Corn plant type regulation gene ZmEXO1 and application

Abstract

This invention provides a maize plant architecture regulating gene ZmEXO1 and its application, belonging to the field of maize molecular breeding technology. The amino acid sequence of ZmEXO1 is shown in SEQ ID NO.7. Studies have found that ZmEXO1 can regulate maize leaf angle and plant height; mutants with reduced or silenced ZmEXO1 expression lead to smaller leaf angles and reduced plant height. Therefore, gene editing of the ZmEXO1 gene or genetic breeding improvement using Zmexo1 low-expression mutants can obtain maize germplasm with improved plant architecture, which is of great significance for enriching maize germplasm and breeding high-yielding, dense-planting-tolerant varieties. This invention also provides a Kasp molecular marker and Kasp primers closely linked to ZmEXO1, which can be used for marker-assisted selection breeding and the discovery of related functional genes, enabling rapid screening and identification of maize germplasm containing ZmEXO1-modified molecules.

Description

Technical Field

[0001] This invention belongs to the field of maize molecular breeding technology, and in particular relates to a maize plant architecture regulating gene ZmEXO1 and its application. Background Technology

[0002] As the world's most important food crop, maize plays a vital role in ensuring food security and meeting market demand. Increasing maize yield is a primary goal, and the key to achieving this goal lies in increasing planting density. However, the limitation of canopy light interception is a major obstacle to achieving high yields and dense planting. Therefore, improving the external morphological structure of maize plants has become an important indicator in plant type breeding. Leaf angle, the angle between the leaf and the main stem, is one of the key agronomic traits affecting maize plant type and yield. A smaller leaf angle helps reduce canopy light interception, thereby improving light energy utilization efficiency and planting density, ultimately leading to increased yield.

[0003] In the context of maize reform, increasing planting density has become a major measure to improve yield. However, overly dense planting can lead to lodging, thus affecting yield. Studies have shown a significant correlation between maize lodging and field planting density. As planting density increases, competition among plants intensifies, and excessively rapid stem growth weakens the plants, reducing their resistance to lodging. Under such circumstances, strong winds or heavy rains can cause widespread lodging, resulting in yield reduction. Therefore, promoting semi-dwarf, compact maize varieties can not only increase planting density but also reduce lodging.

[0004] Currently, several genes associated with dwarfing and reduced leaf angle have been cloned, but mutants of these genes often have negative effects on the overall plant. For example, genes such as na1 and dwarf1 may lead to excessive dwarfing, while liguleless mutants (lg2 ​​and lgn) may cause excessively upright leaf angles, making them difficult to apply in actual production. Therefore, in the process of establishing maize plant architecture, it is necessary to further explore and utilize more genes related to semi-dwarf, compact plant architecture. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a maize plant architecture regulating gene ZmEXO1 and its application. Studies have found that ZmEXO1 has the function of regulating maize leaf angle and plant height, providing a target gene for improving maize plant architecture.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a maize plant architecture regulating gene ZmEXO1, the amino acid sequence of which is shown in SEQ ID NO.7.

[0008] This invention provides a nucleic acid molecule encoding the aforementioned ZmEXO1.

[0009] Preferably, the nucleotide sequence of the ZmEXO1 is shown in SEQ ID NO.6.

[0010] This invention provides an application of the above-mentioned ZmEXO1 or nucleic acid molecule in regulating maize plant architecture.

[0011] Preferably, the plant type includes one or both of leaf angle and plant height; the application includes reducing or silencing the expression level of ZmEXO1 to reduce maize plant height and decrease maize leaf angle.

[0012] This invention provides a method for improving maize plant architecture, including the step of reducing or silencing the expression level of the aforementioned ZmEXO1 in the recipient plant.

[0013] Preferably, the method for reducing or silencing the expression level of ZmEXO1 in the recipient plant according to claim 1 includes: using mutant plants with reduced or silenced ZmEXO1 as donor parents, and using superior maize inbred lines as recurrent parents, performing backcrossing and self-crossing to obtain plants with improved maize plant type; the superior maize inbred lines include PH6WC or PH4CV.

[0014] The present invention provides a Kasp molecular marker closely linked to the above-mentioned ZmEXO1, wherein the Kasp molecular marker is C>T at position 4014 of the nucleotide sequence of the above-mentioned ZmEXO1, and the nucleotide sequence of the Kasp molecular marker is shown in SEQ ID NO. 8.

[0015] The present invention provides a Kasp primer that is closely linked to the above-mentioned ZmEXO1, and the nucleotide sequence of the Kasp primer is shown in SEQ ID NO.3 to 5.

[0016] This invention provides the application of the above-mentioned Kasp molecular markers or Kasp primers in screening or identifying maize plant type or identifying ZmEXO1 genotype.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides a maize plant architecture regulating gene, ZmEXO1, and its application. Research has found that ZmEXO1 can regulate maize leaf angle and plant height; mutants with reduced or silenced ZmEXO1 expression lead to smaller leaf angles and reduced plant height. Therefore, gene editing of the ZmEXO1 gene or genetic breeding improvement using low-expression Zmexo1 mutants can yield new germplasm that improves maize plant architecture, which is of great significance for enriching new maize germplasm and breeding high-yielding, dense-planting-tolerant varieties.

[0019] The present invention also provides a Kasp molecular marker and Kasp primers that are closely linked to ZmEXO1, which can be used for molecular marker-assisted selection breeding and mining of related functional genes, and can quickly screen and identify maize germplasm modified with ZmEXO1 molecules. Attached Figure Description

[0020] Figure 1 The results of phenotypic identification of the Zmexo1 mutant are shown below: A: Comparison of plant architecture between wild-type and Zmexo1 mutants at the pollination stage. The white arrows from top to bottom indicate the positions of the first leaf above the ear, the ear leaf, and the first leaf below the ear, respectively. The scale bar is 20 cm. B: Schematic diagram of DNA Sanger sequencing results for wild-type and Zmexo1 mutants. C: Statistical analysis of plant height differences between wild-type and Zmexo1 mutants at the pollination stage. D: Statistical analysis of leaf angle differences between wild-type and mutants at the pollination stage. E: Schematic diagram of KASP identification results for wild-type and Zmexo1 mutants. The horizontal axis FAM represents wild-type, and the vertical axis HEX represents mutant. Figure 1 The data in B to C represent the mean ± standard deviation. The significance was determined by a two-tailed Student's t-test, and the p-value was <0.001.

[0021] Figure 2 Differences in cytological morphology and cell wall composition of the leaf auricle between wild-type and mutant Zmexo1 were observed. A: Transmission electron microscopy observation of the auricle cell wall structure of wild-type and mutant Zmexo1, scale bar (top): 5 μm; scale bar (bottom): 200 nm; B: Determination of the thickness of the auricle cell wall in wild-type and mutant Zmexo1; C: Determination of the main components of the cell wall in the leaf auricle of wild-type and mutant Zmexo1, namely cellulose, hemicellulose, pectin, and lignin. Figure 2 The data in B to C represent the mean ± standard deviation. The significance was determined by a two-tailed Student's t-test, and the p-value was <0.001.

[0022] Figure 3 As a result of genetic improvement of superior backbone inbred lines, A:PH6WC and PH6WC Zmexo1 Statistics on plant height differences between groups; B: PH6WC and PH6WC Zmexo1 Statistical analysis of leaf angle differences between groups; C: PH4CV and PH4CV Zmexo1 Statistical analysis of plant height differences between groups; D: PH4CV and PH4CV Zmexo1 Statistical analysis of leaf angle differences among groups Figure 3 The data A to D in the table represent the mean ± standard deviation. The significance was determined by a two-tailed t-test, with *p<0.05 and **p<0.01. Detailed Implementation

[0023] This invention provides a maize plant architecture regulating gene ZmEXO1, the amino acid sequence of which is shown in SEQ ID NO.7.

[0024] The ZmEXO1 gene of this invention is a gene that regulates maize plant architecture. Studies have found that reducing or silencing the expression level of ZmEXO1 can reduce the leaf angle and plant height of maize.

[0025] This invention provides a nucleic acid molecule encoding the aforementioned ZmEXO1.

[0026] In this invention, the nucleotide sequence of ZmEXO1 is shown in SEQ ID NO.6.

[0027] This invention provides an application of the above-mentioned ZmEXO1 or nucleic acid molecule in regulating maize plant architecture.

[0028] In this invention, the plant type preferably includes one or both of leaf angle and plant height; the application includes reducing or silencing the expression level of ZmEXO1 to reduce maize plant height and decrease maize leaf angle.

[0029] This invention provides a method for improving maize plant architecture, including the step of reducing or silencing the expression level of the aforementioned ZmEXO1 in the recipient plant.

[0030] In the above method, the reduction or silencing of ZmEXO1 expression in the recipient plant can be achieved through gene editing or genetic breeding. As a preferred embodiment, the method for reducing or silencing ZmEXO1 expression in the recipient plant includes: using a mutant plant with reduced or silenced ZmEXO1 as the donor parent, and using a superior maize inbred line as the recurrent parent, performing backcrossing and self-crossing to obtain plants with improved maize plant type; the superior maize inbred line includes PH6WC or PH4CV. The improved maize plant type exhibits a semi-dwarf, compact plant type.

[0031] The present invention provides a Kasp molecular marker closely linked to the above-mentioned ZmEXO1, wherein the Kasp molecular marker is C>T at position 4014 of the nucleotide sequence of the above-mentioned ZmEXO1, and the nucleotide sequence of the Kasp molecular marker is shown in SEQ ID NO. 8.

[0032] The Kasp molecular marker developed in this invention can accelerate the identification of ZmEXO1 genotypes and increase identification accuracy. This molecular marker can also be used to screen or identify maize plant types, such as one or both of leaf angle and plant height.

[0033] The present invention provides a Kasp primer that is closely linked to the above-mentioned ZmEXO1, and the nucleotide sequence of the Kasp primer is shown in SEQ ID NO.3 to 5.

[0034] This invention uses maize genomic DNA as a template, amplifies it using the aforementioned Kap primers, and detects mutations at position 4014 of the ZmEXO1 nucleotide sequence through sequencing. The Kap primers of this invention can rapidly identify the ZmEXO1 genotype, and based on the ZmEXO1 genotype, screen or identify maize plant types. When the C-base mutation at position 4014 of the ZmEXO1 nucleotide sequence is changed to T, it indicates that the maize plant type being tested is a semi-dwarf, compact type.

[0035] This invention provides the application of the above-mentioned Kasp molecular markers or Kasp primers in screening or identifying maize plant type or identifying ZmEXO1 genotype.

[0036] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] Functional validation of ZmEXO1, a candidate target gene for regulating maize plant architecture

[0040] 1. Materials and Methods

[0041] 1.1 Test Materials

[0042] Inbred line B73 (wild type) and a Zmexo1 gene loss mutant, Zmexo1 (EMS4-08055b, denoted as mutant), were ordered from the maize EMS mutant library.

[0043] 1.2 Test Methods

[0044] 1.2.1 Field trials and phenotypic identification

[0045] In the spring of 2023, a BC1F2 population containing 207 individual plants was constructed by self-pollination of (Zmexo1×B73)×B73 BC1F1 in Wuhan (30°N, 114°E). Plant height traits of each individual plant were measured at the maize maturity stage, including agronomic traits such as plant height, ear height, and leaf angle. The t-test was used to test the significance of differences in agronomic traits between the mutant and the wild type.

[0046] 1.2.2 Genotyping

[0047] Genotyping analysis involves DNA extraction and PCR amplification. The CTAB method was used to extract single-plant DNA from fresh, young leaves.

[0048] The extracted single-strain DNA was used as a template for PCR amplification, that is, PCR amplification was performed according to the PCR reaction system prepared in Table 1 and the PCR reaction procedure in Table 2.

[0049] Table 1 PCR reaction system

[0050] Element Volume (μL) 2×PCR Mix 5 DNA 1 Upstream primer (10 μM) 0.2 Downstream primer (10 μM) 0.2 <![CDATA[ddH2O]]> 3.6

[0051] The upstream primer F is 5'-ATCATCTACGACTTCCTGCT-3' (SEQ ID NO.1); the downstream primer R is 5'-CTGGTCGATCTTGAGGTTGC-3' (SEQ ID NO.2).

[0052] Table 2 PCR reaction procedure

[0053]

[0054] Genotyping of individual plants was performed using a combination of 1% agarose gel electrophoresis and Sanger sequencing.

[0055] 1.2.3 Molecular Marker Development

[0056] To further accelerate genotype identification and increase accuracy, and to facilitate subsequent genotype detection under different inbred line backgrounds, Kasp molecular markers were designed for the SNPs of the mutant Zmexo1, and the samples were sent to the company for sequencing verification.

[0057] The primers designed using the Kasp molecular markers are as follows:

[0058] The upstream primer for detecting wild type is: Kasp-EXO1-F1(FAM):5'-GAAGGTGACCAAGTTCATGCTTGGAGGGCTTCTGCACGAGCC-3' (SEQ ID NO.3); the 5' end of the upstream primer for detecting wild type is labeled with the FAM fluorescent group.

[0059] Detection of wild-type downstream primer: Kasp-EXO1-R:5'-CGTACTCGGGCTGGTGGAAC-3' (SEQ ID NO.4);

[0060] The upstream primer for detecting the mutant is: Kasp-EXO1-F2(HEX):5'-GAAGGTCGGAGTCAACGGATTTGGAGGGCTTCTGCACGAGCT-3':(SEQ ID NO.5); the 5' end of the upstream primer for detecting the mutant is labeled with the HEX fluorescent group;

[0061] The downstream primer for detecting the mutant is: Kasp-EXO1-R:5'-CGTACTCGGGCTGGTGGAAC-3' (SEQ ID NO.4).

[0062] The test sample was simultaneously detected using the upstream and downstream primers for detecting wild type and mutant as shown in SEQ ID NO.3-5. Genomic DNA was extracted from fresh young leaves of the test sample using the CTAB method. Using the genomic DNA of the test sample as a template, amplification was performed according to the reaction system in Table 3 and the amplification program in Table 4. If only FAM fluorescence was detected in the test sample, it was a homozygous wild-type plant; if both FAM and HEX fluorescence were detected in the test sample, it was a heterozygous wild-type plant; if only HEX fluorescence was detected in the test sample, it was a homozygous mutant plant.

[0063] Table 3. PCR reaction system for genotype detection

[0064] Element Volume (μL) FLu-Arms 2x PCR Mix 5μL Upstream typing primer F1 (10 μM) 0.1μL Upstream typing primer F2 (10 μM) 0.1μL Downstream universal primer R (10 μM) 0.3μL DNA template 2.75μL sterile water to 10μL

[0065] Table 4. PCR amplification procedures for genotype detection

[0066]

[0067] 2. Results and Analysis

[0068] Sanger sequencing results showed that, compared with the wild type, the mutant Zmexo1 had a single-base mutation that caused the 450th codon (CAG) encoding glutamine to become a stop codon (TAG), resulting in premature termination of transcription and indicating loss of gene function in the mutant Zmexo1. Figure 1 (B in the text). The SNP (Kasp molecular marker) of the mutant Zmexo1 is C>T at position 4014 of the nucleotide sequence of the Zm00001d018106 gene.

[0069] The nucleotide sequence of the ZmEXO1 gene, also known as Zm00001d018106, is as follows:

[0070] C AGTGCGGCCTGCACGGCGCGGAGTCCGGCGCCGGCGCCGCCTCCGCGTACGTCTGGGTCGGCAACTCGGCCGCGCAGTGCCCCGGCCAGTGCGCGTGGCCGTTCCACCAGCCCGAGTACGGCCCGCAGACCCCGCCCCTCGTCCCGCCTAACGGCGACGCCGCCGTCGACGGCATGGTCGTCAGCCTCGCCACCGCGTTCGCCGGCGCCGTCACCAACCCGTTCCGCGACGCCTACTACCAGGGCTCCAGCGACGCGCCCCTGGAGGCCGCCACGGCGTGCCCCGGACAGTTCGGCAGCGGCTCGTACCCGGGGTACCCCGGCAACCTCAAGATCGACCAGGCCAGCGGTGCCAGCTACAACGCCAATGGCGCGCAGGGGAGGAAATACCTCCTCCCCGCGCTCTACAACCCTTCCACTTCTGCCTGCAGCACGCTGGTCTAG(SEQ ID NO.6).

[0071] The amino acid sequence of ZmEXO1, i.e., the Zm00001d018,106 gene, is as follows:

[0072] MASPHSCFRGWQSTSHVALALVSVLILSSARLSAGGRSLLELYKPPASAILTYHNGAVLQGRIPVSIIWYGRFTPAQKAVVTDFLGSLTTAAASPLAPSPSVSQWWSTIDQLYLSKARGRGSGGGGGGARVALVGQATDEGCSLGKRLTLAQLPQLAARAGPRKGGIALVLTAQDVGVDGFCTSRCGLHGSDARAPGTAYVWVGNSATQCPGQCAWPFHRPLYGPQTPALVPPSGDVGADGMVINIASMVAGAVTNPFRDGFYQGDKDAPLEAATACTGVYGSGAYPGFAALVLLLSSAQLSTGARRRMELYQPDPADMLSYHNGSVLHSDIFVSVLWYGSFTQVQKAIIYDFLLSLTTTPQAASPSVAEWWDMIEQQYLSKAAQTTKHTTTRVMLDTTQVSSDDGCSMGTSLTLAQVSALAARARPRKGGVALVLTAQDVAVEGFCTSQCGLHGAESGAGAASAYVWVGNSAAQCPGQCAWPFHQPEYGPQTPPLVPPNGDAAVDGMVVSLATAFAGAVTNPFRDAYYQGSSDAPLEAATACPGQFGSGSYPGYPGNLKIDQASGASYNANGAQGRKYLLPALYNPSTSACSTLV(SEQ ID NO.7).

[0073] The nucleotide sequence of the Kasp molecular marker is as follows:

[0074] ATCTACGACTTCCTGCTCTCGCTCACCACCACGCCCCAGGCCGCCTCGCCGTCCGTCGCGGAGTGGTGGGACATGATCGAGCAGCAGTACCTGTCCAAGGCGGCGCAGACCACCAAGCATACCACCACCCGGGTGATGCTGGACACCACCCAGGTGTCGTCCGACGACGGCTGCTCCATGGGCACGTCCCTCACCCTGGCGCAGGTCTCCGCCCTCGCCGCGCGGGCCAGGCCCAGGAAGGGCGGCGTCGCGCTGGTGCTCACGGCGCAGGACGTCGCCGTGGAGGGCTTCTGCACGAGC YAGTGCGGCCTGCACGGCGCGGAGTCCGGCGCCGGCGCCGCCTCCGCGTACGTCTGGGTCGGCAACTCGGCCGCGCAGTGCCCCGGCCAGTGCGCGTGGCCGTTCCACCAGCCCGAGTACGGCCCGCAGACCCCGCCCCTCGTCCCGCCTAAC GGCGACGCCGCCGTCGACGGCATGGTCGTCAGCCTCGCCACCGCGTTCGCCGGCGCCGTCACCAACCCGTTCCGCGACGCCTACTACCAGGGCTCCAGCGACGCGCCCCTGGAGGCCGCCACGGCGTGCCCCGGACAGTTCGGCAGCG(SEQ ID NO.8). Among them, the Y in the nucleotide sequence of the Kasp molecular marker represents C / T.

[0075] The Kasp molecular marker refers to the C>T mutation at position 301 of the sequence shown in SEQ ID NO.8. A T at position 301 indicates a semi-dwarf compact plant type (reduced plant height and smaller leaf angle), while a C at position 301 indicates a wild type.

[0076] Field observations of this EMS allelic mutant system revealed segregation between normal plant type and mutant plant types characterized by dwarfism and reduced leaf angle. Backcrossing the Zmexo1 mutant plants with B73 resulted in F1 offspring exhibiting normal plant height and leaf angle. Self-crossing in the F2 generation showed segregation between wild-type and mutant phenotypes. Figure 1 (A) in the middle.

[0077] The agronomic traits of plant height and leaf angle of 12 wild-type and mutant plants were statistically analyzed. The results showed that the plant height and leaf angle of the mutant Zmexo1 were significantly reduced. Figure 1 (C and D in the text).

[0078] To further verify the co-segregation of the Zmexo1 mutation site and phenotype, KASP primers were designed targeting a single-base mutation site at amino acid position 450. Field sampling DNA identification results showed that the genotype and phenotype both conformed to a segregation ratio of 1:2:1 (homozygous wild type: heterozygous wild type: homozygous mutant). All phenotypes and genotypes were mutually corresponding, indicating that the Zmexo1 mutant phenotype and genotype co-segregate and that this gene participates in the establishment of maize plant architecture. Figure 1 (E in the text)

[0079] Example 2

[0080] Cytological analysis of the pulvinus of wild-type and mutant Zmexo1

[0081] 1. Materials and Methods

[0082] 1.1 Transmission electron microscopy observation of cell walls in the leaf sheath of maize

[0083] The mutant in this embodiment is the wild type in the F2 generation population after self-crossing in Example 1, and the mutant is the genotype homozygous mutant Zmexo1 in the F2 generation population after self-crossing in Example 1.

[0084] To investigate the cellular mechanisms underlying the difference in leaf angle between wild-type and homozygous mutant Zmexo1, transmission electron microscopy (TEM) was performed on cells from the leaf pulvinus. Transverse sections of the fourth leaf (not yet unfolded) at the V2 stage of maize were collected and fixed with 2% (v / v) glutaraldehyde in 50 mM pH 7.2 phosphate buffer overnight at 4°C. After fixation, the tissues were post-fixed in 2% (v / v) OsO4 for 2 h. After washing in phosphate buffer, the tissues were dehydrated in ethanol, impregnated with Araldite / Embed 812 resin, and finally polymerized in Araldite / Embed 812 resin. Electron microscopy was performed using a Reichert-Jung ultramicrotome to cut 90 nm thick sections, which were mounted on a Formal-coated gold grid and stained with uranium acetate and lead citrate. The microscopy was performed using a HI TACHI, H-7650 scanning electron microscope at Huazhong Agricultural University. Cell size was measured and calculated using ImageJ.

[0085] 1.2 Quantitative analysis of the main cell wall components in the pulvinus of maize leaves

[0086] The water-soluble pectin, ionic pectin, covalent pectin, soluble hemicellulose, insoluble hemicellulose, and cellulose are extracted stepwise by utilizing the solubility of different cell wall components, and the concentration of the substances is then measured by spectrophotometry; the lignin content is usually determined based on the acid depolymerization method.

[0087] 1.2.1 Sample Pretreatment

[0088] 1. Remove soluble substances:

[0089] (1) Weigh 2g of tissue samples from the leaf pulvinus of each wild type and mutant, add 80% ethanol solution, and heat in a 70℃ water bath for 1h;

[0090] (2) Centrifuge at 10,000 rpm for 10 min, discard the supernatant, and repeat twice;

[0091] (3) Wash the residue with distilled water, dry it, and obtain a dry sample for later use.

[0092] 2. Removal of lipids and proteins:

[0093] (1) Soak the dried sample in a neutral detergent solution of 2% SDS and heat at 70°C for 30 min to remove lipids and proteins;

[0094] (2) Wash until neutral and dry to obtain crude cell wall extract, which is the pretreated sample.

[0095] 1.2.2 Determination of pectin content

[0096] 1. Extraction:

[0097] (1) Add 0.5g of pretreated sample to 20mL of pH 4.0 0.5% ammonium oxalate solution;

[0098] (2) Heat at 85℃ for 1 hour, stirring to promote dissolution;

[0099] (3) After centrifugation, collect the supernatant to obtain supernatant 1, while retaining precipitate 1.

[0100] 2. Concentration determination (phenol-sulfuric acid method):

[0101] (1) Take 1 mL of supernatant 1 and add 1 mL of 5% phenol solution;

[0102] (2) Quickly add 5 mL of concentrated sulfuric acid, let it stand and cool, and then measure the absorbance at a wavelength of 490 nm.

[0103] (3) Compare with the standard curve to calculate the pectin content.

[0104] 1.2.3 Determination of hemicellulose content

[0105] 1. Extraction:

[0106] (1) Add the precipitate 1 after pectin extraction to 20 mL of 4% NaOH solution (containing 1% NaBH4);

[0107] (2) Heat in an 80℃ water bath for 2 hours, stirring constantly;

[0108] (3) Centrifuge and collect the supernatant as hemicellulose extract, while retaining the sediment.

[0109] 2. Concentration determination (DNS method):

[0110] (1) Take 1 mL of hemicellulose extract, add 3 mL of DNS solution, and react in a boiling water bath for 5 min;

[0111] (2) After cooling, dilute with distilled water and measure absorbance at a wavelength of 540 nm.

[0112] (3) Calculate the hemicellulose content by comparing it with the reducing sugar standard curve.

[0113] 1.2.4 Determination of cellulose content

[0114] 1. Extraction:

[0115] (1) Add the precipitate 2 after hemicellulose extraction to 72% H2SO4 solution and stir at room temperature for 2 hours;

[0116] (2) Dilute the acid solution to 4%, heat in a water bath for 1 hour, centrifuge, and collect the cellulose supernatant;

[0117] 2. Concentration determination (DNS method):

[0118] The concentration of reducing sugars in cellulose supernatant was determined using the DNS method, which is the same as that for hemicellulose.

[0119] 1.2.5 Determination of Lignin Content

[0120] (1) Weigh out 0.30g of sample from the leaf pulvinus and record it as W1. Wrap it in filter paper and place it in a Soxhlet extractor for extraction with benzene-ethanol (67:33, v / v) for 4 hours. The extracted powder is then air-dried in a fume hood.

[0121] (2) Untie the filter paper, transfer the air-dried material completely into a 250mL Erlenmeyer flask, add 10.0mL of 67.0% (v / v) sulfuric acid, place it on a shaker, and shake horizontally at 120r / min for 1.5h at 30℃;

[0122] (3) Add 200 mL of dH2O, hydrolyze at 120 °C for 1 h;

[0123] (4) Filter the hydrolysate with a G3 crucible filter to fully transfer the residue into the crucible filter;

[0124] (5) Dilute the filtrate to a final volume of V = 250.0 mL. Take 1.0 mL into a glass colorimetric tube and dilute it 10 times with 2.88% (v / v) sulfuric acid (depending on the sample), ensuring the absorbance is between 0.2 and 0.7. Record the dilution factor as D. Measure the OD value using a UV spectrophotometer at a wavelength of 205 nm. The blank is 2.88% sulfuric acid.

[0125] The content of acid-soluble lignin is calculated according to the following formula:

[0126] ASL%=A×D×V / (1000×K×W1)×100

[0127] A: Absorbance; D: Dilution factor; V: Total volume of filtrate; K: Absorption coefficient of acid-soluble lignin, taken as 110; W1: Sample mass.

[0128] (6) Wash the residue in the crucible filter from step 4 with dH2O until the pH is neutral;

[0129] (7) The residue and filter were dried at 60°C to constant weight, cooled to room temperature in a desiccator, and weighed to obtain W2 (residue + filter);

[0130] (8) Place the weighed filter and residue into a Margaritt furnace, preheat at 200℃ for 30 min, then ashing at 575±25℃ for 4 h, remove and cool in a dryer for 30 min, weigh to obtain W3 (ash + filter).

[0131] (9) The content of acid-insoluble lignin is:

[0132] AIL% = (W2 - W3) × 100 / W1

[0133] (10) The total lignin content of the sample is:

[0134] Lignin(%)=AIL(%)+ASL(%)

[0135] Note: The ash content in the sample is (W3-W4)×100 / W1, and the mass of the clean and dry crucible is W4.

[0136] 2. Results and Analysis

[0137] First, transverse observation of the pulvinus tissue of wild-type and homozygous mutant Zmexo1 at the fully expanded second leaf stage of maize seedlings using transmission electron microscopy revealed that, compared to the wild-type, the pulvinus cells of the Zmexo1 mutant exhibited distorted morphology, reduced cell wall thickness, and frequent breakpoints, indicating that the cell wall structural homeostasis in the mutant was disrupted, suggesting possible changes in cell wall composition. Figure 2 Therefore, equal amounts of leaf sheaths from the wild type and the Zmexo1 mutant were weighed and dried using a colorimetric chemical assay to determine the cell wall components. Statistical results showed that compared to the wild type, the mutant cell wall contained significantly lower levels of major components, including cellulose, hemicellulose, pectin, and lignin. These results preliminarily reveal that the ZmEXO1 gene plays an important regulatory role in the composition and structural remodeling of the maize leaf sheath cell wall. Figure 2 (C in the middle).

[0138] Example 3: Genetic Improvement of Inbred Lines

[0139] Genetic improvement of different superior maize inbred lines was carried out using SNP markers linked to mutant traits developed in Example 1 (see section 1.2.3 of Example 1).

[0140] 1. Materials and Methods

[0141] 1.1 Test Materials

[0142] The semi-dwarf compact mutant Zmexo1 (B73 background) is a superior maize inbred line: PH6WC and PH4CV.

[0143] The semi-dwarf compact mutant Zmexo1 is the genotype homozygous mutant Zmexo1 in the F2 generation population after self-crossing in Example 1.

[0144] 1.2 Test Methods

[0145] 1.2.1 Field Trials

[0146] From 2023 to 2024, genetic improvement of maize inbred lines was carried out using hybridization and molecular marker-assisted selection backcross methods; backcrossing and selection identification of each generation were carried out at two locations in Sanya and Shenzhen.

[0147] 1.2.2 Phenotypic Identification

[0148] Ten plants were planted per row. The leaf angles of the leaf above the ear, the leaf at the ear position, and the leaf below the ear, as well as the plant height, were measured for all individual plants (10 days after corn flowering). The measured traits were taken three times and the average value was taken.

[0149] 1.2.3 Genotype Analysis

[0150] Genotyping analysis involves DNA extraction and PCR amplification. Single-strain DNA is extracted using the CTAB method and then sent to the company for Kasp sequencing identification.

[0151] 2. Results and Analysis

[0152] 2.1 Genetic Improvement of Inbred Lines

[0153] Using the semi-dwarf compact mutant Zmexo1 as the donor parent and two superior inbred lines as recurrent parents, backcrossing and self-crossing were performed to obtain the BC1F2 generation (denoted as PH6WC) of (Zmexo1×PH6WC)×PH6WC. Zmexo1 ) and (Zmexo1×PH4CV Zmexo1 )×PH4CV's BC1F2 generation (denoted as PH4CV) Zmexo1 During the backcrossing process, molecular marker-assisted selection (MAS) was performed using the molecular markers obtained in Example 1. Simultaneously, combined with field observations of agronomic traits and marker identification, improved inbred lines with enhanced leaf angle and plant height were initially obtained in the BC1F2 generation (with the unimproved inbred lines PH6WC and PH4CV as controls, respectively). Compared with the unimproved inbred lines PH6WC and PH4CV, the improved line PH6WC... Zmexo1 and PH4CV Zmexo1Significantly reduced differences were observed in both leaf angle and plant height, two important plant architecture-related traits (see...). Figure 3 ).

[0154] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for reducing the expression level of the maize plant architecture regulating gene ZmEXO1 to decrease maize plant height and / or reduce the leaf angle, characterized in that... The nucleic acid sequence of ZmEXO1 is shown in SEQ ID NO.

6.

2. The silenced maize plant architecture regulating gene ZmEXO1 is used to reduce maize plant height and / or decrease the leaf angle, characterized in that... The nucleic acid sequence of ZmEXO1 is shown in SEQ ID NO.

6.

3. A method for improving maize plant architecture, characterized in that, The step includes reducing the expression level of ZmEXO1 as described in claim 1 in the recipient plant.

4. The method according to claim 3, characterized in that, The method for reducing the expression level of ZmEXO1 in the recipient plant according to claim 1 includes: using a mutant plant with reduced ZmEXO1 as the donor parent and a superior maize inbred line as the recurrent parent, performing backcrossing and selfcrossing to obtain a plant with improved maize plant type; the superior maize inbred line includes PH6WC or PH4CV.

5. A method for improving maize plant architecture, characterized in that, The step includes silencing the expression level of ZmEXO1 as described in claim 1 in the recipient plant.

6. The method according to claim 5, characterized in that, The method for reducing the expression level of ZmEXO1 in the recipient plant according to claim 1 includes: using a mutant plant with silenced ZmEXO1 as the donor parent and a superior maize inbred line as the recurrent parent, performing backcrossing and selfcrossing to obtain a plant with improved maize plant type; the superior maize inbred line includes PH6WC or PH4CV.

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