound that specifically hybridizes with one or more nucleic acids encoding LC4 is used to regulate LC4 expression. The specific hybridization of the oligomer with its target nucleic acid interferes with the normal function of the nucleic acid. The regulation of the function of the target nucleic acid by such a compound that specifically hybridizes with the target nucleic acid is generally referred to as "antisense".

[0077] It should be understood that the methods for downregulating the target gene / target protein in plants are not limited to those listed above. After knowing the functions of the LC4 and the signal pathway in which LC4 participates (preferably, also including its upstream genes and downstream genes), a variety of methods well-known to those skilled in the art can be used to regulate the expression or activity of the LC4 or to regulate the related upstream genes or downstream genes of LC4. For example, a variety of methods well-known to those skilled in the art can be used to overexpress LC4 or its upstream genes or downstream genes.

[0078] Methods for upregulating LC4 expression in plants may include: transferring the coding gene of LC4, or an expression construct (including an expression cassette) or vector containing the coding gene, into a plant. In addition, gain-of-function mutations can also be made to LC4 or its coding gene; the expression of the coding gene of LC4 can be promoted by expressing an enhanced promoter or a tissue-specific promoter; or the expression of the coding gene of LC4 can be promoted by an enhancer. It should be understood that other methods for upregulating LC4 expression in plants should also be included in the present invention.

[0079] In the present invention, the upregulating molecules of LC4 include agonists, promoters, stimulants, etc., and these terms can be used interchangeably. The upregulating molecules of LC4 refer to any substances that can increase the activity of LC4, enhance the stability of LC4, upregulate the expression of LC4, increase the effective action time of LC4, or promote the transcription and translation of the LC4 gene. These substances can all be used in the present invention as substances useful for upregulating LC4, and thus can be used to exert regulatory effects. They can be biomolecules, compounds, small chemical molecules, etc. The biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

[0080] The present invention also provides a method for screening substances (potential substances) that regulate the phenotype of gramineous plants, including: (1) adding a candidate substance to a test system containing LC4; (2) detecting the system and observing the expression or activity of LC4 therein; if the expression or activity of LC4 decreases (a statistically significant decrease), it indicates that the candidate substance can be used to increase the tiller number, reduce the plant height, promote a compact plant type, and promote early flowering.

[0081] Methods for screening substances that act on a target using a protein, gene, or a specific region thereof as the target are well known to those skilled in the art, and these methods can all be used in the present invention. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Those skilled in the art know how to select suitable screening methods according to the type of substance to be screened.

[0082] A variety of conventional techniques can be used to identify the transcription or expression of genes in a system. These techniques include but are not limited to: oligonucleotide hybridization techniques (such as probes), polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, etc. A variety of techniques well known to those skilled in the art can be used to detect the interaction between proteins and the strength of the interaction, such as co-immunoprecipitation techniques, GST precipitation techniques, phage display techniques, or yeast two-hybrid systems. Protein localization is also a well-known technique in the art.

[0083] In addition, the bimolecular fluorescence complementation (BiFC) assay of tobacco can also be used to analyze protein-protein interactions. The main principle is as follows: There are many specific sites on the loop structure between the two β-sheets of fluorescent proteins (such as YFP, GFP, Luciferase, etc.) where foreign proteins can be inserted without affecting the fluorescence activity of the fluorescent proteins. The BiFC technique makes use of this property of the fluorescent protein family, splitting the fluorescent protein into two non-fluorescent active molecular fragments, which are then fused with the target proteins for expression respectively. If the two target proteins approach each other due to physical interactions, the two molecular fragments of the fluorescent protein will approach each other spatially, reforming an active fluorescent group and emitting fluorescence.

[0084] Through large-scale screening, a class of potential substances that specifically act on LC4 or the signal pathways involving it and regulate the phenotypes of gramineous plants can be obtained.

[0085] In addition, after the function of LC4 is known, it can be used as a molecular marker for screening excellent plant varieties.

[0086] After the function of LC4 is known, substances or potential substances that can regulate the phenotypes of gramineous plants (phenotypes) by modulating this mechanism can also be screened based on this new discovery.

[0087] According to the new discovery of the present invention, a method for directional selection or identification of plants is provided, including: identifying the expression or sequence characteristics of LC4 protein or its gene in a test plant; if the LC4 protein or its gene in the test plant has low expression (including no expression) or low activity (including no activity), then it is a plant with a high tiller number, low plant height, compact plant type, and early flowering (compared with the average level of this type of plant). This method can be applied to early judgment, such as identifying plant seeds, buds or seedlings.

[0088] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions such as those described in Molecular Cloning: A Laboratory Manual edited by J. Sambrook et al., Science Press, or according to the conditions recommended by the manufacturer.

[0089] Materials and Methods

[0090] I. Construction of the vector of the CRISPR-Cas system

[0091] 1. Select a suitable CRISPR-Cas12 system

[0092] The preliminary analysis of the inventors showed that compared with the deletion-based editing method of target genes that can be efficiently achieved using the CRISPR-Cas12 system, while achieving trait improvement, it is beneficial to the promotion of rice varieties. This system was selected in the subsequent examples.

[0093] 2. gRNA Design and Synthesis

[0094] Design gRNA according to the sequence of the target gene, select appropriate gRNA to ensure that the gRNA has high specificity and avoid off-target effects. Generally, a 20-base-long sequence is selected, and the PAM sequence is NGG.

[0095] The sequence of gRNA: TTTGAGCATGGTTCAATCAAAGATGAT (SEQ ID NO:3).

[0096] Synthesize the DNA sequence of gRNA, which can be achieved by synthesizing oligonucleotides or by cloning into a vector for expression.

[0097] 2. Vector Construction

[0098] To detect the efficiency of the CRISPR-Cas12 system and the stability of the genetic phenotype after gene deletion, the transformation of the lc4-CRISPR-Cas12 vector in rice was carried out using ZH11 as the background (obtaining the lc4-cr2 material).

[0099] The methods for vector construction and transformation are as follows:

[0100] Insert the gRNA into the gRNA expression framework of the pU6-gRNA vector. After digestion with restriction enzymes, insert the synthesized gRNA sequence into the vector to obtain lc4-CRISPR-Cas12.

[0101] Based on genetic verification, select the rice varieties to be improved as the chassis varieties for transformation, and obtain 3 Huhan series (Huhan 1505, Huhan 106, and Huhan 1516) and 3 main cultivated varieties (Daohuaxiang 2, Daohuaxiang Yangzhou, and Songjing 6) as the chassis transformation varieties for the transformation of the lc4-CRISPR-Cas12 vector.

[0102] The T-DNA region can be used to enhance the transformation efficiency, and corresponding resistance genes can be set as selection markers (such as glyphosate or ampicillin resistance genes).

[0103] 3. Verification of the Vector

[0104] Verify whether the gRNA sequence is correctly inserted through restriction enzyme analysis and sequencing.

[0105] II. Rice Genetic Transformation

[0106] 1. Rice tissue culture and dedifferentiation

[0107] Prepare the chassis variety, select suitable seedlings or embryos, usually select seedlings with 2 - 3 leaves or anthers for culture.

[0108] Cut the tissue into pieces of appropriate size and place them in a medium containing plant growth regulating substances (2,4 - D, BAP) for dedifferentiation.

[0109] 2. Agrobacterium - mediated method

[0110] Transform the constructed CRISPR - Cas12 vector into Agrobacterium tumefaciens strain EHA105.

[0111] Infect rice tissue with Agrobacterium. Contact the Agrobacterium carrying the vector with the recipient tissue of rice (such as embryos or leaf sections), and induce Agrobacterium to transfer the T - DNA region into rice cells under appropriate conditions.

[0112] Screen positive transformants with a selective medium containing antibiotics. Common antibiotics such as kanamycin, ampicillin, etc.

[0113] 3. Screening of transformed plants

[0114] Screen the transformed rice in a selective medium. Promote the growth and development of the transformed rice seedlings through conventional culture.

[0115] 4. Regeneration of transgenic rice

[0116] Cultivate the screened transformed embryos or cells into regenerable rice plants.

[0117] Conduct regeneration culture on an appropriate medium to induce the formation of complete rice plants.

[0118] 5. PCR and sequencing verification

[0119] Extract the genomic DNA of the transformed rice plants and detect the integration of Cas12 and gRNA by PCR.

[0120] Further verify whether the target gene has been successfully edited by sequencing.

[0121] 6. Progeny analysis and phenotype observation

[0122] Self - cross the transformed plants, observe the phenotypes of the progeny and further sequence to detect the gene editing effect, check whether the expected gene mutation phenotypes are successfully obtained, and use them for large - scale yield determination.

[0123] III. Gene sequence information

[0124] Lc4 gene sequence (SEQ ID NO:1):

[0125] ATGGGGGAGGTGGCGGCGCTGCGGCAGCTGGTCGGCGAGGTGCAGGAGCTCTGGGACCTC

[0126] TACGGCGCCAACTCCCACCCCCTCCCAAGGTGGTATTTACTGGACTTTGAGCATGGTTCA

[0127] ATCAAAGATGATCATTGTAGAGCAAGGACTGGATACAACTCAGAATTACTAAAGATCATG

[0128] GAAGCTAACCAATCTCCTCCTCGCAAGCGCTCACGGAGGGACAAAAACCGTGAGAAAGCA

[0129] CCCAACTCAAACTCAACTGAAGAAATGCAACAGGAGATTTGGAGTGAGTTCCCTGGAGAC

[0130] CTTTTTGAAACCGTTGTTGCAAGACTTCCAGTTGCTGCAATTTTCCGATTTCGCACTGTT

[0131] TGCCGGAATTGGTATTCTATGTTGGGCTCAGAAAGTTTCTCTCAGCAGTACTCAGAAGTT

[0132] CCACAGAGGCTGCCATGGTTCTATACAATCACCCATGAGAATGCCAGCAACAATGTAGCG

[0133] ATGTATGACCCTTCGCTGAAAAAATGGCACCACCCATCAGTTCCCCTGGCTCCTGCAAAG

[0134] ATAGTAATTCCAGTGGCATCTGCAGGTGGCCTTGTCTGTTTATTGGATCTTAGCCACAGG

[0135] AACTTCTACATATGCAATCCGCTAACACAATCACTCAAGGAAATTCCGCGCAGGTCAGTC

[0136] CAGGCATGGTCAAGAGTGGCAGTAGGGATGGTGATGAACGGAGGAACCTCTAATGAAGGT

[0137] TACAAAGTAATGTGGTTAGGAAATGATGGGAATTATGAAGTCTATGATTCTATGAAGAAT

[0138] ATGTGGTCTTGTCCAGGCACTTTTCCTCCAAGCATCAAACTTCCGCTTGCTCTAAATTTT

[0139] AGGTCACAGCCTGTGGCGGTTGGCAGCATGCTATACTTCATGTGTGCAGAACCAGAGGGT

[0140] GTTTTGTCGTATGATGTAAGCACTGGGATATGGAGACAATTCGTCATCCCACTGCCACTT

[0141] CATCTGACTGACCACACACTTGCCGAGTTCCAGGGAAGGGTTATGCTGGTGGGTCTGCTC

[0142] TGCAAAAATGCAGCGACATGTGTCTGCATTTGGGAGTTGCAGAAGATGACTCTCCTCTGG

[0143] AAGGAGGTGGACAGAATGCCAAATATCTGGTGCTTAGAATTCTACGGTAAGCACATGAAG

[0144] ATGACATGCCTGGGCAACAGTGGTTTGCTCATGCTCTCCTTGAAGGCGAAGCGGATGAAC

[0145] CGCCTCGTGACATACAACCTTTTGAACAAGGAGTGGCAGAAGGTTCCTGATTGCATGCTC

[0146] CCATGCAGCCGCAAAAAGCAGTGGATAGCATGTGGCACAGCATTTGGTCCATGCCCCTCT

[0147] GCCTTGGCCTGA

[0148] The sequence of the protein encoded by the lc4 gene (SEQ ID NO: 2):

[0149] MGEVAALRQLVGEVQELWDLYGANSHPLPRWYLLDFEHGSIKDDHCRARTGYNSELLKIM

[0150] EANQSPPRKRSRRDKNREKAPNSNSTEEMQQEIWSEFPGDLFETVVARLPVAAIFRFRTV

[0151] CRNWYSMLGSESFSQQYSEVPQRLPWFYTITHENASNNVAMYDPSLKKWHHPSVPLAPAK

[0152] IVIPVASAGGLVCLLDLSHRNFYICNPLTQSLKEIPRRSVQAWSRVAVGMVMNGGTSNEG

[0153] YKVMWLGNDGNYEVYDSMKNMWSCPGTFPPSIKLPLALNFRSQPVAVGSMLYFMCAEPEG

[0154] VLSYDVSTGIWRQFVIPLPLHLTDHTLAEFQGRVMLVGLLCKNAATCVCIWELQKMTLLW

[0155] KEVDRMPNIWCLEFYGKHMKMTCLGNSGLLMLSLKAKRMNRLVTYNLLNKEWQKVPDCML

[0156] PCSRKKQWIACGTAFGPCPSALA*

[0157] Example 1: Analysis of phenotypes related to plant type compactness

[0158] First, using the CRISPR / Cas9 technology, the inventors established a gene-editing material of lc4-cas9 (lc4-cr1) with rice ZH11 as the transformation background, planted it and analyzed its phenotype. The editing type of the gene-editing material is Figure 1 the clip insertion marked in

[0159] The results showed that the rice gene-editing material (lc4-cr1) exhibited an obvious phenotype of a compact plant type, thus determining that the LC4 gene has a regulatory effect on plant type compactness.

[0160] Example 2: Analysis of phenotypes related to plant height, tillering, and early flowering

[0161] Considering that as few or no exogenous bases are introduced in the new variety approval of gene editing materials, the inventors constructed a novel gene editing vector lc4-cas12 and transformed it again using rice ZH11 as the background. As a result, a series of gene editing materials of lc4-cas12 (lc4-cr2) were obtained, including Figure 2 the gene editing lines with three-base knockout as exemplified. Meanwhile, the inventors also prepared overexpression lines (LC4-OE) overexpressing the lc4 gene for comparison.

[0162] Phenotype observation showed that compared with the wild type ZH11 and the overexpression lines (LC4-OE), these novel gene editing materials (lc4-cr2) not only showed a phenotype of compact plant type, but also showed phenotypes such as significantly reduced plant height, decreased tillering and early flowering, as shown in Figure 2 and Figure 3 .

[0163] Therefore, in addition to verifying the regulation of the compact plant type of LC4, its new regulatory role was also discovered.

[0164] Example 3. Trait optimization of water-saving and drought-resistant rice

[0165] Based on the phenotypic analysis of the above gene editing materials and after clarifying the new regulatory role of LC4, the inventors further attempted to improve the traits of different chassis varieties by gene editing of LC4, with the expectation of obtaining new rice germplasms with a compact plant type, which are convenient for reasonable close planting and can improve the yield per unit land area.

[0166] The inventors used three water-saving and drought-resistant rice varieties (Shanghai Agrobiological Gene Center) Huhan 1505 (Hh1505), Huhan 106 (Hh106) and Huhan 1516 (Hh1516) as chassis varieties and attempted gene editing with the lc4-cas12 vector. Multiple gene editing lines were selected for each chassis variety for phenotypic analysis.

[0167] Analysis after planting showed that the gene-edited Huhan 1505 (lc4-cr / Hh1505), Huhan 106 (lc4-cr / Hh106) and Huhan 1516 (lc4-cr / Hh1516) all showed obvious phenotypes of compact plant type, reduced plant height and early flowering. The analysis results of the gene editing materials of each variety (each represented by three plants: -1, -2, -3) are shown in Figure 4 , Figure 5 and Figure 6 .

[0168] Taking the gene-edited material lc4-cr / Hh1505 of Huhang 1505 as an example for yield measurement and analysis. The preliminary yield measurement data in Songjiang, Shanghai in 2024 showed that lc4-cr / Hh1505 exhibited an obvious advantage of increased yield under high planting density, with a significant increase in yield per unit land area.

[0169] The improvement of these traits makes the plant type of water-saving and drought-resistant rice more compact, reduces the plant height, decreases vegetative growth, and also increases its lodging resistance. The tiller number increases significantly, and the earlier flowering time also enables the new rice to be on the market earlier due to the shorter total growth cycle. The earlier listing of the new rice significantly improves its market value.

[0170] Example 3. Trait optimization of local main cultivars

[0171] The inventors used the local main cultivars with plant types to be improved, Daohuaxiang 2 (DH2), Daohuaxiang Yangzhou (DHY), and Songgeng 6 (Sg6) (all three rice lines were obtained from the China National Rice Research Institute) as the chassis, carried out gene editing of the lc4-cas12 vector, and performed sequencing analysis on the edited materials to clarify the editing sites.

[0172] At the same time, further phenotypic analysis showed that the gene-edited materials not only had a compact plant type, but also showed significant reduction in plant height and different degrees of increase in tiller number. See Figure 7 、 Figure 8 and Figure 9 (Three plants of each main cultivar are represented: -1, -2, -3).

[0173] The improvement of the plant type traits of the three materials can also achieve reasonable close planting per unit land area and increase the yield per unit land area.

[0174] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims. At the same time, all the documents mentioned in the present invention are cited in this application as references, just as if each document is cited separately as a reference.

Claims

1. Application of a LC4 down-regulatory molecule to optimize the phenotype of grass plants; in, The optimization of the gramineous plant phenotype includes: increasing the number of tillers, reducing plant height, promoting compact plant shape, and promoting early flowering.

2. The use according to claim 1, characterized in that The down-regulating molecules down-regulate the expression or activity of LC4, including: reagents for causing loss-of-function mutation of LC4, reagents for knocking out or silencing LC4, and reagents for inhibiting the activity of LC4; preferably include: CRISPR gene editing reagents for LC4, homologous recombination reagents, site-directed mutagenesis reagents, or interfering molecules that specifically interfere with the expression of the gene encoding LC4.

3. The use according to claim 2, characterized in that The down-regulating molecule is a CRISPR gene editing reagent; preferably, it is a CRISPR / Cas12 reagent; more preferably, the CRISPR / Cas12 reagent includes gRNA, and the gRNA includes: TTTGAGCATGGTTCAATCAAAGATGAT.

4. The use according to claim 1, 2 or 3, characterized in that: Phenotypic optimized grass plants have one or more of the following properties: increased number of plants per unit area, lodging resistance, increased yield per unit area, and a short growth cycle; Preferably, the grass plant is a water-saving and drought-resistant plant.

5. An application of endogenous LC4 of a gramineous plant as a target gene for identifying the phenotype of a gramineous plant, or as a target gene for targeted screening of plants; wherein, The phenotypes include: tiller number, plant height, plant compactness, and flowering period.

6. A method for optimizing the phenotype of a grass plant, comprising: Reducing the expression or activity of LC4 in grass plants; The optimization of the phenotype of grass plants includes: increasing the number of tillers, reducing plant height, promoting compact plant shape, and promoting early flowering.

7. The method according to claim 6, characterized in that The method comprises: performing a loss-of-function mutation on LC4 in a plant expressing LC4, knocking out or silencing a gene encoding LC4, or inhibiting the activity of LC4.

8. The method according to claim 7, characterized in that The loss-of-function mutation of LC4 includes: targeted modification of the coding gene of LC4 to reduce its function / activity; preferably, causing the coding gene to undergo frameshifting so that translation is terminated prematurely; preferably, causing the coding gene to undergo frameshifting so that translation is terminated prematurely.

9. The method according to claim 8, characterized in that The method comprises: performing gene editing by using a CRISPR system to knock out the coding gene of LC4; silencing LC4 by using an interfering molecule that specifically interferes with the expression of the coding gene of LC4; knocking out the coding gene of LC4 by a homologous recombination method; or constructing a plant screening library formed by gene insertion and deletion, mutation deletion or deletion for the purpose of down-regulating LC4 in plants, and screening plants with down-regulated LC4 therefrom; Preferably, the method is implemented with a CRISPR gene editing reagent; preferably, the CRISPR gene editing reagent is a CRISPR / Cas12 reagent; more preferably, the CRISPR / Cas12 reagent includes a gRNA, and the gRNA includes: TTTGAGCATGGTTCAATCAAAGATGAT.

10. The use according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9, characterized in that: The LC4 is derived from or the grass plants include: rice, wheat, millet, foxtail millet, corn, sorghum, millet, barley, rye, oats, and Brachypodium distichum; preferably, the grass plant is rice; more preferably, the rice includes water-saving and drought-resistant varieties.

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