Proteins that regulate ear height in maize, their encoding genes, and their applications

Abstract

This invention discloses a protein and related biological materials, as well as the application of the protein, or substances regulating the expression of the protein-encoding gene, or substances regulating the activity or content of the protein, in regulating ear height and relative ear height in plants; the protein is a protein with the amino acid sequence SEQ ID No. 1. Mutant maize plants encoding this protein-encoding gene were obtained through the Chinese EMS mutant library. The results of identification and comparison between homozygous plants obtained by continuous self-pollination of the mutant lines and the maize inbred line B73 showed that the mutation of the target gene can reduce the ear height of the maize inbred line, thus proving that the gene Zm00001d011140 has an important biological function in the ear height phenotype.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically involving proteins that regulate ear height in maize, their encoding genes, and their applications. Background Technology

[0002] Maize (Zea mays L.) is one of the most important food, feed, and energy crops, and its production security plays an extremely important role in national food security. Ear height is a major factor influencing maize plant architecture; appropriately lowering ear height can improve lodging resistance and adaptability to higher planting densities, thus contributing to increased yield and production security. Therefore, identifying genes controlling maize plant architecture for basic research or molecular design breeding is of great significance for developing new maize varieties. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to quickly and accurately regulate the height of plant spikelets.

[0004] To solve the above-mentioned technical problems, the present invention provides the application of a protein or a substance that regulates the expression of the protein-encoding gene or a substance that regulates the activity or content of the protein, wherein the application may be any of the following:

[0005] D1) The application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in regulating the height of plant spikelets.

[0006] D2) The application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in the preparation of products that regulate the height of plant spikes.

[0007] D3) Application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in the cultivation of plants with low ear position.

[0008] D4) The use of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in the preparation of products that cultivate plants with low ear position.

[0009] D5) Application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in plant breeding.

[0010] The protein may be A1), A2), or A3):

[0011] A1) The amino acid sequence of this protein is that of SEQ ID No. 1;

[0012] A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1.

[0013] A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0014] The protein mentioned above is derived from corn.

[0015] SEQ ID No.1 consists of 2449 amino acid residues.

[0016] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0017] The protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag, and / or SUMO protein tag, etc.

[0018] Furthermore, in the aforementioned application, the protein is derived from corn.

[0019] Furthermore, in the aforementioned application, the substance regulating the expression of the protein-coding gene or the substance regulating the activity or content of the protein is a biological material, which may be any of the following:

[0020] B1) Nucleic acid molecules that encode the above proteins;

[0021] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0022] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0023] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0024] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0025] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0026] B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

[0027] Furthermore, in the above application, the nucleic acid molecule described in B1) can be any of the following DNA molecules shown in b1) to b3):

[0028] b1) The coding sequence of the coding strand is the DNA molecule shown in positions 130-7479 of SEQ ID No. 2;

[0029] b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 3;

[0030] b3) has 75% or more identity with the nucleotide sequence defined by b1) or b2) and is a DNA molecule encoding the protein of claim 1;

[0031] In the above applications, identity refers to the similarity between amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0032] In the above applications, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0033] Furthermore, the plant mentioned in the above applications is any one of the following P1)-P5):

[0034] P1) Monocotyledons;

[0035] P2) Plants of the order Poales;

[0036] P3) Gramineae plants;

[0037] P4) Plants of the genus *Zea*;

[0038] P5) Corn.

[0039] In the above applications, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the gene transcription level; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of gene translation; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0040] In the above applications, the regulation of gene expression can be achieved by inhibiting or reducing gene expression, which can be achieved by gene knockout or gene silencing.

[0041] Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by altering its DNA sequence.

[0042] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0043] In the above applications, the substance regulating gene expression can be a reagent that inhibits or reduces the expression of the gene. The reagent that inhibits or reduces the expression of the gene can be a gene knockout reagent, such as a reagent that knocks out the gene through homologous recombination or a reagent that knocks out the gene through CRISPR-Cas9. The reagent that inhibits or reduces the expression of the gene can contain a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0044] To address the aforementioned technical problems, the present invention also provides the aforementioned protein and / or biological material.

[0045] To address the aforementioned technical problems, the present invention also provides a method for reducing the ear height of maize, the method comprising F1) or F2):

[0046] F1) Reduce ear height by knocking out the genes encoding the above proteins in maize;

[0047] F2) Maize with the genotype GG at 140,251,860 bp of the maize genome V4 version was selected as the parent for breeding.

[0048] Furthermore, in the method, knocking out the gene encoding the protein in maize can be achieved by performing at least one of the following mutations on the DNA molecule in maize with the nucleotide sequence shown in SEQ ID No. 3:

[0049] M1) The cytosine C at position 5936 of sequence 3 in the maize genome is mutated to thymine T. This mutation causes the cytosine C at position 3682 of sequence 2 in the sequence listing of the gene encoding the above protein to be mutated to thymine T. This mutation changes the codon tgg in the coding sequence to tga, thereby causing the coding protein to terminate prematurely.

[0050] M2) The guanine G at position 2813 of sequence 3 in the maize genome is mutated to adenine A. This mutation causes the guanine G at position 2727 of sequence 2 in the sequence listing of the gene encoding the above protein to be mutated to adenine A. This mutation changes the codon tgg in the coding sequence to tga, thereby causing the coding protein to terminate prematurely.

[0051] In the above method, the corn may include a maize inbred line.

[0052] In the above methods, the knockout can be performed using EMS mutation or a CRISPR / Cas9 system.

[0053] The present invention also provides the application of the above method in the creation of maize mutant plants and / or maize breeding.

[0054] Based on EMS mutation, mutant maize plants encoding the above-mentioned protein were obtained. The results of phenotypic identification and comparison between the homozygous plants obtained by self-pollination of the mutant lines and the wild type showed that the Zm00001d011140 gene mutation can reduce the ear height of maize inbred lines, thus proving that the gene Zm00001d011140 has an important biological function in the ear height phenotype of maize.

[0055] Compared with the prior art, the present invention has the following advantages:

[0056] (1) After gene mutation of the gene encoding the Zm00001d011140 protein of the present invention, the ear height of maize was significantly reduced.

[0057] (2) This invention provides a more precise, efficient and safe technical method for creating maize mutant plants and / or for maize breeding, and realizes the precise improvement of ear height, which can promote the commercial maize breeding process, overcome the shortcomings of traditional breeding, shorten the breeding cycle and not affect other traits. Attached Figure Description

[0058] Figure 1 This is the locus where EP was located on chromosome 8.

[0059] Figure 2 A comparison of ear height phenotypes between 468 inbred lines with haplotype AA and 1101 inbred lines with haplotype GG.

[0060] Figure 3 The sequence alignment results are for the target region of the EMS mutant.

[0061] Figure 4 The Zm00001d011140 gene mutant material and its ear height and relative ear height phenotype. Detailed Implementation

[0062] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0064] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0065] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0066] The maize inbred line B73 was obtained from the National Germplasm Resource Bank.

[0067] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0068] Example 1: Identification of the Target Gene

[0069] 1.1 Determination of ear height and genome-wide association analysis in maize

[0070] 1604 maize inbred lines were planted in the field using a completely randomized block design with two replicates. Each line was planted in a single row, 3 meters long and 0.6 meters wide, with 13 maize plants per row. Plant height (PH) and ear height (EH) at maturity were measured, and the average of all measurements within the plot was taken, in cm. The relative ear height was calculated using the formula: EP = EH / PH.

[0071] Plant height (PH) and ear height (EH) of mature maize were measured. The average value of the measurement data of all plants in the plot was taken, and the plant height and ear height data were used to calculate the value.

[0072] 1.2 Identification of the gene Zm00001d011140 related to ear height in maize

[0073] Genome-wide association analysis identified a distinct signal peak on chromosome 8 controlling ear height and relative ear height, such as... Figure 1 As shown, the SNPs significantly associated with relative ear height are located at 140, 251, 860 bp (maize genome V4 version), and this associated site is located at 862 bp in the promoter of the Zm00001d011140 gene.

[0074] Further haplotype analysis of the 1604 materials used in this study revealed two distinct haplotypes. The AA haplotype showed significantly higher ear height and relative ear height compared to the GG haplotype. From these two haplotypes, materials carrying the AA haplotype (Chang 7-2) and the GG haplotype (Zheng 58) were selected for qRT-PCR to detect the relative expression level of the Zm00001d011140 gene in different haplotype materials. The results showed that the relative expression level of the Zm00001d011140 gene in the higher ear-height material (Chang 7-2) was significantly higher than that in the lower ear-height material (Zheng 58). Figure 2 As shown in Figure B. Therefore, we believe that Zm00001d011140 is a candidate gene controlling ear height. Based on this, we conducted functional verification of this gene.

[0075] The genomic sequence of the Zm00001d011140 gene is a DNA molecule as shown in SEQ ID No. 3, its coding sequence is a DNA molecule as shown in SEQ ID No. 2, and its encoded amino acid sequence is a protein as shown in SEQ ID No. 1. The encoded protein is named Zm00001d011140 protein or protein Zm00001d011140.

[0076] Example 2: Amplification and recovery sequencing of the Zm00001d011140 genome and its coding genes.

[0077] 2.1 Obtaining the full-length genome and cDNA of candidate genes

[0078] 2.1.1 Preparation of plant materials required for amplifying candidate genes

[0079] Select plump maize inbred line B73 seeds and plant them in flowerpots filled with nutrient soil, with 3 seedlings per pot. Place them in a light incubator (28℃, light). When the seedlings reach the six-leaf-one-heart stage, collect leaves. Use some leaves to extract genomic DNA, and use the other part of the leaves to extract RNA, which is then stored at -80℃.

[0080] 2.1.2 Extraction of genomic DNA from maize materials

[0081] 1) Quickly place the leaves into a sterilized mortar and grind them thoroughly with liquid nitrogen, adding liquid nitrogen continuously during the process. After grinding, add the liquid nitrogen to a 2.0ml centrifuge tube, filling the tube to 1 / 3 full.

[0082] 2) Preheat the pre-prepared CTAB extraction solution in a 65°C water bath. Then add 800 μl of preheated CTAB buffer to the centrifuge tube from the previous step and shake vigorously to mix thoroughly.

[0083] 3) Place the mixed extract in a 65℃ constant temperature water bath for 30 minutes, shaking it every 10 minutes to ensure a full reaction.

[0084] 4) Take out the centrifuge tube that has undergone sufficient reaction, add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1), mix slowly for 15 min, and let stand for 10 min.

[0085] 5) Place the centrifuge tubes into the centrifuge and centrifuge at 12,000 rpm for 20 minutes.

[0086] 6) Carefully aspirate the supernatant into another clean 2.0 ml centrifuge tube, add an equal volume of pre-cooled (-20℃) isopropanol, gently mix, and place the centrifuge tube in a -20℃ freezer for 30 minutes until a large amount of white precipitate forms.

[0087] 7) Use a sterilized pipette tip to remove the white precipitate and place it in another clean centrifuge tube. Add 500 μL of 75% ethanol and rinse 2-3 times.

[0088] 8) Centrifuge, discard 75% ethanol, remove excess ethanol with a pipette tip, and dry at room temperature. Dissolve the DNA in 1×TE.

[0089] 9) Add RNase for purification (final concentration 10ug / ul), and incubate at 37℃ for 1 hour.

[0090] 10) Add an equal volume of phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) for extraction once, centrifuge at high speed (12000 rpm) for 10 min, carefully aspirate the supernatant, and then extract once more with an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1), centrifuge at high speed (12000 rpm) for 10 min.

[0091] 12) Precipitate the DNA with pre-cooled anhydrous ethanol and centrifuge (12000 rpm) for 10 min.

[0092] 13) Discard the ethanol, let it dry completely to avoid inhibiting the downstream PCR reaction, add an appropriate amount of TE to dissolve, and store in a -20℃ refrigerator for later use.

[0093] 2.1.3 Extraction and purification of total RNA from maize materials

[0094] Total RNA was extracted from plants using the Gene-better Polysaccharide-Polyphenol Plant Total RNA Extraction Kit (with gDNA filter). The specific procedure is as follows (all experimental instruments used below were sterilized at high temperature to remove RNase):

[0095] 1) Pour liquid nitrogen into an RNase-free mortar that has been treated at 180℃, then take out the frozen leaves from the -76℃ ultra-low temperature freezer, add liquid nitrogen and grind them thoroughly, continuously replenishing liquid nitrogen during the process until they are fully ground.

[0096] 2) Before adding the sample to the centrifuge tube, immerse the 1.5 ml RNase-free centrifuge tube completely in liquid nitrogen for quick freezing. Then quickly add the ground sample. Do not add too much sample, generally add to 1 / 3 (about 50 mg). Add 500 μL of lysis buffer and 50 μL of PLANTaid to the centrifuge tube and vortex for 20 seconds to ensure complete lysis.

[0097] 3) Centrifuge the lysate at 13,000 rpm for 10 minutes to precipitate unlysed fragments and PLANTaid bound with polysaccharides and polyphenols.

[0098] 4) Transfer the supernatant of the lysate to a new centrifuge tube. Add an equal volume of anhydrous ethanol to the supernatant and immediately mix by pipetting; do not centrifuge.

[0099] 5) Add the mixture (less than 720 μL each time, which can be added in two batches) to a genomic DNA filter, centrifuge at 13,000 rpm for 2 min, and discard the waste liquid.

[0100] 6) Place the genomic DNA filter in a clean 2ml centrifuge tube, add 500ul of lysis buffer RLT PLUS to the filter, centrifuge at 13000rpm for 30s, collect the filtrate, estimate the filtration volume accurately with a pipette, and add 0.5 times the volume of anhydrous ethanol. Precipitation may occur at this time, but it will not affect the extraction process. Immediately mix by pipetting and do not centrifuge.

[0101] 7) Immediately add the mixture to an adsorption column RA, centrifuge at 13000 rpm for 2 min, and discard the waste liquid.

[0102] 8) Add 700 μL of protein removal solution RW1, let stand at room temperature for 1 min, centrifuge at 13000 rpm for 30 s, and discard the waste liquid.

[0103] 9) Add 500 μL of wash buffer RW, centrifuge at 13000 rpm for 30 seconds and discard the waste liquid. Add 500 μL of wash buffer RW again and repeat once. Place the adsorption column RA back into the empty collection tube, centrifuge at 13000 rpm for 2 minutes to remove as much wash buffer as possible to avoid residual ethanol in the wash buffer inhibiting the downstream reaction.

[0104] 10) Take out the RA adsorption column, put it into an RNase-free centrifuge tube, add 30-50 μL of RNase-free water in the middle of the adsorption membrane, let it stand at room temperature for 1 minute, and centrifuge at 13,000 rpm for 1 minute.

[0105] The quality of the extracted RNA was determined by 1% agarose gel electrophoresis for 18 min, and the concentration of the extracted RNA was determined by spectrophotometer. An appropriate amount of RNA was used for purification and reverse mixing, and the remaining RNA was stored at -76℃ for long-term storage.

[0106] 2.1.4. Reverse transcription to synthesize first-strand cDNA

[0107] Using the total RNA extracted in section 2.1.3 as a template, cDNA was synthesized by reverse transcription according to the Transgen One-Step gDNA Removal and cDNA Synthesis SuperMix manual. The procedure is as follows:

[0108] 20 μL reverse transcription reaction mixture (placed in a 0.2 mL RNase-free centrifuge tube)

[0109]

[0110] Incubate at 50℃ for 30 min, then heat at 85℃ for 5 s to inactivate the enzyme, and quickly transfer to ice. Dilute the cDNA stock solution 5-fold, perform PCR amplification using the internal control gene GADPH, check the reverse conversion efficiency, and store at -20℃ for later use.

[0111] 2.1.5 Amplification of candidate genomic DNA

[0112] Download the candidate gene sequence corresponding to B73 of the reference genome from the Maize GDB database (https: / / www.maizegdb.org / ). Primers were designed using Primer5 software in the 5'UTR and 3'UTR regions of the genome. The full-length candidate genome DNA was amplified using the genomic DNA extracted in section 2.1.2 as a template. The amplification system is as follows:

[0113]

[0114] The primer sequences are as follows:

[0115] Primer 1 5'-GCCGGCTTTGCAGAGATCGAC-3';

[0116] Primer 2 5'-TTGCACAACAGCTTCCATAGA-3';

[0117] Primer 3 5'-GTCCATCCATATAGCTGCGAAA-3'

[0118] Primer 4 5'-ATGCACAATTAAGCATTCACTG-3';

[0119] Primer 5 5'-GTCAAGTGTTGTTAAGCTGATAC-3';

[0120] Primer 6 5'-GCAAGCAGAACAGAGTCGCTCT-3';

[0121] Primer 7 5'-GCGTCTACACTCATATGACT-3';

[0122] Primer 8 5'-GTCTACAATCCTATCACAGACAT-3';

[0123] Primer 9 5'-CATGTCTATTTCCATTTCCCGA-3';

[0124] Primer 10 5'-GCAATTAGACAAAAGCCACGTAT-3';

[0125] Primer 11 5'-GATCTGGAGGGCCTTGGAG-3';

[0126] Primer 12 5'-CTAACACCAGCTTCATGTTCA-3';

[0127] Primer 13 5'-GCTTCTGTGTGGGTTCGG-3';

[0128] Primer 14 5'-TCGACGCTAAATGAACTCTGTCC-3';

[0129] Primer 15 5'-CAAACTTTACTGCAACCAGT-3';

[0130] Primer 16 5'-TCTTCAATCCGTGCTTGGGTT-3';

[0131] Primer 17 5'-GTTGCCTGCAGTCCAATCGC-3';

[0132] Primer 18 5'-ATCCATCGCATTTTCTAGCAT.

[0133] The PCR amplification procedure is as follows:

[0134]

[0135] 2.1.6 Amplification of full-length cDNA of candidate genes

[0136] Primers were designed based on the amplified genomic DNA sequence. Using the cDNA obtained in section 2.1.4 as templates for gene amplification, primers were designed to amplify the full-length cDNA sequence of the gene. The amplification system is as follows:

[0137]

[0138]

[0139] The primer sequences are as follows:

[0140] Primer 19 5'-ATGGGGGAGCGAGACGCG-3';

[0141] Primer 20 5'-CAGAAGGTGCCCAACACAAAC-3';

[0142] Primer 21 5'-CTCAACTCTCATACCGACAATGC-3';

[0143] Primer 22 5'-GTCAAGGAGCTCAACAATGTCTG-3';

[0144] Primer 23 5'-AACAACATTTCTACCAAGGGGT-3';

[0145] Primer 24 5'-CATGCAGTAGAATTGGTGTCCT-3';

[0146] Primer 25 5'-TGAATGTCACCCTCTCTGCC-3';

[0147] Primer 26 5'-CTCAAGTTGTTGCTGGAGGATCA-3';

[0148] Primer 27 5'-GCATTTGGAATTCCAGGCTAT-3';

[0149] Primer 28 5'-TTAACGCTGATATGCTGCAGCG-3';

[0150] The PCR amplification procedure is as follows:

[0151]

[0152] 2.2 Recovery and sequencing of candidate genomic DNA and cDNA fragments

[0153] 2.2.1 Recovery of candidate genomic DNA and cDNA fragments

[0154] For fragment recovery, refer to the instruction manual of the FastPure Gel DNAExtraction Mini Kit from Beijing Novizan Biotechnology Co., Ltd.:

[0155] 1) Add 1g of agarose to 100ml of TAE solution, heat to boiling in a microwave oven, then add 10ul of 10000×GelStain dye, pour into a plate with a comb, and prepare a 1% agarose gel. Perform agarose gel electrophoresis (125v) on the PCR products of 2.1.5 and 2.1.6 with loading buffer added for 25min. Under long-wave ultraviolet light, cut the target bands and put them into 1.5ml centrifuge tubes.

[0156] 2) Add 400 μL of Buffer GDP to the centrifuge tube for sol-gel preparation.

[0157] 3) Place the centrifuge tubes in a 55°C water bath and incubate. Invert the tubes every 5 minutes to mix until the gel is completely melted and the solution turns pale yellow. Allow the solution to stand at room temperature until it cools down to room temperature before proceeding to the next step of the reaction.

[0158] 4) Transfer the solution from the centrifuge tube in the previous step into the adsorption column, put the centrifuge tube into the centrifuge, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and then put the adsorption column back into the empty collection tube.

[0159] 5) Add 300 μL of Buffer GDP to the adsorption column, let stand for 1 min, add the centrifuge tube to the centrifuge, centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0160] 6) Place the adsorption column in the collection tube, add 700 μL of Buffer GW to the adsorption column, centrifuge at 12,000 rpm for 30 seconds; discard the filtrate. Wash twice.

[0161] 7) Place the adsorption column back into the empty collection tube and centrifuge at 12,000 rpm for 2 minutes.

[0162] 8) Remove the adsorption column and place it in a clean 1.5ml sterile centrifuge tube. Let it stand at room temperature for 5 minutes. Add 25ul of elution buffer to the middle of the adsorption membrane. Let it stand at room temperature for 5 minutes. Centrifuge at 12000rpm for 2 minutes and collect the precipitate.

[0163] 9) Repeat step 8.

[0164] 2.2.2 Sequencing of candidate genomic DNA and cDNA fragments

[0165] Referring to the instructions for Tiangen Biotech's pLB zero-background rapid cloning kit, the operating steps are as follows:

[0166] 1) Ligate the recovered DNA fragment obtained in 2.2.1 to the pLB vector, and gently tap the centrifuge tube to mix the reaction solution. The ligation system is as follows:

[0167]

[0168] 2) Place the mixed reaction solution in a 22°C constant temperature metal bath and react for 15 minutes. After the reaction is complete, place the centrifuge tube on ice for subsequent conversion experiments.

[0169] 3) Prepare LB agarose plates containing ampicillin at a final concentration of 100 ug / ml, and place the plates at 37℃ for 20 min.

[0170] 4) Add 10 μL of the ligation product to 100 μL of E. coli TOP10 competent cells (the competent cells were taken out of the -80℃ freezer and placed on ice. The ligation product was added when the cells were just thawed). Gently tap the cells to mix. Incubate on ice for 30 min.

[0171] 5) Then place the centrifuge tubes in a 42°C water bath for 90 seconds, and immediately place them in an ice bath for 5 minutes.

[0172] 6) Add 500 μL of LB (antibiotic-free) medium to the centrifuge tube and incubate at 150 rpm and 37°C for 60 min with shaking to allow the bacteria to recover.

[0173] 7) Mix the bacterial culture in the centrifuge tube thoroughly, add 100 μL to LB solid agar medium containing ampicillin, and gently spread the bacterial culture with a sterile bent glass rod. After the surface of the plate is dry, invert the plate and incubate at 37°C for 12 hours.

[0174] 8) Remove the plate, pick the milky white positive single clones onto 600 μl of LB liquid medium (containing 100 ug / ml ampicillin antibiotic), and incubate at 37℃ with shaking for 8 h. Perform bacterial PCR using pLB vector universal primers. The clones identified as positive by agarose gel electrophoresis are sequenced for verification, and the plasmids are extracted for subsequent experiments.

[0175] The universal primer sequences for the pLB vector are as follows:

[0176] Forward Primer: 5'-CGACTCACTATAGGGAGAGCGGC-3';

[0177] Reverse Primer: 5'-AAGAACATCGATTTTCCATGGCAG-3'.

[0178] The amino acid sequence, coding sequence, and genomic sequence of the Zm00001d011140 protein are shown in Table 1.

[0179] Table 1:

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] Example 3: Functional Verification of the Zm00001d011140 Gene

[0190] 3.1 Obtaining the Zm00001d011140 gene mutant maize

[0191] Through the China EMS Mutant Library, the website is ( https: / / www.elabcaas.cn / memd / public / index.html# / pages / search / geneid By searching for the gene number Zm00001d011140, the relevant mutants of this gene were found to be EMS-1 and EMS-2.

[0192] The EMS-1 mutant has a mutation at 140258537bp on chromosome 8 (maize genome V4 version), where cytosine C is mutated to thymine T (i.e., cytosine C at position 5936 of sequence 3 in the sequence listing is mutated to thymine T, resulting in the coding sequence, i.e., cytosine C at position 3682 of sequence 2 in the sequence listing is mutated to thymine T). This mutation changes the codon cga to tga, thereby causing premature termination of the encoded protein.

[0193] The EMS-2 mutant has a mutation at 140255414bp on chromosome 8 (maize genome V4 version), where guanine G is mutated to adenine A (i.e., guanine G at position 2813 of sequence 3 in the sequence listing is mutated to adenine A, resulting in the coding sequence, i.e., guanine G at position 2727 of sequence 2 in the sequence listing is mutated to adenine A). This mutation changes the codon tgg to tga, thus causing premature termination of the encoded protein.

[0194] Partial genomic sequences of the EMS-1 and EMS-2 mutants are shown in Table 2:

[0195] Table 2:

[0196]

[0197] 3.2 Genotype and Phenotype of the Zm00001d011140 mutant line

[0198] Seeds of maize inbred line B73(WT), and seeds of B73 mutant lines EMS-1 and EMS-2 were planted in two rows for each material, with a row length of 3 meters, a plant spacing of 0.25 meters, a row spacing of 0.6 meters, and three replicates.

[0199] Leaves of B73 and EMS-1 and EMS-2 mutant materials were collected for DNA extraction. DNA extraction was performed using the same method as described in 2.1.2. The Zm00001d011140 gene mutant plants were detected at the DNA level. Genomic DNA was extracted from the mutant plants and used as a template. Single-plant PCR identification of the EMS-1 mutant was performed using forward prime3 and reverse prime3 primers, while single-plant PCR identification of the EMS-2 mutant was performed using forward prime4 and reverse prime4 primers. Wild-type plant genomic DNA and ddH2O served as negative controls. The reaction system is as follows:

[0200]

[0201] The nucleotide sequences of Forward prime3 and Reverse prime3 are as follows:

[0202] Forward prime3:5'-CTTAGGGTGATGGACGATTT-3';

[0203] Reverse prime3:5'-AGTGGCTATGTTGGTCTGTTA-3';

[0204] Forward prime4:5'-GGAAGAAGGTAAGGGAACT-3';

[0205] Reverse prime4:5'-GGGAAATGGGAAATAGACA-3'.

[0206] The amplification reaction program was as follows: Round 1: denaturation at 95℃ for 5 min; Round 2: denaturation at 95℃ for 10 sec, annealing at 60℃ for 15 sec, extension at 72℃ for 30 sec, 35 cycles; Round 3: extension at 72℃ for 5 min. After the program, the target band was recovered and sequenced by 2.0% agarose gel electrophoresis. The sequencing results are as follows: Figure 3As shown. The sequencing results of the EMS mutant were compared with those of wild-type B104, and the results were consistent. Compared with wild-type B104:

[0207] The EMS-1 mutant mutates cytosine C at position 5936 of sequence 3 in the sequence listing to thymine T, resulting in a mutation of cytosine C to thymine T at position 3682 of sequence 2 in the coding sequence. This mutation changes the codon cga to tga, thus causing premature termination of the encoded protein.

[0208] The EMS-2 mutant mutates by changing guanine G at position 2813 of sequence 3 in the sequence listing to adenine A, resulting in a change of guanine G to adenine A at position 2727 of sequence 2 in the coding sequence. This mutation changes the codon tgg to tga, thus causing premature termination of the encoded protein.

[0209] The ear height of homozygous EMS-1 and EMS-2 mutant lines was statistically analyzed. The results showed that, compared to wild-type B73, the ear height and relative ear height of the homozygous EMS-1 and EMS-2 mutant lines were decreased. Figure 4 ). Figure 4 Image A shows the ear height phenotype of homozygous EMS-1 and EMS-2 mutant lines; Figure 4 Figure B shows the statistical results and significance analysis of ear height and relative ear height of maize homozygous lines of EMS-1 and EMS-2 mutants. The results indicate that mutation of the Zm00001d011140 gene can significantly reduce ear height and relative ear height of maize; thus proving that the target gene has an important biological function in regulating ear height of maize.

[0210] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of a protein, a biological material regulating the expression of the protein-encoding gene, or a biological material regulating the activity or content of the protein, characterized in that: The application is any one of the following: D1) Application in lowering the corn ear position in high-altitude regions; D2) Application in the preparation of reagents that lower the ear height of maize; D3) Application in cultivating maize with low ear position; D4) Application in the preparation of reagents for cultivating maize with low ear position; Application of D5 in maize breeding; The protein is either A1 or A2 as follows: A1) A protein with the amino acid sequence shown in SEQ ID No. 1; A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1); The biomaterial is any one of the following: B1) The nucleic acid molecule that encodes the protein; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1) or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1) or transgenic plant organs containing the expression cassette described in B2).

2. The application according to claim 1, characterized in that: The protein is derived from corn.

3. The application according to claim 1 or 2, characterized in that: B1) The nucleic acid molecule described below is a DNA molecule as shown in b1) or b2): b1) The coding sequence of the coding strand is the DNA molecule shown in positions 130-7479 of SEQ ID No. 2; b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No.

3.

4. A method for reducing the height of corn ears, characterized in that: The method reduces ear height by knocking out the gene encoding the protein described in claim 1 in maize.

5. The method according to claim 4, characterized in that, The method for knocking out the gene encoding the protein in claim 1 in maize involves performing at least one of the following mutations on a DNA molecule in maize with the nucleotide sequence shown in SEQ ID No. 3: M1) Mutate cytosine C to thymine T at position 5936 of sequence 3 in the maize genome; M2) mutates guanine G at position 2813 of sequence 3 in the maize genome to adenine A.

6. The application of the method according to claim 4 or 5 in the preparation of maize mutant plants and / or maize breeding.

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