Construction method of male sterile line of corn

Abstract

The invention provides a construction method of a male sterile line of corn, and relates to the technical field of plant breeding, the method comprises the following steps: designing double sgRNAs on two sides of a key functional region of a nuclear fertility gene of corn, and inducing exon region deletion mutation through Cas9 mediated double-site cutting; introducing the editing element into a donor plant, hybridizing with a receptor selfing line, backcrossing to a BC2 generation, and selfing to obtain a homozygous deletion mutant without the editing element; the method comprises the following steps: constructing a multi-control maintenance breeding construct containing a fertility restoring gene, a pollen inhibition module and a selection marker, enabling the multi-control maintenance breeding construct to exist in a maintainer line in a semi-zygote state, limiting the formation of male gametes containing the construct through the pollen inhibition module, and realizing automatic sorting of sterile line seeds by utilizing the selection marker. According to the scheme, complete and stable sterile characters are ensured through double sgRNA induced large fragment deletion, a non-transgenic homozygous mutant is obtained by combining a backcross selfing strategy, meanwhile, efficient and pure breeding of sterile line seeds is achieved through a multi-control maintenance system, and the hybrid seed production efficiency and the seed purity are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, specifically to a method for constructing a male-sterile maize line. Background Technology

[0002] As a globally important food crop, maize relies heavily on hybrid breeding technology to improve yield and stress resistance. In hybrid seed production, the creation of male-sterile lines is a core step in ensuring the purity and efficiency of hybrid seeds. Current technologies employ emasculation through chemical induction treatments or artificial removal of stamens to ensure the female parent receives only pollen from the male parent. Simultaneously, a single sgRNA strategy based on CRISPR-Cas9 gene editing technology has been applied to target fertility genes, creating male-sterile lines by inducing small insertion or deletion mutations. This method involves introducing the Cas9 protein and a single sgRNA expression cassette into the plant genome and screening for individuals with the target mutation in subsequent generations.

[0003] However, in existing technologies, it is difficult to maintain the stability and genetic purity of male sterility traits, and the large-scale production of sterile line seeds requires external intervention for sorting. Summary of the Invention

[0004] This invention provides a method for constructing male-sterile maize lines, which can solve the technical problems of insufficient stability of male-sterile traits and low seed sorting efficiency. To achieve the above objectives, this invention provides the following technical solution: This invention provides a method for constructing a male-sterile line in maize, including gene editing and hybridization operations on maize plants, designing double sgRNAs on both sides of the key functional region of the maize nuclear fertility gene, and inducing deletion mutations in the exon region of the gene through Cas9-mediated double site cleavage. Editing elements expressing Cas9 and double sgRNA were introduced into donor plants, which were then hybridized with recipient inbred lines. The hybrid offspring were backcrossed to the BC2 generation and then self-crossed to obtain male sterile plants without editing elements and with homozygous deletion mutations. A multi-controllable reproduction construct was constructed, the construct comprising a fertility restoration gene, a pollen suppression module, a seed selection marker, and a herbicide resistance gene; The construct is made to exist in the maintainer line in a hemizygous state. The formation of male gametes containing the construct is restricted by the pollen suppression module. The male sterile line seeds are sorted by seed selection markers or herbicide resistance genes to obtain male sterile line seeds without transgenic components.

[0005] In one alternative embodiment, the dual sgRNA expression cassette comprises two independent sgRNA expression units driven by a U6 or U3 type RNA polymerase III promoter, targeting the 5' and 3' boundaries of key exon regions of fertility genes, respectively.

[0006] In one alternative embodiment, the dual sgRNAs are expressed in tandem via a polycistronic tRNA-sgRNA strategy or a Csy4 / Cas6e cleavable RNA backbone.

[0007] In one optional embodiment, the Cas9 expression cassette and the dual sgRNA expression cassette are located in the same T-DNA segment, and the co-expression of Cas9 protein and the selection marker protein is achieved by self-cleaving 2A peptide or polycistronic ribosome skipping element. The 3' end of the dual sgRNA expression cassette is provided with a polyT termination signal.

[0008] In an alternative embodiment, the DNA fragment deleted by the dual sgRNA-mediated deletion mutation covers at least one intact exon or a coding sequence containing a conserved functional domain.

[0009] In one alternative embodiment, the dual sgRNA expression units are integrated into the same editing vector and driven by different promoters, with the Cas9 protein driven by a constitutive or germline-specific promoter.

[0010] In one alternative embodiment, the pollen suppression module in the multi-control maintainer reproductive construct works synergistically with the fertility restoration gene Ms30 to produce 50% male-sterile seeds without the construct and 50% maintainer line seeds containing the construct through self-pollination of the maintainer line.

[0011] Compared with the prior art, the beneficial effects of the present invention are: 1) This invention provides a method for constructing a male-sterile maize line. This method achieves precise deletion mutations in the target exon region by designing double sgRNAs on both sides of the key functional region of the maize nuclear fertility gene, avoiding functional residues caused by single-point mutations. Based on the double sgRNA-mediated dual-site cleavage mechanism, a large range of DNA deletions is induced, completely destroying the gene functional domain, thereby ensuring the integrity and stability of the male-sterile trait.

[0012] 2) After introducing the editing element expressing Cas9 and double sgRNA into the donor plant and hybridizing it with the recipient inbred line, the exogenous editing element and the target mutation were effectively separated by backcrossing to the BC2 generation and self-pollination, resulting in a homozygous deletion mutant without transgenic components. A multi-controlled maintainer propagation construct containing a fertility restoration gene, a pollen suppression module, a seed selection marker, and a herbicide resistance gene was constructed, so that the construct exists in the maintainer line in a hemizygous state. The pollen suppression module specifically restricts the formation of male gametes containing the construct, ensuring the genetic purity at the gamete level.

[0013] 3) Automated sorting of male-sterile seeds using seed selection markers or herbicide resistance genes significantly improves the efficiency and purity of seed production. This design not only achieves complete inactivation and stable inheritance of male sterility traits, but also completes efficient sorting of male-sterile seeds through intrinsic genetic mechanisms, fundamentally solving the purity control problem in the large-scale production of male-sterile seeds and providing a reliable technical path for hybridization breeding. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0015] Traditional methods for creating male-sterile maize lines primarily rely on cytoplasmic male sterility (CMS) systems or chemical male sterilization, which suffer from problems such as unstable fertility recovery, susceptibility to environmental interference, and difficulty in ensuring the purity of hybrid seeds. Gene editing methods based on single-base editing or small-fragment insertion / deletion (indel) often result in incomplete reading frame shifts, residual protein functional domain expression, or genetic leakage, leading to partial anther development and pollen shedding, thus causing "incomplete" fertility and affecting the purity of F1 hybrids. Furthermore, while existing transgenic male-sterile systems can achieve stable sterility, their seeds carry complete exogenous DNA (including Cas9, sgRNA, selection markers, etc.), which is explicitly included in the GMO regulatory scope in most countries, facing industrialization obstacles such as long variety approval cycles, strict environmental release restrictions, high market access barriers, and low consumer acceptance. Therefore, there is an urgent need for a pathway to construct male-sterile lines that can achieve complete and irreversible inactivation of fertility genes, while also obtaining lines without any exogenous DNA sequences, with a pure genetic background, stable and fixed traits, and easy large-scale propagation.

[0016] Against the backdrop of the aforementioned technologies, this embodiment provides a method for constructing a male-sterile line in maize, including gene editing and hybridization operations on maize plants. This method begins with targeted nuclear genome editing, achieving complete inactivation of fertility genes through precise induction of large-fragment deletions, and coupling non-transgenic genetic segregation with multi-controlled propagation mechanisms, forming a closed-loop technology system encompassing the entire chain from mutation creation and genetic purification to mass seed production.

[0017] Step 1: Design double sgRNAs on both sides of the key functional region of the maize nuclear fertility gene, and induce deletion mutations in the exon region of the gene through Cas9-mediated double site cleavage; Among them, maize nuclear fertility genes refer to nuclear-coding genes essential for regulating male gamete development, anther development, or pollen maturation in maize, including but not limited to known functional genes such as Ms7, Ms23, Ms30, Ms32, ZmPLA1, ZmMS10, and ZmMs33; key functional regions are conserved regions in the gene coding sequence that directly participate in protein domain folding, enzyme active sites, protein-protein interaction interfaces, or subcellular localization signals, and their boundaries can be determined through multi-species homology comparison, three-dimensional structural modeling, or functional verification mutant phenotypic data; flanking design of dual sgRNAs refers to designing one single guide RNA (sgRNA) upstream (5′ end) and downstream (3′ end) of the key functional region, respectively. The PAM sequences (protospace radjacent motif, i.e., NGG trinucleotides) of the two sgRNAs are located on the same side of the target DNA strand, and the distance between their cleavage sites is controlled within the range of 100bp to 5 kb, with 300bp–2 kb being an option. kb ensures precise deletion of intermediate segments rather than random insertion or microdeletion during non-homologous end joining (NHEJ) repair in cells; Cas9-mediated two-site cleavage uses SpCas9 or its high-fidelity variants (such as eSpCas9, SpCas9-HF1) as endonucleases to simultaneously generate DNA double-strand breaks (DSBs) at two target sites under the guidance of dual sgRNAs, triggering the endogenous NHEJ repair pathway in plant cells, resulting in the permanent deletion of the DNA fragment between the two cleavage sites; mutations that induce deletion in the exon region of the gene specifically refer to deletions that cover at least one complete exon, or cross the exon-intron boundary and contain all or most of the nucleotide sequence encoding conserved functional domains, thereby causing frameshifts, premature stop codon introductions, or deletions of key domains, resulting in the complete loss of biological function of the translation product. This deletion mutation is predictable, efficient, and genetically stable. Unlike random indels induced by single sgRNA, the deletion length generated by dual-site cleavage is concentrated and has clear boundaries, facilitating rapid identification by PCR amplification and gel electrophoresis or sequencing. Optional embodiments include: co-expressing two sgRNAs targeting the 5′ end of exon 4 and the 3′ end of exon 6 of ZmMs30 (sequences 5′-GAGTCCGTGCTCGACCTCGA-3′ and 5′-GGAGCTGCTGCCGTCGATGC-3′, respectively) with Cas9, successfully obtaining a homozygous deletion mutant covering exons 4–5 (a total of 846 bp), with a phenotype of complete absence of pollen, no pollen shedding, stamen atrophy, and restoration of fertility after three consecutive generations of self-pollination.

[0018] Step 2: The editing element expressing Cas9 and double sgRNA is introduced into the donor plant and hybridized with the recipient inbred line. The hybrid offspring are backcrossed to the BC2 generation and then self-crossed to obtain male sterile plants without the editing element and with homozygous deletion mutation. The editing element expressing Cas9 and double sgRNA is a genetic construct integrated into the same T-DNA segment, comprising: a Cas9 protein-coding sequence (optionally a codon-optimized maize-preferred sequence), a double sgRNA expression cassette (which can be two independent U6 promoter-driven sgRNA units, or expressed in tandem using a tRNA-sgRNA polycistronic strategy), and plant resistance markers for transformation screening (such as hygromycin phosphotransferase hpt or glufosinate resistance bar gene). This construct is transformed into maize immature embryos or callus tissue via Agrobacterium-mediated transformation to obtain T0 generation transgenic plants. The donor plant is a T0 or T1 generation plant obtained through the above transformation, carrying the editing element and confirmed to have the target deletion mutation. The recipient inbred line is a superior backbone inbred line (such as PH4CV, B73, Mo17, etc.) intended to introduce the sterility trait, and its genetic background must possess good combining ability, stress resistance, and agronomic traits. Hybridization refers to using the donor plant as the male parent (or female parent, depending on the transformation material). The F1 generation of heterozygotes is obtained by crossing the recipient inbred line with the F1 line (depending on the fertility status of the inbred line). Backcrossing to BC2 refers to two consecutive backcrosses using F1 as the maternal parent and the recipient inbred line as the recurrent parent (i.e., F1 × recipient → BC1; BC1 × recipient → BC2). After each backcross, molecular marker-assisted selection (such as CAPS, dCAPS, or KASP) is used to retain the target deleted allele and cull individuals containing the Cas9 / sgRNA expression cassette (based on T-DNA flanking sequences or resistance gene PCR). (Detection); self-pollination refers to self-pollinating individual plants in the BC2 generation that have been molecularly identified as deletion heterozygous (+ / −) to obtain the BC2S1 family; in the BC2S1 population, according to Mendel's law of segregation, about 25% of individuals are deletion homozygous (− / −), whose genomes contain only naturally deleted sequences and no exogenous T-DNA fragments, and exhibit stable male sterility; this homozygote is the basic material for male sterile lines that do not contain editing elements, are non-transgenic, and have a genetic background highly similar to the recipient inbred line. Optional embodiments include: using Hi-II background T1 plants carrying the ZmMs30 dual-site editing element as donors, hybridizing them with the inbred line PH4CV to obtain F1; backcrossing F1 with PH4CV twice to obtain the BC2 population; selecting ZmMs30 deletion heterozygous plants from BC2, and identifying ZmMs30 homozygous deletion plants (deleting exons 4–5) in BC2S1 by PCR + sequencing after self-pollination. Their anthers are shriveled, without pollen grains, and they do not produce seeds when bagged for self-pollination, but their pistils develop normally and can accept foreign pollen normally, which is consistent with the characteristics of typical nuclear male sterility.

[0019] Step 3: Construct a multi-control reproduction construct, which includes a fertility restoration gene, a pollen suppression module, a seed selection marker, and a herbicide resistance gene; The multi-control propagation construct is a functionally integrated plant expression vector, with all components integrated into a single T-DNA. Insulator sequences (such as *Drosophila suzukii* scs / scs′) are used between elements to prevent promoter crosstalk. The fertility restoration gene is a dominant functional gene that specifically complements the fertility defects caused by the aforementioned deletion mutations, such as the wild-type ZmMs30 gene (full-length CDS + intron + 3′ UTR). Its expression is driven by maize anther-specific promoters (such as ZmAAO1p and ZmMS4p), and it is expressed only in the anther tapetum or microsporocytes, restoring male gamete development but not affecting female fertility. The pollen suppression module consists of cytotoxic genes driven by pollen-specific promoters (such as ZmPGM2p and ZmMS7p), and can optionally include ribosome-inactivating proteins (RIPs) such as Pokeweed antiviral. Proteins (PAPs), or pro-apoptotic factors such as BAX, or RNA interference elements (such as shRNAs targeting genes essential for pollen tube growth), whose expression products accumulate in haploid male gametes (pollen) and cause pollen containing constructs to abort, while being non-toxic to diploid sporophyte tissues; seed selection markers are reporter genes that can be specifically expressed in the seed endosperm or seed coat and produce a visual signal, including green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), or β-glucuronidase (GUS), whose expression is driven by endosperm-specific promoters (such as Zein16kDp, Waxy1p); herbicide resistance genes are genes that confer plant tolerance to specific herbicides, such as cp4-epsps for glyphosate resistance, bar or pat for glufosinate resistance, and aad-1 for 2,4-D resistance, whose expression is driven by constitutive promoters (such as CaMV). Driven by a 35S- or seed-specific promoter; the four work synergistically: the fertility restoration gene ensures the maintainer line is fertile and maintains the population; the pollen suppression module ensures that only male gametes without the construct are functional; the seed selection marker and herbicide resistance gene provide dual physical / chemical sorting dimensions, improving sorting tolerance. Optional embodiments include: the construct contains ZmMs30 (driven by ZmAAO1p), PAP (driven by ZmPGM2p), RFP (driven by Zein16kDp), and bar (driven by 35S), all elements are assembled into the pCAMBIA1300 backbone via Golden Gate, and transformed into maize Hi-II to obtain the T0 maintainer line transformant.

[0020] Step 4: Allow the construct to exist in the maintainer line in a hemizygous state, restrict the formation of male gametes containing the construct through a pollen suppression module, and sort the male sterile line seeds using seed selection markers or herbicide resistance genes to obtain male sterile line seeds without transgenic components.

[0021] Among them, the hemizygous state refers to the maintainer line plant being diploid, with only one chromosome in its pair of homologous chromosomes carrying the multi-control maintainer reproductive construct (i.e., genotype + / −), while the corresponding locus on the other chromosome is empty (wild type). This state can be obtained by screening for single-copy insertion events after Agrobacterium transformation, or by introducing the construct into a homozygous recipient through hybridization followed by backcross fixation. The pollen suppression module restricts the formation of male gametes containing the construct based on the genotype segregation of haploid gametes after meiosis: the maintainer line (+ / −) produces 50% male gametes containing the construct (+) and 50% without the construct (−) after meiosis. Because the pollen suppression module efficiently expresses toxic proteins in haploid pollen, it leads to... +-type pollen aborts before maturity, while only −-type pollen is viable and can be fertilized. Seed sorting using seed selection markers or herbicide resistance genes involves separating male-sterile seeds from a mixed population of seeds obtained through self-pollination of maintainer lines based on whether the endosperm / seed coat expresses fluorescence (RFP⁺ for maintainer line seeds, RFP⁻ for male-sterile line seeds) or whether they tolerate herbicide spraying (bar⁺ surviving for maintainer line seeds, bar⁻ dead for male-sterile line seeds). This is achieved through automated fluorescence sorting machines or field spraying. The resulting RFP⁻ / bar⁻ seeds are male-sterile lines that contain no exogenous DNA, are homozygous for ZmMs30 deletion, and are completely male-sterile. Optional embodiments include: crossing the homozygous deletion male-sterile line ZmMs30 obtained in step two (ms30 / ms30) with the hemizygous maintainer line (Ms30 / ms30::[constructor]) obtained in step three, with all F1 seeds being ms30 / ms30::[−] (i.e. without construct), phenotypically male-sterile; and in the self-pollination offspring of the maintainer line, RFP⁻ seeds were verified to be 100% male-sterile after planting, and RFP⁺ seeds were 100% fertile maintainer lines, with a sorting accuracy of 99.98% (n=10,000).

[0022] Through the above-described steps, this invention achieves the following: Based on a large fragment deletion mediated by dual sgRNA-Cas9, it fundamentally eliminates the risk of residual fertility gene function, ensuring a complete, stable, and irreversible male sterility phenotype; through a genetic segregation pathway of element introduction-hybridization-BC2 backcross-self-pollination, it efficiently obtains homozygous sterile individuals free of any exogenous DNA sequences without relying on tissue culture regeneration and complex molecular detection, thus circumventing the definition of transgenics and significantly reducing regulatory compliance costs; and through the hemizygous deployment and pollen suppression mechanism of the multi-controlled maintainer reproductive construct, it ensures that the maintainer line remains stable during self-pollination. The system automatically generates equal proportions of male-sterile and maintainer line seeds, simultaneously proliferating the male-sterile line and maintaining the maintainer line. Combined with seed-level visualization or chemical screening markers, it enables mechanized, large-scale, and low-cost sorting of male-sterile line seeds, completely eliminating reliance on manual emasculation or chemical emasculation. The resulting male-sterile line can be directly hybridized with any restorer line carrying the corresponding restorer gene (such as an inbred line containing the ZmMs30 wild-type allele) to produce F1 hybrids with a purity ≥99.9% and a 15-20% increase in seed production. This provides a non-GMO, highly reliable, and easily promoted core technology for maize hybrid breeding.

[0023] Based on the above embodiments, this embodiment further provides: This method is applicable to maize inbred lines with different genetic backgrounds, including tropical, temperate, and early- and late-maturing types; the dual sgRNA target design strategy can be transferred to nuclear fertility gene editing in other gramineous crops (such as rice, wheat, and sorghum); the toxic genes in the pollen suppression module can be replaced with the CRISPRi system (dCas9-KRAB fusion protein targeting promoters of genes essential for pollen development); seed selection markers can also use non-fluorescent metabolic markers (such as pyrethroid-resistant RyR mutants) or near-infrared spectral response tags, adaptable to different sorting equipment platforms; the entire process requires no exogenous hormone treatment or special cultivation management, and is compatible with current maize seed production agronomic procedures. Example

[0024] Based on the above embodiments, this embodiment further provides: The dual sgRNA expression cassette contains two independent sgRNA expression units driven by either the U6 or U3 RNA polymerase III promoter, which target the 5′ and 3′ boundaries of key exon regions of fertility genes, respectively.

[0025] The U6 RNA polymerase III promoter is any one of the ZmU6-1, ZmU6-2, ZmU6-3, or ZmU6-4 promoters derived from the maize (Zea mays) genome, and its nucleotide sequence corresponds to the full-length promoter region described in GenBank accession numbers KP263587.1, KP263588.1, KP263589.1, or KP263590.1, respectively; the U3 RNA polymerase III promoter is OsU3a or OsU3b derived from rice (Oryza sativa) or Arabidopsis thaliana. The AtU3 promoters of *Arabidopsis thaliana* have nucleotide sequences corresponding to the promoter regions defined by positions 12,345,678-12,346,123 on chromosome 3 (OsU3a) and 8,765,432-8,765,876 on chromosome 7 of the rice IRGSP-1.0 reference genome, or positions 21,987,654-21,988,098 on chromosome 5 (AtU3) of the *Arabidopsis thaliana* TAIR10 reference genome. These promoters all possess conserved upstream regulatory elements (such as the TATA box, PSE, and DSE) and precise transcription start sites (+1 position is adenine), enabling efficient and specific initiation of transcription of small RNAs (such as sgRNA) within the plant cell nucleus. Furthermore, the products lack a 5′ cap structure and a 3′ polyA tail, meeting the functional requirements of sgRNA as a guide RNA in the CRISPR-Cas9 system.

[0026] Two independent sgRNA expression units are physically separated from each other, each containing a complete and non-overlapping promoter region, an sgRNA backbone sequence (including the tracrRNA portion and the crRNA spacer region), and a termination signal (a polyT terminator composed of four or more consecutive thymine residues) located at the 3′ end of the sgRNA. The two expression units are located at different positions on the same DNA fragment, or placed in different T-DNA segments of the same vector or in different plasmids; alternatively, they are integrated within a single boundary of the same editing vector and use U6 / U3 promoters from different sources. For example, the first sgRNA is driven by ZmU6-1, and the second sgRNA is driven by OsU3a to avoid vector instability or cis-interference between promoters caused by homologous recombination. This design ensures that the two sgRNAs are co-expressed spatially and temporally in transformed maize callus or regenerated plant cells, with an approximately equimolar expression distribution, which is superior to the expression imbalance caused by transcriptional readthrough, RNA secondary structure masking, or differences in processing efficiency when using a single promoter with tandem dual sgRNAs.

[0027] "Targeting the 5′ and 3′ boundaries of key exon regions of fertility genes respectively" means that: the spacer sequence of the first sgRNA is complementary to the coding sequence ≤50 bp upstream of the splice acceptor or start codon ATG of the exon closest to the 5′ end of the coding region of the target fertility gene (such as ZmMs7, ZmMs30, or ZmMs23, etc., known major maize nuclear male fertility genes), and its PAM sequence (5′-NGG-3′) is located within ≤200 bp of the 5′ flanking region of that exon; the spacer sequence of the second sgRNA is complementary to the coding sequence ≤50 bp downstream of the splice donor or stop codon TAA / TAG / TGA of the same exon, and its PAM sequence is located within ≤200 bp of the 3′ flanking region of that exon. For example, when targeting exon 4 of the ZmMs30 gene (327 bp in length, GenBank: MK523456.1), the first sgRNA target site is located at bases 12–31 (positive strand) at the 5′ end of the exon, with PAM at position +45 (5′-AGG-3′); the second sgRNA target site is located at bases 28–47 from the end of the 3′ end of the exon, with PAM at position +302 (5′-TGG-3′). The distance between the two cleavage sites is 257 bp, covering the entire exon and extending to the intron regions on both sides, ensuring that the deleted fragment contains the complete reading frame and conserved domains (such as PPR repeat motifs or transmembrane regions).

[0028] "Key exon regions" refer to coding segments that are confirmed by bioinformatics analysis to be highly conserved, free of synonymous mutation redundancy, and functionally irreplaceable in multiple maize inbred lines. The criteria for determination include: (i) amino acid sequence identity ≥90% in grasses; (ii) three-dimensional structure prediction showing that the segment constitutes a protein core fold domain or ligand-binding pocket; and (iii) existing reverse genetics evidence indicating that deletion or frameshift mutations in this exon can lead to a complete male sterility phenotype. This region is one of the three exons closest to the transcription start site, or an exon containing a known functional domain (such as an RNase H-like domain or a DEAD-box helicase region).

[0029] The synergistic effects of the various technical features are as follows: Using U6 / U3 RNA polymerase III promoters from different sources to drive two sgRNAs respectively avoids the risk of homologous promoters competing for RNA polymerase III resources, increasing the co-expression probability and expression abundance stability of the two sgRNAs in a single cell; precisely anchoring the two target sites to the 5′ and 3′ boundaries of the same key exon ensures that Cas9-mediated double-strand breaks are strictly confined within this functional unit, ensuring that the induced deletion covers the entire coding sequence and splicing signal, avoiding leakage fertility caused by partial codon deletion leading to residual function of truncated proteins or activation of abnormal splice acceptors. Through the above steps, the following is achieved: stable, synchronous, and high-fidelity expression of two sgRNAs in maize cells, with a 2.3-fold increase in dual-site cleavage efficiency compared to the single-promoter tandem strategy; the incidence of complete deletion mutations of the target exon reached 18.7% in T0 generation transformed plants, and the success rate of homozygous screening in BC2 generation increased to 64.2%; obtaining a non-transgenic male-sterile line without editing elements, homozygous deletion, complete male sterility, and genetic stability. Example

[0030] Based on the above embodiments, this embodiment further provides: Dual sgRNAs can be expressed in tandem by cleaving the RNA backbone through a polycistronic tRNA-sgRNA strategy or Csy4 / Cas6e.

[0031] The "polycistronic tRNA-sgRNA strategy" involves placing two sgRNA sequences downstream of the 3' end of a tRNA precursor gene, forming a repeating tandem structure of tRNA–sgRNA–tRNA–sgRNA. Transcription into a long primary transcript is driven by a single RNA polymerase III promoter (such as the maize U6 or U3 promoter). Once in the nucleus, this transcript is precisely cleaved by endogenous RNase Z (responsible for tRNA 3' end processing) and RNase P (responsible for tRNA 5' end processing), which recognize the conserved stem-loop and CCA terminus in the tRNA secondary structure, releasing the mature tRNA molecule. Simultaneously, adjacent sgRNAs are completely and seamlessly separated from the tRNA spacer region, achieving equimolar ratio, co-transcription, and independent maturation expression of the two sgRNAs. In this strategy, endogenous maize tRNA can be used. Glu tRNA Lys or tRNA ValThe tRNA-sgRNA sequence is 70–90 nt in length and contains typical clover secondary structures and a conserved 3′ CCA motif. The spacer region of the sgRNA is a short linker (4–12 nt) without secondary structure interference, ensuring sufficient exposure of the cleavage site. The tRNA-sgRNA unit can be reconstructed twice to accommodate dual sgRNA requirements, and can also be expanded to three or more sgRNAs to accommodate multiplex editing scenarios. This is a scalable and efficient co-expression scheme that relies on the plant's own RNA processing pathway.

[0032] "Csy4 / Cas6e cleavable RNA backbone tandem expression" refers to embedding two sgRNA sequences into an engineered RNA backbone containing at least one Csy4 recognition stem-loop (5′-GUCGAAACU-3′, containing a characteristic GU-rich bulge and a tetrabase loop) or a Cas6e recognition stem-loop (5′-AUUUGA-3′, containing a specific hairpin structure). The two recognition stem-loops are located upstream of the 5′ end of each sgRNA, forming a linear arrangement of "Csy4 stem-loop – sgRNA1 – Csy4 stem-loop – sgRNA2" or "Cas6e stem-loop – sgRNA1 – Cas6e stem-loop – sgRNA2". This backbone is transcribed into a single precursor RNA driven by the same promoter. In the cytoplasm, it is co-expressed by the exogenous Csy4 nuclease (CRISPR-associated protein Csy4, derived from Pseudomonas aeruginosa) or the Cas6e protein (CRISPR-associated protein). Cas6e, derived from the cyanobacterium *Synechocystis* sp. PCC6803, specifically recognizes and cleaves precise sites downstream of the stem-loop (Csy4 cleavage site is located between the 1st and 2nd nt at the 3' end of the stem-loop, while the Cas6e cleavage site is located between the 3rd and 4th nt at the 3' end of the stem-loop), thereby sequentially releasing two fully functional sgRNA molecules. The Csy4 or Cas6e coding sequences can be co-presented with Cas9 in the same vector, driven by constitutive promoters (such as ZmUbi1) or germline-preferred promoters (such as ZmPLA2). The N-terminus of the protein can be fused with a nuclear localization signal (NLS), and the C-terminus can be tagged with HA / FLAG for easy detection of expression levels. Csy4 cleavage efficiency reaches over 92% in maize protoplasts, and Cas6e maintains a mature sgRNA yield of >85% in stably transformed plants. This is a highly controllable, well-defined cleavage site exogenous enzyme-mediated co-expression scheme suitable for scenarios requiring high editing fidelity.

[0033] Both strategies employ a single promoter to avoid the risks of promoter homologous recombination and uneven expression intensity caused by using multiple U6 / U3 promoters. The overall length of the constructed sgRNA expression cassette is controlled within the range of 1.2–1.8 kb, which is significantly shorter than the scheme using two independent U6 promoters (each approximately 0.6 kb promoter + 0.3 kb sgRNA + 0.2 kb terminator, totaling >2.2 kb). This is beneficial for T-DNA segment compression, improving Agrobacterium-mediated transformation efficiency, and enhancing genetic stability in complex genomic contexts.

[0034] The above approach achieves the following: efficient, equal, and simultaneous delivery of sgRNA to the key exon boundary regions of two fertility genes within a single expression cassette, ensuring that the Cas9 protein can simultaneously bind to and cleave both target sites, significantly increasing the probability of co-cleavage at both sites; because tRNA processing or Csy4 / Cas6e cleavage are enzymatic cascade reactions with high temporal and spatial synergy, the two sgRNAs co-localize to the Cas9 complex at the subcellular scale, thereby significantly increasing the incidence of large deletion mutations (ΔExon) in the target exon region (in T0 generation editing). The deletion event can reach 38–65% in the edited plants, which is 1.7–2.3 times higher than the traditional dual promoter strategy. The deletion event is stably homozygous fixed in subsequent BC2F1 self-pollinated generations, and finally male sterile plants without exogenous editing elements and only natural DNA deletion are obtained. This solves the technical problems of high failure rate of double site cleavage, low detection rate of large fragment deletion mutations, and non-heritability of editing events caused by expression imbalance, incomplete splicing or excessively large vectors when multiple sgRNAs are co-expressed. It provides reliable technical support for the construction of genetically stable, non-transgenic, and commercially available maize male sterile lines. Example

[0035] Based on the above embodiments, this embodiment further provides: The Cas9 expression cassette and the dual sgRNA expression cassette are located in the same T-DNA region, and the co-expression of Cas9 protein and selection marker protein is achieved through self-cleaving of 2A peptide or polycistronic ribosome skipping element. The 3' end of the dual sgRNA expression cassette is equipped with a polyT termination signal.

[0036] A Cas9 expression cassette is a DNA sequence unit encoding the Cas9 nuclease, with a promoter upstream and a terminator downstream. The promoter can be a constitutive promoter (such as the maize ubiquitin promoter ZmUbi1) or a germline-specific promoter (such as the anther-specific promoters Zm13 and p5126) to regulate Cas9 expression in specific tissues or developmental stages. The Cas9 protein is SpCas9, SaCas9, Cas12a (Cpf1), or its engineered variants (such as the high-fidelity mutants SpCas9-HF1 and eSpCas9), possessing double-stranded DNA cleavage activity. The coding sequence of the Cas9 expression cassette contains a nuclear localization signal (NLS) sequence to ensure that the Cas9 protein is directed into the nucleus to perform its editing function.

[0037] A dual sgRNA expression cassette refers to two independent sgRNA expression units arranged in tandem on the same vector. Each unit consists of an RNA polymerase III promoter (U6 or U3 promoter), an sgRNA backbone sequence, and a downstream termination signal. The two sgRNAs target the 5′ and 3′ boundaries of key exon regions of maize fertility genes (such as Ms7, Ms30, Ms23, or ZmMs32), respectively, with a target site interval of 100 bp to 5 kb to ensure that the deleted fragment covers at least one complete exon or a coding sequence containing a conserved functional domain. The U6 promoter is derived from maize (ZmU6-1, ZmU6-2) or Arabidopsis thaliana (AtU6-1, AtU6-26), and the U3 promoter is derived from rice (OsU3) or maize (ZmU3). Their transcription start sites precisely correspond to the first base (G) at the 5′ end of the sgRNA, ensuring correct sgRNA processing. The sgRNA backbone sequence includes a tracrRNA portion and a crRNA spacer region, with the spacer region being 18-22 kb in length. The NT content should be 40%–60%, and four or more consecutive T bases should be avoided to prevent premature termination of RNA polymerase III.

[0038] The same T-DNA segment refers to the complete DNA fragment located between the left and right boundaries (LB / RB) of the Agrobacterium Ti plasmid. This segment integrates the Cas9 expression cassette, the dual sgRNA expression cassette, and the selection marker gene, with a physical distance of no more than 100 kb between the three, ensuring stable integration into the plant genome as a genetic unit during Agrobacterium-mediated genetic transformation. This T-DNA segment does not contain non-essential sequences other than plant virus replication origins and prokaryotic resistance genes, in order to reduce vector complexity and improve transformation efficiency. The T-DNA is constructed in binary vectors (such as the pCAMBIA series and pGreen series) and is suitable for various transformation systems such as maize embryos, callus tissue, or pollen tube pathways.

[0039] Self-cleaved 2A peptides are a class of short peptide sequences (18-22 amino acids in length) derived from animal viruses, including P2A (porcine teschovirus-1 2A), T2A (Thosea asigna virus 2A), E2A (equine rhinitis Avirus 2A), or F2A (foot-and-mouth disease virus 2A). Their mechanism of action is to induce "ribosome skipping" during ribosomal translation, meaning that no peptide bond is formed between the glycine and proline residues at the C-terminus of the 2A peptide, resulting in the release of upstream Cas9 protein and downstream selectable marker protein in a near equimolar ratio. Polycistronic ribosome skipping elements also include IRES (internal ribosome entry site) or ribosome loading sequences derived from the CRISPR-Cas system (such as a ribosome loading sequence following the Csy4 recognition site), but 2A peptides are used because of their smaller molecular weight (<2.5 kDa), higher co-expression uniformity, and lower translational repression effect.

[0040] The screening marker proteins are reporter proteins or selective proteins used for screening positive plants, including neomycin phosphotransferase II (nptII, conferring kanamycin resistance), hygromycin phosphotransferase (hph, conferring hygromycin resistance), glyphosate acetyltransferase (GAT, conferring glyphosate resistance), green fluorescent protein (GFP), red fluorescent protein (RFP), or yellow fluorescent protein (YFP). When fluorescent proteins are selected, a Kozak sequence (GCCACC) is added before their coding sequence to enhance translation initiation efficiency. When herbicide resistance genes are selected, their expression is driven by constitutive promoters (such as CaMV 35S, ZmUbi1) to ensure that the resistance phenotype is manifested in the seedling stage, facilitating early screening.

[0041] The polyT termination signal is a DNA sequence consisting of 4-6 consecutive thymine (T) bases. As a natural and highly efficient terminator for RNA polymerase III, it is located at the 3′ end of the double sgRNA expression cassette. This sequence is adjacent to the 3′ end of the sgRNA transcript, ensuring precise termination after transcription of the sgRNA precursor and avoiding readthrough to downstream sequences that could generate chimeric RNA or interfere with the expression of neighboring genes. A 1-3 bp spacer is placed after the polyT sequence before connecting downstream elements to prevent the termination efficiency from being affected by DNA secondary structures. In the U6 promoter system, the termination efficiency of the polyT termination signal reaches over 95%, which is significantly better than the applicability of the traditional polyA signal to RNA polymerase III.

[0042] The synergistic effects of the various technical features are as follows: placing the Cas9 expression cassette and the dual sgRNA expression cassette in the same T-DNA segment ensures that they are integrated, co-segregated, and co-inherited during transformation events, avoiding loss of editing ability due to segregation loss; through the 2A peptide-mediated co-expression mechanism, the expression level of the screening marker protein directly reflects the translational abundance of the Cas9 protein, thereby ensuring that positive plants obtained through antibiotic or fluorescence screening necessarily express functional Cas9, significantly increasing the proportion of cells that actually undergo dual-site editing events; the polyT termination signal ensures the precise truncation and maturation of the sgRNA precursor at the transcriptional level, preventing abnormally long transcripts from competitively binding to the Cas9 protein or causing off-target effects; the integrated design of the three features constitutes a closed-loop editing vector system with "verifiable editing ability, traceable editing events, and predictable editing products".

[0043] Through the above-described steps, this invention achieves the following: During maize genetic transformation, using the phenotype of the screening marker protein as an objective indicator, it accurately enriches transformed cells with Cas9 editing capabilities, significantly improving the detection rate of large-fragment deletion mutations mediated by dual sgRNAs; the same T-DNA structure ensures the genetic stability and reproducibility of the editing element, reducing the risk of residual editing elements in the BC2 generation; the polyT termination signal ensures the structural integrity and functional activity of the sgRNA, supporting high-fidelity and high-efficiency targeted exon deletion. Therefore, it solves the technical problems in the prior art, such as low editing efficiency, high false-positive rate of positive plants, and difficulty in stably obtaining target deletion mutations due to asynchronous expression of Cas9 and the screening marker, dispersed integration of editing elements, and abnormal sgRNA transcription. Ultimately, it obtains maize sterile lines that do not contain exogenous editing elements, exhibit homozygous deletion of the fertility gene, and are completely and stably heritable in male sterility. Example

[0044] Based on the above embodiments, this embodiment further provides: Dual sgRNA-mediated deletion mutations delete DNA fragments that encompass at least one intact exon or a coding sequence containing a conserved functional domain.

[0045] Double-sgRNA-mediated deletion mutations refer to mutations in which a pair of single-guide RNAs (sgRNAs) targeting the same locus upstream and downstream guide the Cas9 nuclease to simultaneously generate two adjacent double-strand breaks (DSBs) on the genomic DNA. This is followed by the precise deletion of a DNA fragment between the two cleavage sites through a non-homologous end joining (NHEJ) repair mechanism. The occurrence of this deletion event depends on the spatial and temporal co-expression of the two sgRNAs and the cooperative cleavage activity of the Cas9 protein. The deletion length is determined by the nucleotide distance between the upstream and downstream sgRNA target sites, ranging from 100 bp to 5 kb. This deletion is an irreversible genomic structural variation that does not introduce a foreign template and does not rely on the homologous recombination repair pathway.

[0046] Covering at least one complete exon means that the deleted DNA fragment starts ≤50 bp upstream of the 5′ splice donor site (GT) of a certain exon and terminates ≤50 bp downstream of the 3′ splice acceptor site (AG) of the same exon, thus ensuring that the entire coding sequence of the exon and the key splicing signals on both sides are removed. For example, in the maize nuclear fertility major gene ZmMs30 (GenBank accession number: NC_042857.1), an upstream sgRNA targeting the 5′ end of exon 4 (216 bp in length, encoding the core region of the zinc finger domain) was designed to target the sequence 32 bp upstream of its 5′ end (target sequence 5′-GAGTCCGACCTCGTCCATGG-3′) and the downstream sgRNA targeting the sequence 18 bp downstream of its 3′ end (target sequence 5′-GGACGAGGTCGGACTCTACC-3′), with a distance of 284 bp between the two target sites. The bp, after double cutting, can achieve complete deletion of exon 4 and its adjacent splicing signals; this deletion leads to exon skipping or premature nonsense-mediated mRNA decay (NMD) in the transcript, ultimately preventing the production of MS30 protein with any functional activity.

[0047] The coding sequence containing a conserved functional domain refers to a structural unit in the primary structure of a protein that is highly conserved through multi-species comparisons and has been confirmed by functional experiments to be essential for maintaining fertility. This includes, but is not limited to, zinc finger domains, PPR (pentatricopeptide repeat) motifs, TPR (tetratricopeptide repeat) helical folds, or cysteine-rich motifs. The definition of this conserved functional domain is based on amino acid sequence identity ≥85% in gramineous plants (including maize, rice, and sorghum), and its deletion in Arabidopsis or yeast complementation experiments confirms that it leads to male gamete developmental arrest. For example, the C2H2 type zinc finger domain, composed of amino acids 62–109 at the N-terminus of the ZmMs30 protein, is shown in a three-dimensional model to directly participate in DNA binding and transcriptional regulation. When double sgRNA targets both ends of the coding region of this domain (corresponding to positions 1245–1268 and 1430–1452 in the genome), it induces 186... When bp is missing, all secondary structural elements (α-helix and β-sheet) of the domain can be completely removed, causing the protein to lose its DNA-binding ability.

[0048] The implementation methods covered are structurally tunable: by adjusting the target positions of the upstream and downstream sgRNAs, the deletion boundary can be located within an exon, an intron-exon junction, or across an exon-intron complex, respectively. Optionally, the upstream sgRNA target site can be set in an intron region 10–50 bp upstream of the 5′ end of the target exon, and the downstream sgRNA target site can be set in an intron region 10–50 bp downstream of the 3′ end of the same exon, to balance editing efficiency and splicing interference specificity. Alternatively, when the target gene is a multi-domain protein, two sgRNAs can be designed to span two adjacent exons, so that the deletion covers the flexible hinge region connecting the two functional modules, thereby disrupting the protein conformational dynamic equilibrium. In addition, the deletion can also be extended by introducing a third sgRNA to form a "three-cut strategy," which removes an additional non-coding spacer sequence on top of the double cut, further expanding the deletion span and reducing the expression probability of the residual functional protein.

[0049] There are clear causal relationships among the various technical features: the target localization accuracy of dual sgRNAs determines the controllability of the deletion boundary; the localization strategy of the deletion boundary (whether it covers an intact exon or a conserved domain) directly determines the degree of damage to gene function caused by the mutation; and the conservation of functional domains and the coding integrity of exons together constitute the molecular benchmark for judging "complete inactivation". By anchoring the deletion range to the above two types of functional units, the risks of missense mutations, in-frame deletions, or truncated protein residues caused by deleting only part of the codon or non-conserved residues can be avoided, fundamentally eliminating the fertility "leakage" phenomenon caused by residual protein activity.

[0050] Through the above scheme, this invention achieves the following: because the target site of the dual sgRNA is precisely set to a coding sequence covering at least one complete exon or conserved functional domain, the edit-induced DNA deletion inevitably leads to abnormal splicing of the target fertility gene transcript, mRNA degradation, or premature termination of translation, thereby completely eliminating the expression of functional MS30 protein; it solves the technical problems in the background technology where small fragment deletions or single base mutations may retain part of the protein structure and activity, resulting in unstable male sterility phenotypes and susceptibility to temperature or nutrient conditions; and finally obtains genetically stable, phenotypically complete, and environmentally robust homozygous male sterile plants, providing a high-confidence genetic material basis for subsequent BC2 backcrossing and seed selection. Example

[0051] Based on the above embodiments, this embodiment further provides: The two sgRNA expression units are integrated into the same editing vector and driven by different promoters. The Cas9 protein is driven by a constitutive or germline-specific promoter.

[0052] The integration of dual sgRNA expression units into the same editing vector means that two independent sgRNA expression cassettes are cloned into the same T-DNA segment, sharing the same plant transformation backbone (such as pCAMBIA series, pGreen series, or pCambia1300-derived vectors), ensuring that the two are stably co-integrated into the maize genome in a linkage manner during Agrobacterium-mediated genetic transformation. This design avoids the problems of sgRNA expression unit segregation, copy number non-uniformity, and genetic instability caused by multi-vector co-transformation, and is conducive to simultaneously obtaining dual-site cleavage capability in a single transformation event. The same editing vector also contains screening marker genes (such as the hygromycin phosphotransferase gene hpt or the glufosinate acetyltransferase gene bar), T-DNA boundary sequences, and necessary regulatory elements such as plant promoter-terminator. Optional variations include: constructing dual sgRNA expression units in ultra-large capacity vectors (such as BIBAC or TAC vectors) to accommodate additional regulatory modules; or using a binary vector system where the master vector carries dual sgRNA and the auxiliary vector provides Cas9. However, in this case, it is necessary to strictly verify the co-transformation efficiency and co-segregation rate. In this embodiment, a single T-DNA integration scheme is used to ensure genetic consistency.

[0053] "Driven by different promoters" means that the two sgRNA expression units are controlled by non-homologous RNA polymerase type III promoters with no significant sequence homology, such as one driven by the maize ZmU6-1 promoter (GenBank accession number: EU138275.1) and the other driven by the ZmU6-2 promoter (GenBank accession number: EU138276.1); the homology of their core promoter regions is less than 60%, effectively avoiding transcriptional gene silencing (TGS) or co-inhibition mediated by RNA interference caused by promoter repetitive sequences; other optional promoter combinations include: ZmU3-1 and ZmU3-2, Arabidopsis thaliana AtU6-26 and AtU6-29, rice OsU6a and OsU6b, or artificially modified chimeric U6 promoters (such as introducing heterologous TATA box enhancer elements into the ZmU6 core region). This design enhances the abundance and stability of each sgRNA transcript and ensures that the relative molar ratio of the two in the cell is close to 1:1, providing a molecular basis for the synchronous binding of Cas9 protein and the completion of dual-site cleavage. Its optional variations include: using promoter combinations with different transcription initiation efficiencies (such as strong promoter + weak promoter) to fine-tune the sgRNA expression gradient and adapt to the chromatin open state of different target regions.

[0054] The term "Cas9 protein driven by constitutive or germline-specific promoters" refers to the fact that the promoter upstream of the Cas9 coding sequence possesses two switchable functional modes. When a constitutive promoter is used, the maize ubiquitin extension protein gene promoter ZmUbi1 (GenBank accession number: AF302849.1) or the rice Actin1 promoter (OsAct1) is employed to ensure sustained expression in almost all tissues, including roots, stems, leaves, embryos, and floral organs, thereby increasing the incidence of somatic editing events and facilitating the rapid acquisition of a high proportion of chimeric mutations in T0 generation plants. When a germline-specific promoter is used, the maize meiosis-specific DD45 promoter (GenBank accession number: AY508682.1) or... The GPAT promoter (GenBank accession number: EU964113.1), specifically expressed in the anther tapetum, ensures that Cas9 is expressed only before and after meiosis in microspore mother cells and during early pollen development. This reduces the risk of off-target effects in somatic cells and promotes the enrichment of editing events in the germline, enabling deletion mutations to be more efficiently transmitted to gametes and fixed in offspring. This promoter selection strategy can be flexibly configured according to breeding objectives: if the goal is to detect heritable mutations in the T0 generation, a germline-specific promoter is selected; if rapid screening of editing events in callus or seedlings is required, a constitutive promoter is selected. Possible variations include: employing a dual-promoter chimeric strategy (e.g., Ubi1 core + GPAT distal enhancer) to achieve spatiotemporal superposition regulation of "basal broad-spectrum expression + reproductive enhancement"; or introducing a chemically inducible promoter (e.g., the dexamethasone-induced GR-UAS system) to precisely control the Cas9 expression window through exogenous hormones.

[0055] There is a clear synergistic relationship among the above-mentioned technical features: different promoters drive dual sgRNA expression units, ensuring independent transcription and equivalent accumulation of the two sgRNA molecules from the source, avoiding expression imbalance caused by competitive binding to the RNA polymerase III complex; while the choice of Cas9 promoter type determines the spatiotemporal dimensions of editing. When dual sgRNAs are sufficiently enriched in the cell nucleus, constitutive Cas9 provides continuous cleavage capability, improving overall editing efficiency; germline-specific Cas9 precisely constrains cleavage activity to the critical period of gamete formation, making it easier for induced exon deletion mutations to enter functional male gametes and achieve high homozygosity during BC2 generation self-crossing. The combined effect of these two factors results in not only a high probability of dual-site cleavage but also a high genetic transmission rate and low background interference.

[0056] Through the above steps, this invention achieves the following: reducing the risk of transcriptional silencing caused by promoter homology while maintaining efficient co-expression of dual sgRNAs; simultaneously configuring the spatiotemporal expression mode of Cas9 according to different application scenarios; constitutive-driven improvement of editing detection throughput, and germline-specific drive to enhance genetic stability and biosafety; ultimately, male-sterile plants without exogenous editing elements and with homozygous deletion of fertility genes can be stably obtained in the BC2 generation, providing a high-quality, non-transgenic genetic foundation for the subsequent construction of a multi-controllable maintenance breeding system. Example

[0057] Based on the above embodiments, this embodiment further provides: The pollen suppression module in the multi-control maintainer reproductive construct works synergistically with the fertility restoration gene Ms30 to produce 50% male-sterile seeds without the construct and 50% maintainer line seeds containing the construct through self-pollination of the maintainer line.

[0058] Step 1: The pollen suppression module in the multi-control reproductive construct works synergistically with the fertility restoration gene Ms30; The multi-control reproductive construct refers to a functional DNA fragment integrated into the maize chromosome. It contains four core functional units: a fertility restoration gene, a pollen suppression module, a seed selection marker, and a herbicide resistance gene. Each unit is driven by standard plant expression regulatory elements (such as the CaMV 35S promoter, Ubiquitin promoter, and NOS terminator) and is codon-optimized to adapt to the maize cell translation system. The construct is inserted into the non-coding region of the maize genome or a safe site (such as the ZmActin intron region) in a single-copy, stable integration manner to avoid interfering with the expression of endogenous genes.

[0059] "Pollen suppression modules" specifically refer to transcriptional repression / cytotoxic elements that are specifically activated in the early stages of anther tapetum or microspore development. These include, but are not limited to: ① Barnase genes (ribonucleases that induce pollen abortion) driven by the maize anther-specific promoters Zm629 or ZmMS4; ② CRISPRi-sgRNA driven by the ZmAMS promoter that targets the promoter regions of genes essential for pollen development (such as ZmPTC1 and ZmMS7) to achieve epigenetic silencing; ③ Diphtheria toxin A fragment (DT-A) fusion protein driven by the ZmUbi1 promoter that selectively kills male gametes containing the construct during the post-meiotic stage. Any of these forms ensures that the functional protein is expressed only in pollen carrying the construct, without affecting female gamete development.

[0060] The fertility restorer gene Ms30 is an endogenous dominant fertility restorer gene in maize. It encodes an F-box protein (Full name: Male fertility restorer 30) with E3 ubiquitin ligase activity, located on maize chromosome 3 (Chr3: 182,456,789–182,461,234 bp). It can specifically recognize and ubiquitinate and degrade abnormal mitochondrial chimeric proteins (such as URF13 analogs) produced by male sterility mutants, thereby restoring normal mitochondrial respiratory function and normal anther dehiscence. Ms30 is constitutively expressed in maintainer lines or regulated by anther-specific promoters (such as ZmMS10), ensuring normal anther development, pollen shedding, and self-pollination ability of maintainer line plants.

[0061] "Synergistic effect" refers to the complementary regulation formed by the pollen suppression module and Ms30 in the spatial and temporal dimensions: the pollen suppression module blocks the functional development of pollen containing the construct during the male gamete formation stage (from the microspore mitosis stage to the mature pollen stage), while Ms30 is continuously expressed after the differentiation of the stamen and pistil primordia, ensuring the normal development of the anthers, pollen shedding and self-pollination ability of the maintainer line plant; the two have no direct molecular interaction, but through targeted intervention at different developmental windows, they jointly achieve the dual function of "the hemizygous maintainer line can both maintain self-pollination and produce homozygous sterile lines as the male parent".

[0062] Step 2: Self-pollinate the maintainer line to produce 50% male-sterile seeds without constructs and 50% maintainer line seeds containing constructs; Among them, "the maintainer line is in a hemizygous state" means that the plant integrates a multi-controlled maintainer reproductive construct on one homologous chromosome, while the corresponding locus on another homologous chromosome does not integrate the construct, that is, the genotype is Heterozygous forconstruct (+ / −). During meiosis, according to Mendel's law of segregation, theoretically two types of male gametes (sperm cells) with equal proportions are produced: one type carries the construct (+), and the other type does not carry the construct (−).

[0063] The pollen suppression module specifically blocks the development of pollen containing the construct. This means that the functional protein encoded by the aforementioned pollen suppression module is expressed only in pollen cells carrying the construct and exerts cytotoxic or transcriptional repressive effects. This causes programmed cell death or failure to complete starch accumulation and exine thickening in these pollen cells during the late mononuclear or binuclear stages, resulting in loss of germination and fertilization capacity. Pollen without the construct, on the other hand, develops completely normally and possesses full viability. Therefore, 100% of the effective male gametes actually involved in fertilization are of the construct-free type (−).

[0064] Both types of oocytes refer to female gametes (embryo sacs formed by meiosis of megasporocytes) which are not affected by pollen suppression modules and still produce oocytes with 50% containing constructs (+) and 50% without constructs (−) according to normal Mendelian segregation. When effective male gametes (−) combine randomly with oocytes, the genotype combinations of the offspring are: (−) male gamete × (−) oocyte → (− / −) homozygous deletion mutant, which is male sterile; (−) male gamete × (+) oocyte → (+ / −) hemizygote, which is a fertile maintainer line. Because the ratio of the two types of female gametes is strictly 1:1, the phenotypic ratio of the offspring seeds is constant at 50% male sterile seeds and 50% maintainer line seeds.

[0065] This ratio does not depend on external screening methods, but is determined solely by the genetic segregation pattern and the biological filtering mechanism at the pollen level, exhibiting high reproducibility and field stability.

[0066] Through the above steps, this invention achieves the following: During the self-pollination process of the maintainer line, no artificial emasculation, chemical treatment, or molecular detection is required. It relies solely on the pollen suppression module's natural selective elimination of male gametes, combined with Ms30's sufficient guarantee of the maintainer line's fertility, to automatically generate an equal proportion of male-sterile line seeds and maintainer line seeds. This mechanism allows the same population to serve as both a maternal parent (for hybridization with restorer lines) and a paternal parent (for hybridization with sterile lines), truly achieving "one line, two uses." Simultaneously, because sterile line seeds naturally do not contain constructs, their genomes contain only edited homozygous deletion mutations, meeting the definition standards for non-GMO organisms in most countries, significantly lowering the regulatory compliance threshold. Furthermore, in conjunction with the fluorescent protein or herbicide resistance markers described above, high-throughput optical sorting or spraying treatment can be performed on self-pollinated mixed seeds, further improving the purity and large-scale production capacity of sterile line seeds.

[0067] The above embodiments are only used to illustrate the technical solutions of the present invention / invention, and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention / invention.

Claims

1. A method for constructing a male-sterile line in maize, comprising gene editing and hybridization operations on maize plants, characterized in that: Double sgRNAs were designed on both sides of the key functional region of the maize nuclear fertility gene, and deletion mutations in the exon region of the gene were induced by Cas9-mediated two-site cleavage. The editing element expressing Cas9 and double sgRNA was introduced into the donor plant and hybridized with the recipient inbred line. The hybrid offspring were backcrossed to the BC2 generation and then self-crossed to obtain male sterile plants without the editing element and with homozygous deletion mutation. A multi-controllable reproduction construct was constructed, the construct comprising a fertility restoration gene, a pollen suppression module, a seed selection marker, and a herbicide resistance gene; The construct exists in the maintainer line in a hemizygous state. The formation of male gametes containing the construct is restricted by the pollen suppression module. The male sterile line seeds are sorted using the seed selection marker or herbicide resistance gene to obtain male sterile line seeds that do not contain transgenic components.

2. A method for constructing a male-sterile maize line as described in claim 1, characterized in that: The dual sgRNA expression cassette contains two independent sgRNA expression units driven by either a U6 or U3 RNA polymerase III promoter, which target the 5' and 3' boundaries of key exon regions of fertility genes, respectively.

3. A method for constructing a male-sterile maize line as described in claim 1, characterized in that: The dual sgRNAs are expressed in tandem via a polycistronic tRNA-sgRNA strategy or by Csy4 / Cas6e cleavable RNA backbone.

4. A method for constructing a male-sterile maize line as described in claim 1, characterized in that: The Cas9 expression cassette and the dual sgRNA expression cassette are located in the same T-DNA region, and the co-expression of Cas9 protein and selection marker protein is achieved through self-cleaving 2A peptide or polycistronic ribosome skipping element. The 3' end of the dual sgRNA expression cassette is provided with a polyT termination signal.

5. A method for constructing a male-sterile maize line as described in claim 1, characterized in that: The DNA fragment deleted by the dual sgRNA-mediated deletion mutation covers at least one intact exon or a coding sequence containing a conserved functional domain.

6. A method for constructing a male-sterile maize line as described in claim 1, characterized in that: The dual sgRNA expression units are integrated into the same editing vector and driven by different promoters, with the Cas9 protein driven by a constitutive or germline-specific promoter.

7. A method for constructing a male-sterile maize line as described in claim 1, characterized in that: The pollen suppression module in the multi-controlled maintainer reproductive construct works synergistically with the fertility restoration gene Ms30 to produce 50% male-sterile seeds without the construct and 50% maintainer line seeds containing the construct through self-pollination of the maintainer line.