Use of zmrlr1 gene and encoded protein thereof in regulating development of corn root system

Abstract

Disclosed is a use of a ZmRLR1 gene and an encoded protein thereof in regulating the development of a corn root system. By overexpressing the ZmRLR1 protein of a genetically modified plant, the number of lateral root primordia, the total length of lateral roots, and the total lateral root density of the root system in the seedling stage are all significantly increased, the number of root systems and the size of the root systems of the fifth layer of crown roots in the maturation stage are significantly increased, and the weight of the rhizosphere soil is increased.

Description

Application of ZmRLR1 gene and the encoded protein thereof in regulating maize root development

[0001] The present application claims priority to the prior application filed on September 9, 2024 with the China National Intellectual Property Office and with the patent application number 202411255777.2, and the title of the invention is "Application of ZmRLR1 gene and the encoded protein thereof in regulating maize root development". The prior application is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, and more particularly to the application of ZmRLR1 gene and the encoded protein thereof in regulating maize root development. BACKGROUND

[0003] Maize is the first grain crop in China, and its annual output accounts for 23% of the global output. It is of great importance to food security, economic development and energy crisis relief. In recent years, due to the gap between the growth of domestic maize consumption and the stagnation of production, the import of maize in China has increased rapidly. Increasing maize yield is a major demand of the current maize industry. Increasing maize planting density is an important measure to increase maize yield. However, the increase of maize planting density will increase the risk of maize lodging. As an important organ for plants to absorb water and nutrients, good root architecture helps to enhance the lodging resistance and nutrient efficient absorption and utilization efficiency of crops. Especially, lateral roots, as an important organ for plants to absorb water, nutrients and respond to stress, play a crucial role in adapting to various environmental conditions. Therefore, exploring key genes that regulate maize root development is of great significance for breeding new maize varieties with lodging resistance. SUMMARY

[0004] The present application aims to provide the application of ZmRLR1 gene and the encoded protein thereof in regulating maize root development.

[0005] In a first aspect, the present application provides a ZmRLR1 gene, wherein the polynucleotide of the ZmRLR1 gene is represented by (a), (b), (c) or (d):

[0006] (a) a polynucleotide as represented in SEQ ID No: 1; or

[0007] (b) a polynucleotide capable of hybridizing to the complement of SEQ ID No: 1 under stringent hybridization conditions, wherein the encoded protein still has the function of regulating maize root development;

[0008] (c) a polynucleotide having at least 90% or more homology with the polynucleotide represented by SEQ ID No: 1; or

[0009] (d) a polynucleotide mutant obtained by deletion, substitution or insertion of one or more bases in the polynucleotide shown in SEQ ID No: 1, and the protein encoded by the polynucleotide mutant still has the function of regulating the root system development of corn.

[0010] The second aspect of the present application provides a ZmRLR1 protein, the amino acid sequence of the ZmRLR1 protein is shown in (a), (b) or (c):

[0011] (a) the amino acid sequence shown in the sequence table SEQ ID No: 2; or

[0012] (b) an amino acid having at least 90% or above homology with the amino acid shown in SEQ ID No: 2; or

[0013] (c) a protein mutant obtained by deletion, substitution or insertion of one or more amino acids in the protein shown in SEQ ID No: 2, and the protein still has the function of regulating the root system development of corn.

[0014] The third aspect of the present application provides a primer for amplifying any fragment of the ZmRLR1 gene.

[0015] The fourth aspect of the present application provides a recombinant expression vector containing the ZmRLR1 gene.

[0016] The fifth aspect of the present application provides the application of the ZmRLR1 gene or the ZmRLR1 protein in regulating the root system development of corn.

[0017] Further, when the expression level of the ZmRLR1 gene or the content or activity of the ZmRLR1 protein is increased, the root system of the corn plant is increased, the total length of the root system of the fifth layer of crown root is increased, and the weight of the rhizosphere soil is increased; or when the expression level of the ZmRLR1 gene or the content or activity of the ZmRLR1 protein is reduced, the root system of the corn plant is reduced, the total length of the root system of the fifth layer of crown root is reduced, and the weight of the rhizosphere soil is reduced.

[0018] The sixth aspect of the present application provides a method for increasing the root system of corn, overexpressing the ZmRLR1 gene in the corn plant.

[0019] The seventh aspect of the present application provides a corn breeding method, the method comprising the following steps:

[0020] S1) providing corn breeding material to be improved;

[0021] S2) increasing the expression level of the ZmRLR1 gene in the corn breeding material;

[0022] S3) obtaining corn breeding material with improved root system phenotype.

[0023] Further, the root phenotype improvement comprises at least one of:

[0024] 1) an increase in the number of root primordia, the total length of lateral roots and the total lateral root density of the root system at the seedling stage, as compared to the corn breeding material to be improved;

[0025] 2) an increase in the number of root systems and the size of the root systems of the fifth layer of crown roots at the maturation stage, as compared to the corn breeding material to be improved; or

[0026] 3) an increase in the weight of the rhizosphere soil, as compared to the corn breeding material to be improved.

[0027] Further, the expression level of the ZmRLR1 gene is increased by any one of:

[0028] transgenesis, hybridization, backcrossing, selfing or vegetative reproduction;

[0029] The transgenesis preferably comprises one or more of the following methods: Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated.

[0030] In an eighth aspect, the present application provides a corn plant or corn plant material, wherein the corn plant or corn plant material is obtained by the corn breeding method.

[0031] In a ninth aspect, the present application provides a product made from the corn plant or corn plant material obtained by the above method.

[0032] Further, the product is a food, a feed, an industrial raw material or a biofuel.

[0033] Further, the food is selected from the group consisting of corn flour, corn oil, corn starch or corn syrup. The feed is an animal feed composition comprising an ingredient derived from the corn plant or a part thereof.

[0034] In a tenth aspect, the present application provides a method for preparing the product, comprising the steps of: a) providing the corn plant or corn plant material obtained by the method; and b) processing the corn plant or corn plant material to make a food, a feed, an industrial raw material or a biofuel.

[0035] Further, the processing step comprises physical processing, chemical processing or biological processing.

[0036] The application has the beneficial effects that the application finds that the corn ZmRLR1 gene and the encoded protein thereof can regulate the root system development of corn. The number of lateral root primordia, the total length of lateral roots and the total lateral root density of the root system of the ZmRLR1 protein overexpression transgenic plant at the seedling stage are significantly increased, the number of root system and the size of the root system of the fifth layer of crown roots at the mature stage are significantly increased, and the weight of the rhizosphere soil is increased. The more developed corn root system helps to improve the lodging resistance and nutrient absorption and utilization efficiency of corn. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Relative expression of ZmRLR1 in each tissue part of corn at the seedling stage and the mature stage

[0038] Figure 2 Root system phenotype of the mutant at the seedling stage and the mature stage; a is rlr1 ems Figure 2 Root system phenotype of the mutant at the seedling stage and the mature stage; a is rlr1 ems Figure 2 Root system phenotype of the mutant at the seedling stage and the mature stage; a is rlr1 ems Figure 2 Root system phenotype of the mutant at the seedling stage and the mature stage; a is rlr1 ems Figure 2 Root system phenotype of the mutant at the seedling stage and the mature stage; a is rlr1 ems Figure 2 Root system phenotype of the mutant at the seedling stage and the mature stage; a is rlr1 ems Figure 2 Root system phenotype of the mutant at the seedling stage and the mature stage; a is rlr1 ems Figure 2 Root system phenotype of the mutant at the seedling stage and the mature stage; a is rlr1 ems Figure 2 Root system phenotype of the mutant at the seedling stage and the mature stage; a is rlr1

[0039] Figure 3 Construction and identification of the zmrlr1 knockout mutant; a is the target site of the zmrlr1 knockout mutant; b is the editing type of the zmrlr1 knockout mutant.

[0040] Figure 4 Identification of the ZmRLR1 overexpression transgenic plant.

[0041] Figure 5 Root system phenotype analysis of the ZmRLR1 knockout mutant and the overexpression transgenic plant; a is the number of lateral root primordia of the main root after hydroponics for 7 days; b is the lateral root density of the root system after hydroponics for 7 days; c is the total length of lateral roots of the root system after hydroponics for 7 days; d is the root system phenotype at the mature stage; e is the total length of the fifth layer of crown roots at the mature stage; f is the weight of the rhizosphere soil at the mature stage. DETAILED DESCRIPTION

[0042] TERMS

[0043] The following definitions and methods are provided to better define the present application and to guide working persons in the art in the practice of the present application. Unless otherwise defined, the terms are understood according to the conventional usage of the terms by those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards and other published publications cited by the present application are incorporated herein in their entirety as references.

[0044] As used herein, "maize" is intended to mean any maize plant and includes all plant parts, plant cells, plant organs, plant protoplasts, plant cell and tissue cultures of the genus Zea, a plant cell that is intact, has the capability for subsequent development into a whole plant according to biology, and that is derived from any plant part, including embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, roots, root tips, anthers, and the like. Genetically modified maize plants are plants of which a heritable trait has been introduced by genetic engineering methods. In particular, the term "maize" as used herein refers to plants of the species Zea mays L. and related species thereof, including all plant parts, plant cells, plant organs, plant protoplasts, plant cell and tissue cultures of the genus Zea, a plant cell that is intact, has the capability for subsequent development into a whole plant according to biology, and that is derived from any plant part, including embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, roots, root tips, anthers, and the like.

[0045] In this application, the words "comprise," "comprises," and "comprising" or any variation thereof, will be understood to imply a non- limiting inclusion of the elements, numbers or steps recited and any associated description.

[0046] In this application, the words "increase," "increasing," or "significantly increase," "significantly increasing" mean that the respective trait of the subject plant or subject plant cell is improved, e.g., by 1%, 5%, 10% or more, as compared to a control.

[0047] A "subject plant" or "subject plant cell" refers to a plant or plant cell in which a genetic modification has taken effect, or a progeny cell of such a modified plant or cell that contains the modification. A "control" or "control plant" or "control plant cell" provides a reference point against which the altered phenotype of a subject plant or plant cell is measured. A negative or control plant can include, for example: (a) a wild-type plant or cell, i.e., a plant or cell of the same genotype as the starting material from which a subject plant or cell was genetically modified; (b) a plant or plant cell of the same genotype as the starting material but transformed with an empty construct, i.e., a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene; (c) a plant or plant cell that is a non-transformed segregant of a subject plant or plant cell; (d) a plant or plant cell that is genetically identical to the subject plant or plant cell but has not been exposed to a condition or stimulus that induces expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.

[0048] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxyl orientation. The amino acids can be referred to by either their commonly accepted single-letter codes or by accepted three-letter codes. Similarly, nucleotides can be referred to by their commonly accepted single-letter codes. Numeric ranges are inclusive of the numbers defining the range.

[0049] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In one preferred embodiment, for comparison purposes, the length of an aligned reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The comparison of sequences and the determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. In one preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm which has been encapsulated in the Needleman- Wunsch alignment function in the Needle program of EMBOSS (The European Molecular Biology Open Software Suite) version 5.0.0 or later. EMBOSS is freely available from the EMBOSS homepage. Alignment parameters used with the Needleman-Wunsch algorithm can not be uniform for all alignment situations, but can vary. Suitable alignment parameters and the

[0050] As used herein, "nucleic acid" includes reference to deoxyribonucleotides or ribonucleotides in either single- or double-stranded form, and unless otherwise indicated, includes known analogues of natural nucleotides that have similar binding properties (e.g., peptide nucleic acids) to natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to natural nucleotides.

[0051] As used herein, the term "encoding" or "encoded" in the context of a specified nucleic acid refers to the inherent property of that nucleic acid, as opposed to the specified protein, to direct the synthesis of the protein by virtue of its nucleotide sequence. The term "encoding" or "encoded" is used technically in the sense of by definition. As used herein, "full-length sequence" in reference to a specified polynucleotide or its encoded protein refers to the entire nucleic acid sequence or the entire amino acid sequence that has the natural (non-synthetic) endogenous sequence. The full-length polynucleotide encodes the full-length, catalytically active form of the specified protein.

[0052] The terms "polypeptide," "polypeptides," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The terms are used in the context of amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids. The terms are also used in the context of naturally occurring amino acid polymers. The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively "protein"). The amino acid can be a naturally occurring amino acid and, unless otherwise limited, can include known analogs of naturally occurring amino acids that can function in a similar manner.

[0053] Experimental materials used in the following examples: Maize inbred lines: B73, CAL50; strains: E. coli strain DH5a, Agrobacterium strain GV3101; overexpression vector is CUB.

[0054] Example 1 Real-time fluorescent quantitative ZmRLR1 specifically expresses in maize root system

[0055] Select the seed roots, primary roots and aboveground parts of maize inbred line B73 grown in water for 7 days, and the samples of root system, leaf, stem, ear and seed of field mature period. Extract total RNA from the aboveground part, generate first strand cDNA by reverse transcription, use the first strand cDNA as a template, use the primer pair composed of 5'-CATCCTCTTGTCTTGCATATGC-3'(SEQ ID No: 3) and 5'-CTCGGAACAACACAACATACAG-3'(SEQ ID No: 4) to perform real-time fluorescent quantitative PCR amplification, and identify the relative expression level of ZmRLR1 gene. Select Zmactinl gene as the internal reference gene (the primer pair for identifying the internal reference gene is composed of 5'-CAATGGCACTGGAATGGT-3'(SEQ ID No: 5) and 5'-ATCTTCAGGCGAAACACG-3'(SEQ ID No: 6)). Use Applied Biosystems 7500 Real Time PCR system (ABI, USA) to perform real-time fluorescent quantification, set 3 repeats in parallel for each experiment. Calculate the relative expression amount by 2- ΔΔCT The results are shown in Figure 1, and ZmRLR1 specifically expresses in the primary roots of maize seedling stage and the root system of mature stage.

[0056] Example 2 Root system phenotype analysis of ZmRLR1 EMS mutant

[0057] Two EMS mutants in B73 background were obtained from the EMS induced mutation database of maize (http: / / elabcaas.cn / memd / public / index.html / # / ). The mutations of G to A and C to T in the exons of the mutants caused the premature termination of the coding sequence, and the mutants were named rlr1 ems #1 and rlr1 ems #2 (Figure 2a). The wild type and rlr1 ems mutants were treated with water culture for 7 days, and it was found that the number of lateral root primordia (Figure 2b), the total lateral root density (Figure 2c) and the length of the main root of the rlr1 ems mutant were significantly reduced (Figure 2d). Under field conditions, the rlr1 ems mutant had smaller root system (Figure 2e), the total length of the fifth layer of crown root was significantly reduced (Figure 2f), and the weight of rhizosphere soil was reduced (Figure 2g).

[0058] Example 3 Construction and identification of ZmRLR1 knockout mutant

[0059] Two target sites (5'-GACGTACGACGCCAAGACG-3' (SEQ ID No: 7) and 5'-CGTGGGCGATCAACCCGGC-3' (SEQ ID No: 8)) were designed in the exon region of the ZmRLR1 gene, and the CAL50 background material was transformed by using the CRISPR / Cas9 editing technology to construct the zmrlr1 knockout mutant (Figure 3a). By using PCR amplification and first-generation sequencing with primer pairs consisting of 5'-GGCAAGGCTTTCTTCATACCAC-3' and 5'-TGTTGTGTCATCTCTCTCCCGC-3', two homozygous mutant types, rlr1 #1 and rlr1 #2, were obtained. As shown in Figure 3b, rlr1 #1 lacks 79 bp, and rlr1 #2 lacks 80 bp.

[0060] Example 4 ZmRLR1 overexpression transgenic plants

[0061] The full-length sequence of ZmRLR1 was obtained by PCR amplification using the cDNA of B73 as template, using the upstream primer 5'-CTCTAGAGGATCGGTCACCATGGCCTACGCGACGCAGCAT-3' (SEQ ID No: 9) and the downstream primer 5'-GGGCCCGCGGTACGGTGACCCATCGCCAAGGAACCTGCCAAT-3' (SEQ ID No: 10), and purifying and recovering a DNA fragment of about 830 bp. The CUB vector was cut by the restriction enzyme BamH I, and the above DNA fragment was inserted into the linearized CUB vector by In-Fusion (Clontech, product catalog number 639648), to obtain the recombinant vector CUB-ZmRLR1-EGFP-MYC. The vector was transformed into the maize inbred line CAL50 by the method of agrobacterium infection of young embryos, to obtain ZmRLR1 overexpression transgenic plants (Zm RLR1 OE#1 and OE#2). The expression of ZmRLR1 in the ZmRLR1 overexpression transgenic plants was detected by fluorescence quantitative PCR using a primer pair consisting of 5'-CGTAGTCAACACCTACAACGT-3' (SEQ ID No: 11) and 5'-GCTGCAGCTTCCCGTAGAT-3' (SEQ ID No: 12), and the results are shown in Figure 4. The transcriptional expression level of the two transformation events Zm RLR1 OE#1 and OE#2 was increased by 9.5-fold and 11-fold, respectively, compared with the wild type.

[0062] Example 5 Root phenotype analysis of ZmRLR1 knockout mutants and overexpression transgenes

[0063] The root phenotypes of ZmRLR1 knockout mutants and overexpression transgenes were observed under water culture growth for 7 days and under field maturation, respectively. The results are shown in Figure 5. Compared with the wild type, the number of lateral root primordia (Figure 5a), the total length of lateral roots (Figure 5b), and the total lateral root density of the main root of the ZmRLR1 overexpression transgenic plants were all significantly increased; the number of lateral root primordia (Figure 5a), the total length of lateral roots (Figure 5b), and the total lateral root density of the main root of the rlr1 knockout mutants were all significantly decreased (Figure 5c). Under field conditions, compared with the wild type, the root system of the ZmRLR1 overexpression transgenic plants was larger (Figure 5d), the total length of the root system of the fifth layer of crown roots was significantly increased (Figure 5e), and the weight of the rhizosphere soil was increased (Figure 5f); the root system of the rlr1 knockout mutants was smaller (Figure 5d), the total length of the root system of the fifth layer of crown roots was significantly decreased (Figure 5e), and the weight of the rhizosphere soil was decreased (Figure 5f).

[0064] The coding region DNA sequence of ZmRLR1 (SEQ ID No: 1):

[0065] Protein sequence of ZmRLR1 (SEQ ID No: 2):

Claims

1. A ZmRLR1 gene characterized in that, The polynucleotide of the ZmRLR1 gene is as shown in (a), (b), (c) or (d): (a) the polynucleotide as shown in SEQ ID No: 1; or (b) a polynucleotide capable of hybridizing to the complement of SEQ ID No: 1 under stringent hybridization conditions, and the protein encoded by the polynucleotide still has the function of regulating the root development of maize; (c) a polynucleotide homologous to the polynucleotide as shown in SEQ ID No: 1 by at least 90% or above; or (d) a polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide as shown in SEQ ID No: 1, and the protein encoded by the polynucleotide mutant still has the function of regulating the root development of maize.

2. A ZmRLR1 protein characterized in that, The amino acid sequence of the ZmRLR1 protein is as shown in (a), (b) or (c): (a) the amino acid sequence as shown in SEQ ID No: 2; or (b) an amino acid homologous to the amino acid as shown in SEQ ID No: 2 by at least 90% or above; or (c) a protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein as shown in SEQ ID No: 2, and the protein still has the function of regulating the root development of maize.

3. A primer for amplifying any fragment of the ZmRLR1 gene of claim 1.

4. A recombinant expression vector containing the ZmRLR1 gene of claim 1.

5. Use of the ZmRLR1 gene of claim 1 or the ZmRLR1 protein of claim 2 in regulating the root development of maize. Further, when the expression level of the ZmRLR1 gene or the content or activity of the ZmRLR1 protein is increased, the root system of the maize plant is increased, the total length of the root system of the fifth layer of crown root is increased, and the weight of the rhizosphere soil is increased; or when the expression level of the ZmRLR1 gene or the content or activity of the ZmRLR1 protein is decreased, the root system of the maize plant is decreased, the total length of the root system of the fifth layer of crown root is decreased, and the weight of the rhizosphere soil is decreased.

6. A method of increasing corn root mass, comprising, Overexpressing the ZmRLR1 gene in the maize plant in vivo.

7. A method for breeding maize, the method comprising the following steps: S1) providing a maize breeding material to be improved; S2) increasing the expression level of the ZmRLR1 gene in the maize breeding material; S3) obtaining a maize breeding material with improved root system phenotype. Further, the improved root system phenotype comprises at least one of the following: 1) the number of root primordia, the total length of lateral roots and the total lateral root density of the root system at the seedling stage are all increased compared with the maize breeding material to be improved; 2) the number and size of the root system of the fifth layer of crown root at the mature stage are increased compared with the maize breeding material to be improved; or 3) the weight of the rhizosphere soil is increased compared with the maize breeding material to be improved. Further, the expression level of the ZmRLR1 gene is increased by any of the following methods: transgenation, hybridization, backcrossing, selfing or vegetative propagation; The transgene preferably comprises: one or more of Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated method.

8. A maize plant or maize plant material, wherein said maize plant or maize plant material is obtained by the method of claim 7.

9. A product made from a maize plant or maize plant material obtained by the method of claim 7, Further, said product is a food, a feed, an industrial raw material or a biofuel; Further, said food is selected from the group consisting of corn meal, corn oil, corn starch or corn syrup; said feed is an animal feed composition comprising an ingredient derived from said maize plant or part thereof.

10. A method of preparing the product of claim 9, characterized in that, comprising the steps of: a) providing a maize plant or part thereof obtained by the method of claim 8; b) processing said maize plant or part thereof into a food, a feed, an industrial raw material or a biofuel; Further, said processing step comprises a physical processing, a chemical processing or a biological processing.

Images