ZmXT6 protein regulating maize husk number, its encoding gene, and its application in regulating maize husk number

Abstract

The present invention discloses the ZmXT6 protein, encoding gene and its application in regulating the number of corn bracts. The present invention obtains mutant corn plants with the encoding gene of the ZmXT6 protein knocked out through a knockout experiment based on the CRISPR-Cas9 system. The homozygous plants obtained by self-pollination of positive plants and the phenotypic identification results of the wild type show that the corn inbred line with the encoding gene of the ZmXT6 protein knocked out has more bracts, which shows that the gene encoding the ZmXT6 protein has an important biological function in regulating the number of bracts. Therefore, it is feasible for the present invention to apply the ZmXT6 protein and the gene encoding the ZmXT6 protein to regulate the number of corn bracts. It can not only provide a new gene resource for improving the number of corn bracts, but also provide a new direction for improving the number of corn bracts and its breeding from the molecular level.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering technology, in particular to a method for regulating the number of corn husks. ZmXT6 Protein, encoding gene and its application in regulating maize husk number. Background Art

[0002] Corn is the largest grain crop cultivated in my country, with a total area of ​​650 million mu (approximately 1.5 million hectares). Mechanical corn harvesting is a key measure for achieving efficient corn production and reducing costs. However, the corn varieties currently promoted in my country generally suffer from slow kernel dehydration and high moisture content at harvest, severely hindering the promotion and application of mechanical corn harvesting. Factors influencing corn moisture content during maturity include the maturity of the variety, the characteristics of the cob husk, and the dehydration rate of the kernels during the late filling phase. The husk, the tissue that encases the female ear, directly influences cob dehydration and indirectly affects kernel moisture loss, making it a key trait that must be prioritized in corn breeding for high yield and suitability for mechanical harvesting. Previous studies have shown that the morphological and structural characteristics of the corn husk are the most direct factor affecting the dehydration rate of the corn cob during the late filling phase. Fewer husks are more conducive to kernel dehydration.

[0003] The corn husk also serves as a protective organ for the corn ear, providing a suitable environment for ear development and kernel filling. First, the husk maintains a suitable temperature for kernel development. Especially in the late growth period of corn, when frost damage may occur before harvest, the husk prevents heat loss and effectively mitigates frost damage caused by falling temperatures. The husk prevents pests and diseases from entering the ear, reducing or preventing their occurrence and protecting the kernels from bird predation. Thus, the husk plays a crucial role in corn ear development. However, no research has yet been conducted on regulating husk number at the molecular level. Summary of the Invention

[0004] Therefore, based on the above background, the present invention has discovered genes related to the number of corn bracts, and applied them to regulate the number of corn bracts, so as to provide a new direction for the improvement of the corn bract number trait and its breeding at the molecular level.

[0005] The technical solutions of the present invention are as follows:

[0006] One of the objects of the present invention is to provide:

[0007] Regulating the number of corn husks ZmXT6 Protein, ZmXT6 The protein is at least one selected from A1) to A3):

[0008] A1) a protein with the amino acid sequence shown in SEQ ID No. 1;

[0009] A2) a protein having at least 80% identity to the protein of A1) and having the same function as the protein of A1) obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence of SEQ ID No. 1;

[0010] A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0011] above ZmXT6 Protein comes from corn.

[0012] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0013] A protein tag is a polypeptide or protein that is fused with a target protein using in vitro DNA recombination techniques to facilitate expression, detection, tracing, and / or purification of the target protein. Examples of protein tags include Flag, His, MBP, HA, myc, GST, and / or SUMO tags.

[0014] A second object of the present invention is to provide:

[0015] ZmXT6 A protein-encoding gene, wherein the coding gene is a DNA molecule shown in any one of the following items b1) to b3):

[0016] b1) a DNA molecule having a coding sequence of the coding strand as shown in SEQ ID No. 2;

[0017] b2) a DNA molecule whose nucleotide sequence is shown in SEQ ID No. 3;

[0018] b3) having 75% or more identity with the nucleotide sequence defined in b1) or b2) and encoding the above-mentioned ZmXT6 Protein DNA molecules.

[0019] In the above, identity refers to amino acid sequence or nucleotide sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, the blastp program can be used with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained.

[0020] In the above applications, the above 75% 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.

[0021] The third object of the present invention is to provide:

[0022] Biological material, the biological material is a substance that regulates the expression of the coding gene, or regulates the ZmXT6 A substance that measures protein activity or content.

[0023] In the above, the substance that regulates the expression of the coding gene may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the cell nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0024] Furthermore, the biological material is any one of the following B1) to B3):

[0025] B1) the nucleic acid molecule;

[0026] B2) Inhibit or reduce the above ZmXT6 Nucleic acid molecules expressed by protein-coding genes;

[0027] or inhibit or reduce the ZmXT6 Nucleic acid molecules that are active for proteins;

[0028] B3) An expression cassette, a recombinant vector, a recombinant microorganism, or a transgenic plant cell line containing the nucleic acid molecule described in B2).

[0029] Furthermore, the nucleic acid molecule in B2) is a DNA molecule that expresses a gRNA targeting the protein-coding gene shown in A1), or is a gRNA targeting the protein-coding gene described in A1).

[0030] A fourth object of the present invention is to provide:

[0031] The ZmXT6 The protein, or the application of the biomaterial, wherein the application includes any one of the following D1) to D5):

[0032] D1) Application in regulating the number of corn husks;

[0033] D2) Application in the preparation of products for regulating the number of corn husks;

[0034] D3) Use in the preparation of products for cultivating corn with a high number of husks;

[0035] D4) Application in identifying, screening, or assisting in breeding corn plants, lines, or varieties with a high number of bracts;

[0036] D5) Application in corn breeding or assisted breeding to increase the number of bracts.

[0037] Furthermore, in D1) and D2), the number of corn husks is regulated by gene knockout or gene silencing to inhibit or reduce ZmXT6 The expression of protein-encoding genes increases the number of corn husks.

[0038] Gene knockout refers to the inactivation of a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene through changes in the DNA sequence.

[0039] Specifically, the gene knockout is targeted knockout of the above-mentioned ZmXT6 Protein encoding gene, and obtain knockout mutant plants.

[0040] The CRISPR / Cas9 system includes expressing the target ZmXT6 Protein-encoding gene knockout vector.

[0041] The nucleotide sequence of the knockout target is shown in SEQ ID No. 5:

[0042] TGGAAGATAACCCTGTTGGG.

[0043] Gene silencing refers to the phenomenon of suppressing or under-expressing a gene without damaging the original DNA. Gene silencing prevents or reduces gene expression without altering the DNA sequence. Gene silencing can occur at two levels: transcriptional gene silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates a gene by specifically inhibiting target RNA after gene transcription. This includes antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational inhibition.

[0044] In the above application, the regulation of ZmXT6 Substances that inhibit or reduce the expression of protein-coding genes ZmXT6 An agent that inhibits or reduces expression of a gene encoding a protein ZmXT6 The agent for inhibiting gene expression of a protein may be an agent for knocking out the gene, such as an agent for knocking out the gene by homologous recombination, or an agent for knocking out the gene by CRISPR-Cas9. The agent for inhibiting or reducing gene expression may comprise a polynucleotide targeting the gene, such as siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0045] The fifth object of the present invention is to provide:

[0046] A method for cultivating corn with a high number of bracts, the specific steps of which are as follows:

[0047] Selected encoding ZmXT6 The nucleic acid molecules of proteins are used as knockout targets to construct CRISPR / Cas9 The vector was knocked out, and mutants were obtained through Agrobacterium-mediated transformation. After screening, corn strains with a high number of bracts were cultivated.

[0048] The fifth object of the present invention is to provide:

[0049] A corn breeding method, during breeding, the corn with the high bract number trait obtained above is used as the germplasm parent.

[0050] The above technical solution has the following beneficial effects:

[0051] The present invention obtained knockout results through a knockout experiment based on the CRISPR-Cas9 system. ZmXT6The results of phenotypic identification of the homozygous plants obtained by self-pollination of positive plants and the wild type showed that the corn inbred lines with the ZmXT6 protein coding gene knocked out had more bracts, which indicated that the ZmXT6 protein coding gene had an important biological function in regulating the number of bracts.

[0052] Therefore, the present invention will ZmXT6 It is feasible to use proteins and their encoding genes to regulate the number of corn husks. It can not only provide new genetic resources for improving the husk number trait of corn, but also provide a new direction for improving the husk number trait of corn and its breeding at the molecular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0054] Figure 1 These are the results of association analysis of bract number and its haplotype analysis.

[0055] Figure 2 Schematic diagram of the conserved protein structure.

[0056] Figure 3 Schematic diagram of the construction of the recombinant vector CPB-sgRNA and the vector map of the recombinant vector.

[0057] Figure 4 The genotype and phenotype of the mutant plants.

[0058] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0059] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0060] The present invention will be further described below with reference to the accompanying drawings.

[0061] The maize B104 used in the following examples is described in "Char, SN, Neelakandan, AK, Nahampun, H., Frame, B., Main, M., Spalding, MH, Becraft, PW, Meyers, BC, Walbot, V., Wang, K., Yang, B. (2016). An Agrobacterium-delivered CRISPR / Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnology Journal, 15(2), 257-268." Available from the US National Plant Germplasm System (https: / / npgsweb.ars-grin.gov / gringlobal / search).

[0062] The CPB vector was provided by Xie Chuanxiao's research group at the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, and is disclosed in the document "Zhao, Y., Zhang, C., Liu, W. et al. An alternative strategy for targeted gene replacement in plants using a dual-sgRNA / Cas9 design. Sci Rep 6, 23890(2016)." The public can obtain the above-mentioned biological material from the applicant. The obtained biological material is only for use in repeating the experiments of the present invention and cannot be used for other purposes.

[0063] Fast-T1 Escherichia coli competent cells were purchased from Nanjing Novozymes Biotech Co., Ltd. with the product number C505-03.

[0064] EHA105 Agrobacterium competent cells were purchased from Beijing Biomed Gene Technology Co., Ltd. with the product number BC303.

[0065] The following examples use Excel 2020 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. T test is used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates an extremely significant difference, and P < 0.001 (***) indicates an extremely significant difference.

[0066] The following examples use Excel 2020 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. T test is used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates an extremely significant difference, and P < 0.001 (***) indicates an extremely significant difference. Example

[0067] 1. Determination of target genes

[0068] 1) Determination of bract number and genome-wide association analysis

[0069] A total of 1,604 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 plants per row. Before harvest, the number of husks on the corn ears was measured.

[0070] 2) Genes related to corn husk number ZmXT6 Identification

[0071] Through genome-wide association analysis, seven signals significantly associated with bract number were located on chromosome 8, such as Figure 1 As shown in a. Further analysis of the signal revealed that among these 7 sites, 5 significant sites were located in the promoter region of Zm00001d011959 and 2 were located within the gene. Haplotype analysis found that the population material could be divided into 4 haplotypes (Hap1-Hap4) using 7 SNPs, as shown in Figure 1 b. Among them, Hap1 and Hap2 have more bracts, while Hap3 and Hap4 have fewer bracts. There are significant differences between Hap1 and Hap2 and Hap3 and Hap4, as shown in Figure 1 c. Furthermore, 12 materials with different numbers of bracts were selected from the 1604 materials used in this study. Bract tissues were selected for transcriptome analysis at the V9 stage, and the expression level (FPKM) of the Zm00001d011959 gene in maize material plants was detected. It was found that the expression level of Zm00001d011959 was significantly negatively correlated with the number of bracts, as shown in Figure 3. Figure 1 d. Therefore, Zm00001d011959 is considered a candidate gene for controlling bract number. Based on this, functional verification of this gene was carried out.

[0072] The Zm00001d011959 gene is 3866 bp long and has 4 exons, encoding 421 amino acids. Using the protein domain prediction software SMART website, a search revealed that the Zm00001d011959 gene contains an Exostosin domain and belongs to the GT47 gene family. This gene was annotated as β-1,4 glycosyltransferase 6 by MaizeGDB and was named ZmXT6 base because. ZmXT6 The genomic sequence of the 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 ZmXT6 protein or protein ZmXT6.

[0073] The amino acid sequence shown in SEQ ID No. 1 (ZmXT6 protein amino acid sequence (421AA)) is as follows: MRRWVLAIAIPILAAASAAALFLGAEAQAVQQGHQTERISGSAGDVLEDNPVGRLKVYVYDLPSKYNKKLVKKDPRCLNHMFAAEIFMHRFLLSSAVRTFNPEEADWFYTPVYATCDLTPSGLPLPFKSPRMMRSAIELIATNWPYWNRSEGADHFFVTPHDFGACFHYQEEKAIGRGILPLLQRATLVQTFG QKNHVCLKGGSITIPPFAPPQKMQAHLIPLDTPRSIFVYFRGLFYDTSNDPEGGYYARGARASVWENFKNNPLFDISTDHPPTYYEDMQRSVFCLCPLGWAPWSPRLVEAVVFG CIPVIIADDIVLPFADAIPWEEIGVFVAEEDVPKLDSILTSIPTDVILRKQRLLANPAMKQAMLFPQPAQAGDAFHQILNGLARKLPHGDNVFLKPGERVLNWTAGPPGDLKPW.

[0074] The nucleotide sequence shown in SEQ ID No. 2 (coding sequence of ZmXT6 protein (1266 bp)) is shown below:

[0075]

[0076] The bold portion of the nucleotide sequence shown in SEQ ID No. 2 above is the knockout target sequence, which is the sequence shown in SEQ ID No. 5.

[0077] The nucleotide sequence shown in SEQ ID No.3 ( ZmXT6 The genomic sequence (3866 bp) is shown below:

[0078]

[0079] 2. Build ZmXT6 CRISPR / Cas9 knockout maize lines

[0080] 1) Build ZmXT6 Gene knockout vector

[0081] ①CPB vector linearization

[0082] Use HindIII endonuclease to cut the CPB vector in a 37℃ water bath to obtain the CPB linearized vector. The enzyme digestion system is as follows:

[0083] Plasmid 1ug

[0084] HindⅢ1ul

[0085] Buffer 10ul

[0086] Incubate in a 37°C water bath for 3 h, then cut and recover the gel.

[0087] ② ZmXT6 Gene target gene screening

[0088] A target list was generated using an online target prediction website (http: / / crispor.tefor.net / ), and the target was selected as follows: 5′-TGGAAGATAACCCTGTTGGG-3′ (SEQ ID No. 5)

[0089] ③ Construction of sgRNA expression cassette

[0090] The sgRNA expression cassette is shown in SEQ ID No. 4 and was sent to a biological company for sequence synthesis.

[0091] The nucleotide sequence shown in SEQ ID No.4 ( ZmXT6 The sgRNA expression cassette for the gene knockout target is as follows:

[0092] TCACGCTGCACTGCACAATCGGGAATTCGTAATCATGTCAaAATTGGCCCTTACAAAATAGCTAGACGTGCAGGTGGCTGGATGTGCGCTCCCTGAATATCAACTTGTGTCTCCTCCGATTCAGTCCGCAGATGAAACTTGGTAATAACTGCAGCTGATCCGTCGTCATTCATGCTATGCAGGGGATTCGATCTTCAGCATGTGCAGTGCAGGCAACAATCTACG TTGTCTGGGCTTGCGATAGGTACACGACCACGAGGGAAGGCAACGCGTGATGTATGGGCCGCGCCTAAGCATCCAGCCCACGCGGGCGTGCGCGTCGTCGCTACGGCTTGCGGGGGAAGGGATCAAGGGACGAACCGAGAACTAGTACCAGACCGGCCAGCGAGCATTGCAGACACCGGCTTATAAGTTCAGCTGCGACCACCGCTCCTGGAAGATAACCCTGTTGGG GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA GTCGGTGCTTTTTTT aAGCTTGGCACTGGCCGTCGTTTTACAAC.

[0093] The bold portion of the nucleotide sequence shown in SEQ ID No. 4 above is the U6 promoter; the nucleotide sequence of the sgRNA is the underlined portion.

[0094] ④ Connect the CPB vector linear fragment obtained in step ① and the sgRNA expression cassette fragment obtained in step ③ by homologous recombination. The connection principle is in-fusion. The reagent used is Clone Smarter Technologies' Seamless Assembly Cloning Kit. The connection system is as follows:

[0095] 2*Assembly Mix 5uL

[0096] 2uL of CPB linearized vector

[0097] sgRNA expression cassette fragment 3uL

[0098] Total 10uL

[0099] The mixture was gently mixed and reacted at 50°C for 15 minutes to obtain the recombinant vector CPB-sgRNA.

[0100] ⑤Conversion

[0101] Thaw Fast-T1 competent cells on ice, add 10uL of the recombinant vector obtained in step ④ to 50uL of competent cells, gently flick the centrifuge tube to mix, place on ice for 30 minutes, then heat shock in a 42°C water bath for 30 seconds, then immediately transfer to ice to cool for 2 minutes, add 450uL of room temperature LB medium, then incubate in a 37°C shaker at 220rpm for 1 hour, then take 100uL of cells and evenly spread them on an LB plate containing kanamycin resistance, and incubate in a 37°C incubator overnight.

[0102] 2) Obtaining EHA105 / CPB-sgRNA

[0103] The recombinant vector CPB-sgRNA was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which was named EHA105 / CPB-ZmXT6.

[0104] The schematic diagram of the construction of the recombinant vector CPB-ZmXT6 and the vector map of the recombinant vector are shown in Figure 2. Figure 3 shown.

[0105] 3) T0 generation knockout ZmXT6 Obtaining genetically modified corn

[0106] Using Agrobacterium-infected maize embryos, the EHA105 / CPB-ZmXT6 prepared in step 2) was transformed into B104 to generate T0-generation transgenic maize. Basta was applied to the leaves of the T0-generation transgenic maize plants. Plants with normal leaf growth (resistant seedlings) were designated as T0-generation transgenic plants. Transgenic T0 plants were self-pollinated and harvested at maturity to generate T1-generation transgenic seeds, including knockout lines KO#1 and KO#2 mutants.

[0107] 4) Knockout ZmXT6 Genotype and phenotype of strains

[0108] Take the leaves of the transgenic plants for DNA extraction. DNA level detection ZmXT6 Gene knockout mutant-positive plants. Genomic DNA from knockout mutant plants was extracted and used as a template, with cas959-F and cas959-R as primers for amplification.

[0109] ddH2O 20μL

[0110] 2×Mix 25μL

[0111] cas959-F (10 pmol / μL) 2 μL

[0112] cas959-R (10 pmol / μL) 2 μL

[0113] Template 1 μL

[0114] Total volume 50 μL

[0115] Among them, the nucleotide sequences of cas959-F and cas959-R are:

[0116] cas959-F:CTTCTACCTGGCACACT SEQ ID No. 6.

[0117] cas959-R:ATCTCAGCAAACAT SEQ ID No.7.

[0118] The amplification reaction procedure was as follows: first round: denaturation at 95°C for 5 minutes; second round: denaturation at 95°C for 10 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 15 seconds, for 35 cycles; third round: extension at 72°C for 5 minutes. After the completion of the procedure, the PCR products were sent to the company for sequencing. Two types of mutants (KO#1 and KO#2) were detected, such as Figure 4 As shown in a. KO#1 has a base deletion, resulting in a frameshift and premature termination of the encoded protein; KO#2 has a base insertion, resulting in a frameshift and premature termination of the encoded protein.

[0119] Seeds of the corn inbred line B104 (WT) and T2 generation seeds of the knockout strain were planted in one row of each material, with a row length of 3 meters, a plant spacing of 0.25 meters, and a row spacing of 0.6 meters. Three replicates were used to investigate the number of bracts in the T2 generation plants. The results showed that KO#1 and KO#2 had significantly more bracts than wild-type B104 ( Figure 4 bc). The results show that ZmXT6 Knockout of the gene can significantly increase the number of corn husks, thus proving that the target gene has an important biological function in regulating the number of corn husks. ZmXT6 It is feasible to use protein genes or nucleic acid molecules to regulate the number of corn husks.

[0120] The present invention and its embodiments are described above. This description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs structures and embodiments similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. Application of biomaterials, characterized in that, The application includes any one of the following D1) to D5): D1) Application in increasing the number of corn husks; D2) Use in the preparation of products for increasing the number of corn husks; D3) Use in the preparation of products for cultivating corn with a high number of husks; D4) Application in identifying, screening, or assisting in breeding corn plants, lines, or varieties with a high number of bracts; D5) Application in corn breeding or assisted breeding to increase the number of husks; The biological material is a substance that inhibits or reduces the expression of the coding gene, or inhibits or reduces ZmXT6 Protein-expressing substances; described ZmXT6 The protein is selected from at least one of A1) and A3): A1) a protein with an amino acid sequence as shown in SEQ ID NO. 1; A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1); The coding gene is a DNA molecule as shown in any one of the following items b1) and b2): b1) a DNA molecule having a coding sequence of the coding strand as shown in SEQ ID NO. 2; b2) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.

3.

2. The use of the biomaterial according to claim 1, characterized in that: The biological material is any one of the following B2) and B3): B2) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the gene according to claim 1; or inhibit or reduce the ZmXT6 Nucleic acid molecules expressing proteins; B3) An expression cassette, a recombinant vector, a recombinant microorganism, or a transgenic plant cell line containing the nucleic acid molecule described in B2).

3. The use of the biomaterial according to claim 2, characterized in that: The nucleic acid molecule in B2) is a DNA molecule that expresses a gRNA targeting the protein-coding gene shown in A1) of claim 1, or is a gRNA targeting the protein-coding gene shown in A1) of claim 1.

4. The use of the biomaterial according to claim 1, characterized in that: Increasing the number of corn husks in D1) and D2) is achieved by inhibiting or reducing the expression of the coding gene according to claim 1 by gene knockout or gene silencing to increase the number of corn husks.

5. The use of the biomaterial according to claim 4, characterized in that: The gene knockout is to target and knock out the coding gene according to claim 1 by using the CRISPR / Cas9 system to obtain a knockout mutant plant; The nucleotide sequence of the knockout target is shown in SEQ ID NO.5: TGGAAGATAACCCTGTTGGG.

6. A method for cultivating corn with a high number of bracts, characterized in that: The specific operations are as follows: Select the code of claim 1 ZmXT6 The nucleic acid molecules of proteins are used as knockout targets to construct CRISPR / Cas9 The vector was knocked out, and mutants were obtained through Agrobacterium-mediated transformation. After screening, corn strains with a high number of bracts were cultivated.

7. A corn breeding method, characterized in that: During breeding, the corn with a high number of bracts obtained according to claim 6 is used as the germplasm parent.

Images