WO2023016097A1 - 水稻细胞质雄性不育恢复基因osrf19及其应用 - Google Patents

水稻细胞质雄性不育恢复基因osrf19及其应用 Download PDF

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WO2023016097A1
WO2023016097A1 PCT/CN2022/100423 CN2022100423W WO2023016097A1 WO 2023016097 A1 WO2023016097 A1 WO 2023016097A1 CN 2022100423 W CN2022100423 W CN 2022100423W WO 2023016097 A1 WO2023016097 A1 WO 2023016097A1
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rice
gene
cytoplasmic male
osrf19
restorer
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English (en)
French (fr)
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张启发
王乃元
周发松
蒋海潮
欧阳亦聃
陈美容
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华中农业大学
中国种子集团有限公司
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Priority to CN202280054250.6A priority Critical patent/CN118159657A/zh
Publication of WO2023016097A1 publication Critical patent/WO2023016097A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This application relates to the technical field of plant genetic engineering, in particular to the cloning, isolation, functional verification and application of a rice cytoplasmic male sterility restorer gene OsRf19, and its application in rice improvement.
  • male reproductive system During the development of plants, due to the influence of environmental conditions or their own genetic mutations, the male reproductive system degenerates and cannot produce pollen or the pollen produced lacks normal functions, while a biological phenomenon in which the female reproductive system develops normally is called male sterility.
  • Crop varieties or lines with genetic characteristics of male sterility are called sterile lines.
  • the hybrids produced by crossing some crop varieties (strains) with sterile lines can restore normal fertility, and these crop varieties (strains) are called restorer lines.
  • CMS lines and restorer lines have important production and application value in crop heterosis utilization and cross breeding.
  • male sterility includes nuclear sterility controlled by nuclear genes and cytoplasmic male sterility (cytoplasmic male sterility, referred to as CMS) controlled by cytoplasmic genes.
  • cytoplasmic male sterility referred to as CMS
  • nuclear male sterility genes mainly include pms3 and tms5, etc., and their fertility is affected by light and temperature.
  • a two-line breeding method for rice is proposed. The two-line breeding method flexibly utilizes the fertility conversion characteristics of the GMS line, and has many advantages such as simple sterility genetic behavior and wide recovery sources.
  • the genetic basis of three-line hybrid breeding is the cytoplasmic male sterile line and its maintainer and restorer lines.
  • the protein encoded by the specific gene in the mitochondrial genome of the sterile line can cause male sterility.
  • the maintainer line has normal cytoplasm, its nuclear genome is the same as that of the sterile line, and its pollen is normally fertile and self-fertile. The hybridization of the maintainer line and the sterile line can produce sterile line seeds, so that the sterile line can reproduce.
  • the nuclear genome of the restorer line carries the gene of restorer function, and its pollen is normally fertile and self-fertilizing.
  • the restorer line was crossed with the sterile line to produce hybrid F1 seeds, which were normally fertile.
  • Three-line hybrid breeding is an important embodiment and guarantee to make full use of heterosis and ensure the safety of food production.
  • the cytoplasmic male sterility of rice mainly includes “Yebai”, “Baotai”, “Honglian” and other types, and the hybrid rice in my country mainly uses the “Yebai” type of sterility.
  • CMS-WA is an indica-type cytoplasmic male sterile line. It is the earliest type of three-line hybrid rice in China, and the most hybrid combinations have been prepared. It is the most important rice used in China's production at present. CMS type. Cytoplasmic sterile types such as Yinshui type, Gang type, D type and dwarf type, which have been bred in recent years with an increasing trend in combination and extension area, also have a similar recovery relationship with wild type sterile lines.
  • the wild sabotage male sterile line is sporophytic sterility. It is believed that the wild sabotage cytoplasmic male sterility and restoration are affected by two pairs of major genes, and may also be modified by minor genes. Most studies reported that the inheritance of the wild-abrupt male sterility restoration gene is controlled by two pairs of genes, which are independently inherited and have certain interaction effects. Yao (1997) conducted a genetic study on fertility restoration using wild-type Zhenshan 97A and Minghui 63, and concluded that the wild-type restorer line Minghui 63 had 2 pairs of dominant restorer genes. Subsequently, Rf4 was located and cloned using the genetic populations constructed by IR24 and Minghui 63. At the same time, it was found through sequence analysis that it encodes a protein with a PPR (pentatricopeptide repeat proteins) structure (Tang et al.2014; Kazama et al.2014).
  • the rice red lotus-type male sterile cytoplasm (CMS-HL) is derived from the wild Oryza sativa, which is an indica-type cytoplasmic male sterile line.
  • CMS-HL The rice red lotus-type male sterile cytoplasm
  • the red lotus type and wild abortive type were obviously different in the relationship between recovery and preservation, genetic characteristics, and cytological characteristics of pollen abortion, and the red lotus type was a typical gametophyte sterile type.
  • Huang Qingyang et al. (1999) located the restorer gene Rf-5 of red lotus type male sterility on chromosome 10 through SSR markers, and the genetic distance from RM258 was 7.8cM.
  • F 1 plants carrying two non-allelic restorer genes had higher seed setting rate under stress conditions than F 1 plants carrying only one restorer gene (Huang et al., 2012).
  • the japonica rice sterile line widely used in production is mainly Baotai-type cytoplasmic male sterile (CMS-BT), which belongs to the gametophytic sterile type.
  • CMS-BT cytoplasmic male sterile
  • Japanese scholar Shinjyo (1975) conducted a more detailed study on the selection and inheritance of the three lines of Baotai type, and believed that the restoration of the Baotai type male sterile line was controlled by a pair of dominant genes.
  • Akagi et al. used two near-isogenic lines as materials, and detected the co-dominant marker OSRRf linked to the restorer gene Rf-1 through ISSR molecular markers. The marker is located on chromosome 10, and the genetic distance from Rf-1 is 3.7 ⁇ 1.1 cM.
  • Komori et al. (2003) selected 9 known RFLP markers linked to Rf-1 on the 10th chromosome, and used a segregation population containing 1024 individuals to fine-map the Baotai type restorer gene. The result was that Rf-1 was fine-mapped In the interval between S12564Tsp509I and C1361MwoI, Komori et al. subsequently cloned the Baotai type cytoplasmic male sterility restorer gene Rf-1 by map-based cloning ( Komori et al., 2004).
  • the male sterile restorer line of Xinzhiyuan was bred, and the combination and utilization of the three lines of Xinzhiyuan were realized (Wang Naiyuan, 2006b).
  • the sterile line bred by using the new male sterile cytoplasm source has a wide range of maintainer lines, which can not only expand the source of maintainer lines to various fields such as mid-season rice, late rice, and high-quality rice, but also break the genetic limitations of the early indica rice system in the Yangtze River Basin. Greatly improved the breeding potential of CMS (Wang Naiyuan et al., 2008a).
  • the resources of new male sterile restorer lines in conventional varieties are limited, and current research shows that the restorer gene is controlled by a pair of dominant genes (Wang Naiyuan et al., 2008b).
  • CMS-FA new mass-derived male sterile line
  • the restorer gene can be quickly and effectively transferred to excellent breeding resources, thereby greatly Broadening the sources of restorer lines and making full use of heterosis will help ensure food security in my country.
  • the purpose of this application is to isolate and clone a cytoplasmic male sterile fertility restorer gene OsRf19 from rice, aiming to improve the recovery ability of rice in hybridization with sterile lines through the research and application of the fertility restorer gene OsRf19 , to provide new genetic resources for rice genetic breeding.
  • This application utilizes map-based cloning to isolate and clone a gene OsRf19 that controls the recovery of rice new cytoplasmic male sterility, and provides new gene resources for three-line breeding of rice.
  • the present application relates to the following technical solutions.
  • the present application provides a rice cytoplasmic male sterility restorer gene OsRf19, which comprises the nucleotide sequence shown in SEQ ID NO:1.
  • the present application provides a protein encoded by the rice cytoplasmic male sterility restorer gene OsRf19, the protein comprising the amino acid sequence shown in SEQ ID NO: 4 or the amino acid shown in SEQ ID NO: 4 Sequence composition.
  • the present application provides a nucleic acid encoding the protein described in the second aspect.
  • the present application provides an expression vector comprising the rice cytoplasmic male sterility restorer gene OsRf19 described in the first aspect or the nucleic acid described in the third aspect.
  • the present application provides a method for creating a rice restorer line, which includes the rice cytoplasmic male sterility restorer gene OsRf19 described in the first aspect or the nucleic acid described in the third aspect or the expression vector described in the fourth aspect introduced into rice varieties.
  • the present application provides a method for restoring fertility of rice cytoplasmic male sterile lines, comprising:
  • the transgenic restorer line is crossed with the rice cytoplasmic male sterile line to produce hybrid seeds with normal fertility.
  • the present application provides the rice cytoplasmic male sterile restorer gene OsRf19 described in the first aspect or the nucleic acid described in the third aspect in rice breeding or creating a rice restorer line or restoring the fertility of a rice cytoplasmic male sterile line use in .
  • the present application provides a method for producing rice hybrid seeds, which includes: using the rice cytoplasmic male sterile line as the female parent and using the rice restorer line containing the cytoplasmic male sterility restorer gene OsRf19 as the male parent for hybridization , producing rice hybrid seeds, wherein the gene OsRf19 comprises the nucleotide sequence shown in SEQ ID NO: 1 or the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4.
  • Figure 1 is the map-based cloning of the OsRf19 gene of the present application, and Figure a shows the position of OsRf19 on the rice chromosome 10 genetic linkage map; Figures b and c use two mapping populations to fine-map OsRf19 respectively.
  • the numbers between the markers represent the recombination times between each marker and the OsRf19 site.
  • Fig. 2 is a map of the OsRf19 transgene complementary verification of the present application, that is, the map of the functional vector used is pCAMBIA1300.
  • Fig. 3 is a microscopic examination of pollen iodine staining and a phenotype diagram of seed setting rate of the T1 generation transgenic individual plants of the present application.
  • Figure a is the identification of transgenic plants by molecular markers;
  • Figure b is the phenotype of the whole plant of transgene negative (left) and transgene positive (right);
  • Figure c is the spikelet setting rate of transgene negative (left) and transgene positive (right) Phenotype;
  • Figure d is the microscopic examination of the pollen of the transgenic negative plants;
  • Figure e is the microscopic examination of the pollen of the transgenic positive plants.
  • Fig. 4 is the phenotype of the OsRf19 transgenic positive plant of the present application and the new quality source male sterile line Xinqin 1A hybridized.
  • Figure a is the pollen iodine staining phenotype of the CMS Xinqian 1A (left) and the transgenic positive plant crossed with the Xinqian-derived CMS F 1 plant (right);
  • Figure b is the CMS Xinqian 1A (left) and The spikelets of the F 1 plants (right) of transgenic positive plants crossed with the new plastid-derived male sterile line.
  • Fig. 5 is the RT-PCR detection figure of the OsRf19 gene expression characteristic of the present application.
  • the upper part is the expression of the target gene OsRf19 in each tissue; the lower part is the expression of the internal reference gene Actin1 in each tissue.
  • rice is any rice plant and includes all plant varieties that can be bred with rice.
  • plant or “plant” includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant bushes, and plants or plant parts Intact plant cells, such as plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like.
  • Rice as used herein includes paddy rice, upland rice, indica rice, japonica rice, early rice, late rice, glutinous rice, glutinous rice, rice with hairy leaves and rice with glabrous leaves (Oryza sativa).
  • sterile line also known as “male sterile line” or “cytoplasmic male sterile line” refers to a rice line whose pollen is sterile and which can transmit this characteristic to the offspring.
  • CMS-FA new cytoplasmic sterile line
  • CMS-FA new cytoplasmic sterile line
  • CMS-FA new cytoplasmic sterile line
  • the resource utilization of the maintainer line of the cytoplasm reaches 55.5%, which is more extensive than that of the wild type (CMS-WA) maintainer line resource utilization of 20%, it is also called the broad-preservation cytoplasmic male sterile line, and the wide-preservation type male sterile line Line or wide-protected sterile line, for example, please refer to the Chinese invention patent document CN1954666B.
  • single nucleotide polymorphism or "SNP” or “SNP marker” or “SNP site” refers to a nucleotide sequence present in the genomic sequence of a chromosome, based on the nucleotide sequence
  • a change in the sequence of a polynucleotide resulting from a difference (a change in a single nucleotide—A, T, C, or G), resulting in chromosomal genome diversity that allows for different alleles (such as alleles from two different individuals) genes) or different individuals are distinguished from each other.
  • the change may occur within the coding or non-coding region of the gene (e.g., at or near a promoter region, or an intron) or in an intergenic region.
  • InDel refers to an insertion or deletion, which specifically refers to a difference in the whole genome, a certain number of nucleotide insertions or deletions in the genome of an individual relative to a standard control (Jander et al., 2002).
  • SSR Simple Sequence Repeats
  • Microsatellite DNA is a class of repeating units consisting of several nucleotides (generally 1 to 6) long Tandem repeats of ten nucleotides. The sequences flanking each SSR are generally relatively conserved single-copy sequences.
  • the term "gene homologous to OsRf19" refers to a gene derived from the same species or a different species from rice OsRf19 and having a similar function.
  • Gene editing or “genome edting” used in this article is an emerging and more precise genetic engineering technology that can modify specific target genes in the genome of an organism.
  • Gene editing refers to the ability to "edit” a target gene at a specific point to modify a specific DNA segment.
  • Gene editing relies on genetically engineered nucleases, also known as “molecular scissors”, to generate site-specific double-strand breaks (DSBs) at specific locations in the genome, inducing organisms to undergo non-homologous end joining (NHEJ) or homologous Source-derived recombination (HR) is used to repair DSBs because this repair process is error-prone, resulting in targeted mutations.
  • NHEJ non-homologous end joining
  • HR homologous Source-derived recombination
  • CRISPR/Cas9 refers to an endonuclease that uses an RNA guide strand to target the endonuclease cleavage site. See, Jinek et al., Science 337 :816-821 (2013); Cong et al., Science (January 3, 2013); and Mali et al., Science (January 3, 2013). There are three different types of CRISPR/Cas9 systems discovered so far, type I, type II and type III, which are present in approximately 40% of sequenced eubacteria and 90% of sequenced archaea.
  • the composition of type II is relatively simple, with Cas9 protein and guide RNA (gRNA) as the core composition, and it is also the most deeply studied type at present.
  • gRNA Cas9 protein and guide RNA
  • CRISPR is transcribed into a long RNA precursor (pre-crRNA), and then processed into a series of short mature crRNAs containing conserved repeat sequences and spacers, Finally, it recognizes and binds to the complementary exogenous DNA sequence to play a cutting role.
  • the cleavage site of CRISPR/Cas9 is located at the NGG site in the 5'-GG-N18-NGG-3' characteristic region of the PAM region (Protospacer Adjacent Motif) adjacent to the downstream of the crRNA complementary sequence, and the sequence of this characteristic is in each The 128bp random DNA sequence repeats once.
  • CRISPR/Cas12a used herein is a novel class of CRISPR-Cas system. Compared with Cas9 protein, Cas12a protein is more accurate and safer. When CRISPR/Cas9 works, the Cas9 protein recognizes the PAM sequence (genetic code written by RNA), and unwinds part of the double helix through gRNA. In this process, some mismatches may occur, but this combination is irreversible. In this regard, Cas12a is much smarter. When it is looking for a "target", it will conduct single-base recognition of the DNA sequence along the way. If it finds a base that does not match well, it will leave and search again.
  • PAM sequence genetic code written by RNA
  • the Cas12a protein When the PAM sequence is found, the Cas12a protein will form a semi-closed R-loop (R-loop) with the PAM sequence. When the correct sequence is recognized, it will be completely combined into a closed R-loop, so this combination It is reversible, which also reflects its safer side.
  • R-loop semi-closed R-loop
  • transcriptional activator-like effector nucleosidase or “TAL effector nucleosidase” or “TALEN” as used herein refers to a class of TAL effector nucleosidases produced by fusing the TAL effector DNA binding domain to the DNA cleavage domain. Artificial restriction endonucleases.
  • ZFN zinc finger nuclease
  • the DNA recognition domain is a tandem structure of 3 to 4 ZFs, each ZF contains about 30 amino acids, is fixed by a zinc ion, can recognize and bind a specific triplet base, and the DNA splicing domain consists of non-specific nucleic acids
  • the endonuclease Fok I carboxy-terminal 96 amino acid residues. Each Fok I monomer is connected with a ZFP to form a ZFN and recognizes a specific site. When the two recognition sites are at an appropriate distance (6-8 bp), the two monomer ZFNs interact to produce restriction enzymes Function, forming double-strand breaks, thereby mediating DNA-directed cleavage.
  • meganuclease refers to a homing endonuclease capable of recognizing nucleic acid sequences of 14-40 bases in length. Some meganucleases can tolerate small homing site sequence differences, and the large recognition region still ensures high specificity of these enzymes, which in turn keeps low levels of non-specific cleavage within the genome and low toxicity . Meganucleases are encoded by open reading frames within mobile sequences of self-splicing RNA intron or self-splicing intron sequences.
  • DddA-derived cytosine base editor used in this article is a new base editor created in 2020 by a research team led by the Broad Institute of Harvard University and Ruqian Liu of the Massachusetts Institute of Technology.
  • C to T transition from DNA in mitochondria This was achieved by creating a new gene-editing technique called base editing, which converts a single nucleotide base into another without damaging the DNA.
  • base editing a new gene-editing technique called base editing, which converts a single nucleotide base into another without damaging the DNA.
  • this technique also has its limitations. It is not restricted to C to T conversions, but mainly to TC motifs, making it an efficient TC-TT converter. That means it can only correct 9 of 90 confirmed disease-causing mitochondrial point mutations, or 10 percent.
  • the A-to-G transition of mitochondrial DNA was long thought to be impossible.
  • TALED transcriptional activator-like effector-associated deaminase
  • the first component is a transcriptional activator-like effector that targets DNA sequences.
  • the second component is TadA8e, a protein that promotes A to G Transforming adenine deaminase.
  • the third component, DddAtox is a cytosine deaminase that makes DNA more accessible to TadA8e.
  • the application provides the nucleotide sequence of the OsRf19 gene and the protein encoded by it, its nucleotide sequence is shown in the sequence table SEQ ID NO: 1, 2 and 3, wherein SEQ ID NO: 1 is an intron-free An open reading frame (ORF) of 2376 bases, SEQ ID NO: 2 is the sequence containing the promoter regulatory element and the 5' untranslated region, and SEQ ID NO: 3 is the sequence of the 3' untranslated region.
  • SEQ ID NO:4 The sequence of the protein encoded by the above gene OsRf19 is shown in SEQ ID NO:4.
  • the sequence consists of 791 amino acids and has a functional domain composed of 19 PPR repeat units (pentatricopeptide repeat), and its biological function is to restore the fertility of plant cytoplasmic male sterility.
  • a typical PPR is a repeating unit consisting of 35 amino acid residues.
  • more than two PPR units in the same direction form a functional domain, and the amino acid sequences of different PPR units differ to varying degrees.
  • the present application relates to the following technical solutions.
  • the present application provides a rice cytoplasmic male sterility restorer gene OsRf19 or a functional variant thereof, the restorer gene OsRf19 comprising the nucleotide sequence shown in SEQ ID NO:1.
  • SEQ ID NO: 1 is an open reading frame of 2376 bases without introns.
  • Those skilled in the art can determine and/or obtain the functional variant of the rice cytoplasmic male sterility restorer gene OsRf19 by conventional methods. Compared with the original gene, the functional variant may have one or more nucleotide deletions, additions and/or substitutions, but still retains the function of the original gene, for example, still encodes a protein with the same function.
  • the functional variant may have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% of the sequence shown in SEQ ID NO: 1 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity and encode proteins with the same function. Therefore, SEQ ID NO: 1 includes both the nucleotide sequence shown in SEQ ID NO: 1 itself and functional variants of the nucleotide sequence shown in SEQ ID NO: 1.
  • the term "functional variant” refers to substantially similar sequences. With respect to nucleotide sequences, functional variants include those sequences that, due to the degeneracy of the genetic code, encode proteins of the same function. Allelic variants such as naturally occurring allelic variants can be identified using well known molecular biology techniques such as polymerase chain reaction (PCR) and hybridization techniques.
  • PCR polymerase chain reaction
  • At least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 Genes from other rice varieties that have %, 95%, 96%, 97%, 98%, 99% or higher identity and also encode a functional OsRf19 protein are included in the "functional variants" defined in the present application. Identity is determined by the sequence alignment program described herein, using default parameters. The sequence of a functional variant of a nucleotide may differ from the nucleotide sequence by as few as 1-15 nucleotides, as few as 1-10 (eg 6-10), as few as 5, as few as 4 , 3, 2 or even 1 nucleotide.
  • genes homologous to OsRf19 or functional variants of OsRf19 can be easily obtained by the following methods: (a) By comparing with the database, obtain the homology of OsRf19 that has been published but whose function is unknown Gene; (b) use the OsRf19 gene fragment as a probe to screen the rice genome library to obtain positive clones and sequencing; (c) design oligonucleotide primers according to the sequence information of SEQ ID NO:1, and use the PCR method to obtain a positive clone from the genome of rice or wild rice The OsRf19 gene fragment is amplified and sequenced; (d) obtained by molecular biology method modification based on the sequence of SEQ ID NO:1; or (e) obtained by chemical synthesis with reference to the sequence of SEQ ID NO:1.
  • the rice cytoplasmic male sterility restorer gene OsRf19 consists of SEQ ID NO: 1, 2 and 3, wherein SEQ ID NO: 1 is an open reading frame of 2376 bases without introns (ORF), SEQ ID NO: 2 is the sequence containing the promoter regulatory element and the 5' untranslated region, and SEQ ID NO: 3 is the sequence of the 3' untranslated region.
  • the rice cytoplasmic male sterility restorer gene OsRf19 consists of SEQ ID NO:1.
  • the application provides a protein or a functional variant thereof, the protein is encoded by the rice cytoplasmic male sterility restorer gene OsRf19, and the protein comprises the amino acid sequence shown in SEQ ID NO: 4 or represented by SEQ ID NO: 4 The amino acid sequence composition shown in ID NO:4.
  • the functional variant may have one or more amino acid deletions, additions and/or substitutions (such as conservative substitutions), but still retains the function of the original protein, such as restoring the cytoplasmic male sterile line of rice. Fertility function.
  • the functional variant may have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% of the sequence shown in SEQ ID NO: 4 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity and still retain the function of the original protein. Therefore, SEQ ID NO: 4 not only includes the amino acid sequence shown in SEQ ID NO: 4 itself, but also includes functional variants of the amino acid sequence shown in SEQ ID NO: 4.
  • the term "functional variant” includes polypeptides derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N- and/or C-terminus of the native protein or by adding one or more Amino acids are added to the N-terminal and/or C-terminal of the natural protein; one or more amino acids are deleted or added at one or more positions of the natural protein; or one or more amino acids are substituted at one or more positions of the natural protein multiple amino acids.
  • the term "functional variant” includes biologically active fragments of native proteins that contain a sufficient number of contiguous amino acid residues that retain the biological activity of the native protein, eg, have OsRf19 protein function. Such functions may be different or improved relative to the native protein, or may be unchanged, as long as the OsRf19 protein function is retained. Identity is determined by the sequence alignment program described herein, using default parameters. The difference between the active variant sequence of the protein and the protein can be as little as 1-15 amino acid residues, as little as 1-10 (such as 6-10), as little as 5, as little as 4, 3, 2 or even 1 amino acid residue.
  • the protein consists of the amino acid sequence shown in SEQ ID NO:4.
  • the present application provides a nucleic acid encoding the protein described in the second aspect.
  • the nucleic acid may be a codon-optimized nucleic acid suitable for expression in a host cell.
  • a codon-optimized nucleic acid suitable for expression in a host cell.
  • Methods for codon optimization depending on the host cell used are well known to those skilled in the art.
  • the present application provides an expression vector comprising the rice cytoplasmic male sterility restorer gene OsRf19 described in the first aspect or the nucleic acid described in the third aspect.
  • prokaryotic cloning vectors include plasmids from E. coli such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4.
  • Prokaryotic vectors also include derivatives of phage DNA such as M13 and other filamentous single-stranded DNA phages.
  • An example of a vector that can be used in yeast is the 2 ⁇ plasmid.
  • Suitable vectors for expression in mammalian cells include the following well-known derivatives: SV-40, adenovirus, retrovirus-derived DNA sequences as well as those derived from functional mammalian vectors (such as those described above) and functional plasmids and Combinatorial shuttle vectors for phage DNA.
  • Expression vectors useful in the present invention contain at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. Control sequences are inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operator and promoter region of bacteriophage lambda, the control region of the fd coat protein, the glycolytic promoter of yeast, e.g.
  • the present application provides a method for creating a rice restorer line, which includes the rice cytoplasmic male sterility restorer gene OsRf19 described in the first aspect or the nucleic acid described in the third aspect or the expression vector described in the fourth aspect introduced into rice varieties.
  • the introducing is by gene editing.
  • the rice variety is selected from the group consisting of rice, upland rice, indica rice, japonica rice, early rice, late rice, sticky rice, glutinous rice, rice with hairy leaves and rice with glabrous leaves.
  • the gene editing is performed by one or more selected from: CRISPR/Cas9, CRISPR/Cpf1, CRISPR/Cas12a, TALEN, meganuclease, ZFN, DddA-derived cytosine bases Editor and TALED.
  • said gene editing is performed by CRISPR/Cas9.
  • said gene editing is performed by CRISPR/Cas9.
  • the present application provides a method for restoring fertility of rice cytoplasmic male sterile lines, comprising:
  • the rice cytoplasmic male sterile line is a new cytoplasmic male sterile line.
  • the rice cytoplasmic male sterile line is a rice sterile line containing the sterility gene orf182.
  • the rice sterile line used is a new plastid sterile line containing the mitochondrial sterile gene orf182.
  • the new plastid male sterile line containing the mitochondrial sterile gene orf182 is a sporophytic male sterile line with no pollen abortion, complete abortion and extremely stable fertility.
  • the gene sequence of the mitochondrial sterility gene orf182 is shown in SEQ ID NO:9.
  • the introducing is by gene editing.
  • the rice variety and the rice cytoplasmic male sterile line are each selected from the group consisting of rice, upland rice, indica rice, japonica rice, early rice, late rice, sticky rice, glutinous rice, rice with hairy leaves and rice without hairy leaves .
  • the gene editing is performed by one or more selected from: CRISPR/Cas9, CRISPR/Cpf1, CRISPR/Cas12a, TALEN, meganuclease, ZFN, DddA-derived cytosine bases Editor and TALED.
  • said gene editing is performed by CRISPR/Cas9.
  • said gene editing is performed by CRISPR/Cas9.
  • the present application provides the rice cytoplasmic male sterile restorer gene OsRf19 described in the first aspect or the nucleic acid described in the third aspect in rice breeding or creating a rice restorer line or restoring the fertility of a rice cytoplasmic male sterile line use in .
  • the rice cytoplasmic male sterile line is a new cytoplasmic male sterile line.
  • the rice cytoplasmic male sterile line is a rice sterile line containing the sterility gene orf182.
  • the rice sterile line used is a new plastid sterile line containing the mitochondrial sterile gene orf182.
  • the sterility gene orf182 comprises the nucleotide sequence shown in SEQ ID NO: 9.
  • OsRf19 similar gene described in this application is defined as having one or several nucleotide differences compared with the nucleotide sequence of SEQ ID NO: 1, including changes, deletions, or insertions of several bases, and its A gene whose function of the expression product is equivalent to that of the expression product of OsRf19.
  • the present application provides a method for producing rice hybrid seeds, which includes: using the rice cytoplasmic male sterile line as the female parent and using the rice restorer line containing the cytoplasmic male sterility restorer gene OsRf19 as the male parent for hybridization , producing rice hybrid seeds, wherein the gene OsRf19 comprises the nucleotide sequence shown in SEQ ID NO: 1 or the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4.
  • the rice cytoplasmic male sterile line is a new cytoplasmic male sterile line.
  • the rice cytoplasmic male sterile line is a rice sterile line containing the sterility gene orf182.
  • the rice sterile line used is a new plastid sterile line containing the mitochondrial sterile gene orf182.
  • the sterility gene orf182 comprises the nucleotide sequence shown in SEQ ID NO: 9.
  • the rice restorer line and the rice cytoplasmic male sterile line are each selected from the group consisting of rice, upland rice, indica rice, japonica rice, early rice, late rice, sticky rice, waxy rice, rice with hairy leaves, and rice without hairs rice.
  • the words "comprising”, “comprising” and “containing” mean “including but not limited to”, and are not intended to exclude other parts, additives, components, or steps.
  • Example 1 Preliminary localization of rice new quality source male sterility restoration gene OsRf19 (also known as RFFA)
  • F 1 generation of crossing the new plasma-derived male sterile line (CMS-FA) and the restorer line is normally fertile, and the fertility restoration (fertility) gene is dominantly inherited.
  • F 2 Fertility is separated from the generation: sterility is suitable for 3:1, and the fertility restoration (fertility) gene is controlled by a pair of dominant genes (Wang Naiyuan et al., 2008b).
  • the female parent of the three-line hybrid rice Jinnong 2 You 3 (see the website of the Chinese rice variety and its pedigree database: https://www.ricedata.cn/variety/varis/605314.htm) is the male sterile line Jinnong 2A (See the website of the Chinese rice variety and its pedigree database: https://www.ricedata.cn/variety/varis/607151.htm), the male parent is the restorer line Jinhui 3 (see the website of the Chinese rice variety and its pedigree database: https://www.ricedata.cn/variety/varis/609760.htm).
  • Jinnong 2A is a new mass-derived male sterile line (CMS-FA)
  • Jinhui 3 is a CMS-FA restorer line, which contains the rice CMS-FA restorer gene and is named OsRf19.
  • Map-based cloning was used to map the gene to two molecular markers Rf1D6 (Chr10 : 18.828Mb) and Rf1D7 (Chr10: 18.894Mb) and about 66kb region (rice TIGR/MSU annotation version 6.1, http://rice.plantbiology.msu.edu/), contains Rf1a, does not contain Rf1b ( Figure 1) .
  • Example 3 Construction of whole-genome BAC library of rice Jinhui No. 3 and sequencing of target BAC
  • SEQ ID NO: 1 is an open reading frame (ORF) of 2376 bases without introns derived from the OsRf19 gene of rice Jinhui 3 that was isolated and cloned.
  • SEQ ID NO: 2 is the sequence of the OsRf19 gene including the promoter regulatory element and the 5' untranslated region.
  • SEQ ID NO: 3 is the sequence of the 3' untranslated region of the OsRf19 gene.
  • SEQ ID NO: 4 is the amino acid sequence of the isolated and cloned OsRf19 gene derived from rice Jinhui 3.
  • Embodiment 4 Creation of new quality-derived male sterile line 93-11A of rice
  • Jinnong 2A was used as the female parent and rice indica variety 93-11 (see the website of the Chinese rice variety and its pedigree database: https://www. ricedata.cn/variety/varis/600611.htm) was used as the male parent to obtain F 1 hybrids. Then the hybrid was planted, and the BC 7 F 1 plant was obtained after continuous backcrossing with the hybrid for 7 generations with the variety 93-11 as the recurrent male parent. After backcrossing, the nuclear genome DNA was basically consistent with that of variety 93-11 and the cytoplasm of Jinnong 2A was obtained. We called this rice material Xinzhi 93-11A.
  • 93-11 and Xinqin 93-11A were investigated at flowering and maturity respectively, and it was found that 93-11 was fertile by iodine pollen staining, while Xinqin 93-11A was aborted by iodine pollen staining; Fruiting was normal, but when Xinzhi 93-11A was mature, it could not be fruiting normally, and the fruiting rate was 0. Through the above method, we obtained a stable male sterile line material Xinqin 93-11A.
  • CTCC China Center for Type Culture Collection
  • Embodiment 5 the transgenic complementation test of OsRf19
  • a 5071bp fragment containing promoter, coding region and downstream termination sequence was amplified from the subclone of Jinhui 3 by PCR technique.
  • the PCR reaction system is as follows: a 50 ⁇ l reaction contains 1x reaction buffer, 200 ⁇ M dNTPs, 100 ng subcloned DNA, 0.3 ⁇ M each primer, and 1.0 U KOD FX polymerase. Reaction procedure, Step 1: 95°C for 5 min; Step 2: 95°C for 20 S, 55°C for 30 S, 72°C for 5 min (30 cycles); Step 3: 72°C for 7 min; Step 4: 25°C for 1 min. After the reaction, the PCR product was purified.
  • the vector pCAMBIA1300 (CAMBIA Company, Canberra, Australia, FIG. 2 ) was cut with restriction endonucleases BamHI and PstI
  • the PCR product also cut with restriction endonucleases BamHI and PstI was ligated to the vector.
  • the correct cloning vector without mutation was selected and introduced into Agrobacterium EHA105. Callus was induced from the mature seeds of the rice Xinqianyuan CMS line Xinqian 93-11A on the induction medium.
  • the abbreviated names of reagents and plant hormones used in the culture medium are as follows: 6-BA (6-BenzylaminoPurine, 6-benzyl adenine); CN (Carbenicillin, carbenicillin); KT (Kinetin, kinetin); NAA (Napthalene acetic acid, naphthalene acetic acid); IAA (Indole-3-acetic acid, indole acetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid); AS (Acetosringone, acetyl syringone); CH (Casein Enzymatic Hydrolysate, hydrolyzed casein); HN (Hygromycin B, hygromycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide); N6 max (N6 macroelement solution); N6 mix ( N6 trace element composition solution); MS max (MS macro element composition solution
  • N6 max medium macroelement mother solution prepared according to 10 times concentrated solution (10X):
  • the above reagents were dissolved at room temperature and made up to 1000 ml with distilled water, and stored at room temperature.
  • Vitamin storage solution prepared according to 100X concentrated solution:
  • MS medium macroelement mother solution (MS max mother solution) (prepared according to 10X concentrated solution):
  • the above reagents were dissolved at room temperature, and the volume was adjusted to 1000 ml with distilled water, and stored at room temperature.
  • the above reagents were dissolved at room temperature, and the volume was adjusted to 1000 ml with distilled water, and stored at room temperature.
  • Pre-differentiation medium (japonica rice can skip this step):
  • LA medium with corresponding resistance selection (the preparation of LA medium refers to J. Sambrook et al., Molecular Cloning Experiment Guide, the third edition, Jin Dongyan et al. (translation), Science Press, 2002, Beijing)
  • Agrobacterium EHA105 the strain comes from the Agrobacterium strain publicly used by CAMBIA Company for two days with a temperature of 28° C.
  • Embodiment 6 Expression analysis of OsRf19
  • the expression of this gene was analyzed by RT-PCR.
  • the primer sequences used by RT-PCR are shown in the following SEQ ID NO:7 and SEQ ID NO:8.
  • SEQ ID NO: 7 5'-GATGTACTTTGCAAGTCAGG-3'
  • SEQ ID NO: 8 5'-CCTTCTTTGCAAAGATTGCT-3'
  • Example 7 Restorer line containing restorer gene OsRf19 restores fertility of sterile line
  • the rice restorer line Jinhui No. 3 (the parent of the approved variety Jinnong 3 You No. 3, which can be obtained from China Seed Group Co., Ltd.), Huazhan-RFFA (both containing the restorer gene OsRf19) and rice non- Breeding lines Jinnong 3A (the parent of the approved variety Jinnong 3 You 3, which can be obtained from China Seed Group Co., Ltd.) and Xinzhi 1A (variety right number: CNA20162267.2) (both contain the sterile gene orf182)
  • OsRf19 restores the fertility of the sterile line containing the sterility gene orf182.
  • the restorer line Huazhan-RFFA used in this example was crossed with Jinhui 3 as the donor parent and Huazhan as the recipient parent, and after four times of backcrossing, the restorer gene OsRf19 was introduced into the recipient parent And get.
  • the seeds of the restoration line Huazhan-RFFA were sent to the China Collection Center for Typical Cultures of Wuhan University in Wuhan, China on June 20, 2022.
  • the preservation information is as follows:
  • CTCC China Center for Type Culture Collection
  • the existing Yebai-type restorer lines Minghui 63 and Gui 99 were used as male parents to cross with Jinnong 3A and Xinzhi 1A respectively to obtain F1 generation hybrids. Then 4 combined hybrids were planted in the field to investigate the seed setting rate. The results showed that the seed setting rate of the hybrids of Minghui 63 and Gui 99 with Jinnong 3A and Xinqin 1A were 0. This experiment proves that the wild-type restorer line does not have the ability to restore the new quality sterile line.

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Abstract

提供了一个水稻细胞质雄性不育恢复基因OsRf19、及其编码的蛋白、包含OsRf19基因的表达载体，以及利用上述基因和表达载体构建水稻恢复系以及使水稻细胞质雄性不育系恢复育性和生产水稻杂交种种子的方法和相关用途。

Description

[根据细则37.2由ISA制定的发明名称]　水稻细胞质雄性不育恢复基因OSRF19及其应用 技术领域
[根据细则91更正 13.07.2022]　
本申请涉及植物基因工程技术领域，具体涉及一个控制水稻细胞质雄性不育育性恢复基因OsRf19的克隆、分离、功能验证及其在水稻改良中的应用。
背景技术
植物在发育过程中，由于受环境条件影响或自身遗传突变导致雄性生殖系统退化不能产生花粉或者产生的花粉缺乏正常功能，而雌性生殖系统发育正常的一种生物学现象称为雄性不育。具有雄性不育遗传特征的作物品种或品系称为不育系。而有些作物品种(品系)与不育系杂交产生的杂交种能恢复正常的育性，这些作物品种(品系)称为恢复系。不育系和恢复系在作物杂种优势利用和杂交育种上有重要的生产应用价值。
目前，雄性不育及其育性恢复的研究已经在多种农作物如小麦、玉米、油菜和水稻(Oryza sativa)中报道，在生产上已大规模用于杂交种生产。雄性不育包括由细胞核基因控制的核不育和由细胞质基因控制的细胞质雄性不育(cytoplasmic male sterility,简称CMS)。在水稻中细胞核雄性不育基因主要有pms3和tms5等，其育性受光照和温度的影响，在此基础上提出了水稻两系法育种途径。两系法育种灵活地利用了核不育系的育性转换特点,具有不育性遗传行为简单、恢复源广等诸多优势。但两系核不育系的育性容易受光温条件波动的影响,给不育系的繁殖和杂交种的制种造成了很多困难。三系法杂交育种的遗传基础是细胞质雄性不育系及其保持系和恢复系。其中不育系线粒体基因组中的特异基因编码的蛋白能导致雄性不育。保持系具有正常的细胞质，其核基因组与不育系基因组相同，其花粉正常可育，可自交结实。保持系与不育系杂交能产生不育系种子，使不育系能繁殖下去。恢复系的核基因组中携带有恢复功能的基因，其花粉正常可育，可自交结实。恢复系与不育系杂交产生杂种F 1种子，其育性正常可育。三系杂交育种是充分利用杂种优势，保证粮食生产安全的重要体现与保证。
水稻的细胞质雄性不育主要有“野败”、“包台”、“红莲”等类型，我国杂交水稻主要是利用“野败”型不育。野败型不育系(CMS-WA)为籼型细胞质雄性不育系，是中国三系杂交稻中应用最早的类型，配制的杂交组合也最多，是目前中国生产上应用的最主要的水稻不育系类型。近年来所培育的组合和推广面积呈上升趋势的印水型、冈型、D型和矮败型等胞质不育类型，也与野败型不育系具有相似的恢保关系。野败型不育系为孢子体不育，研究认为，野败型细胞质雄性不育性和恢复性受两对主效基因作用的同时，还可能受微效基因的修饰作用。多数研究报道认为野败型雄性不育恢复基因的遗传受2对基因控制，这2对基因间独立遗传，并存在一定的互作效应。Yao(1997)等利用野败型珍汕97A和明恢63进行育性恢复的遗传研究，结果认为野败型恢复系明恢63具有2对显性恢复基因。随后分别用IR24和明恢63构建的遗传群体定位并克隆了Rf4同时，通过序列分析发现它编码一种PPR(pentatricopeptide repeat  proteins)结构的蛋白(Tang et al.2014；Kazama et al.2014)。
水稻红莲型雄性不育胞质(CMS-HL)来源于红芒野生稻，为籼型细胞质雄性不育系类型。但红莲型和野败型在恢保关系、遗传特点、花粉败育的细胞学特征上明显不同，红莲型属于典型的配子体不育类型。对于红莲型恢复基因的定位，黄青阳等(1999)通过SSR标记，将红莲型雄性不育的恢复基因Rf-5定位在第10染色体上，与RM258的遗传距离为7.8cM。Huang等(2012)通过粤泰A和9311构建了F 2和BC 1F 1群体把红莲型细胞质雄性不育的两个恢复基因Rf5和Rf6分别定位于水稻第10染色体上标记RM6469和RM25661之间以及水稻第8染色体上标记RM3710和RM22242之间；并通过研究发现在仅携带Rf5或Rf6一个恢复基因的F 1植株中50％的花粉粒可育，而同时携带Rf5和Rf6的杂种中75％花粉育性正常。携带两个非等位恢复基因的F 1植株在逆境条件下，其结实率高于只携带一个恢复基因的F 1植株(Huang et al.,2012)。Hu等在2012年克隆了红莲型细胞质雄性不育恢复基因Rf5(Hu et al.,2012)，Huang等则在2015年克隆了红莲型细胞质雄性不育另一个恢复基因Rf6(Huang et al.,2015)，并对他们的调控机理进行研究。
目前生产上广泛应用的粳稻不育系主要为包台型细胞质雄性不育(CMS-BT)，属配子体不育类型。日本学者Shinjyo(1975)对包台型三系的选育及遗传作过比较详细的研究，认为包台型雄性不育系的恢复性受1对显性基因控制。1996年，Akagi等以2个近等基因系为材料，通过ISSR分子标记检测到共显性标记OSRRf与恢复基因Rf-1连锁，该标记位于第10染色体上，与Rf-1的遗传距离为3.7±1.1cM。Komori等(2003)选取第10染色体已知的9个与Rf-1连锁的RFLP标记，用含有1024个个体的分离群体，对包台型恢复基因进行了精细定位，结果将Rf-1精细定位于S12564Tsp509I和C1361MwoI区间内，随后Komori等通过图位克隆的方法克隆了包台型细胞质雄性不育回复基因Rf-1(Komori et al.,2004)。华南农大刘耀光课题组在2006年发现包台型细胞质雄性不育恢复基因Rf-1位点包含两个恢复基因Rfla和Rflb，并对水稻CMS-BT细胞质雄性不育机理和育性恢复功能进行了研究(Wang et al.,2006)。
我国杂交稻育种虽然取得了巨大的进步，但也长期存在一些需要解决的问题，主要原因之一就是目前生产上所用的不育细胞质主要为野败型细胞质，而其育性的恢复需要Rf3和Rf4基因同时存在时才能达到生产上的要求，这就导致新的恢复系选育难道较大。另一方面，野败型细胞质雄性不育系的育性不够稳定，在高温天气时会出现自交结实，制种时出现假杂种，导致种子纯度不过关(席建民等，2011；葛小平等，2012)。许多优良的稻种资源无法作为野败型保持系亲本利用，造成亲本间遗传基础狭窄，大大限制了产量提高的潜力。王乃元等在研究水稻雄性不育性在异源胞质背景的遗传表达过程中，从普通野生稻中发现一种新型的雄性不育细胞质，命名为CMS-FA型细胞质，育成了系列新质源不育系，拓宽了优质米杂交稻的育种途径(王乃元，2006a)。在此研究基础上育成了新质源雄性不育恢复系，实现了新质源三系配套和利用(王乃元，2006b)。利用新型雄性不育细胞质源而培育的不育系具有广泛的保持系来源，不仅可以将保持系来源扩大到中稻、晚稻、优质稻等各个领域，而且打破了长江流域早 籼稻系统的遗传局限，大大提高了不育系育种潜力(王乃元等，2008a)。与保持系不同，常规品种中新型雄性不育恢复系资源有限，目前研究表明该恢复基因由1对显性基因控制(王乃元等，2008b)。基于此种考虑，通过对能够恢复新质源雄性不育系(CMS-FA)的恢复基因进行克隆，结合分子育种工具，可以快速有效地将恢复基因转移到优良的育种资源中去，从而大大拓宽恢复系的来源，充分利用杂种优势，有利于保障我国粮食安全。
发明内容
[根据细则91更正 13.07.2022]　
本申请的目的是从水稻中分离克隆一个细胞质雄性不育的育性恢复基因OsRf19，旨在通过对育性恢复基因OsRf19的研究和应用，在与不育系杂交配组方面改良水稻的恢复能力，为水稻遗传育种提供新的基因资源。
[根据细则91更正 13.07.2022]　
本申请利用图位克隆的方法分离克隆一个控制水稻新质源型细胞质雄性不育育性恢复的基因OsRf19，为水稻的三系配套育种提供新的基因资源。具体而言，本申请涉及以下技术方案。
[根据细则91更正 13.07.2022]　
在第一方面，本申请提供了一种水稻细胞质雄性不育恢复基因OsRf19，其包含SEQ ID NO：1所示的核苷酸序列。
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在第二方面，本申请提供了一种蛋白质，其由水稻细胞质雄性不育恢复基因OsRf19编码，所述蛋白质包含SEQ ID NO：4所示的氨基酸序列或者由SEQ ID NO：4所示的氨基酸序列组成。
在第三方面，本申请提供了一种核酸，其编码第二方面所述的蛋白质。
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在第四方面，本申请提供了一种表达载体，其包含第一方面所述的水稻细胞质雄性不育恢复基因OsRf19或第三方面所述的核酸。
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在第五方面，本申请提供了创制水稻恢复系的方法，其包括将第一方面所述的水稻细胞质雄性不育恢复基因OsRf19或第三方面所述的核酸或第四方面所述的表达载体引入水稻品种中。
在第六方面，本申请提供了使水稻细胞质雄性不育系恢复育性的方法，其包括：
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将第一方面所述的水稻细胞质雄性不育恢复基因OsRf19或第三方面所述的核酸或第四方面所述的表达载体引入水稻品种中以产生转基因恢复系；和
使所述转基因恢复系与所述水稻细胞质雄性不育系杂交，从而产生育性正常的杂交种子。
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在第七方面，本申请提供了第一方面所述的水稻细胞质雄性不育恢复基因OsRf19或第三方面所述的核酸在水稻育种或创制水稻恢复系或使水稻细胞质雄性不育系恢复育性中的用途。
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在第八方面，本申请提供了一种生产水稻杂交种种子的方法，其包括：以水稻细胞质雄性不育系为母本并以包含细胞质雄性不育恢复基因OsRf19的水稻恢复系为父本杂交，生产水稻杂交种种子，其中所述基因OsRf19包含SEQ ID NO：1所示的核苷酸序列或包含编码SEQ ID NO：4所示的氨基酸序列的核苷酸序列。
附图说明
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图1是本申请的OsRf19基因的图位克隆，图a为OsRf19在水稻第10染色体遗传连锁图上的位置；图b和c分别利用两个作图群体来精细定位OsRf19。标记间的数字代表各标记与OsRf19位点间发生的重组次数。
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图2是本申请的OsRf19转基因互补验证的载体图，即所用的功能性载体为pCAMBIA1300的图谱。
图3是本申请的T 1代转基因单株的花粉碘染镜检和结实率表型图。图a为分子标记对转基因植株的鉴定；图b为转基因阴性(左)和转基因阳性(右)的整个植株表型；图c为转基因阴性(左)和转基因阳性(右)的小穗结实率表型；图d为转基因阴性植株花粉碘染镜检图；图e为转基因阳性植株花粉镜检图。
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图4是本申请的OsRf19转基因阳性植物与新质源不育系新质1A杂交后代表型。图a为不育系新质1A(左)和转基因阳性植物与新质源不育系杂交F 1植株(右)的花粉碘染表型；图b为不育系新质1A(左)和转基因阳性植物与新质源不育系杂交F 1植株(右)的小穗。
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图5是本申请的OsRf19基因表达特征的RT-PCR检测图。上面为目的基因OsRf19在各组织中的表达；下面为内参基因Actin1在各组织中的表达。
发明详述
定义
如本文所用，“水稻”是任何水稻植株并包括可以与水稻育种的所有植物品种。如本文所用，“植株”或“植物”，包括整株植物、植物细胞、植物器官、植物原生质体、植物可以从中再生的植物细胞组织培养物、植物愈伤组织、植物丛和植物或植物部分中完整的植物细胞，所述植物部分例如胚、花粉、胚珠、种子、叶、花、枝、果实、茎杆、根、根尖、花药等。如本文所用的水稻，包括水稻、旱稻、籼稻、粳稻、早稻、晚稻、粘稻、糯稻、稻叶有毛稻和稻叶无毛(光身稻)稻。
如本文所用，术语“不育系”又称为“雄性不育系”或“细胞质雄性不育系”，是指花粉不育并能将该特征遗传给后代的水稻品系。
如本文所用，术语“新质源不育系(CMS-FA)”是指从福建野生稻(O.rufipogon)研发成功且带有福建野生稻雄性不育细胞质的雄性不育系，即具有特定CMS-FA细胞质背景的不育系，简称CMS-FA或新质源不育系。因该细胞质的保持系资源利用达到55.5％，比野败型(CMS-WA)保持系资源利用20％更加广泛，因而又被称为广保型细胞质雄性不育系，广保型雄性不育系或广保型不育系，例如，请参见中国发明专利文献CN1954666B。
如本文所用，术语“单核苷酸多态性”或者“SNP”或者“SNP标记”或者“SNP位点”是指存在于染色体的基因组序列中的核苷酸序列，基于核苷酸序列的差异(单个核苷酸——A、T、C或G的改变)而引起的多核苷酸序列变化，造成染色体基因组的多样性，进而允许不同等位基因(例如来自两个不同个体的等位基因)或不同个体彼此相区分。该变化可能发生在基因的编码区或非编码区(例如启动子区或其附 近，或者内含子)内或者基因间区域中。
如本文所用，术语“InDel”是指插入或缺失，其具体是指全基因组中的差异，相对标准对照而言，个体的基因组中有一定数量的核苷酸插入或缺失(Jander et al.,2002)。
如本文所用，术语“SSR(Simple Sequence Repeats)”，也称为微卫星DNA(Microsatellite DNA)，是一类由几个核苷酸(一般为1～6个)为重复单位组成的长达几十个核苷酸的串联重复序列。每个SSR两侧的序列一般是相对保守的单拷贝序列。
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如本文所用，术语“与OsRf19同源的基因”是指与水稻OsRf19来源于相同物种或不同物种，并且具有类似功能的基因。
本文使用的术语“基因编辑(gene editing)”或“基因组编辑(genome edting)”，是一种新兴的比较精确的能对生物体基因组特定目标基因进行修饰的一种基因工程技术。基因编辑指能够对目标基因进行定点“编辑”，实现对特定DNA片段的修饰。基因编辑依赖于经过基因工程改造的核酸酶，也称“分子剪刀”，在基因组中特定位置产生位点特异性双链断裂(DSB)，诱导生物体通过非同源末端连接(NHEJ)或同源重组(HR)来修复DSB，因为这个修复过程容易出错，从而导致靶向突变。
本文使用的术语“CRISPR/Cas9”是指使用RNA引导链靶向内切核酸酶切割位点的内切核酸酶。参见，Jinek等人，Science  337:816-821(2013)；Cong等人，Science(2013年1月3日)；和Mali等人，Science(2013年1月3日)。目前发现的CRISPR/Cas9系统有三种不同类型即I型、II型和III型，它们存在于大约40％已测序的真细菌和90％已测序的古细菌中。其中II型的组成较为简单，以Cas9蛋白以及向导RNA(gRNA)为核心组成，也是目前研究得最深入的类型。当细菌抵御噬菌体等外源DNA入侵时，在前导区的调控下，CRISPR被转录为长的RNA前体(pre-crRNA)，然后加工成一系列短的含有保守重复序列和间隔区的成熟crRNA，最终识别并结合到与其互补的外源DNA序列上发挥剪切作用。CRISPR/Cas9的剪切位点位于crRNA互补序列下游临近的PAM区(Protospacer Adjacent Motif)的5’-GG-N18-NGG-3’特征区域中的NGG位点，而这种特征的序列在每128bp的随机DNA序列中就重复出现一次。
本文使用的术语“CRISPR/Cas12a”是一类新型的CRISPR-Cas系统。相较于Cas9蛋白来说，Cas12a蛋白更具准确度，同时也更安全。CRISPR/Cas9工作时，Cas9蛋白识别PAM序列(RNA编写的遗传密码)，通过gRNA解开部分双螺旋，在这个过程中，Cas9蛋白一旦找到匹配较好的序列时，便会紧密贴合在该段DNA上，而在这个过程中，也许会出现一些错配的现象，但这种结合是不可逆的。而在这一方面，Cas12a就显得机智许多，它在寻找“靶标”时，会对沿途的DNA序列进行单碱基的识别，如若发现有匹配不好的碱基，便会离开重新寻找，在寻找到PAM序列时，Cas12a蛋白会与PAM序列形成一种半封闭的R-环(R-loop)，当识别到正确的序列时，才会完全结合成封闭的R-环，所以这种结合是可逆的，这也体现出了它更安全的一面。
本文使用的术语“转录激活子样效应器核苷酶”或“TAL效应器核苷酶”或“TALEN”是指一类通过使TAL效应器DNA结合结构域与DNA切割结构域融合而产生的人工限制内切核酸酶。
本文使用的术语“锌指核酸酶(ZFN)”由一个DNA识别域以及一个DNA剪切域组成。DNA识别域为3～4个ZF串联结构，每个ZF约含30个氨基酸，被1个锌离子所固定，可识别并结合1个特异的三联体碱基，DNA剪切域由非特异性核酸内切酶Fok I羧基端的96个氨基酸残基组成。每个Fok I单体与1个ZFP相连构成1个ZFN，并识别特定的位点，当2个识别位点相距恰当的距离时(6～8bp)，2个单体ZFN相互作用产生酶切功能，形成双链断裂，从而介导DNA定点剪切。
本文使用的术语“大范围核酸酶(meganuclease)”是指能够识别14-40碱基长度的核酸序列的归巢核酸内切酶。一些大范围核酸酶可以容忍小的归巢位点序列差异，大的识别区域仍然能够保证这些酶的高度特异性，而这反过来又可保持低水平的基因组内非特异性裂解和较低的毒性。大范围核酸酶由自我剪接RNA内含子或自我剪接蛋白内含子序列的移动序列内的开放阅读框架编码。
本文使用的术语“DddA衍生的胞嘧啶碱基编辑器”，是2020年，由美国哈佛大学博德研究所和麻省理工学院刘如谦领导的研究团队创建的一种新的碱基编辑器，可从线粒体中的DNA进行C到T转换。这是通过创造一种称为碱基编辑的新基因编辑技术来实现的，该技术将单个核苷酸碱基转化为另一个碱基而不会破坏DNA。但是，这种技术也有其局限性。它不仅仅限于C到T转换，而且主要限于TC基序，使其成为有效的TC-TT转换器。这意味着它只能纠正90个已确认的致病性线粒体点突变中的9个，也就是10％。长期以来，线粒体DNA的A到G转换被认为是不可能的。
本文使用的术语“类转录激活因子效应相关脱氨酶(TALED)”，是由韩国基础科学研究所(IBS)基因组工程中心研究人员开发的一种新的基因编辑平台。TALED是能够在线粒体中进行A到G碱基转换的碱基编辑器。这一发现是长达数十年治愈人类遗传疾病之旅的结晶。这种新碱基编辑器极大地扩展了线粒体基因组编辑的范围。这不仅可为建立疾病模型作出巨大贡献，还可为开发治疗方法作出巨大贡献。值得注意的是，其在人类mtDNA中能够进行A到G的转化可纠正90种已知致病性突变中的39种，约为43％。”研究人员通过融合三种不同的成分创造了TALED。第一个组分是转录激活子样效应子，它能够靶向DNA序列。第二个组分是TadA8e，一种用于促进A到G转化的腺嘌呤脱氨酶。第三个组分DddAtox，是一种使DNA更容易被TadA8e获取的胞嘧啶脱氨酶。
具体实施方案
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本申请提供了OsRf19基因的核苷酸序列及其编码的蛋白，其核苷酸序列如序列表SEQ ID NO:1、2和3所示，其中SEQ ID NO:1为一个没有内含子的2376个碱基的开放读码框(ORF)，SEQ ID NO:2为含有启动子调控元件和5’端非翻译区的序列， SEQ ID NO:3为3’端非翻译区序列。上述基因OsRf19的编码的蛋白质的序列如SEQ ID NO:4所示。该序列由791个氨基酸组成，具有由19个PPR重复单元(pentatricopeptide repeat)组成的功能结构域，其生物学功能为恢复植物细胞质雄性不育的育性。典型的PPR是由35个氨基酸残基构成的重复单元，在不同的含PPR蛋白中以2个以上的同向PPR单位形成功能结构域，不同的PPR单位的氨基酸序列存在不同程度的差异。
具体而言，本申请涉及如下技术方案。
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在第一方面，本申请提供了一种水稻细胞质雄性不育恢复基因OsRf19或其功能变体，所述恢复基因OsRf19包含SEQ ID NO：1所示的核苷酸序列。
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SEQ ID NO：1为一个没有内含子的2376个碱基的开放读码框。本领域技术人员能够通过常规方法确定和/或获得水稻细胞质雄性不育恢复基因OsRf19的功能变体。所述功能变体与原基因相比，可以具有一个或多个核苷酸的缺失、添加和/或取代，但仍然保留原基因的功能，例如仍编码相同功能的蛋白质。所述功能变体与SEQ ID NO：1所示的序列可以具有至少约70％,75％,80％,85％,86％,87％,88％,89％,90％,91％,92％,93％,94％,95％,96％,97％,98％,99％或更高的序列同一性并编码相同功能的蛋白质。因此，SEQ ID NO：1既包括SEQ ID NO：1所示的核苷酸序列本身，也包括SEQ ID NO：1所示的核苷酸序列的功能变体。
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本文所用的术语“功能变体”指基本上相似的序列。对于核苷酸序列而言，功能变体包括由于遗传密码子简并性而编码相同功能的蛋白质的那些序列。诸如天然存在的等位基因变体可以使用公知的分子生物学技术例如聚合酶链式反应(PCR)和杂交技术来鉴定。例如，在本申请中，与基因OsRf19具有至少约70％,75％,80％,85％,86％,87％,88％,89％,90％,91％,92％,93％,94％,95％,96％,97％,98％,99％或更高的同一性并且也编码功能性OsRf19蛋白的来自其他水稻品种的基因，包括在本申请定义的“功能变体”中。同一性的确定是通过本文描述的序列比对程序，所述程序使用默认参数。核苷酸的功能变体的序列与该核苷酸序列的差异可以少至1-15个核苷酸、少至1-10个(例如6-10个)，少至5个，少至4,3,2或甚至1个核苷酸。
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根据本申请提供的OsRf19基因序列信息，可以通过以下方法容易地获得与OsRf19同源的基因或OsRf19的功能变体：(a)通过与数据库比对，获得已经公开但功能未知的OsRf19的同源基因；(b)用OsRf19基因片段为探针筛选水稻基因组文库获得阳性克隆和测序；(c)根据SEQ ID NO:1序列信息设计寡核苷酸引物，用PCR方法从水稻或野生稻的基因组扩增获得OsRf19基因片段和测序；(d)在SEQ ID NO:1序列的基础上用分子生物学方法改造获得；或(e)参照SEQ ID NO:1序列用化学合成的方法获得。
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在一些实施方案中，所述水稻细胞质雄性不育恢复基因OsRf19由SEQ ID NO：1、2和3组成，其中SEQ ID NO:1为一个没有内含子的2376个碱基的开放读码框(ORF)，SEQ ID NO:2为含有启动子调控元件和5’端非翻译区的序列，SEQ ID NO:3 为3’端非翻译区序列。
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在优选的实施方案中，所述水稻细胞质雄性不育恢复基因OsRf19由SEQ ID NO：1组成。
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在第二方面，本申请提供了一种蛋白质或其功能变体，所述蛋白质由水稻细胞质雄性不育恢复基因OsRf19编码，并且所述蛋白质包含SEQ ID NO：4所示的氨基酸序列或者由SEQ ID NO：4所示的氨基酸序列组成。
本领域技术人员能够通过常规方法确定和/或获得所述功能变体。所述功能变体与原蛋白质相比，可以具有一个或多个氨基酸的缺失、添加和/或取代(例如保守性取代)，但仍然保留原蛋白质的功能，例如恢复水稻细胞质雄性不育系的育性的功能。所述功能变体与SEQ ID NO：4所示的序列可以具有至少约70％,75％,80％,85％,86％,87％,88％,89％,90％,91％,92％,93％,94％,95％,96％,97％,98％,99％或更高的序列同一性并仍然保留原蛋白质的功能。因此，SEQ ID NO：4既包括SEQ ID NO：4所示的氨基酸序列本身，也包括SEQ ID NO：4所示的氨基酸序列的功能变体。
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对于蛋白质序列，术语“功能变体”包括衍生自天然蛋白的多肽，所述衍生是通过缺失(所谓的截短)天然蛋白N末端和/或C末端的一个或多个氨基酸或将一个或多个氨基酸添加至所述天然蛋白的N末端和/或C末端；在天然蛋白的一个或多个位点缺失或添加一个或多个氨基酸；或者在天然蛋白的一个或多个位点取代一个或多个氨基酸。因而，就蛋白质而言，术语“功能变体”包括天然蛋白的生物活性片段，其包含保留天然蛋白生物学活性，例如具有OsRf19蛋白功能的足够数目的连续氨基酸残基。这样的功能相对天然蛋白可以是不同的或者是改良的，或者可以是不变的，只要保留了OsRf19蛋白功能。同一性的确定是通过本文描述的序列比对程序，所述程序使用默认参数。蛋白的活性变体序列与该蛋白的差异可以少至1-15个氨基酸残基、少至1-10个(例如6-10个)，少至5个，少至4,3,2或甚至1个氨基酸残基。
在具体的实施方案中，所述蛋白质由SEQ ID NO：4所示的氨基酸序列组成。
在第三方面，本申请提供了一种核酸，其编码第二方面所述的蛋白质。
在优选的实施方案中，所述核酸可以是适合在宿主细胞中表达的密码子优化的核酸。例如根据密码子的简并性，其仍然编码同样的蛋白质。根据所用宿主细胞进行密码子优化的方法是本领域技术人员公知的。
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在第四方面，本申请提供了一种表达载体，其包含第一方面所述的水稻细胞质雄性不育恢复基因OsRf19或第三方面所述的核酸。
可以使用任何合适的表达载体。例如，原核克隆载体包括来自大肠杆菌的质粒，如colEl、pCRl、pBR322、pMB9、pUC、pKSM和RP4。原核载体还包括噬菌体DNA如M13和其它丝状单链DNA噬菌体的衍生物。可用于酵母的载体的实例是2μ质粒。用于在哺乳动物细胞中表达的合适载体包括以下众所周知的衍生物：SV-40、腺病毒、逆转录病毒衍生的DNA序列以及衍生自功能性哺乳动物载体(如上述那些)和功能性质粒和噬菌体DNA的组合的穿梭载体。
另外的真核表达载体为本领域已知的(例如，P J.Southern&P.Berg,J.Mol.Appl.Genet,1:327-341(1982)；Subramani等人,Mol.Cell.Biol,1:854-864(1981)；Kaufinann&Sharp,"Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene,"J.Mol.Biol,159:601-621(1982)；Kaufhiann&Sharp,Mol.Cell.Biol,159:601-664(1982)；Scahill等人,"Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells,"Proc.Nat'l Acad.Sci USA,80:4654-4659(1983)；Urlaub&Chasin,Proc.Nat'l Acad.Sci USA,77:4216-4220,(1980)，将其全部通过引用并入本文)。
可用于本发明的表达载体含有至少一个表达控制序列，其与待表达的DNA序列或片段可操作连接。将控制序列插入载体中以控制和调节克隆的DNA序列的表达。有用的表达控制序列的实例是lac系统，trp系统，tac系统，trc系统，噬菌体λ的主要操纵子和启动子区，fd外壳蛋白的控制区，酵母的糖酵解启动子，例如3-磷酸甘油酸激酶的启动子，酵母酸性磷酸酶的启动子，例如Pho5，酵母α-交配因子的启动子，以及来源于多瘤病毒、腺病毒、逆转录病毒和猿猴病毒的启动子，例如SV40的早期和晚期启动子和已知控制原核或真核细胞及其病毒或其组合的基因表达的其它序列。
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在第五方面，本申请提供了创制水稻恢复系的方法，其包括将第一方面所述的水稻细胞质雄性不育恢复基因OsRf19或第三方面所述的核酸或第四方面所述的表达载体引入水稻品种中。
在一些实施方案中，所述引入通过基因编辑进行。
在一些实施方案中，所述水稻品种选自水稻、旱稻、籼稻、粳稻、早稻、晚稻、粘稻、糯稻、稻叶有毛稻和稻叶无毛稻。
在一些实施方案中，所述基因编辑通过选自以下的一种或多种进行：CRISPR/Cas9、CRISPR/Cpf1、CRISPR/Cas12a、TALEN、大范围核酸酶、ZFN、DddA衍生的胞嘧啶碱基编辑器和TALED。优选地，所述基因编辑通过CRISPR/Cas9进行。优选地，所述基因编辑通过CRISPR/Cas9进行。
在第六方面，本申请提供了使水稻细胞质雄性不育系恢复育性的方法，其包括：
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将第一方面所述的水稻细胞质雄性不育恢复基因OsRf19或第三方面所述的核酸或第四方面所述的表达载体引入水稻品种中以产生转基因恢复系；和使所述转基因恢复系与所述水稻细胞质雄性不育系杂交，从而产生育性正常的杂交种子。
在一些实施方案中，所述水稻细胞质雄性不育系为新质源不育系。具体地，所述水稻细胞质雄性不育系为含有不育基因orf182的水稻不育系。
在具体的实施方案中，采用的水稻不育系是含有线粒体不育基因orf182的新质源不育系。含有线粒体不育基因orf182的新质源不育系为孢子体不育系，无花粉败育，败育彻底，育性极其稳定。线粒体不育基因orf182的基因序列如SEQ ID NO:9所示。
在一些实施方案中，所述引入通过基因编辑进行。
在一些实施方案中，所述水稻品种和所述水稻细胞质雄性不育系各自选自水稻、旱稻、籼稻、粳稻、早稻、晚稻、粘稻、糯稻、稻叶有毛稻和稻叶无毛稻。
在一些实施方案中，所述基因编辑通过选自以下的一种或多种进行：CRISPR/Cas9、CRISPR/Cpf1、CRISPR/Cas12a、TALEN、大范围核酸酶、ZFN、DddA衍生的胞嘧啶碱基编辑器和TALED。优选地，所述基因编辑通过CRISPR/Cas9进行。优选地，所述基因编辑通过CRISPR/Cas9进行。
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在第七方面，本申请提供了第一方面所述的水稻细胞质雄性不育恢复基因OsRf19或第三方面所述的核酸在水稻育种或创制水稻恢复系或使水稻细胞质雄性不育系恢复育性中的用途。
在一些实施方案中，所述水稻细胞质雄性不育系为新质源不育系。具体地，所述水稻细胞质雄性不育系为含有不育基因orf182的水稻不育系。
在具体的实施方案中，采用的水稻不育系是含有线粒体不育基因orf182的新质源不育系。具体地，所述不育基因orf182包含SEQ ID NO：9所示的核苷酸序列。
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本申请所述OsRf19相似基因，是定义为与SEQ ID NO:1的核苷酸序列相比有一个或若干个核苷酸的差别，包括若干个碱基的改变、缺失、或插入，且其表达产物的功能与OsRf19表达产物的功能相当的基因。
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在第八方面，本申请提供了一种生产水稻杂交种种子的方法，其包括：以水稻细胞质雄性不育系为母本并以包含细胞质雄性不育恢复基因OsRf19的水稻恢复系为父本杂交，生产水稻杂交种种子，其中所述基因OsRf19包含SEQ ID NO：1所示的核苷酸序列或包含编码SEQ ID NO：4所示的氨基酸序列的核苷酸序列。
在一些实施方案中，所述水稻细胞质雄性不育系为新质源不育系。具体地，所述水稻细胞质雄性不育系为含有不育基因orf182的水稻不育系。
在具体的实施方案中，采用的水稻不育系是含有线粒体不育基因orf182的新质源不育系。具体地，所述不育基因orf182包含SEQ ID NO：9所示的核苷酸序列。
在一些实施方案中，所述水稻恢复系和所述水稻细胞质雄性不育系各自选自水稻、旱稻、籼稻、粳稻、早稻、晚稻、粘稻、糯稻、稻叶有毛稻和稻叶无毛稻。本说明书和权利要求书中，词语“包括”、“包含”和“含有”意指“包括但不限于”，且并非意图排除其他部分、添加物、组分、或步骤。
应该理解，在本申请的特定方面、实施方案或实施例中描述的特征、特性、组分或步骤，可适用于本文所描述的任何其他的方面、实施方案或实施例，除非与之矛盾。
实施例
以下实施例仅用于说明而非限制本申请范围的目的。
本申请中所使用的水稻植株材料信息可参见中国水稻品种及其系谱数据库(http://www.ricedata.cn/variety/index.htm)。
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实施例1：水稻新质源雄性不育恢复基因OsRf19(也称为RFFA)的初步定位
研究表明，新质源雄性不育系(CMS-FA)与恢复系杂交F 1代正常可育,育性恢 复(可育)基因为显性遗传。F 2[根据细则91更正 13.07.2022]　
代分离出可育：不育适合3:1，育性恢复(可育)基因为1对显性基因控制(王乃元等，2008b)。三系杂交水稻金农2优3号(参见中国水稻品种及其系谱数据库网址：https://www.ricedata.cn/variety/varis/605314.htm)的母本为雄性不育系金农2A(参见中国水稻品种及其系谱数据库网址：https://www.ricedata.cn/variety/varis/607151.htm)，父本为恢复系金恢3号(参见中国水稻品种及其系谱数据库网址：https://www.ricedata.cn/variety/varis/609760.htm)。金农2A为新质源雄性不育系(CMS-FA)，金恢3号为CMS-FA恢复系，含有水稻CMS-FA恢复基因，命名为OsRf19。为了克隆该基因，发明人在海南三亚基地种植金农2优3号自交后代(F 2代)群体约2000株，对其中94株的育性进行考察发现，可育株和不育株分别为74株和20株，符合3:1(x 2＝0.695,P＝0.405)，表明确实为单基因显性遗传。从2000株的群体中随机取10株可育株(可育池)和20株不育株(不育池)叶片分别等量混合抽提基因组DNA，用RICE6K水稻全基因组育种芯片(CN102747138A)进行混合分组分析(BSA，Bulked Segregant Analysis)，结果发现可育池和不育池的基因型主要差异为水稻第10号染色体(Chr10)上18.1-19.9Mb的大约1800kb区域，包含已克隆的CMS恢复基因Rf1a和Rf1b(Wang et al,2006)。在该区域，可育池的多态性SNP位点基因型为杂合，而不育池的基因型为纯合，表明可育为显性。
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实施例2：水稻新质源雄性不育恢复基因OsRf19的精细定位
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为了开发和利用更多的分子标记精细定位OsRf19基因，利用公共数据库中SSR标记(http://www.gramene.org/)，同时利用Illumina新一代测序技术对两亲本金农2A和金恢3号进行全基因组测序(http://www.illumina.com/)，通过序列比对获得SSR、InDel和SNP标记。定位所用的材料为初步定位种植金农2优3号的2096株F 2群体及其自交后代F 3和F 4群体，采用图位克隆的方法，将基因定位于两个分子标记Rf1D6(Chr10：18.828Mb)和Rf1D7(Chr10：18.894Mb)之间约66kb区域(水稻TIGR/MSU注释6.1版，http://rice.plantbiology.msu.edu/)，包含Rf1a，不包含Rf1b(图1)。随后利用金恢3号和华占(参见中国水稻品种及其系谱数据库网址：https://www.ricedata.cn/variety/varis/607962.htm)杂交后以华占为轮回亲本得到的BC 1F 2和BC 2F 2代群体共4059个单株进一步定位恢复基因，将基因定位于分子标记Rf1D3(18.874Mb)和TMRf1M10(18.890Mb)之间约16Kb的区域(水稻TIGR/MSU注释6.1版，http://rice.plantbiology.msu.edu/)。
实施例3：水稻金恢3号全基因组BAC文库的构建和目的BAC的测序
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为了获得定位区间更精确的信息，我们构建了水稻恢复系品种金恢3号的全基因组BAC文库。BAC文库总计41472个克隆，平均插入片段大小约为114kb，覆盖水稻基因组约10.5倍。利用定位区间的连锁标记在BAC文库中共筛选到两个包含目标区间片段的BAC 71-N-20和90-J-22。随后对筛选出的两个BAC进行了测序和分析，在定位标记Rf1D3和TMRf1M10之间预测得到4个候选基因ORF1，ORF2，ORF3和ORF4(图1)。由于ORF2，ORF3和ORF4的转基因互补没有表型，因此，克隆ORF1是OsRf19的唯一 可靠候选基因。
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SEQ ID NO:1是分离克隆的来源于水稻金恢3号的OsRf19基因的没有内含子的2376个碱基的开放读码框(ORF)。
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SEQ ID NO:2为OsRf19基因的含有启动子调控元件和5’端非翻译区的序列。
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SEQ ID NO:3为OsRf19基因的3’端非翻译区序列。
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SEQ ID NO:4是分离克隆的来源于水稻金恢3号的OsRf19基因的氨基酸序列。
实施例4：水稻新质源不育系93-11A的创制
为了获得一个稳定的不育系材料用于后续的转基因互补试验，我们以金农2A为母本，以水稻籼稻品种93-11(参见中国水稻品种及其系谱数据库网址：https://www.ricedata.cn/variety/varis/600611.htm)为父本进行杂交，得到F 1杂交种。然后种下杂交种，继续以品种93-11为轮回父本与杂交种连续回交7代后得到BC 7F 1植株。通过回交后得到了核基因组DNA与品种93-11基本一致而保留了金农2A细胞质的材料，我们把这个水稻材料称为水稻新质93-11A。在水稻开花和成熟时分别对93-11和新质93-11A进行表型考察，发现93-11花粉碘染可育，而新质93-11A花粉碘染败育；93-11在成熟时结实正常，而新质93-11A成熟时不能正常结实，结实率为0。通过上面方法我们得到了一个稳定的不育系材料新质93-11A。
本实施例中得到的水稻不育系新质93-11A(其含有不育基因orf182)的种子于2021年7月28日送交中国.武汉.武汉大学中国典型培养物保藏中心保藏，保藏信息如下：
保藏单位：中国典型培养物保藏中心(CCTCC)
保藏单位地址：中国湖北省武汉市武汉大学
保藏日期：2021年7月28日
培养物名称(分类命名)：水稻种子新质93-11A
保藏编号：CCTCC NO：P202115。
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实施例5：OsRf19的转基因互补试验
根据预测的候选基因全长序列，设计一对带有限制性核酸内切酶BamHI和PstI接头的PCR扩增寡核苷酸引物，其引物序列如SEQ ID NO:5(下划线为限制性内切酶BamHI切点)和SEQ ID NO:6(下划线为限制性内切酶PstI切点)所示。
SEQ ID NO:5：
5’-GAGCTCGGTACCCGG GGATCCTCGGTCCCGTATTTTGAATC-3’
SEQ ID NO:6：
5’-GCCAAGCTTGCATGC CTGCAGTAGAAGAGCAGCTGCACCAA-3’
用PCR技术从金恢3号的亚克隆中将含有启动子、编码区和下游终止序列的长度为5071bp片段扩增出来。PCR反应体系如下：在50μl反应中含有1x反应缓冲液，200μM的dNTPs，100ng亚克隆DNA，引物各0.3μM，KOD FX聚合酶1.0U。反应程序，步骤一：95℃5min；步骤二：95℃20S，55℃30S，72℃5min(30个循环)； 步骤三：72℃7min；步骤四：25℃1min。反应结束后把PCR产物进行纯化。用限制性内切酶BamHI和PstI对载体pCAMBIA1300(CAMBIA公司，澳大利亚堪培拉，图2)进行切割后，将同样经过限制性内切酶BamHI和PstI切割后的PCR产物连接到载体上。挑选出无突变的正确克隆载体导入农杆菌EHA105中。将水稻新质源不育系新质93-11A的成熟种子在诱导培养基上诱导出愈伤组织。把含有目的基因转化载体的EHA105侵染新质93-11A的愈伤组织，经过共培养、筛选具有潮霉素抗性的愈伤、分化、生根、炼苗移栽，得到转基因的水稻小植株。
上述遗传转化的步骤、培养基及其配制方法如下所述：
(1)试剂和溶液缩写
培养基所用到的试剂和植物激素的缩写名称如下：6-BA(6-BenzylaminoPurine，6-苄基腺嘌呤)；CN(Carbenicillin，羧苄青霉素)；KT(Kinetin，激动素)；NAA(Napthalene acetic acid，萘乙酸)；IAA(Indole-3-acetic acid，吲哚乙酸)；2,4-D(2,4-Dichlorophenoxyacetic acid，2,4-二氯苯氧乙酸)；AS(Acetosringone，乙酰丁香酮)；CH(Casein Enzymatic Hydrolysate，水解酪蛋白)；HN(Hygromycin B，潮霉素)；DMSO(Dimethyl Sulfoxide，二甲基亚砜)；N6 max(N6大量元素成分溶液)；N6 mix(N6微量元素成分溶液)；MS max(MS大量元素成分溶液)；MS mix(MS微量元素成分溶液)
(2)溶液配方
1)N6 max培养基大量元素母液(按照10倍浓缩液(10X)配制)：
Figure PCTCN2022100423-appb-000001
将上述试剂逐一溶解，然后室温下用蒸馏水定容至1000毫升，室温储存。
2)N6 min培养基微量元素母液(按照100倍浓缩液(100X)配制)：
Figure PCTCN2022100423-appb-000002
将上述试剂在室温下溶解并用蒸馏水定容至1000毫升，室温储存。
3)铁盐(Fe 2+EDTA)贮存液(按照100X浓缩液配制)：
将3.73克乙二铵四乙酸二钠(Na 2EDTA·2H 2O)和2.78克FeSO 4·7H 2O分别溶解，混合并用蒸馏水定容至1000毫升，至70℃温浴2小时，4℃保存备用。
4)维生素贮存液(按照100X浓缩液配制)：
Figure PCTCN2022100423-appb-000003
加蒸馏水定容至1000毫升，4℃保存备用。
5)MS培养基大量元素母液(MS max母液)(按照10X浓缩液配制)：
Figure PCTCN2022100423-appb-000004
将上述试剂在室温下溶解，并用蒸馏水定容至1000毫升，室温储存。
6)MS培养基微量元素母液(MS min母液)(按照100X浓缩液配制)：
Figure PCTCN2022100423-appb-000005
将上述试剂在室温下溶解，并用蒸馏水定容至1000毫升，室温储存。
7)2,4-D贮存液(1毫克/毫升)的配制：
称取2,4-D 100毫克，用1毫升1N氢氧化钾溶解5分钟，然后加10毫升蒸馏水溶解完全后定容至100毫升，于室温下保存。
8)6-BA贮存液(1毫克/毫升)的配制：
称取6-BA 100毫克，用1毫升1N氢氧化钾溶解5分钟，然后加10毫升蒸馏水溶解完全后定容至100毫升，室温保存。
9)NAA贮存液(1毫克/毫升)的配制：
称取NAA 100毫克，用1毫升1N氢氧化钾溶解5分钟，然后加10毫升蒸馏水溶解完全后定容至100毫升，4℃避光保存。
10)IAA贮存液(1毫克/毫升)的配制：
秤取IAA 100毫克，用1毫升1N氢氧化钾溶解5分钟，然后加10毫升蒸馏水溶解完全后定容至100毫升，4℃避光保存。
11)葡萄糖贮存液(0.5克/毫升)的配制：
秤取葡萄糖125克，然后用蒸馏水溶解定容至250毫升，灭菌后4℃保存备用。
12)AS贮存液的配制：
秤取AS 0.392克，加入DMSO 10毫升溶解，分装至1.5毫升离心管内，-20℃保存备用。
13)1N氢氧化钾贮存液配制：
秤取氢氧化钾5.6克，用蒸馏水溶解定容至100毫升，室温保存备用。
(3)用于水稻遗传转化的培养基配方：
1)诱导培养基
Figure PCTCN2022100423-appb-000006
加蒸馏水至900毫升，1N氢氧化钾调节pH值到5.9，煮沸并定容至1000毫升，分装到50毫升三角瓶(30毫升/瓶)，封口后按常规方法灭菌(例如121℃下灭菌15分钟，下述的培养基灭菌方法与本培养基的灭菌方法相同)。
2)继代培养基：
Figure PCTCN2022100423-appb-000007
加蒸馏水至900毫升，1N氢氧化钾调节pH值到5.9，煮沸并定容至1000毫升，分装到50毫升三角瓶(30毫升/瓶)，封口，按上述方法灭菌。
3)预培养基(粳稻可以不做这一步)：
Figure PCTCN2022100423-appb-000008
加蒸馏水至250毫升，1N氢氧化钾调节pH值到5.6，封口，按上述方法灭菌。
使用前加热溶解培养基并加入5毫升葡萄糖贮存液和250微升AS贮存液，分装倒入培养皿中(25毫升/皿)。
4)悬浮培养基：
Figure PCTCN2022100423-appb-000009
加蒸馏水至100毫升，调节pH值到5.4，分装到两个100毫升的三角瓶中，封口，按上述方法灭菌。
使用前加入1毫升无菌葡萄糖贮存液和100微升AS贮存液。
5)共培养基：
Figure PCTCN2022100423-appb-000010
Figure PCTCN2022100423-appb-000011
加蒸馏水至250毫升，1N氢氧化钾调节pH值到5.6，封口，按上述方法灭菌。
使用前加热溶解培养基并加入5毫升葡萄糖贮存液和250微升AS贮存液，分装倒入培养皿中(25毫升/每皿)。
6)筛选培养基：
Figure PCTCN2022100423-appb-000012
加蒸馏水至250毫升，调节pH值到6.0，封口，按上述方法灭菌。
使用前溶解培养基，加入250微升HN(50毫克/毫升)和400微升CN(10克CN/36毫升水)分装倒入培养皿中(25毫升/皿)。(注：第一次筛选培养基羧苄青霉素浓度为400毫克/升，第二次及以后筛选培养基羧苄青霉素浓度为250毫克/升)。
7)预分化培养基(粳稻可以不做这一步)：
Figure PCTCN2022100423-appb-000013
加蒸馏水至250毫升，1N氢氧化钾调节pH值到5.9，封口，按上述方法灭菌。
使用前溶解培养基，250微升HN(50毫克/毫升)250微升CN(250毫克/毫升)，分装倒入培养皿中(25毫升/皿)。
8)分化培养基：
Figure PCTCN2022100423-appb-000014
加蒸馏水至900毫升，1N氢氧化钾调节pH值到6.0。
煮沸并用蒸馏水定容至1000毫升，分装到100毫升三角瓶(50毫升/瓶)，封口，按上述方法灭菌。
9)生根培养基
Figure PCTCN2022100423-appb-000015
加蒸馏水至900毫升，用1N氢氧化钾调节pH值到5.8。
煮沸并用蒸馏水定容至1000毫升，分装到生根管中(25毫升/管)，封口，按上述方法灭菌。
(4)农杆菌介导的遗传转化步骤：
4.1愈伤诱导
1)将成熟的中花11水稻种子去壳，然后依次用70％的乙醇处理1分钟，0.15％氯化汞(HgCl 2)种子表面消毒15分钟；
2)用灭菌水洗种子4-5次；
3)将8-10粒种子放在诱导培养基上；
4)将接种后的培养基置于黑暗处培养4-5周，温度26±1℃。
4.2愈伤继代：
挑选亮黄色、紧实且相对干燥的胚性愈伤，放于继代培养基上黑暗下培养2周，温度25±1℃。
4.3预培养：
挑选紧实且相对干燥的胚性愈伤，放于预培养基上黑暗下培养2周，温度26±1℃。
4.4农杆菌培养：
1)在带有对应抗性选择的LA培养基(LA培养基的配制参照J.萨姆布鲁克等，分子克隆实验指南，第三版，金冬雁等(译)，科学出版社，2002，北京)上划线预培养农杆菌EHA105(该菌株来自CAMBIA公司公开使用的农杆菌菌株)两天，温度28℃；
2)将农杆菌转移至悬浮培养基里，28℃摇床上培养2-3小时。
4.5农杆菌侵染：
1)将预培养的愈伤转移至灭好菌的瓶子内；
2)调节农杆菌的悬浮液至OD600为0.8-1.0；
3)将愈伤在农杆菌悬浮液中浸泡30分钟；
4)转移愈伤至灭菌好的滤纸上吸干；然后放置在共培养基上培养3天，温度19-20℃。
4.6愈伤洗涤和选择培养：
1)灭菌水洗涤愈伤至看不见农杆菌；
2)浸泡在含400毫克/L羧苄青霉素(CN)的灭菌水中摇30分钟；
3)转移愈伤至灭菌好的滤纸上吸干；
4)转移愈伤至筛选培养基上选择培养2-3次，每次2周至长出好的抗性愈伤。
4.7分化：
1)将抗性愈伤转移至预分化培养基上于黑暗处培养5-7天；
2)转移预分化培养的愈伤至分化培养基上，每瓶平均分布三个独立的愈伤，光照下培养5周-6周至长出大的苗子，温度26℃。
4.8生根与炼苗：
1)剪掉分化时产生的老根；
2)将其转移至生根培养基中光照下培养2-3周至长出大的苗子后，去掉封口膜加部分自来水炼苗一周再移栽，温度26℃。
4.9移栽
洗掉根上的残留培养基，将具有良好根系的幼苗转入温室，同时在最初的几天保持水分湿润，等长势良好再移栽至大田。
[根据细则91更正 13.07.2022]　
本次实验共获得独立转基因互补T 0代水稻植株14株，包括7株阳性单株和7株阴性单株。将阳性植株种植在大田中，待水稻抽穗开花时取花粉用碘化钾染色镜检，T 0代植株的可育花粉率达到80％以上，且自交小穗结实率达到60％以上；而对照不育系的可育花粉率为零，自交不结实。收获T 0代植株的种子后将其种植在田间继续观察T 1代表型。在成熟期进行表型考察，结果如图3所示，T 1代的群体中出现了可育和不育的分离，可育单株的结实率都在70％以上，而不育单株的结实率为零。本实验的结果说明，将恢复基因OsRf19转化导入到原来不含有功能型OsRf18的不育品种，可创制新的恢复系。此实验也证明了该基因的生物学功能为恢复细胞质雄性不育的育性。本申请将这个从水稻恢复系基因组的OsRf19座位克隆获得的基因确认为目的基因，即细胞质雄性不育恢复基因OsRf19。
[根据细则91更正 13.07.2022]　
我们将转OsRf19基因的T 1代阳性植株与新质源不育系新质1A(参见中国水稻品种及其系谱数据库网址：https://www.ricedata.cn/variety/varis/621300.htm)进行杂交后得到F 1代植株。在武汉田间种植F 1代植株和不育系新质1A，待水稻抽穗开花时取花粉用碘化钾染色镜检，待水稻成熟后取小穗进行结实率考察。结果如图4所示，不育系新质1A的花粉用碘化钾染色后为不育，结实率为零；F 1代植株花粉用碘化钾染色后为可育，结实率在80％以上。此实验也证明了转入恢复基因OsRf19后创制的恢复系能够恢复新质源不育系的育性。
[根据细则91更正 13.07.2022]　
实施例6：OsRf19的表达分析
用RT-PCR对该基因的表达进行了分析。RT-PCR使用的引物序列如以下的SEQ ID NO:7和SEQ ID NO:8所示。
SEQ ID NO:7：5’-GATGTACTTTGCAAGTCAGG-3’
SEQ ID NO:8：5’-CCTTCTTTGCAAAGATTGCT-3’
[根据细则91更正 13.07.2022]　
RT-PCR分析结果如图5所示。图5中的结果表明，金恢3号中的OsRf19基因在根、茎、叶和幼穗等各种组织中均有表达，这种表达即组成型表达，因此该基因属于组成型表达的基因。
[根据细则91更正 13.07.2022]　
实施例7：含有恢复基因OsRf19的恢复系对不育系育性的恢复
[根据细则91更正 13.07.2022]　
本实施例采用水稻恢复系金恢3号(是审定品种金农3优3号的亲本，可以在中国种子集团有限公司获取)和华占-RFFA(二者均含有恢复基因OsRf19)以及水稻不育系金农3A(是审定品种金农3优3号的亲本，可以在中国种子集团有限公司获取)和新质1A(品种权号：CNA20162267.2)(二者均含有不育基因orf182)的配制组合来证明恢复基因OsRf19对含有不育基因orf182的不育系育性的恢复。
[根据细则91更正 13.07.2022]　
本实施例中采用的恢复系华占-RFFA是以金恢3号为供体亲本，以华占为受体亲本进行杂交，再经过4次回交后把恢复基因OsRf19导入进了受体亲本中而得到的。恢复系华占-RFFA的种子于2022年6月20日送交中国.武汉.武汉大学中国典型培养物保藏中心保藏，保藏信息如下：
保藏单位：中国典型培养物保藏中心(CCTCC)
保藏单位地址：中国湖北省武汉市武汉大学
保藏日期：2022年6月20日
培养物名称(分类命名)：水稻种子华占-RFFA
保藏编号：CCTCC NO：P202117。
为了证明恢复系的恢复能力，我们采用新质源恢复系华占-RFFA和金恢3号分别与新质源不育系金农3A和新质1A进行杂交。对杂交F1代进行结实率考察。结果显示恢复系华占-RFFA和金恢3号与金农3A和新质1A分别杂交后F1代结实率在79.6％—85.8％之间(表1)。
表1
Figure PCTCN2022100423-appb-000016
此外，本实施例还以现有的野败型恢复系明恢63和桂99为父本分别与金农3A和新质1A进行杂交得到F1代杂交种。然后在田间种植4个组合的杂交种进行结实率考察，结果显示明恢63和桂99分别与金农3A和新质1A杂交后代结实率为0。通过本实验证明野败型恢复系对新质源不育系不具有恢复能力。

Claims (13)

  1. [根据细则91更正 13.07.2022]　
    一种水稻细胞质雄性不育恢复基因OsRf19，其包含SEQ ID NO：1所示的核苷酸序列。
  2. [根据细则91更正 13.07.2022]　
    如权利要求1所述的细胞质雄性不育恢复基因OsRf19，其由SEQ ID NO：1、2和3所示的核苷酸序列组成或者由SEQ ID NO：1所示的核苷酸序列组成。
  3. [根据细则91更正 13.07.2022]　
    一种蛋白质，其由水稻细胞质雄性不育恢复基因OsRf19编码，所述蛋白质包含SEQ ID NO：4所示的氨基酸序列或者由SEQ ID NO：4所示的氨基酸序列组成。
  4. 一种核酸，其编码权利要求3所述的蛋白质。
  5. [根据细则91更正 13.07.2022]　
    一种表达载体，其包含权利要求1或2所述的水稻细胞质雄性不育恢复基因OsRf19或权利要求4所述的核酸。
  6. [根据细则91更正 13.07.2022]　
    创制水稻恢复系的方法，其包括将权利要求1或2所述的水稻细胞质雄性不育恢复基因OsRf19或权利要求4所述的核酸或权利要求5所述的表达载体引入水稻品种中。
  7. 如权利要求6所述的方法，所述水稻品种选自水稻、旱稻、籼稻、粳稻、早稻、晚稻、粘稻、糯稻、稻叶有毛稻和稻叶无毛稻，任选地，所述引入通过基因编辑进行，任选地，所述基因编辑通过选自以下的一种或多种进行：CRISPR/Cas9、CRISPR/Cas12a、TALEN、大范围核酸酶、ZFN、DddA衍生的胞嘧啶碱基编辑器和TALED。
  8. [根据细则91更正 13.07.2022]　
    使水稻细胞质雄性不育系恢复育性的方法，其包括：
    将权利要求1或2所述的水稻细胞质雄性不育恢复基因OsRf19或权利要求4所述的核酸或权利要求5所述的表达载体引入水稻品种中以产生转基因恢复系；和
    使所述转基因恢复系与所述水稻细胞质雄性不育系杂交，从而产生育性正常的杂交种子。
  9. [根据细则91更正 13.07.2022]　
    一种生产水稻杂交种种子的方法，其包括：以水稻细胞质雄性不育系为母本并以包含细胞质雄性不育恢复基因OsRf19的水稻恢复系为父本杂交，生产水稻杂交种种子，
    其中所述基因OsRf19包含SEQ ID NO：1所示的核苷酸序列或包含编码SEQ ID NO：4所示的氨基酸序列的核苷酸序列。
  10. 如权利要求8或9所述的方法，其中所述水稻品种、所述水稻细胞质雄性不育系和所述水稻恢复系各自选自水稻、旱稻、籼稻、粳稻、早稻、晚稻、粘稻、糯稻、稻叶有毛稻和稻叶无毛稻，任选地，所述引入通过基因编辑进行，任选地，所述基因编辑通过选自以下的一种或多种进行：CRISPR/Cas9、CRISPR/Cas12a、TALEN、大范围核酸酶、ZFN、DddA衍生的胞嘧啶碱基编辑器和TALED。
  11. 如权利要求8或9所述的方法，其中所述水稻细胞质雄性不育系为新质源不育系，任选地，所述新质源不育系为含有不育基因orf182的水稻不育系。
  12. 如权利要求11的方法，其中所述不育基因orf182包含SEQ ID NO：9所示 的核苷酸序列。
  13. [根据细则91更正 13.07.2022]　
    权利要求1或2所述的水稻细胞质雄性不育恢复基因OsRf19或权利要求4所述的核酸在水稻育种或创制水稻恢复系或使水稻细胞质雄性不育系恢复育性中的用途，任选地，其中所述水稻细胞质雄性不育系为新质源不育系，任选地，所述水稻细胞质雄性不育系为含有不育基因orf182的水稻不育系，任选地，所述不育基因orf182包含SEQ ID NO：9所示的核苷酸序列。
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