A maize gene ZmMYB99 and application thereof in regulating plant height and lodging resistance

Abstract

The application discloses a corn gene ZmMYB99 and application thereof in regulation of plant height and lodging resistance, and belongs to the technical field of plant genetic engineering.The nucleotide sequence of the ZmMYB99 gene is shown as SEQ ID NO.1, and the amino acid sequence of the expression protein thereof is shown as SEQ ID NO.2.The ZmMYB99 gene provided by the application can regulate the plant height and the lodging resistance of corn, and is of great significance for breeding corn with lodging resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering and molecular biology, in particular to a corn gene ZmMYB99 and its application in regulating plant height and lodging resistance. BACKGROUND

[0002] Lodging is a major problem in maize production, which results in yield loss, quality reduction and increased harvesting cost (Cao et al., 2013). Plant height is an important agronomic trait affecting crop yield and lodging, and is one of the important indicators for evaluating excellent maize varieties (Salas Fernandez et al., 2009). Overly high plant height will cause planting density to decrease, lodging resistance to decrease, and harvesting quality to decrease, while overly low plant height will affect the biomass and growth structure of the whole population (Peiffer et al., 2014). Therefore, it is very important to explore genes regulating maize plant height and lodging resistance.

[0003] The gene ZmMYB99 involved in the present application is a MYB transcription factor composed of 314 amino acids. This family has various physiological and biochemical functions, and is widely involved in the development and metabolic regulation of plants. The present application studies the molecular biological function of the gene in regulating maize plant height and lodging resistance, and provides a gene resource for breeding new maize varieties with lodging resistance. SUMMARY

[0004] The present application aims to provide a corn gene ZmMYB99 and its application in regulating plant height and lodging resistance.

[0005] The present application achieves the above-mentioned purpose by adopting the following technical solutions:

[0006] The first object of the present application is to provide a corn gene ZmMYB99, wherein the nucleotide sequence of the ZmMYB99 gene is shown as SEQ ID NO. 1.

[0007] As a further improvement of the present application, the encoded protein of the ZmMYB99 gene has an amino acid sequence shown as SEQ ID NO. 2.

[0008] The second object of the present application is to provide an application of the above-mentioned ZmMYB99 gene in regulating maize plant height and lodging resistance.

[0009] As a further improvement of the present application, the gene ZmMYB99 overexpression regulates maize plant height to decrease and the ability of lodging resistance to increase.

[0010] The third object of the present application is to provide an overexpression vector, which is obtained by introducing the above corn gene ZmMYB99 into a vector.

[0011] As a further improvement of the present application, the plasmid is NEWMOL.

[0012] The fourth object of the present application is to provide a method for breeding corn with lodging resistance and dwarf plant height, which overexpresses the corn ZmMYB99 gene to obtain a ZmMYB99 gene overexpression corn plant with lodging resistance and dwarf plant height.

[0013] As a further improvement of the present application, the method comprises the following steps:

[0014] (1) constructing a recombinant plasmid NEWMOL-ZmMYB99 containing the corn ZmMYB99 gene;

[0015] (2) introducing the recombinant plasmid NEWMOL-ZmMYB99 into an Agrobacterium competent cell, using Agrobacterium-mediated infection to invade corn callus, culturing to obtain corn seedlings, and culturing and breeding to obtain corn plants with dwarf plant height and small leaf angle after sequencing.

[0016] The present application has the following beneficial effects: the present application overexpresses the ZmMYB99 gene in the corn B104 inbred line, and obtains a ZmMYB99 overexpression line. The ZmMYB99 gene provided by the present application can regulate corn plant height and lodging resistance, and has important significance for breeding lodging-resistant corn. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is the identification of wild type B104 and corn overexpressing ZmMYB99 gene and plant height phenotype; Figure 1 A, detection of the expression amount of the ZmMYB99 gene in wild type B104 and overexpression plants; B, plant height phenotype of wild type B104 and corn overexpressing ZmMYB99 gene;

[0018] Figure 2 A, plant height statistics of wild type B104 and corn overexpressing ZmMYB99 gene; B, ear height statistics of wild type B104 and corn overexpressing ZmMYB99 gene; C, lodging index statistics of wild type B104 and corn overexpressing ZmMYB99 gene;

[0019] Figure 3 A, determination of the cell wall components of wild type B104 and corn overexpressing ZmMYB99 gene; B, analysis of the expression amount of cell wall synthesis related genes; C, determination of the stem strength of wild type B104 and corn overexpressing ZmMYB99 gene.

[0020] Figure 4 Figure of overexpression vector containing ZmMYB99 gene. DETAILED DESCRIPTION

[0021] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the present application is further described below in combination with specific drawings,

[0022] 1. Materials

[0023] The method used in this embodiment is a conventional method known to those skilled in the art, and the reagents and other materials used are commercially available products, unless otherwise specified. The primers used are indicated when first used, and the same primers used thereafter are the same as the first indicated content.

[0024] 2. Methods

[0025] 2.1 Obtaining of ZmMYB99 overexpression lines and identification of their expression amounts

[0026] 2.1.1 Obtaining of ZmMYB99 overexpression lines

[0027] The present application recombines the ZmMYB99 gene (the DNA sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2) into the overexpression vector NEWMOL (purchased from Invitrogen Biotechnology Co., Ltd.), obtains the ZmMYB99 overexpression vector, and the vector map of the constructed vector is shown as Figure 4 The constructed overexpression vector is transferred into Agrobacterium, the target gene is transferred into the mature callus of B104 by using the Agrobacterium infection technology of corn transgenic technology, and through the processes of co-culture, screening and differentiation and rooting, the transgenic T0 generation corn seedlings are obtained, and the positive corn seedlings are identified by Bar test paper.

[0028] The present application obtains the ZmMYB99 functional overexpression plant by transferring the ZmMYB99 gene into corn B104, and detects three overexpression plants, ZmMYB99-OE#1, ZmMYB99-OE#3 and ZmMYB99-OE#5, through sequencing analysis.

[0029] 2.1.2 Identification of expression amounts of ZmMYB99 overexpression lines

[0030] Total RNA of corn was extracted (using Novagen FastPure Universal Plant Total RNA Isolation Kit), reverse-transcribed into cDNA (using Novagen HiScript IV RT SuperMix for qPCR (+gDNA wiper) kit), and then qRT-PCR experiment was performed (according to the instructions of Novagen ChamQ SYBR qPCR Master Mix). The primers used in the qRT-PCR experiment were as follows:

[0031] SEQ ID NO. 3: 5'-TGTCGGAGAACCTGGGATAC-3';

[0032] SEQ ID NO. 4: 5'-ATTCCAAGCCGAAGTCGAGA-3';

[0033] As shown in Figure 1 A, it was found that the expression levels of ZmMYB99 gene in the three overexpression lines were significantly higher than that in wild type B104, indicating that we obtained corn lines overexpressing ZmMYB99 gene. After normal cultivation and breeding, transgenic T1 generation plants were obtained, and it was found that compared with wild type B104, ZmMYB99-OE#1, ZmMYB99-OE#3 and ZmMYB99-OE#5 had different degrees of dwarfing, and the results are shown in Figure 1 B.

[0034] 2.2 Evaluation of plant height, ear height and lodging resistance of ZmMYB99 overexpression lines

[0035] 2.2.1 Measurement of plant height of wild type B104 and three overexpression lines and statistics, as shown in Figure 2 A, it was found that the plant height of the three overexpression lines was significantly lower than that of wild type B104, indicating that overexpression of ZmMYB99 gene in corn could reduce the plant height of corn.

[0036] 2.2.2 Measurement of plant height H (cm), fresh weight W (g) and stem breaking force F (N) of wild type B104 and three overexpression lines, and calculation of lodging index according to the following formula:

[0037] Lodging index = H x W / (F / 9.8 x 1000 x 5) x 100

[0038] As shown in Figure 2As shown in C, the statistical results show that the lodging index of the three overexpression lines is significantly lower than that of wild type B104, indicating that the ZmMYB99 overexpression lines are more resistant to lodging, and that overexpression of ZmMYB99 gene in maize makes maize more resistant to lodging.

[0039] In addition, lower ear height is beneficial to plant resistance to lodging, and the present application also measures and statistics the ear height of wild type B104 and ZmMYB99 gene overexpression maize, as shown in B. Figure 2 As shown in B, it is found that the ear height of the three overexpression lines is significantly lower than that of wild type B104, which also improves the resistance to lodging.

[0040] 2.3 Cell wall component determination and cellulose synthesis related gene expression analysis of overexpression lines

[0041] 2.3.1 Cell wall component determination

[0042] The present application determines the components of the cell wall by the method in NB / T 34057 "Determination of Chemical Components of Lignocellulosic Biomass Raw Materials" Part 5: Determination of Cellulose, Hemicellulose, Pectin and Lignin. As shown in Figure 3 The results show that the cellulose content of the three overexpression lines is significantly higher than that of wild type B104, and the hemicellulose and lignin do not change significantly.

[0043] 2.3.2 Expression analysis of cellulose synthesis related gene ZmCesA12

[0044] ZmCESA12 (Zm00001d020531) is an important gene in cellulose synthase and is closely related to cellulose synthesis. ZmCESA12 may be a downstream regulatory gene of ZmMYB99.

[0045] Total RNA of corn is extracted (using Nuoyuan FastPure Universal Plant Total RNA Isolation Kit), reverse transcribed into cDNA (using Nuoyuan HiScript IV RT SuperMix for qPCR (+gDNAwiper) kit), and then qRT-PCR experiment is carried out (according to Nuoyuan ChamQ SYBR qPCR Master Mix instructions), the primers used in ZmCESA12-qRT-PCR experiment are (the primers are designed from Zhang et al 2024, Characterization of ZmCesAs for Secondary Cell Wall Biosynthesis in Maize),

[0046] ZmCesA12:

[0047] SEQ ID NO.5: 5'-CACCGCTGATATGGGAGTAGA-3';

[0048] SEQ ID NO.6: 5'-CGAACCGCTTCTCGAAGTTCA-3';

[0049] like Figure 3 As shown in Figure B, the gene expression level of ZmCESA12 was significantly increased in all three ZmMYB99 overexpression lines. This indicates that overexpression of the ZmMYB99 gene may regulate maize cellulose synthesis by modulating genes related to cellulose synthesis, thereby controlling maize plant height and lodging resistance. Overexpression of ZmMYB99 increased cellulose content and the expression level of synthesis genes, and there was a trend towards increased stem strength.

[0050] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A maize gene ZmMYB99 In the use for modulating plant height and resistance to lodging in maize, characterized in that, The nucleotide sequence of the corn gene ZmMYB99 is shown as SEQ ID NO. 1; the gene ZmMYB99 overexpression regulates the decrease of corn plant height and the increase of resistance to lodging.

2. Use according to claim 1, characterized in that, The gene ZmMYB99 The encoded protein has an amino acid sequence as shown in SEQ ID NO.

2.

3. A method of growing a lodging resistant and short stature corn plant, comprising, A maize gene of nucleotide sequence as shown in SEQ ID NO. 1 ZmMYB99 Overexpressed to obtain anti-lodging and dwarf plant height ZmMYB99 Gene overexpressed maize plant.

4. The method for breeding lodging-resistant and short-stalked maize according to claim 3, characterized by, comprising the steps of: (1) Constructing a recombinant plasmid containing a maize ZmMYB99 gene NEWMOL-ZmMYB99 ; (2) The recombinant plasmid is NEWMOL-ZmMYB99 The cells were transferred into competent Agrobacterium cells and infected with maize callus tissue using Agrobacterium-mediated inoculation. Maize seedlings were obtained by culturing and, after correct sequencing, were cultured and propagated to obtain lodging-resistant and short-statured maize plants.

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