ZmXT6 protein for regulating and controlling corn bract number, coding gene and application of ZmXT6 protein in regulating and controlling corn bract number

Abstract

The invention discloses a ZmXT6 protein for regulating and controlling the number of corn bracts, a coding gene and application of the ZmXT6 protein in regulating and controlling the number of corn bracts, and a mutant corn plant with the coding gene of the ZmXT6 protein knocked out is obtained through a knockout experiment based on a CRISPR-Cas9 system. A phenotype identification result of a homozygous plant obtained by selfing a positive plant and a wild type shows that the corn inbred line with the coding gene of the ZmXT6 protein knocked out has more bracts, so that the gene for coding the ZmXT6 protein has an important biological function in regulating the bract number. Therefore, the application of the ZmXT6 protein and the gene encoding the ZmXT6 protein to regulation and control of the corn bract number is feasible, a new gene resource can be provided for improvement of the corn bract number character, and a new direction can be provided for improvement of the corn bract number character from the molecular level and breeding of the corn bract number character.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and particularly to a ZmXT6 protein for regulating the number of maize husks, a coding gene thereof, and an application thereof in regulating the number of maize husks. Background Art

[0002] Maize is the food crop with the largest planting area in China, and its planting area has reached 650 million mu. Maize mechanical grain harvesting is the key measure to achieve high-efficiency production of maize and reduce costs. However, the maize varieties currently promoted in China generally have problems such as too slow grain dehydration rate and too high water content at the harvesting stage, which seriously restricts the popularization and application of maize mechanical grain harvesting. The factors affecting the water content of maize at maturity include variety maturity, ear husk traits, and the self-dehydration rate of grains in the late grain filling stage; among them, the husk is the tissue that wraps the maize female ear, directly affects the dehydration of the ear, and indirectly affects the loss of grain moisture, and is the key trait that needs to be solved first in high-yield and machine-harvestable maize breeding. Existing research shows that the morphological and structural characteristics of maize husks are the most direct factors affecting the late dehydration rate of maize ears. Fewer husk numbers are more conducive to maize grain dehydration.

[0003] At the same time, the maize husk is also the protective organ of the ear on the maize plant, providing a suitable environment for the development of the ear and the grain filling of maize. First of all, the husk provides a suitable temperature for grain development. Especially in the late growth stage of maize, when frost damage may occur during harvesting, the husk can prevent heat loss and effectively reduce the frost damage caused by temperature drop. The husk prevents diseases and insects from entering the ear, reduces or prevents the occurrence of pests and diseases, and protects maize grains from being pecked by birds. Thus, it can be seen that the husk plays an important role in the development of maize ears. At present, there is no research on regulating the number of maize husks 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 maize husks through research and applied them to regulate the number of maize husks, so as to provide a new direction for the improvement of the trait of the number of maize husks and its breeding at the molecular level.

[0005] The technical solution of the present invention is as follows:

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

[0007] A ZmXT6 protein for regulating the number of maize husks, wherein the ZmXT6 protein is selected from at least one of A1) to A3):

[0008] A1) A protein with an amino acid sequence as shown in SEQ ID No.1;

[0009] A2) A protein that has more than 80% identity with the protein shown in A1) and has the same function, which is obtained by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1;

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

[0011] The above-mentioned ZmXT6 protein is derived from maize.

[0012] The above-mentioned protein can be artificially synthesized, or its coding gene can be synthesized first and then obtained by biological expression.

[0013] The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein by using in vitro DNA recombination technology for the purpose of facilitating the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag, and / or a SUMO protein tag, etc.

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

[0015] The coding gene of the ZmXT6 protein, and the coding gene is a DNA molecule shown in any one of the following b1) to b3):

[0016] b1) A DNA molecule whose coding sequence of the coding strand is shown in SEQ ID No.2;

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

[0018] b3) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) or b2) and encodes the above-mentioned ZmXT6 protein.

[0019] In the above, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined by using homology search sites on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0020] In the above application, the identity of more than 75% may 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] A third object of the present invention is to provide:

[0022] A biological material, which is a substance for regulating the expression of the coding gene, or a substance for regulating the activity or content of the ZmXT6 protein.

[0023] In the above, the substance for regulating the expression of the coding gene may be a substance that performs at least one of the following six regulations: 1) regulation at the gene transcription level; 2) regulation after gene transcription (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of the RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the 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 by the gene).

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

[0025] B1) The nucleic acid molecule according to claim 2;

[0026] B2) A nucleic acid molecule that inhibits or reduces the expression of the coding gene of the above-mentioned ZmXT6 protein;

[0027] Or a nucleic acid molecule that inhibits or reduces the activity of the ZmXT6 protein described in claim 1;

[0028] B3) An expression cassette or recombinant vector or recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule 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) of claim 1, or is a gRNA targeting the protein coding gene described in A1) of claim 1.

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

[0031] The application of the above-mentioned ZmXT6 protein, or the above-mentioned biological material, and the application includes any one of the following D1) to D5):

[0032] D1) Use in regulating the number of husks in maize;

[0033] D2) Use in preparing a product for regulating the number of husks in maize;

[0034] D3) Use in preparing a product for cultivating maize with a high number of husks;

[0035] D4) Use in identifying or screening or assisting in breeding maize plants or lines or varieties with a high number of husks;

[0036] D5) Use in maize breeding or assisting breeding for increasing the number of husks.

[0037] Furthermore, in D1) and D2), regulating the number of husks in maize is achieved by gene knockout or gene silencing to inhibit or reduce the expression of the coding gene of the ZmXT6 protein, thereby increasing the number of husks in maize.

[0038] The so-called gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by changing the DNA sequence.

[0039] Specifically, the gene knockout is achieved by targeting and knocking out the coding gene of the above-mentioned ZmXT6 protein through the CRISPR / Cas9 system to obtain knockout mutant plants.

[0040] The CRISPR / Cas9 system includes a knockout vector expressing the coding gene targeting the ZmXT6 protein.

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

[0042] TGGAAGATAACCCTGTTGGG.

[0043] The so-called gene silencing refers to the phenomenon of gene non-expression or low-expression without damaging the original DNA. Gene silencing makes the gene non-expressed or low-expressed on the premise of not changing the DNA sequence. Gene silencing can occur at two levels. One is transcriptional gene silencing caused by DNA methylation, heterochromatinization, and position effects, etc. The other is post-transcriptional gene silencing, that is, at the post-transcriptional level of the gene, the target gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, quelling, RNA interference (RNAi), and translational inhibition mediated by microRNA (miRNA), etc.

[0044] In the above application, the substance that regulates the expression of the coding gene of the ZmXT6 protein can be a reagent that inhibits or reduces the expression of the gene encoding the ZmXT6 protein. The reagent that inhibits or reduces the expression of the gene encoding the ZmXT6 protein can be a reagent that knocks out the gene, such as a reagent that knocks out the gene by homologous recombination or a reagent that knocks out the gene by CRISPR-Cas9. The reagent that inhibits or reduces the gene expression can contain 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 maize with a high number of husk leaves, and the specific operation is as follows:

[0047] Select the nucleic acid molecule encoding the ZmXT6 protein as the knockout target, construct a CRISPR / Cas9 knockout vector, obtain mutants through Agrobacterium-mediated transformation, and after screening, cultivate a maize line with a high number of husk leaves.

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

[0049] A maize breeding method, when breeding, the maize with the trait of a high number of husk leaves obtained above is used as a germplasm parent.

[0050] Adopting the above technical solution, the beneficial effects are as follows:

[0051] Through knockout experiments based on the CRISPR-Cas9 system, the present invention obtained mutant maize plants with the coding gene of the ZmXT6 protein knocked out. The identification results of the homozygous plants obtained by self-crossing the positive plants and the wild-type phenotype showed that: the maize inbred line with the coding gene of the ZmXT6 protein knocked out has more husk leaves, which indicates that the coding gene of the ZmXT6 protein has an important biological function in regulating the number of husk leaves.

[0052] Therefore, it is feasible to apply the ZmXT6 protein and its coding gene to regulate the number of maize husk leaves. It not only provides a new gene resource for improving the trait of the number of maize husk leaves, but also provides a new direction for improving the trait of the number of maize husk leaves and its breeding at the molecular level. Description of the Drawings

[0053] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0054] Figure 1 For the correlation analysis results of bract numbers and their haplotype analysis.

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

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

[0057] Figure 4 For the genotypes of mutant plants and the phenotypes of mutant plants. Detailed implementation manners

[0058] The present invention will be further described in detail below in conjunction with the detailed implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0059] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

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

[0061] The maize B104 in the following embodiments is described in the literature "Char, S.N., Neelakandan, A.K., Nahampun, H., Frame, B., Main, M., Spalding, M.H., Becraft, P.W., Meyers, B.C., 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." It can be obtained from the U.S. National Plant Germplasm System (https: / / npgsweb.ars-grin.gov / gringlobal / search).

[0062] The CPB vector was provided by the research group of Xie Chuanxiao from the Institute of Crop Science, Chinese Academy of Agricultural Sciences and was disclosed in the literature "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 biological materials from the applicant. The obtained above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0063] The Fast-T1 Escherichia coli competent cells were purchased from Nanjing Novoprotein Scientific Inc., and the product number is C505-03.

[0064] The EHA105 Agrobacterium competent cells were purchased from Beijing Bomed Gene Technology Co., Ltd., and the product number is BC303.

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

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

[0067] Examples:

[0068] 1. Determination of target gene

[0069] 1) Determination of the number of husk leaves and genome-wide association analysis

[0070] 1604 maize inbred lines were planted in the field using a completely randomized block design with 2 replicates. Each material was planted in a single row with a row length of 3 m and a row width of 0.6 m, and 13 maize plants were planted in each row. Before maize harvest, the number of husk leaves on the maize ears was investigated.

[0071] 2) Identification of the gene ZmXT6 related to the number of maize husk leaves

[0072] Through genome-wide association analysis, 7 signals significantly associated with the number of husk leaves were mapped on chromosome 8, such as Figure 1As shown in Figure a. Further analysis of the signals revealed that among these 7 loci, 5 significant loci were located in the promoter region of Zm00001d011959, and 2 were located within the gene. Haplotype analysis showed that 7 SNPs could be used to divide the population materials into 4 haplotypes (Hap1 - Hap4), as Figure 1 shown in Figure b. Among them, Hap1 and Hap2 had more bracts, while Hap3 and Hap4 had fewer bracts. There were significant differences between Hap1 and Hap2 and Hap3 and Hap4, as Figure 1 shown in Figure c. Further, 12 materials with different numbers of bracts were selected from the 1604 materials used in this study. At the V9 stage, bract tissues were selected for transcriptome analysis to detect the expression level (FPKM) of the Zm00001d011959 gene in maize plants. It was found that the expression level of Zm00001d011959 was significantly negatively correlated with the number of bracts, as Figure 1 shown in Figure d. Therefore, it can be considered that Zm00001d011959 is a candidate gene controlling the number of bracts. On this basis, functional verification of this gene was carried out. The full length of the Zm00001d011959 gene is 3866 bp, with a total of 4 exons, encoding 421 amino acids. Using the protein domain prediction software SMART website for searching, it was found 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 named ZmXT6 gene. The genomic sequence of the ZmXT6 gene is the DNA molecule shown in SEQ ID No.3, its coding sequence is the DNA molecule shown in SEQ ID No.2, and its encoded amino acid sequence is the protein 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 (amino acid sequence of ZmXT6 protein (421AA)) is as follows: MRRWVLAIAIPILAAASAAALFLGAEAQAVQQGHQTERISGSAGDVLEDNPVGRLKVYVYDLPSKYNKKLVKKDPRCLNHMFAAEIFMHRFLLSSAVRTFNPEEADWFYTPVYATCDLTPSGLPLPFKSPRMMRSAIELIATNWPYWNRSEGADHFFVTPHDFGACFHYQEEKAIGRGILPLLQRATLVQTFGQKNHVCLKGGSITIPPFAPPQKMQAHLIPLDTPRSIFVYFRGLFYDTSNDPEGGYYARGARASVWENFKNNPLFDISTDHPPTYYEDMQRSVFCLCPLGWAPWSPRLVEAVVFGCIPVIIADDIVLPFADAIPWEEIGVFVAEEDVPKLDSILTSIPTDVILRKQRLLANPAMKQAMLFPQPAQAGDAFHQILNGLARKLPHGDNVFLKPGERVLNWTAGPPGDLKPW。

[0074]

[0075]

[0076] 2. Construction of CRISPR / Cas9 Knockout Maize Lines of ZmXT6 Gene

[0077] 1) Construction of the knockout vector of ZmXT6 gene

[0078] ① Linearization of CPB vector

[0079] The vector CPB was digested with HindⅢ endonuclease in a 37℃ water bath to obtain the linearized CPB vector. The digestion system is as follows:

[0080] 1 μg of plasmid

[0081] 1 μL of HindⅢ

[0082] 10 μL of Buffer

[0083] Incubate in a 37℃ water bath for 3 h, and then recover the gel slice by cutting.

[0084] ② Screening of target genes of ZmXT6 gene

[0085] A target list was generated through the online target prediction website (http: / / crispor.tefor.net / ), and the selected target is as follows: 5′-TGGAAGATAACCCTGTTGGG-3′ (SEQ ID No.5)

[0086] ③ Construction of sgRNA expression cassette

[0087] The sgRNA expression cassette is shown in SEQ ID No.4 and was sent to a biological company for sequence synthesis.

[0088] The nucleotide sequence shown in SEQ ID No.4 (sgRNA expression cassette for the ZmXT6 gene knockout target) is as follows:[[]]

[0089]

[0090] The bold part in the nucleotide sequence shown in SEQ ID No.4 above is the U6 promoter; the nucleotide sequence of sgRNA is the underlined part.

[0091] ④ Ligate the linear fragment of the CPB vector obtained in step ① and the sgRNA expression cassette fragment obtained in step ③, and perform ligation by the method of homologous recombination. The principle of ligation is in-fusion, and the reagent used is the Seamless Assembly Cloning Kit of Clone Smarter Technologies. The ligation system is as follows:[[]]

[0092] 5 μL of 2*Assembly Mix

[0093] 2 μL of CPB linearized vector

[0094] 3 μL of sgRNA expression cassette fragment

[0095] Total 10 μL

[0096] Gently mix and react at 50 °C for 15 minutes to obtain the recombinant vector CPB-sgRNA respectively.

[0097] ⑤ Transformation

[0098] Melt Fast-T1 competent cells on ice. Add 10 μL of the recombinant vector obtained in step ④ to 50 μL of competent cells respectively. Gently flick the centrifuge tube to mix evenly, place on ice for 30 minutes, then perform heat shock in a 42 °C water bath for 30 seconds, immediately transfer to ice for cooling for 2 minutes, add 450 μL of LB medium at room temperature, then culture in a shaker at 37 °C and 220 rpm for 1 hour. Then take 100 μL of the cells and evenly spread them on an LB plate containing kanamycin resistance, and culture overnight in a 37 °C incubator.

[0099] 2) Obtaining EHA105 / CPB-sgRNA

[0100] Introduce the recombinant vector CPB-sgRNA into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, named EHA105 / CPB-ZmXT6.

[0101] The construction schematic diagram of the overloaded vector CPB-ZmXT6 and the vector map of the recombinant vector are as Figure 3 shown.

[0102] 3) Obtaining T0 generation maize with ZmXT6 gene knockout

[0103] Adopt the transformation method of Agrobacterium-mediated infection of maize immature embryos. Transfer EHA105 / CPB-ZmXT6 prepared in 2) into B104 to obtain T0 generation transgenic maize. Apply Basta on the leaves of T0 generation transgenic maize plants. The plants (resistant seedlings) with normal leaf growth are T0 generation transgenic plants. The transgenic T0 plants are self-crossed, harvested after maturity, and T1 generation transgenic seeds including knockout line KO#1 mutant plants and KO#2 mutant plants are obtained.

[0104] 4) Genotype and phenotype of ZmXT6 knockout lines

[0105] Take the leaves of transgenic plants for DNA extraction. Detect positive plants of ZmXT6 gene knockout mutants at the DNA level. Extract genomic DNA of knockout mutant plants, use it as a template, and perform amplification reaction with cas959-F and cas959-R as primers.

[0106]

[0107] Among them, the nucleotide sequences of cas959-F and cas959-R are respectively:

[0108] cas959-F: CTTCTACCTGGCACACT SEQ ID No.6.

[0109] cas959-R: ATCTCAGCAGCAAACAT SEQ ID No.7.

[0110] The amplification reaction procedure is as follows: The first round: denaturation at 95°C for 5 min; the second round: denaturation at 95°C for 10 sec, annealing at 58°C for 15 sec, extension at 72°C for 15 sec, 35 cycles; the third round: extension at 72°C for 5 min. After the program ends, send the PCR product to the company for sequencing. Two types of mutants (KO#1 and KO#2) were detected, as Figure 4 shown in a. KO#1 has a single-base deletion, resulting in a frameshift and premature termination of the encoded protein; KO#2 has a single-base insertion, resulting in a frameshift and premature termination of the encoded protein.

[0111] Seeds of maize inbred line B104 (WT) and T2-generation seeds of the knockout lines were planted with 1 row for each material, the row length was 3 m, the plant spacing was 0.25 m, and the row spacing was 0.6 m, with 3 replicates. Investigate the number of husk leaves of T2-generation plants. The results showed that compared with the wild type B104, the number of husk leaves of KO#1 and KO#2 was significantly increased ( Figure 4 b-c). The results indicate that knockout of the ZmXT6 gene can significantly increase the number of husk leaves in maize; thus proving that the target gene has an important biological function in regulating the number of husk leaves in maize. It is feasible to apply the target gene, that is, the gene or nucleic acid molecule encoding the ZmXT6 protein, to the regulation of the number of husk leaves in maize.

[0112] The above describes the present invention and its implementation manners. Such description is not restrictive. What is shown in the drawings is only one of the implementation manners of the present invention, and the actual structure is not limited thereto. All in all, if those of ordinary skill in the art are inspired by it and without departing from the gist of the present invention, they design similar structural manners and embodiments without creative efforts, which shall fall within the protection scope of the present invention.

Claims

1. The ZmXT6 protein that regulates the number of maize husks, characterized in that, The ZmXT6 protein is selected from at least one of A1) to A3): A1) A protein with an amino acid sequence as shown in SEQ ID No.1; A2) A protein obtained by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1, having more than 80% identity with the protein shown in A1) and having the same function; A3) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of A1) or A2).

2. The coding gene of the ZmXT6 protein according to claim 1, characterized in that, The coding gene is a DNA molecule shown in any one of b1) to b3) as follows: b1) A DNA molecule with a coding sequence of the coding strand as shown in SEQ ID No.2; b2) A DNA molecule with a nucleotide sequence as shown in SEQ ID No.3; b3) A DNA molecule having 75% or more identity with the nucleotide sequence defined by b1) or b2) and encoding the ZmXT6 protein described in claim 1.

3. A biological material, characterized in that, The biological material is a substance that regulates the expression of the coding gene described in claim 2, or a substance that regulates the activity or content of the ZmXT6 protein described in claim 1.

4. The biomaterial according to claim 3, wherein, The biological material is any one of the following B1) to B3): B1) The coding gene described in claim 2; B2) A nucleic acid molecule that inhibits or reduces the expression of the coding gene described in claim 2; or a nucleic acid molecule that inhibits or reduces the activity of the ZmXT6 protein described in claim 1; B3) An expression cassette or recombinant vector or recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule in B2).

5. The biological material according to claim 4, wherein the nucleic acid molecule in B2) is a DNA molecule that expresses a gRNA targeting the protein coding gene shown in A1) in claim 1, or is a gRNA targeting the protein coding gene described in A1) in claim 1.

6. Use of the ZmXT6 protein according to claim 1, or the biomaterial according to any one of claims 3 to 5, characterized in that, The application includes any one of the following D1) to D5): D1) Application in regulating the number of maize husks; D2) Application in preparing a product for regulating the number of maize husks; D3) Application in preparing a product for cultivating maize with a high number of husks; D4) Application in identifying or screening or assisting in breeding maize plants or lines or varieties with a high number of husks; D5) Application in maize breeding or assisting breeding for increasing the number of husks.

7. The application according to claim 6, wherein In D1) and D2), regulating the number of maize husks is to increase the number of maize husks by gene knockout or by gene silencing to inhibit or reduce the expression of the coding gene described in claim 2.

8. The application according to claim 7, wherein The gene knockout is to target and knockout the coding gene described in claim 2 through the CRISPR / Cas9 system to obtain a knockout mutant plant; The nucleotide sequence of the knockout target is as shown in SEQ ID No.5 as follows: TGGAAGATAACCCTGTTGGG.

9. A cultivation method for corn with a high number of husk leaves, characterized in that, The specific operation is as follows: Select the nucleic acid molecule encoding the ZmXT6 protein described in claim 2 as the knockout target, construct a CRISPR / Cas9 knockout vector, obtain mutants through Agrobacterium-mediated transformation, and after screening, cultivate a maize line with a high number of husks.

10. A corn breeding method, characterized in that, When breeding, the maize with a high number of husk leaves obtained in claim 9 is used as a germplasm parent.

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