ZmMARD1 gene and its application in enhancing maize lodging resistance

Abstract

This invention discloses ZmMARD1 Genes and their application in enhancing maize lodging resistance. ZmMARD1 The polynucleotide sequence of the gene is shown in SEQ ID No: 1, and the amino acid sequence of the ZmMARD1 protein is shown in SEQ ID No: 2. This invention utilizes overexpression in maize. ZmMARD1 Genetic analysis revealed that transgenic maize plants exhibited increased stem tensile strength, puncture resistance, and lignin content, enhancing their lodging resistance and laying the foundation for developing lodging-resistant maize varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving ZmMARD1 Genes and their application in enhancing maize's resistance to lodging. Background Technology

[0002] Corn is an important food, feed, and energy crop. In recent years, with the continuous improvement of corn production levels, corn lodging has become a frequent occurrence. Corn lodging not only reduces corn yield but also seriously affects mechanized corn harvesting. Corn lodging is a complex symptom influenced by multiple factors. In addition to environmental factors such as water, nutrients, ecological climate, and pests and diseases, the chemical composition of the crop stem itself also significantly affects its lodging resistance. The composition of the cell wall is closely related to lodging resistance. The cell wall is mainly composed of cellulose, hemicellulose, pectin, lignin, polysaccharides, and proteins. Cellulose is the main component of the cell wall, and its content is directly related to the development of mechanical tissues and closely related to stem bending stress. However, cellulose itself has a very high content in the cell wall, leaving little room for further improvement. Lignin is distributed in the cell walls of lignified mechanical and vascular tissues of plants. As the second largest high-molecular-weight biopolymer after cellulose, it plays an important role in stem stiffness and support and can increase the plant's resistance to pests and diseases. Meanwhile, the morphology and anatomical characteristics of the stem also affect its lodging resistance. The thicker the sclerenchyma subepithelial layer, the higher the stem's rigidity and the stronger its lodging resistance. However, the regulatory mechanism of sclerenchyma subepithelial cell development in maize stems is still unclear.

[0003] The main structural unit of lignin is the hydroxycinnamyl alcohol monomer, which mainly includes three monomers: coniferyl alcohol (G), sinigrin (S), and coumarin (H). In Arabidopsis thaliana, LAC17 and LAC4 It is mainly expressed in the intervascular bundle fiber tissue, and also LAC4 It can also be expressed in vascular bundle fiber tissue. lac4 / lac17 The double-gene knockout mutants exhibited reduced intervascular fiber lignification, collapsed stem vascular bundle cells, and a 20-40% decrease in lignin content. This is the first study to confirm that laccase can participate in the lignin synthesis process within plants. These results suggest that increasing stem lignin content can enhance stem strength and improve lodging resistance. Summary of the Invention

[0004] The purpose of this invention is to provide ZmMARD1 Genes and their application in enhancing maize's resistance to lodging.

[0005] A sort of ZmMARD1 Genes, the ones mentioned ZmMARD1The gene polynucleotides are shown in (a), (b), (c), or (d):

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

[0007] (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 enhancing the lodging resistance of maize;

[0008] (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or

[0009] (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 enhancing the lodging resistance of maize.

[0010] A ZmMARD1 protein, the amino acid sequence of which is shown in (a), (b), or (c):

[0011] (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or

[0012] (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or

[0013] (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 enhancing the lodging resistance of maize.

[0014] Includes the ZmMARD1 The carrier of genes.

[0015] Includes the ZmMARD1 Engineered bacteria that act as vectors for genes.

[0016] The detection ZmMARD1 Primers for any segment of a gene.

[0017] The ZmMARD1 Application of genes in breeding lodging-resistant maize varieties.

[0018] A method to enhance the lodging resistance of maize is to increase the expression level of ZmMARD1 protein in maize.

[0019] Beneficial effects of the present invention: The present invention enables overexpression in maize ZmMARD1Genetic analysis revealed that transgenic maize plants exhibited increased stem tensile strength, puncture resistance, and lignin content, enhancing their lodging resistance and laying the foundation for developing lodging-resistant maize varieties. Attached Figure Description

[0020] Figure 1 for OE #1 OE #2 and OE In the #3 strain of plants ZmMARD1 Relative gene expression levels.

[0021] Figure 2 Wild type and ZmMARD1 Plant height of overexpressing transgenic lines.

[0022] Figure 3 Wild type and ZmMARD1 Stem tensile strength of overexpressing transgenic lines.

[0023] Figure 4 Wild type and ZmMARD1 Lignin staining and signal intensity in the stems of overexpressing transgenic lines.

[0024] Figure 5 Wild type and ZmMARD1 The stem puncture force and stem cortex thickness of the overexpressing transgenic lines. Detailed Implementation

[0025] 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.

[0026] The experimental materials used in the following examples are: maize inbred line B73; strains: Escherichia coli strain DH5α and Agrobacterium strain GV3101; overexpression vector CUB.

[0027] Example 1 ZmMARD1 Preparation and identification of overexpression transgenic plants

[0028] I. Construction of recombinant plasmids

[0029] Total RNA was extracted from maize B73 and reverse transcribed to obtain cDNA. Using this cDNA as a template, PCR amplification was performed using the upstream primer: 5'-GGTCGACTCTAGAGGATCCATGGCAACTGATTCCTCAGCT-3' and the downstream primer: 5'-GCTCGGTACCCGGGGATCCACGGATCAGTTGGAAAATCC-3', yielding the PCR amplification product. Approximately 800 bp of DNA fragment was purified and recovered. ZmMARD1 Full-length sequence. Utilizing restriction endonucleases. BamH I cut the CUB vector and inserted the above DNA fragment into the linearized CUB vector using In-Fusion (Clontech, catalog number 639648) to obtain the recombinant vector CUB-ZmMARD1.

[0030] II. Preparation of Transgenic Plants

[0031] The research focuses on maize variety B73. Maize variety B73, also known as the wild-type plant, is represented by WT in the figure.

[0032] Recombinant plasmid pCUB-ZmMARD1 was introduced into background plants to obtain T0 generation transgenic plants. T0 generation transgenic plants were self-pollinated to obtain T1 generation plants, and T1 generation plants were then self-pollinated to obtain T2 generation plants. Homozygous transgenic lines were obtained from the T2 generation and named [the transgenic line is missing from the original text]. ZmMARD1 Gene overexpression lines. Three were randomly selected. ZmMARD1 Gene overexpression lines ( OE #1 OE #2 and OE Subsequent experiments were conducted on strain #3.

[0033] III. Real-time quantitative PCR detection

[0034] The experimental plants were: T2 generation plants of the #1, #2 and #3 lines, and background plants.

[0035] Three plants were used for each line, and the average value of the results was taken.

[0036] Leaves were collected from experimental plants grown under hydroponic conditions for 7 days (counting from the start of germination). Total RNA was extracted and amplified by PCR using the upstream primer: 5'-CTTGTGGAAGACGGTCTGATG-3' and the downstream primer: 5'-GGAGACGCAGGATGATATGC-3'. ZmMARD1 Relative gene expression levels. The Ubi gene was used as an internal reference gene (upstream primer: 5'-GCTGCCGATGTGCCTGCGTCG-3' and downstream primer: 5'-CTGAAAGACAGAACATAATGAGCACAG-3').

[0037] See results Figure 1 Compared to wild-type plants, OE #1 OE #2 and OE In the #3 strain of plants ZmMARD1 The relative expression level of the gene was significantly increased.

[0038] Example 2 ZmMARD1 Overexpression of genetically modified maize does not affect plant height

[0039] Combine wild type and ZmMARD1 Gene overexpression lines ( OE #1 OE #2 OE The #3 strain was planted at the transgenic base. Each material was sown in 2 rows, with a row length of 3 m, a row spacing of 60 cm, and a plant spacing of 25 cm. The plant height of each strain was measured during the flowering period.

[0040] See results Figure 2 Wild type and ZmMARD1 There was no difference in plant height among the overexpressing transgenic lines.

[0041] Example 3 ZmMARD1 Overexpression of transgenic maize significantly increases stalk tensile strength

[0042] During the corn flowering period, lodging resistance was measured in wild-type and... ZmMARD1 Gene overexpression lines ( OE #1 OE #2 OE The lodging resistance of each plant was assessed by measuring the stem tension at 50 cm above the ground for strain #3.

[0043] See results Figure 3 Compared to wild-type plants, ZmMARD1 The stem tensile strength of the overexpressing transgenic lines increased significantly.

[0044] Example 4 ZmMARD1 Overexpression significantly increases the lignin content in the stems of transgenic maize.

[0045] The lodging resistance of plants is significantly correlated with the lignin content of their stems. Under acidic conditions, phloroglucinol can react with total lignin to form pink or dark red substances, a method that can visually and quickly reflect changes in lignin content. During the flowering period of maize, stem samples from the third elongated internode of the aboveground part were selected, and the lignin content of each plant's stem was assessed using the phloroglucinol staining method.

[0046] See results Figure 4 Compared to wild-type plants, ZmMARD1The lignin staining signal in the stems of the overexpressing transgenic lines was significantly increased; the relative signal intensity of lignin staining was also analyzed using ImageJ. ZmMARD1 The staining intensity of the stem epidermis of the overexpressing plants was higher than that of the wild type.

[0047] Example 5 ZmMARD1 Overexpression of transgenic maize significantly increased stalk cortex cell thickness and stalk puncture force.

[0048] During the corn flowering period, the piercing force of the stem at the third elongated internode of the aboveground part was measured using a stem strength tester (YYD-1). At the same time, the stem epidermis was selected and the thickness of the thick-walled cells of the stem epidermis was observed under a microscope.

[0049] See results Figure 5 Compared to wild-type plants, ZmMARD1 The stem puncture force and stem cortex thickness of the overexpressing transgenic lines were significantly increased.

[0050] 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. ZmMARD1 The use of genes in breeding maize varieties resistant to lodging, characterized in that, The ZmMARD1 The polynucleotide sequence of the gene is shown as SEQ ID No:

1.

2. A method of enhancing lodging resistance in corn, characterized by, increasing the expression level of a ZmMARD1 protein in a maize plant; the amino acid sequence of the ZmMARD1 protein is shown as SEQ ID No: 2.

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