Application of zinc-dependent DNA binding protein ZmPLATZ9 in regulation and control of corn plant height

Abstract

The invention discloses application of zinc-dependent DNA (deoxyribonucleic acid) binding protein ZmPLATZ9 in regulation and control of corn plant height, belongs to the field of plant genetic engineering and corn molecular breeding, and finds that the ZmPLATZ9 gene has a biological function of regulating and controlling the corn plant height and lodging resistance for the first time. In corn, by constructing a ZmPLATZ9 gene CRISPR / Cas9 knockout vector, compared with a wild type, the plant height and the ear height of an obtained homozygous strain of a ZmPLATZ9 knockout transgenic plant are obviously reduced, that is, by knocking out the ZmPLATZ9 gene, the plant height of the corn can be reduced, the ear height of the corn can be reduced, and the lodging resistance of the corn can be improved. The method can be used for cultivating a new semi-dwarf corn variety, and provides a thought for creating a new high-yield lodging-resistant corn variety.

Description

Technical Field

[0001] The present invention belongs to the fields of plant genetic engineering and maize molecular breeding, and particularly relates to the application of a zinc-dependent DNA binding protein ZmPLATZ9 in regulating maize plant height. Background Art

[0002] Maize (Zea mays L.) is an annual herbaceous plant of the Gramineae family, originating from central Mexico and evolving from wild teosinte. It is one of the crops with the largest planting area at home and abroad. Since the Green Revolution, high-yield, semi-dwarf varieties have been widely applied to crops such as wheat, rice, and maize, resulting in a rapid increase in grain yield, solving the food problem of half of the population in developing countries and avoiding large-scale food crises. The increase in maize yield is closely related to plant height, planting density, leaf area index, photosynthesis, etc. In recent years, researchers have discovered the potential to regulate the levels of endogenous hormones in plants through genetic engineering means and developed reasonable plans to increase grain yield. Currently, more than 60 dwarf genes are known, and most of them have been cloned. However, most of them show extreme phenotypes and are difficult to be used for production and high yield. Therefore, analyzing the molecular mechanism of regulating maize plant height is not only beneficial to basic scientific research but also beneficial to increasing crop yield.

[0003] The zinc-dependent DNA binding protein PLATZ (Plant A / T-rich protein and Zinc-binding protein), which was first identified in peas and only exists in plants, plays an indispensable role in the process of plant growth and development. It has been shown that PLATZs genes play an important role in regulating plant architecture. The Arabidopsis PLATZ family gene ORE15 (ORESARA15) regulates leaf size by directly interacting with the GRF family genes GRF1 / GRF4. Overexpression of the rice PLATZ family gene GL6 resulted in a significant phenotype of increased plant height. In addition, PLATZ transcription factors also play important roles in promoting the transition of poplar stems from primary growth to secondary growth and the synthesis of secondary cell walls in immature tissues of sugarcane. A total of 17 genes have been identified as members of the PLATZ gene family in maize. However, the research on PLATZ genes in maize is very limited, and there has never been a report on direct evidence that PLATZ genes are expressed in the intercalary meristem or affect plant height and ear height in maize. Summary of the Invention

[0004] Aiming at the current research status, the purpose of the present invention is to provide the application of a zinc-dependent DNA binding protein ZmPLATZ9 in regulating maize plant height.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, there is provided the use of the ZmPLATZ9 gene in any one of the following (1)-(4):

[0007] (1) Regulating the plant height of maize;

[0008] (2) Regulating the ear height of maize;

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

[0010] (4) Cultivating new varieties of maize suitable for mechanical harvesting;

[0011] The ZmPLATZ9 gene is a DNA molecule shown in any one of the following i)-iii):

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

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

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

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

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

[0017] (B) Regulating the size of cells in the internodes of maize.

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

[0019] (1) Regulating the plant height of maize;

[0020] (2) Regulating the ear height of maize;

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

[0022] (4) Cultivating new varieties of maize suitable for mechanical harvesting;

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

[0024] In the third aspect of the present invention, there is provided the use of the knockout recombinant expression vector or genetically engineered bacterium of the above ZmPLATZ9 gene in any one of the following (1)-(4):

[0025] (1) Regulate the plant height of maize;

[0026] (2) Regulate the ear height of maize;

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

[0028] (4) Breed new varieties of maize suitable for mechanical harvesting.

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

[0030] Increasing the plant height of maize by overexpressing the ZmPLATZ9 gene or enhancing the expression of the ZmPLATZ9 gene.

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

[0032] Increasing the ear height of maize by overexpressing the ZmPLATZ9 gene or enhancing the expression of the ZmPLATZ9 gene.

[0033] The ear height refers to the height from the ground surface to the stem node where the uppermost ear is borne. By appropriately reducing the ear height, the overall center of gravity of the plant can be lowered, avoiding the problem that the moment of the ear weight borne by the stem is too large due to a high ear position, reducing the stem load, and lowering the risk of breakage, thereby significantly improving the wind resistance and lodging resistance of maize.

[0034] Modern maize harvesters have certain requirements for the ear height. A high ear position may cause the ears to be missed or collide and fall off during the harvesting process. Reducing the ear height can reduce such losses. Therefore, a lower ear position is more conducive to mechanical picking, reducing ear omission or damage, and improving the harvesting efficiency. Therefore, new varieties of maize suitable for mechanical harvesting can be bred by regulating the ear height of maize.

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

[0036] Advantages of the present invention:

[0037] The present invention first discovers that the ZmPLATZ9 gene has the biological function of regulating maize plant height and lodging resistance. In maize, by constructing a CRISPR / Cas9 knockout vector for the ZmPLATZ9 gene, the homozygous lines of the ZmPLATZ9 knockout transgenic plants obtained have significantly reduced plant height and ear height compared with the wild type, that is: by knocking out the ZmPLATZ9 gene, the plant height and ear height of maize can be reduced, and the lodging resistance of maize can be improved. The present invention provides direct evidence for the ZmPLATZ9 gene to regulate maize plant height and ear height, which can be used for the cultivation of new semi-dwarf maize varieties and provides ideas for creating new high-yield and lodging-resistant maize materials. Description of the Drawings

[0038] Figure 1 To use CRISPR / Cas9 to knockout the ZmPLATZ9 gene specifically expressed in the internode meristem below the ear; among them Figure 1 In A, it is a schematic diagram of the ZmPLATZ9 gene structure and the knockout position. The black bar in the figure represents the exon of the ZmPLATZ9 gene, the black line represents the intron, a dual-target system is adopted, and the red rectangle represents the PAM site; Figure 1 In B, it is the sequencing detection peak map 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), causing a change in the protein sequence; ZmPLATZ9-KO#2 has a 1bp insertion and a 2bp deletion, causing a change in the protein sequence.

[0039] Figure 2 For the plant height traits of the wild type (B104) and the knockout mutants, among them Figure 2 In A, it is the plant height phenotype diagram of three plants. From left to right are the wild type B104, the knockout mutants ZmPLATZ9-KO#1, and ZmPLATZ9-KO#2; Figure 2 In B, it is the statistical histogram of the plant heights of the three plants; Figure 2 In C, it is the statistical histogram of the ear heights of the three plants. Detailed Embodiments

[0040] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0041] As mentioned above, plant height, as an important agronomic trait affecting maize plant type, is an important factor in achieving lodging resistance and increasing yield in maize. By appropriately reducing the plant height of maize and cultivating excellent dwarf maize germplasm resources, it helps to achieve high and stable yields of maize, increase the total maize output and economic benefits. The ear height of maize refers to the height from the ground to the stem node where the uppermost ear is borne. Reducing the ear height of maize is one of the important goals of modern maize breeding and cultivation, and it is of great significance to optimize the plant structure, enhance the density tolerance of maize plants, and cultivate new maize varieties suitable for mechanized production.

[0042] The PLATZ gene is involved in plant abiotic stress in wheat and is also related to cell differentiation in plant tissues. In maize, the ZmPLATZ9 protein is usually involved in the regulation of RNAPIII-mediated transcription of small non-coding RNAs and is associated with endosperm development. Currently, there is no report on the direct evidence of the PLATZ gene in maize being related to the expression of the intercalary meristem or affecting plant height and ear height.

[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. By constructing a knockout mutation method, the present invention obtained two homozygous transgenic lines with two editing methods. 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] MIMQAMWKPGWLEALDTQKFFVACSFHEHAKKNEKNICCLDCCTSICPHCVAAHRAHRLLQVRRYVYHDVVRLEDLEKLIDCSSVQSYTINSSKVVFLKKRPQNRQFKGSGNICTSCDRSLQEPYFHCSLDCKVEYILRQKKKLSAYLRPCKTLQLGPDFFIPHDADDDTTHSTLVDVDEPMGSSDSENLSVPCTNFVRKKRSGPYICARSANRVSEEDMATNMSRRKGVPQRSPLC。

[0049] In the present invention, CRISPR / Cas9 is used to knockout the gene ZmPLATZ9 specifically expressed in the intercalary meristem of the internode below the ear. By means of transgenic technology, a knockout vector is constructed, then introduced into Agrobacterium, and the maize plants are transformed by Agrobacterium-mediated method to obtain ZmPLATZ9 knockout transgenic plants (ZmPLATZ9-KO#1, ZmPLATZ9-KO#2).

[0050] By statistically analyzing the agronomic traits of the ZmPLATZ9 knockout transgenic plants, it is found that compared with the wild-type plants, the plant height and ear height of the homozygous lines of ZmPLATZ9-KO#1 and ZmPLATZ9-KO#2 are significantly shorter, thus proving that ZmPLATZ9 can regulate the plant height and ear height of maize. The research results of the present invention provide direct evidence for regulating the plant height and ear height of maize, and can be used for the cultivation of new semi-dwarf maize varieties, providing ideas for creating new high-yield and lodging-resistant maize materials.

[0051] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in combination with specific embodiments. The following detailed descriptions are all illustrative and are intended to provide further descriptions of the present application, rather than limiting the scope of the present invention.

[0052] The test materials not specifically described in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. For those not specified in the embodiments of the present invention regarding specific experimental conditions and methods, they are usually in accordance with conventional conditions.

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

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

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

[0056] (1) Target site design: Two specific 19-bp sequences were selected on the CDS sequence of the target gene. The sequences were used for target prediction on the website (http: / / crispor.tefor.net / ), and appropriate target sites were selected for primer design.

[0057] (2) PCR amplification: Using pBUE411 as the template, the designed primers (Table 1) were used to amplify the target fragment according to the PCR amplification system (Table 2). Subsequently, the PCR products were recovered and purified. The pBUE411 empty plasmid was linearized with the KspAI restriction endonuclease from Thermo Scientific. Subsequently, recombinant ligation was carried out according to Table 3, incubated at 50 °C for 30 min, and then placed on ice. After the ligation was completed, all the ligation products were transferred into Escherichia coli TOP10 competent cells and cultured at 37 °C overnight with inversion. Subsequently, single colonies were 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 1 μL Primer CAP-R 1 μL Primer MT1T2-F0 0.5 μL Primer MT1T2-R0 0.5 μL 2×Phanta Flash Master Mix (Dye Plus) 10 μL Vector template 1 μL <![CDATA[ddH2O]]> Make up to 20 μL

[0063] Table 3: Recombinant ligation reaction system

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

[0065] (3) Agrobacterium competent cell transformation: Take out 50 μL of Agrobacterium EHA105 competent cells from the -80 °C refrigerator and insert them into ice to melt for 5 min. Take 5 μL of the ligation product and add it to a centrifuge tube, gently flick to mix evenly, and let it stand on ice for 5 min. Quick-freeze it in liquid nitrogen for 5 min, then immediately treat it in a 37 °C water bath for 5 min, and quickly insert it into ice for an ice bath for 5 min. Transfer 1 mL of YEP into a centrifuge tube, place it in a 28 °C shaking incubator for 1 h, and set the rotation speed to 180 rpm. Then use a centrifuge to centrifuge at 5000 rpm for 1 min. In a laminar flow hood, discard most of the supernatant, resuspend it, and transfer the bacterial solution to the pre-prepared YEP medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampicin). After evenly spreading and sealing, invert the plate and culture it in a 28 °C incubator for 48 - 72 h for identification.

[0066] Using maize inbred line B104 as the material, Agrobacterium-mediated genetic transformation was carried out.

[0067] Screen the T0 generation, identify the target gene mutations induced by CRISPR / Cas9, through self-crossing and segregation from T0 to T2 generations, herbicide screening, collect the leaves of the transplanted and survived plants to extract DNA and perform PCR detection.

[0068] Finally, two different editing types of CRISPR / Cas9 transgenic knockout lines that can stably inherit without the Cas9 vector backbone were obtained, and they were named ZmPLATZ9-KO#1 and ZmPLATZ9-KO#2 respectively. Sequence alignment with the wild type showed that ZmPLATZ9-KO#1 had a 1-bp deletion and a single-base substitution (C→A) between two PAM sites; ZmPLATZ9-KO#2 had a 1-bp insertion and a 2-bp deletion between two PAM sites. This resulted in changes in the ZmPLATZ9 protein structure, leading to the loss of ZmPLATZ9 function ( Figure 1 ).

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

[0070] The plant height and ear height phenotypes of maize wild type (B104) and the obtained ZmPLATZ9-KO#1 and ZmPLATZ9-KO#2 knockout mutants were statistically analyzed. The results showed that compared with the WT, the plant height and ear height of the mature plants of the two homozygous editing lines ZmPLATZ9-KO#1 and ZmPLATZ9-KO#2 were significantly decreased ( Figure 2)。The results of agronomic trait investigation showed that knocking out the ZmPLATZ9 gene could reduce the plant height and ear height of maize, thereby improving the lodging resistance of maize. This invention provides direct evidence for the regulation of maize plant height and ear height by the ZmPLATZ9 gene.

[0071] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of the ZmPLATZ9 gene in any of the following (1)-(4): (1) Regulate corn plant height; (2) Regulating the height of corn ears; (3) Cultivate new corn varieties that are resistant to lodging; (4) Cultivate new corn varieties suitable for mechanized harvesting; The ZmPLATZ9 gene is a DNA molecule as shown in any one of the following i)-iii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i); 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.

2. The use according to claim 1, characterized in that: The ZmPLATZ9 gene regulates corn plant height through at least one of the following (A) or (B): (A) Regulates the cell number of maize internodes; (B) Regulation of maize internode cell size.

3. Use of the protein encoded by the ZmPLATZ9 gene in any of the following (1)-(4): (1) Regulate corn plant height; (2) Regulating the height of corn ears; (3) Cultivate new corn varieties that are resistant to lodging; (4) Cultivate new corn varieties suitable for mechanized harvesting; The amino acid sequence of the protein encoded by the ZmPLATZ9 gene is shown in SEQ ID NO.

2.

4. Use of the knockout recombinant expression vector or genetically engineered bacteria of the ZmPLATZ9 gene of claim 1 in any one of the following (1)-(4): (1) Regulate corn plant height; (2) Regulating the height of corn ears; (3) Cultivate new corn varieties that are resistant to lodging; (4) Cultivate new corn varieties suitable for mechanized harvesting.

5. A method for regulating corn plant height, characterized in that: The following steps are involved: The height of maize plants was reduced by knocking out the ZmPLATZ9 gene or inhibiting its expression; The height of corn plants can be increased by overexpressing the ZmPLATZ9 gene or enhancing the expression of the ZmPLATZ9 gene.

6. A method for regulating corn ear height, characterized in that: The following steps are involved: The height of corn ears can be reduced by knocking out the ZmPLATZ9 gene or inhibiting the expression of the ZmPLATZ9 gene; The height of corn ear can be increased by overexpressing the ZmPLATZ9 gene or enhancing the expression of the ZmPLATZ9 gene.

7. A method for breeding lodging-resistant corn varieties, characterized in that: The following steps are involved: By knocking out the ZmPLATZ9 gene in the wild-type corn inbred line, a new lodging-resistant corn variety with reduced plant height was obtained.

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