A breeding method for improving biomass and grain yield of corn by site-directed base mutation of corn ZmGRF1 gene and application thereof

Abstract

The invention discloses a method for producing corn by site-directed base mutation ZmGRF1 The present invention relates to the field of plant genetic engineering and provides a method for increasing corn biomass and / or grain yield, comprising: modifying the wild type shown in SEQ ID No. 1 in the corn genome to improve the biomass and / or grain yield of the corn. ZmGRF1 The nucleotides 1322 and 1323 of the gene are mutated from G to A, thereby increasing the biomass and / or grain yield of corn. The present invention is of great significance for cultivating high-yield corn varieties and providing material accumulation for corn breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering, and in particular to a method for improving the biomass and grain yield of corn by site-directed base mutation ZmGRF1 The present application relates to the field of plant genetic engineering, and in particular to a method for improving the biomass and grain yield of corn by site-directed base mutation BACKGROUND

[0002] Gene editing technology can precisely modify gene sequences to achieve the improvement and regulation of target traits, and has the advantages of convenient operation, precise targeting, and wide adaptability, which has a revolutionary impact on the global seed industry technology iteration and industrial pattern. CRISPR / Cas9 is derived from the immune system of bacteria, which recognizes and cuts foreign DNA through crRNA-guided Cas9 protein. This system relies on single guide RNA (sgRNA) to precisely locate the target site, and induces DSB to trigger the cell repair mechanism to achieve gene editing.

[0003] Base editors (BEs) can achieve target point mutation without generating DNA double-strand breaks (DSBs) or providing homologous repair templates, thereby enabling efficient editing in cells lacking homology-directed repair (HDR). The two types of BEs initially developed include cytosine base editors (CBEs) and adenine base editors (ABEs). CBEs contain cytidine deaminase (such as APOBEC1) and uracil glycosylase inhibitor (UGI), which mediate C to T base conversion. ABEs use tRNA-specific deaminase TadA designed by directed evolution to achieve A to G conversion. Due to the high predictability of its editing results, base editing technology provides an efficient and precise tool for plant genome engineering, enabling researchers and agronomists to quickly create new plant mutants, significantly better than traditional breeding methods. For example, by mutating the ALS gene of plants at a specific point, resistance to sulfonylurea and imidazolinone herbicides can be conferred. CBEs and ABEs technology has been successfully applied in a variety of crops. Gao et al. confirmed that BE3 base editor can efficiently induce point mutations in wheat, corn and rice. In addition, multiple studies have shown that ABEs exhibit extremely high editing efficiency in rice. In Arabidopsis thaliana, researchers introduced the Y85H mutation in the FT protein by base editing technology, successfully delaying flowering time. These studies fully demonstrate that base editing technology is a powerful tool for precise modification of plant genomes, enabling the creation of crop lines with excellent agronomic traits through targeted mutation of specific genes. Corn is an important global food and feed crop, and its yield and quality improvement is of great significance to food security. Plant growth regulators (PGRs) play a crucial role in regulating plant growth and development, and their mutations can lead to changes in plant morphology, flowering time, and other traits. For example, the mutation of the FT gene in Arabidopsis thaliana can delay flowering time, and the mutation of the ZCN8 gene in maize can increase plant height and yield. However, traditional breeding methods are time-consuming and labor-intensive, and it is difficult to achieve precise modification of specific genes. Base editing technology provides a new approach to precisely modify plant genomes and create new crop lines with improved agronomic traits. GRF) gene family plays a key role in plant growth and development, especially in leaf development, flower organ formation, seed development and stress response. In recent years, with the rapid development of molecular biology technology, GRF The functional analysis of genes and their potential application in crop breeding have gradually become research hotspots. GRF The function of genes in maize and their application in breeding have important theoretical and practical implications. Growth-regulating factors (GRFs) are a class of plant-specific transcription factors first discovered in rice and Arabidopsis, comprising 12 and 9 family members, respectively. GRF proteins contain two highly conserved domains at their N-termini: the QLQ domain and the WRC domain. The QLQ domain mediates binding to GRF-interacting factors (GIFs), while the WRC domain functions as a DNA-binding domain (DBD). The C-terminal region of GRF proteins varies in length and composition and possesses transcriptional activation functions. GRFs regulate gene expression by recognizing and binding specific cis-acting elements (GTEs) in downstream target genes through the WRC domain. For example, AtGRF7 represses DREB2A expression by binding to the GTE (TGTCAGG), while AtGRF9 binds to the CTGACA sequence in the ORG3 promoter. Recent studies have demonstrated that GRF transcription factors play important roles in plant growth, development, and stress responses. GRF family members are widely involved in the formation of plant organs, including roots, stems, leaves, and flowers, as well as in the regulation of stress adaptation, by regulating cell division, organ development, and hormone signaling pathways. In plant organ development, GRF transcription factors influence root architecture by regulating cell division and elongation. Furthermore, GRFs interact with the auxin signaling pathway to regulate root gravitropism and lateral root development. In stem and leaf development, GRF transcription factors influence leaf size and morphology by regulating cell proliferation and expansion. Furthermore, GRFs regulate floral organ development through interactions with hormone signaling pathways, such as gibberellins and cytokinins. Regarding stress adaptation, GRF transcription factors enhance plant tolerance to stress by regulating the expression of antioxidant enzyme genes and hormone signaling pathways. For example, OsGRF7 in rice enhances plant tolerance to drought stress by regulating the ABA (abscisic acid) signaling pathway.

[0004] In summary, GRF transcription factors play an important role in plant growth and development and stress responses, with their functions involved in multiple aspects, including cell division, organ development, hormone signaling pathways, and stress adaptation. Research on GRF genes in maize can further reveal the functional diversity of GRFs in different plant species and their molecular regulatory mechanisms. This provides important insights into the molecular mechanisms of plant growth and development and offers potential genetic resources for breeding high-yield and stress-tolerant crop varieties.

[0005] Therefore, a method for creating high-yield maize germplasm by precisely targeting ZmGRF1 gene editing technology is provided. SUMMARY

[0006] The purpose of the present application is to provide a method for improving the biomass and grain yield of maize by site-directed base mutation. ZmGRF1 The present application relates to a breeding method for improving the biomass and grain yield of maize and application thereof.

[0007] In a first aspect, the present application claims a method for improving the biomass and / or grain yield of maize.

[0008] The method for improving the biomass and / or grain yield of maize claimed by the present application can comprise the following steps: mutating the wild-type ZmGRF1 gene in the maize genome from G to A at positions 1322 and 1323 of the nucleotide sequence of the wild-type ZmGRF1 gene, thereby achieving improved biomass and / or grain yield of maize. ZmGRF1 The method for improving the biomass and / or grain yield of maize claimed by the present application can comprise the following steps: mutating the wild-type ZmGRF1 gene in the maize genome from G to A at positions 1322 and 1323 of the nucleotide sequence of the wild-type ZmGRF1 gene, thereby achieving improved biomass and / or grain yield of maize.

[0009] In the maize genome, the nucleotide sequence of the wild-type ZmGRF1 gene is as shown in SEQ ID No. 1. ZmGRF1 In the maize genome, the nucleotide sequence of the wild-type ZmGRF1 gene is as shown in SEQ ID No. 1. ZmGRF1 In the maize genome, the nucleotide sequence of the wild-type ZmGRF1 gene is as shown in SEQ ID No. 1. ZmGRF1 In the maize genome, the nucleotide sequence of the wild-type ZmGRF1 gene is as shown in SEQ ID No. 1.

[0010] In the method, the mutation of the wild-type ZmGRF1 gene in the maize genome from G to A at positions 1322 and 1323 of the nucleotide sequence can be achieved by introducing a CRISPR / nCas9BE gene editing vector into the maize.

[0011] The CRISPR / nCas9BE gene editing vector is used to express a cytosine base editor; the cytosine base editor targets a target sequence in the wild-type ZmGRF1 gene, and the target sequence is as shown in SEQ ID No. 3.

[0012] Further, the CRISPR / nCas9BE gene editing vector can express a fusion protein formed by fusing nCas9(D10A), APOBEC1 deaminase and UGI protein, and the CRISPR / nCas9BE gene editing vector can express gRNA targeting the target sequence.

[0013] Furthermore, the amino acid sequence of the fusion protein may be as shown in SEQ ID No.6; the spacer sequence of the gRNA is the reverse complementary sequence of positions 1-20 of SEQ ID No.3.

[0014] In one embodiment of the present invention, the full sequence of the CRISPR / nCas9BE gene editing vector is obtained by connecting SEQ ID No. 4 and SEQ ID No. 5 end to end in sequence.

[0015] The method is also a method of breeding corn varieties with increased biomass and / or grain yield.

[0016] The method is also a method for increasing corn plant height and / or ear length and / or 100-grain weight and / or single-ear grain yield.

[0017] The method is also a method for breeding corn varieties with increased plant height and / or ear length and / or 100-grain weight and / or single-ear grain yield.

[0018] In a second aspect, the present invention claims protection for any of the following biological materials:

[0019] (A1) the gRNA described in the first aspect above;

[0020] (A2) a cytosine base machine consisting of the gRNA described in the first aspect and the fusion protein described in the first aspect;

[0021] (A3) the CRISPR / nCas9BE gene editing vector described in the first aspect above;

[0022] (A4) A recombinant bacterium containing the CRISPR / nCas9BE gene editing vector described in the first aspect above.

[0023] In (A4), the recombinant bacterium may be a recombinant Agrobacterium.

[0024] In one embodiment of the present invention, the recombinant bacteria is Agrobacterium EHA105 containing the CRISPR / nCas9BE gene editing vector.

[0025] In a third aspect, the present invention claims protection for the use of the biomaterial described in the second aspect above in any of the following:

[0026] (B1) increasing maize biomass and / or grain yield;

[0027] (B2) Breeding maize varieties with increased biomass and / or grain yield;

[0028] (B3) increasing corn plant height and / or ear length and / or 100-kernel weight and / or kernel yield per ear;

[0029] (B4) Cultivate corn varieties with increased plant height and / or ear length and / or 100-kernel weight and / or kernel yield per ear.

[0030] In a fourth aspect, the present invention claims protection for any of the following biological materials:

[0031] (C1) A protein having an amino acid sequence as shown in SEQ ID No. 7.

[0032] (C2) a nucleic acid molecule encoding the protein described in (C1);

[0033] (C3) An expression cassette, recombinant vector or recombinant bacterium containing the nucleic acid molecule described in (C2).

[0034] Among them, the protein mentioned in (C1) is ZmGRF1 E163K Protein, that is, the wild-type ZmGRF1 protein is obtained by mutating the 163rd amino acid from E to K. Wherein, the wild-type ZmGRF1 protein is SEQ ID No.1 ( ZmGRF1 The genomic sequence of the gene) or SEQ ID No.2 ( ZmGRF1 The protein is encoded by the nucleotide sequence shown in the CDS sequence of the gene. Accordingly, the nucleic acid molecule in (C2) is the protein encoded by the nucleotide sequence shown in SEQ ID No.1 ( ZmGRF1 The nucleotides 1322 and 1323 of the DNA molecule shown in SEQ ID No. 2 ( ZmGRF1 The CDS sequence of the gene is shown in Figure 1, resulting from the substitution of G for A at positions 486 and 487 of the DNA molecule.

[0035] In a fifth aspect, the present invention claims protection for the use of the biomaterial described in the fourth aspect above in any of the following:

[0036] (B1) increasing maize biomass and / or grain yield;

[0037] (B2) Breeding maize varieties with increased biomass and / or grain yield;

[0038] (B3) increasing corn plant height and / or ear length and / or 100-kernel weight and / or kernel yield per ear;

[0039] (B4) Cultivate corn varieties with increased plant height and / or ear length and / or 100-kernel weight and / or kernel yield per ear.

[0040] In the above-mentioned related aspects, in one embodiment of the present invention, the corn is specifically the corn inbred line ZC01.

[0041] Compared with the prior art, the present invention discloses a method for producing corn by site-directed mutation using gene editing technology. ZmGRF1 The invention relates to a method for genetically improving corn biomass and grain yield. The invention is of great significance for cultivating high-yield corn varieties and providing material accumulation for corn breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the phylogenetic tree of the ZmGRF1 protein in the present invention.

[0043] Figure 2 Plasmid map of the CRISPR / nCas9BE base editing vector constructed in the present invention.

[0044] Figure 3 The present invention screened ZmGRF1 Sequencing peak diagram of homozygous gene mutants.

[0045] Figure 4 The homozygous mutant material ZmGRF1 screened by the present invention E163K The results of the plant and ear phenotypic investigation.

[0046] Figure 5 The homozygous mutant material ZmGRF1 screened by the present invention E163K The survey results of plant height, ear length, 100-grain weight and single-ear grain weight were obtained. DETAILED DESCRIPTION

[0047] 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.

[0048] 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.

[0049] The materials used in the embodiments of the present invention are as follows:

[0050] 1. Plant materials

[0051] The plant material used in the present invention is the maize inbred line ZC01, and the genetic transformation material and the progeny of the transformed material were commissioned by China National Seed Group Co., Ltd. The maize inbred line ZC01 is described in the article "Honglin Wang, et al. Pollen self-elimination CRISPR / Cas genome editing prevents transgenic pollen dispersal in maize. Plant Commun. 2023 Nov 13;4(6):100637" and is available to the public from the applicant for use only in repeating the experiments of the present invention and for no other use.

[0052] 2. Vectors and strains

[0053] Vector: The gene editing vector maintained by our laboratory is the CUB backbone vector. The CUB backbone vector is described in "Yanmin Li, et al. Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize." Acta Agronomica Sinica, 2020, Vol. 3, No. 3, pp. 449-456. The CUB backbone vector is available from the applicant for use solely in replicating the experiments described in this invention and is not for any other use.

[0054] Bacterial strain: Trans1-T1 Phage Resistant Chemically Competent Cell (Beijing Quanshijin Biotechnology Co., Ltd., CD501-02).

[0055] 3. Enzymes

[0056] High-fidelity PCR enzyme KAPA HiFi HotStart ReadyMixPCR Kit (Beijing Juhuatech Technology Co., Ltd., KK2602);

[0057] Homologous recombination enzyme NEBuilder® HiFi DNA Assembly Master Mix (Beijing Bailingke Biotechnology Co., Ltd., E2621L);

[0058] HindIII restriction endonuclease (Beijing Bailingke Biotechnology Co., Ltd., R0104).

[0059] 4. Test kit

[0060] QIAquick Gel Extraction Kit (250) (Beijing Qiangxinborui Biotechnology Co., Ltd., 28706);

[0061] AxyPrep Plasmid Extraction Kit (Axygen, AP-MN-P-250).

[0062] Example 1, Maize ZmGRF1 E163K Acquisition of mutants

[0063] 1. Corn ZmGRF1 Acquisition of genes

[0064] By comparing the GRF protein sequences of different species with the phylogenetic tree ( Figure 1 ) analysis to obtain the homologous gene of maize ZmGRF1 (Gene ID: Zm00001eb251820), its nucleotide sequence in the maize genome is shown in SEQ ID No. 1, and its corresponding CDS sequence is shown in SEQ ID No. 2.

[0065] 2. Acquisition of CRISPR / nCas9BE base editing vector

[0066] The nucleotide sequence 5'-tttccacaggctttcttgaacgg-3' (SEQ ID No. 3) in the maize ZmGRF1 gene was selected as the target site for base editing using CRISPR / nCas9BE-mediated precision editing.

[0067] The CRISPR / nCas9BE base editing vector was constructed using the gene editing basic vector CUB preserved in our laboratory as the backbone vector ( Figure 2 ).

[0068] The CRISPR / nCas9BE base editing vector components mainly include:

[0069] (1) The fusion gene expression cassette generated by the nCas9(D10A) fused with APOBEC1 deaminase and uracil glycosylase inhibitor UGI, driven by the promoter of the maize Ubiquitin gene (the amino acid sequence of the fusion protein encoded by the expression cassette is shown in SEQ ID No. 6), is terminated by the NOS terminator. (2) A nuclear localization signal sequence (NLS) is added to the N-terminus of the APOBEC1 deaminase sequence. (3) APOBEC1 deaminase is linked to the N-terminus of nCas9(D10A) via a 16-amino acid linker peptide, and a UGI is linked to the C-terminus of nCas9(D10A). (4) The esgRNA is initiated by the RNA polymerase III class III promoter ZmU6-2 and terminated with a poly T.

[0070] The target site sequence was inserted between the ZmU6-2 promoter and the esgRNA backbone sequence by homologous recombination. The target site amplification fragment ZmU6-2 and esgRNA amplification primers are as follows:

[0071] U6-HF: 5'-CGGGTCACGCTGCACTGCAGAAGCTTCTAATTGGCCCTTACAAAAT-3';

[0072] U6-HR: 5'-TTCAAGAAAGCCTGTGGAAACGGAGCGGTGGTCGCAGCTGAAC-3';

[0073] esgHF: 5'-GTTTCCACAGGCTTTCTTGAAGTTTTAGAGCTAGAAATAGCAAG-3';

[0074] esgHR: 5'-CGCTGCACTGCAGGCATGCAAGCTTAAAAAAAGCACCGAC-3'.

[0075] After homologous recombination, the plasmid with correct sequencing is the CRISPR / nCas9BE base editing vector.

[0076] The complete sequence of the final CRISPR / nCas9BE base editing vector was obtained by concatenating SEQ ID No. 4 and SEQ ID No. 5 in sequence. Sequence ID No. 4 contains the U6-2 promoter at positions 9691-10085, the sgRNA (spacer) coding sequence at positions 10086-10106, and the Ub promoter at positions 96-2091. Sequence ID No. 5 contains the APOBEC1 deaminase coding gene at positions 2226-2912, the nCas9 coding gene at positions 2961-7061, the UGI coding gene at positions 7062-7334, and the NOS terminator at positions 7417-7669.

[0077] 3. Genetic transformation of maize embryos

[0078] The CRISPR / nCas9BE base editing vector obtained above (resulting from SEQ ID No. 4 and SEQ ID No. 5 linked end-to-end) was introduced into the Agrobacterium tumefaciens EHA105 strain. Embryo infection, co-cultivation, callus induction, seedling differentiation, and rooting induction were performed to obtain positively transformed seedlings. The specific steps are as follows:

[0079] Agrobacterium-mediated genetic transformation of maize

[0080] (1) Processing of receptor materials

[0081] Soak the seeds of corn inbred line ZC01 in water at 37℃ for 4h, germinate the seeds in a plate at 28℃ for 48h, and then place the germinated seeds in pots filled with nutrient soil. Ten days after pollination, select the young embryos as the callus induction material. Sterilize the young ears of corn with 75% (volume fraction) ethanol for 10min in the operating table, rinse them with sterile water, and then dry them. Cut off half of the grains with a scalpel, and then separate the young embryos with tweezers and place them in sterile water for use.

[0082] (2) Callus induction and subculture

[0083] Place the young embryos in N6 medium (BINDER, AA958) and cultivate them in the dark at 28℃ for one week. Cut the callus that grows vigorously and has bright color into small pieces, and then place them in N6 medium for continuous cultivation. Subculture them every two weeks to maintain the good state of the callus, and select the callus that grows well for use.

[0084] (3) Agrobacterium infection

[0085] Take out the preserved Agrobacterium (which has introduced the CRISPR / nCas9BE base editing vector), inoculate it for shaking, and when the OD 600 value is about 0.8, centrifuge it at 5000r / min for 10min, collect the bacterial cells, suspend them in infection buffer (1L of infection buffer is obtained by uniformly mixing 4g of N6 medium basic salt medium containing N6 vitamins, 2mg of 2,4-D, 100mg of inositol, 0.7g of L-proline, 68.4g of sucrose, 36g of glucose, 1mL of AgNO3 with a concentration of 10mg / mL, 1mL of As with a concentration of 100mol / L, and water, and the pH is 5.2), so that the OD 600 value is about 0.5, and then shake it at 28℃ and 150r / min for 0.5h to obtain the infection solution. Soak the callus that grows well selected in (2) in the infection buffer for 1h, and then transfer it to the infection solution containing Agrobacterium for 15min, dry it, and obtain the callus after infection.

[0086] (4) Co-culture and recovery culture

[0087] The infected callus was placed in a co-culture medium (1 L co-culture medium was prepared by mixing 4 g of N6 medium basal salts containing N6 vitamins, 2 mg 2,4-D, 30 g sucrose, 8 g agar, 1 mL of 10 mg / mL AgNO3, 1 mL of 100 mol / L As, 3 mL of 100 mg / mL L-cysteine ​​and water, pH 5.8), cultured at 20°C for 3 days, and then transferred to a recovery medium (1 L recovery medium was prepared by mixing 4 g of N6 medium basal salts containing N6 vitamins, 2 mg 2,4-D, 0.7 g L-proline, 30 g sucrose, 0.5 g MES, 4 g phytagel, 1 mL of 10 mg / mL AgNO3, 1 mL of 250 mg / mL cephalosporin and water, pH 5.8) for 10 days. Then, the callus was transferred to a recovery medium supplemented with glufosinate-ammonium to select positive callus.

[0088] (5) Differentiation, redifferentiation, rooting, and seedling hardening

[0089] The above positive callus was transferred to embryoid induction medium (1L embryoid induction medium is obtained by mixing 4.43g MS medium basal salt (containing inositol) containing MS vitamins, 0.25mg 2,4-D, 30g sucrose, 5mg6-BA, 4g plant gel, 1mL Cefo with a concentration of 250mg / mL and water, pH5.8), cultured in the dark for 2 weeks, and then transferred to differentiation medium (1L differentiation medium is obtained by mixing 4.43g MS medium basal salt (containing inositol) containing MS vitamins, 30g sucrose, 4g plant gel, 1mL Cefo with a concentration of 250mg / mL and water, pH5.8), and transferred to rooting medium (1L rooting medium is obtained by mixing 2.215g 1 / 2MS, 30g sucrose, 51.55mg MS vitamins, obtained by mixing 4g of plant gel and water, pH 5.8) take root, grow to a certain height, expose to air for 3 days, and transplant.

[0090] (6) Bar test strips to screen positive plants

[0091] Take a plant leaf of about 3 cm, put it into a tube and grind it thoroughly, add 500 μl of buffer, and insert it into a Bar test strip (Shanghai Youlong Biotechnology Co., Ltd., catalog number: EnviroLogix AS03). The plant with a positive band is the T0 generation positive transgenic plant.

[0092] 4. Maize ZmGRF1 E163K Acquisition and analysis of mutants

[0093] T0-generation positive transgenic plants were obtained through Agrobacterium-mediated genetic transformation of maize immature embryos. Genomic DNA from leaves was extracted, and target site sequences were amplified and Sanger sequencing was performed. The study found that the following mutations occurred in the ZmGRF1 gene:

[0094] SEQ ID No.1( ZmGRF1 The 1322nd and 1323rd bases of the genomic sequence of the gene (corresponding to the SEQ ID No. ZmGRF1 The 486th and 487th bases of the CDS sequence of the gene mutated from G to A. The obtained material was named ZmGRF1 E163K ( Figure 3 ).

[0095] Mutated ZmGRF1 E163K The amino acid sequence is shown in SEQ ID No. 7, and compared with before mutation, the 163rd amino acid is mutated from E to K.

[0096] The T0 generation was test-crossed to obtain the T1 generation, which was then self-crossed to obtain ZmGRF1 without transgenic element insertion and homozygous for the mutation site. E163K Mutant material.

[0097] ZmGRF1 E163K The only difference between the mutant material and the wild-type material ZC01 is that the 1322nd and 1323rd bases of the ZmGRF1 gene (SEQ ID No. 1) in the genome of the wild-type material ZC01 are mutated from G to A, and the rest of the sequence remains unchanged.

[0098] Example 2, Maize ZmGRF1 E163K Phenotypic investigation of mutant materials

[0099] The homozygous ZmGRF1 obtained in Example 1 E163K The mutants were sown in the field and their plant height phenotypes were measured 7 days after pollination. Ear length, 100-grain weight, and single-ear yield phenotypes were measured after harvest. The wild-type maize inbred line ZC01 was used as a control. Data from 10 plants per group were collected, and the results were averaged.

[0100] The results are as follows Figure 4 and Figure 5 As shown, ZmGRF1 E163K Compared with the wild-type material ZC01, the mutant material had significantly increased silage-related phenotypes such as plant height and ear length, as well as grain yield (including 100-grain weight and grain yield per ear).

[0101] The results showed that: ZmGRF1The mutation of nucleotides 1322 and 1323 of the gene (SEQ ID No. 1) from G to A can increase maize biomass and / or grain yield. E163K Helps increase corn biomass and / or grain yield.

[0102] 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 further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for increasing corn biomass and / or grain yield, comprising the following steps: mutating nucleotides 1322 and 1323 of the wild-type ZmGRF1 gene in the corn genome from G to A, thereby increasing corn biomass and / or grain yield; In the maize genome, the wild type ZmGRF1 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The method according to claim 1, wherein: In the method, the nucleotides 1322 and 1323 of the wild-type ZmGRF1 gene in the corn genome are mutated from G to A by introducing a CRISPR / nCas9BE gene editing vector into the corn; The CRISPR / nCas9BE gene editing vector is used to express a cytosine base editor; the cytosine base editor targets the wild-type ZmGRF1 The target sequence in the gene is shown as SEQ ID No.

3.

3. The method according to claim 2, wherein: The CRISPR / nCas9BE gene editing vector can express a fusion protein formed by the fusion of nCas9 (D10A), APOBEC1 deaminase and UGI protein, and the CRISPR / nCas9BE gene editing vector can express gRNA targeting the target sequence.

4. The method according to claim 3, wherein: The amino acid sequence of the fusion protein is shown in SEQ ID No.

6.

5. The method according to claim 3, wherein: The spacer sequence of the gRNA is the reverse complementary sequence of positions 1-20 of SEQ ID No.

3.

6. The method according to any one of claims 2 to 5, wherein: The full sequence of the CRISPR / nCas9BE gene editing vector is obtained by connecting SEQ ID No. 4 and SEQ ID No. 5 end to end in sequence.

7. Biological material, any of the following: (C1) a protein having an amino acid sequence as shown in SEQ ID No. 7; (C2) a nucleic acid molecule encoding the protein described in (C1); (C3) An expression cassette, recombinant vector or recombinant bacterium containing the nucleic acid molecule described in (C2).

8. The biomaterial according to claim 7, wherein: The nucleic acid molecule is obtained by replacing nucleotides 1322 and 1323 of the DNA molecule shown in SEQ ID No. 1 from G to A, or by replacing nucleotides 486 and 487 of the DNA molecule shown in SEQ ID No. 2 from G to A.

9. Use of the biomaterial according to claim 7 or 8 in any of the following: (B1) increasing maize biomass and / or grain yield; (B2) Breeding maize varieties with increased biomass and / or grain yield; (B3) increasing corn plant height and / or ear length and / or 100-kernel weight and / or kernel yield per ear; (B4) Cultivate corn varieties with increased plant height and / or ear length and / or 100-kernel weight and / or kernel yield per ear.

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