Method for improving biomass and / or grain yield of corn through site-directed base mutation of corn ZmGRF3 gene

Abstract

The invention discloses a method for improving biomass and / or grain yield of corn through site-specific base mutation of a corn ZmGRF3 gene. The invention relates to the field of plant genetic engineering, and provides a method for improving corn biomass and / or grain yield, which comprises the following step of: mutating nucleotides at positions 506, 508 and 509 of a wild-type ZmGRF3 gene as shown in SEQ ID No.1 in a corn genome from G to A so as to improve the corn biomass and / or grain yield. The invention has important significance for cultivating high-yield and high-quality silage corn varieties and providing material accumulation for corn breeding.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and in particular to a method for increasing corn biomass and / or grain yield by site-directed base mutation of corn ZmGRF3 gene. Background Art

[0002] Genome editing is a technology for targeted and precise modification of the genome, mainly including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR / Cas). The CRISPR / Cas system is roughly divided into three types: type I, type II and type III. Among them, the type II CRISPR / Cas9 system is relatively simple in composition, requiring only three components: Cas9 protein, tracrRNA and crRNA to function, and is highly efficient and easy to operate, making it the most widely used genome editing system. The characteristic protein of the type II CRISPR / Cas system is Cas9, which has two nuclease domains, RuvC and HNH, which are responsible for cutting the two strands of the target DNA, thereby causing double-strand breaks in the target DNA. The HNH domain is responsible for cutting the DNA strand complementary to the crRNA, and the cutting site is located 3bp upstream of the protospacer adjacent motif (PAM), while the RuvC domain is responsible for cutting the non-complementary strand, and the cutting site is located 3-8bp upstream of the PAM. Cas9 protein has the function of processing and producing crRNA and cutting exogenous nucleic acids. crRNA combines with tracrRNA through base pairing to form a tracrRNA / crRNA complex. Researchers can use tracrRNA and crRNA as two guide RNAs (gRNAs) or fuse the two together to form a single guide RNA (sgRNA). sgRNA can bind to Cas9 nuclease and guide Cas9 to the target site on the genome for cutting.

[0003] The CRISPR / Cas9 system causes double-strand breaks (DSBs) in the target gene DNA, activating the DNA damage repair mechanism in the cell, and then causing deletions, knock-ins, or gene insertions and specific base changes when there is a donor template. However, it is difficult to achieve efficient and stable single-base mutations in DNA repair caused by DSBs, so it is particularly important to develop a technology that can accurately and efficiently achieve single-base replacements. In addition, concerns about the genotoxicity of DNA double-strand breaks and the low efficiency of homology-directed repair (HDR) have further promoted the development of "second-generation" CRISPR technologies, which mediate genome editing without relying on the formation of DNA double-strand breaks and HDR. The most representative of these technologies are base editors (BE) and prime editors (PE). Scientists have developed several different base editors, the most commonly used of which are cytosine base editors (CBE) and adenine base editors (ABE). These base editors do not rely on the generation of DSBs when working, and do not require the participation of donor DNA.

[0004] The first DNA base editing system achieves the conversion of C•G base pairs to T•A base pairs by causing the target cytosine to deaminize to uracil. In order to precisely limit the deamination to a specific small target region in the genome, the researchers used APOBEC1 cytidine deaminase, which uses single-stranded DNA (ssDNA) as a substrate and cannot affect double-stranded DNA (dsDNA). The deaminase contained in APOBEC1 is fused with an inactivated Cas9 (nCas9) from Streptococcus pyogenes to produce base editor 1 (BE1). When BE1 binds to homologous DNA, nCas9 denatures a specific position in the DNA double helix, then generates an R loop, and the DNA strand that is not paired with the guide RNA becomes a single-stranded bubble. This feature enables BE1 to efficiently deaminize local cytosine, and its deamination activity window is limited to a window of about 5bp in the size of the ssDNA generated by nCas9 (positions approximately between 4-8, with PAM as positions 21-23). After fusion with nCas9, the effective molar concentration of APOBEC1 is high, allowing BE1 to deaminize cytosine in the sequence. nCas9BE is a class of base editing vectors that fuse nCas9 with APOBEC1. It has been publicly released as a free nuclease for academic and commercial use.

[0005] corn( Zea maysL.) is a major food crop and industrial raw material. my country is one of the world's major corn producers. Corn plays a vital role in national production and stable economic development. The general goals of corn genetic improvement are high yield, high quality, stable yield, suitable growth period and adaptability to mechanization. Among them, high yield has always been the most important breeding goal. Therefore, how to ensure the increase of corn yield has become a key issue in current corn breeding.

[0006] Transcription factors control gene expression, thereby regulating the growth and development patterns of plants. The identification and functional characterization of transcription factors are prerequisites for analyzing biological life activities. A class of plant-specific transcription factors, growth-regulating factors (GRFs), are important regulatory factors for plant growth and development. GRF proteins contain two highly conserved QLQ and WRC domains in the N-terminal half. The QLQ domain is composed of highly conserved Gln-Leu-Gln (QX3LX2Q) and adjacent residues. The QLQ domain provides an interface for interaction with GIF, and this functional domain is very similar to the SWI2 / SNF2 protein in yeast. The WRC domain is composed of three conserved spaced Cys and one His residue (CX9CX10CX2H, referred to as the C3H motif), which serves as a DNA binding domain. As transcription factors with WRC and DBD domains, GRFs are expected to bind to specific cis-elements in downstream target genes and regulate the expression of downstream target genes. GRFs transcription factors mainly regulate the growth and development of plant roots, stems, leaves, and flowers, as well as the adaptability of plants to adverse stress and the life span of plants.

[0007] Therefore, a method for precise targeting using gene editing technology is provided. ZmGRF3 The method of carrying out base mutation of genes and creating high-yield and high-quality silage corn germplasm is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0008] The purpose of the present invention is to provide a site-directed base mutation ZmGRF3 Methods and applications of genetically improved high-yield and high-quality silage corn varieties.

[0009] In a first aspect, the present invention claims a method for increasing corn biomass and / or grain yield.

[0010] The method for increasing corn biomass and / or grain yield claimed in the present invention may include the following steps: ZmGRF3 The nucleotides at positions 506, 508 and 509 of the gene are mutated from G to A, thereby increasing the biomass and / or grain yield of corn; In the maize genome, the wild type ZmGRF3The nucleotide sequence of the gene is shown in SEQ ID No. 1. ZmGRF3 The CDS sequence of the gene is shown in SEQ ID No. 2. Mutating the 506th, 508th and 509th nucleotides of the wild-type ZmGRF3 gene (i.e., SEQ ID No. 1) in the maize genome from G to A corresponds to the CDS sequence equivalent to mutating the 394th, 396th and 397th nucleotides of SEQ ID No. 2 from G to A.

[0011] In the method, the wild type in the corn genome can be modified by introducing a CRISPR / nCas9BE gene editing vector into the corn. ZmGRF3 The nucleotides 506, 508, and 509 of the gene mutated from G to A; The CRISPR / nCas9BE gene editing vector is used to express a cytosine base editor; the cytosine base editor targets the wild-type ZmGRF3 The target sequence in the gene is shown as SEQ ID No.3.

[0012] Furthermore, 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 a 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 in sequence end to end.

[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 leaf area 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 leaf area 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: (A1) the gRNA described in the first aspect above; (A2) a cytosine base machine consisting of the gRNA described in the first aspect and the fusion protein described in the first aspect; (A3) the CRISPR / nCas9BE gene editing vector described in the first aspect above; (A4) A recombinant bacterium containing the CRISPR / nCas9BE gene editing vector described in the first aspect above.

[0019] In (A4), the recombinant bacterium may be a recombinant Agrobacterium.

[0020] In one embodiment of the present invention, the recombinant bacteria is Agrobacterium EHA105 containing the CRISPR / nCas9BE gene editing vector.

[0021] 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: (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 leaf area and / or ear length and / or 100-grain weight and / or grain yield per ear; (B4) Breed corn varieties with increased plant height and / or leaf area and / or ear length and / or 100-grain weight and / or single-ear grain yield.

[0022] In a fourth aspect, the present invention claims protection for any of the following biological materials: (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, a recombinant vector or a recombinant bacterium containing the nucleic acid molecule described in (C2).

[0023] Among them, the protein in (C1) is ZmGRF3 V132I / E133K Protein, that is, the 132nd amino acid of the wild-type ZmGRF3 protein is mutated from V to I, and the 133rd amino acid is mutated from E to K. Wherein, the wild-type ZmGRF3 protein is obtained by SEQ ID No.1 ( ZmGRF3 The genomic sequence of the gene) or SEQ ID No.2 ( ZmGRF3 The protein is encoded by the nucleotide sequence shown in SEQ ID No.1 ( ZmGRF3The 506th, 508th and 509th nucleotides of the DNA molecule shown in SEQ ID No. 2 ( ZmGRF3 The CDS sequence of the gene is obtained by replacing the 394th, 396th and 397th nucleotides of the DNA molecule by G.

[0024] 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: (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 leaf area and / or ear length and / or 100-grain weight and / or grain yield per ear; (B4) Breed corn varieties with increased plant height and / or leaf area and / or ear length and / or 100-grain weight and / or single-ear grain yield.

[0025] Corn biomass is a key factor in determining silage yield. The higher the biomass, the more corn material can be used for silage, and the higher the silage yield will be.

[0026] In the above-mentioned related aspects, in an embodiment of the present invention, the corn is specifically the corn inbred line ZC01.

[0027] Compared with the prior art, the present invention provides a method for producing corn by site-directed mutation using gene editing technology. ZmGRF3 The method for genetically improving corn biomass and grain yield is of great significance for cultivating high-yield and high-quality silage corn varieties and providing material accumulation for corn breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the phylogenetic tree of the ZmGRF3 protein in the present invention.

[0029] Figure 2 The present invention screened ZmGRF3 Sequencing peak diagram of homozygous gene mutants.

[0030] Figure 3 The homozygous mutant material ZmGRF3 screened by the present invention V132I / E133K The results of the plant, leaf and ear phenotype investigation.

[0031] Figure 4 The homozygous mutant material ZmGRF3 screened by the present invention V132I / E133K The survey results of plant height, ear length, 100-grain weight and single-ear grain weight were presented. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] The materials used in the embodiments of the present invention are as follows: 1. Plant materials The plant material used in the present invention is the maize inbred line ZC01, and China National Seed Group Corporation was commissioned to carry out genetic transformation materials and progeny materials of the transformation materials. The maize inbred line ZC01 is recorded 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", which can be obtained by the public from the applicant and can only be used for repeating the experiments of the present invention and cannot be used for other purposes.

[0035] 2. Vectors and strains Vector: The basic gene editing vector preserved by this laboratory is the CUB backbone vector. The CUB backbone vector is recorded in the article "Yanmin Li, et al. Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize. Acta Agronomica Sinica, 2020, No. 3, P. 449-456", which can be obtained by the public from the applicant and can only be used to repeat the experiments of the present invention and cannot be used for other purposes.

[0036] Bacterial strain: Trans1-T1 Phage Resistant Chemically Competent Cell (Beijing Quanshijin Biotechnology Co., Ltd., CD501-02).

[0037] 3. Enzymes High-fidelity PCR enzyme KAPA HiFi HotStart ReadyMixPCR Kit (Beijing Juhuatech Technology Co., Ltd., KK2602); Homologous recombination enzyme NEBuilder ® HiFi DNA Assembly Master Mix (Beijing Bailingke Biotechnology Co., Ltd., E2621L); HindⅢ restriction endonuclease (Beijing Bailingke Biotechnology Co., Ltd., R0104).

[0038] 4. Test kit QIAquick Gel Extraction Kit (250) (Beijing Qiangxinborui Biotechnology Co., Ltd., 28706); AxyPrep Plasmid Extraction Kit (Axygen, AP-MN-P-250).

[0039] Example 1, Maize ZmGRF3 V132I / E133K Acquisition of mutants 1. Corn ZmGRF3 Acquisition of genes By comparing the GRF protein sequences of different species with the phylogenetic tree ( Figure 1 ) analysis to obtain the homologous gene of maize ZmGRF3 (Gene number: Zm00001eb193180), 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.

[0040] 2. Acquisition of CRISPR / nCas9BE base editing vector Select corn ZmGRF3 The nucleotide sequence 5'-tttccacaggctttcttgaacgg-3' (SEQ ID No. 3) in the gene was used as the target site. CRISPR / nCas9BE-mediated precision editing was used for base editing.

[0041] The CRISPR / nCas9BE base editing vector was constructed using the gene editing basic vector CUB preserved in our laboratory as the backbone vector.

[0042] CRISPR / nCas9BE base editing vector elements mainly include: (1) The fusion gene expression cassette obtained by fusion of nCas9(D10A) with APOBEC1 deaminase and uracil glycosylase inhibitor UGI driven by the promoter of maize Ubiquitin gene (the amino acid sequence of the fusion protein encoded by the expression cassette is shown in SEQ ID No.6), with NOS terminator as the terminator. (2) A nuclear localization signal sequence NLS is added to the N-terminus of the APOBEC1 deaminase sequence. (3) APOBEC1 deaminase is connected to the N-terminus of nCas9(D10A) with a 16-amino acid linker peptide, and a UGI is connected to the C-terminus of nCas9(D10A). (4) esgRNA is driven by RNA polymerase class III promoter ZmU6-2 and terminated with poly T.

[0043] 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: U6-HF: 5'-CGGGTCACGCTGCACTGCAGAAGCTTCTAATTGGCCCTTACAAAAT-3' (SEQ IDNo. ​​8); U6-HR: 5'-TTCAAGAAAGCCTGTGGAAACGGAGCGGTGGTCGCAGCTGAAC-3' (SEQ ID No. 9); esgHF: 5'-GTTTCCACAGGCTTTCTTGAAGTTTTAGAGCTAGAAATAGCAAG-3' (SEQ IDNo. ​​10); esgHR: 5'-CGCTGCACTGCAGGCATGCAAGCTTAAAAAAAGCACCGAC-3' (SEQ ID No. 11).

[0044] After homologous recombination, the plasmid with correct sequencing is the CRISPR / nCas9BE base editing vector.

[0045] The final constructed CRISPR / nCas9BE base editing vector sequence is obtained by connecting SEQ ID No. 4 and SEQ ID No. 5 in sequence. The 9691-10085th positions of SEQ ID No. 4 are U6-2 promoter, the 10086-10106th positions are the coding sequence of sgRNA (spacer sequence), the 96-2091th positions of SEQ ID No. 5 are Ub promoter, the 2226-2912th positions are the coding gene of APOBEC1 deaminase, the 2961-7061th positions are the nCas9 coding gene, the 7062-7334th positions are the UGI coding gene, and the 7417-7669th positions are the NOS terminator.

[0046] 3. Genetic transformation of maize embryos The CRISPR / nCas9BE base editing vector (the one obtained by connecting SEQ ID No.4 and SEQ ID No.5 in sequence) was introduced into the Agrobacterium EHA105 strain; embryo infection, co-cultivation, callus induction, seedling differentiation, and rooting induction were performed to finally obtain positive transformation seedlings. The specific steps are as follows: (1) Processing of receptor materials Soak the seeds of the maize inbred line ZC01 in water at 37℃ for 4h, germinate them in a plate at 28℃ for 48h, place the germinated seeds in a pot filled with nutrient soil for cultivation, and select young embryos as callus induction materials 10 days after pollination. Sterilize the young ears of maize with 75% (volume fraction) ethanol for 10min on the operating table, rinse with sterile water, and dry them. Cut off half of the kernel with a scalpel, then separate the young embryos with tweezers and place them in sterile water for later use.

[0047] (2) Callus induction and subculture Place the young embryos in N6 medium (BINDER, AA958) and culture in the dark at 28°C for one week. Cut the vigorously growing and brightly colored callus into small pieces and place them on N6 medium for continued culture. Subculture every two weeks to keep the callus in good condition and select the callus with good growth for later use.

[0048] (3) Agrobacterium infection Take out the stored Agrobacterium (with CRISPR / nCas9BE base editing vector introduced), inoculate and shake the bacteria, and wait for OD 600The value was about 0.8, and the mixture was centrifuged at 5000r / min for 10min. The bacteria were collected and suspended in infection buffer (1L infection buffer was prepared by mixing 4g of N6 medium basal salt containing N6 vitamins, 2mg 2,4-D, 100mg inositol, 0.7g L-proline, 68.4g sucrose, 36g glucose, 1mL of 10mg / mL AgNO3, 1mL of 100mol / L As and water, pH 5.2) to make OD 600 The value is about 0.5, and then oscillate at 28℃ and 150r / min for 0.5h to obtain the infection solution. The callus tissue with good growth selected in (2) is soaked in the infection buffer for 1h, then transferred to the infection solution containing Agrobacterium and soaked for 15min, and dried to obtain the infected callus tissue.

[0049] (4) Co-culture and recovery culture The infected callus was placed in a co-culture medium (1L of co-culture medium was prepared by mixing 4g of N6 medium basal salt containing N6 vitamin, 2mg 2,4-D, 30g of sucrose, 8g of agar, 1mL of 10mg / mL AgNO3, 1mL of 100mol / L As, 3mL of 100mg / mL L-cysteine ​​and water, pH5.8), cultured at 20°C for 3 days, transferred to a recovery medium (1L of recovery medium was prepared by mixing 4g of N6 medium basal salt containing N6 vitamin, 2mg 2,4-D, 0.7g of L-proline, 30g of sucrose, 0.5g of MES, 4g of plant gel, 1mL of 10mg / mL AgNO3, 1mL of 250mg / mL cephalosporin and water, pH5.8), cultured for 10 days, and then transferred to a recovery medium with glufosinate added to screen for positive callus.

[0050] (5) Differentiation, redifferentiation, rooting, and seedling hardening The positive callus tissue 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, 5mg 6-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 vitamin, obtained by mixing 4g of plant gel and water, pH 5.8) to take root, grow to a certain height, expose to the air for 3 days, and then transplant the seedlings.

[0051] (6) Bar test strips to screen positive plants Take a plant leaf of about 3 cm, put it into a tube and grind it thoroughly, add 500 μl of buffer, insert it into a Bar test strip (Shanghai Youlong Biotechnology Co., Ltd., catalog number: EnviroLogix AS03), and the plant with a positive band is the T0 generation positive transgenic plant.

[0052] 4. Maize ZmGRF3 V132I / E133K Acquisition and analysis of mutants T0 positive transgenic plants were obtained by Agrobacterium-mediated genetic transformation of maize embryos, and leaf genomic DNA was extracted, and the target site sequences were amplified and Sanger sequencing was performed. ZmGRF3 The following mutations occurred in the gene: SEQ ID No.1( ZmGRF3 The 506th, 508th and 509th bases of the genomic sequence of the gene (corresponding to SEQ ID No.2 ZmGRF3 The 394th, 396th and 397th bases of the CDS sequence of the gene mutated from G to A. The obtained material was named ZmGRF3 V132I / E133K ( Figure 2 ).

[0053] Mutated ZmGRF3 V132I / E133K The amino acid sequence is shown in SEQ ID No.7. Compared with before mutation, the 132nd amino acid mutated from V to I, and the 133rd amino acid mutated from E to K.

[0054] The T0 generation was tested to obtain the T1 generation, which was then further selfed to obtain ZmGRF3 without transgenic element insertion and homozygous for the mutation site.V132I / E133K Mutant material.

[0055] ZmGRF3 V132I / E133K The mutant material is different from the wild-type material ZC01 in that the wild-type material ZC01 genome is replaced by ZmGRF3 The 506th, 508th and 509th bases of the gene (SEQ ID No. 1) mutated from G to A, and the rest of the sequence remained unchanged.

[0056] Example 2, Maize ZmGRF3 V132I / E133K Phenotypic investigation of mutant materials The homozygous ZmGRF3 obtained in Example 1 V132I / E133K The mutant material was sown in the field, and the plant height and leaf area phenotypes were investigated 7 days after pollination. The ear length, 100-grain weight and single ear yield phenotypes were investigated after harvest. At the same time, the maize inbred line ZC01 was used as the wild type control. Data of 10 plants were counted in each group, and the results were averaged.

[0057] The results are as follows Figure 3 and Figure 4 As shown, ZmGRF3 V132I / E133K Compared with the wild-type material ZC01, the mutant material had significantly increased silage-related phenotypes such as plant height, leaf area, and ear length, as well as grain yield (including 100-grain weight and grain yield per ear).

[0058] The results showed that: ZmGRF3 The nucleotides 506, 508 and 509 of the gene (SEQ ID No. 1) are mutated from G to A, which can increase the biomass and / or grain yield of corn. V132I / E133K Helps increase corn biomass and / or grain yield.

[0059] 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 increasing corn biomass and / or grain yield, comprising the following steps: ZmGRF3 The nucleotides at positions 506, 508 and 509 of the gene are mutated from G to A, thereby increasing the biomass and / or grain yield of corn; In the maize genome, the nucleotide sequence of the wild-type ZmGRF3 gene is shown as SEQ ID No.

1.

2. The method according to claim 1, characterized in that: In the method, the wild type in the corn genome is modified by introducing a CRISPR / nCas9BE gene editing vector into the corn. ZmGRF3 The nucleotides 506, 508, and 509 of the gene mutated from G to A; The CRISPR / nCas9BE gene editing vector is used to express a cytosine base editor; the cytosine base editor targets the wild-type ZmGRF3 The target sequence in the gene is shown as SEQ ID No.

3.

3. The method according to claim 2, characterized in that: 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 a gRNA targeting the target sequence.

4. The method according to claim 3, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ ID No.6; and / or The spacer sequence of the gRNA is the reverse complementary sequence of positions 1 to 20 of SEQ ID No.

3.

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

6. Biomaterial, characterized in that: The biological material is any one of the following (A1)-(A4): (A1) The gRNA described in claim 3 or 4; (A2) a cytosine base machine consisting of the gRNA described in claim 3 or 4 and the fusion protein described in claim 3 or 4; (A3) The CRISPR / nCas9BE gene editing vector described in any one of claims 2 to 5; (A4) A recombinant bacterium containing the CRISPR / nCas9BE gene editing vector described in any one of claims 2 to 5.

7. Use of the biomaterial according to claim 6 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 leaf area and / or ear length and / or 100-grain weight and / or grain yield per ear; (B4) Breed corn varieties with increased plant height and / or leaf area and / or ear length and / or 100-grain weight and / or single-ear grain yield.

8. Biomaterial, characterized in that: The biological material is any one of the following (C1)-(C3): (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, a recombinant vector or a recombinant bacterium containing the nucleic acid molecule described in (C2).

9. The biomaterial according to claim 8, characterized in that: The nucleic acid molecule is obtained by replacing nucleotides at positions 506, 508 and 509 of the DNA molecule shown in SEQ ID No.1 by G with A, or by replacing nucleotides at positions 394, 396 and 397 of the DNA molecule shown in SEQ ID No.2 by G with A.

10. Use of the biomaterial according to claim 8 or 9 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 leaf area and / or ear length and / or 100-grain weight and / or grain yield per ear; (B4) Breed corn varieties with increased plant height and / or leaf area and / or ear length and / or 100-grain weight and / or single-ear grain yield.

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