Male sterile maintainer line plant and application thereof

Abstract

The invention belongs to the field of plant genetic breeding and seed production. In particular, the present application relates to nucleic acid molecules, vectors, and host cells (e.g., Agrobacterium tumefaciens) for producing male sterile maintainer plants. The present application also relates to a male sterile maintainer plant, a method of producing said male sterile maintainer plant, and the use of said plant for propagating male sterile plants and male sterile maintainer plants. In addition, the invention also provides a recombinant DNA molecule and a method for detecting a specific male sterility maintainer line plant.

Description

Technical Field

[0001] This application belongs to the field of plant genetics and breeding, and seed production. Specifically, this application relates to nucleic acid molecules, vectors, and host cells (e.g., Agrobacterium) for producing male-sterile maintainer lines. This application also relates to a male-sterile maintainer line, a method for producing the male-sterile maintainer line, and the use of the plant for propagating male-sterile and male-sterile maintainer lines. Furthermore, this application provides methods for detecting recombinant DNA molecules in specific male-sterile maintainer lines. Background Technology

[0002] Heterosis refers to a biological phenomenon in which hybrid offspring surpass their parents in multiple traits such as yield, quality, adaptability, stress resistance, growth vigor, and reproductive capacity. It is prevalent in nature. The common method for producing hybrids is as follows: female and male parents are planted together; the tassels of the female parents are removed, while the tassels of the male parents are retained; the seeds are harvested from the female parents, which are the hybrids.

[0003] Plants in nature exhibit three types of pollination: self-pollination, cross-pollination, and interpollination. Self-pollination refers to the phenomenon where pollen from one plant pollinates its own pistil. In hermaphroditic plants, this can be further divided into self-pollination (e.g., beans), neighboring flower pollination, and monopodial cross-pollination. Self-pollination occurs between the stamens and pistils of the same flower. Neighboring flower pollination occurs between different flowers within the same inflorescence (individual plant). Monopodial cross-pollination occurs between different flowers on the same plant. Some plants have stamens and pistils that do not reside in the same flower, and some even do not reside on the same plant, making self-pollination impossible. Their pistils can only receive pollen from other flowers; this is called cross-pollination. Crops with a natural hybridization rate higher than 50% and exhibiting self-pollination depression are classified as interpodial crops.

[0004] Maize is monoecious, with male and female flowers located on different parts of the plant. Maize reproduces through both self-pollination and cross-pollination. Natural pollination occurs when wind carries pollen from the male tassel to the silks of the female tassel. In maize breeding, homozygous inbred lines are typically developed first. Two inbred lines are then crossed, and the yield, stress resistance, and other characteristics of the hybrid offspring are evaluated to determine their commercial potential. Each inbred line may possess one or more desirable traits lacking in the other inbred line, or supplement one or more undesirable traits. The first generation seeds from the cross between the two inbred lines are the F1 generation seeds. Germination of the F1 seeds yields F1 plants, which are more robust and have higher biomass than the two parent inbred lines.

[0005] Hybrids (F1) can be produced by manually detasseling the female parent. This involves removing the unpollenized male ears of the female parent (which can be sown alternately with the male parent in the field, e.g., five rows of female parent and one row of male parent), while retaining the male ears. Subsequently, by isolating the female parent from foreign corn pollen, the female ears will only accept pollen from the male parent, resulting in the hybrid (F1) seeds, which can be used for agricultural production. However, in actual hybrid production, environmental changes may cause the plants to develop tassels again after detasseling, or detasseling may be incomplete. Both situations can lead to self-pollination of the female parent, resulting in the production of hybrids mixed with seeds from the female parent's inbred lines. The yield of the female parent's inbred lines is far lower than that of the hybrids, making these seeds substandard products. This affects farmers' income and the reputation of seed companies, potentially leading to compensation liabilities for the seed companies. Alternatively, machines can be used to detassel the female parent. Machine detasseling has essentially the same reliability as manual detasseling, but it is faster and cheaper. However, most emasculation machines cause more damage to the plant than manual emasculation. Therefore, there is currently no satisfactory emasculation method. People are still looking for cheaper and more thorough alternatives.

[0006] Stable male-sterile systems provide a simple and efficient means of emasculation. In some cases, the arduous process of emasculation can be avoided by using male-sterile systems. This method involves three main materials: (1) the male-sterile line (also simply called the sterile line): which is male-sterile material; (2) the male-sterile maintainer line (also simply called the maintainer line): which provides pollen to the sterile line, ensuring that the offspring of the sterile line remain sterile; and (3) the male-sterile restorer line (also simply called the restorer line): which restores the fertility of the sterile line. Crossing the sterile line with the restorer line produces F1, which is the hybrid used in agricultural production.

[0007] Male sterility in plants can be classified into three types: cytoplasmic male sterility, nuclear male sterility, and nucleo-cytoplasmic interaction male sterility. Cytoplasmic male sterility is inherited through cytoplasm, typically represented by single cytoplasmic genes S and N, indicating male sterility and fertility respectively; however, this type is difficult to apply in agricultural production. Nuclear male sterility is inherited through the nucleus; in most cases, male sterility is controlled by a pair of recessive genes (msms), while normal fertility is controlled by a relatively dominant gene (MsMs) or (Msms). Nucleo-cytoplasmic interaction male sterility (CMS) plants exhibit nucleo-cytoplasmic interaction inheritance. In short, male sterility only occurs when the cytoplasm contains the sterility gene S and the nucleus contains a homozygous sterility gene (rfrf). If the cytoplasm contains the fertile gene N, the plant will exhibit male fertility regardless of whether the gene in the nucleus is the fertile gene (RfRf) or the sterile gene (rfrf). Similarly, if the nucleus contains either the fertile gene (RfRf) or (Rfrf), the plant will exhibit male fertility regardless of whether the gene in the cytoplasm is the fertile gene N or the sterile gene S.

[0008] Nucleocytoplasmic male sterility (CMS) materials have been reported for breeding. In this method, the male sterile line has a genetic makeup of S(rfrf) and cannot produce normal pollen, but can be used as a female parent in hybridization. The maintainer line has a genetic makeup of N(rfrf), and the F1 generation produced by crossing it with the male sterile line will still remain male sterile, i.e.: S(rfrf)(♀) × N(rfrf) → S(rfrf)(male sterile). The restorer line has a genetic makeup of S(RfRf) or N(RfRf), and the F1 generation produced by crossing it with the male sterile line will be restored to male fertility, i.e.: S(rfrf)(♀) × S(RfRf) → S(Rfrf)(F1)(fertile), or S(rfrf)(♀) × N(RfRf) → S(Rfrf)(F1)(fertile). The resulting F1 plants are self-pollinated to produce F2, which can also be widely used in agricultural production. Male-sterile lines eliminate the need for manual emasculation, saving labor, reducing seed costs, and ensuring seed purity. Currently, crops such as rice, corn, sorghum, onions, castor beans, sugar beets, and rapeseed are using nucleo-cytoplasmic male-sterile lines for hybrid seed production. Nucleo-cytoplasmic male-sterile lines for other crops are also under extensive research. However, the CMS system also has its drawbacks: firstly, some CMS materials have been observed to be susceptible to disease; secondly, restorer lines are relatively difficult to find. These problems hinder the widespread application of the CMS system in seed production.

[0009] In nuclear male sterility systems, the nuclear gene controlling male sterility is usually recessive, and the plant only exhibits male sterility when homozygous (msms). However, since male-sterile plants cannot self-pollinate, male-sterile offspring (msms) can only be obtained by crossing them with heterozygous plants (Msms). However, in the fruit ears of offspring from heterozygous plants (Msms) and male-sterile plants (msms), both male-sterile seeds (msms) and fertile heterozygous seeds (Msms) coexist, and it is impossible to distinguish which are sterile and which are fertile; this distinction is only made after sowing when the plant sheds pollen. This limits the widespread application of nuclear male sterility systems in seed production.

[0010] In recent years, methods for maintaining male-sterile plants using transgenic techniques have been reported (US6743968). This method involves: first, constructing a transgenic vector containing a pollen cell lethal gene and a dominant gene that restores fertility; then, transferring this vector into male-sterile plants, where it exists in a heterozygous state. Due to the presence of the fertility-restoring gene, the transgenic plant is male-fertile. Furthermore, when it is crossed with a male-sterile plant, the pollen containing the fertility-restoring gene (Msms) also contains the lethal gene, thus causing the pollen containing the fertility-restoring gene to abort. Therefore, the transgenic plant can only produce pollen (ms) without the fertility-restoring gene, which can be crossed with female gametes (ms) of a male-sterile plant. The resulting offspring are all recessive homozygous individuals (msms). That is, when such a plant is crossed with a male-sterile plant, its offspring retain the homozygous recessive state of the recessive male-sterile plant. However, the drawback of the above method is that, due to the existence of heteroandrogenic fertilization (the embryo and endosperm are formed by fertilization of sperm that develop from different male gametophytes, i.e., the sperm that form the embryo and endosperm have different genotypes), a certain proportion of fertile seeds will still be obtained from the offspring seeds after screening the endosperm. Furthermore, these fertile seeds are difficult to distinguish from sterile seeds, and cannot fully meet the needs of actual production.

[0011] Furthermore, methods for constructing male-sterile maintainer lines and maintaining the sterility of male-sterile plants have been reported using vectors containing both Ms45 and mn1 RNAi (Chinese Patent ZL201210406155.6). However, due to factors such as heteroandrogenic fertilization, seeds obtained through endosperm phenotype screening may contain the target gene in the embryo but not in the endosperm, making these plants fertile. These fertile seeds are difficult to distinguish from sterile seeds and can only be identified when the plants shed pollen. Removing fertile plants at pollen shedding will affect the purity of the hybrids produced, failing to fully meet the needs of practical production. Therefore, it is necessary to establish a method for identifying sterile and fertile plants after sowing and before pollen shedding, so as to promptly remove mixed fertile plants.

[0012] Therefore, how to improve nuclear male sterility systems to conveniently maintain the sterility of male-sterile lines, and how to obtain highly pure male-sterile offspring, are urgent problems to be solved in breeding. More efficient methods for propagating male-sterile lines still need to be developed in this field. Summary of the Invention

[0013] In transgenic plants or their seeds, the expression of target genes (genes introduced through genetic transformation within the transgenic plant) and the effectiveness of trait regulation are influenced by the interaction between the target genes and regulatory elements, and this interaction is more pronounced in plants containing two or more target genes. The inventors of this application, through careful design and extensive experiments, obtained a nucleic acid molecule that, after insertion into the plant genome, resulted in multiple target genes being at ideal expression (or non-expression) levels and effectively regulating the traits of the transgenic plant. Furthermore, the expression and genetic stability of target genes in transgenic plants are also affected by the genomic location of the nucleic acid molecule insertion. Therefore, the inventors of this application also provide the transgenic plant SSMM-15 with an optimal insertion site. SSMM-15, as a maintainer line, can be harvested simultaneously and its offspring can be effectively distinguished when crossed with male-sterile plants. Thus, the inventors have completed this invention.

[0014] Nucleic acid molecules

[0015] Therefore, in a first aspect, this application provides an isolated nucleic acid molecule comprising:

[0016] A first polynucleotide comprising a nucleotide sequence of a restoration gene capable of restoring male fertility in plants that are male-sterile due to a male-sterile gene.

[0017] The second polynucleotide contains a nucleotide sequence that includes a silencing element capable of targeting the Mn1 gene and / or the Sh1 gene; and

[0018] The third polynucleotide contains the nucleotide sequence of the Lc gene.

[0019] As used herein, the term "silencing element" refers to a polynucleotide capable of reducing or eliminating the level or expression of a target polynucleotide or its encoded polypeptide. In this document, the "target polynucleotide" can be any gene or sequence whose expression level is desired to be reduced in a transgenic plant. In some embodiments, the target polynucleotide is the Mn1 gene and the Sh1 gene. Therefore, in some embodiments, the silencing element is a silencing element capable of targeting the Mn1 gene and the Sh1 gene.

[0020] In some embodiments, the silencing element is selected from: sense inhibitory elements, antisense inhibitory elements, hairpin inhibitory elements, double-stranded RNA, siRNA, amiRNA, miRNA, or any combination thereof.

[0021] In some embodiments, the silencing element comprises a sense repressor and an antisense repressor capable of targeting the Mn1 and Sh1 genes.

[0022] In some embodiments, the silencing element comprises a nucleotide sequence as shown in SEQ ID NO:14-17.

[0023] In some embodiments, the silencing element comprises a sense repressor, a hairpin repressor, and an antisense repressor capable of targeting the Mn1 and Sh1 genes.

[0024] In some embodiments, the silencing element comprises a nucleotide sequence as shown in SEQ ID NO:14-18.

[0025] In some embodiments, the silencing element comprises, from 5' to 3', the following elements in sequence: a sense repressor element for the Mn1 gene, a sense repressor element for the Sh1 gene, a hairpin repressor element, an antisense repressor element for the Sh1 gene, and an antisense repressor element for the Mn1 gene.

[0026] In some embodiments, the silencing element has a nucleotide sequence as shown in SEQ ID NO:13.

[0027] In some embodiments, the male sterility gene is a recessive male sterility gene that causes male sterility in plants when homozygous.

[0028] In some embodiments, the male sterility gene is selected from ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, ms10, ms11, ms12, ms13, ms14, ms15, ms16, ms17, ms18, ms19, ms20, ms21, ms22, ms23, ms24, ms25, ms26, ms27, ms28, ms29, ms30, ms31, ms32, ms33, ms34, ms35, ms36, ms37, ms38, ms43, ms45, ms47, ms48, ms49, ms50, ms52, and any combination thereof. In some embodiments, the male sterility gene is ms45.

[0029] In some embodiments, the restoration gene is selected from Ms1, Ms2, Ms3, Ms4, Ms5, Ms6, Ms7, Ms8, Ms9, Ms10, Ms11, Ms12, Ms13, Ms14, Ms15, Ms16, Ms17, Ms18, Ms19, Ms20, Ms21, Ms22, Ms23, Ms24, Ms25, Ms26, Ms27, Ms28, Ms29, Ms30, Ms31, Ms32, Ms33, Ms34, Ms35, Ms36, Ms37, Ms38, Ms43, Ms45, Ms47, Ms48, Ms49, Ms50, Ms52, and any combination thereof. In some embodiments, the restoration gene is Ms45.

[0030] It is readily understood that in the embodiments of this application, the restoration gene should correspond to the male sterility gene, thereby enabling it to salvage the sterility trait caused by the male sterility gene. Preferably, in this application, the male sterility gene is a recessive gene, and the restoration gene can be a dominant allele of the recessive gene, capable of restoring male fertility in the plant.

[0031] In some embodiments, the male infertility gene is ms45, and the fertility restoration gene is Ms45.

[0032] In some embodiments, the Lc gene encodes a protein with the amino acid sequence SEQ ID NO:6. In some embodiments, the Mn1 gene encodes a protein with the amino acid sequence SEQ ID NO:10. In some embodiments, the Sh1 gene encodes a protein with the amino acid sequence SEQ ID NO:12. In some embodiments, the Ms45 gene encodes a protein with the amino acid sequence SEQ ID NO:2.

[0033] As used herein, the term "operable link" refers to a functional connection between elements (e.g., expression regulatory elements and regulated genes). Typically, an operable link means that the linked nucleic acid sequences are contiguous and within the same reading frame.

[0034] In some embodiments, the first polynucleotide further comprises one or more expression regulatory elements operatively linked to the nucleotide sequence of the restored gene.

[0035] In some embodiments, the second polynucleotide further comprises one or more expression regulatory elements operatively linked to the nucleotide sequence of the silencing element.

[0036] In some embodiments, the third polynucleotide further comprises one or more expression regulatory elements operatively linked to the nucleotide sequence of the Lc gene.

[0037] In some embodiments, the expression regulation element is selected from promoters, enhancers, terminators, or any combination thereof.

[0038] In some embodiments, the promoter is selected from: constitutive promoters, inducible promoters, tissue-preferred promoters, tissue-specific promoters, growth-phase-preferred promoters, or any combination thereof.

[0039] In some implementations, the promoter is derived from corn or microorganisms (e.g., viruses).

[0040] In some implementations, the nucleic acid molecule as described above comprises:

[0041] The first polynucleotide contains an operatively linked promoter derived from the maize Ms45 gene and the nucleotide sequence of Ms45.

[0042] The second polynucleotide contains an operatively linked promoter from the maize-derived Mn1 gene and a nucleotide sequence capable of targeting silencing elements of both the Mn1 and Sh1 genes; and

[0043] The third polynucleotide contains an operable 35S promoter derived from cauliflower mosaic virus (CaMV) and a nucleotide sequence of the Lc gene.

[0044] In some implementations, the first polynucleotide, the second polynucleotide, and the third polynucleotide are covalently linked, with or without the linker nucleotide.

[0045] In some embodiments, the length of the linker nucleotide is no more than 10 kb, no more than 5 kb, no more than 1 kb, no more than 500 bp, no more than 100 bp, no more than 50 bp, no more than 10 bp, no more than 5 bp, or shorter.

[0046] In some implementations, the first polynucleotide, the second polynucleotide, and the third polynucleotide are genetically linked.

[0047] In some embodiments, the nucleotide sequence of the Ms45 gene is SEQ ID NO:1. In some embodiments, the nucleotide sequence of the silencing element targeting the Mn1 and Sh1 genes is SEQ ID NO:13. In some embodiments, the nucleotide sequence of the Lc gene is SEQ ID NO:5.

[0048] In some embodiments, the nucleotide sequence of the promoter of the Ms45 gene derived from maize is SEQ ID NO:3. In some embodiments, the nucleotide sequence of the promoter of the Mn1 gene derived from maize is SEQ ID NO:19. In some embodiments, the nucleotide sequence of the 35S promoter derived from cauliflower mosaic virus (CaMV) is SEQ ID NO:7.

[0049] In some embodiments, the nucleic acid molecule as described above further comprises a fourth polynucleotide, which contains the nucleotide sequence of a selectable marker gene.

[0050] Selective marker genes that can be used in the embodiments of this application include, but are not limited to, neomycin resistance genes (e.g., genes encoding neomycin phosphotransferase), hygromycin resistance genes (e.g., genes encoding hygromycin phosphotransferase), chloramphenicol resistance genes, streptomycin resistance genes, spectinomycin resistance genes, bleomycin resistance genes, sulfonamide resistance genes, bromobenzonitrile resistance genes, glyphosate resistance genes, diammonium phosphate resistance genes, and glufosinate resistance genes.

[0051] In some embodiments, the selection marker gene is a herbicide resistance gene. In some embodiments, the herbicide resistance gene is the bar gene.

[0052] In some implementations, the selection marker gene is derived from an organism other than maize (e.g., a plant, an animal, or a microorganism).

[0053] In some embodiments, the bar gene encodes a protein with the amino acid sequence SEQ ID NO:21.

[0054] In some embodiments, the fourth polynucleotide, as described above, comprises an operatively linked 35S enhanced promoter derived from cauliflower mosaic virus (CaMV) and a nucleotide sequence of the bar gene.

[0055] In some embodiments, the first, second, third, and fourth polynucleotides are covalently linked, with or without the linker nucleotide.

[0056] In some embodiments, the length of the linker nucleotide is no more than 10 kb, no more than 5 kb, no more than 1 kb, no more than 500 bp, no more than 100 bp, no more than 50 bp, no more than 10 bp, no more than 5 bp, or shorter.

[0057] In some implementations, the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are genetically linked.

[0058] In some embodiments, the nucleotide sequence of the bar gene is SEQ ID NO:20.

[0059] In some implementations, the nucleotide sequence of the 35S enhanced promoter derived from cauliflower mosaic virus (CaMV) is SEQ ID NO:22.

[0060] In some embodiments, the nucleic acid molecule as described above, wherein the first polynucleotide further comprises a terminator derived from the Ms45 gene of maize that is operatively linked to the nucleotide sequence of Ms45.

[0061] In some embodiments, the second polynucleotide further comprises a terminator derived from Agrobacterium tumefaciens encoding a carmine synthase gene, operatively linked to a nucleotide sequence capable of targeting silencing elements of the Mn1 and Sh1 genes.

[0062] In some embodiments, the third polynucleotide further comprises a terminator derived from Agrobacterium tumefaciens encoding a gene encoding carmine synthase, which is operatively linked to the nucleotide sequence of the Lc gene.

[0063] In some embodiments, the fourth polynucleotide further comprises a 35S PolyA terminator derived from cauliflower mosaic virus (CaMV) that is operatively linked to the nucleotide sequence of the bar gene.

[0064] In some embodiments, the nucleotide sequence of the terminator of the Ms45 gene derived from maize is SEQ ID NO:4.

[0065] In some embodiments, the nucleotide sequence of the terminator encoding the carmine synthase gene derived from Agrobacterium tumefaciens is SEQ ID NO:8.

[0066] In some embodiments, the nucleotide sequence of the 35SPolyA terminator derived from cauliflower mosaic virus (CaMV) is SEQ ID NO:23.

[0067] In some embodiments, the nucleotide sequence of the nucleic acid molecule is SEQ ID NO:24.

[0068] carrier

[0069] In a second aspect of this application, a carrier is provided that comprises the isolated nucleic acid molecules as described above.

[0070] In some embodiments, the vector may be a cloning vector, a transfer vector, or an expression vector. In some embodiments, the vector is a plasmid (e.g., pCAMBIA3301), a granule, a bacteriophage, etc. In some embodiments, the vector is capable of expressing the isolated nucleic acid molecules as described above in plant cells (e.g., maize).

[0071] host cells

[0072] In a third aspect of this application, a host cell is provided, comprising, as described above, isolated nucleic acid molecules or as described above, a vector.

[0073] In some embodiments, the host cell is an Agrobacterium cell (e.g., Agrobacterium EHA105) or a plant cell (e.g., corn). In some embodiments, the plant cell is a monocotyledonous or dicotyledonous plant cell. In some embodiments, the plant cell is a cell selected from the following plants: corn (Zea mays), rapeseed (Brassica napus), rice (Oryza sativa), Arabidopsis thaliana, barley (Hordeum vulgare), wheat (Triticumaestivum), sorghum (Sorghum bicolor), soybean (Glycinemax), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), cotton (Gossypium hirsutum), sunflower (Helianthus annuus), or sugarcane (Saccharum officinarum).

[0074] This application also provides a tissue culture of the host cell and protoplasts produced from the tissue culture.

[0075] Recombinant DNA molecules

[0076] As used herein, the term "recombinant DNA molecule" refers to a DNA molecule that does not exist in nature. Typically, said recombinant DNA molecules are artificially generated and are produced by a combination of at least two DNA molecules that do not coexist in nature. For example, a DNA molecule composed of a combination of at least two heterologous DNA molecules. It is understood that after inserting the isolated nucleic acid molecule as described in the first aspect into a transgenic plant, a recombinant DNA sequence, i.e., said recombinant DNA molecule, will be generated near the insertion site. The wild-type plant corresponding to the transgenic plant will not contain the sequence of said recombinant DNA molecule.

[0077] Therefore, in a fourth aspect of this application, a recombinant DNA molecule is provided, the recombinant DNA molecule comprising all or part of the sequence of the isolated nucleic acid molecule as described in the first aspect and at least part of the sequence of the adjacent plant genomic DNA. In some embodiments, the recombinant DNA molecule further comprises a repair sequence resulting from genetic transformation integration.

[0078] In some embodiments, the recombinant DNA molecule comprises the sequence shown in SEQ ID NO:38.

[0079] In some embodiments, the recombinant DNA molecule comprises the sequences shown in SEQ ID NO:37 and SEQ ID NO:38; and / or the sequences shown in SEQ ID NO:39 and SEQ ID NO:38. In some embodiments, the recombinant DNA molecule comprises the sequence shown in SEQ ID NO:36.

[0080] Plant or plant seeds

[0081] In a fifth aspect of this application, a plant or plant seed is provided, wherein the plant or plant seed contains, in its genome, isolated nucleic acid molecules as described in the first aspect, or recombinant DNA molecules as described in the fourth aspect.

[0082] In some embodiments, the plant or plant seed further contains the male sterility gene (e.g., ms45) in its genome. In some embodiments, the male sterility gene is a homozygous recessive male sterility gene (e.g., ms45ms45).

[0083] In some embodiments, the isolated nucleic acid molecule is integrated into the genome of the plant or seed. In some embodiments, the isolated nucleic acid molecule is integrated into the genome of the plant or seed on the same or different chromosome as the male sterility gene. In some embodiments, the nucleic acid molecule is present in a heterozygous form in the genome of the plant or seed. As used herein, the term "present in a heterozygous form" has the meaning commonly understood by those skilled in the art. For example, it can mean that there are no identical alleles at the gene locus corresponding to the location where the nucleic acid molecule is integrated into the plant genome (i.e., heterozygous). For example, the nucleic acid molecule is present in only one chromatid, while its sister chromatids do not contain the nucleic acid molecule. In some embodiments, the nucleic acid molecule is present in a homozygous form in the genome of the plant or seed. As used herein, the term "present in a homozygous form" has the meaning commonly understood by those skilled in the art. For example, it can mean that there are identical alleles at the gene locus corresponding to the location where the nucleic acid molecule is integrated into the plant genome (i.e., homozygous).

[0084] The nucleic acid molecule can be integrated into the genome of the plant or plant seed using any method known to those skilled in the art. Such methods include, but are not limited to, stable transformation methods, transient transformation methods, virus-mediated methods, and Agrobacterium-mediated methods.

[0085] In some embodiments, the plant or seed is male-fertile. In some embodiments, the plant or seed can be used as a maintainer line for a male-sterile plant containing the male-sterile gene.

[0086] In some embodiments, the plant or seed is a monocotyledonous or dicotyledonous plant or seed. In some embodiments, the plant or seed is a corn, rapeseed, rice, Arabidopsis thaliana, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugarcane plant or seed.

[0087] Corn transformant SSMM-15

[0088] In some embodiments, the plant or plant seed is corn or corn seed.

[0089] In some embodiments, the representative sample of the maize or maize seed is maize transformant SSMM-15, which has the accession number CGMCC No. 46085. In some embodiments, the maize transformant SSMM-15 comprises a heterozygous SSMM-15 transformant sequence.

[0090] It is understood that the maize or maize seeds of the present invention may also contain one or more additional target gene traits, particularly traits introduced by crossing the maize transformant SSMM-15 with another maize plant containing the additional target gene trait. Such traits include, but are not limited to, increased insect resistance, increased nutrient use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, and / or increased herbicide tolerance, wherein said traits are measured relative to maize plants lacking such target gene traits.

[0091] Therefore, in some embodiments, the plant or plant seed contains nucleic acid molecules as described above, as well as one or more additional target genes.

[0092] In some embodiments, the one or more additional target genes are introduced into the plant or seed by transformation (e.g., Agrobacterium-mediated transformation) or by hybridization of the maize transformant SSMM-15 with another maize plant containing the one or more additional target genes.

[0093] In some embodiments, the plant has one or more traits regulated by one or more additional target genes compared to its corresponding wild-type plant.

[0094] In some embodiments, the trait is selected from increased insect resistance, increased nutrient use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production and / or increased herbicide tolerance, or any combination thereof.

[0095] Products

[0096] In a sixth aspect of this application, an article is provided comprising a plant or plant seed as described in the fifth aspect.

[0097] In some embodiments, the article comprises genomic DNA of the plant or plant seed.

[0098] In some embodiments, the article comprises genomic DNA of maize transformant SSMM-15 with accession number CGMCC No. 46085.

[0099] In some embodiments, the article is selected from one or more of the following: corn ears, hulled corn, corn silks, corn pollen, corn grits, corn flour, crushed corn, cornmeal, corn oil, corn starch, corn syrup, corn malt, corn sugar, corn syrup, margarine produced from corn oil, unsaturated corn oil, saturated corn oil, corn flakes, popcorn, ethanol and / or juice produced from corn, dried distillers grains (DDGS) from corn fermentation, animal feed derived from corn, cosmetics, and fillers.

[0100] Oligonucleotide molecules or combinations thereof

[0101] In a seventh aspect of this application, an oligonucleotide molecule or combination thereof is provided, the oligonucleotide molecule comprising a sequence complementary to at least a portion of the sequence of the recombinant DNA molecule as described above.

[0102] In some embodiments, the oligonucleotide molecule may be a primer and / or probe capable of specifically binding to a target nucleic acid sequence (e.g., at least a partial sequence of a recombinant DNA molecule as described above), and thus can be used to identify the maize transformant SSMM-15. The detection of the presence of the maize transformant SSMM-15 can be performed using methods known in the art, such as thermal amplification of nucleic acids or nucleic acid hybridization techniques (such as Northern blotting and Southern analysis).

[0103] probe

[0104] In some embodiments, the oligonucleotide molecule is an oligonucleotide probe capable of detecting the recombinant DNA molecule as described above.

[0105] In this document, the term "probe" refers to an oligonucleotide molecule that is complementary to a target nucleic acid sequence (e.g., at least a partial sequence of a recombinant DNA molecule as described above) and can be used in hybridization detection methods. In this invention, probes not only comprise deoxyribonucleic acid or ribonucleic acid, but also include polyamides and other probe materials that specifically bind to the target nucleic acid sequence, and the detection of this binding can be used to detect the presence of the target nucleic acid sequence. Probes may be linked to conventional detectable markers or reporter molecules, such as radioisotopes, ligands, chemiluminescent agents, or enzymes.

[0106] In some embodiments, the oligonucleotide probe comprises a sequence complementary to SEQ ID NO:38. In some embodiments, the oligonucleotide probe comprises a sequence complementary to SEQ ID NO:38 and a sequence complementary to at least a portion of the sequence in SEQ ID NO:37. In some embodiments, the oligonucleotide probe comprises a sequence complementary to SEQ ID NO:38 and a sequence complementary to at least a portion of the sequence in SEQ ID NO:39.

[0107] The length of the probe is generally at least about 11 nucleotides, at least about 18 nucleotides, at least about 24 nucleotides, or at least about 30 nucleotides or longer. In some embodiments, the length of the oligonucleotide probe is 11-20 nt, 21-30 nt, 31-40 nt, 41-50 nt, 51-60 nt, 61-70 nt, 71-80 nt, 81-90 nt, 91-100 nt or longer.

[0108] Primers

[0109] In some embodiments, the oligonucleotide molecule or combination thereof is an oligonucleotide primer or primer pair capable of amplifying the recombinant DNA molecule as described above.

[0110] In this document, the term "primer" is an oligonucleotide molecule capable of annealing or hybridizing with a target nucleic acid sequence (e.g., at least a partial sequence of a recombinant DNA molecule as described above) to produce an amplification product. A pair of primers may be used together with template DNA (such as a maize genomic DNA sample) in an amplification reaction (such as polymerase chain reaction (PCR)) to produce an amplification product whose DNA sequence corresponds to the sequence of the template DNA located between the two sites where the primer hybridizes with the template. Primers are typically designed to hybridize with complementary target nucleic acid sequences to form a heterozygote between the primer and the target nucleic acid sequence. In this document, the term "primer pair" refers to two primers using opposite strands that bind to double-stranded nucleotide segments for amplifying the nucleotide segment between them.

[0111] In some embodiments, the oligonucleotide primer pair includes a first oligonucleotide primer containing a sequence complementary to at least a portion of the sequence in SEQ ID NO:37, and a second oligonucleotide primer containing a sequence complementary to at least a portion of the sequence in SEQ ID NO:39.

[0112] In some embodiments, the sequence of the first oligonucleotide primer in the oligonucleotide primer pair is shown in SEQ ID NO:42, and the sequence of the second oligonucleotide primer is shown in SEQ ID NO:43.

[0113] The primers are generally at least about 11 nucleotides, at least about 18 nucleotides, at least about 24 nucleotides, or at least about 30 nucleotides or longer. In some embodiments, the oligonucleotide primers are 11-20 nt, 21-30 nt, 31-40 nt, 41-50 nt, 51-60 nt, 61-70 nt, 71-80 nt, 81-90 nt, 91-100 nt or longer.

[0114] The methods for designing and using primers and probes are well known in the art. Furthermore, probes and primers can have complete sequence identity with the target nucleic acid sequence or exhibit some mismatch. For oligonucleotide molecules to be used as primers or probes, it is only necessary that the oligonucleotide molecule be sufficiently complementary to the target nucleic acid sequence to form a stable double-stranded structure. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of target gene DNA from the maize transformant SSMM-15 in a sample.

[0115] Reagent test kit

[0116] In an eighth aspect of this application, a kit is provided that comprises oligonucleotide molecules or combinations thereof as described above.

[0117] In some embodiments, the kit contains at least one pair of oligonucleotide primers as described above. In some embodiments, the kit contains at least one oligonucleotide probe as described above.

[0118] Method for detecting maize transformant SSMM-15

[0119] In a ninth aspect of this application, a method is provided for detecting the presence of maize transformant SSMM-15 in a nucleic acid sample derived from maize plants, maize seeds, or maize cells, the method comprising:

[0120] (a) Contact the sample with oligonucleotide molecules or combinations thereof as described in the seventh aspect;

[0121] (b) Perform nucleic acid amplification reaction;

[0122] (c) Detect the amplification product from step (b).

[0123] In some implementations, the presence of the amplification product of step (b) indicates the presence of maize transformant SSMM-15 in the nucleic acid sample.

[0124] Testing applications

[0125] In a tenth aspect of this application, there is provided the use of an oligonucleotide molecule or combination thereof as described in the seventh aspect or a kit as described in the eighth aspect for detecting nucleic acid samples derived from maize plants, maize seeds or maize cells.

[0126] In some embodiments, the representative sample of the maize plant, maize seed or maize cell is maize transformant SSMM-15 with accession number CGMCC No. 46085.

[0127] method

[0128] In the eleventh aspect of this application, a method for obtaining a plant is provided, the method comprising: (1) introducing a nucleic acid molecule as described in the first aspect or a vector as described in the second aspect into a plant cell, and (2) culturing the plant cell into a plant.

[0129] In some implementations, in step (1), Agrobacterium is used to introduce the nucleic acid molecule or vector into plant cells.

[0130] In some embodiments, the plant cells contain the male sterility gene in their genome and are male sterile prior to the introduction of the nucleic acid molecule or vector. In some embodiments, the plant cells contain a homozygous recessive male sterility gene in their genome.

[0131] In some embodiments, in step (1), the nucleic acid molecule is integrated into the genome of the plant cell. In some embodiments, after integration into the genome of the plant cell, the nucleic acid molecule is located on the same or a different chromosome from the male sterility gene.

[0132] In some embodiments, the plant cells are cells of monocotyledonous or dicotyledonous plants.

[0133] In some embodiments, the plant cells are cells selected from the following plants: corn, rapeseed, rice, Arabidopsis thaliana, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugarcane.

[0134] In some embodiments, the plant contains a homozygous recessive male sterility gene and the nucleic acid molecule or vector, and is male-fertile. In some embodiments, the nucleic acid molecule or vector exists in the genome of the plant in a heterozygous form. In some embodiments, the nucleic acid molecule is integrated into the genome of the plant and located on the same or a different chromosome from the male sterility gene.

[0135] In some embodiments, the plant can be used as a maintainer line for a male-sterile plant containing the male-sterile gene. In some embodiments, the plant is the maize transformant SSMM-15.

[0136] In some embodiments, the method further includes:

[0137] (3) The plants from step (2) are used to pollinate male-sterile plants containing the male-sterile gene to produce offspring seeds or plants.

[0138] (4) Screen offspring seeds or plants that show extrinsic traits regulated by the Lc gene and / or Mn1 gene and Sh1 gene.

[0139] In the twelfth aspect of this application, a method is provided for obtaining offspring seeds or plants of male-sterile lines and maintainer lines, the method comprising crossing a plant described in the fifth aspect or a plant obtained by the method described in the eleventh aspect as a male parent with a male-sterile plant containing the male-sterile gene as a female parent, and producing offspring seeds or plants.

[0140] In some implementations, the method includes:

[0141] (1) Providing a male-sterile plant containing the male-sterile gene as the maternal parent. In some embodiments, the male-sterile gene is a homozygous recessive male-sterile gene.

[0142] (2) Provide the plant described in the fifth aspect as the parent plant or the plant obtained by the method of the eleventh aspect.

[0143] (3) Use the plants from step (2) to pollinate the plants from step (1) to produce offspring seeds.

[0144] (4) Optionally, the offspring seeds are cultured into offspring plants.

[0145] Among them, the offspring seeds or plants exhibiting the extrinsic traits regulated by the Lc gene and / or not exhibiting the extrinsic traits regulated by the Mn1 and Sh1 genes are male-fertile and can be used as maintainer lines. Furthermore, the offspring seeds or plants not exhibiting the Lc gene and / or exhibiting the extrinsic traits regulated by the Mn1 and Sh1 genes are male-sterile and can be used as male-sterile lines.

[0146] In some implementations, the method includes:

[0147] (1) Providing a male-sterile plant containing the male-sterile gene as the maternal parent. In some embodiments, the male-sterile gene is a homozygous recessive male-sterile gene.

[0148] (2) Provide the plant described in the fifth aspect as the parent plant or the plant obtained by the method of the eleventh aspect.

[0149] (3) The plants from step (2) are used to pollinate the plants from step (1) to produce two types of offspring seeds. The first type of offspring seeds does not exhibit the extrinsic traits of seeds regulated by the Mn1 and Sh1 genes. The second type of offspring seeds exhibits the extrinsic traits of seeds regulated by the Mn1 and Sh1 genes.

[0150] (4) Separate the first and second offspring seeds, and optionally, cultivate them into the first and second offspring plants respectively.

[0151] Optionally, the method further includes the following steps:

[0152] (5) Remove plants from the first progeny that do not exhibit the extrinsic traits regulated by the Lc gene, thereby leaving the remaining first progeny plants that are male-fertile and can be used as maintainer lines. and / or

[0153] Removing plants from the second progeny that exhibit the extrinsic traits regulated by the Lc gene leaves the remaining second progeny plants male-sterile and usable as male-sterile lines.

[0154] (6) The remaining second-generation plants are pollinated with the remaining first-generation plants to produce further offspring seeds.

[0155] In a thirteenth aspect of this application, a method for preparing hybrid seeds is provided, the method comprising:

[0156] (1) Provides offspring seeds of male-sterile lines obtained by the method described in the twelfth aspect, which exhibit the extrinsic traits of seeds regulated by the Mn1 and Sh1 genes. Also, provides seeds of the target line.

[0157] (2) Sow the offspring seeds of the male-sterile line plant and the seeds of the target line plant in the field to obtain male-sterile line plants and target line plants.

[0158] (3) Remove plants from the male-sterile line that exhibit the extrinsic traits of plants regulated by the Lc gene.

[0159] (4) The remaining male-sterile lines were pollinated with the target line plants.

[0160] (5) Seeds harvested from male-sterile plants are called hybrid seeds.

[0161] use

[0162] In another aspect of this application, the isolated nucleic acid molecule described in the first aspect, or the vector of the second aspect, or the host cell (e.g., plant cell) of the third aspect, is provided for the use of producing maintainer plants.

[0163] In some embodiments, the plant is a monocotyledonous or dicotyledonous plant.

[0164] In some embodiments, the plant is selected from the following: corn, rapeseed, rice, Arabidopsis thaliana, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugarcane.

[0165] In another aspect of this application, there is provided the use of plants obtained by the method of the eleventh aspect or progeny seeds or plants obtained by the method of the twelfth aspect or the plants of the fifth aspect for producing hybrid offspring.

[0166] In some embodiments, the plant is a monocotyledonous or dicotyledonous plant.

[0167] In some embodiments, the plant is selected from the following: corn, rapeseed, rice, Arabidopsis thaliana, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugarcane.

[0168] Isolation uses and methods

[0169] In another aspect of this application, a method for cultivating male-sterile plants is provided, wherein the male-sterile plants are produced by using the plants described in the fifth aspect as the male parent and using male-sterile plants with different genetic backgrounds from the male parent and hybrid vigor from the plants described in the fifth aspect as the female parent, and by crossing the male parent with the female parent.

[0170] The method includes planting the male-sterile line plant and one or more other plants in a growth area.

[0171] In some embodiments, the additional one or more plants are male-fertile. In some embodiments, multiple fertile plants are planted alternately using the male-sterile line. In some embodiments, the male-sterile line is planted between a first type of male-fertile plant and a second type of male-fertile plant.

[0172] Optionally, the method further includes the step of removing offspring plants from the cross between the male and female parent that exhibit the extrinsic traits regulated by the Lc gene, thereby leaving the remaining offspring plants that are male-sterile and exhibit heterosis.

[0173] In another aspect of this application, there is a use of a male-sterile line plant for intercropping one or more other plants; the male-sterile line plant is produced by crossing the male-sterile line plant described in the fifth aspect as the male parent and a male-sterile line plant with a different genetic background from the male parent as the female parent.

[0174] In some embodiments, the additional one or more plants are male-fertile.

[0175] In some implementations, the male-sterile line is planted between a first type of male-fertile plant and a second type of male-fertile plant.

[0176] As used in this article, the term "different genetic background" means that the plants have different evolutionary origins. For example, maizes with different genetic backgrounds will have differences in their genomes and traits that regulate growth, development, and yield. These differences can stem from the genetic diversity of maize varieties. Hybrid vigor can occur when maizes with different genetic backgrounds are crossed, meaning that the hybrid offspring are superior to their parents in certain traits. This advantage may manifest in multiple aspects, such as yield, quality, and resistance.

[0177] Terminology Definition

[0178] As used herein, the term "nucleic acid molecule" refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plasmid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA), as well as DNA or RNA analogs produced using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

[0179] As used herein, the term "target gene" refers to a segment of nucleic acid (e.g., DNA) of interest introduced into a plant through genetic transformation. Typically, target genes are introduced into plants to regulate one or more traits.

[0180] As used herein, the term "extrinsic trait" refers to the heritable, observable phenotypic characteristics exhibited by an individual (including seeds and plants), typically controlled by one or more pairs of alleles. The "extrinsic traits" of seeds and plants primarily include the external morphological characteristics of roots, stems, leaves, flowers, fruits, and seeds, such as seed color, seed size, plant color, stem morphology, and leaf morphology. Generally, the extrinsic traits of seeds primarily refer to their external morphological characteristics, including, for example, seed color or seed size. The extrinsic traits of plants primarily refer to the external morphological characteristics of roots, stems, leaves, flowers, and fruits, including, for example, plant color. Because the Mn1 and Sh1 genes are silenced or mutated, the seed size of plants will be smaller than that of wild-type plants (i.e., plants with normal expression of the Mn1 and Sh1 genes). In this article, the term "extrinsic trait not exhibiting regulation by the Mn1 and Sh1 genes" means that the plant exhibits the trait resulting from the silence or mutation of the Mn1 and Sh1 genes. That is, the seed size of the plant is smaller than that of the wild-type plant (i.e., the plant in which the Mn1 and Sh1 genes are normally expressed).

[0181] As used herein, the term "male sterility" refers to the loss of physiological function in male cells or tissues of a plant. Typically, in sexually reproducing plants (e.g., maize), male sterility manifests as abnormal development of male tissues (e.g., stamens) that fail to produce functional pollen, while female tissues (e.g., pistils) develop normally and can accept normal pollen for fertilization and seed production. As used herein, the term "male sterility gene" refers to a gene that controls the male sterility trait in plants. In this application, preferably, the male sterility gene is a nuclear male sterility gene. In most cases, the nuclear gene controlling male sterility is a recessive gene, which only causes male sterility in plants when homozygous (msms). For example, the maize male sterility line Zheng 58 possesses a homozygous recessive male sterility gene (ms45ms45). In this application, particularly preferably, the male sterility gene is a recessive gene that causes male sterility in plants when homozygous.

[0182] As used herein, the term "restoration gene" refers to a gene capable of restoring male fertility in plants that are male-sterile due to a male-sterile gene. When the restoration gene is introduced into a male-sterile line, the plant will regain male fertility. In this application, when the male-sterile gene is recessive, the restoration gene may be a dominant allele of the recessive gene.

[0183] Several recessive genes that can cause male sterility in plants and their corresponding dominant alleles that can restore male fertility have been reported. For example, several recessive male sterility genes and their corresponding dominant alleles that restore male fertility have been identified in maize, including but not limited to those shown in Table 1 below (Skibbe et al. 2005).

[0184] Table 1. Recessive male sterility genes and their corresponding dominant allelic restorer genes in maize.

[0185]

[0186]

[0187] As used in this article, the term "Lc gene" refers to a gene that can affect plant color by influencing the synthesis of pigments, anthocyanins, etc. For more information, see Ludwig SR et al., Proc Natl Acad Sci, 1989 Sep; 86(18):7092-6; Wang Juan et al., Genomics and Applied Biology, 2009, Vol. 02; and gene number Zm00001d026147. The Lc gene is a regulatory gene related to anthocyanin synthesis. Its heterologous expression in various plants (e.g., maize) can affect anthocyanin synthesis, increase anthocyanin content, and influence seed color. Expression of this gene can cause maize plants to appear purple.

[0188] As used herein, the term "Mn1 gene" refers to a gene that affects endosperm development in seeds, and its expression level can influence seed size. Silencing or mutating this gene will affect normal endosperm development, resulting in smaller seeds, but will not affect germination rate or plant development. For more information, please refer to, for example, Cheng Wh et al., 1996; and the gene number Zm00001d003776.

[0189] As used herein, the term "Sh1 gene" refers to a gene that influences endosperm development in seeds, and its expression level affects seed size. Silencing or mutating this gene will affect normal endosperm development, resulting in smaller seeds, but will not affect germination rate or plant development. For more information, see, for example, Hauptmann RM et al., 1988; and the gene number Zm00001eb374090.

[0190] As used herein, the term "silencing element" refers to a polynucleotide capable of reducing or eliminating the level or expression of a target polynucleotide or its encoded polypeptide. Therefore, it should be understood that "silencing element" as used herein includes polynucleotides such as RNA constructs, DNA constructs encoding these RNA constructs, and expression constructs containing these DNA constructs. In some embodiments, the silencing element is selected from: sense repressor elements, antisense repressor elements, hairpin repressor elements, double-stranded RNA, siRNA, amiRNA, miRNA, or any combination thereof.

[0191] As used herein, antisense repressive elements are sequence complementary to the target polynucleotide, while sense repressive elements are homologous to the target polynucleotide. Typically, sense repressive elements share a significant amount of sequence identity with the target polynucleotide, usually greater than approximately 65%, greater than approximately 85%, and approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Both sense and antisense repressive elements can be of any length, as long as they allow for the repression of the target sequence.

[0192] As used herein, the term "tissue-preferred promoter" refers to a promoter that preferentially initiates transcription in certain plant tissues (e.g., stamens, pollen sacs, filaments, and pollen). As used herein, the term "growth-stage-preferred promoter" refers to a promoter that preferentially initiates transcription in certain growth and developmental stages (e.g., sporogenous tissues, microspores, and microgametophytes). As used herein, the term "tissue-specific promoter" refers to a promoter that specifically initiates transcription only in certain plant tissues.

[0193] As used herein, the term "selection marker gene" refers to a gene whose encoded product enables transformed or transfected host cells to grow normally or exhibit other visual characteristics under selective pressure. Such selective pressure includes, but is not limited to, the addition of selective agents (e.g., antibiotics or herbicides) or nutrient deficiency. As used herein, the term "antibiotic resistance gene" refers to a gene whose encoded product enables transformed or transfected host cells to grow normally or exhibit other visual characteristics under antibiotic selective pressure. As used herein, the term "herbicide resistance gene" refers to a gene whose encoded product enables transformed or transfected host cells to grow normally or exhibit other visual characteristics under herbicide selective pressure.

[0194] As used in this article, the term "heterandrogenic fertilization" refers to the phenomenon in double fertilization where the embryo and endosperm of the same seed are fertilized by sperm from two different male gametophytes. In this case, the embryo and endosperm of the same seed can have different genotypes.

[0195] In this application, the plant may be all or part of the plant, such as roots, stems, leaves, embryos, root tips, pollen or anthers.

[0196] As used herein, the terms “transformation,” “genetic transformation,” or “transgenic” have the same meaning and can be used interchangeably, referring to the act of inserting a foreign nucleic acid fragment (e.g., a target gene) into the genome of a plant cell using plant transformation methods known in the art (e.g., Agrobacterium infection, gene gun method).

[0197] Furthermore, after inserting a foreign nucleic acid fragment into the genome of a plant cell, a new DNA sequence is generated at the insertion site. This sequence consists of the inserted foreign nucleic acid fragment and plant genomic DNA (called "flanking DNA") adjacent to or "side-joined" on either side of the insertion site. This sequence is referred to as the "transformer sequence." In some embodiments, the transformer sequence also includes a repair sequence resulting from genetic transformation.

[0198] In this document, a maize plant containing a "transformer sequence" is referred to as a "maize transformant," and may also be called a "transformer plant" or "transformer line." Generally, the transformant sequence of each maize transformant is unique and specific, and can be easily identified by comparing it with the sequences of other maize plants (e.g., untransformed maize genomic DNA). In some embodiments, the maize transformant SSMM-15 provided in this application contains the sequence shown in SEQ ID NO:36. In some embodiments, the transformant SSMM-15 exists in a heterozygous form in the maize plant, and the male sterility gene (e.g., ms45) exists in a homozygous form; said maize plant can serve as a maintainer line in this document.

[0199] As used in this article, the term "introduction" or "backcrossing" refers to the introduction of a transformant into a plant (e.g., maize) through hybridization.

[0200] Beneficial effects

[0201] The nucleic acid molecule provided in this application contains three to four genes and their corresponding regulatory elements. Different combinations of these genes and regulatory elements affect the expression and expression level of genes in transgenic plants. The inventors of this application provide a nucleic acid molecule that, after being inserted into the genome of a transgenic plant, allows multiple target genes to be expressed (or not expressed) at a relatively ideal level, and can effectively regulate the traits of transgenic plants.

[0202] Furthermore, since the genetic stability of transgenic plants is also affected by the genomic location of nucleic acid molecule insertion, the inventors of this application have also provided a transgenic plant, SSMM-15, with an optimal insertion site. SSMM-15, as a maintainer line, can be harvested simultaneously and its offspring can be effectively distinguished after hybridization with male-sterile plants. Based on this, this application also provides a method for propagating male-sterile plants using the aforementioned maintainer line. Thus, the method and maintainer line provided in this application enable efficient propagation of male-sterile plants, improving breeding efficiency. In addition, by combining the external traits of seeds and plants, the method provided in this application allows for two or more screenings of the hybrid offspring, further improving the purity of both the male-sterile line and maintainer line offspring, increasing breeding efficiency, and enhancing the quality and purity of the resulting hybrid seeds.

[0203] Sequence information

[0204] Information about the sequences involved in this application is shown in Table 2 below.

[0205] Table 2. Sequence Information

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218] In the LAD1-1 to LAD1-4 primers, "V" indicates that it can be selected from any one of G, A, and C; "N" indicates that it can be selected from any one of T, G, A, and C; "B" indicates that it can be selected from any one of T, G, and C; and "D" indicates that it can be selected from any one of G, A, and T. Attached Figure Description

[0219] Figure 1 This is a vector map containing the nucleic acid molecules of this application.

[0220] Figures 2A to 2H The ear performance of SSMM-15 under different genetic backgrounds.

[0221] Figure 3 The color of SSMM-15 seedlings is shown (left is the control, right is the SSMM-15 plant).

[0222] Figures 4A to 4C The male flower phenotypes are SSMM-15 (fertile), B73 inbred line (fertile), and male flower phenotypes of male-sterile plants containing homozygous ms45 (sterile).

[0223] Figure 5 The ear performance after hybridization of the sterile line and the SSMM-15 transformant heterozygous plant.

[0224] Figure 6 The ear performance after self-pollination of homozygous plants of SSMM-15 transformant.

[0225] Figure 7 The ear behavior after self-pollination of SSMM-15 transformant heterozygous plants.

[0226] Figure 8 Phenotype of SSMM-15 maintainer line plants (controls on both sides, SSMM-15 plant in the middle).

[0227] Figure 9 Propagation of male-sterile lines from the SSMM-15 maintainer line (the male-sterile line is on the left, and the SSMM-15 plant is on the right).

[0228] Figure 10 It is used to produce hybrids from male-sterile female lines (with male-sterile lines on both sides and conventional male-sterile inbred lines in the middle).

[0229] Figure 11 A schematic diagram illustrating the propagation of male-sterile lines and hybrid production using SSMM-15.

[0230] Instructions on the Preservation of Biological Materials

[0231] Seeds of maize (Zea mays) SSMM-15 have been deposited in accordance with the Budapest Treaty at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.46085, and the deposit date is August 7, 2024. Detailed Implementation

[0232] The present invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it). Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products. Those skilled in the art will understand that the embodiments are described by way of example and are not intended to limit the scope of protection claimed by the present invention.

[0233] Example 1: Construction of the carrier

[0234] In this embodiment, a recombinant vector was constructed using transgenic technology, utilizing the maize male sterility restorer gene Ms45 (mutation of which will cause male sterility in the plant, gene number Zm00001d047858), the genes Mn1 and Sh1 that can regulate kernel size, and the gene Lc that can regulate plant color.

[0235] 1. Synthesis of Mn1 / Sh1-RNAi fragments that regulate seed size

[0236] The nucleotide sequence of the Mn1 gene is shown in SEQ ID NO:9, and the amino acid sequence of the Mn1 protein it encodes is shown in SEQ ID NO:10. The nucleotide sequence of the Sh1 gene is shown in SEQ ID NO:11, and the amino acid sequence of the Sh1 protein it encodes is shown in SEQ ID NO:12.

[0237] A synthetic Mn1 / Sh1-RNAi fragment was created, with a HindIII restriction site added to the 5' end and a BstEII restriction site added to the 3' end. The Mn1 / Sh1-RNAi fragment (SEQ ID NO:13) comprises an Mn1 interference fragment and a Sh1 interference fragment, wherein the Mn1 / Sh1-RNAi fragment sequentially contains: a positive interference fragment of the Mn1 gene (SEQ ID NO:14), a positive interference fragment of the Sh1 gene (SEQ ID NO:16), an intron of a hairpin structure (SEQ ID NO:18), a negative interference fragment of the Sh1 gene (SEQ ID NO:17), and a negative interference fragment of the Mn1 gene (SEQ ID NO:15).

[0238] Furthermore, transcription of the Mn1 / Sh1-RNAi fragment was initiated using the promoter of the maize endogenous gene Mn1 (SEQ ID NO:19), with a SmaI restriction site added at the 5' end and a HindIII restriction site added at the 3' end. Transcription of the Mn1 / Sh1-RNAi fragment was terminated using the Agrobacterium tumefaciens carmine synthase gene terminator (SEQ ID NO:8).

[0239] 2. Synthesis of Lc genes that regulate plant color

[0240] The nucleotide sequence of the Lc gene is shown in SEQ ID NO:5, and the amino acid sequence of the Lc protein it encodes is shown in SEQ ID NO:6. The Lc expression element was artificially synthesized, with an EcoRI restriction site added to the 5' end and pmlI and SmaI restriction sites added to the 3' end. Furthermore, the 35S promoter (SEQ ID NO:7) was used as the promoter of the Lc gene, and the Agrobacterium tumefaciens carmine synthase gene terminator (SEQ ID NO:8) was used as the terminator of the Lc gene.

[0241] 3. Synthesis of the male infertility restoration gene Ms45

[0242] The male sterility restoration gene Ms45 is derived from the maize variety B73, and its nucleotide sequence is shown in SEQ ID NO:1. The amino acid sequence of the encoded Ms45 protein is shown in SEQ ID NO:2. Based on the sequence, the Ms45 expression element was artificially synthesized, with a pmlI restriction site added to the 5' end and a SmaI restriction site added to the 3' end. Furthermore, the natural promoter of Ms45 from maize was used as the promoter for Ms45, and its nucleotide sequence is shown in SEQ ID NO:3. The natural terminator of Ms45 from maize was used as the terminator for Ms45, and its nucleotide sequence is shown in SEQ ID NO:4.

[0243] 4. Construct a reorganization vehicle

[0244] The recombinant expression vector was constructed using plasmid pCAMBIA3301 (Center for Applied Agro-Molecular Biology, CAMBIA, Australia), which contains the selectable marker gene bar (its nucleotide sequence is shown in SEQ ID NO:20, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:21). Furthermore, the 35S enhanced promoter (SEQ ID NO:22) was used as the promoter for the Bar gene, and the 35S PolyA terminator of cauliflower mosaic virus (CaMV) (SEQ ID NO:23) was used as the terminator for the Bar gene.

[0245] The aforementioned expression elements were integrated into pCAMBIA3301, and the resulting recombinant vector was named pCAMBIA3301-SSMM. Figure 1 As shown in Table 3 below, the elements and their order in the recombinant nucleic acid fragment (T-border sequence, SEQ ID NO:24) contained in pCAMBIA3301-SSMM are shown in Table 3 below.

[0246] Table 3. Elements in the recombinant vector

[0247]

[0248]

[0249] Example 2: Obtaining and Screening Transformants

[0250] Inbred line B73 was planted in the field and bagged when pollen was released. Pollination was then performed, and 9-11 days later, immature embryos were collected from the kernels of the pollinated ears. pCAMBIA3301-SSMM was transformed into Agrobacterium EHA105, and the obtained Agrobacterium was used to infect maize embryos. The infected embryos were placed on a selective medium and screened multiple times to obtain resistant callus tissue. The resistant callus tissue was then used to regenerate seedlings, resulting in transgenic T0 generation plants. After obtaining the transgenic T0 generation, pollen from these T0 generation transgenic plants was used to cross, backcross, and self-pollinate the seed production female parent B73 and the ms45 male-sterile material (obtained from Maize Genetics Cooperation Stock Center, 905I), and phenotypes were observed.

[0251] The specific implementation steps are as follows:

[0252] 1. Obtaining the corn embryo

[0253] 1) Cut off about 1 cm from the top of the F1 generation ears obtained by self-pollination of B73, and insert tweezers into the ears from the top. Then place the ears into a beaker containing sterilization solution. Depending on the actual needs, 4-6 ears can be placed in the same beaker.

[0254] 2) Add approximately 700ml of disinfectant solution (50% bleach or 5.25% sodium hypochlorite, with one drop of Tween 20) to a beaker to soak the kernels. During the 20-minute disinfection process, rotate the kernels occasionally while gently tapping the beaker to remove air bubbles from the kernel surface, thus achieving the best disinfection effect. After disinfection, remove the kernels and place them in a beaker filled with sterile water, wash them three times, and then prepare to remove the embryos.

[0255] 3) Place the sterilized ears of grain on a large petri dish and use a large scalpel to cut off the top of the kernels (1.5-1.8mm).

[0256] 4) Insert the tip of the embryo removal knife between the embryo and the endosperm, and then gently pry the embryo upwards. Use the tip of a small scalpel to gently lift the embryo, ensuring that the embryo is not damaged in any way. Place the embryo's hypocotyl side against the N6E medium containing filter paper. The embryo density is approximately 2x2cm (30 embryos / plate).

[0257] 5) Seal the petri dish with sealing film and incubate in the dark at 28℃ for 2-3 days.

[0258] 2. Infection by Agrobacterium

[0259] 1) The recombinant Agrobacterium EHA105 / pCAMBIA3301-SSMM was cultured on YEP medium (containing 33 mg / L Kana and 100 mg / L Str antibiotics) one week in advance.

[0260] 2) Transfer the above-cultured recombinant Agrobacterium to fresh YEP medium (containing 33 mg / L Kana and 50 mg / L Str) and incubate at 19°C for 3 days.

[0261] 3) After 3 days, pick the recombinant Agrobacterium and put it into a 50ml centrifuge tube containing 5mL of infiltration medium. At the same time, add 100uM of AS (inf+AS) and incubate at room temperature (25℃) and 75rpm for 2-4 hours.

[0262] 4) Infect the embryos: Place the freshly detached embryos into centrifuge tubes containing 2 ml of inf+AS liquid medium, with about 20-100 embryos per tube. Wash twice with this medium, then add 1-1.5 ml of Agrobacterium at a specific concentration (OD550 = 0.3-0.4). Gently invert the centrifuge tubes 20 times, then place them upright in a dark chamber for 5 minutes to ensure that the embryos are completely immersed in the Agrobacterium liquid. Avoid vortexing throughout the process.

[0263] 3. Co-cultivation

[0264] 1) After inoculation, transfer the inoculated corn embryos to a co-culture medium (solutes as shown in Table 3, solvent is water) so that the hypocotyl of the embryos comes into contact with the surface of the medium, and remove excess Agrobacterium from the surface of the medium.

[0265] 2) Seal the petri dish with sealing film and incubate in the dark at 20°C for 3 days.

[0266] 4. Resting incubation

[0267] After co-culturing for 3 days, the embryos were transferred to resting culture medium, and the culture dish was sealed with sealing film and cultured in the dark at 28°C for 7 days.

[0268] 5. Select

[0269] 1) After 7 days, all the immature embryos were transferred to selective medium (solute as shown in Table 3, solvent is water) (35 immature embryos / plate) and cultured for two weeks. The selective medium contained 1.5 mg / L of dipropylamine. After two weeks, subculture was performed, and the concentration of dipropylamine could be increased to 3 mg / L.

[0270] 2) After about 5 weeks of immersion, the cells containing the transformants will grow into visible type II callus.

[0271] 6. Regeneration of transgenic plants

[0272] 1) Grow on regeneration medium for 3 weeks, and then germinate on the regeneration medium (placed in a light culture chamber) to obtain T0 generation transgenic maize.

[0273] 2) When they have grown 3-4 leaves, transfer them to the greenhouse. When they reach the stage of silking and pollination, pollinate them separately.

[0274] 7. Analysis of the obtained transgenic SSMM maize

[0275] Through the above experiments, more than 1,000 maize transformants were obtained. These maize transformants were then subjected to the following tests, and the best transgenic SSMM-15 maize transformants were finally selected. (Some transgenic maize transformants were screened out because the Mn1 / Sh1-RNAi fragment had a relatively low interference efficiency on the Mn1 and Sh1 genes, resulting in an insufficiently significant ratio of large to small kernels; some transgenic maize transformants were screened out because their purple color was not obvious; some transgenic maize transformants were screened out because they could not restore the fertility of the ms45 mutant; and some transgenic maize transformants were screened out because of the rejection phenomenon inherent in maize itself, resulting in low genetic stability of traits after several generations of testing.)

[0276] 1) Genotyping

[0277] Primers were designed for the Ms45 gene, Lc gene, Mn1 / Sh1-RNAi and bar gene, respectively, and PCR analysis was performed to obtain transformants with positive DNA levels for the Ms45 gene, Lc gene, Mn1 / Sh1-RNAi and bar gene, and phenotypic detection was performed.

[0278] 2) Multi-generation representative type testing

[0279] The agronomic traits of the transformed organisms obtained above were identified in three consecutive generations of field trials, including fertility, plant color, and grain size, to determine the stability of the target traits of the transformed organisms.

[0280] Specifically, the aforementioned transgenic SSMM maize (T0 generation transformant lines) were crossed with ms45 homozygous recessive male-sterile material (obtained from Maize Genetics Cooperation Stock Center, 905I) to obtain hybrid offspring (T1 generation transformants), and the phenotypes of the hybrid offspring were examined. The experimental results showed that by backcrossing and introducing the insert into the ms45 homozygous recessive male-sterile material, the fertility of the ms45 homozygous recessive male-sterile material could be restored.

[0281] Furthermore, the T1 generation transformant was crossed with maize B73 to obtain the T2 generation transformant. The T2 generation transformant was then backcrossed with maize B73 (molecular detection of the Ms45 site was required at the seedling stage, and plants with heterozygous Ms45 sites were selected for backcrossing with B73) to obtain the T3 generation transformant. The T3 generation transformant was self-crossed to obtain the T4 generation transformant, which can be used to propagate the male-sterile line (molecular detection of the Ms45 site was required at the seedling stage, and plants with homozygous Ms45 sites were selected). This T4 generation transformant was then crossed with the male-sterile plants in the T4 generation (1 / 4 of the large kernels in the T4 generation ears were homozygous female-sterile plants with the ms45 site mutation) to obtain the T5 generation transformant and the male-sterile line. The small kernels in the T5 generation ears can be used as maintainer lines, and the large kernels can be used as male-sterile lines.

[0282] The results showed that, compared with other transformants, the expression or transcription of the target gene in SSMM-15 was relatively stable across different generations, and SSMM-15 exhibited stable fertility, stable plant color, and stable seed size. Therefore, through the multi-generation evaluation in 1) and 2) above, a superior transformant was finally obtained and named SSMM-15 (with accession number CGMCC No. 46085, and accession date August 7, 2024).

[0283] Example 3: Identification of SSMM-15

[0284] 1. Molecular-level identification of SSMM-15 transformants

[0285] This embodiment analyzes the molecular characteristics of the SSMM-15 transformant and its progeny. First, the SSMM-15 transformant was identified, confirming positive results for the Ms45 gene, Lc gene, Mn1 / Sh1-RNAi, and bar gene. Then, Tail-PCR (Liu YG, Chen Y. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. BioTechniques,2007(5):43.) was used to determine the insertion position of the T-border sequence into the transformant chromosome. Three rounds of PCR amplification were performed, and the specific primer sequences are shown in SEQ ID NO:25-35.

[0286] The amplification product was transferred into competent *E. coli* cells, and single clones were selected and added to 10 μL of sterile water. PCR identification and sequencing were performed. The obtained sequencing results were compared with the T-border sequence and the maize genome to determine the site of integration of the exogenous fragment into the maize genome. Sequences near the site were examined for the presence of genes, and the results were then analyzed.

[0287] The insertion sequence of the SSMM-15 transformant was determined by Tail-PCR, and the specific sequence is shown in SEQ ID NO:36. The sequence shown in SEQ ID NO:36, from 5' to 3', includes: the 3' end sequence of the T-border insertion region (SEQ ID NO:37), the repair sequence generated by genetic transformation integration (SEQ ID NO:38), and the flanking genomic sequence of the insertion region in SSMM-15 (SEQ ID NO:39).

[0288] Furthermore, SSMM-15 transformant-specific detection primer pairs SSMM-15F (SEQ ID NO:42) and SSMM-15R (SEQ ID NO:43) were designed targeting partial sequences in the 3' end sequence of the aforementioned T-border insertion region (SEQ ID NO:40) and partial sequences in the flanking genome sequence (SEQ ID NO:41), respectively. The primer pairs were used to detect T3–T5 generation plants of SSMM-15, confirming the stable integration of the exogenous insertion sequence into the SSMM-15 genome.

[0289] 2. Phenotypic identification of SSMM-15 transformants

[0290] This embodiment identified the phenotypes of the SSMM-15 transformant and its progeny. The results of the target trait identification showed that backcrossing with an insert (containing the Ms45 gene) into homozygous recessive male-sterile ms45 material restored the fertility of the ms45 mutant. Furthermore, the simultaneous introduction of the Mn1 / Sh1-RNAi fragment resulted in smaller grains containing this fragment compared to those without. The presence of the Lc gene also increased anthocyanin content, resulting in purple plants. Therefore, because the SSMM-15 transformant contains fertility restoration genes, grain morphology marker genes, plant color marker genes, and glufosinate selection marker genes, it can cause smaller grains (shriveled, short, narrow, thin) or sunken and wrinkled endosperm, a purple plant color, and tolerance to glufosinate.

[0291] The SSMM-15 transformant showed significant and stable differences in kernel phenotype in maize materials with different genetic backgrounds (kernels were significantly smaller than those before the introduction of the SSMM-15 transformant; normal kernels in plants before the SSMM-15 transformation were subsequently referred to as "large kernels" for distinction). Seedling plants were generally purple (segregation ratio was normal) and stable, and the fertility of ms45 mutant plants was restored. The expression of various traits was stable across generations.

[0292] The following tests confirmed that the following marker traits of the SSMM-15 transformant are stable in maize materials with different genetic backgrounds:

[0293] (1) The seeds are small.

[0294] The SSMM-15 transformant was backcrossed and introduced into maize plants with different genetic backgrounds. Self-pollination of this material produced male-sterile plants with a ms45 / ms45 morphology. Maize plants with different genetic backgrounds containing the SSMM-15 transformant were then crossed with these male-sterile plants. The resulting ears were as follows: Figures 2A to 2H As shown (the maize varieties are inbred lines M101, R25, D15, B73, A632, LH82, LH150, and W64a), the kernels are divided into normal kernels ( Figure 2A The yellow and plump kernels in H are normal kernels) and small kernels ( Figure 2A (The kernels in H are small and white). Observe the kernel size phenotype of the SSMM-15 transformant in maize materials with different genetic backgrounds (the kernels are significantly smaller compared to the kernels before the introduction of the SSMM-15 transformant), count the number and weight of kernels of different sizes, and calculate the segregation ratio.

[0295] The number of normal and small kernels on the above-mentioned ears was counted. 100 kernels were selected from the middle section of each ear, and the number of large and small kernels was counted. The separation ratio was calculated, and the results are shown in Table 4. The ratio of normal kernels to small kernels was 1:1.

[0296] Table 4. Granulocyte-to-size segregation ratio of SSMM-15 under different genetic backgrounds

[0297]

[0298]

[0299] Note: Degrees of freedom = 1. According to the chi-square distribution table, a chi-square value greater than 3.84 (P value less than 0.05) indicates a significant difference.

[0300] The weight of 100 kernels was measured for both normal and small kernels harvested from the above-mentioned ears. The results are shown in Table 5. The average weight of 100 kernels for large kernels was 30.675 g, and the average weight of 100 kernels for small kernels was 14.375 g.

[0301] Table 5. 100-grain weight of SSMM-15 under different genetic backgrounds

[0302]

[0303] (2) During the seedling stage, the anthocyanin content in the plants increases, and the plant color turns purple.

[0304] Normal and small grains harvested from the ears obtained in (1) were planted. It was observed that plants growing from normal grains were green, while plants growing from small grains containing the SSMM-15 transformant were purple. Figure 3 As shown (the plants on the left are all normal green plants, which are the control group; the plants on the right are purple plants, which are SSMM-15 plants).

[0305] (3) Contains the fertility restoration gene Ms45, and the male flowers of the plant are fertile.

[0306] The presence of the fertility restoration gene Ms45 makes the plants containing the SSMM-15 transformant in (2) fertile, and the male flower phenotype is as follows: Figure 4A As shown. Similarly, the fertile B73 inbred line (containing wild-type endogenous Ms45) has the following male flower phenotype. Figure 4B As shown. Male-sterile plants containing homozygous ms45 exhibit the following male flower phenotype: Figure 4C As shown.

[0307] (4) Contains the marker gene bar and has glufosinate tolerance.

[0308] During the seedling stage, SSMM-15 and B73 were sprayed with glufosinate. All SSMM-15 (purple) plants survived and grew normally, while all B73 plants died. This indicates that SSMM-15 has glufosinate tolerance and plants containing SSMM-15 transformants can be screened by spraying glufosinate.

[0309] (5) The marker trait is gene-linked, and plants grown from small seeds containing SSMM-15 are purple in color and fertile.

[0310] Therefore, at the seed stage, fertile seeds containing SSMM-15 and sterile seeds not containing SSMM-15 can be quickly and easily distinguished by seed size. Furthermore, for maintainer seeds mixed in with sterile lines due to mechanical contamination or other reasons, they can be further removed at the seedling stage by observing plant color.

[0311] Example 4: Identification of genetic stability of SSMM-15 offspring

[0312] When a heterozygous SSMM-15 transformant plant is crossed with a male-sterile plant (ms45ms45), two types of offspring are produced. One type is a male-sterile line with normal kernel and plant color (sterile line, genotype ms45ms45), which can be restored to fertility by any wild-type maize (Ms45Ms45). The other type is a fertile line with smaller kernels and a purple plant (maintainer line). This maintainer line is homozygous recessive at the locus controlling male fertility but contains the introduced Ms45 gene, thus the plant is fertile.

[0313] The phenotypic detection and identification results of the transgenic plant offspring are as follows:

[0314] (1) The ears obtained by crossing SSMM-15 transformant heterozygotes with the ms45 male-sterile line showed segregation of large and small kernels, with a large kernel:small kernel ratio of 1:1. The large-kernel ears were the male-sterile lines and could be used for seed production; the small-kernel ears were the maintainer lines and could be used to propagate male-sterile lines, such as... Figure 5 As shown (A in the figure is an exemplary large particle, and B in the figure is an exemplary small particle).

[0315] (2) The ears obtained by self-pollination of homozygous SSMM-15 transformants all had small transgenic kernels. Because they were homozygous lines, their offspring from crosses with sterile lines were all maintainer lines of the transgene. The ear characteristics after self-pollination of homozygous SSMM-15 transformants were as follows: Figure 6 As shown, all the transgenic seeds are small. Since they are homozygous lines, their offspring from crosses with sterile lines are all maintainer lines of the transgenic line.

[0316] (3) The ears obtained by self-pollination of SSMM-15 transformant heterozygous plants showed segregation of large and small kernels, with a large kernel:small kernel ratio of 1:3. The large kernels were non-transgenic sterile lines, while two-thirds of the small kernels were transgenic maintainer lines, such as... Figure 7 As shown.

[0317] (4) SSMM-15 transformant plants can be distinguished by color; SSMM-15 plants have a distinct purple phenotype, while non-transgenic sterile lines are a normal green. Figure 8 As shown (the two sides are controls, and the middle is an SSMM-15 plant).

[0318] (5) Take 30 ears of SSMM-15 homozygous self-pollinated ears, SSMM-15 heterozygous self-pollinated ears and male-sterile line × SSMM-15 heterozygous single plant ears respectively, and count the number of large and small kernels in each ear. The results show that the locus controlling kernel size is a single gene dominant trait. The statistical results are shown in the table below.

[0319] Table 6. Separation ratio of large and small kernels per ear in the transformants.

[0320]

[0321] (6) 10,000 small and 10,000 large kernels from the heterozygous single plant of the male-sterile line × SSMM-15 were used as one replicate, and three replicates were set up. After seedling emergence, the color of small and large kernel plants was counted. The results showed that the proportion of purple seedlings in small kernels was 98.59%-98.62%, and the proportion of green seedlings in large kernels was 99.90%-99.91%. The traits were stable across different generations. The statistical results are shown in the table below.

[0322] Table 7. Transformation plant size and color.

[0323]

[0324] (7) 1000 small and 1000 large kernels from the ear of the male-sterile line × SSMM-15 were used as one replicate, and three replicates were set up. After seedling emergence, green seedlings from the small kernels and purple seedlings from the large kernels were removed. The fertility of the plants was counted when pollen was released. The results showed that all purple seedlings were fertile and all green seedlings were sterile. The traits were stable across different generations, as shown in Table 8 below.

[0325] Table 8. Fertility of Transformed and Propagated Sterile Lines

[0326]

[0327] When selecting the progeny of the transformants, the purple plants sown from small seeds were still able to grow normally under glufosinate selection, indicating that the selection marker gene bar was functioning normally.

[0328] In summary, as verified by this embodiment, the various traits of the SSMM-15 transformants in different generations (e.g., T1 to T5) are all normal and can be stably inherited.

[0329] Example 5: Large-scale propagation of male-sterile lines using male-sterile maintainer lines

[0330] Since sterile lines cannot self-pollinate, recessive homozygous sterile lines are typically used as the female parent and crossed with fertile plants to obtain male-sterile lines. However, the hybrid offspring will contain 50% sterile lines and 50% heterozygous fertile lines. Therefore, the key issue in sterile line seed production is how to efficiently and accurately distinguish between the 50% sterile plants and the 50% fertile plants.

[0331] Crossing the SSMM-15 obtained in Examples 1-3 with a male-sterile line as a maintainer line produces two types of offspring. One type is a male-sterile line with normal kernels and plant structure. This male-sterile line can be restored to fertility by any wild-type maize and used as the female parent for hybrid production. The other type is a fertile line with smaller kernels and purple seedlings (maintainer line). This maintainer line is homozygous recessive at the locus controlling male fertility but contains the introduced fertility-restoring gene, thus the plant is fertile and can be used to propagate male-sterile lines.

[0332] The male-sterile line (ms45 / ms45) and the SSMM-15 maintainer line (pCAMBIA3301-SSMM heterozygous and ms45 / ms45) described above were sown alternately. For every row of maintainer line sown, five rows of male-sterile line were sown, ensuring that no other maize was planted within 300 meters of the seed production area, allowing natural pollination between the male-sterile and maintainer lines in the field. Figure 9 As shown ( Figure 9 The left side of the middle section represents the male-sterile line, and the right side represents the SSMM-15 maintainer line.

[0333] In this situation, the maintainer line can only accept its own pollen, producing two types of offspring: one type exhibits the extrinsic trait of the target gene (e.g., small seeds, purple plants). The target gene in these offspring may be homozygous or heterozygous, making identification difficult. Therefore, these seeds or plants are discarded. The second type, offspring with normal extrinsic traits, does not contain the target gene and can be retained as a sterile line.

[0334] When a sterile line receives pollen from a maintainer line, it will produce two types of offspring: one type will produce offspring exhibiting the extrinsic trait of the target gene (e.g., small seeds, purple plants), and these offspring will be heterozygous for the target gene, thus serving as a maintainer line; the other type will produce offspring with normal extrinsic traits (e.g., yellow seeds, green plants), which will not contain the target gene, and will also serve as a sterile line, with the ratio of the two types of offspring being 1:1. Figure 11 As shown in A. Of the harvested offspring, the maintainer line can be used to continue propagating the sterile and maintainer lines the following year, while most of the sterile lines are used for producer varieties, such as... Figure 11 As shown, the remaining small portion is used to continue propagating sterile and maintainer lines the following year.

[0335] Example 6: Large-scale production of hybrids using male-sterile lines

[0336] After the sterile line material receives pollen from the maintainer line provided in this application, the resulting sterile line is a recessive homozygous sterile line controlled by the cell nucleus. This sterile line can be restored to fertility by any wild-type plant (Ms45 / Ms45). Therefore, by selecting an inbred line (e.g., Chang 7-2) with high combining ability with the male sterile line (e.g., Zheng 58A) for hybridization, hybrids with excellent agronomic traits can be produced.

[0337] In short, maintainer line plants (e.g., Zheng 58B) containing the SSMM-15 transformant and sterile line plants Zheng 58A (ms45 / ms45) are sown alternately, with five rows of sterile line planted for every row of maintainer line planted. This ensures that no other maize is planted within 300 meters of the seed production area, allowing for natural pollination between the sterile and maintainer line plants in the field. Seeds from the offspring of the sterile line plants are collected, and an initial screening is performed based on seed size, separating the sterile line offspring (large seeds) from the maintainer line offspring (small seeds).

[0338] Then, the obtained male-sterile line seed Zheng 58A (as the female parent) and the target maize line seed (as the male parent, Chang 7-2 inbred line) were sown alternately, with 6 rows of male-sterile line seeds sown for every row of target maize line seeds. Figure 10 As shown ( Figure 10 The seed production line consists of male-sterile maternal lines on the left and right sides, and a maternal inbred line in the middle. It is crucial to ensure that no other maternal plants are planted within a 300-meter radius of the seed production site. During the seedling stage, observe the external characteristics of the male-sterile lines and remove any plants exhibiting a purple hue, such as... Figure 11 As shown in the figure. According to statistics, among the 100,000 sterile plants, 253 purple seedlings were removed, that is, the purity of the sterile line propagated was 99.747%.

[0339] After screening, the sterile line Zheng 58A was allowed to undergo natural pollination in the field with the target maize line Chang 7-2. Figure 11 As shown in Figure B, hybrid seeds produced from sterile lines were collected. These hybrid seeds were then sown in the field, and 10,000 plants were randomly selected for gene testing to determine the proportion of hybrid seeds containing the target gene component (pCAMBIA3301-SSMM) derived from the maintainer line, thus assessing the quality (purity) of the hybrid seeds. The results showed that after dual selection using seed size and plant color, the purity of the resulting hybrid seeds reached 100%, meaning that none of the hybrid seeds contained the target gene component derived from the maintainer line.

[0340] Based on this result, it can be determined that by utilizing the male-sterile maintainer line of the present invention and employing dual screening (seed screening and seedling screening), 100% purity of both the progeny sterile line and the progeny maintainer line can be achieved at the seedling stage. The male-sterile maintainer line and seed propagation method of the present invention can be used to produce high-purity offspring seeds of the sterile line and high-purity hybrid seeds.

[0341] Example 7. Novel sterilization isolation material required for seed production fields

[0342] Isolation methods used in maize seed production include spatial isolation, staggered timing, tall crops, natural barriers, and male-sterile hybrids. Because maize seed production sites and times are relatively concentrated, spatial isolation, staggered timing, and natural barrier isolation are relatively difficult, often resulting in seed production fields of different varieties being adjacent to each other. In such cases, planting tall crops and male-sterile hybrids is mainly used to reduce pollen contamination of the maternal parent by the paternal parent in the seed production field, thereby improving the purity of the hybrid.

[0343] This embodiment produces a male-sterile hybrid variety by crossing a conventional male-sterile inbred line (ms45ms45) (as the female parent) with the SSMM-15 transformant plant provided in this application (as the male parent). Further, plants exhibiting the extrinsic traits regulated by the Lc gene are removed from the offspring of the male-female hybrid. Thus, the remaining offspring are male-sterile and exhibit heterosis. This yields a male-sterile hybrid. Because this male-sterile hybrid has stable sterility (100% sterility), it meets the field requirements for isolation in maize seed production fields. Therefore, it is suitable as an isolation material for intercropping to reduce pollen contamination of adjacent female parents in seed production fields of different varieties, thereby improving the purity of the hybrid.

[0344] Furthermore, in this embodiment, the male-sterile hybrid was sown separately from seeds of two different target fertile maize lines, and the cross-contamination of pollen between the two fertile varieties was successfully reduced.

[0345] Example 8. Polymerization of multi-trait transformants to enhance the overall competitiveness of varieties.

[0346] In this embodiment, the SSMM-15 maintainer line with the same genetic background was crossed with 2A-7 (this maize has insect resistance traits, specific information of which is disclosed in CN202011220206.7), followed by self-pollination. Molecular detection was used to screen for plants that were homozygous for 2A-7, homozygous for the fertility recessive locus (ms45ms45), and heterozygous for the SSMM-15 transformant locus, which were then used as maintainer lines. These maintainer lines were further crossed with plants that were homozygous for 2A-7 and homozygous for the fertility recessive locus, which were used as sterile lines. Furthermore, these lines were crossed to propagate insect-resistant sterile lines, which can then be used to produce insect-resistant hybrids.

[0347] The sterile lines produced by propagating the SSMM-15 transformant of the present invention do not involve the introduction of exogenous genes or nucleic acids (genes or nucleic acids introduced through genetic transformation methods). At the same time, the genes controlling fertility are expressed only at specific times and in specific tissues during pollen formation and do not affect the development of other tissues. Therefore, as long as the recovery rate meets the requirements, it will not have an adverse effect on agronomic traits.

[0348] SSMM-15, as a transformant of nuclear male sterility seed production technology with a sterility rate of up to 100%, can be combined with multiple traits such as insect resistance, herbicide tolerance, disease resistance, high yield, and high quality to realize the breeding and promotion of multi-trait varieties, enhance the comprehensive competitiveness of varieties, and realize the industrial upgrading of my country's bio-breeding industry.

Claims

1. An isolated nucleic acid molecule comprising: a first polynucleotide comprising a nucleotide sequence of a restorer gene, the restorer gene being capable of restoring male fertility to a plant that is male sterile due to a male sterility gene; a second polynucleotide comprising a nucleotide sequence of a silencing element capable of targeting the Mn1 gene and / or the Sh1 gene; and a third polynucleotide comprising a nucleotide sequence of an Lc gene. 2.The nucleic acid molecule of claim 1, wherein the silencing element is a silencing element capable of targeting the Mn1 gene and the Sh1 gene; preferably, the silencing element is selected from the group consisting of a sense suppression element, an antisense suppression element, a hairpin suppression element, a double-stranded RNA, an siRNA, an amiRNA, an miRNA, or any combination thereof; preferably, the silencing element comprises a sense suppression element and an antisense suppression element capable of targeting the Mn1 gene and the Sh1 gene; preferably, the silencing element comprises a nucleotide sequence as set forth in SEQ ID NO: 14-17; preferably, the silencing element comprises a sense suppression element, a hairpin suppression element and an antisense suppression element capable of targeting the Mn1 gene and the Sh1 gene; preferably, the silencing element comprises a nucleotide sequence as set forth in SEQ ID NO: 14-18; preferably, the silencing element comprises, in order from 5’ to 3’ direction, a sense suppression element of the Mn1 gene, a sense suppression element of the Sh1 gene, a hairpin suppression element, an antisense suppression element of the Sh1 gene and an antisense suppression element of the Mn1 gene; preferably, the silencing element has a nucleotide sequence as set forth in SEQ ID NO:

13.

3. The nucleic acid molecule of claim 1 or 2, wherein, the male sterility gene is a recessive male sterility gene that causes male sterility in a homozygous state; preferably, the male sterility gene is selected from the group consisting of ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, ms10, ms11, ms12, ms13, ms14, ms15, ms16, ms17, ms18, ms19, ms20, ms21, ms22, ms23, ms24, ms25, ms26, ms27, ms28, ms29, ms30, ms31, ms32, ms33, ms34, ms35, ms36, ms37, ms38, ms43, ms45, ms47, ms48, ms49, ms50, ms52, and any combination thereof;preferably, the male sterility gene is ms45. Preferably, the restorer gene is selected from the group consisting of Ms1, Ms2, Ms3, Ms4, Ms5, Ms6, Ms7, Ms8, Ms9, Ms10, Ms11, Ms12, Ms13, Ms14, Ms15, Ms16, Ms17, Ms18, Ms19, Ms20, Ms21, Ms22, Ms23, Ms24, Ms25, Ms26, Ms27, Ms28, Ms29, Ms30, Ms31, Ms32, Ms33, Ms34, Ms35, Ms36, Ms37, Ms38, Ms43, Ms45, Ms47, Ms48, Ms49, Ms50, Ms52, and any combination thereof; preferably, the restorer gene is Ms45; Preferably, the male sterile gene is ms45, and the restorer gene is Ms45; Preferably, the nucleic acid molecule has one or more features selected from the group consisting of: (1) the Lc gene encodes a protein with an amino acid sequence of SEQ ID NO: 6; (2) the Mn1 gene encodes a protein with an amino acid sequence of SEQ ID NO: 10; (3) the Sh1 gene encodes a protein with an amino acid sequence of SEQ ID NO: 12; (4) the Ms45 gene encodes a protein with an amino acid sequence of SEQ ID NO:

2.

4. The nucleic acid molecule of any one of claims 1-3, which has one or more features selected from the group consisting of: (1) the first polynucleotide further comprises one or more expression regulatory elements operably linked to the nucleotide sequence of the restorer gene; (2) the second polynucleotide further comprises one or more expression regulatory elements operably linked to the nucleotide sequence of the silencing element; (3) the third polynucleotide further comprises one or more expression regulatory elements operably linked to the nucleotide sequence of the Lc gene; Preferably, the expression regulatory elements are selected from the group consisting of a promoter, an enhancer, a terminator, or any combination thereof; Preferably, the promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-preferred promoter, a tissue-specific promoter, a growth stage-preferred promoter, or any combination thereof; Preferably, the promoter is derived from maize.

5. The nucleic acid molecule of claim 4, which comprises: a first polynucleotide comprising a promoter operably linked to a nucleotide sequence of a Ms45 gene derived from maize, and a nucleotide sequence of Ms45; a second polynucleotide comprising a promoter operably linked to a nucleotide sequence of a Mn1 gene derived from maize, and a silencing element capable of targeting the Mn1 gene and the Sh1 gene; and a third polynucleotide comprising a 35S promoter derived from Cauliflower Mosaic Virus (CaMV) operably linked to a nucleotide sequence of a Lc gene; Preferably, the first polynucleotide, the second polynucleotide, and the third polynucleotide are covalently linked, with or without a linker nucleotide. Preferably, the length of the connecting nucleotides is no more than 10 kb, no more than 5 kb, no more than 1 kb, no more than 500 bp, no more than 100 bp, no more than 50 bp, no more than 10 bp, no more than 5 bp, or shorter; Preferably, the first, second, and third polynucleotides are genetically linked; Preferably, the nucleic acid molecule has one or more of the following features: (1) the nucleotide sequence of the Ms45 gene is SEQ ID NO: 1; (2) the nucleotide sequence of the silencing element targeting the Mn1 gene and the Sh1 gene is SEQ ID NO: 13; (3) the nucleotide sequence of the Lc gene is SEQ ID NO: 5; (4) the nucleotide sequence of the promoter of the Ms45 gene derived from maize is SEQ ID NO: 3; (5) the nucleotide sequence of the promoter of the Mn1 gene derived from maize is SEQ ID NO: 19; (6) the nucleotide sequence of the 35S promoter derived from CaMV is SEQ ID NO:

7.

6. The nucleic acid molecule of any one of claims 1-5, further comprising a fourth polynucleotide, which comprises a nucleotide sequence of a selectable marker gene; Preferably, the selectable marker gene is an antibiotic resistance gene or a herbicide resistance gene, for example, a phosphinothricin resistance gene (bar gene); Preferably, the selectable marker gene is derived from an organism other than maize (e.g., a plant, an animal, a microorganism); Preferably, the bar gene encodes a protein with an amino acid sequence of SEQ ID NO:

21.

7. The nucleic acid molecule of claim 6, wherein the fourth polynucleotide comprises a nucleotide sequence of a 35S enhanced promoter derived from CaMV operably linked to the bar gene; Preferably, the first, second, third, and fourth polynucleotides are covalently linked, with or without connecting nucleotides; Preferably, the length of the connecting nucleotides is no more than 10 kb, no more than 5 kb, no more than 1 kb, no more than 500 bp, no more than 100 bp, no more than 50 bp, no more than 10 bp, no more than 5 bp, or shorter; Preferably, the first, second, third, and fourth polynucleotides are genetically linked; Preferably, the nucleotide sequence of the bar gene is SEQ ID NO: 20; Preferably, the nucleotide sequence of the 35S enhanced promoter derived from CaMV is SEQ ID NO:

22.

8. The nucleic acid molecule of any one of claims 5-7, wherein, the first polynucleotide further comprises a terminator of the Ms45 gene derived from maize operably linked to the nucleotide sequence of the Ms45 gene; Preferably, the second polynucleotide further comprises a terminator of the gene encoding nopaline synthase derived from Agrobacterium tumefaciens operably linked to the nucleotide sequence capable of targeting the silencing element of the Mn1 gene and the Sh1 gene; Preferably, the third polynucleotide further comprises a terminator of a gene encoding a nopaline synthase derived from Agrobacterium tumefaciens operably linked to the nucleotide sequence of the Lc gene; Preferably, the fourth polynucleotide further comprises a 35S PolyA terminator derived from CaMV operably linked to the nucleotide sequence of the bar gene; Preferably, the nucleic acid molecule has one or more of the following features selected from: (1) the nucleotide sequence of the terminator of the Ms45 gene derived from maize is SEQ ID NO: 4; (2) the nucleotide sequence of the terminator of the gene encoding a nopaline synthase derived from Agrobacterium tumefaciens is SEQ ID NO: 8; (3) the nucleotide sequence of the 35S PolyA terminator derived from CaMV is SEQ ID NO: 23; Preferably, the nucleic acid molecule has the nucleotide sequence of SEQ ID NO:

24.

9. A vector comprising the isolated nucleic acid molecule of any one of claims 1-8.

10. A host cell comprising the isolated nucleic acid molecule of any one of claims 1-8 or the vector of claim 9. Preferably, the host cell is an Agrobacterium cell or a plant cell. Preferably, the plant cell is a cell of a monocotyledonous or dicotyledonous plant. Preferably, the plant cell is a cell of a plant selected from the group consisting of maize, oilseed rape, rice, Arabidopsis, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugarcane.

11. A recombinant DNA molecule comprising all or part of the sequence of the isolated nucleic acid molecule of any one of claims 1-8 and at least part of the sequence of the plant genomic DNA adjacent thereto. Preferably, the recombinant DNA molecule comprises the sequence set forth in SEQ ID NO:

38. Preferably, the recombinant DNA molecule comprises the sequence set forth in SEQ ID NO: 37 and the sequence set forth in SEQ ID NO: 38; and / or the sequence set forth in SEQ ID NO: 39 and the sequence set forth in SEQ ID NO:

38. Preferably, the recombinant DNA molecule comprises the sequence set forth in SEQ ID NO:

36.

12. A plant or plant seed, wherein, Preferably, the plant or plant seed further comprises in the genome the male sterility gene; preferably, the male sterility gene is a homozygous recessive male sterility gene. Preferably, the isolated nucleic acid molecule is integrated in the genome of the plant or plant seed; preferably, the nucleic acid molecule is integrated in the genome of the plant or plant seed and is located on the same or a different chromosome as the male sterility gene; preferably, the nucleic acid molecule is present in the genome of the plant or plant seed in a heterozygous form. Preferably, the plant or plant seed is male fertile. ​ Preferably, the plant or plant seed is capable of being used as a maintainer line for a male sterile plant comprising the male sterility gene; Preferably, the plant or plant seed is a plant or plant seed of a monocotyledon or a dicotyledon; Preferably, the plant or plant seed is a plant or plant seed of corn, oilseed rape, rice, Arabidopsis, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugarcane; Preferably, the plant or plant seed comprises the nucleic acid molecule of any one of claims 1-8, and one or more additional genes of interest; Preferably, the one or more additional genes of interest are introduced in the plant or plant seed by transformation (e.g., Agrobacterium transformation), or, by crossing corn transformant SSMM-15 with another corn plant comprising the one or more additional genes of interest; Preferably, the plant has one or more traits modulated by the one or more additional genes of interest as compared to its corresponding wild type plant; Preferably, the traits are selected from the group consisting of increased pest resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed mass, improved nutritional quality, hybrid seed production, and / or increased herbicide tolerance, or any combination thereof.

13. The plant or plant seed of claim 12, wherein, The plant or plant seed is corn or a corn seed; Preferably, a representative sample of the corn or corn seed is corn transformant SSMM-15, and the corn transformant SSMM-15 has a deposit number of CGMCC No. 46085.

14. An article of manufacture comprising the plant or plant seed of claim 12 or 13; Preferably, the article of manufacture comprises genomic DNA of the plant or plant seed; Preferably, the article of manufacture comprises genomic DNA of corn transformant SSMM-15 having a deposit number of CGMCC No. 46085. Preferably, the article of manufacture is selected from the group consisting of one or more of a corn ear, a corn on the cob, a corn silk, a corn pollen, a corn grit, a corn meal, a crushed corn, a corn flour, a corn oil, a corn starch, a corn syrup, a corn malt, a corn sugar, a corn syrup, a margarine produced from corn oil, an unsaturated corn oil, a saturated corn oil, a corn chip, a popcorn, an ethanol and / or a liquor produced from corn, a dried distillers grains with solubles (DDGS) produced from corn fermentation, an animal feed from corn, a cosmetic, and a filler.

15. An oligonucleotide molecule or a combination thereof, the oligonucleotide molecule comprising a sequence complementary to at least a portion of the sequence of the recombinant DNA molecule of claim 11; Preferably, the oligonucleotide molecule is used to identify corn transformant SSMM-15.

16. The oligonucleotide molecule or combination thereof of claim 15, wherein, The oligonucleotide molecule is an oligonucleotide probe capable of detecting the recombinant DNA molecule of claim 11; Preferably, the oligonucleotide probe comprises a sequence complementary to SEQ ID NO: 38; Preferably, the oligonucleotide probe comprises a sequence complementary to SEQ ID NO: 38, and a sequence complementary to at least a portion of SEQ ID NO:

37. Preferably, the oligonucleotide probe comprises a sequence complementary to SEQ ID NO: 38, and a sequence complementary to at least part of SEQ ID NO:

39. Preferably, the oligonucleotide probe has a length of 11-20 nt, 21-30 nt, 31-40 nt, 41-50 nt, 51-60 nt, 61-70 nt, 71-80 nt, 81-90 nt, 91-100 nt or longer.

17. The oligonucleotide molecule or combination thereof of claim 15, wherein, The oligonucleotide molecule or combination thereof is an oligonucleotide primer or primer pair capable of amplifying the recombinant DNA molecule of claim 11. Preferably, in the oligonucleotide primer pair, the first oligonucleotide primer comprises a sequence complementary to at least part of SEQ ID NO: 37, and the second oligonucleotide primer comprises a sequence complementary to at least part of SEQ ID NO:

39. Preferably, in the oligonucleotide primer pair, the first oligonucleotide primer has a sequence as set forth in SEQ ID NO: 42, and the second oligonucleotide primer has a sequence as set forth in SEQ ID NO:

43. Preferably, the oligonucleotide primer has a length of 11-20 nt, 21-30 nt, 31-40 nt, 41-50 nt, 51-60 nt, 61-70 nt, 71-80 nt, 81-90 nt, 91-100 nt or longer.

18. A kit comprising the oligonucleotide molecule or combination thereof of any one of claims 15-17. Preferably, the kit comprises at least one pair of oligonucleotide primer pairs of claim 17. Preferably, the kit comprises at least one oligonucleotide probe of claim 16.

19. A method for detecting the presence of corn transformant SSMM-15 in a nucleic acid sample derived from a corn plant, a corn seed or a corn cell, the method comprising: (a) contacting the sample with the oligonucleotide molecule or combination thereof of any one of claims 15-17; (b) performing a nucleic acid amplification reaction; (c) detecting the amplification product of step (b); Preferably, the presence of corn transformant SSMM-15 in the nucleic acid sample is indicated if the presence of the amplification product of step (b) is detected.

20. Use of the oligonucleotide molecule or combination thereof of any one of claims 15-17 or the kit of claim 18 in detecting a nucleic acid sample derived from a corn plant, a corn seed or a corn cell; Preferably, the representative sample of the corn plant, the corn seed or the corn cell is corn transformant SSMM-15 having the accession number CGMCC No. 46085.

21. A method for obtaining a plant, the method comprising, (1) introducing the nucleic acid molecule of any one of claims 1-8 or the vector of claim 9 into a plant cell, and, (2) culturing the plant cell into a plant; Preferably, in step (1), the nucleic acid molecule or the vector is introduced into the plant cell using Agrobacterium. Preferably, the plant cell comprises the male sterile gene in its genome and is male sterile prior to introduction of the nucleic acid molecule or vector; preferably, the plant cell comprises a homozygous recessive male sterile gene in its genome; Preferably, in step (1), the nucleic acid molecule is integrated into the genome of the plant cell; preferably, the nucleic acid molecule, after integration into the genome of the plant cell, is located on a different chromosome than the male sterile gene; Preferably, the plant cell is a cell of a monocotyledonous or dicotyledonous plant; Preferably, the plant cell is a cell of a plant selected from the group consisting of maize, oilseed rape, rice, Arabidopsis, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugarcane; Preferably, the plant comprises a homozygous recessive male sterile gene and the nucleic acid molecule or vector, and is male fertile; preferably, the nucleic acid molecule or vector is present in the genome of the plant in a heterozygous form; preferably, the nucleic acid molecule is integrated in the genome of the plant, and is located on the same or a different chromosome than the male sterile gene; Preferably, the plant is capable of being used as a maintainer plant of a male sterile plant comprising the male sterile gene; Preferably, the method further comprises: (3) pollinating the plant of step (2) with a male sterile plant comprising the male sterile gene, to produce progeny seeds or plants; and (4) screening the progeny seeds or plants for the presence of the Lc gene and / or the presence of the Mn1 gene and Sh1 gene regulated external traits.

22. A method of obtaining progeny seeds or plants of a male sterile line plant and a maintainer plant, the method comprising crossing the plant of claim 12 or 13 or a plant obtained by the method of claim 21 as a male parent with a male sterile plant comprising the male sterile gene as a female parent, and producing progeny seeds or plants; Preferably, the method comprises: (1) providing a male sterile plant comprising the male sterile gene as a female parent; preferably, the male sterile gene is a homozygous recessive male sterile gene; (2) providing the plant of claim 12 or 13 or a plant obtained by the method of claim 21 as a male parent; (3) pollinating the plant of step (2) with the plant of step (1) to produce progeny seeds; (4) optionally, growing the progeny seeds into progeny plants; wherein the progeny seeds or plants that show the Lc gene and / or do not show the Mn1 gene and Sh1 gene regulated external traits are male fertile and can be used as a maintainer line; and the progeny seeds or plants that do not show the Lc gene and / or show the Mn1 gene and Sh1 gene regulated external traits are male sterile and can be used as a male sterile line.

23. The method of claim 22, wherein, The method comprises: (1) providing a male sterile plant comprising the male sterile gene as a female parent; preferably, the male sterile gene is a homozygous recessive male sterile gene; (2) providing the plant of claim 12 or 13 or a plant obtained by the method of claim 21 as a male parent; (3) pollinating the plant of step (2) with the plant of step (1) to produce progeny seeds; (3) pollinating the plants of step (1) with the plants of step (2) to produce two types of progeny seeds; wherein the first type of progeny seeds do not show the external traits of the Mn1 gene and Sh1 gene regulated seeds; and, the second type of progeny seeds show the external traits of the Mn1 gene and Sh1 gene regulated seeds; (4) separating the first and second types of progeny seeds, and optionally, growing them into first and second types of progeny plants, respectively; Optionally, the method further comprises the following steps: (5) removing from the first type of progeny plants those plants that do not show the external traits of the Lc gene regulated plants, whereby the remaining first type of progeny plants are male fertile and can be used as maintainer plants; and / or removing from the second type of progeny plants those plants that show the external traits of the Lc gene regulated plants, whereby the remaining second type of progeny plants are male sterile and can be used as male sterile line plants; (6) pollinating the remaining second type of progeny plants with the remaining first type of progeny plants and producing further progeny seeds.

24. Use of the isolated nucleic acid molecule of any one of claims 1-8 or the vector of claim 9 or the host cell (e.g., plant cell) of claim 10 for producing a maintainer line plant; Preferably, the plant is a monocot or dicot plant; Preferably, the plant is a plant selected from the group consisting of maize, oilseed rape, rice, Arabidopsis, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugarcane.

25. Use of a plant obtained by the method of claim 21 or a progeny seed or plant obtained by the method of claim 22 or 23 or the plant of claim 12 or 13 for producing a hybrid offspring; Preferably, the plant is a monocot or dicot plant; Preferably, the plant is a plant selected from the group consisting of maize, oilseed rape, rice, Arabidopsis, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugarcane.

26. A method of making hybrid seeds, the method comprising: (1) providing progeny seeds of a male sterile line plant obtained by the method of claim 22 or 23, which show the external traits of the Mn1 gene and Sh1 gene regulated seeds; and, providing seeds of a target line plant; (2) field planting the progeny seeds of the male sterile line plant and the seeds of the target line plant to obtain male sterile line plants and target line plants; (3) removing from the male sterile line plants those plants that show the external traits of the Lc gene regulated plants; (4) pollinating the remaining male sterile line plants with the target line plants; (5) harvesting seeds from the male sterile line plants, which are hybrid seeds.

27. A method of growing a male sterile plant produced by crossing a plant of claim 12 or 13 as a male parent, a male sterile plant that is genetically distinct from the genetic background of the male parent as a female parent, and by crossing the male and female parents; the method comprising: planting the male sterile line plants and one or more additional plants in a growing area; Preferably, the one or more additional plants are male fertile; Preferably, the male sterile line plants are spaced apart from the plurality of fertile plants. Preferably, the male sterile line plant is planted between a first male fertile plant and a second male fertile plant; Optionally, the method further comprises the step of removing from the progeny plants resulting from the cross of the male parent and the female parent, plants that show the external traits of the Lc gene regulated plants, whereby the remaining progeny plants are male sterile and have heterosis.

28. Use of a male sterile line plant, planted with one or more additional plants interspaced; wherein, The male sterile plant is produced by crossing the plant of claim 12 or 13 as a male parent with a male sterile plant that is genetically different from the male parent as a female parent; Preferably, the one or more additional plants are male fertile; Preferably, the male sterile line plant is planted between a first male fertile plant and a second male fertile plant.

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