Method for creating maize parthenogenetic haploid inducer line and application thereof

Abstract

This invention discloses a method for creating maize androgenetic haploid inducible lines and its applications. This invention belongs to the field of biotechnology. The substances regulating the expression of protein-coding genes or regulating the activity or content of said proteins can be applied in any of the following ways: U1) application in regulating the induction ability of plant androgenetic haploids; U2) application in preparing products that regulate the induction ability of plant androgenetic haploids; U3) application in cultivating plants with altered androgenetic haploid induction ability; U4) application in preparing products that cultivate plants with altered androgenetic haploid induction ability; U5) application in plant breeding. This invention can identify haploids through the compact, leafless plant phenotype, and further confirm their ploidy using flow cytometry, greatly reducing screening costs and time, and has promising applications.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for creating a maize parthenogenetic haploid induction line and its application. Background Technology

[0002] Genome editing is a technique for targeted and precise modification of the genome, primarily involving zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats / CRISPR-associated nucleases (CRISPR / Cas). The CRISPR-Cas system is an adaptive immune system discovered in prokaryotes that can precisely recognize and cut specific DNA sequences via RNA guidance, thereby achieving genome editing. Based on the different effector proteins, CRISPR-Cas systems are divided into three main categories: Class 1, Class 2, and Class 3. The MAD7 system belongs to the Class 2 type VA CRISPR-Cas (Cas12a / Cpf1) family of CRISPR-Cas systems. Class 2 CRISPR-Cas systems are characterized by utilizing a single protein effector, rather than the multi-protein effector complexes found in Class 1 systems. The CRISPR / Cas12 system is a gene editing tool based on CRISPR (clustered regular-spaced short palindromic repeats) and Cas protein, belonging to the type V CRISPR system.

[0003] Cas12ICS is an engineered nuclease belonging to the Class 2 Type VA CRISPR-Cas system (the Cas12a family). It was optimized for specific organismal codon usage preferences based on the commercially available MAD7 nuclease, aiming to improve its expression efficiency and gene editing success rate in the target host. Molecularly, Cas12ICS is similar to typical Cas12a proteins, relying on a single RuvC domain to cleave double-stranded DNA. This system requires only one crRNA as a guide RNA, eliminating the need for tracrRNA, thus simplifying the editing system. Cas12ICS recognizes specific PAM sequences (TTNs), producing double-strand breaks with sticky ends after cleaving the target DNA. This breakage pattern may theoretically be more conducive to subsequent DNA repair processes, such as precise gene insertion. Like many members of the Cas12a family, Cas12ICS, after successful cleavage and activation of the target DNA, may also exhibit trans-cleavage activity (i.e., the ability to non-specifically cleave surrounding single-stranded DNA), a characteristic that makes it potentially valuable for developing highly sensitive nucleic acid detection tools. After codon optimization, Cas12ICS exhibits high editing efficiency in model plants, with mutation efficiency reaching up to 50% in editing specific genes, providing another efficient tool option for crop genetic improvement and new germplasm creation.

[0004] Therefore, Cas12ICS can be regarded as a gene editing tool with enhanced performance for specific application scenarios, obtained through targeted optimization strategies on the existing Cas12a core architecture.

[0005] corn( Zea mays Maize (L.) is an important food crop in my country, and increasing its yield is of strategic significance to the development of agriculture in my country and the world. Double haploid (DH) technology, based on haploids, has become an important means of improving crop breeding efficiency in modern agriculture and has been successfully applied in a variety of plants. Although genes related to haploid induction are being identified, the genetic basis and molecular mechanism of haploid induction are still not fully understood. With the gradual maturation and improvement of bioinformatics and molecular biology techniques, combining molecular breeding techniques with gene editing methods to aggregate haploid-inducing major genes in maize varieties has become an important means of breeding homozygous inbred lines for maize in the future.

[0006] Haploids, after chromosome doubling, form homozygous diploids, or double haploids. Selecting non-segregating, high-quality offspring from double haploids requires only two generations, significantly shortening the breeding cycle compared to traditional maize inbred line breeding. CRISPR / Cas9 technology enables site-directed mutagenesis of target genes, holding significant application value in crop breeding. Ye Xingguo and Wang Ke's team used CRISPR / Cas9 technology to mutate wheat... TaMTL Gene knockout was discovered, and simultaneous knockout TaMTL-4A, TaMTL-4B and TaMTL-4D The haploid induction rate of multiple mutants of genes can reach 11.8%-31.6%. Mao Long et al. also achieved haploid induction rates in wheat through editing... TaMTL The team successfully created a haploid inducible line and preliminarily elucidated the mechanism by which this gene mutation induces haploid production. Chen Shaojiang's team discovered that knocking out this gene in wheat... PLA The team obtained a wheat haploid inducible line and used CRISPR / Cas9 gene editing technology to modify the Arabidopsis thaliana gene. DMP8 and DMP9 Gene knockout successfully created a haploid inducible line of Arabidopsis thaliana. Meanwhile, Chen Shaojiang's team also used gene editing technology to knock out corn in tomatoes. ZmMTL By using homologous genes, they successfully obtained a haploid inducible line for tomato. Lin Hao et al. used gene editing technology to... MtDMP8 and MtDMP9 The haploid induction rate of double mutants obtained by gene knockout was 0.29%-0.82%. Studies have found that knocking out maize in millet... ZmMTL homologous genes SiMTL It can also produce haploid inducible lines, and the frequency of haploid occurrence in self-crossed offspring is 2.7%.

[0007] In 2018, Khanday et al. used CRISPR / Cas9 technology to perform site-specific gene editing in rice. BBM1, BBM2 and BBM3 They obtained double- or triple-mutated plants and found that the BBM1-ee transgenic plants had inducible ability. In 2019, Wang Kejian et al. used the same technique to study the genes in rice. REC8 , PAIR1, OSD1 and MTL Multiple editing was performed, and a haploid inducible line for rice was successfully obtained. CRISPR / Cas9 technology was then used in maize to... MTLGene mutations can yield maize parthenogenetic haploid inducible lines. Recently, Qi et al. screened for a highly efficient system for targeted gene transcriptional activation regulation by fusing the CRISPR / Cas9 system with different transcriptional activation effector elements. They then activated the expression of the BABYBOOM gene in maize live oocytes using this system, and the results showed that the maternal haploid content in the transformed material reached 3.6%, making parthenogenesis possible in maize. Although the technology combining gene editing and double haploidization has significant application value in the targeted improvement of maize, existing maize inducible lines are mainly parthenogenetic haploid inducible lines, such as CAU5 and CHOI1. Highly efficient androgen-producing haploid inducible lines are lacking in breeding research. The average induction rate of existing maize parthenogenetic haploid inducible lines is around 8%, indicating potential for further improvement.

[0008] corn indeterminate gametophyte 1 (IG1) It is a gene encoding a protein with the LOB domain, which is considered crucial for the development of lateral organs in plants and closely associated with androgenetic induction of haploidy. ZmIG1 In the mutant embryo sac, the prolongation of the proliferation period indicates that the wild type... IG1 Functions promote the transformation of the embryonic sac from proliferation to differentiation; mutations IG1 The blastocyst contains an extra egg cell, an extra central cell, and a polar nucleus within the central cell. IG1 The phenotype of mutant embryo sacs suggests that cell identity is determined based on location. For example, the ability of extra cells and nuclei to function as egg cells or polar nuclei appears to depend on their location within the embryo sac. Many of these defective embryo sacs produce aberrant seeds due to their abnormal structure. These aberrations include heterozygotes, endosperm abnormalities, and early seed abortion. Maize endosperm development is highly sensitive to deviations from the normal 2 maternal:1 paternal genome ratio in the endosperm. Therefore, when an embryo sac with three polar nuclei is fertilized by a standard haploid pollen grain, the resulting endosperm is miniature, and when an embryo sac with four or more polar nuclei is fertilized by a haploid pollen grain, the resulting endosperm aborts and shrivels early in development. When the induction line carries… IG1 When genes are involved, abnormal embryonic development often occurs, such as the production of anucleate oocytes, which develop into haploid embryos after fusing with sperm cells, thus achieving the production of androgenetic haploids.

[0009] ZmDMP It is highly expressed and located in the plasma membrane during the later stages of pollen development. If a missense mutation occurs due to a single TC base substitution in the membrane protein encoding the DUF679 domain, it can also trigger haploid induction.

[0010] This study is specifically aimed at ZmIG1, ZmDMP Genes were used to knock out the major gene inducing parthenogenesis haploidy in maize using gene editing technology. Zm00001d042560, Zm00001eb372320After generating stable mutations, they aggregate, and the induction efficiency is evaluated to create new maize germplasm. Summary of the Invention

[0011] The main problem to be solved by this invention is to create new maize parthenogenetic haploid germplasm.

[0012] To address the aforementioned problems, this invention provides the application of substances that regulate the expression of protein-coding genes or substances that regulate the activity or content of said proteins in regulating the ability of plants to induce parthenogenesis and haploidy.

[0013] The application provided by this invention is the use of substances that regulate the expression of protein-coding genes or substances that regulate the activity or content of said proteins in any of the following: 1) Application in regulating the ability of plants to induce parthenogenesis and haploidy; 2) Application in the preparation of products that regulate the ability of plants to induce parthenogenesis and haploidy; 3) Application in cultivating plants with altered ability to induce androgenetic haploid reproduction; 4) Application in the preparation of products from plants with altered ability to induce androgenetic haploid reproduction; 5) Applications in plant breeding; The protein is any of the following proteins: G1) A composition consisting of a protein whose amino acid sequence is SEQ ID No:1 and a protein whose amino acid sequence is SEQ ID No:4; G2) A protein whose amino acid sequence is SEQ ID No:1 or a protein whose amino acid sequence is SEQ ID No:4; G3) Proteins obtained by substituting and / or deleting and / or adding amino acid residues of G1) and G2) have more than 75% identity with the protein shown in A1) and have functions related to regulating the ability of plants to induce parthenogenesis haploids. G4) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of G1) or G2).

[0014] The protein whose amino acid sequence is SEQ ID No:1 described above is named IG1.

[0015] The protein with the amino acid sequence described above, SEQ ID No:4, is named DMP.

[0016] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0017] In the aforementioned proteins, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0018] The protein mentioned in the above applications is derived from corn ( Zea mays L.).

[0019] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of the protein-encoding gene, which encodes the proteins IG1 and DMP.

[0020] In the above applications, the substances that regulate the expression of protein-coding genes and the substances that regulate the activity or content of the protein can be biological materials related to the protein, and the biological materials can be any of the following: c1) The nucleic acid molecule that encodes the protein described above; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-coding genes mentioned above; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

[0021] In the above-mentioned biological materials, c1) the nucleic acid molecule is any of the following DNA molecules: d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No:3; d2) The coding region sequence is the DNA molecule shown in SEQ ID No:2 in the sequence listing; d3) The nucleotide sequence is the DNA molecule shown in SEQ ID No:6; d4) The coding region sequence is the DNA molecule shown in SEQ ID No:5 in the sequence listing.

[0022] The nucleic acid molecule mentioned in e1 of this article can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0023] The sgRNA can be sgRNA1 or sgRNA2.

[0024] The target sequence of sgRNA1 is located at positions 12-31 of SEQ ID No:2 and positions 488-507 of SEQ ID No:3.

[0025] The target sequence of the sgRNA2 is located at positions 132-152 of SEQ ID No:5 and positions 315-335 of SEQ ID No:6.

[0026] In the above e3), the recombinant vector can be a plant gene editing vector. The plant gene editing vector can be the vector CRISPR / CAS12ICS.

[0027] The microorganism mentioned in e4) above can be Agrobacterium. The Agrobacterium is EHA105.

[0028] Those skilled in the art can readily mutate the nucleotide sequences encoding the proteins IG1 and DMP of this invention using known methods, such as directed evolution or point mutation. Artificially modified nucleotides that possess 75% or more of the nucleotide sequence identity with the proteins IG1 and DMP isolated in this invention, as long as they encode and function as proteins IG1 and DMP, are derived from and equivalent to the nucleotide sequences of this invention.

[0029] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0030] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0031] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0032] The vectors described herein are known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cos plasmids), Ti plasmids, or viral vectors.

[0033] Existing plant expression vectors can be used to construct structures containing the aforementioned... IG1 and DMPRecombinant gene expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0034] The microorganisms mentioned in this article may be yeast, bacteria, algae, or fungi. Among them, bacteria may originate from the genus *Escherichia* (…). Escherichia Erwinia ( Erwinia Agrobacterium tumefaciens ( ), Agrobacterium tumefaciens Agrobacterium Flavobacterium ( Flavobacterium) Alkalophytum genus ( Alcaligenes ), Pseudomonas ( Pseudomonas ), Bacillus spp. ( Bacillus (e.g., Agrobacterium tumefaciens EHA105).

[0035] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0036] This invention also provides a method for regulating the ability of plants to induce parthenogenetic haploids.

[0037] The method for regulating the ability of plants to induce parthenogenetic haploids provided by the present invention includes regulating the ability of plants to induce parthenogenetic haploids by simultaneously regulating the expression of the encoding genes of proteins IG1 and DMP or regulating the activity and / or content of said proteins.

[0038] In this invention, the regulation can be increased, enhanced, or raised. The regulation can also be decreased, weakened, or reduced.

[0039] This invention also provides a method for cultivating plants with altered ability to induce androgenetic haploid reproduction.

[0040] The method for cultivating plants with altered androgenic haploid induction ability provided by the present invention includes downregulating or inhibiting or reducing the expression levels of the encoding genes of the proteins IG1 and DMP in the target plant, and / or downregulating or inhibiting or reducing the activity and / or content of the encoding genes of the proteins, to obtain plants with altered androgenic haploid induction ability.

[0041] In the above cultivation method, the downregulation, inhibition, or reduction of the activity and / or content of the protein in the target plant, or / and the expression level of the gene encoding the protein can be obtained by introducing nucleic acid molecules containing genes encoding the proteins IG1 and DMP that inhibit, reduce, or silence the expression of the protein, into the recipient plant, thereby obtaining a target plant with enhanced ability to induce parthenogenesis haploidy.

[0042] In the above method, firstly, single mutant plants encoding the gene for protein IG1 or DMP are knocked out. Then, the homozygous mutants of the two are crossed to obtain the F1 generation in a heterozygous state. Subsequently, the F1 generation is self-crossed, and according to the law of segregation, double mutant homozygous materials with homozygous mutations in both alleles are obtained by screening.

[0043] In one embodiment of the present invention, the method for cultivating plants with altered androgenic haploid induction ability includes the following steps: 1) Construct recombinant expression vectors containing DNA molecules shown in SEQ ID No:1 and SEQ ID No:4, respectively; 2) The recombinant expression vector constructed in step 1) was transferred into recipient plants to obtain single mutant plants with the coding genes of IG1 or DMP knocked out, respectively. 3) The single mutant plants obtained in step 2) are hybridized to obtain double mutant homozygous materials with homozygous mutations in both alleles. The double mutant homozygous materials are transgenic plants with altered androgenic haploid induction ability.

[0044] The importation refers to the use of recombination methods, including but not limited to Agrobacterium (…). Agrobacterium Introduced methods include mediated transformation, bio-projectile methods, electroporation, in-planta techniques, and more.

[0045] By using any vector capable of guiding the expression of exogenous genes in plants, the coding genes or gene fragments for knocking out proteins IG1 and DMP provided in this invention can be introduced into plant cells or recipient plants to obtain transgenic cell lines and transgenic plants with altered ability to induce parthenogenesis haploidy. Expression vectors carrying the coding genes for knocking out proteins IG1 and DMP can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0046] In this invention, the purpose of plant breeding may include cultivating plants with enhanced ability to induce androgenetic haploids.

[0047] In this invention, the proteins and / or the biological materials described above are also within the scope of protection claimed by this invention.

[0048] In this invention, the plant may be a dicotyledonous plant.

[0049] In the above applications or methods, the dicotyledonous plant may be N1, N2, N3, or N4. N1) Monocotyledons; N2) Plants of the order Poales; N3) Gramineae plants; N4) Plants of the genus *Zea*; N5) Corn.

[0050] In this article, the maize referred to may be the maize inbred line KN5585 or a compact maize plant. lg1 .

[0051] This invention uses harvesting induction lines and recipient materials to combine F1 generation plants, sowing the F1 generation in flowerpots, and observing the phenotype of plants at the 3-4 leaf stage to screen and identify haploids. Compared with conventional techniques, this invention does not require haploid ploidy verification of all plants. Haploids can be identified by the phenotype of compact plants without a ligule, and their ploidy can be further verified by flow cytometry, which greatly reduces screening costs and time. Attached Figure Description

[0052] Figure 1 To construct gene editing vectors and mutant plants.

[0053] Figure 2 This represents the F1 generation phenotype of leafless lingula plants.

[0054] Figure 3 For flow cytometry ploidy verification.

[0055] Figure 4 To evaluate the induction rate of androgenetic haploids. Detailed Implementation

[0056] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0058] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0059] The maize inbred line KN5585 in the following examples is described in: Liu HJ, Jian LM, et al. High-throughput CRISPR / Cas9 mutagenesis streamlines trait gene identification in maize. Plant Cell. 2020;32(5):1397-1414. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0060] The compact maize plant in the following examples lg1 This has been documented in: Li C, Liu C, Qi X, Wu Y, FeiX, Mao L, Cheng B, Li X, Xie C. RNA-guided Cas9 as an in vivo desired-target mutator in maize. Plant Biotechnol J. 2017 Dec;15(12):1566-1576. The biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0061] The gene editing vectors CPB-CAS12ICS, pEASY®-BluntSimpleCloning Vector, and Escherichia coli strain Trans1-T1 used in the following examples were purchased from Beijing TransGen Biotech Co., Ltd. Hind III restriction endonuclease (NEB), KOD Plus, and KOD FX high-fidelity PCR amplification enzymes were mainly purchased from Beijing Bailinke Biotechnology Co., Ltd., Beijing Liuhetong Trade Co., Ltd., and other companies.

[0062] Example 1: Creation of knockout plants IG1 The coding sequence (CDS) of the gene in maize variety B73 is SEQ ID No:2, encoding the IG1 protein with the amino acid sequence SEQ ID No:1. The genomic gene encoding the IG1 protein in the genomic DNA of maize variety B73 is shown in SEQ ID No:3 of the sequence listing.

[0063] DMP The coding sequence (CDS) of the gene in maize variety B73 is SEQ ID No:5, encoding the DMP protein with the amino acid sequence SEQ ID No:4. The genomic gene encoding the DMP protein in the genomic DNA of maize variety B73 is shown in SEQ ID No:6 of the sequence listing.

[0064] 1. Construction of CRISPR / CAS12ICS knockout vector CRISPR / CAS12ICS knockout vectors such as Figure 1 In the middle A, the maize endogenous U6-2 promoter is used to initiate sgRNA expression. Reference genome sequence B73_RefGen_v4 is used. IG1 ( Zm00001d042560 )Gene, DMP ( Zm00001eb372320 Gene design gRNA1: 5'-GGTGCCGGCGCCATCGGGGT-3' (SEQ ID No:7, corresponding to positions 12-31 of SEQ ID No:2 and positions 488-507 of SEQ ID No:3) and gRNA2: 5'-gctggccaacttcctccccac-3' (SEQ ID No:8, corresponding to positions 132-152 of SEQ ID No:5 and positions 315-335 of SEQ ID No:6).

[0065] The gRNA sequence was verified in the KN5585 receptor by Sanger sequencing. Sequences of key elements such as the promoter are shown in Appendix 1. The nucleotide sequence of the CRISPR / CAS12ICS knockout vector is SEQ ID. No:9 (used for knockout). IG1 (gene) and SEQ ID. No:10 (for knockout) DMP Gene).

[0066] 2. Maize genetic transformation (1) A CRISPR / CAS12ICS knockout vector was constructed and introduced into BMEHA105 Agrobacterium competent cells (Beijing Bomeide Gene Technology Co., Ltd., catalog number: BC303-01) to obtain recombinant Agrobacterium EHA105 / CRISPR / CAS12ICS.

[0067] (2) The recombinant Agrobacterium EHA105 / CRISPR / CAS12ICS obtained in step (1) was cultured using N6 liquid culture medium (dissolving: slowly add the dry powder to about 600-800 mL of distilled water while stirring. Adjusting volume: adjust the volume to 1 liter with distilled water. Dispensing and sterilization: dispense the culture medium into suitable containers and then immediately autoclave at 115℃ for 30 minutes). 600nm The recombinant Agrobacterium bacterial suspension was centrifuged at 5000 rpm for 10 min to obtain bacterial cells. The cells were then resuspended in a prepared infection buffer (1 L of infection buffer was prepared by mixing 4 g of N6 salt containing N6 vitamin, 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 (10 mg / mL), 1 mL of As (100 mol / L), and water, pH 5.2). 600nm The value was around 0.5, and then the mixture was shaken at 28 °C and 150 r / min for 0.5 h to obtain the inoculum.

[0068] (3) Soak the callus tissue of the maize inbred line KN5585 with good growth in the infection buffer for 1 hour, then transfer it to the infection solution prepared in step (2) and soak it for 15 minutes, and then air dry.

[0069] (4) Place the infected callus tissue from step (3) into a co-culture medium (1L of co-culture medium is prepared by mixing 4g of N6 salt containing N6 vitamin, 2mg of 2,4-D, 30g of sucrose, 8g of agar, 1mL of AgNO3 (10mg / mL), 1mL of As (100mol / L), 3mL of L-cysteine ​​(100mg / mL) and water, pH 5.8), and culture at 20℃ for 3 days. Then transfer it to a recovery medium (1L of recovery medium is prepared by mixing 4g of N6 salt, 1ml of N6 vitamin 1000×, 1.5mg of... The sample was prepared by mixing 2,4-D, 0.7g L-proline, 30g sucrose, 5μM AgNO3, 0.5g MES, 100mg cefotaxime, 100mg vancomycin, 8g agar and water (pH 5.8), and cultured at 28℃ for 10 days. Then, it was transferred to recovery medium containing 1.5mg / L glufosinate and cultured in the dark at 28℃ for 7 days. Positive callus tissue was then screened.

[0070] (5) Transfer the positive callus obtained in step (4) to embryoid induction medium (1L of embryoid induction medium is prepared by mixing 4.43g MS salt containing MS vitamin (containing inositol), 0.25mg 2,4-D, 30g sucrose, 5mg 6-BA, 4g plant gel, 1mL Cefo (250mg / mL) and water, pH 5.8), and culture in the dark for 2 weeks. Then transfer to differentiation medium (1L of differentiation medium is prepared by mixing 4.43g MS salt containing MS vitamin (containing inositol), 30g sucrose, 4g plant gel, 1mL Cefo (250mg / mL) and water, pH 5.8). After green shoots emerge, transfer to rooting medium (1L of rooting medium is prepared by mixing 2.215g 1 / 2 MS, 30g sucrose, 51.55mg 6-BA, 4g plant gel, 1mL Cefo (250mg / mL) and water). MSvitamin (4g plant gel and water, pH 5.8) rooted, grew to a certain height, and was cultured in the air for 3 days before transplanting.

[0071] (6) Take about 3 cm of plant leaves, grind them thoroughly in a tube, add 500 μl of buffer (from bar gene test strip), insert bar gene test strip (Beijing Aochuang Jinbiao Biotechnology Co., Ltd., product number: A07-13-413), and plants that show positive bands are T0 generation positive plants.

[0072] 3. Verification of genetically modified components DNA was extracted and purified using a plant genomic DNA extraction kit (Tiangen, China). T-DNA was detected by PCR using a bar test strip (Agdia, Cat. #STX14200 / 0012, US) and the SpCAS12ICS gene. CAS12ICS PCR amplification used forward primers (5'-tgttctgggtgatgaagttg-3') and reverse primers (5'-tacacctacgacaagtactt-3').

[0073] The PCR program consisted of 35 cycles: 94 °C for 3 min; 95 °C for 30 s, 58 °C for 30 s, and 68 °C for 20 s; followed by a final extension at 68 °C for 10 min. The PCR reaction mixture consisted of: 10x Buffer 5 μL; 2 mM dNTP 5 μL; 25 mM MgSO4 2 μL; forward and reverse primers (10 μM) 1.5 μL each; DNA Template 2 μL; KOD plus (1 U / μL) 1 μL; and ddH2O 32 μL.

[0074] 4. Mutation detection Primers 5'-cagcatccggctgccat-3' and 5'-tgacagcacgattgacacca-3' were designed based on the target location of the knockout vector. Target gene amplification was performed on the corresponding plants using PCR, and the mutation type was analyzed using DSDecode software after sequencing. The PCR reaction system and procedure were the same as those used for transgenic component verification.

[0075] Compared to the wild type, for IG1 Genes, mutants IG1 Two homologous chromosomes in (-20, -20) IG1 The genes all underwent the following mutations: IG1 The mutation "5'-GTCGGTGCCGGCGCCATCGGGGTCGGTGATCACCGTGGCGTC-3'" (corresponding to positions 8-53 of SEQ ID No:2 and positions 484-527 of SEQ ID No:3) in the gene to "5'-GTCGGTGCCGGCGCCATCGGTC-3'" causes a frameshift due to nucleotide deletion, leading to premature termination of translation and resulting in... IG1 Protein function loss, thus IG1 Gene knockout; sequencing results of the mutation site and surrounding nucleotides are shown in [link to gene knockout sequence]. Figure 1 C.

[0076] Compared to the wild type, for DMP Genes, mutants DMPTwo homologous chromosomes (+1,+1) DMP The genes all underwent the following mutations: DMP The mutation “5'-CCTCCCCACGGGCACGCTGC-3'” (corresponding to positions 143-163 of SEQ ID No:5 and positions 326-346 of SEQ ID No:6) in the gene to “5'-CCTCCCACACGGGCACGCTGC-3'” causes a frameshift, leading to premature termination of translation and loss of DMP protein function, thus knocking out the DMP gene. Sequencing results of this mutation site and its surrounding nucleotides are shown in [link to sequencing data]. Figure 1 C.

[0077] 5. Single mutant hybridization In obtaining IG1 and DMP After a single-gene mutation homozygous line is formed, the homozygous line is negative and does not contain transgenic elements, and the mutation site can be stably inherited.

[0078] Using KN5585_IG1(-20,-20) as the female parent and KN5585_DMP(+1,+1) as the male parent, the two materials were crossed to obtain the F1 generation, which was allele heterozygous. Subsequently, the F1 generation was self-crossed, and according to the law of segregation, double mutant homozygous materials with both alleles homozygous were obtained through screening. IG1 (-20, -20) × DMP (+1, +1).

[0079] Example 2: Evaluation of mutant polymerization and induction rate of dual-mutant materials 1. Evaluation of Induction Rate The compact plant type created by the research group in the early stage was selected. lg1 As a receptor material, utilizing lg1 It is specifically highly expressed in the leaf pulvinus region of grasses. Its deletion homozygous mutants all have phenotypes with defects in auricle and ligule development and extremely compact plant type as markers for selecting haploids. The proportion of recessively inherited compact plant type plants is calculated in the offspring.

[0080] The stability of the induction system was tested by crossbreeding in two regions, Beijing (BJ) and Hainan (HN). The maternal induction line obtained in Example 1 was used... IG1 (-20, -20) × DMP (+1, +1) and parent lg1 The F1 generation was sown in seedling trays, and the plant phenotype at the 3-4 leaf stage was observed. Plants with the same phenotype as the male parent and without the ligule feature were selected. The ploidy of the plants without the ligule was then verified by flow cytometry.

[0081] 2. Flow cytometry verification The experimental steps for flow cytometry ploidy verification are as follows: First, prepare a single-cell suspension: collect cells, wash with PBS, and pass through a cell sieve (e.g., 40 μm). Second, fix the cells: resuspend the cells in pre-chilled 70% ethanol and fix at -20°C for at least 2 hours or overnight. Third, stain: centrifuge to discard the ethanol, wash once with PBS, resuspend in staining solution containing PI (50 μg / mL) and RNase A (100 μg / mL), and incubate at 37°C in the dark for 30 minutes. Finally, filter the stained cells into flow cytometry tubes and immediately load them into the instrument.

[0082] After repeated screening and selection over four consecutive seasons, it was found that: when IG1 Single gene mutant IG1 (-20, -20) and DMP Single gene mutant DMP When (+1, +1) was used as the inducing maternal parent, no haploids appeared in the offspring; while IG1×DMP polymer materials IG1 (-20, -20) × DMP (+1, +1) and lg1 A plant without a leaf ligule was found in the hybrid offspring. Figure 2 These plants showed significantly weaker growth than the diploid control at the same time point, consistent with the phenotypic characteristics of haploids. Flow cytometry analysis revealed that the relative DNA content of the plants without a leaf ligule was half that of the diploid control, confirming them as haploids. Figure 3 ).

[0083] The results of the induction efficiency assessment show that ( Figure 4 ):by IG1(-20,-20)×DMP(+1,+1) The polymeric material (named E1) was used as the induction parent material, achieving an induction efficiency of 2.09‰-2.81‰. Furthermore, this study also demonstrated the use of... lg1 As a receptor material, it can ensure the stability of the induction system and the high efficiency of screening.

[0084] 3. SNP microarray sequencing to verify genome origin A certain number of haploids were obtained through hybridization induction. To verify the genomic origin of the haploids, SNP microarray technology was used for sequencing to obtain the genomic chromosome sequences of the haploids and their parents.

[0085] Comparative analysis revealed that the similarity between the maternal inducing line and the haploid was almost identical to that between the inducing line and the paternal recipient material. The similarity between the haploid and the paternal line was over 99%, consistent with the genetic characteristic that the genome of the androgenetic haploid is entirely derived from the paternal line. Therefore, it can be determined that the haploid obtained by hybridization belongs to the androgenetic haploid, successfully verifying that the inducing line material created using gene editing technology has the ability to induce androgenetic haploids.

[0086] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a substance that regulates the expression of a protein-coding gene or a substance that regulates the activity or content of said protein in any of the following: Application of U1 in regulating the ability of plant parthenogenesis haploid induction; Application of U2 in the preparation of products that regulate the ability of plant parthenogenesis haploid induction; U3) Application in cultivating plants with altered ability to induce androgenetic haploidy; U4) Application in the preparation of products from plants whose ability to induce parthenogenesis haploidy has been altered; Application of U5 in plant breeding; The protein is any of the following proteins: G1) A composition consisting of a protein whose amino acid sequence is SEQ ID No:1 and a protein whose amino acid sequence is SEQ ID No:4; G2) A protein whose amino acid sequence is SEQ ID No:1 or a protein whose amino acid sequence is SEQ ID No:4; G3) Proteins obtained by substituting and / or deleting and / or adding amino acid residues of G1) and G2) have more than 75% identity with the protein shown in A1) and have functions related to regulating the ability of plants to induce parthenogenesis haploids. G4) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of G1) or G2).

2. The application according to claim 1, characterized in that: The protein is derived from corn.

3. The application according to claim 1 or 2, characterized in that: The substance is a biomaterial related to the protein, and the biomaterial is any one of the following: c1) A nucleic acid molecule encoding the protein described in claim 1 or 2; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding gene as described in claim 1 or 2; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

4. The application according to claim 3, characterized in that: c1) The nucleic acid molecule is any of the following DNA molecules. d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No:3; d2) The coding region sequence is the DNA molecule shown in SEQ ID No:2 in the sequence listing; d3) The nucleotide sequence is the DNA molecule shown in SEQ ID No:6; d4) The coding region sequence is the DNA molecule shown in SEQ ID No:5 in the sequence listing.

5. The application according to claim 3, characterized in that: e3) The recombinant vector is a DNA molecule with nucleotide sequences shown in SEQ ID No:9 and SEQ ID No:

10.

6. A method for regulating the ability of plants to induce parthenogenesis haploids, characterized in that: This includes regulating the activity and / or content of the protein described in claim 1 or 2 in the target plant, and / or regulating the expression level of the gene encoding the protein described in claim 1 or 2, to regulate the plant's ability to induce parthenogenetic haploids.

7. A breeding method for cultivating plants with altered ability to induce androgenetic haploidy, comprising regulating the activity and / or content of the protein described in claim 1 or 2 in the target plant, and / or regulating the expression level of the gene encoding the protein described in claim 1 or 2, to obtain plants with altered ability to induce androgenetic haploidy.

8. The method according to claim 7, characterized in that: The regulation of the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and the expression level of the gene encoding the protein described in claim 1 or 2, includes introducing a nucleic acid molecule containing a gene encoding the protein described in claim 1 or 2 that inhibits, reduces, or silences its expression into the recipient plant, thereby obtaining a target plant with altered ability to induce androgenetic haploidy; wherein the gene encoding the protein described in claim 1 or 2.

9. The protein as described in claim 1 or 2 and / or the biomaterial as described in claim 3 or 4.

10. The application according to any one of claims 1-4, and the method according to any one of claims 5-8, characterized in that: The plant is any one of the following: N1) Monocotyledons: N2) Plants of the order Poales; N3) Gramineae plants; N4) Plants of the genus *Zea*; N5) Corn.

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