A method for improving maize biomass and / or grain yield by site-directed base mutation of maize ZmGRF3 gene

Abstract

The present invention discloses a method for improving maize biomass and / or grain yield by site-directed base mutation of the maize ZmGRF3 gene. The present invention relates to the field of plant genetic engineering and provides a method for improving maize biomass and / or grain yield, including: mutating the nucleotides at positions 506, 508, and 509 of the wild-type ZmGRF3 gene shown in SEQ ID No.1 in the maize genome from G to A, thereby achieving the improvement of maize biomass and / or grain yield. The present invention is of great significance for cultivating high-yield and high-quality silage maize varieties and accumulating materials for maize breeding.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and specifically relates to a method for increasing maize biomass and / or grain yield by site-directed base mutation of maize 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 categories: type I, type II, and type III. Among them, the composition of the type II CRISPR / Cas9 system is relatively simple, and only three components, namely Cas9 protein, tracrRNA, and crRNA, are required to function. Moreover, it has high efficiency and easy operation, and has become the most widely used genome editing system at present. The characteristic protein of the type II CRISPR / Cas system is Cas9. The Cas9 protein has two nuclease domains, RuvC and HNH. These domains are responsible for cleaving both strands of the target DNA, thereby causing double-strand breaks in the target DNA. Among them, the HNH domain is responsible for cleaving the DNA strand complementary to crRNA, and the cleavage site is located 3 bp upstream of the protospacer adjacent motif (PAM). The RuvC domain is responsible for cleaving the non-complementary strand, and the cleavage site is located 3-8 bp upstream of the PAM. The Cas9 protein also has the functions of processing crRNA and cleaving exogenous nucleic acids. crRNA binds to 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). The sgRNA can bind to the Cas9 endonuclease and guide Cas9 to the genome to cleave the target site.

[0003] The CRISPR / Cas9 system induces double-strand breaks (DSBs) in the target gene DNA, activates the DNA damage repair mechanism in cells, and then generates 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 through DNA repair induced by DSBs. Therefore, it is particularly important to develop a precise technology that can efficiently achieve single-base substitutions. 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 ones are base editors (BE) and prime editors (PE). Scientists have developed several different base editors, and the most commonly used are cytosine base editors (CBE) and adenine base editors (ABE). These base editors do not rely on the generation of DSBs during operation and do not require the participation of donor DNA.

[0004] The first DNA base editing system achieved the conversion of C•G base pairs to T•A base pairs by promoting the deamination of target cytosine to uracil. To precisely confine the deamination to a specific small target region in the genome, researchers used APOBEC1 cytidine deaminase, which acts on single-stranded DNA (ssDNA) and cannot affect double-stranded DNA (dsDNA). The deaminase contained in APOBEC1 was fused with inactivated Cas9 (nCas9) from Streptococcus pyogenes to generate base editor 1 (BE1). When BE1 binds to homologous DNA, nCas9 denatures a specific position in the DNA double strand, and then generates an R-loop. The DNA strand that does not pair with the guide RNA becomes a single-stranded bubble. This property enables BE1 to efficiently perform local cytosine deamination, and its deamination activity window is limited to a window of approximately 5 bp in size of the ssDNA generated by nCas9 (positions approximately between 4 - 8, numbered as positions 21 - 23 with PAM). After fusion with nCas9, the effective molar concentration of APOBEC1 is relatively high, enabling BE1 to deaminate cytosine in the sequence. nCas9BE is a type of base editing vector that fuses nCas9 with APOBEC1. It has been publicly released as a free nuclease for academic and commercial use.

[0005] Maize ( Zea maysMaize (Zea mays L.) is a major food crop and industrial raw material, and China is one of the major maize-producing countries in the world. Maize plays a crucial role in national production and stable economic development. The general objectives of maize genetic improvement are high yield, good quality, stable yield, suitable growth period, and adaptability to mechanization. Among them, high yield has always been one of the most important breeding objectives in breeding. Therefore, how to ensure the increase in maize yield has become the key issue in current maize breeding.

[0006] Transcription factors control gene expression and thus regulate the growth and development patterns of plants. The identification and functional characterization of transcription factors are prerequisite steps for analyzing the biological activities of organisms. A class of plant-specific transcription factors, growth-regulating factors (GRFs), are important regulators of plant growth and development. GRF proteins contain two highly conserved QLQ and WRC domains in the N-terminal half. The QLQ domain consists of the 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 consists of three conservatively spaced Cys and one His residue (CX9CX10CX2H, abbreviated 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. GRF transcription factors mainly regulate the growth and development of plant roots, stems, leaves, and floral organs, as well as regulate the adaptability of plants to stress and the lifespan of plants.

[0007] Therefore, providing a method for precise targeted ZmGRF3 gene base mutation using gene editing technology to create high-yield and high-quality silage maize germplasm is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0008] The object of the present invention is to provide a method and application for site-directed base mutation ZmGRF3 gene improvement to cultivate high-yield and high-quality silage maize varieties.

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

[0010] The method for increasing maize biomass and / or grain yield claimed by the present invention may include the following steps: mutating the 506th, 508th, and 509th nucleotides of the wild-type ZmGRF3 gene in the maize genome from G to A, thereby achieving an increase in maize biomass and / or grain yield;

[0011] In the maize genome, the wild-typeZmGRF3 The nucleotide sequence of the gene is shown in SEQ ID No.1. The wild-type ZmGRF3 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 mutating the 394th, 396th, and 397th nucleotides of SEQ ID No.2 from G to A in the CDS sequence.

[0012] In the method, the mutation of the 506th, 508th, and 509th nucleotides of the wild-type ZmGRF3 gene in the maize genome from G to A can be achieved by introducing a CRISPR / nCas9BE gene editing vector into the maize;

[0013] The CRISPR / nCas9BE gene editing vector is used to express a cytosine base editor; the cytosine base editor targets the target sequence in the wild-type ZmGRF3 gene, and the target sequence is shown in SEQ ID No.3.

[0014] Furthermore, 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 a gRNA targeting the target sequence.

[0015] Even further, the amino acid sequence of the fusion protein can be as shown in SEQ ID No.6; the spacer sequence of the gRNA is the reverse complementary sequence of the 1st - 20th positions of SEQ ID No.3.

[0016] In one embodiment of the present invention, the full sequence of the CRISPR / nCas9BE gene editing vector is obtained by sequentially connecting the head and tail of SEQ ID No.4 and SEQ ID No.5.

[0017] The method is also a method for cultivating maize varieties with increased biomass and / or grain yield.

[0018] The method is also a method for increasing the plant height and / or leaf area and / or ear length and / or 100 - kernel weight and / or single - ear grain yield of maize.

[0019] The method is also a method for cultivating maize varieties with increased plant height and / or leaf area and / or ear length and / or 100 - kernel weight and / or single - ear grain yield.

[0020] In the second aspect, the present invention claims any of the following biological materials:

[0021] (A1) The gRNA described in the first aspect above;

[0022] (A2) A cytosine base editor composed of the gRNA described in the first aspect above and the fusion protein described in the first aspect above;

[0023] (A3) The CRISPR / nCas9BE gene editing vector described in the first aspect above;

[0024] (A4) A recombinant bacterium containing the CRISPR / nCas9BE gene editing vector described in the first aspect above.

[0025] In (A4), the recombinant bacterium can be a recombinant Agrobacterium.

[0026] In one embodiment of the present invention, the recombinant bacterium is Agrobacterium EHA105 containing the CRISPR / nCas9BE gene editing vector.

[0027] In a third aspect, the present invention claims the use of the biological material described in the second aspect above in any of the following:

[0028] (B1) Increasing the biomass and / or grain yield of maize;

[0029] (B2) Cultivating maize varieties with increased biomass and / or grain yield;

[0030] (B3) Increasing the plant height and / or leaf area and / or ear length and / or 100-grain weight and / or single-ear grain yield of maize;

[0031] (B4) Cultivating maize varieties with increased plant height and / or leaf area and / or ear length and / or 100-grain weight and / or single-ear grain yield.

[0032] In a fourth aspect, the present invention claims the biological material shown in any of the following:

[0033] (C1) A protein with an amino acid sequence shown in SEQ ID No. 7;

[0034] (C2) A nucleic acid molecule encoding the protein described in (C1);

[0035] (C3) An expression cassette or recombinant vector or recombinant bacterium containing the nucleic acid molecule described in (C2).

[0036] Among them, the protein described in (C1) is the ZmGRF3 V132I / E133K protein, which is obtained by mutating the 132nd amino acid of the wild-type ZmGRF3 protein from V to I and the 133rd amino acid from E to K. Among them, the wild-type ZmGRF3 protein is the one encoded by SEQ ID No. 1 (ZmGRF3 the genomic sequence of the gene) or SEQ ID No. 2 ( ZmGRF3 the protein encoded by the nucleotide sequence shown in the CDS sequence of the gene). Accordingly, the nucleic acid molecule described in (C2) is obtained by replacing the nucleotides at positions 506, 508, and 509 of the DNA molecule shown in SEQ ID No. 1 ( ZmGRF3 the genomic sequence of the gene) with A, or is obtained by replacing the nucleotides at positions 394, 396, and 397 of the DNA molecule shown in SEQ ID No. 2 ( ZmGRF3 the CDS sequence of the gene) with A.

[0037] Fifthly, the present invention claims the application of the biological material described in the fourth aspect above in any one of the following:

[0038] (B1) increasing the biomass and / or grain yield of maize;

[0039] (B2) cultivating maize varieties with increased biomass and / or grain yield;

[0040] (B3) increasing the plant height and / or leaf area and / or ear length and / or 100-kernel weight and / or single-ear grain yield of maize;

[0041] (B4) cultivating maize varieties with increased plant height and / or leaf area and / or ear length and / or 100-kernel weight and / or single-ear grain yield.

[0042] Maize biomass is a key factor determining the silage yield. The higher the biomass, the more maize material available for silage, and the higher the final silage feed yield.

[0043] In one embodiment of the present invention in the above relevant aspects, the maize is specifically the maize inbred line ZC01.

[0044] Compared with the prior art, the present invention provides a method for improving the biomass and grain yield of maize by using gene editing technology to site-specifically mutate the ZmGRF3 gene of maize. The present invention is of great significance for cultivating high-yield and high-quality silage maize varieties and accumulating materials for maize breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the phylogenetic tree of the ZmGRF3 protein in the present invention.

[0046] Figure 2 It is the ZmGRF3 sequencing peak map of the homozygous mutant of the gene screened in the present invention.

[0047] Figure 3 It is the homozygous mutant material ZmGRF3 screened in the present inventionV132I / E133K Phenotypic investigation results of plants, leaves and ears

[0048] Figure 4 The homozygous mutant material ZmGRF3 screened for the present invention V132I / E133K Investigation results of plant height, ear length, 100-grain weight and grain weight per ear traits Specific implementation manners

[0049] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0050] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0051] The materials used in the embodiments of the present invention are as follows:

[0052] 1. Plant materials

[0053] The plant material used in the present invention is the maize inbred line ZC01, and the applicant entrusts China National Seed Group Co., Ltd. to conduct genetic transformation materials and the progeny materials of the transformed materials. The maize inbred line ZC01 is recorded in the article "Honglin Wang, etal.Pollen self-elimination CRISPR / Cas genome editing prevents transgenicpollen dispersal in maize. Plant Commun. 2023 Nov 13;4(6):100637", and the public can obtain it from the applicant and can only be used for repeating the experiments of the present invention and cannot be used for other purposes.

[0054] 2. Vectors and strains

[0055] Vector: The basic vector for gene editing 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.

[0056] Bacterial strain: Trans1-T1 Phage Resistant Chemically Competent Cell (Beijing Quanshijin Biotechnology Co., Ltd., CD501-02).

[0057] 3. Enzymes

[0058] High-fidelity PCR enzyme KAPA HiFi HotStart ReadyMixPCR Kit (Beijing Juhuatech Technology Co., Ltd., KK2602);

[0059] Homologous recombination enzyme NEBuilder ® HiFi DNA Assembly Master Mix (Beijing Bailingke Biotechnology Co., Ltd., E2621L);

[0060] HindⅢ restriction endonuclease (Beijing Bailingke Biotechnology Co., Ltd., R0104).

[0061] 4. Test kit

[0062] QIAquick Gel Extraction Kit (250) (Beijing Qiangxinborui Biotechnology Co., Ltd., 28706);

[0063] AxyPrep Plasmid Extraction Kit (Axygen, AP-MN-P-250).

[0064] Example 1, Maize ZmGRF3 V132I / E133K Acquisition of mutants

[0065] 1. Corn ZmGRF3 Acquisition of genes

[0066] 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 ID: Zm00001eb193180), whose nucleotide sequence in the maize genome is shown in SEQ ID No.1, and the corresponding CDS sequence is shown in SEQ ID No.2.

[0067] 2. Obtaining of CRISPR / nCas9BE Base Editing Vector

[0068] Select the nucleotide sequence 5'-tttccacaggctttcttgaacgg-3' (SEQ ID No.3) in the maize ZmGRF3 gene as the target site. Use CRISPR / nCas9BE-mediated precise editing for base editing.

[0069] Construct the CRISPR / nCas9BE base editing vector using the gene editing basic vector CUB stored in this laboratory as the backbone vector.

[0070] The main components of the CRISPR / nCas9BE base editing vector include:

[0071] (1) A fusion gene expression cassette obtained by fusing nCas9(D10A) 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 this expression cassette is shown in SEQ ID No.6), with NOS terminator as the terminator. (2) Add a nuclear localization signal sequence NLS to the N-terminus of the APOBEC1 deaminase sequence. (3) Connect the APOBEC1 deaminase to the N-terminus of nCas9(D10A) with a 16-amino acid linker peptide, and connect a UGI to the C-terminus of nCas9(D10A). (4) The esgRNA is driven by the RNA polymerase III promoter ZmU6-2 and terminated with poly T.

[0072] Insert the target site sequence 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:

[0073] U6-HF: 5'-CGGGTCACGCTGCACTGCAGAAGCTTCTAATTGGCCCTTACAAAAT-3' (SEQ ID No.8);

[0074] U6-HR: 5'-TTCAAGAAAGCCTGTGGAAACGGAGCGGTGGTCGCAGCTGAAC-3' (SEQ ID No.9);

[0075] esgHF: 5'-GTTTCCACAGGCTTTCTTGAAGTTTTAGAGCTAGAAATAGCAAG-3' (SEQ ID No.10);

[0076] esgHR: 5'-CGCTGCACTGCAGGCATGCAAGCTTAAAAAAAGCACCGAC-3' (SEQ ID No.11).

[0077] After homologous recombination, the plasmid with correct sequencing is the CRISPR / nCas9BE base editing vector.

[0078] The full sequence of the finally constructed CRISPR / nCas9BE base editing vector is obtained by sequentially connecting the head and tail of SEQ ID No.4 and SEQ ID No.5. The 9691-10085th positions of SEQ ID No.4 are the U6-2 promoter, the 10086-10106th positions are the coding sequence of sgRNA (spacer sequence), the 96-2091st positions of SEQ ID No.5 are the Ub promoter, the 2226-2912th positions are the coding gene of APOBEC1 deaminase, the 2961-7061st positions are the nCas9 coding gene, the 7062-7334th positions are the UGI coding gene, and the 7417-7669th positions are the NOS terminator.

[0079] 3. Genetic transformation of maize immature embryos

[0080] The CRISPR / nCas9BE base editing vector (obtained by sequentially connecting the head and tail of SEQ ID No.4 and SEQ ID No.5) is introduced into Agrobacterium tumefaciens strain EHA105; the processes of embryo excision and infection, co-culture, callus induction, shoot differentiation, and root induction are carried out, and finally positive transformed seedlings are obtained. The specific steps are as follows:

[0081] (1) Treatment of receptor materials

[0082] Soak the seeds of maize inbred line ZC01 in water at 37°C for 4 h, germinate them in a petri dish at 28°C for 48 h, place the germinated seeds in a pot filled with nutrient soil for cultivation, and select immature embryos as materials for callus induction 10 days after pollination. Disinfect the maize young ears with 75% (volume fraction) ethanol for 10 min on the operating table, rinse them thoroughly with sterile water, and dry them. Cut half of the grain with a scalpel, and then separate the immature embryos with forceps and place them in sterile water for use.

[0083] (2) Callus induction and subculture

[0084] Place the immature embryos on N6 medium (BINDER, AA958) and culture them in the dark at 28°C for one week. Cut the vigorously growing and brightly colored callus into small pieces, place them on N6 medium and continue to culture, subculture every two weeks to maintain the good state of the callus, and select the well-growing callus for future use.

[0085] (3)Agrobacterium infection

[0086] Take out the preserved Agrobacterium (which has been introduced with the CRISPR / nCas9BE base editing vector), inoculate and shake the bacteria. When the OD 600 value is about 0.8, centrifuge at 5000 r / min for 10 min, collect the bacterial cells, and suspend the cells with infection buffer (1 L of infection buffer is obtained by mixing 4 g of N6 medium basal salts containing N6 vitamins, 2 mg of 2,4-D, 100 mg of inositol, 0.7 g of L-proline, 68.4 g of sucrose, 36 g of glucose, 1 mL of AgNO3 with a concentration of 10 mg / mL, 1 mL of As with a concentration of 100 mol / L and water, pH 5.2) to make the OD 600 value about 0.5. Then shake at 28°C and 150 r / min for 0.5 h to obtain the infection solution. Immerse the well-growing callus selected in (2) in the infection buffer for 1 h, then transfer it to the infection solution containing Agrobacterium and soak for 15 min, and air-dry to obtain the infected callus.

[0087] (4)Co-culture and recovery culture

[0088] Place the infected callus on the co-culture medium (1 L of co-culture medium is obtained by mixing 4 g of N6 medium basal salts containing N6 vitamins, 2 mg of 2,4-D, 30 g of sucrose, 8 g of agar, 1 mL of AgNO3 with a concentration of 10 mg / mL, 1 mL of As with a concentration of 100 mol / L, 3 mL of L-cysteine with a concentration of 100 mg / mL and water, pH 5.8), culture at 20°C for 3 days, transfer to the recovery medium (1 L of recovery medium is obtained by mixing 4 g of N6 medium basal salts containing N6 vitamins, 2 mg of 2,4-D, 0.7 g of L-proline, 30 g of sucrose, 0.5 g of MES, 4 g of plant gel, 1 mL of AgNO3 with a concentration of 10 mg / mL, 1 mL of cefotaxime with a concentration of 250 mg / mL and water, pH 5.8) and culture for 10 days, then transfer to the recovery medium added with glufosinate to screen for positive callus.

[0089] (5)Differentiation, redifferentiation, rooting, and acclimatization

[0090] 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.

[0091] (6) Bar test strips to screen positive plants

[0092] 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.

[0093] 4. Maize ZmGRF3 V132I / E133K Acquisition and analysis of mutants

[0094] Through Agrobacterium-mediated genetic transformation of maize embryos, T0-generation positive transgenic plants were obtained, leaf genomic DNA was extracted, target site sequences were amplified, and Sanger sequencing was performed. The study found that ZmGRF3 The following mutations occurred in the gene:

[0095] 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 ).

[0096] Mutated ZmGRF3 V132I / E133K The amino acid sequence is shown in SEQ ID No. 7. Compared with before mutation, the 132nd amino acid mutates from V to I, and the 133rd amino acid mutates from E to K.

[0097] The T1 generation was obtained by test cross of the T0 generation, and then further self-crossed to obtain ZmGRF3 without transgenic element insertion and with homozygous mutation sites. V132I / E133K Mutant materials.

[0098] ZmGRF3 V132I / E133K Compared with the wild-type material ZC01, the mutant material of ZmGRF3 only differs in that the 506th, 508th, and 509th bases of the 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. ZmGRF3 The 506th, 508th, and 509th bases of the gene (SEQ ID No.1) were mutated from G to A, and the rest of the sequence remained unchanged.

[0099] Example 2. Phenotypic investigation of maize ZmGRF3 V132I / E133K Phenotypic investigation of mutant materials

[0100] The homozygous ZmGRF3 mutant materials obtained in Example 1 were sown in the field. The plant height and leaf area phenotypes were investigated 7 days after pollination, and 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. The data of 10 plants were statistically analyzed in each group, and the results were averaged. V132I / E133K The data of 10 plants were statistically analyzed in each group, and the results were averaged.

[0101] The results are as Figure 3 and Figure 4 shown. Compared with the wild-type material ZC01, the mutant material of ZmGRF3 showed significant increases in the phenotypes related to silage such as plant height, leaf area, and ear length, as well as grain yield (including 100-grain weight and single-ear grain yield). V132I / E133K The results showed that mutating the 506th, 508th, and 509th nucleotides of the wild-type gene (SEQ ID No.1) in the maize genome from G to A could increase the biomass and / or grain yield of maize. That is, the mutated ZmGRF3

[0102] contributed to the increase in the biomass and / or grain yield of maize. ZmGRF3 gene (SEQ ID No.1) from G to A can increase the biomass and / or grain yield of maize. That is, the mutated ZmGRF3 V132I / E133K contributes to the increase in the biomass and / or grain yield of maize.

[0103] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

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 A target sequence in a gene, wherein the target sequence is shown in SEQ ID No. 3; 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.

3. The method according to claim 2, 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.

4. The method according to claim 2 or 3, 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.

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

6. Use of the biomaterial according to claim 5 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.

7. 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).

8. The biomaterial according to claim 7, 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.

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 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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