Application of zinc-dependent DNA binding protein ZmPLATZ9 in regulating corn plant height

Abstract

The application discloses application of a zinc-dependent DNA binding protein ZmPLATZ9 in regulating corn plant height, and belongs to the field of plant genetic engineering and corn molecular breeding.The application first finds that the ZmPLATZ9 gene has a biological function of regulating corn plant height and lodging resistance capacity.In corn, a ZmPLATZ9 gene CRISPR / Cas9 knockout vector is constructed, and a homozygous strain of a ZmPLATZ9 knockout transgenic plant obtained by the vector has a plant height and ear position height obviously lower than those of a wild type, that is, the plant height and ear position height of corn can be reduced by knocking out the ZmPLATZ9 gene, and the lodging resistance capacity of corn is improved.The application provides direct evidence for the ZmPLATZ9 gene regulating corn plant height and ear position height, and can be used for cultivating a semi-dwarf corn new variety and providing a train of thought for creating a high-yield and lodging-resistant corn new variety.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and maize molecular breeding, specifically involving the application of a zinc-dependent DNA-binding protein, ZmPLATZ9, in regulating maize plant height. Background Technology

[0002] Maize (Zea mays L.) is an annual herbaceous plant belonging to the Gramineae family, originating in central Mexico and evolved from wild fodder. It is one of the most widely cultivated crops both domestically and internationally. Since the Green Revolution, high-yield, semi-dwarf varieties have been widely used in crops such as wheat, rice, and maize, leading to a rapid increase in grain production and solving the food problem for half of the population in developing countries, thus preventing large-scale food crises. Increased maize yield is closely related to plant height, planting density, leaf area index, and photosynthesis. In recent years, researchers have discovered the potential to regulate the levels of endogenous hormones in plants through genetic engineering, and have developed rational strategies to increase grain yield. Currently, more than 60 dwarf genes are known, most of which have been cloned. However, most of them exhibit extreme phenotypes, making them difficult to utilize and achieve high yields. Therefore, elucidating the molecular mechanisms regulating maize plant height is beneficial not only for basic scientific research but also for improving crop yields.

[0003] PLATZ (Plant A / T-rich protein and Zinc-binding protein), first identified in peas and found only in plants, plays an indispensable role in plant growth and development. Studies have shown that PLATZ genes play a crucial role in regulating plant architecture. In Arabidopsis, the PLATZ family gene ORE15 (ORESARA15) regulates leaf size through direct interaction with the GRF family genes GRF1 / GRF4. Overexpression of the PLATZ family gene GL6 in rice resulted in a significant increase in plant height. Furthermore, PLATZ transcription factors play important roles in promoting the transition from primary to secondary growth in poplar stems and in the synthesis of secondary cell walls in immature sugarcane tissues. Seventeen genes in maize have been identified as belonging to the PLATZ gene family. However, research on PLATZ genes in maize is limited, and direct evidence of PLATZ gene expression in intercalary meristems or its influence on plant height and ear height has never been reported. Summary of the Invention

[0004] In view of the current research status, the purpose of this invention is to provide an application of the zinc-dependent DNA-binding protein ZmPLATZ9 in regulating maize plant growth.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the use of the ZmPLATZ9 gene in any of the following (1)-(4):

[0007] (1) Regulating maize plant height;

[0008] (2) Regulate the height of corn ears;

[0009] (3) Develop new lodging-resistant maize varieties;

[0010] (4) Develop new corn varieties suitable for mechanized harvesting;

[0011] The ZmPLATZ9 gene is a DNA molecule represented by any of the following i)-iii):

[0012] i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0013] ii) DNA molecules other than those in i) encoding the amino acid sequence shown in SEQ ID NO. 2;

[0014] iii) A DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii) and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.2.

[0015] The ZmPLATZ9 gene regulates maize plant height through at least one of the following pathways (A) or (B):

[0016] (A) Regulates the number of cells in maize internodes;

[0017] (B) Regulates the size of internode cells in maize.

[0018] In a second aspect, the present invention provides the use of the protein encoded by the ZmPLATZ9 gene in any of the following (1)-(4):

[0019] (1) Regulating maize plant height;

[0020] (2) Regulate the height of corn ears;

[0021] (3) Develop new lodging-resistant maize varieties;

[0022] (4) Develop new corn varieties suitable for mechanized harvesting;

[0023] The amino acid sequence of the protein encoded by the ZmPLATZ9 gene is shown in SEQ ID NO.2.

[0024] In a third aspect, the present invention provides the application of the above-mentioned ZmPLATZ9 gene knockout recombinant expression vector or genetically engineered bacteria in any of the following (1)-(4):

[0025] (1) Regulating maize plant height;

[0026] (2) Regulate the height of corn ears;

[0027] (3) Develop new lodging-resistant maize varieties;

[0028] (4) Develop new corn varieties suitable for mechanized harvesting.

[0029] In a fourth aspect, the present invention provides a method for regulating maize plant height, comprising the following steps: reducing maize plant height by knocking out the ZmPLATZ9 gene or inhibiting the expression of the ZmPLATZ9 gene;

[0030] Maize plant height can be increased by overexpressing or enhancing the expression of the ZmPLATZ9 gene.

[0031] In a fifth aspect, the present invention provides a method for regulating the ear height of maize, comprising the following steps: reducing the ear height of maize by knocking out the ZmPLATZ9 gene or inhibiting the expression of the ZmPLATZ9 gene;

[0032] Maize ear height can be increased by overexpressing or enhancing the expression of the ZmPLATZ9 gene.

[0033] Ear height refers to the height from the ground surface to the uppermost node on which the ear grows. By appropriately lowering the ear height, the overall center of gravity of the plant can be lowered, avoiding the problem of excessive torque caused by the weight of the ear on the stem due to a high ear height. This reduces the load on the stem, lowers the risk of breakage, and thus significantly improves the corn's resistance to wind and lodging.

[0034] Modern corn harvesters have certain requirements for ear height. High ear height may lead to missed harvesting or ears falling off during harvesting. Lowering the ear height can reduce such losses. Therefore, lower ear height is more conducive to mechanized harvesting, reducing missed or damaged ears and improving harvesting efficiency. Thus, by controlling the ear height, new corn varieties suitable for mechanized harvesting can be developed.

[0035] In a sixth aspect, the present invention provides a method for breeding lodging-resistant maize varieties, comprising the following steps: obtaining a new lodging-resistant maize variety with reduced plant height by knocking out the ZmPLATZ9 gene in a wild-type maize variety.

[0036] The beneficial effects of this invention are:

[0037] This invention is the first to discover that the ZmPLATZ9 gene has a biological function in regulating maize plant height and lodging resistance. In maize, by constructing a CRISPR / Cas9 knockout vector for the ZmPLATZ9 gene, homozygous ZmPLATZ9 knockout transgenic plants showed significantly reduced plant height and ear height compared to the wild type. This demonstrates that knocking out the ZmPLATZ9 gene can reduce maize plant height and ear height, thereby improving lodging resistance. This invention provides direct evidence for the ZmPLATZ9 gene's regulation of maize plant height and ear height, and can be used for the breeding of semi-dwarf maize varieties, providing a framework for creating high-yielding, lodging-resistant maize materials. Attached Figure Description

[0038] Figure 1 To utilize CRISPR / Cas9, the gene ZmPLATZ9, specifically expressed in the intermeristor of the lower node, was knocked out; among which... Figure 1 A in the figure is a schematic diagram of the structure of the ZmPLATZ9 gene and the location of the knockout. The black bars in the figure represent the exons of the ZmPLATZ9 gene, the black lines represent the introns, a dual-target system is used, and the red rectangles represent the PAM sites. Figure 1 Figure B shows the sequencing peaks of two transgenic knockout lines obtained by two editing methods of the ZmPLATZ9 gene. ZmPLATZ9-KO#1 has a 1bp deletion and a single base substitution (C→A), which causes changes in the protein sequence; ZmPLATZ9-KO#2 has a 1bp insertion and a 2bp deletion, which also causes changes in the protein sequence.

[0039] Figure 2 The plant height trait was compared between the wild-type (B104) and knockout mutants. Figure 2 The figure in A is a phenotypic diagram of plant height for three plants. From left to right, they are wild-type B104, knockout mutants ZmPLATZ9-KO#1 and ZmPLATZ9-KO#2. Figure 2 B represents the plant height statistics of the three plants in the bar chart; Figure 2 The bar chart in section C shows the ear height of the three plants. Detailed Implementation

[0040] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0041] As mentioned earlier, plant height, as an important agronomic trait affecting maize plant architecture, is a crucial factor in achieving lodging resistance and increasing yield. Appropriately reducing maize plant height and cultivating superior dwarf maize germplasm resources can help achieve high and stable maize yields, increasing total maize production and economic benefits. Maize ear height refers to the height from the ground surface to the node where the uppermost ear grows. Reducing maize ear height is one of the important goals of modern maize breeding and cultivation. Optimizing plant structure, enhancing maize plant density tolerance, and cultivating new maize varieties suitable for mechanized production are of great significance.

[0042] The PLATZ gene is involved in abiotic stress in wheat and is also related to cell differentiation in plant tissues. In maize, the ZmPLATZ9 protein is typically involved in the regulation of RNAPIII-mediated small non-coding RNA transcription and is associated with endosperm development. Currently, there is no direct evidence reported regarding PLATZ gene expression in intercalary meristems or its influence on plant height or ear height in maize.

[0043] Based on this, the present invention provides the application of the zinc-dependent DNA-binding protein ZmPLATZ9 in regulating maize plant height and ear height. The present invention obtained two homozygous transgenic lines with different editing methods by constructing a knockout mutation. The CDS sequence of the ZmPLATZ9 gene is 714 bp long, and the specific nucleotide sequence is shown in SEQ ID NO.1, as follows:

[0044] SEQ ID NO.1:

[0045] ATGATCATGCAGGCAATGTGGAAGCCAGGATGGCTAGAGGCCCTTGACACACAGAAGTTCTTCGTAGCATGCTCTTTCCATGAGCATGCCAAGAAGAACGAGAAGAACATCTGTTGCCTTGACTGCTGCACTAGCATCTGCCCACACTGTGTGGCAGCACACCGTGCACACAGGCTCCTGCAGGTGCGGCGATACGTCTACCATGACGTTGTCCGGCTGGAGGACCTGGAGAAGCTTATTGATTGCTCTAGTGTTCAGTCTTATACTATTAACAGCTCTAAGGTTGTTTTCCTGAAGAAGAGACCACAGAATAGGCAATTCAAGGGTTCAGGGAATATCTGCACCTCCTGCGACAGGAGCCTTCAAGAACCGTATTTCCACTGCTCTCTGGATTGCAAGGTAGAGTATATACTACGACAGAAGAAAAAATTGTCAGCATATTTGCGCCCATGCAAGACCTTGCAGCTTGGCCCTGATTTCTTCATTCCTCATGATGCTGATGACGACACAACTCACTCAACCCTTGTTGATGTTGATGAGCCCATGGGATCATCGGACTCGGAGAATTTGAGTGTGCCGTGCACAAATTTTGTTCGGAAAAAACGGAGTGGACCATATATTTGTGCACGGTCTGCAAACAGAGTGTCTGAAGAAGACATGGCCACAAATATGAGCAGAAGGAAAGGGGTTCCTCAGAGATCGCCTTTGTGCTAA。

[0046] The amino acid sequence of the protein encoded by the ZmPLATZ9 gene is shown in SEQ ID NO.2, as follows:

[0047] SEQ ID NO.2:

[0048] MIMQAMWKPGWLEALDTQKFFVACSFHEHAKKNEKNICCLDCCTSICPHCVAAHRAHRLLQVRRYVYHDVVRLEDLEKLIDCSSVQSYTINSSKVVFLKKRPQNRQFKGSGNICTSCDR SLQEPYFHCSLDCKVEYILRQKKKLSAYLRPCKTLQLGPDFFIPHDADDDTTHSTLVDVDEPMGSSDSENLSVPCTNFVRKKRSGPYICARSANRVSEEDMATNMSRRKGVPQRSPLC.

[0049] This invention uses CRISPR / Cas9 to knock out the ZmPLATZ9 gene specifically expressed in the intermeristor of the lower ear node. A knockout vector is constructed through transgenic methods, which is then introduced into Agrobacterium and transformed into maize plants using Agrobacterium-mediated transformation to obtain ZmPLATZ9 knockout transgenic plants (ZmPLATZ9-KO#1 and ZmPLATZ9-KO#2).

[0050] Statistical analysis of the agronomic traits of ZmPLATZ9 knockout transgenic plants revealed that the homozygous lines ZmPLATZ9-KO#1 and ZmPLATZ9-KO#2 exhibited significantly shorter plant height and ear height compared to wild-type plants. This demonstrates that ZmPLATZ9 can regulate maize plant height and ear height. The findings of this invention provide direct evidence for regulating maize plant height and ear height, and can be used to cultivate semi-dwarf maize varieties, offering insights for creating high-yielding, lodging-resistant maize materials.

[0051] In order to enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. The following detailed description is illustrative and is intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0052] The experimental materials used in the embodiments of this invention, unless otherwise specified, are all conventional experimental materials in the art and can be purchased through commercial channels. Where specific experimental conditions and methods are not specified in the embodiments of this invention, conventional conditions are generally followed.

[0053] Example 1: Construction of ZmPLATZ9-CRISPR / Cas9 vector and acquisition of knockout plants

[0054] Using maize B104 inbred line recipient material, a CRISPR / Cas9 knockout vector for the ZmPLATZ9 gene was constructed with pBUE411-2gR as the vector backbone. Genetic transformation of maize immature embryos was carried out by Agrobacterium-mediated transformation, and ZmPLATZ9 transgenic knockout lines were obtained.

[0055] The steps for constructing the ZmPLATZ9 gene CRISPR / Cas9 knockout vector are as follows:

[0056] (1) Target design: Select two 19bp specific sequences on the CDS sequence of the target gene, predict the target on the website (http: / / crispor.tefor.net / ), select appropriate targets, and design primers.

[0057] (2) PCR amplification: Using pBUE411 as a template, the target fragment was amplified using the designed primers (Table 1) according to the PCR amplification system (Table 2). The PCR products were then recovered and purified. The pBUE411 empty vector plasmid was linearized using KspAI restriction endonuclease from Thermo Scientific. Recombinant ligation was then performed according to Table 3, incubated at 50°C for 30 min, and then placed on ice. After ligation, all ligation products were transformed into *E. coli* TOP10 competent cells and cultured upside down at 37°C for 16 h. Single colonies were then selected, positive clones were screened, and sent to the company for sequencing verification.

[0058] Table 1: Primer Design

[0059]

[0060]

[0061] Table 2: PCR amplification system

[0062] reaction solution components Dosage Primer CAP-F 1μL Primer CAP-R 1μL Primer MT1T2-F0 0.5μL Primer MT1T2-R0 0.5μL 2×Phanta Flash Master Mix(Dye Plus) 10μL Carrier template 1μL <![CDATA[ddH2O]]> Add to 20μL

[0063] Table 3: Recombinant Linkage Reaction System

[0064] Components Volume (μL) Linearization pBUE411 3.5μL Target fragment 6.5μL 2×ClonExpress Mix 10μL

[0065] (3) Agrobacterium competent cell transformation: Take 50 μL of Agrobacterium EHA105 competent cells from a -80℃ freezer and thaw them in ice for 5 min. Add 5 μL of ligation product to a centrifuge tube, gently tap to mix, and let stand on ice for 5 min. Quickly freeze in liquid nitrogen for 5 min, then treat in a 37℃ water bath for 5 min, and quickly immerse in ice for 5 min. Transfer 1 mL of YEP to a centrifuge tube and incubate in a 28℃ shaker for 1 h at 180 rpm. Then centrifuge at 5000 rpm for 1 min. In a clean bench, discard most of the supernatant, resuspend the bacterial culture, and transfer it to a pre-prepared YEP medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampin). Spread evenly, seal the plate, and invert it in a 28℃ incubator for 48-72 h before identification.

[0066] Agrobacterium-mediated genetic transformation was performed using maize inbred line B104 as the material.

[0067] T0 generation was screened to identify target gene mutations induced by CRISPR / Cas9. Herbicide screening was conducted through self-pollination from T0 to T2 generation. DNA was extracted from leaves of transplanted and surviving plants and tested by PCR.

[0068] Two different edit types were ultimately obtained, and both stably inherited CRISPR / Cas9 transgenic knockout lines lacking the Cas9 vector backbone were named ZmPLATZ9-KO#1 and ZmPLATZ9-KO#2, respectively. Sequence alignment with the wild type revealed that ZmPLATZ9-KO#1 exhibited a 1 bp deletion and a single base substitution (C→A) between two PAM sites; ZmPLATZ9-KO#2 exhibited a 1 bp insertion and a 2 bp deletion between two PAM sites. This resulted in alterations to the ZmPLATZ9 protein structure, leading to loss of ZmPLATZ9 function. Figure 1 ).

[0069] Example 2: Observation and statistics of traits of wild-type maize (B104) and ZmPLATZ9 knockout mutant.

[0070] Statistical analysis of plant height and ear height phenotypes in wild-type maize (B104) and the obtained ZmPLATZ9-KO#1 and ZmPLATZ9-KO#2 knockout mutants revealed that, compared to WT, the mature plant height and ear height of the two homozygous edited lines, ZmPLATZ9-KO#1 and ZmPLATZ9-KO#2, were significantly reduced. Figure 2The results of agronomic trait investigation showed that knocking out the ZmPLATZ9 gene can reduce maize plant height and ear height, thereby improving maize lodging resistance. This invention provides direct evidence for the regulation of maize plant height and ear height by the ZmPLATZ9 gene.

[0071] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. Knockout ZmPLATZ9 The application of genes in any of the following (1)-(4): (1) Reduce corn plant height; (2) Reduce the height of the corn ear; (3) Develop new lodging-resistant maize varieties; (4) Develop new corn varieties suitable for mechanized harvesting; The ZmPLATZ9 A gene is a DNA molecule as shown in i) or ii) below: i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.

2.

2. Inactivation ZmPLATZ9 The application of the gene-encoded protein in the following (1) or (2): (1) Reduce corn plant height; (2) Reduce the height of the corn ear; The ZmPLATZ9 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. ZmPLATZ9 The use of gene knockout recombinant expression vectors or genetically engineered bacteria containing said recombinant expression vectors in any of the following (1)-(4): (1) Reduce corn plant height; (2) Reduce the height of the corn ear; (3) Develop new lodging-resistant maize varieties; (4) Develop new corn varieties suitable for mechanized harvesting; The ZmPLATZ9 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. A method for regulating maize plant height, characterized in that, Includes the following steps: By knocking ZmPLATZ9 Gene or repression ZmPLATZ9 Gene expression is used to reduce maize plant height; The ZmPLATZ9 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. A method for regulating the height of maize ears, characterized in that, Includes the following steps: By knocking ZmPLATZ9 Gene or repression ZmPLATZ9 Gene expression is used to reduce ear height in maize; The ZmPLATZ9 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. A method for breeding lodging-resistant maize varieties, characterized in that, Includes the following steps: By knocking out wild-type maize inbred lines ZmPLATZ9 Genes were used to obtain new lodging-resistant maize varieties with reduced plant height; The ZmPLATZ9 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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