Application of ZmRLR1 gene and encoded protein thereof in regulation and control of corn root development

Abstract

The invention discloses an application of a ZmRLR1 gene and an encoded protein thereof in regulation and control of corn root development. The nucleotide sequence of the ZmRLR1 gene is shown as a sequence table SEQ ID No: 1, and the protein sequence of the ZmRLR1 gene is shown as a sequence table SEQ ID No: 2. The root lateral root primordium number, the lateral root total length and the total lateral root density of the ZmRLR1 protein overexpression transgenic plant in the seedling stage are all remarkably increased, the root number and the root size of the fifth-layer crown root in the mature stage are remarkably increased, and the weight of rhizosphere soil is increased.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to ZmRLR1 Application of genes and their encoded proteins in regulating maize root development. Background Art

[0002] Improving corn yield is a major demand of the current corn industry. Increasing corn planting density is an important measure to increase corn yield. However, increasing corn planting density will increase the risk of corn lodging. The root system is an important organ for plants to absorb water and nutrients. A good root system configuration helps to enhance the crop's resistance to lodging and the efficient absorption and utilization of nutrients. In particular, lateral roots, as important organs for plants to absorb water, nutrients and cope with stress, play a vital role in adapting to various environmental conditions. Therefore, discovering the key genes that regulate corn root development is of great significance for breeding new lodging-resistant corn varieties. Summary of the invention

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

[0004] ZmRLR1 Gene, ZmRLR1 The polynucleotide of the gene is shown as (a), (b), (c) or (d): (a) a polynucleotide as shown in SEQ ID No: 1; or (b) a polynucleotide that can hybridize with the complementary sequence of SEQ ID No: 1 under stringent hybridization conditions, and the protein encoded by the polynucleotide still has the function of regulating the development of corn root system; (c) a polynucleotide having at least 90% homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, and the protein encoded by the polynucleotide mutant still has the function of regulating the development of corn root system.

[0005] ZmRLR1 protein, wherein 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 in the sequence listing; or (b) an amino acid having at least 90% homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids in the protein shown in SEQ ID No: 2, and the protein still has the function of regulating the development of corn root system.

[0006] Amplify the ZmRLR1 Primers for any fragment of a gene.

[0007] Containing the ZmRLR1 Recombinant gene expression vector.

[0008] Said ZmRLR1 Application of the gene or the ZmRLR1 protein in regulating corn root development.

[0009] Said ZmRLR1 The gene or ZmRLR1 protein enlarges the root system of corn plants, increases the total length of the root system of the fifth crown root layer, and increases the weight of the rhizosphere soil.

[0010] A method for increasing the root system of corn, ZmRLR1 Gene overexpression in maize plants.

[0011] Beneficial effects of the present invention: The present invention finds that corn ZmRLR1 The gene and its encoded protein can regulate the development of maize root system. The number of lateral root primordia, total length of lateral roots and total lateral root density of transgenic plants overexpressing ZmRLR1 protein at the seedling stage were significantly increased, the number and size of roots in the fifth crown root at maturity were significantly increased, and the weight of rhizosphere soil was increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 for ZmRLR1 Relative expression of various tissues in maize seedling and maturity stages.

[0013] Figure 2 for rlr1 ems Root phenotypes of mutants at seedling and mature stages; In the figure, a is rlr1 ems Schematic diagram of the mutation site of the mutant; b is rlr1 ems The number of lateral root primordia of the main root of the mutant after 7 days of hydroponics; c is rlr1 ems The lateral root density of the mutant after 7 days of hydroponics; d is rlr1 ems The total length of the lateral roots of the mutant after 7 days of hydroponics; e is rlr1 ems Root phenotype of mutant at mature stage; f is rlr1 ems Total length of the fifth crown root of the mutant at maturity; g isrlr1 ems Rhizosphere soil weight of mutants at maturity.

[0014] Figure 3 zmrlr1 Construction and identification of knockout mutants; In the figure, a is zmrlr1 The target site of the knockout mutant; b is zmrlr1 Editing type of knockout mutants.

[0015] Figure 4 for ZmRLR1 Identification of overexpressing transgenic plants.

[0016] Figure 5 for ZmRLR1 Root phenotypic analysis of knockout mutants and overexpressing transgenics; In the figure, a is the number of lateral root primordia of the main root after 7 days of hydroponics; b is the lateral root density of the root system after 7 days of hydroponics; c is the total length of the lateral roots of the root system after 7 days of hydroponics; d is the root phenotype at maturity; e is the total length of the fifth layer of crown roots at maturity; f is the weight of rhizosphere soil at maturity. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0018] The experimental materials used in the following examples are: maize inbred line materials: B73, CAL50; strains: Escherichia coli strain DH5α, Agrobacterium strain GV3101; and the overexpression vector is CUB.

[0019] Example 1 Real-time fluorescence quantification ZmRLR1 Specific expression in maize roots The seminal roots, primary roots and aerial parts of maize inbred line B73 grown in hydroponics for 7 days, as well as the roots, leaves, stems, female ears and grains at maturity in the field were selected. Total RNA of aerial parts was extracted and reverse transcribed to generate first-strand cDNA. The first-strand cDNA was used as a template and the 5'-CATCCTCTTGTCT The primer pair consisting of TGCATATGC-3' and 5'-CTCGGAACAACACAACATACAG-3' was used for real-time fluorescence quantitative PCR amplification and identification. ZmRLR1 The relative expression level of the gene. Zmactin1 The gene is an internal reference gene (the primer pair used to identify the internal reference gene is composed of 5'-CAATGGCACTGGAATGG T-3' and 5'-ATCTTCAGGCGAAACACG-3'). Real-time fluorescence quantification was performed using the Applied Biosystems 7500 RealTime PCR system (ABI, USA), and three replicates were set for each parallel experiment. -ΔΔCT The relative expression was calculated by the method.

[0020] Results Figure 1 , ZmRLR1 It is specifically expressed in the primary roots of maize seedlings and mature roots.

[0021] Example 2 ZmRLR1 Root phenotype analysis of EMS mutants From the Maize EMS-Induced Mutation Database ( http: / / elabcaas.cn / memd / public / index.html# / ) obtained two EMS mutants in the B73 background. The mutants had mutations from G to A and C to T in the exons, causing the coding sequence to terminate prematurely. This mutant was named rlr1 ems #1 and rlr1 ems #2( Figure 2 a). The wild type and rlr1 ems The mutant was treated with hydroponics for 7 days and the results showed that compared with the wild type, rlr1 ems The number of lateral root primordia in the mutant taproot ( Figure 2 b) Total lateral root density ( Figure 2 c) and length were significantly reduced ( Figure 2 d). Under field conditions, compared with the wild type, rlr1 ems The mutant had a smaller root system ( Figure 2 e), the total root length of the fifth crown root was significantly reduced ( Figure 2 f), the weight of rhizosphere soil decreased ( Figure 2 g).

[0022] Example 3 ZmRLR1 Knockout mutant construction and identification exist ZmRLR1 Two target sites (5'-GACGTACGACGCCAAGACG-3' and 5'-CGTGGGCGATCAACCCGGC-3') were designed in the exon region of the gene, and CRISPR / Cas9 editing technology was used to transform the CAL50 background material to construct zmrlr1 Knockout mutants ( Figure 3a). By using the primer pair consisting of 5'-GGCAAGGCTTTCTTCATACCAC-3' and 5'-TGTTGTGTCATCTCTCTCCCGC-3', two homozygous mutation types were obtained by PCR amplification and first-generation sequencing. rlr1 #1 and rlr1 #2. The result is as follows Figure 3 As shown in b, rlr1 #1 is missing 79 bp, rlr1 #2 is missing 80bp.

[0023] Example 4 ZmRLR1 Overexpressing transgenic plants Using B73 cDNA as a template, PCR amplification was performed using the upstream primer 5'-CTCTAGAGGATCGGTCACCATGGCCTACGCGACGCAGCAT-3' and the downstream primer 5'-GGGCCCGCGGTACGGTGACCCATCGCCAAGGAACCTGCCAAT-3', and a DNA fragment of about 830 bp was purified and recovered to obtain ZmRLR1 The CUB vector was cut with restriction endonuclease BamH I, and the DNA fragment was inserted into the linearized CUB vector by In-Fusion (Clontech, catalog number 639648) to obtain the recombinant vector CUB-ZmRLR1-EGFP-MYC. The vector was transformed into maize inbred line CAL50 by Agrobacterium infection of immature embryos to obtain ZmRLR1 Overexpressing transgenic plants ( Zm RLR1 OE#1 and OE#2 ). The primer pair consisting of 5'-CGTAGTCAACACCTACAACGT-3' and 5'- GCTGCAGCTTCCCGTAGAT-3' was used for fluorescence quantitative PCR detection. ZmRLR1 Overexpression in transgenic plants ZmRLR1 The results of expression are shown in Figure 4 , Zm RLR1 OE#1 and OE#2 Compared with the wild type, the expression levels of the two transformation events increased by 9.5 and 11 times.

[0024] Example 5 ZmRLR1 Root phenotype analysis of knockout mutants and overexpressing transgenics Observe separately ZmRLR1 Root phenotypes of knockout mutants and overexpressing transgenic plants at 7 days of hydroponic growth and field maturity. Figure 5 As shown, compared with the wild type, ZmRLR1 The number of lateral root primordia in the main root of the transgenic plants overexpressing Figure 5a) Total length of lateral roots ( Figure 5 b) 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 significantly reduced ( Figure 5 c). Under field conditions, compared with the wild type, ZmRLR1 The root system of the overexpressing transgenic plants became larger ( Figure 5 d), the total root length of the fifth crown root increased significantly ( Figure 5 e), the weight of rhizosphere soil increased ( Figure 5 f); rlr1 The knockout mutant has a smaller root system ( Figure 5 d), the total root length of the fifth crown root was significantly reduced ( Figure 5 e), the weight of rhizosphere soil decreased ( Figure 5 f).

[0025] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. ZmRLR1 A gene characterized by Said ZmRLR1 The polynucleotide of the gene is shown as (a), (b), (c) or (d): (a) a polynucleotide as shown in SEQ ID No: 1; or (b) a polynucleotide that can hybridize with the complementary sequence of SEQ ID No: 1 under stringent hybridization conditions, wherein the protein encoded by the polynucleotide still has the function of regulating the development of corn root system; (c) a polynucleotide having at least 90% homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, and the protein encoded by the polynucleotide mutant still has the function of regulating the development of corn root system.

2. ZmRLR1 protein, characterized in that The amino acid sequence of the ZmRLR1 protein is shown in (a), (b) or (c): (a) the amino acid sequence shown in SEQ ID No: 2 in the sequence listing; or (b) an amino acid having at least 90% homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids in the protein shown in SEQ ID No: 2, and the protein still has the function of regulating the development of corn root system.

3. Amplification of claim 1 ZmRLR1 Primers for any fragment of a gene.

4. Containing the composition according to claim 1 ZmRLR1 Recombinant gene expression vector.

5. The method according to claim 1 ZmRLR1 Use of the gene or the ZmRLR1 protein according to claim 2 in regulating corn root development.

6. According to claim 5 ZmRLR1 The use of the gene or the ZmRLR1 protein according to claim 2 in regulating the development of corn root system, characterized in that: Said ZmRLR1 The gene or ZmRLR1 protein enlarges the root system of corn plants, increases the total length of the root system of the fifth crown root layer, and increases the weight of the rhizosphere soil.

7. A method for increasing the root system of corn, characterized in that: Will ZmRLR1 Gene overexpression in maize plants.

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