Maize plant type regulation gene ZmEXO1 and application thereof

Abstract

The invention provides a corn plant type regulation gene ZmEXO1 and application, and belongs to the technical field of corn molecular breeding. The amino acid sequence of the ZmEXO1 is as shown in SEQ ID NO.7. The research finds that the ZmEXO1 can regulate and control the corn leaf included angle and the plant height, and the corn leaf included angle is reduced and the plant height is reduced due to the fact that a mutant with the expression quantity of the ZmEXO1 reduced or silenced. Therefore, the germplasm capable of improving the corn plant type can be obtained by carrying out gene editing on the ZmEXO1 gene or carrying out genetic breeding improvement by utilizing the Zmeso1 low-expression mutant, and the method has important significance on enriching the corn germplasm and cultivating density-tolerant high-yield varieties. The invention further provides a Kasp molecular marker and a Kasp primer which are closely linked with the ZmEXO1, and the Kasp molecular marker and the Kasp primer can be used for molecular marker-assisted selective breeding and mining of related functional genes and can be used for rapidly screening and identifying ZmEXO1 molecular improvement-containing corn germplasm.

Description

Technical Field

[0001] The invention belongs to the technical field of corn molecular breeding, and in particular relates to a corn plant type regulating gene ZmEXO1 and an application thereof. Background Art

[0002] As the world's most important food crop, corn plays an important role in ensuring food security and meeting market demand. Increasing corn yield is the primary goal, and the key to achieving this goal is to increase planting density. However, the limitation of canopy light interception rate is the main obstacle to achieving high-yield and dense planting, so improving the external morphological structure of corn plants has become an important indicator for plant breeding. Leaf angle, that is, the angle between the leaf and the main stem, is one of the key agronomic traits that affect corn plant type and yield. A smaller leaf angle helps to reduce the canopy light interception rate, thereby improving light energy utilization efficiency and planting density, and ultimately achieving an increase in yield.

[0003] In the context of corn reform, increasing planting density has become the main measure to increase yields. However, over-dense planting may cause corn to lodging, thus affecting yield. Studies have shown that there is a significant correlation between corn lodging and field planting density. As planting density increases, plant competition intensifies, and the rapid growth of stems makes the plants thin and weak, and their resistance to lodging decreases. In this case, once strong winds or heavy rains occur, natural disasters will cause large-scale lodging, resulting in reduced yields. Therefore, the promotion of new semi-dwarf and compact corn varieties can not only increase planting density, but also reduce lodging.

[0004] Currently, many genes related to dwarfing and reducing leaf angle have been cloned, but mutants of these genes often have a negative impact on the overall plant. For example, genes such as na1 and dwarf1 may cause excessive dwarfing of the plant, while liguleless mutants (lg2 ​​and lgn) may make the leaf angle too upright, making them difficult to use in actual production. Therefore, in the process of establishing corn plant type, it is necessary to further explore and utilize more genes related to semi-dwarf compact plant type. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a corn plant type regulatory gene ZmEXO1 and its application. Studies have found that ZmEXO1 has the function of regulating corn leaf angle and plant height, providing a target gene for improving corn plant type.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a corn plant type regulating gene ZmEXO1, and the amino acid sequence of the ZmEXO1 is shown in SEQ ID NO.7.

[0008] The present invention provides a nucleic acid molecule encoding the above ZmEXO1.

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

[0010] The present invention provides an application of the above ZmEXO1 or nucleic acid molecule in regulating corn plant type.

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

[0012] The present invention provides a method for improving corn plant type, comprising the step of reducing or silencing the expression level of the above ZmEXO1 in a receptor plant.

[0013] Preferably, the method for reducing or silencing the expression level of ZmEXO1 in claim 1 in a receptor plant comprises: using a mutant plant with reduced or silenced ZmEXO1 as a donor parent and an excellent corn inbred line as a recurrent parent, performing backcrossing and selfing to obtain a plant with improved corn plant type; the excellent corn inbred line comprises PH6WC or PH4CV.

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

[0015] The present invention provides a Kasp primer tightly linked to the above ZmEXO1, and the nucleotide sequence of the Kasp primer is shown in SEQ ID NOs. 3-5.

[0016] The present invention provides the use of the above-mentioned Kasp molecular marker or Kasp primer in screening or identifying corn plant types or identifying ZmEXO1 genotypes.

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

[0018] The present invention provides a corn plant type regulating gene ZmEXO1 and its application. Studies have shown that ZmEXO1 can regulate the corn leaf angle and plant height, which is manifested in that mutants with reduced or silenced expression of ZmEXO1 will cause the corn leaf angle to become smaller and the plant height to decrease. Therefore, by gene editing the ZmEXO1 gene or using Zmexo1 low-expression mutants for genetic breeding improvement, new germplasm with improved corn plant type can be obtained, which is of great significance for enriching new corn germplasm and cultivating new varieties with high density resistance and high yield.

[0019] The present invention also provides a Kasp molecular marker and Kasp primer tightly linked to ZmEXO1, which can be used for molecular marker-assisted selection breeding and mining related functional genes, and can quickly screen and identify corn germplasm containing ZmEXO1 molecule improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The identification results of the Zmexo1 mutant phenotype, including: A: comparison of plant types between the wild type and mutant Zmexo1 at the corn powder shedding stage, the white arrows indicate the positions of the first leaf on the ear, the ear leaf, and the first leaf under the ear from top to bottom, respectively, and the scale is 20 cm; B: schematic diagram of DNA Sanger sequencing results of the wild type and mutant Zmexo1; C: statistical difference in plant height between the wild type and mutant Zmexo1 at the corn powder shedding stage; D: statistical difference in leaf angle between the wild type and mutant at the corn powder shedding stage; E: schematic diagram of KASP identification results of the wild type and mutant Zmexo1, the horizontal axis FAM represents the wild type, the vertical axis HEX represents the mutant, where Figure 1 Data in B to C represent mean ± standard deviation, and significance was determined by two-tailed Student's t test, ***p < 0.001;

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

[0022] Figure 3 It is the result of genetic improvement of excellent backbone inbred lines. Zmexo1 The difference of plant height between the two groups; B: PH6WC and PH6WC Zmexo1 The leaf angle difference statistics of the groups; C: PH4CV and PH4CV Zmexo1 The difference of plant height between the two groups; D: PH4CV and PH4CV Zmexo1 The leaf angle difference statistics of the groups, Figure 3 Data in A to D represent mean ± standard deviation, and significance was determined by two-tailed t-test, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0023] The present invention provides a corn plant type regulating gene ZmEXO1, and the amino acid sequence of the ZmEXO1 is shown in SEQ ID NO.7.

[0024] The ZmEXO1 of the present invention is a gene for regulating corn plant type. Studies have found that the corn leaf angle can be reduced and the plant height can be lowered by reducing or silencing the expression level of ZmEXO1.

[0025] The present invention provides a nucleic acid molecule encoding the above ZmEXO1.

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

[0027] The present invention provides an application of the above ZmEXO1 or nucleic acid molecule in regulating corn plant type.

[0028] In the present 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 corn plant height and reduce corn leaf angle.

[0029] The present invention provides a method for improving corn plant type, comprising the step of reducing or silencing the expression level of the above ZmEXO1 in a receptor plant.

[0030] In the above method, the method of reducing or silencing the expression level of the above ZmEXO1 in the receptor plant can be carried out by gene editing or genetic breeding. As a preferred embodiment, the method of reducing or silencing the expression level of the above ZmEXO1 in the receptor plant comprises: using a mutant plant that reduces or silences ZmEXO1 as a donor parent and an excellent corn inbred line as a recurrent parent, performing backcrossing and selfing to obtain a plant with improved corn plant type; the excellent corn inbred line includes PH6WC or PH4CV. The plant with improved corn plant type exhibits a semi-dwarf compact plant type.

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

[0032] The Kasp molecular marker developed by the present invention can accelerate the ZmEXO1 genotype identification speed and increase the identification accuracy, and the molecular marker can also be used to screen or identify corn plant types, such as one or both of leaf angle and plant height.

[0033] The present invention provides a Kasp primer tightly linked to the above ZmEXO1, and the nucleotide sequence of the Kasp primer is shown in SEQ ID NOs. 3-5.

[0034] The present invention uses corn genomic DNA as a template, utilizes the Kasp primers for amplification, and detects the mutation of the 4014th position of the nucleotide sequence of ZmEXO1 by sequencing. The Kasp primers of the present invention can quickly identify the ZmEXO1 genotype, and screen or identify the corn plant type according to the situation of the ZmEXO1 genotype. When the 4014th base C of the nucleotide sequence of ZmEXO1 mutates to T, it indicates that the plant type of the corn to be tested is a semi-dwarf compact plant type.

[0035] The present invention provides the use of the above-mentioned Kasp molecular marker or Kasp primer in screening or identifying corn plant types or identifying ZmEXO1 genotypes.

[0036] In the present 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 are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] Example 1

[0039] Functional verification of the candidate target gene ZmEXO1 for plant architecture regulation in maize

[0040] 1. Materials and methods

[0041] 1.1 Test Materials

[0042] The inbred line B73 (wild type) and a ZmEXO1 gene loss-of-function 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 constructed by self-pollination of (Zmexo1×B73)×B73 BC1F1 was planted in Wuhan (30°N, 114°E), containing 207 individuals. The plant height traits of each individual plant were measured at the corn maturity stage, including agronomic traits such as plant height, ear height, and leaf angle. The t-test was used to test the significance of the differences in agronomic traits between the mutant and the wild type.

[0046] 1.2.2 Genotype identification

[0047] Genotype analysis involves DNA extraction and PCR amplification. The CTAB method is used to extract single plant DNA from fresh young leaves to obtain single plant DNA.

[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 program 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] Among them, the upstream primer F: 5'-ATCATCTACGACTTCCTGCT-3' (SEQ ID NO.1); the downstream primer R: 5'-CTGGTCGATCTTGAGGTTGC-3' (SEQ ID NO.2).

[0052] Table 2 PCR reaction procedure

[0053]

[0054] The genotype of each strain was detected by combining 1% agarose gel electrophoresis and Sanger sequencing.

[0055] 1.2.3 Molecular marker development

[0056] In order to further speed up the identification of genotypes and increase the accuracy, and to facilitate the subsequent detection of genotypes in different inbred lines, Kasp molecular markers were designed for the SNP of the mutant Zmexo1, and the markers were sent to the company for sequencing verification.

[0057] The primers designed by the Kasp molecular marker are as follows:

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

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

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

[0061] Downstream primer for detecting mutants: Kasp-EXO1-R: 5'-CGTACTCGGGCTGGTGGAAC-3' (SEQ ID NO. 4).

[0062] That is, the wild-type upstream and downstream primers and mutant upstream and downstream primers shown in SEQ ID NO.3 to 5 are used to simultaneously detect the sample to be tested, and the genomic DNA is extracted from the fresh young leaves of the sample to be tested according to the CTAB method. The genomic DNA of the sample to be tested is used as a template and amplified according to the reaction system in Table 3 and the amplification program in Table 4 below. If only FAM fluorescence is detected in the sample to be tested, it is a homozygous wild-type plant; if both FAM fluorescence and HEX fluorescence are detected in the sample to be tested, it is a heterozygous wild-type plant; if only HEX fluorescence is detected in the sample to be tested, it is 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 to10μL

[0065] Table 4 PCR amplification procedure for genotype detection

[0066]

[0067] 2. Results and Analysis

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

[0069] Among them, the nucleotide sequence of ZmEXO1, i.e., Zm00001d018106 gene, is as follows:

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

[0071] The amino acid sequence of ZmEXO1, i.e., the Zm00001d018106 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, wherein a T at position 301 indicates a semi-dwarf compact plant type (reduced plant height and reduced leaf angle), and a C at position 301 indicates a wild type.

[0076] When the EMS allele mutant system was planted in the field, it was observed that the normal plant type and the mutant plant type with dwarfed plant height and reduced leaf angle were separated. The F1 offspring of the mutant Zmexo1 plants backcrossed to B73 all showed normal plant height and leaf angle plant type, and the F2 generation population after self-pollination showed separation of wild-type phenotype and mutant phenotype ( Figure 1 A).

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

[0078] To further verify the co-segregation of the Zmexo1 mutation site and phenotype, KASP primers were designed for the single base mutation site at amino acid position 450. The results of field sampling DNA identification showed that the genotype and phenotype were in accordance with the segregation ratio of 1:2:1 (homozygous wild type: heterozygous wild type: homozygous mutant). All phenotypes and genotypes corresponded to each other, indicating that the mutant phenotype and genotype of Zmexo1 were co-segregated and that the gene was involved in the establishment of maize plant type ( Figure 1 E).

[0079] Example 2

[0080] Cytological analysis of wild-type and Zmexo1 mutants in the pulvinar

[0081] 1. Materials and methods

[0082] 1.1 Transmission electron microscopy observation of cell wall in corn leaf pulvinus

[0083] The mutant in this example is the wild type in the F2 generation population after selfing in Example 1, and the mutant is the genotype homozygous mutant Zmexo1 in the F2 generation population after selfing in Example 1.

[0084] In order to investigate the cytological mechanism of the difference in leaf angle between wild type and homozygous mutant Zmexo1, transmission electron microscopy was performed on cells in the pulvinus. The fourth leaf that had not yet unfolded in the V2 period of maize was selected for transverse section sampling of the pulvinus. The tissue was fixed with 2% (v / v) glutaraldehyde in 50mM pH7.2 phosphate buffer at 4°C overnight. After fixation, the tissue was postfixed in 2% (v / v) OsO4 for 2h. After washing in phosphate buffer, the tissue was dehydrated in ethanol, infiltrated with Araldite / Embed 812 resin, and finally polymerized in Araldite / Embed 812 resin. Electron microscopy was performed using a Reichert-jung ultramicrotome to cut 90nm thick sections, mounted on formar-coated gold grids, and stained with uranyl acetate and lead citrate. The electron microscopy platform of Huazhong Agricultural University was completed using a HI TACHI, H-7650 scanning electron microscope. Cell size statistics were measured and calculated using Image J.

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

[0086] The solubility of different components of the cell wall is used to extract water-soluble pectin, ionic pectin, covalent pectin, soluble hemicellulose, insoluble hemicellulose and cellulose in steps, and then the concentration of the material components is measured by spectrophotometry; the determination of lignin content is usually based on the acid depolymerization method.

[0087] 1.2.1 Sample pretreatment

[0088] 1. Removal of soluble substances:

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

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

[0091] (3) Wash the residue with distilled water and dry it to 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 2% SDS and heat at 70°C for 30 min to remove lipids and proteins;

[0094] (2) Washing to neutrality and drying to obtain a crude cell wall extract, which is the pretreated sample.

[0095] 1.2.2 Determination of pectin content

[0096] 1. Extraction:

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

[0098] (2) Heat at 85°C for 1 h, stirring to promote dissolution;

[0099] (3) After centrifugation, the supernatant is collected to obtain supernatant 1, while the precipitate 1 is retained.

[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 and calculate the pectin content.

[0104] 1.2.3. Determination of hemicellulose content

[0105] 1. Extraction:

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

[0107] (2) Heat in a water bath at 80°C for 2 h with constant stirring;

[0108] (3) Collect the supernatant by centrifugation as the hemicellulose extract and retain the pellet 2.

[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 the absorbance at a wavelength of 540 nm;

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

[0113] 1.2.4 Determination of cellulose content

[0114] 1. Extraction:

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

[0116] (2) diluting the acid solution to 4%, heating in a water bath for 1 h, and then centrifuging to collect the cellulose supernatant;

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

[0118] The reducing sugar concentration in the supernatant of cellulose was determined by DNS method, which was the same as that of hemicellulose.

[0119] 1.2.5 Determination of lignin content

[0120] (1) Weigh 0.30 g of the leaf pulvinus sample, record it as W1, wrap it with filter paper, put it into a Soxhlet extractor and extract it with benzene-ethanol (67:33, v / v) for 4 h. The extracted powder is air-dried in a fume hood;

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

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

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

[0124] (5) The filtrate is made up to V = 250.0 mL, 1.0 mL is taken into a glass colorimetric tube, and diluted 10 times (depending on the sample) with 2.88% (v / v) sulfuric acid to ensure that the absorbance is between 0.2 and 0.7. The dilution factor is recorded as D. The OD value is measured by ultraviolet spectrophotometer with a wavelength of 205 nm and a blank of 2.88% sulfuric acid;

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

[0126] ASL%=A×D×V / (1000×K×W1)×100

[0127] A: absorption value; D: dilution factor; V: total volume of filtrate; K: absorption coefficient of acid-soluble lignin, taken as 110; W1: sample mass.

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

[0129] (7) The residue and the 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 in a Marsh furnace, preheat at 200°C for 30 min, then ash at 575±25°C for 4 h, take out and cool in a dryer for 30 min, and 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, we observed the pulvinus tissue of the wild-type and homozygous Zmexo1 mutants in the second fully expanded leaf at the seedling stage by transmission electron microscopy. We found that compared with the wild-type, the auricle cells of the Zmexo1 mutants were distorted in morphology, with reduced cell wall thickness and frequent breakage points, indicating that the homeostasis of the cell wall structure in the mutants was disrupted, suggesting that the composition of the cell wall may have changed ( Figure 2 A and B in the figure), so the wild type and Zmexo1 mutant were weighed with equal amounts of leaf pillows and dried and dehydrated to measure the components of the cell wall. The statistical results showed that compared with the wild type, the contents of the main components of the mutant cell wall were significantly reduced, including cellulose, hemicellulose, pectin, and lignin. The above results preliminarily revealed that the ZmEXO1 gene plays an important regulatory role in the composition and structural remodeling of the corn ear cell wall ( Figure 2 C).

[0138] Example 3: Genetic Improvement of Inbred Lines

[0139] Genetic improvement of different elite maize inbred lines using the SNP markers linked to the mutant trait developed in Example 1 (see Section 1.2.3 of Example 1)

[0140] 1. Materials and methods

[0141] 1.1 Test Materials

[0142] Semi-dwarf compact mutant Zmexo1 (B73 background), two elite maize inbred lines: PH6WC and PH4CV.

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

[0144] 1.2 Test methods

[0145] 1.2.1 Field trials

[0146] From 2023 to 2024, genetic improvement of corn inbred lines will be carried out using hybridization and molecular marker-assisted selection backcrossing methods; backcrossing and selection identification of each generation will be carried out in Sanya and Shenzhen respectively.

[0147] 1.2.2 Phenotypic identification

[0148] There were 10 plants in each 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 (10 days after corn flowering) of all individual plants were measured. The measured traits were measured three times and the average value was taken.

[0149] 1.2.3 Genotype analysis

[0150] Genotype analysis involves DNA extraction and PCR amplification. The DNA of a single strain 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] The semi-dwarf mutant Zmexo1 was used as the donor parent and the two superior inbred lines were used as recurrent parents. Backcrossing and selfing were performed to obtain the BC1F2 generation of (Zmexo1×PH6WC)×PH6WC (denoted as PH6WC Zmexo1 ) and (Zmexo1×PH4CV Zmexo1 )×PH4CV BC1F2 generation (denoted as PH4CV Zmexo1 ), and molecular marker assisted selection (MAS) was performed with the molecular markers obtained in Example 1 during backcrossing. At the same time, combined with the investigation of agronomic traits in the field and marker identification, inbred lines with improved leaf angle and plant height were initially obtained in the BC1F2 generation (with unimproved inbred lines PH6WC and PH4CV as controls, respectively); compared with unimproved inbred lines PH6WC and PH4CV, the improved line PH6WC Zmexo1 and PH4CV Zmexo1There were significant differences in leaf angle and plant height, two important plant-type-related traits (see Figure 3 ).

[0154] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A maize plant type regulating gene ZmEXO1, characterized in that: The amino acid sequence of ZmEXO1 is shown in SEQ ID NO.

7.

2. A nucleic acid molecule encoding the ZmEXO1 according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of ZmEXO1 is shown in SEQ ID NO.

6.

4. Use of ZmEXO1 according to claim 1 or the nucleic acid molecule according to claim 2 or 3 in regulating corn plant type.

5. The use according to claim 4, characterized in that: 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 corn plant height and reduce corn leaf angle.

6. A method for improving corn plant type, characterized in that: The method comprises the steps of reducing or silencing the expression level of ZmEXO1 according to claim 1 in a receptor plant.

7. The method according to claim 6, characterized in that The method for reducing or silencing the expression level of ZmEXO1 in claim 1 in a receptor plant comprises: using a mutant plant with reduced or silenced ZmEXO1 as a donor parent and an excellent corn inbred line as a recurrent parent, performing backcrossing and selfing to obtain a plant with improved corn plant type; the excellent corn inbred line comprises PH6WC or PH4CV.

8. A Kasp molecular marker tightly linked to the ZmEXO1 of claim 1, characterized in that: The Kasp molecular marker is C>T at position 4014 of the nucleotide sequence of ZmEXO1 described in claim 1, and the nucleotide sequence of the Kasp molecular marker is shown in SEQ ID NO.

8.

9. A Kasp primer tightly linked to the ZmEXO1 of claim 1, characterized in that: The nucleotide sequences of the Kasp primers are shown in SEQ ID NOs. 3-5.

10. Use of the Kasp molecular marker according to claim 8 or the Kasp primer according to claim 9 in screening or identifying corn plant types or identifying the ZmEXO1 genotype according to claim 1.

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