ZmRLR1 gene and its coded protein in regulating corn root development

Abstract

The application discloses ZmRLR1 application of a gene and a coded protein in regulating corn root development. ZmRLR1 The nucleotide sequence of the gene is shown in the sequence table SEQ ID No: 1, and the protein sequence is shown in the sequence table SEQ ID No: 2. The number of lateral root primordia, the total length of lateral roots and the total lateral root density of the seedling stage of the ZmRLR1 protein overexpression transgenic plant are significantly increased, the number and 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.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving ZmRLR1 Application of genes and their encoded proteins in regulating maize root development. Background Technology

[0002] Increasing maize yield is a major demand in the current maize industry. Increasing maize planting density is an important measure to improve yield. However, increased planting density also increases the risk of lodging. Roots, as vital organs for water and nutrient absorption, contribute to enhanced lodging resistance and efficient nutrient absorption and utilization. Lateral roots, in particular, play a crucial role in adapting to various environmental conditions, acting as essential organs for water and nutrient absorption and stress management. Therefore, identifying key genes regulating maize root development is of great significance for breeding new lodging-resistant maize varieties. Summary of the Invention

[0003] The purpose of this invention is to provide ZmRLR1 Application of genes and their encoded proteins in regulating maize root development.

[0004] ZmRLR1 Genes, the ones mentioned ZmRLR1 The gene polynucleotides are shown in (a), (b), (c), or (d):

[0005] (a) A polynucleotide as shown in SEQ ID No: 1; or

[0006] (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under strict hybridization conditions, and the protein encoded by the polynucleotide still has the function of regulating maize root development;

[0007] (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or

[0008] (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the function of regulating maize root development.

[0009] ZmRLR1 protein, the amino acid sequence of which is shown in (a), (b), or (c):

[0010] (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or

[0011] (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or

[0012] (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the function of regulating maize root development.

[0013] Amplification of the ZmRLR1 Primers for any segment of a gene.

[0014] Containing the ZmRLR1 Recombinant gene expression vectors.

[0015] The ZmRLR1 Application of the gene or the ZmRLR1 protein in regulating maize root development.

[0016] The ZmRLR1 The gene or ZmRLR1 protein increases the root system of maize plants, increases the total root length of the fifth crown root, and increases the weight of the rhizosphere soil.

[0017] A method to increase the root system of corn, ZmRLR1 The gene is overexpressed in the maize plant.

[0018] Beneficial effects of the present invention: The present invention discovers that corn ZmRLR1 Genes and their encoded proteins can regulate maize root development. Transgenic plants overexpressing ZmRLR1 protein showed a significant increase in the number of lateral root primordia, total lateral root length, and total lateral root density during the seedling stage. At maturity, the number and size of the fifth crown root layer were significantly increased, and the weight of the rhizosphere soil also increased. Attached Figure Description

[0019] Figure 1 for ZmRLR1 The relative expression of different tissues in maize during the seedling and maturity stages.

[0020] Figure 2 for rlr1 ems Root phenotypes of mutants at seedling and mature stages;

[0021] In the diagram, 'a' represents... rlr1 ems Schematic diagram of mutation sites in mutants; b is rlr1 ems The number of lateral root primordia on the primary root of the mutant after 7 days of hydroponics; c is rlr1 ems Lateral root density of the mutant after 7 days of hydroponics; d is rlr1 ems The total length of lateral roots of the mutant after 7 days of hydroponics; e is rlr1ems The root phenotype of the mutant at maturity; f is rlr1 ems Total length of the fifth crown root layer at maturity of the mutant; g is rlr1 ems The weight of the rhizosphere soil at the mature stage of the mutant.

[0022] Figure 3 zmrlr1 Construction and identification of knockout mutants;

[0023] In the diagram, 'a' represents... zmrlr1 The target site for knocking out mutants; b is zmrlr1 Editing type for knockout mutants.

[0024] Figure 4 for ZmRLR1 Identification of overexpressing transgenic plants.

[0025] Figure 5 for ZmRLR1 Root phenotype analysis of knockout mutants and overexpressing transgenes;

[0026] In the figure, a represents the number of lateral root primordia of the taproot after 7 days of hydroponics; b represents the lateral root density of the root system after 7 days of hydroponics; c represents the total length of the lateral roots of the root system after 7 days of hydroponics; d represents the root phenotype at maturity; e represents the total length of the fifth layer of crown roots at maturity; and f represents the weight of the rhizosphere soil at maturity. Detailed Implementation

[0027] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0028] The experimental materials used in the following examples were: maize inbred lines: B73, CAL50; strains: Escherichia coli strain DH5α, Agrobacterium strain GV3101; and overexpression vector: CUB.

[0029] Example 1 Real-time fluorescence quantification ZmRLR1 Root-specific expression in maize

[0030] Samples were collected from seed roots, primary roots, and aboveground parts of maize inbred line B73 grown hydroponically for 7 days, as well as from roots, leaves, stems, ears, and kernels at field maturity. Total RNA was extracted from the aboveground parts and reverse transcribed to generate first-strand cDNA. Using this first-strand cDNA as a template, 5'-CATCCTCTTGTCT was used for transcribing.

[0031] A primer pair consisting of TGCATATGC-3' and 5'-CTCGGAACAACACAACATACAG-3' was used for real-time quantitative PCR amplification to identify... ZmRLR1 Relative gene expression levels. (Selection) Zmactin1 The gene is an internal reference gene (the primer pair used to identify the internal reference gene consists of 5'-CAATGGCACTGGAATGG).

[0032] (Composed of T-3' and 5'-ATCTTCAGGCGAAACACG-3'). Real-time fluorescence quantification was performed using an Applied Biosystems 7500 RealTime PCR system (ABI, USA), with three replicates per experiment. -ΔΔCT The relative expression level is calculated using this method.

[0033] See results Figure 1 , ZmRLR1 Specific expression in primary roots during the seedling stage and root system during the mature stage of maize.

[0034] Example 2 ZmRLR1 EMS mutant root phenotypic analysis

[0035] From the maize EMS induced mutation database ( http: / / elabcaas.cn / memd / public / index.html# / Two EMS mutants under the B73 background were obtained. These mutants exhibited G-to-A and C-to-T mutations in exons, respectively, causing premature termination of the coding sequence. This mutant was named... rlr1 ems #1 and rlr1 ems #2 Figure 2 a). Wild type and rlr1 ems After hydroponic treatment for 7 days, the mutant strain was found to be superior to the wild type. rlr1 ems The number of lateral root primordia in the primary root of the mutant ( Figure 2 b) Total lateral root density ( Figure 2 c) and length were both significantly reduced ( Figure 2 d). Under field conditions, compared to the wild type, rlr1 ems The mutant's root system is smaller ( Figure 2 e), the total root length of the fifth layer of crown roots is significantly reduced ( Figure 2 f), the weight of the rhizosphere soil decreased ( Figure 2 g).

[0036] Example 3 ZmRLR1 Construction and identification of knockout mutants

[0037] exist ZmRLR1Two target sites (5'-GACGTACGACGCCAAGACG-3' and 5'-CGTGGGCGATCAACCCGGC-3') were designed in the exon regions of the gene. CRISPR / Cas9 editing technology was used to transform CAL50 background material to construct... zmrlr1 Knockout mutant ( Figure 3 a). Using primer pairs consisting of 5'-GGCAAGGCTTTCTTCATACCAC-3' and 5'-TGTTGTGTCATCTCTCTCCCGC-3', two homozygous mutation types were obtained through PCR amplification and first-generation sequencing, namely... rlr1 #1 and rlr1 #2. The result is as follows: Figure 3 As shown in b. rlr1 #1 is missing 79bp. rlr1 #2 is missing 80bp.

[0038] Example 4 ZmRLR1 overexpression transgenic plants

[0039] Using B73 cDNA as a template, PCR amplification was performed using the upstream primer 5'-CTCTAGAGGATCGGTCACCATGGCCTACGCGACGCAGCAT-3' and the downstream primer 5'-GGGCCCGCGGTACGGTGACCCATCGCCAAGGAACCTGCCAAT-3'. A DNA fragment of approximately 830 bp was purified and recovered. ZmRLR1 Full-length sequence. The CUB vector was digested with the restriction endonuclease BamHI, and the DNA fragment was inserted into the linearized CUB vector via In-Fusion (Clontech, catalog number 639648) to obtain the recombinant vector CUB-ZmRLR1-EGFP-MYC. This vector was transformed into the maize inbred line CAL50 using Agrobacterium tumefaciens infection of immature embryos to obtain... ZmRLR1 Overexpressing transgenic plants ( Zm RLR1 OE#1 and OE#2 Using primer pairs consisting of 5'-CGTAGTCAACACCTACAACGT-3' and 5'-GCTGCAGCTTCCCGTAGAT-3', quantitative real-time PCR was used to detect... ZmRLR1 In overexpressing transgenic plants ZmRLR1 The expression situation, the results are shown in Figure 4 , Zm RLR1 OE#1 and OE#2 Compared to wild type, the two transformation events showed a 9.5-fold and 11-fold increase in transcriptional expression levels, respectively.

[0040] Example 5 ZmRLR1Root phenotype analysis of knockout mutants and overexpression transgenes

[0041] Observe separately ZmRLR1 Root phenotypes of knockout mutants and overexpressing transgenes at 7 days of hydroponic growth and at field maturity. Results are as follows. Figure 5 As shown, compared to the wild type, ZmRLR1 The number of lateral root primordia in the main root of overexpressing transgenic plants ( Figure 5 a) Total length of lateral roots ( Figure 5 b) Both total lateral root density and total lateral root density increased significantly; rlr1 The number of lateral root primordia in the primary root of the knockout mutant ( Figure 5 a) Total length of lateral roots ( Figure 5 b) and total lateral root density were both significantly reduced ( Figure 5 c). Under field conditions, compared to the wild type, ZmRLR1 The roots of overexpressing transgenic plants are larger ( Figure 5 d), the total root length of the fifth layer of crown roots increased significantly ( Figure 5 e), the weight of the rhizosphere soil increases ( Figure 5 f); rlr1 The root system of the knockout mutant is smaller. Figure 5 d), the total root length of the fifth layer of crown roots decreased significantly ( Figure 5 e), the weight of the rhizosphere soil decreases ( Figure 5 f).

[0042] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. 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 all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of the ZmRLR1 gene or ZmRLR1 protein in upregulating maize root development, characterized in that, The nucleotide sequence of the ZmRLR1 gene is shown in SEQ ID No: 1, and the amino acid sequence of the ZmRLR1 protein is shown in SEQ ID No:

2.

2. The application according to claim 1, characterized in that, The ZmRLR1 gene or ZmRLR1 protein increases the root system of maize plants, increases the total root length of the fifth crown root, and increases the weight of the rhizosphere soil.

3. A method for increasing the root system of corn, characterized in that, The ZmRLR1 gene was overexpressed in maize plants, and the nucleotide sequence of the ZmRLR1 gene is shown in SEQ ID No: 1.

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