Method for creating corn germplasm with accelerated embryo development and increased oil content in grains through mutation

Abstract

The invention discloses a method for creating a corn germplasm with accelerated embryo development and increased oil content in grains through mutation. The method comprises the following steps: transforming a corn inbred line B73-329 by adopting recombinant agrobacterium containing a recombinant plasmid pBUE411-HO, editing an HO gene, causing HO gene mutation after the HO gene is subjected to gene editing, and causing loss of protein HO activity when the HO genes of two homologous chromosomes are mutated; the loss of protein HO activity forms transgenic maize with increased embryo area and / or oil content in kernels. The amino acid sequence of the protein HO is as shown in SEQ ID NO: 1. Therefore, the transgenic corn with increased embryo area and / or oil content in grains can be obtained by performing gene editing on the HO gene of the corn. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for creating corn germplasm with accelerated embryo development and increased oil content in kernels through mutation. Background Art

[0002] Corn (Zeamays L.) belongs to the genus Zea mays of the Poaceae family. It is an important food crop and feed crop, and its production areas are spread all over the country. In 2012, my country's corn production exceeded rice and became the largest food crop. In 2020, my country became the world's second largest corn producer after the United States. Corn occupies a very important position in national food security. It has important uses in food, feed and industry, and has high economic value. With the development of production and the continuous improvement of living standards, people's demand for corn is also increasing.

[0003] The kernel is the most economically valuable organ of corn, which is mainly composed of three parts: embryo, endosperm and seed coat. The embryo and endosperm are both nutrient storage organs of corn kernels, storing a large amount of nutrients such as starch, protein and oil. The corn embryo accounts for about 9% of the weight of the kernel, but accumulates about 85% of the oil in the kernel. Corn oil is rich in carotene, vitamin D3, choline and other nutrients, which provide the nutrients needed by the human body and promote human development. In addition, corn oil is rich in unsaturated fatty acids and vitamin E, which help improve blood lipid patterns and reduce the incidence of cardiovascular diseases. Therefore, studying the regulatory factors in the embryo development and oil accumulation process of corn is crucial to the genetic improvement of corn germplasm. Summary of the invention

[0004] The purpose of the present invention is to create corn germplasm with accelerated embryo development and / or increased oil content in kernels.

[0005] The present invention firstly protects a method for cultivating transgenic corn, which may include the following steps: reducing the activity and / or expression level of protein HO in starting corn to obtain transgenic corn;

[0006] The transgenic corn has faster embryo development and / or increased oil content in the kernels compared to the starting corn;

[0007] The protein HO is a1) or a2) or a3) or a4):

[0008] a1) the amino acid sequence is the protein shown in SEQ ID No: 1;

[0009] a2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1);

[0010] a3) a protein related to maize embryo development and / or oil content in kernels obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein shown in a1) or a2);

[0011] a4) A protein having 80% or more homology with the amino acid sequence defined in a1), which is derived from corn and is related to corn embryo development and / or oil content in kernels.

[0012] The specific labels of the proteins in a2) above are shown in Table 1.

[0013] Table 1. Sequences of tags

[0014] Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tagII 8 WSHQ c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

[0015] In the protein described in a3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0016] The protein in a3) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0017] The coding gene of the protein in a3) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID No: 2 or SEQ ID No: 3, and / or performing missense mutation of one or several base pairs, and / or connecting the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0018] In the above method, the reduction of the activity and / or expression level of the protein HO in the starting corn can be achieved by mutating the gene encoding the protein HO in the starting corn (ie, the HO gene).

[0019] In the above method, the gene encoding the protein HO in the mutated corn can be mutated to HO / -2bp, HO / -18bp / +13bp or HO / -3bp / +19bp.

[0020] The HO / -2bp is a DNA molecule obtained by deleting the A at position 939 and the T at position 940 from the 5' end of SEQ ID No: 3, while keeping the other nucleotide sequences of SEQ ID No: 3 unchanged.

[0021] The HO / -18bp / +13bp is a DNA molecule obtained by deleting 18 nucleotides from positions 937 to 954 from the 5' end of SEQ ID No: 3, inserting 13 nucleotides shown in SEQ ID No: 5, and keeping the other nucleotide sequences of SEQ ID No: 3 unchanged.

[0022] The HO / -3bp / +19bp is a DNA molecule obtained by deleting the 3 nucleotides from positions 938 to 940 from the 5' end of SEQ ID No: 3 and inserting the 19 nucleotides shown in SEQ ID No: 6 while keeping the other nucleotide sequences of SEQ ID No: 3 unchanged.

[0023] In the above method, the gene encoding the protein HO in the mutated starting corn is achieved by introducing a gene editing system into the starting corn; the gene editing system includes a recombinant expression vector; the recombinant expression vector contains a DNA molecule expressing a gRNA targeting the gene encoding the protein HO.

[0024] In the above method, the target sequence of the gRNA may be as shown in SEQ ID No: 3, from position 935 to position 953 from the 5' end.

[0025] In the above method, the recombinant expression vector may be a recombinant plasmid pBUE411-HO. The recombinant plasmid pBUE411-HO may be a recombinant plasmid obtained by inserting the double-stranded DNA molecule shown in SEQ ID No: 4 into the recognition site of the restriction endonuclease BsaI of the pBUE411-BG vector.

[0026] The present invention also protects the use of a gene encoding any of the above-mentioned proteins HO (i.e., HO gene) in mutated starting corn in cultivating transgenic corn; compared with the starting corn, the embryo development of the transgenic corn is accelerated and / or the oil content in the grain is increased.

[0027] The gene encoding any of the above-mentioned proteins HO (i.e., HO gene) may be a DNA molecule of (b1) or (b2) or (b3) or (b4) or (b5):

[0028] (b1) a DNA molecule whose coding region is shown in SEQ ID NO: 3;

[0029] (b2) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2;

[0030] (b3) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 3;

[0031] (b4) a DNA molecule that hybridizes with the DNA molecule defined in (b1) or (b2) or (b3) under stringent conditions and encodes the protein HO;

[0032] (b5) A DNA molecule derived from corn and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the DNA molecule defined in (b1) or (b2) or (b3) and encoding the protein HO.

[0033] The stringent conditions are hybridization and membrane washing twice at 68°C in a 2×SSC, 0.1% SDS solution, each for 5 min, and hybridization and membrane washing twice at 68°C in a 0.5×SSC, 0.1% SDS solution, each for 15 min.

[0034] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0035] Among them, SEQ ID NO: 2 consists of 10521 nucleotides, and SEQ ID NO: 3 consists of 2085 nucleotides. The nucleotides of SEQ ID NO: 2 or SEQ ID NO: 3 encode the amino acid sequence shown in SEQ ID NO: 1.

[0036] A person skilled in the art can easily mutate the nucleotide sequence encoding the protein HO of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 70% or higher identity with the nucleotide sequence of the protein HO isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein HO.

[0037] The term "identity" as used herein refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 70% or more, or 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein HO of the present invention encoding the amino acid sequence shown in SEQ ID NO: 1. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0038] In the above application, the mutation may be to mutate the HO gene shown in SEQ ID No: 3 to HO / -2bp, HO / -18bp / +13bp or HO / -3bp / +19bp.

[0039] The HO / -2bp is a DNA molecule obtained by deleting the A at position 939 and the T at position 940 from the 5' end of SEQ ID No: 3, while keeping the other nucleotide sequences of SEQ ID No: 3 unchanged.

[0040] The HO / -18bp / +13bp is a DNA molecule obtained by deleting 18 nucleotides from positions 937 to 954 from the 5' end of SEQ ID No: 3, inserting 13 nucleotides shown in SEQ ID No: 5, and keeping the other nucleotide sequences of SEQ ID No: 3 unchanged.

[0041] The HO / -3bp / +19bp is a DNA molecule obtained by deleting the 3 nucleotides from positions 938 to 940 from the 5' end of SEQ ID No: 3 and inserting the 19 nucleotides shown in SEQ ID No: 6 while keeping the other nucleotide sequences of SEQ ID No: 3 unchanged.

[0042] In the above application, the material of the gene encoding the HO protein in corn that is mutated may be the following B1) or B2):

[0043] B1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein HO;

[0044] B2) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule described in B1).

[0045] In the above application, the nucleic acid molecule in B1) may be a DNA molecule expressing a gRNA targeting the gene encoding the protein HO or a gRNA targeting the gene encoding the protein HO.

[0046] In the above application, the target sequence of the gRNA may be as shown in SEQ ID No: 3, from position 935 to position 953 from the 5' end.

[0047] In the above application, the recombinant vector in B2) can be a recombinant plasmid pBUE411-HO. The recombinant plasmid pBUE411-HO can be a recombinant plasmid obtained by inserting the double-stranded DNA molecule shown in SEQ ID No: 4 into the recognition site of the restriction endonuclease BsaI of the pBUE411-BG vector.

[0048] Any of the above-mentioned starting corns can be the corn inbred line B73-329.

[0049] Any of the above-mentioned transgenic corns may be KO-1, KO-2 or KO-3 mentioned in the examples.

[0050] Any of the above-mentioned grains may be mature grains.

[0051] Any of the above-mentioned accelerated embryo development is manifested as an increase in embryo area.

[0052] Experiments have shown that the HO gene can be edited by transforming the corn inbred line B73-329 with a recombinant Agrobacterium containing the recombinant plasmid pBUE411-HO. After gene editing, the HO gene can cause HO gene mutation. When the HO genes of the two homologous chromosomes mutate, the protein HO activity can be lost; the loss of protein HO activity forms transgenic corn with increased embryo area and / or oil content in the kernel. The method of the present invention can be used to edit the HO gene of corn and obtain transgenic corn with increased embryo area and / or oil content in the kernel. It can be seen that protein HO can regulate corn embryo development and the oil content in mature kernels. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 To identify the structure of the HO gene and the mutation types of the HO gene in the HO homozygous mutant strains KO-1, KO-2 and KO-3.

[0054] Figure 2 The embryo area statistics of the HO homozygous mutant and the maize inbred line B73-329. A is the phenotype of the embryo in the ear on the 16th day after pollination, Bar = 1 mm; B is the embryo area statistics of the embryo in the ear on the 16th day after pollination; the values ​​are shown as mean ± standard deviation, and the two-tailed t test was used to analyze the significant difference (** is P < 0.01).

[0055] Figure 3 The statistical results of oil content in mature grains of HO homozygous mutants and maize inbred line B73-329. A is the statistical results of oil content in mature grains of maize inbred lines B73-329, KO1 and KO2 harvested in Hainan; B is the statistical results of oil content in mature grains of maize inbred lines B73-329 and KO3 harvested in Beijing; the values ​​are shown as mean ± standard deviation, and the two-tailed t-test was used to analyze significant differences (** is P<0.01). DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0057] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0058] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0059] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0060] The pBUE411-BG vector is described in the following literature: Xing, HL, Dong, L., Wang, ZP, Zhang, HY, Han, CY, Liu, B., Wang, XC, and Chen, QJ (2014). A CRISPR / Cas9 toolkit for multiplex genome editing in plants. Bmc Plant Biology 14. The pBUE411-BG vector has resistance to phosphinothricin (also known as glufosinate resistance).

[0061] Maize inbred line B73-329 is recorded in the following literature: Zhang M, Cao Y, Wang Z, et al. Aretrotransposon in an HKT1 family sodium transporter causes variation of leaf Na+exclusion and salt tolerance in maize[J]. New Phytologist, 2017. The name of maize inbred line B73-329 in the literature is maize B73-329.

[0062] Example 1. Discovery of protein HO and its encoding gene

[0063] After a large number of experiments, the inventors of the present application discovered the HO gene from the genome of the corn inbred line B73 (hereinafter referred to as corn B73).

[0064] The nucleotide sequence of the HO gene in the genomic DNA of corn B73 is shown in SEQ ID NO:2.

[0065] The nucleotide sequence of the HO gene in the cDNA of corn B73 is shown in SEQ ID NO:3.

[0066] The HO gene encodes the protein HO. The amino acid sequence of the protein HO is shown in SEQ ID NO:1.

[0067] Example 1: Mutation to create new corn germplasm with increased embryo area and oil content in kernels

[0068] 1. Acquisition and identification of HO homozygous mutants

[0069] In this example, a target site was selected for the experiment. The target site sequence was: 5'-GTCTATTACCTGAGGGGAG-3' (i.e., positions 935-953 from the 5' end of SEQ ID No: 3), and the corresponding target gene was the HO gene.

[0070] 1. Obtaining the recombinant plasmid pBUE411-HO

[0071] (1) Primer HO-CAS9-F: 5'-TTTTACTATACCCACCGCAAC-3' and primer HO-CAS9-R: 5'-CTCAGGATAATCGCTTCCC-3' were designed and synthesized according to the target site, and primer HO-CAS9-F and primer HO-CAS9-R were diluted to 100 μM with deionized water to obtain primer HO-CAS9-F dilution solution and primer HO-CAS9-R dilution solution; then, an annealing reaction was performed to form an annealed target site fragment.

[0072] The annealing program was: 95°C for 10 min, 89°C for 7-8 min, 83°C for 7-8 min, 77°C for 7-8 min, 71°C for 7-8 min, and stored at 16°C.

[0073] (2) The pBUE411-BG vector and the annealed target fragment were connected to obtain the recombinant plasmid pBUE411-HO.

[0074] The ligation system is 15 μL, consisting of 2 μL annealed target fragment, 2 μL pBUE411-BG vector, 1.5 μL 10×NEBT4Buffer (a product of NEB, catalog number M0202S), 1.5 μL 10×BSA (a product of NEB, catalog number M0202S), 1 μL restriction endonuclease BsaI (NEB), 1 μL T4 Ligase (a product of NEB, catalog number M0202S) and 6 μL ddH2O.

[0075] The connection procedure was: 37°C for 5 h; 50°C for 5 min; 80°C for 10 min.

[0076] The recombinant plasmid pBUE411-HO was sequenced. The sequencing results showed that the recombinant plasmid pBUE411-HO was obtained by inserting the double-stranded DNA molecule shown in SEQ ID No: 4 into the recognition site of the restriction endonuclease BsaI of the pBUE411-BG vector.

[0077] 2. Acquisition and identification of HO homozygous mutants

[0078] Since corn is a diploid plant, when Cas9 starts to cut specific genes, both alleles on the two homologous chromosomes in the same cell may be edited, resulting in the same or different types of mutations, so the two alleles in one plant are regarded as two gene editing events. Homozygous mutant strains refer to the same mutation in the HO genes of the two homologous chromosomes of the plant. Bi-allelic mutant strains refer to the HO genes of the two homologous chromosomes of the plant that have mutated but in different forms. Heterozygous mutant strains refer to the HO gene of one of the two homologous chromosomes of the plant that has mutated and the HO gene of the other homologous chromosome has not mutated. Wild type refers to the HO genes of the two homologous chromosomes of the plant that have not mutated.

[0079] (1) The recombinant plasmid pBUE411-HO was transferred to Agrobacterium tumefaciens EHA105 by liquid nitrogen freezing to obtain recombinant Agrobacterium.

[0080] (2) The recombinant Agrobacterium was transformed into the maize inbred line B73-329, and after screening, differentiation and rooting, the T0 generation pseudo-transgenic maize was obtained. For specific transformation methods, please refer to the following literature: Wei Xiaoyu, Shao Shidi, Sun Su, et al., Establishment of Agrobacterium-mediated genetic transformation system of maize immature embryos, Journal of Jilin Agricultural University, 2017(06)640-647; Huang Lu, Wei Zhiming, Agrobacterium-mediated genetic transformation of maize [J], Acta Biologica Sinica, 1999(04).

[0081] (3) Using the genomic DNA of the T0 generation transgenic corn leaves as templates, PCR amplification was performed using a primer pair consisting of primer F: 5'-TTTTACTATACCCACCGCAAC-3' and primer R: 5'-CTCAGGATAATCGCTTCCC-3' to obtain the corresponding PCR amplification products. The PCR amplification products were subjected to Sanger sequencing. The sequencing results were compared with the Cas9 target sequence of the HO gene (shown in SEQ ID No: 3), and the mutation types were counted. Several heterozygous mutant strains were obtained.

[0082] (4) The heterozygous mutant strains obtained in step (3) are self-pollinated to obtain seeds, namely T1 generation seeds, and the plants grown from the T1 generation seeds are T1 generation plants; the T1 generation plants are self-pollinated to obtain seeds, namely T2 generation seeds, and the plants grown from the T2 generation seeds are T2 generation plants.

[0083] (5) Using the genomic DNA of the leaves of the T2 generation plants as templates, PCR amplification was performed using a primer pair consisting of primer F: 5'-TTTTACTATACCCACCGCAAC-3' and primer R: 5'-CTCAGGATAATCGCTTCCC-3' to obtain the corresponding PCR amplification products. The PCR amplification products were subjected to Sanger sequencing. The sequencing results were compared with the Cas9 target sequence of the HO gene (shown in SEQ ID No: 3), and the mutation types were counted.

[0084] A total of three HO homozygous mutants were obtained, named KO-1, KO-2 and KO-3. The mutation types of the HO gene in maize inbred lines B73-329, KO-1, KO-2 and KO-3 are shown in Figure 1 (WT is maize inbred line B73-329).

[0085] The HO genes of the two homologous chromosomes of KO-1 underwent the same mutation, specifically, a deletion of two nucleotides "AT" occurred in the HO genes on both homologous chromosomes (i.e., the 939th to 940th positions from the 5' end of SEQ ID No: 3 were deleted), which caused a frameshift and resulted in the functional loss of protein HO.

[0086] The HO genes of the two homologous chromosomes of KO-2 underwent the same mutation, specifically, an 18-nucleotide deletion of "CTATTACCTGAGGGGAGT" (i.e., deletion of positions 937 to 954 from the 5' end of SEQ ID No: 3) and an insertion of 13 nucleotides of TGATCATCATTTA (SEQ ID No: 5) occurred in the HO genes of both homologous chromosomes, causing a frameshift and resulting in the functional loss of the protein HO.

[0087] The HO genes of the two homologous chromosomes of KO-3 underwent the same mutation, specifically, the HO genes on both homologous chromosomes had a deletion of three nucleotides "TAT" (i.e., deletion of positions 938 to 940 from the 5' end of SEQ ID No: 3), and an insertion of 19 nucleotides ATCATTTATTCGTGCGAAA (SEQ ID No: 6), which caused a frameshift and resulted in the functional loss of protein HO.

[0088] 3. Phenotypic observation of embryo in grains of HO homozygous mutants and statistics of embryo area

[0089] 1. Plant the corn to be tested (corn inbred lines B73-329, KO-1, KO-2 or KO-3) and self-pollinate; take the ears on the 16th day after pollination, peel off 30 kernels from the middle third of each ear, and peel out the embryo.

[0090] 2. After completing step 1, randomly select 10 embryos and take photos under a dissecting microscope.

[0091] Results Figure 2 Middle A (WT is maize inbred line B73-329).

[0092] 3. After completing step 1, use ImageJ software to calculate the embryo area and perform significance analysis.

[0093] The statistical results are shown in Figure 2 Zhong B (WT is corn inbred line B73-329).

[0094] The above results showed that compared with the maize inbred line B73-329, the embryo area of ​​HO homozygous mutant strains (such as KO-1, KO-2, and KO-3) was significantly increased.

[0095] This shows that protein HO negatively regulates the embryo development of corn kernels, that is, inhibiting the expression of protein HO leads to a significant increase in embryo area.

[0096] 4. Detection of Oil Content in Mature Grains of HO Homozygous Mutant

[0097] Since protein HO negatively regulates the embryonic development of corn kernels, the inventors of the present application also detected the oil content in mature kernels of HO homozygous mutants. The specific steps are as follows:

[0098] 1. Plant the tested corn (corn inbred lines B73-329, KO-1 or KO-2) in Hainan in 2022, self-pollinate, and harvest mature corn kernels in February 2023. Then use a nuclear magnetic resonance oil content detector (product of Newmai Technology, model NMI20-015V-I) to detect the oil content in each mature kernel. Test more than 30 mature kernels and take the average value of the results.

[0099] Test results see Figure 3 A (WT is maize inbred line B73-329). The results showed that the oil content in mature grains of HO homozygous mutants (such as KO-1 and KO-2) was significantly increased compared with maize inbred line B73-329.

[0100] 2. Plant the corn to be tested (corn inbred line B73-329 or KO-3) in Beijing in the summer of 2023, self-pollinate, and harvest mature kernels in September 2023. Then use a nuclear magnetic resonance oil content detector to test the oil content in each mature kernel. Test more than 30 mature kernels and take the average value of the results.

[0101] Test results see Figure 3 The results showed that the oil content in mature grains of KO-3 was significantly increased compared with that of maize inbred line B73-329. It can be seen that protein HO controls the oil content in mature grains by regulating embryo development.

[0102] These results suggest that HO protein negatively regulates the oil content in mature corn kernels, that is, inhibiting the expression of HO protein leads to a significant increase in the oil content in mature kernels.

[0103] KO-1, KO-2 and KO-3 are new maize germplasms created by mutation. Compared with the maize inbred line B73-329, the embryo area of ​​KO-1, KO-2 and KO-3 is significantly increased, and the oil content in mature grains is also significantly increased.

[0104] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for cultivating transgenic corn, comprising the following steps: reducing the activity and / or expression level of protein HO in starting corn to obtain transgenic corn; The transgenic corn has faster embryo development and / or increased oil content in the kernels compared to the starting corn; The protein HO is a1) or a2) or a3) or a4): a1) the amino acid sequence is the protein shown in SEQ ID No: 1; a2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1); a3) a protein related to maize embryo development and / or oil content in kernels obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein shown in a1) or a2); a4) A protein having 80% or more homology with the amino acid sequence defined in a1), which is derived from corn and is related to corn embryo development and / or oil content in kernels.

2. The method according to claim 1, characterized in that: The reduction of the activity and / or expression level of the protein HO in the starting corn is achieved by mutating the gene encoding the protein HO in the starting corn.

3. The method according to claim 2, characterized in that: The coding gene of the protein HO in the mutated corn is mutated to HO / -2bp, HO / -18bp / +13bp or HO / -3bp / +19bp as shown in SEQ ID No: 3; The HO / -2bp is a DNA molecule obtained by deleting the A at position 939 and the T at position 940 from the 5' end of SEQ ID No: 3, while keeping the other nucleotide sequences of SEQ ID No: 3 unchanged; The HO / -18bp / +13bp is a DNA molecule obtained by deleting 18 nucleotides from positions 937 to 954 from the 5' end of SEQ ID No: 3, inserting 13 nucleotides shown in SEQ ID No: 5, and keeping the other nucleotide sequences of SEQ ID No: 3 unchanged; The HO / -3bp / +19bp is a DNA molecule obtained by deleting the 3 nucleotides from positions 938 to 940 from the 5' end of SEQ ID No: 3 and inserting the 19 nucleotides shown in SEQ ID No: 6 while keeping the other nucleotide sequences of SEQ ID No: 3 unchanged.

4. The method according to claim 2 or 3, characterized in that: The coding gene of the protein HO in the mutated starting corn is achieved by introducing a gene editing system into the starting corn; the gene editing system includes a recombinant expression vector; the recombinant expression vector contains a DNA molecule expressing a gRNA targeting the coding gene of the protein HO.

5. The method according to claim 4, characterized in that: The target sequence of the gRNA is shown in SEQ ID No: 3 at positions 935-953 from the 5' end.

6. Application of a material that mutates the gene encoding the protein HO in the original corn in breeding transgenic corn; compared with the original corn, the embryo development of the transgenic corn is accelerated and / or the oil content in the kernel is increased; The protein HO is a1) or a2) or a3) or a4): a1) the amino acid sequence is the protein shown in SEQ ID No: 1; a2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1); a3) a protein related to maize embryo development and / or oil content in kernels obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein shown in a1) or a2); a4) A protein having 80% or more homology with the amino acid sequence defined in a1), which is derived from corn and is related to corn embryo development and / or oil content in kernels.

7. The use according to claim 6, characterized in that: The mutation is to mutate the HO gene shown in SEQ ID No: 3 to HO / -2bp, HO / -18bp / +13bp or HO / -3bp / +19bp; The HO / -2bp is a DNA molecule obtained by deleting the A at position 939 and the T at position 940 from the 5' end of SEQ ID No: 3, while keeping the other nucleotide sequences of SEQ ID No: 3 unchanged; The HO / -18bp / +13bp is a DNA molecule obtained by deleting 18 nucleotides from positions 937 to 954 from the 5' end of SEQ ID No: 3, inserting 13 nucleotides shown in SEQ ID No: 5, and keeping the other nucleotide sequences of SEQ ID No: 3 unchanged; The HO / -3bp / +19bp is a DNA molecule obtained by deleting the 3 nucleotides from positions 938 to 940 from the 5' end of SEQ ID No: 3 and inserting the 19 nucleotides shown in SEQ ID No: 6 while keeping the other nucleotide sequences of SEQ ID No: 3 unchanged.

8. The use according to claim 6 or 7, characterized in that: The material of the gene encoding the protein HO in corn that is mutated is as follows B1) or B2): B1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein HO; B2) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule described in B1).

9. The use according to claim 8, characterized in that: B1) The nucleic acid molecule is a DNA molecule that expresses a gRNA targeting the gene encoding the protein HO or is a gRNA targeting the gene encoding the protein HO.

10. The use according to claim 9, characterized in that: The target sequence of the gRNA is shown in SEQ ID No: 3 at positions 935-953 from the 5' end.

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