WO2014187312A1 - 植物新型保持系及不育系建立及其用途 - Google Patents
植物新型保持系及不育系建立及其用途 Download PDFInfo
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- WO2014187312A1 WO2014187312A1 PCT/CN2014/077967 CN2014077967W WO2014187312A1 WO 2014187312 A1 WO2014187312 A1 WO 2014187312A1 CN 2014077967 W CN2014077967 W CN 2014077967W WO 2014187312 A1 WO2014187312 A1 WO 2014187312A1
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Classifications
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- 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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
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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
- A01H1/022—Genic fertility modification, e.g. apomixis
- A01H1/023—Male sterility
Definitions
- the invention relates to the field of plant molecular biology and breeding.
- embodiments of the invention relate to transgenic plants containing seed production techniques.
- the present invention relates to fertility restoration of homozygous recessive nuclear male sterility plants and uses thereof.
- the present invention relates to a method for constructing a plant maintainer line and a breeding male sterile line, and more particularly, the present invention relates to a construct, a plant cell, tissue or organ, a method for breeding a plant male sterile line, A method for restoring male fertility of a plant-sterile plant, a method for preparing a plant seed, a method for preparing a hybrid plant, and a use of a plant male-sterile line for preparing a hybrid rice.
- Crossbreeding is the main way to breed new varieties, and it is the most important method for modern breeding.
- the creation, utilization and industrialization of hybrids are the focus of competition in the international agricultural multinational group seed market. Due to the genetic recombination caused by hybridization, the offspring will have excellent trait genotypes controlled by the combination of parents, produce additive effects, and use some genes to interact to form new individuals with super-parents.
- Crop crossbreeding has great potential for development and has become the main way to increase food production. In recent decades, heterosis has been widely used as a means of increasing crop yield, improving crop quality, and improving crop resistance to insects, disease, and stress. Breeding with heterosis has become the main breeding method for many crops.
- Effective control of crop self-pollination and fertilization is the key to obtaining high-purity hybrid F1 seeds and thus utilizing crop heterosis.
- the key issues that must be addressed in cross-breeding are: (1) obtaining available sterile lines: generally male infertility (controlled by cytoplasmic infertility or recessive nuclear sterility genes); (2) hybrid matching: infertility It can be combined with the corresponding male plants to produce hybrid progeny with excellent traits; (3) Breeding of sterile lines: Sterile lines can restore fertility under certain conditions to maintain them. Therefore, the breeding of crop male sterile lines is a key link in the utilization of heterosis.
- the three-line hybrid japonica rice is not strong, and the sterile cytoplasm is relatively single, and there is a potential danger of some devastating pest and disease outbreak.
- the "two-line” hybrid rice is not restricted by the relationship between restorer lines and maintainer lines, and the genetic diversity of the parents is significantly improved.
- the speed of breeding high-yield hybrid rice combinations is significantly accelerated, which promotes the research and production of super hybrid rice.
- the sterile lines used in the "two-line” crosses are mostly "light-temperature-sensitive" sterile lines, and their fertility is affected by temperature and light in the environment.
- Maize is the earliest to use heterosis, and hybrids are the most popular crops in the world. Maize is a male and female cross-crop crop with high reproductive coefficient. When the hybrid is produced, the parent and the female parent are planted in proportion in the isolation area. When the female tassel is barely exposed, the male parent pollen is freely pollinated and crossed.
- the utilization of maize hybridization and the application of hybrids in production have led to dramatic changes in corn production levels.
- there is a large difference in the genetic basis of parent breeding in the production of maize hybrids and thus affects the main breeding objectives such as high yield, stable yield, stress resistance, and early maturity.
- the mother-to-male work is cumbersome and incomplete, which in turn affects the yield and quality of the hybrids, and needs to be solved urgently.
- Heterosis is also used in the production of dicotyledonous plants such as tobacco and canola. Rape is the world's third largest oil crop, and China's total rapeseed production and area accounts for one-third of the world's total. It is the largest rapeseed producer, and China's rapeseed hybrid utilization is at the world's leading level.
- Crossbreeding commonly used in rapeseed is also based on cytoplasmic infertility and nuclear infertility. There are “three-line” and "two-line” cross breeding. Up to now, there are three main cytoplasmic male sterile lines in production.
- cytoplasmic male sterile line Porima cytoplasmic male sterile line, Shaanxi 2A CMS and radish cytoplasmic male sterile line OgU CMS.
- cytoplasmic male sterility has been widely used, it is still technically affected by restoration source limitation, fertility stability, and cytoplasmic simplification. Therefore, as in rice, the same germplasm resource utilization rate is low in the development of rapeseed hybrids, and the purity of hybrids is low.
- the invention aims to solve the bottleneck problem of the above-mentioned plant cross breeding technology. To this end, it is an object of the present invention to provide a method for efficiently constructing a novel stable plant maintainer line and a male sterile line, thereby expanding the application of plant germplasm resources in cross breeding, and eliminating the process of hybrid seed production. Risk, improve the purity of hybrid seeds.
- the technology uses a recessive nuclear gene-controlled male sterile line and transgenic technology to link the fertility restorer gene, the pollen lethal gene, and the herbicide resistance gene to a homozygous recessive nuclear male sterile plant (sterile line).
- Fertility restorer gene The function restores the fertility of the transgenic plants; but to the later stages of pollen development, the pollen lethal gene inactivates the pollen-containing pollen, and only the pollen-retaining activity does not carry the transgene.
- the transgenic plants produce two types of seeds after selfing: non-transgenic sterile lines, no herbicide resistance, can be screened by herbicides; fertile seeds carrying transgenics, herbicide resistant, can pass herbicides The screening survives, that is, the maintainer line, which can be used for pollination of the sterile line with the sterile line.
- the method for breeding a sterile line comprising: (a) providing a first plant, the first plant being a sterile line, the sterile line containing a homozygous recessive allele Breeding the gene; (b) introducing into the second plant a construct comprising a homozygous recessive allelic sterility gene, the construct comprising: (i) a first nucleotide sequence a protein that functionally complements the homozygous recessive plant sterility of the plant when expressed in the first or second plant; ( ⁇ ) a second nucleotide sequence whose expression inhibits the second The formation or function of a male gamete in the plant such that the male gamete produced in the second plant containing the recessive allele does not contain the construct; (iii) a third nucleotide sequence, the sequence Expression of the encoded product can be used to select a plant cell having the construct; and (c) fertilizing the first plant with the male game
- the first nucleotide sequence is a fertility restoration gene selected from the group consisting of 0sFG2, BrMS2, MSP1, PAIR1, PAIR2, ZEP1, MELL, PSS1, TDR, UDT1, GAMYB4, PTC1, API5, WDA1, CYP704B2, MS26, A group consisting of MS22, DPW, MAD S3, 0SC6, RIP1, CSA or AID1.
- the first nucleotide sequence is operably linked to a fourth nucleotide sequence that directs expression to a male plant cell, or the fourth nucleotide sequence is only It has a function in the presence of an inducing substance or an inducing condition.
- the fourth nucleotide sequence is selected from the group consisting of 0sFG2, BrMS2, MSP1, PAIR1, PAIR2, ZEP1, MELL, PSS1, TDR, UDT1, GAMYB4, PTC1, API5, WDA1, CYP704B2, MS26, MS22, DPW A group consisting of male tissue regulatory sequences of MADS3, 0SC6, RIP1, CSA, A dish, 5126, Ms26, Ms22 or Ms45.
- the second nucleotide sequence is selected from the group consisting of a DAM methylase gene, a Zea mays alpha amylase gene, a cytotoxin encoding gene, or a combination of Barnase and Barstar.
- the second nucleotide sequence is operably linked to a fifth nucleotide sequence that directs expression of a male gamete.
- the fifth nucleotide sequence is selected from the group consisting of a polygalacturonase 47 gene, a Zml3 gene, a pectin methylesterase gene, a calmodulin-binding protein gene, an actin depolymerizing factor gene, A group consisting of the prolfi l in gene and the regulatory region of the sulfated pentapeptide phytosulphokine gene.
- the above third nucleotide sequence is specifically a mutant of ALS gene having herbicide resistance.
- the mutant of the ALS gene described in the rice is a Trp548, Ala96 and/or Ser627 mutation, preferably with a Trp548Cys, Trp548Met, Ala96Val, Ala96Thr and/or Ser627Asn mutation; wherein the ALS gene mutant is in a canola
- the mutations are Alal07, Alal90, Trp559 and/or Ser638 mutations.
- the third nucleotide sequence is operably linked to a sixth nucleotide sequence which is a constitutive expression promoter. More specifically, wherein the sixth nucleotide sequence is a 35s promoter.
- the present invention also provides a method for producing a seed from a plant having a female gamete and a male gamete, the method comprising: (a) introducing into the first plant the following construct, the first plant containing a pure A recessive allelic sterility gene, the construct comprising: (i) a first nucleotide sequence that, when expressed in the first plant, functionally complements the homozygous recessiveness of the plant a plant sterility trait; (ii) a second nucleotide sequence whose expression inhibits the formation or function of a male gamete in the first plant such that it is produced in the first plant containing the recessive allele
- the male gamete does not contain the construct; (iii) a third nucleotide sequence, the expression of which encodes the product can be used to select a plant cell having the construct; (b) autologous fertilization of the plant; (c) producing a seed comprising the construct; characterized in that the third nucleo
- Fertility restorer genes restore male fertile plants to fertility, pollen loss
- the live gene (BrSZl/Pr2 combination) inactivates the pollen-containing pollen, ie, loses fertility
- the herbicide resistance gene is used for the sorting of the transgenic seed, the maintainer line and the non-transgenic seed, ie the sterile line, wherein the non-transgenic seed is
- the herbicide is screened, and the transgenic seeds are used as a maintainer line, which is cross-pollinated with the sterile line and continuously breeds the sterile line.
- the invention provides a construct.
- the construct comprises: a first expression cassette comprising a rape male sterile restorer gene; a second expression cassette comprising a pollen lethal gene; and a third expression cassette comprising a herbicide resistance gene.
- the aforementioned construct is introduced into a canola by a conventional technique, such as Agrobacterium-mediated stencil method, to obtain a transgenic plant.
- a third aspect of the invention provides a method of breeding a rape male sterile line.
- the above-mentioned construct was transferred into a homogenous recessive male sterile plant of Brassica napus to obtain a transgenic rapeseed plant, and the transgenic rapeseed plant was able to produce a fertile male gamete which did not contain the transgene, and obtained a foreign gene by selfing. Seeds and do not carry foreign genes Seed. Seeds that do not carry foreign genes are not resistant to herbicides and can be screened by herbicides. Seed-tolerant herbicides carrying foreign genes can survive. As a maintainer, they are cross-pollinated with male sterile lines to breed sterile lines. .
- a fourth aspect of the invention provides a method of restoring fertility of a rapeseed sterile plant.
- the method comprises: introducing the construct described above into a canola homozygous recessive male sterile plant.
- a fifth aspect of the invention provides a method of preparing a canola seed.
- the method comprises the steps of: introducing the construct described above into a canola plant; self-fertilizing the canola plant to obtain a seed comprising the construct described above.
- a sixth aspect of the invention provides a transgenic plant, i.e., a transgenic rapeseed plant.
- the transgenic plant is obtained by introducing the aforementioned construct into a rapeseed homozygous recessive male sterile plant.
- the inventors use a rice recessive infertility mutant as a transforming receptor material, and transform the three functional genes closely linked to the above-mentioned sterile line: the fertility restoring gene restores the male sterile rice plant Fertility, pollen lethal gene Um-AAD inactivates pollen-containing pollen, ie, loses fertility; herbicide resistance gene is used for sorting of transgenic seeds, ie, maintainer lines and non-transgenic seeds, ie, sterile lines, where non-GM seeds Screened by herbicides, the transgenic seeds are used as a maintainer line, pollinated with the sterile line, and the source continuously produces the sterile line.
- the fertility restoring gene restores the male sterile rice plant Fertility
- pollen lethal gene Um-AAD inactivates pollen-containing pollen, ie, loses fertility
- herbicide resistance gene is used for sorting of transgenic seeds, ie, maintainer lines and non-transgenic seeds, ie
- the aforementioned construct is introduced into rice by conventional techniques, such as Agrobacterium-mediated transformation of immature embryos or mature embryos, to obtain transgenic plants.
- a method of breeding a male sterile line of rice is provided.
- the above-mentioned constructs were transferred into rice homozygous recessive male sterile plants to obtain transgenic rice plants, and transgenic rice plants were able to produce fertile male gametes without transgenes, and obtained foreign genes by selfing. Seeds and seeds that do not carry foreign genes. Seeds that do not carry foreign genes are not resistant to herbicides and can be screened by herbicides. Seed-tolerant herbicides carrying foreign genes can survive and serve as maintainer lines, which are cross-pollinated with male sterile lines for sterile lines. Reproduction.
- a method of restoring fertility of a rice sterile plant comprises: introducing the construct described above into a rice homozygous recessive male sterile plant.
- a method of preparing rice seeds comprises the steps of introducing the previously described construct into a rice plant; self-fertilizing the rice plant to obtain a seed comprising the construct described above.
- a twelfth aspect of the invention provides a transgenic plant, i.e., a transgenic rice plant.
- the transgenic plant is introduced into a rice homozygous recessive male sterile plant by introducing the aforementioned construct acquired.
- This new hybrid breeding technology has significant advantages: (1) The sterile line created by the technology is stable and unaffected by the environment, which removes the constraints of environmental factors on cross breeding and eliminates the potential risks in production; (2) The plant recessive nuclear sterility gene used is suitable for most species of the plant, so that the resource utilization of heterosis is greatly improved; (3) termination of fertilization process of transgenic pollen, preventing exogenous gene drift To other plants;
- the sterile lines produced do not contain genetically modified genes, eliminating people's concerns about genetically modified crops; (5) using herbicide genes to conveniently separate sterile seeds from fertile seeds, facilitating access to maintainer lines, and further breeding with sterile lines The pollination hybrid breeding of sterile seed ensures the purity of sterile seeds.
- the technology involves three linked functional elements: pollen lethal gene, herbicide resistance gene, and fertility restorer gene.
- the time, level and lethal efficiency of the selected pollen lethal gene in pollen are directly related to the successful application of the technology. Therefore, the present invention utilizes a pollen-specific promoter to drive the pollen inactivation gene to be specifically expressed during pollen maturation.
- the effect of specific pollen lethality is achieved; the male sterility plant controlled by the plant endogenous promoter is used to restore the corresponding plant male sterility plants, so that the transgenic plants can be restored to fertility; the transgenic plants can be conveniently used by using the herbicide resistance gene.
- the two types of seeds produced after selfing are separated: sterile seeds that do not carry transgenic, have no herbicide resistance, can be screened by herbicides, carry transgenic fertile seeds, have herbicide resistance, and can survive as a maintainer Therefore, the maintainer system can be conveniently and continuously obtained through self-crossing; the maintainer system further pollinates with the sterile line to restore the sterile line, and the sterile line that does not contain the transgene is obtained, so that the sterile line is propagated; The sterile line thus obtained can be crossed with any parent and used for hybrid seed. produce.
- the novel fertility control vector constructed by the present invention is used to transform the plant male sterile plants, and the obtained transgenic plant plants are restored to fertility, and the pollen fertility and the herbicide resistance of the seeds all exhibit the expected results.
- FIG. 1 is a schematic diagram showing the structure of a plant expression vector pBnSI according to an embodiment of the present invention, wherein P1 is a pollen-specific promoter P1 derived from the Arabidopsis genome, BrSZl is a bacterial-derived barnase gene coding region, and T1 is from the south
- the terminator of the Rbcs gene P2 is the constitutive promoter 35S promoter from tobacco mosaic virus, Pr2 is a synthetic barstar gene from bacteria, T2 is the 35S terminator, and BrMS2 is the fertility restorer gene expression of rapeseed. Box BrMS2, T3 represents the synthetic rbcs 3A terminator, P4 is the NOS promoter, Pr4 is the herbicide resistance gene, and T4 is the Nos terminator.
- FIG. 2 is a view showing a rapeseed maintainer pollen grain and aborted pollen grain (infertile) according to an embodiment of the present invention.
- Alexander staining results where A represents the staining result of the transgenic plant pollen; B is the staining result of the wild type plant pollen.
- Figure 3 shows the herbicide resistance screening of the seeds of the rapeseed line.
- FIG. 4 is a schematic diagram showing the structure of a plant expression vector pOsSI according to one embodiment of the present invention
- PG47 is a pollen-specific expression promoter derived from the maize genome
- TP-Zm-AAl is a corn-derived Zm- fused with a brittle- ⁇ peptidase sequence.
- AAl gene coding region In2-1 is the terminator from the maize genome
- P5 is the promoter from the rice genome
- 0sFG2 is the rice fertility restoration gene expression cassette
- T5 is the terminal term derived from the rice genome
- Ubi is from A constitutive expression promoter of the maize genome
- Pr5 is a herbicide resistant gene
- T3 is a synthetic terminator.
- Fig. 5 is a result of I2-KI staining of rice fertile pollen grains and abortive pollen grains according to an embodiment of the present invention.
- Figure 6 Screening of Herbicide Resistant for Seeds of Rice Maintaining Lines
- Figure 7 shows the technical route for creating a maintainer line, breeding a sterile line, and producing hybrids. Detailed description of the invention:
- the present invention was completed based on the following findings of the inventors:
- the inventors respectively used rapeseed and rice nuclear recessive mutants as transforming receptor materials to transform three closely related target genes into infertile mutants, respectively.
- the fertility restorer gene can restore the transformation to physical activity
- the pollen inactivating gene can inactivate the pollen containing the foreign gene, that is, the insemination ability is lost
- the herbicide resistance gene can be used for the transgenic seed and the non-transgenic seed.
- Screening and sorting non-GM seeds are screened by herbicides because they do not contain herbicide resistance genes; transgenic seed herbicides can survive by herbicide screening, and are used as a source of continuous and stable pollination of maintainer lines and sterile lines.
- the Brassica napus recessive brms2/brms2 and brmsl/brmsl mutants can be used as transforming receptor materials, and the closely linked three sets of target genes can be transformed into the sterile line: Fertility restoration
- the gene BrMS2 can restore the transformation to sports.
- the pollen inactivating gene BrSZl/Pr2 combination can inactivate the pollen containing the foreign gene, that is, lose the ability to inseerge, and the herbicide resistance gene Pr4 is used for the screening of transgenic seeds and non-transgenic seeds. Non-transgenic seeds are screened by herbicides because they do not contain herbicide resistance genes.
- Transgenic seeds containing herbicide-tolerant genes can survive by herbicide screening and serve as a source of continuous and stable propagation of pollination lines between maintainer lines and sterile lines. Sterile line.
- the rice nuclear recessive sterility Osfg2/ OS fg2 mutant can be used as a transforming receptor material, and the three closely linked genes can be transformed into the sterile line: the fertility restorer gene 0sFG2 can transform the sport Restoration, the pollen-inactivated gene Zm-AAl can inactivate pollen containing foreign genes, ie, lose fertility, and the herbicide-resistant gene Pr5 is used for screening of transgenic seeds and non-transgenic seeds. Non-transgenic seeds do not contain herbicides.
- the gene is screened by the herbicide, and the genetically modified seed containing the herbicide-tolerant gene can survive by herbicide screening, and is used as a source of continuous and stable breeding of the sterile line between the maintainer line and the sterile line. Because the technology uses biotechnology to produce non-GM products, it solves the bottleneck problem in the process of plant hybrid seed production, that is, the low utilization rate of the three-line method and the instability of the sterile line in the two-line method.
- the invention proposes a construct.
- the construct comprises: a first expression cassette, the first expression cassette comprising a first nucleic acid molecule, the first nucleic acid molecule encoding a rapeseed or rice male sterility recovery gene; a second expression cassette, The second expression cassette contains a second nucleic acid molecule, the second nucleic acid molecule encodes a pollen inactivating gene; a third expression cassette, the third expression cassette contains a third nucleic acid molecule, and the third nucleic acid molecule encodes a herbicide Resistance gene.
- the construct can effectively introduce rapeseed, rice male sterility recovery gene and pollen inactivating gene into rapeseed, rice plants such as homozygous recessive male sterile plants, and screened by herbicides to obtain foreign sources.
- the fertile strain of the gene acts as a maintainer, so that the sterile line can be easily propagated by pollination with the sterile line, and the maintainer is continuously produced by selfing.
- plants that do not carry foreign genes can be used as parents in hybrid seed production. Therefore, it can be effectively used for crossbreeding of rapeseed and rice.
- the form of the construct is not particularly limited, and according to a specific example of the present invention, it may be at least one of a plasmid, a phage, an artificial chromosome, a cosmid, and a virus.
- the construct (sometimes also referred to as an expression vector, genetic vector or vector) is in the form of a plasmid.
- the plasmid has the advantages of simple operation, can carry a large fragment, and is easy to handle and handle.
- the form of the plasmid is also not particularly limited, and may be either a circular plasmid or a linear plasmid, that is, it may be single-stranded or double-stranded.
- a Ti vector may be employed, for example, the first, second and third expression cassettes may be arranged between the left and right boundaries of the T-DNA of the expression vector pOsSI or pBnSI.
- the first, second and third expression cassettes can be transformed into recipient plants by Agrobacterium-mediated transformation methods, such as rapeseed brms2 and brmsl recessive nuclear male sterility mutants or rice osfg2 recessive nuclear males. Breeding mutants.
- rapeseed or rice transformed lines can be obtained.
- the transformant strain thus obtained has the following characteristics: (1) The transformation site is always heterozygous in each generation, so half of the pollen does not contain the foreign gene, and half of the pollen contains the exogenous gene, and the pollen containing the foreign gene is inactivated ( That is, the ability to insemination is lost), so the foreign gene is transmitted to the next generation only through the female gametes, and does not drift through the pollen into the environment; (2) The transformant is self-sufficient, and the fertile seed (containing the herbicide resistance gene) And sterile seeds (without herbicide resistance genes) The ratio is 1:1. Two types of seeds can be distinguished by herbicide screening, in which fertile plants (with foreign genes) are resistant to herbicide screening and survived as a maintainer, which can be crossed with sterile lines.
- Pollination is convenient and continuous breeding of sterile lines.
- fertile plants can also easily and continuously produce maintainer lines through self-crossing, while sterile lines that are propagated by cross-breeding between maintainer lines and sterile lines ( (GMO-free) is used as a parent for hybrid seed production; (3) Because the sterile line does not contain a transgene, the hybrid seed produced by it does not contain a transgene, and the commercial grain produced by this hybrid is more free of transgenic , thus eliminating the hidden dangers of genetically modified organisms.
- the new hybrid breeding system provides a practical and technological breakthrough for making full use of plant heterosis.
- nucleic acid may be any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, the length of which is not subject to any Special restrictions.
- the nucleic acid is preferably DNA because DNA is more stable and easier to manipulate than RNA.
- Brassica napus is tetraploid, containing AA genome and CC genome.
- Rape nuclear recessive nuclear sterility is usually controlled by two pairs of genes.
- the fertility of recessive genic male sterile material S45AB is regulated by two recessive genes (BnMS1 and BnMS2).
- BnMS1 and BnMS2 two recessive genes
- Infertility occurs when both genes are recessively homozygous.
- the sterility trait of recessive genic male sterile material 9012AB is controlled by the double gene (BnMs3, BnMs4).
- the type of the rapeseed male sterility recovery gene is not particularly limited.
- the rape male sterile restorer gene encodes a protein having the amino acid sequence of SEQ ID NO: 1, and the rapeseed male sterility recovery gene can be used as BrMS2, which can be used as a rapeseed Wild type fertility restorer gene of brms2 and brmsl homozygous mutant (complete male sterility).
- the rape male sterile restorer gene has the nucleotide sequence shown in SEQ ID NO: 2, and the endogenous male sterility gene BnMS2 (The nucleotide sequence thereof is shown in SEQ ID NO: 3)
- the male sterility recovery gene in the vector of the present invention has 8 single base mutations, 6 of which are synonymous mutations and 2 are missense mutations. , resulting in changes in the amino acids encoded by the two (positions 179 and 297). This sequence is effective in restoring the fertility of Brassica brms2/brms2 and brmsl/brmsl sterile recipient plants.
- the first expression cassette of the rapeseed transformation vector may further comprise: a first promoter, the first promoter being operably linked to the first nucleic acid molecule; and the first a terminator, the first terminator being operably linked to the first nucleic acid molecule.
- the types of the first promoter and the first terminator are not particularly limited.
- the sequences of the endogenous promoter, the 0RF region and the termination region of BrMS2 can be used, all of which are wild rapeseed genome sequences.
- the first promoter has the nucleotide sequence set forth in SEQ ID NO: 4.
- the first terminator has a nucleotide sequence as set forth in SEQ ID NO: 5.
- the inventors have surprisingly found that the combination of the promoter and the terminator can be further marked The efficiency of expressing the corresponding protein is increased, thereby improving the efficiency of constructing the maintainer using the construct, and more effectively restoring the fertility of the rapebred brms2 and brmsl sterile recipient plants.
- the type of the rape pollen inactivating gene is not particularly limited.
- Known pollen inactivation genes can encode carbohydrate degradation or modification enzymes, amylases, debranching enzymes and pectinase, auxin, rol B, cytotoxin, diphtheria toxin, DAM methylase, pro Or a hormone, or may be selected from a prokaryotic regulatory system.
- a prokaryotic regulatory system for example, Mariani, et al., Nature Vol. 347; pp.
- the rolB gene of Agrobacterium rhizogenes encodes an enzyme that interferes with the metabolism of auxin by releasing free sputum from indole- ⁇ - ⁇ . Estruch, et al., ⁇ J. Vol. 11 : pp. 3125 (1991) and Spena, et al., Theor. Appl. Genet.; Vol. 84: pp. 520 (1992), the rolB gene in tobacco Pollen sac-specific expression produces plants with pollen sac, in which the production of pollen is greatly reduced, and the rolB gene is an example of a gene that can be used to control pollen production.
- the nucleotide sequence of the rolB gene is disclosed in Sl ightom, et al., J. Biol. Chem. Vol. 261: pp. 108 (1985).
- the DNA molecule encoding the diphtheria toxin gene can be obtained from American Type Culture Colection (Rockvi l le, MD), ATCC No. 39359 or ATCC No. 67011.
- American Type Culture Colection Rockvi l le, MD
- ATCC No. 39359 ATCC No. 67011
- Fabijanski, et al., ⁇ . P. Appl. No. 90902754. 2 "Molecular Methods of Hybrid Seed Production".
- the DAM methylase gene is used in the methods discussed in U.S. Patent No.
- the pollen inactivating gene BrSZ1 encodes a protein having an amino acid sequence as shown in SEQ ID NO: 6, whereby a Barnase protein can be encoded.
- the pollen inactivating gene BrSZ1 has a nucleotide sequence as shown in SEQ ID NO : 7.
- the second expression cassette further comprises: a second promoter operably linked to the second nucleic acid molecule, the second promoter being a pollen-specific promoter; And a second terminator operably linked to the second nucleic acid molecule.
- the construct may further comprise an expression cassette encoding a Pr2 protein, wherein the Pr2 encodes a protein of the amino acid sequence of SEQ ID NO : 8, thereby The Barstar protein is encoded, and the promoter of the expression cassette is P2 and the terminator is T2 sequence as shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, to constitute a P2 :: Pr2 :: T2 expression cassette.
- the second expression cassette can efficiently encode a pollen-inactivated protein, and the pollen-specific promoter can enable the target gene (pollen inactivating gene) to be targeted to a specific cell, while the Pr2 expression cassette encodes Pr2.
- the protein can inhibit the leakage expression of BrSZl in tissues other than pollen, and finally can inactivate the pollen-containing pollen, but does not cause an unexpected phenotype of other tissues and organs of the plant.
- This design inactivates all transgenic pollen containing this gene, can not be inseminated, and can strictly prevent biosafety problems such as gene drift. Inactivated pollen cannot be pollinated with other plants or weeds around, so the transgene cannot drift through the pollen to the environment. .
- the rapeseed transformed construct may further comprise: a third expression cassette, the third expression cassette comprising a third nucleic acid molecule, the third nucleic acid molecule encoding a herbicide resistance gene,
- the type of herbicide resistance gene is not particularly limited. Thus, it is convenient to determine whether the plant contains the gene introduced by the construct by the expression of the herbicide resistance gene.
- at least one selected from the group consisting of glyphosate resistant, glufosinate resistant, paraquat resistant, and imidazolinone resistant genes may be employed as a screening gene.
- the Pr4 protein can be used as a herbicide-tolerant gene, and the nucleotide GH of the 319 gene becomes A in comparison with the gene of the wild-type canola plant, resulting in the encoded amino acid site. From Ala to Thr, the Brassica napus containing the mutated gene has herbicide resistance, and overexpression of the mutated gene can confer transgenic plants with imidazolinone herbicide resistance.
- the third expression cassette further comprises: a third promoter, the third promoter is operably linked to the third nucleic acid molecule, and the third promoter is constitutive a promoter; a third terminator operably linked to the third nucleic acid molecule.
- the third promoter P4 has a nucleotide sequence as shown in SEQ ID NO: 16.
- the third terminator T3 has a nucleotide sequence as set forth in SEQ ID NO:11.
- a construct according to an embodiment of the present invention can be used to carry out genetic transformation using non-transgenic recessive nuclear male sterile rapeseed (brms2/brms2 and brmsl/brmsl) as receptors for transformation.
- rapeseed maintainer line containing four exogenous genes Pr4, BrMS2, BrSZl, Pr2 closely linked below was obtained.
- the insertion of the foreign gene is not linked to the endogenous male sterility loci (brms2/brms2 and brmsl/brmsl), so the resulting transgenic rapeseed maintainer contains independent homozygous brms2 and brmsl recessive sterility sites and miscellaneous The foreign gene integration site.
- the aforementioned construct can be introduced into cells, tissues or organs of canola by conventional techniques such as Agrobacterium-mediated method to obtain a sample which can be subsequently used for research and hybridization.
- the invention proposes a canola cell, tissue or organ.
- the canola cell, tissue or organ Contains the constructs described above.
- the canola cell, tissue or organ is derived from a rapeseed homozygous recessive male sterile plant.
- the rapeseed homozygous recessive male sterile plant comprises a homozygous recessive allele of the BrMS2 gene.
- the canola cells, tissues or organs of the present invention can be effectively used for constructing a maintainer line and breeding a male sterile line.
- the features and advantages described above with respect to the constructs also apply to the canola cells, tissues or organs and will not be described again.
- the type of the rice male sterility recovery gene is not particularly limited.
- the rice male sterility recovery gene encodes a protein having the amino acid sequence set forth in SEQ ID NO: 18. That is, the rice male sterility recovery gene which can be used is 0sFG2, and thus, it can be used as a wild type fertility restorer gene of the rice receptor osfg2 homozygous mutant (complete male sterility).
- the protein encoded by the 0sFG2 gene is specifically expressed during the anther development stage.
- the rice male sterility recovery gene has a nucleotide sequence as shown in SEQ ID NO: 19, which is capable of efficiently making rice osfg2/ OS Fertility of fg2 sterile recipient plants was restored.
- the homozygous mutant of rice receptor osfg2 was obtained by EMS mutagenesis of Huanghuazhan.
- the 1688 locus of the coding region of this gene was changed from G to A, resulting in the corresponding
- the amino acid at position 563 of the encoded protein sequence is changed from glycine (G) to aspartic acid (D).
- the first expression cassette of the rice transformation vector may further comprise: a first promoter, the first promoter being operably linked to the first nucleic acid molecule, the A promoter is a male gamete-specific promoter; and a first terminator, the first terminator being operably linked to the first nucleic acid molecule.
- the types of the first promoter and the first terminator are not particularly limited.
- the sequence of the endogenous promoter, the 0RF region and the termination region of the 0sFG2 can be used, all of which are wild rice genome sequences.
- the first promoter has a nucleotide sequence as set forth in SEQ ID NO: 20.
- the first terminator has a nucleotide sequence as set forth in SEQ ID NO:21.
- the type of rice pollen-inactivated gene is not particularly limited.
- the pollen inactivating gene encodes a protein having the amino acid sequence set forth in SEQ ID NO: 22.
- the ⁇ -amylase encoded by Zm-AAl can be encoded.
- the pollen inactivating gene has a nucleotide sequence as shown in SEQ ID NO: 23. Thereby, the efficiency of expressing the corresponding protein can be further improved.
- the second expression cassette further comprises: a second promoter operably linked to the second nucleic acid molecule, the second promoter being a pollen-specific promoter; And a second terminator, the second terminator and the second nucleic acid The molecules are operatively linked.
- a sequence encoding a peptide may be further included in the second expression cassette, whereby the second expression cassette can efficiently encode a pollen inactivating protein having a peptide, thereby enabling The gene of interest (pollen inactivating gene) can be targeted to specific organelles.
- the sequence encoding the leader peptide has the nucleotide sequence shown in SEQ ID NO: 24 (the sequence encoding the leader peptide (TP) from the brittle- ⁇ gene of maize).
- the expressed protein can be effectively targeted to the amyloplast, and the starch in the pollen is decomposed, thereby depriving the pollen, losing the ability to fertilize, and inactivating the transgenic pollen.
- the gene is driven by the maize pollen-specific promoter PG47, and the sequence of the coding peptide (TP) derived from the brittle- ⁇ gene of maize and the terminator IN2-1 are expressed.
- the rice transformed construct may further comprise: a third expression cassette, the third expression cassette comprising a third nucleic acid molecule, the third nucleic acid molecule encoding a herbicide resistant gene,
- the type of herbicide resistance gene is not particularly limited. Thus, it is convenient to determine whether the plant contains the gene introduced by the construct by the expression of the herbicide resistance gene.
- at least one selected from the group consisting of glyphosate resistant, glufosinate resistant, paraquat resistant, and imidazolinone resistant genes may be employed as a screening gene.
- Pr5 protein can be used as a herbicide resistance gene, which is a site of OsALS 548 (nucleotides 1642, 1643 from TG to AT, resulting in 548 amino acids from Trp to Met).
- the mutated rice gene with herbicide resistance, the rice gene overexpressing the mutation can make the transgenic plant have imidazolinone herbicide resistance.
- the third expression cassette further comprises: a third promoter, the third promoter is operably linked to the third nucleic acid molecule, and the third promoter is constitutive a promoter; a third terminator operably linked to the third nucleic acid molecule.
- the third promoter has a nucleotide sequence as set forth in SEQ ID NO: 25.
- the third terminator has a nucleotide sequence as set forth in SEQ ID NO: 11.
- a construct according to an embodiment of the present invention can be used to perform genetic transformation and integration by using non-transgenic recessive nuclear male sterile rice (osfg2/ OS fg2) as a receptor for transformation.
- the rice maintainer line contains the following closely linked three exogenous genes Pr5, 0sFG2, Zm-AAl.
- the insertion of the foreign gene is not linked to the endogenous male sterility locus ( OS fg2/ OS fg2), so the resulting transgenic rice maintainer contains an independent homozygous osfg2 Recessive sites and heterozygous foreign genes (including the 0SFG2 gene) integration site.
- the aforementioned construct can be introduced into cells, tissues or organs of rice by a conventional technique such as Agrobacterium-mediated method to obtain a sample which can be subsequently used for research and hybridization.
- the invention provides a rice cell, tissue or organ.
- the rice cell, tissue or organ contains the construct described above.
- the rice cell, tissue or organ is derived from a rice homozygous recessive male sterile plant.
- the rice homozygous recessive male sterile plant comprises a homozygous recessive allele of the 0sFG2 gene.
- the rice cell, tissue or organ of the present invention can be effectively used for constructing a maintainer line and breeding a male sterile line.
- the features and advantages described above with respect to the constructs also apply to the rice cells, tissues or organs and will not be described again.
- the invention provides a method of constructing a rape or rice male sterile line.
- the method comprises: introducing the rapeseed or rice construct described above into a first rapeseed or rice homozygous recessive male sterile plant to obtain a gene carrying a foreign gene A second rape or rice plant, the second rapeseed or rice plant is capable of producing a fertile male gamete, and the foreign gene in the second rapeseed or rice plant is in a heterozygous state, so half of the second rapeseed or rice plant has no pollen Containing foreign genes, half contain foreign genes, pollen containing foreign genes is inactivated (ie, lost fertility).
- the first rapeseed or rice homozygous recessive male sterile plant comprises a homozygous recessive allele of the BrMSl/BrMS2 or 0sFG2 gene.
- the step of sorting by herbicide screening that is, by detecting whether rapeseed or rice seeds are resistant to herbicides, can be sorted to distinguish whether or not they carry foreign genes.
- the invention provides a method for restoring male fertility in rapeseed or rice sterile plants.
- the method comprises: introducing the rapeseed or rice construct described above into a rapeseed or rice homozygous recessive male sterile plant.
- the rapeseed or rice homozygous recessive male sterile plant comprises a homozygous recessive allele of the BrMSl/BrMS2 or 0sFG2 gene.
- the invention provides a method of preparing a rapeseed or rice seed.
- the method comprises the steps of: introducing the rapeseed or rice construct described above into a canola or rice plant, respectively; and self-fertilizing the canola or rice plant to obtain the aforementioned The seed of the construct.
- the rape or rice plant is a rapeseed or rice homozygous recessive male sterile plant.
- the rapeseed or rice homozygous recessive male sterile plant comprises a homozygous recessive allele of the BrMS1/BrMS2 or 0sFG2 gene.
- the invention provides a method for the preparation of hybrid rapeseed or rice.
- the method employs a rapeseed or a rice male sterile line constructed by a method of constructing a rapeseed or a rice male sterile line.
- the hybridization of the rapeseed or the rice male sterile line of the present invention can be further utilized to improve the efficiency of hybridization of rapeseed or rice.
- the invention provides the use of a rapeseed or rice male sterile line for the preparation of hybrid seeds.
- the rapeseed or rice male sterile line is constructed by a method of constructing a rapeseed or a rice male sterile line in advance.
- the rapeseed or the rice male sterile line of the present invention can be further hybridized with the restorer line, and an excellent hybridization combination can be screened to improve the efficiency of the hybrid seed production.
- each expression cassette was able to work well when it was transformed into rapeseed alone, and achieved the desired design effect. Further inventors constructed the following expression vectors.
- the herbicide resistance gene is referred to the Chinese patent 201310116228. 2, the present invention is incorporated herein by reference.
- the pBnSI vector shown in Figure 1 was constructed by assembling the various elements of the BrSZl, Pr2, BrMS2 and Pr4 expression cassettes.
- the vector comprises four expression cassettes, wherein the first expression cassette is a barnase expression cassette consisting of a pollen deliberate expression promoter P1 from the Arabidopsis genome, as shown in SEQ ID NO: 4 of the Sequence Listing, of bacterial origin Barnase gene coding region BrSZl, as shown in SEQ ID NO: 7 in the list, and termination of the Arabidopsis Rbcs gene
- the sub-T1 as shown in SEQ ID NO: 5 of the Sequence Listing, constitutes a first expression cassette PI: : BrSZl: : T1 ;
- the second expression cassette is a barstar expression cassette consisting of a constitutive promoter derived from tobacco mosaic virus 35S promoter P2, as shown in SEQ ID NO: 13 in the Sequence Listing, artificially synthesized barstar gene Pr2 from bacteria, as shown
- the fourth expression cassette is a herbicide-resistant gene expression cassette, and is composed of the NOS promoter P4, as shown in SEQ ID NO: 16 in the Sequence Listing.
- herbicide resistance gene Pr4 as shown in SEQ ID NO: 17 in the sequence listing, No s terminator T4, as shown in SEQ ID NO: 11 of the Sequence Listing, constitutes the fourth expression cassette P4::Pr4::T4.
- the plasmid PBnSI was transferred into Agrobacterium tumefaciens EHA105 strain by electroporation, and the rapeseed containing homozygous brms2 and brmsl recessive sterile locus was genetically transformed by Agrobacterium-mediated method to obtain 22 single-copy transgenic plants.
- material The rapeseed transforming receptor material containing homozygous brms2 and brmsl recessive sterility loci is Brassica napus L.
- Example 3 Pollen fertility testing of transgenic rapeseed plants
- Example 2 Analysis of 22 single-copy transgenic rapeseed (containing homozygous brms2 and brmsl recessive male sterile sites) obtained in Example 2 showed no significant morphological differences between transgenic plants and non-transgenic control plants, but pollen Fertility is significantly different.
- the transgenic plant material obtained by transforming the pBnSI construct into rapeseed was tested for pollen staining rate, and the pollen staining rate of wild type rape was detected (Fig. 2).
- the method used is as follows: In the flowering stage of rapeseed, a single plant is randomly selected from the transgenic rapeseed plants and their wild-type control plants, each plant takes a flower, and each flower takes an anther, placed in the center of the slide, and drops. Add a drop of 1% Alexander solution, release the pollen with tweezers and anatomic needle, cover with a cover slip, observe under the microscope, count the number of pollen pollen and the total number of pollen, dye the orange-red for fertile pollen, green for Aborted pollen (Figure 2 shows the fertile pollen grains and sterile pollen grains after dyeing).
- the pollen staining rate of transgenic rapeseed plants was analyzed. The results showed that the orange-red pollen of the control plants accounted for 98% ⁇ 100%.
- the ratio of normal pollen to abortive pollen was close to 1:1, indicating that the constructed Maintaining system
- barnase gene On the one hand, the expression of the barnase gene is driven by a pollen-specific promoter, and on the other hand, the 35S constitutive promoter drives the barstar gene.
- barnase is highly expressed in pollen and can kill the male gametes, thereby achieving pollen abortion.
- the expression leakage of barnase in other plant tissues other than pollen can be completely inhibited by the constitutively expressed barstar, so the other phenotypes of the plant are completely normal.
- T1 generation seeds of 22 single-copy transgenic rapeseed plants (containing homozygous brms2 and brmsl recessive male sterile sites) obtained in Example 2 were investigated for herbicide-tolerant segregation ratios, and the results showed that these seeds all showed 1 : 1 separation ratio (Fig. 3), that is, the herbicide-tolerant seed carrying the foreign gene and the herbicide-resistant seed not carrying the foreign gene showed a 1:1 separation, indicating that the components of the vector provided by the present invention are expressed as a whole Achieve the desired pollen inactivation function and seed screening marker function.
- Example 5 Construction of a rice expression vector pOsSI:
- the pOsSI vector shown in Fig. 4 was constructed by assembling the respective elements of the Zm-AA1, 0sFG2 and Pr5 expression cassettes.
- the vector comprises three expression cassettes, wherein the first expression cassette is a Zm-AAl expression cassette consisting of a pollen-specific expression promoter PG47 from the maize genome, as shown by SEQ ID NO: 28 in the sequence listing, fused brittle a maize-derived Zm-AAl gene coding region of the -1 peptide sequence, as set forth in SEQ ID NO: 29 of the Sequence Listing, and terminator In2-1 from the maize genome, as set forth in SEQ ID NO: 30 of the Sequence Listing
- the first expression cassette is PG47 :: TP::Zm-AAl:: In2-1 ;
- the second expression cassette is the rice fertility restorer gene 0sFG2 expression cassette, which is composed of the promoter P5 from the rice genome, such as the sequence listing.
- the second expression cassette is constructed as a second expression cassette P5 : : 0sFG2 : : T5 ;
- the third expression cassette is a herbicide resistance gene expression cassette consisting of a constitutive expression promoter Ubi from the maize genome, as set forth in SEQ ID NO: 25 of the Sequence Listing Show that the herbicide resistance gene Pr5, as in the sequence listing SEQ ID NO: 27, and the artificially synthesized terminator T3, as set forth in SEQ ID NO: 15 of the Sequence Listing, constitutes the fourth expression cassette P6: : Pr5 : : T6.
- the plasmid pOsSI was transferred into Agrobacterium tumefaciens strain AglO by electroporation, and the rice containing homozygous osfg2 recessive sterile locus was genetically transformed by Agrobacterium-mediated method to obtain 32 single-copy transgenic plants.
- the specific transforming receptor material is rice Huanghuazhan variety.
- Example 6 The analysis of 32 single-copy transgenic rice plants (containing homozygous osfg2 recessive male sterile sites) obtained in Example 6 showed that there was no obvious morphological difference between transgenic plants and non-transgenic control plants, but pollen fertility obviously different.
- the transgenic plant material obtained by transforming the pOsSI construct into rice was tested for pollen staining rate, and the pollen staining rate of wild type rice was detected (Fig. 5).
- the method used is as follows: In the flowering stage of rice, a single plant is randomly selected from the transgenic rice plants and their wild-type control plants, each plant takes a flower, and each flower takes an anther, placed in the center of the slide, and drops. Add a drop of 1% I2-KI solution, release the pollen with tweezers and anatomic needle, cover with a cover slip, observe under the microscope, count the number of pollenable pollen and the total number of pollen, and color the dark blue for fertile pollen. The inability to color the aborted pollen (Figure 5 shows the dyed fertile pollen grains and sterile pollen grains). The pollen staining rate of transgenic rice plants was analyzed.
- T1 generation seeds of 32 single-copy transgenic rice plants (containing homozygous osfg2 recessive male sterile sites) obtained in Example 6 were investigated for herbicide-tolerant segregation ratio, and the results showed that these seeds all showed 1:1.
- the separation ratio (Fig. 6) that is, the herbicide-tolerant seed carrying the foreign gene and the herbicide-resistant seed not carrying the foreign gene showed a 1:1 separation, indicating that the components of the vector provided by the present invention were expressed as a whole as expected. Pollen inactivation function and seed screening marker function.
- Example 9 Technical route for creating maintainer lines, breeding lines, and producing hybrids
- Fig. 7 The technique involves three Linked functional elements: pollen lethal gene, herbicide resistance gene, fertility restorer gene.
- the pollen lethal gene selected The time, level and lethal efficiency of pollen expression are directly related to the successful application of this technology. Therefore, the present invention utilizes a specific promoter in the late pollen development to drive the pollen inactivation gene to specifically express in pollen maturation, and achieve specific pollen death.
- Seed-like separation sterile seeds that do not carry transgenic, no herbicide resistance, can be screened by herbicides, carry transgenic fertile seeds, have herbicide resistance, can survive as a maintainer, and thus can be conveniently
- the line is continuously obtained from the maintainer line; the maintainer is further pollinated with the sterile line to restore the sterile line, and the sterile line that does not contain the transgene is obtained, so that the sterile line can be propagated; Hybrid with any restorer parent, used for the production of hybrid seeds.
- the novel fertility regulating vector constructed by the invention is used to transform the plant male sterile plant, and the obtained transgenic plant plant recovers the fertility, and the pollen fertility and the herbicide resistance of the seed all exhibit the expected results.
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Abstract
本发明提供了一种植物保持系和不育系及其用途,将育性恢复基因、花粉致死基因、抗除草剂基因同时转入到纯合隐性核雄性不育植株中,该转基因植株自交后产生两类种子:不携带转基因的不育系种子,没有除草剂抗性,可通过除草剂筛除;携带转基因的可育种子,即保持系,具有除草剂抗性,可与不育系授粉杂交用于不育系的繁殖。本发明创制的不育系育性稳定,不受环境影响,且该植物隐性核不育基因适用于绝大多数植物品种。
Description
植物新型保持系及不育系建立及其用途 技术领域
本发明涉及植物分子生物学和育种领域。 具体地, 本发明的实施方案涉及含有制种技 术的转基因植物。 更具体地, 本发明涉及纯合隐性核雄性不育植株的育性恢复及其用途。 进一步本发明涉及构建植物保持系及繁殖雄性不育系的方法, 更具体地, 本发明涉及一种 构建体, 一种植物细胞、 组织或器官, 一种繁殖植物雄性不育系的方法, 一种恢复植物不 育植株雄性育性的方法, 一种制备植物种子的方法, 一种用于制备杂交植物的方法, 以及 植物雄性不育系在制备杂交水稻中的用途。
背景技术
杂交育种是选育新品种主要途径, 是近代育种最重要的方法, 杂交种的创制、 利用 和产业化是国际农业跨国集团种业市场竞争的焦点。 由于杂交引起基因重组, 后代会出 现组合双亲控制的优良性状基因型, 产生加性效应, 并利用某些基因互作, 形成具超亲 类型新个体。 作物杂交育种具有巨大的发展潜力, 已经成为提高粮食产量的主要途径。 近几十年来, 杂种优势作为一种提高作物产量、 改良作物品质、 提高作物抗虫、 抗病、 抗逆的手段已被广泛利用。利用杂种优势育种已成为许多作物的主要育种方法。而有效 控制作物自花授粉、 受精是获得高纯度杂交 F1种子、 从而利用作物杂种优势的关键。 而杂交育种中必须要解决的关键问题是 (1 ) 获得可用的不育系: 一般为雄性不育 (由 细胞质不育或隐性核不育基因控制) ; (2 ) 杂交配组: 不育系可与相应的父本植物组 合生产具有优良性状的杂交后代; (3 ) 不育系的繁殖: 不育系能在一定条件下恢复育 性使其得到保持。 因此, 作物雄性不育系的选育是杂种优势利用的关键环节。
上世纪七、八十年代, 以袁隆平为代表的中国科学家们利用水稻野败型细胞质不育 基因资源创制了 "三系"杂交水稻, 使水稻产量提高了近 20%, 被称为第二次 "绿色革 命", 为保障我国粮食供应起到了非常重要的作用, 使我国的水稻育种与生产技术处于 世界领先水平,也向世人展示了杂种优势的强大作用。水稻杂交育种中常用的是"三系" 和 "两系"杂交。 "三系 "杂交需要有特定的恢复系和保持系, 育种程序和生产环节复 杂, 选育新不育系及新组合的周期长、 效率低, 种质资源的利用率低于 5%。 另外, 三 系杂交粳稻优势不强,不育细胞质较单一,存在某种毁灭性病虫害暴发的潜在危险。 "两 系"杂交水稻由于不受恢复系、保持系之间关系的制约, 亲本的遗传多样性得到明显改 善, 选育出高产杂交稻组合的速度明显加快, 促进了超级杂交稻的研究和生产。 但目前
"两系"杂交中采用的不育系多为 "光温敏"不育系, 其育性受环境中的温度和光照影 响。 这些环境因素的不稳定会直接影响杂交种子的纯度和数量, 加大制种风险, 严重时 会使企业和农民造成重大经济损失, 限制 "两系" 杂交稻的大面积推广。 而且利用目前 技术所能选用的两系杂交稻不育系十分有限,例如粳稻品种中几乎没有很好的两系杂交 组合, 限制了品种资源的充分利用。 因而, 培育不受环境影响且可自主繁殖的稳定不育 系已成为限制 "两系" 杂交技术广泛应用的技术瓶颈。
玉米是利用杂种优势最早, 且杂交种在世界上普及推广最成功的作物。玉米是雌雄 异花作物, 繁殖系数高的作物, 杂交种生产时, 在隔离区内父、 母本按比例种植, 母本 雄穗刚露出时拔掉,父本花粉自由授粉杂交。玉米杂交优势的利用及生产上应用杂交种, 使得玉米生产水平产生了巨大变化。但是, 玉米杂交种生产中存在着常用育种的亲本遗 传基础差异不够大, 并因此影响到主要育种目标如高产、 稳产、 抗逆、 早熟等的尽快实 现。 同时, 在杂交种生产过程中, 存在着母本去雄工作繁重且不彻底、 进而影响杂交种 产量和质量不稳定的问题, 急待解决。
杂种优势在双子叶植物如烟草和油菜的生产中也得到应用。油菜是世界的第三大油 料作物, 而中国油菜总产和面积占世界三分之一, 是最大的油菜生产国, 中国的油菜杂 种优势利用处于世界领先水平。油菜中常用的杂交育种也是基于细胞质不育和细胞核不 育两大类, 存在 "三系"和 "两系 "杂交育种, 到目前为止, 生产上应用最多的油菜细 胞质雄性不育系主要有三个: 波里马细胞质不育系, 陕 2A CMS和萝卜细胞质不育系 OgU CMS。 尽管细胞质雄性不育系已得到广泛应用, 但在技术上依旧受到恢复源限制、 育性 稳定性、 胞质单一化等方面的影响。 因此, 与水稻中一样, 油菜杂交种开发中存在同样 的种质资源利用率低、 杂交种纯度较低的问题。
因而, 目前的植物杂交育种技术仍有待改进。
发明内容
本发明旨在解决上述植物杂交育种技术瓶颈问题。为此, 本发明的一个目的在于提 供一种具有能够有效构建新型稳定的植物保持系和雄性不育系的方法,从而扩展植物种 质资源在杂交育种中的应用, 消除杂交制种过程中的风险, 提高杂交种子纯度。
围绕如何提高不育系的稳定性及打破杂交品种资源利用的局限性,并进一步解决现 有制种技术复杂且成本高等技术瓶颈问题, 我们建立了一种新的杂交育种技术。该技术 利用隐性核基因控制的雄性不育系和转基因技术, 将育性恢复基因、 花粉致死基因、 抗 除草剂基因连锁, 同时转入到纯合隐性核雄性不育植株(不育系) 中, 育性恢复基因的
功能使转基因植株恢复育性; 但到花粉发育后期, 花粉致死基因使含有转基因的花粉失 活, 只有不携带转基因的花粉保留活性。 该转基因植株自交后产生两类种子: 不携带转 基因的不育系种子, 没有除草剂抗性, 可通过除草剂筛除; 携带转基因的可育种子, 具 有除草剂抗性, 可以通过除草剂筛选存活下来, 即保持系, 可与不育系授粉杂交用于不 育系的繁殖。
本发明所提供的繁殖不育系的方法, 所述方法包括: (a) 提供第一植株, 所述第一植 株为不育系, 所述不育系含有一个纯合隐性的等位不育基因; (b) 向第二植株中引入下述 构建体, 所述第二植株含有一个纯合隐性的等位不育基因, 所述构建体包含: (i ) 第一核 苷酸序列, 当其在所述第一或第二植株中表达时, 能功能性互补所述植株的纯合隐性植物 不育性状; (ϋ ) 第二核苷酸序列, 其表达抑制所述第二植株中雄性配子的形成或功能, 从 而使得在含有所述隐性等位基因的所述第二植株中产生的雄配子不含所述构建体; (iii ) 第三核苷酸序列,该序列编码产物的表达能用于选择具有所述构建体的植物细胞;以及(c ) 用所述第二植株的所述雄性配子使所述第一植株受精,以繁殖保持了所述第一植株纯合 隐性等位状态的后代; 进一步的, 上述的第三核苷酸序列选自抗草甘膦、 抗草丁膦、 抗 百草枯、 抗草铵膦、 抗莠去津、 抗溴苯腈、 抗 2, 4-D、 抗咪唑啉酮或抗磺酰脲类等除 草剂的基因构成的组。
具体地, 上述第一核苷酸序列为育性恢复基因, 选自 0sFG2、 BrMS2、 MSP1、 PAIR1、 PAIR2、 ZEP1、 MELL, PSS1、 TDR、 UDT1、 GAMYB4、 PTC1、 API5、 WDA1、 CYP704B2 , MS26、 MS22、 DPW、 MAD S3 , 0SC6、 RIP1、 CSA或 AID1构成的组。 其中所述的第一核苷酸序列与 第四核苷酸序列可操作地相连, 所述第四核苷酸序列指导偏好于雄性植物细胞的表达, 或者所述的第四核苷酸序列仅在存在诱导物质或诱导条件时才具有功能。更具体的, 所 述的第四核苷酸序列选自 0sFG2、 BrMS2、 MSP1、 PAIR1、 PAIR2、 ZEP1、 MELL、 PSS1、 TDR、 UDT1、 GAMYB4、 PTC1、 API5、 WDA1、 CYP704B2 , MS26、 MS22、 DPW、 MADS3、 0SC6、 RIP1、 CSA、 A皿、 5126、 Ms26、 Ms22或 Ms45的雄性组织调控序列构成的组。
具体地, 上述第二核苷酸序列选自 DAM甲基化酶基因、 Zea mays α淀粉酶基因、 细 胞毒素编码基因、或 Barnase与 Barstar的组合序列构成的组。所述的第二核苷酸序列与 第五核苷酸序列可操作地相连, 所述第五核苷酸序列指导偏好于雄性配子的表达。更具 体地, 所述的第五核苷酸序列选自聚半乳糖醛酸酶 47基因、 Zml3基因、 果胶甲基酯酶基 因、 钙调蛋白结合蛋白基因、 肌动蛋白解聚因子基因、 prolfi l in基因和硫酸化五肽 phytosulphokine基因的调控区域构成的组。
具体地, 上述第三核苷酸序列具体为具有除草剂抗性的 ALS基因突变体。 其中所述 的 ALS基因突变体在水稻中的突变为 Trp548、Ala96和 /或 Ser627突变,优选带有 Trp548Cys、 Trp548Met, Ala96Val、 Ala96Thr和 /或 Ser627Asn突变; 其中所述的 ALS基因突变体在油菜 中的突变为 Alal07、 Alal90、 Trp559和 /或 Ser638突变。 具体地, 其中所述的第三核苷酸序 列与第六核苷酸序列可操作地连接,所述第六核苷酸序列为组成型表达启动子。更具体地, 其中所述的第六核苷酸序列为 35s启动子。
本发明还提供了一种方法, 用于从具有雌性配子和雄性配子的植株产生种子,所述方 法包括: (a) 向第一植株中引入下述构建体, 所述第一植株含有一个纯合隐性的等位不育 基因, 所述构建体包含: (i ) 第一核苷酸序列, 当其在所述第一植株中表达时, 能功能性 互补所述植株的纯合隐性植物不育性状; (ii ) 第二核苷酸序列, 其表达抑制所述第一植株 中雄性配子的形成或功能, 从而使得在含有所述隐性等位基因的所述第一植株中产生的雄 配子不含所述构建体; (iii ) 第三核苷酸序列, 该序列编码产物的表达能用于选择具有所 述构建体的植物细胞; (b ) 使所述植株自体受精; 以及 (c ) 产生含有所述构建体的种子; 其特征在于所述的第三核苷酸序列选自抗草甘膦、 抗草丁膦、 抗百草枯、 抗草铵膦、 抗 莠去津、 抗溴苯腈、 抗 2, 4-D、 抗咪唑啉酮或抗磺酰脲类等除草剂的基因构成的组。
具体地, 本发明是基于下列发现而完成的:
发明人以油菜核隐性不育 和 Zwm5 突变体为转化受体材料, 将紧密连锁的 3 组功能基因转化至上述不育系: 育性恢复基因使雄性不育油菜植株恢复育性, 花粉失活 基因 (BrSZl/Pr2组合)使含有转基因的花粉失活, 即失去授精能力;除草剂抗性基因用 于转基因种子即保持系和非转基因种子即不育系的分拣,其中非转基因种子被除草剂筛 除, 转基因种子则用作保持系, 与不育系授粉杂交, 来源源不断地繁殖不育系。
本发明提供了一种构建体。 该构建体包括: 第一表达盒, 含有油菜雄性不育恢复基 因; 第二表达盒, 含有花粉致死基因; 第三表达盒, 含有除草剂抗性基因。 利用该构建 体, 能够有效地将油菜育性恢复基因和花粉致死基因引入到油菜不育系植株中, 从而杀 死带转基因成分的花粉并恢复油菜的育性。
本发明的第二方面, 通过常规技术, 例如农杆菌介导的蘸花法, 将前述构建体引入 到油菜中, 得到转基因植株。
本发明的第三方面提供了一种繁殖油菜雄性不育系的方法。 将前面所述的构建体转 入到油菜纯合隐性雄性不育植株中, 获得转基因油菜植株, 转基因油菜植株能够产生可 育的不含有转基因的雄性配子, 通过自交, 得到携带外源基因的种子和不携带外源基因
的种子。 其中不携带外源基因的种子不抗除草剂, 可以被除草剂筛除, 携带外源基因的 种子抗除草剂可以存活, 作为保持系, 与雄性不育系授粉杂交, 进行不育系的繁殖。
本发明的第四方面提供了一种恢复油菜不育植株育性的方法。根据本发明的实施例, 该方法包括: 将前面所述的构建体引入到油菜纯合隐性雄性不育植株中。
本发明的第五方面提供了一种制备油菜种子的方法。根据本发明的实施例, 该方法 包括以下步骤: 将前面所述的构建体引入到油菜植株中; 将所述油菜植株自体受精, 以 获得含有前面所述的构建体的种子。
本发明的第六方面提供了一种转基因植株, 即转基因油菜植株。根据本发明的实施 例,所述转基因植株是通过将前面所述的构建体引入到油菜纯合隐性雄性不育植株中获 得的。
本发明的第七方面, 发明人以水稻核隐性不育 突变体为转化受体材料, 将紧 密连锁的 3个功能基因转化至上述不育系:育性恢复基因 使雄性不育水稻植株恢 复育性, 花粉致死基因 Um-AAD 使含有转基因的花粉失活, 即失去授精能力;除草剂 抗性基因用于转基因种子即保持系和非转基因种子即不育系的分拣,其中非转基因种子 被除草剂筛除, 转基因种子则用作保持系, 与不育系授粉杂交, 来源源不断地生产不育 系。
本发明的第八方面, 通过常规技术, 例如农杆菌介导的幼胚或成熟胚转化, 将前述 构建体引入到水稻中, 得到转基因植株。
本发明的第九方面, 提供了一种繁殖水稻雄性不育系的方法。 将前面所述的构建体 转入到水稻纯合隐性雄性不育植株中, 获得转基因水稻植株, 转基因水稻植株能够产生 可育的不含有转基因的雄性配子, 通过自交, 得到携带外源基因的种子和不携带外源基 因的种子。 其中不携带外源基因的种子不抗除草剂, 可以被除草剂筛除, 携带外源基因 的种子抗除草剂可以存活,并作为保持系,与雄性不育系授粉杂交,进行不育系的繁殖。
本发明的第十方面, 提供了一种恢复水稻不育植株育性的方法。 根据本发明的实施 例, 该方法包括: 将前面所述的构建体引入到水稻纯合隐性雄性不育植株中。
本发明的第十一方面, 提供了一种制备水稻种子的方法。 根据本发明的实施例, 该 方法包括以下步骤:将前面所述的构建体引入到水稻植株中;将所述水稻植株自体受精, 以获得含有前面所述的构建体的种子。
本发明的第十二方面提供了一种转基因植株, 即转基因水稻植株。根据本发明的实 施例,所述转基因植株是通过将前面所述的构建体引入到水稻纯合隐性雄性不育植株中
获得的。
这种新型的杂交育种技术具有显著的优越性: (1 )该技术创制的不育系育性稳定, 不受环境影响, 解除了环境因素对杂交育种的制约, 消除了生产上的潜在风险; (2 ) 所利用的植物隐性核不育基因适用于该种植物的绝大多数品种,使杂种优势的资源利用 大幅提高; (3 )终止携带转基因的花粉的受精过程, 防止外源基因漂移到其它植物中;
( 4 ) 所产生的不育系不含转基因, 消除人们对转基因作物的顾虑; (5 ) 利用除草剂基 因方便地将不育种子和可育种子分开, 方便获得保持系, 进一步与不育系授粉杂交繁殖 不育系种子, 保证了不育种子的纯度。
该技术涉及三个连锁的功能元件: 花粉致死基因, 抗除草剂基因, 育性恢复基因。 其中所选用的花粉致死基因在花粉中表达的时间、水平及致死效率都直接关系到该技术 能否成功应用,因此本发明利用花粉特异性启动子驱动花粉失活基因在花粉成熟期特异 表达, 实现了特异性花粉致死的效果;利用植物内源启动子控制的雄性不育恢复核基因 转化对应的植物雄性不育植株, 使转基因植株恢复育性; 利用抗除草剂基因可以方便地 将转基因植株自交后产生的两类种子分开:不携带转基因的不育种子,没有除草剂抗性, 可通过除草剂筛除, 携带转基因的可育种子, 具有除草剂抗性, 可以作为保持系存活下 来, 由此可以通过自交方便地、 源源不断地得到保持系; 保持系进一步与不育系授粉杂 交, 使不育系恢复结实, 得到不含有转基因的不育系, 使不育系得到繁殖; 由此得到的 不育系既可以与任意父本杂交, 用于杂交种子的生产。利用本发明构建的新型育性调控 载体转化植物雄性不育植株, 得到的转基因植物植株恢复育性, 其花粉育性和种子的除 草剂抗性均表现预期的结果。
附图说明
本发明的上述和 /或附加的方面和优点从结合下面附图对实施例的描述中将变得明 显和容易理解, 其中:
图 1为根据本发明一个实施例的植物表达载体 pBnSI的结构示意图, 其中 P1为来 自拟南芥基因组的花粉特意表达启动子 Pl, BrSZl为细菌来源的 barnase基因编码区, T1 为来自于拟南芥 Rbcs基因的终止子, P2为来自于烟草花叶病毒的组成型启动子 35S启动子, Pr2为人工合成的来自于细菌的 barstar基因, T2为 35S终止子, BrMS2为油菜育性恢复基 因表达框 BrMS2, T3表示人工合成的 rbcs 3A终止子, P4为 N0S启动子, Pr4为抗除草剂 基因, T4为 Nos终止子。
图 2为根据本发明一个实施例的油菜保持系可育花粉粒和败育花粉粒(不可育)的
Alexander染色结果,其中 A表示转基因植株花粉的染色结果; B为野生型植株花粉的染 色结果。
图 3 为油菜保持系结实的种子的抗除草剂筛选。
图 4 为根据本发明一个实施例的植物表达载体 pOsSI 的结构示意图, PG47为来自 玉米基因组的花粉特异表达启动子; TP-Zm-AAl表示融合了 brittle-Ι导肽序列的玉米来源 的 Zm-AAl基因编码区; In2-1为来自于玉米基因组的终止子; P5为来自于水稻基因组的启 动子; 0sFG2表示水稻育性恢复基因表达框; T5表示来源于水稻基因组的终止子; Ubi为来 自玉米基因组的组成型表达启动子; Pr5为抗除草剂基因; T3为人工合成的终止子。
图 5 为根据本发明一个实施例的水稻可育花粉粒和败育花粉粒的 I2-KI染色结果。 图 6 为水稻保持系结实的种子的抗除草剂筛选
图 7 为创制保持系、 繁殖不育系和生产杂交种的技术路线。 发明详细描述:
下面详细描述本发明的实施例。 下面通过参考附图描述的实施例是示例性的, 旨在用 于解释本发明, 而不能理解为对本发明的限制。
本文提到的所有参考文献都通过弓 I用并入本文。
除非有相反指明, 本文所用的所有技术和科学术语都具有与本发明所属领域普通技术 人员通常所理解的相同的含义。 除非有相反指明, 本文所使用的或提到的技术是本领域普 通技术人员公知的标准技术。 材料、 方法和例子仅作阐述用, 而非加以限制。
本发明是基于发明人的下列发现而完成的: 发明人分别以油菜、 水稻核隐性不育突变 体为转化受体材料, 通过分别将紧密连锁的 3 组目标基因转化至不育突变体中, 其中, 育 性恢复基因可使转化受体育性恢复, 花粉失活基因可使含有外源基因的花粉失活, 即失去 授精能力, 除草剂抗性基因可以用于转基因种子和非转基因种子的筛选分拣, 非转基因种 子由于不含有抗除草剂基因而被除草剂筛除; 转基因种子抗除草剂可以通过除草剂筛选存 活下来, 用作保持系与不育系杂交授粉来源源不断地、 稳定地繁殖不育系。 例如, 根据本 发明的一个实施例, 可以以油菜核隐性不育 brms2/brms2和 brmsl/brmsl突变体为转化受 体材料, 将紧密连锁的 3组目标基因转化至不育系: 育性恢复基因 BrMS2可使转化受体育 性恢复, 花粉失活基因 BrSZl/Pr2 组合可使含有外源基因的花粉失活, 即失去授精能力, 抗除草剂基因 Pr4用于转基因种子和非转基因种子的筛选, 非转基因种子由于不含有抗除 草剂基因而被除草剂筛除, 转基因种子含有抗除草剂基因可以通过除草剂筛选存活下来, 用作保持系与不育系授粉杂交来源源不断地、 稳定地繁殖不育系。 再例如, 根据本发明的
另一个实施例, 可以以水稻核隐性不育 Osfg2/O Sfg2 突变体为转化受体材料, 将紧密连锁 的 3个目标基因转化至不育系: 育性恢复基因 0sFG2可使转化受体育性恢复, 花粉失活基 因 Zm-AAl可使含有外源基因的花粉失活, 即失去授精能力, 抗除草剂基因 Pr5用于转基因 种子和非转基因种子的筛选, 非转基因种子由于不含有抗除草剂基因而被除草剂筛除, 转 基因种子含有抗除草剂基因可以通过除草剂筛选存活下来, 用作保持系与不育系授粉杂交 来源源不断地、 稳定地繁殖不育系。 由于该技术利用生物技术生产非转基因产品, 解决了 植物杂交制种过程中面临的瓶颈问题, 即三系法资源利用率低而两系法中不育系育性不稳 定的问题。
由此, 在本发明的一个实施例, 本发明提出了一种构建体。 根据本发明的实施例, 该 构建体包括: 第一表达盒, 所述第一表达盒含有第一核酸分子, 所述第一核酸分子编码油 菜或水稻雄性不育恢复基因; 第二表达盒, 所述第二表达盒含有第二核酸分子, 所述第二 核酸分子编码花粉失活基因; 第三表达盒, 所述第三表达盒含有第三核酸分子, 所述第三 核酸分子编码除草剂抗性基因。 利用该构建体, 能够有效地将油菜、 水稻雄性不育恢复基 因和花粉失活基因分别引入到油菜、 水稻植株例如纯合隐性雄性不育植株中, 通过除草剂 筛选, 从而得到携带外源基因的可育株作为保持系, 从而可以方便地通过与不育系授粉杂 交以繁殖不育系, 并通过自交源源不断地生产保持系。 另外, 不携带外源基因的植株可以 用作杂交制种之中的亲本。 由此, 可以有效地用于油菜、 水稻杂交育种。
在本文中, 构建体的形式不受特别限制, 根据本发明的具体示例, 其可以为质粒、 噬 菌体、人工染色体、粘粒(Cosmid)、病毒的至少一种。根据本发明的具体示例, 构建体(有 时也称为表达载体、遗传载体或载体)呈质粒的形式。质粒作为遗传载体, 具有操作简单, 可以携带较大片段的性质, 便于操作和处理。 质粒的形式也不受特别限制, 既可以是环形 质粒, 也可以是线性质粒, 即可以是单链的, 也可以是双链的。 本领域技术人员可以根据 需要进行选择。 根据本发明的实施例, 可以采用 Ti载体, 例如可以采用将第一、 第二和第 三表达盒设置在表达载体 pOsSI或 pBnSI的 T-DNA之左右边界之间。 由此, 可以通过农杆 菌介导的转化方法将第一、 第二和第三表达盒转化至受体植株, 例如油菜 brms2 和 brmsl 隐性核雄性不育突变体或水稻 osfg2 隐性核雄性不育突变体中。 由此, 可以获得油菜或水 稻转化株系。如此获得的转化株系有如下特点: (1 )转化位点在各世代始终处于杂合状态, 因此有一半花粉不含外源基因, 一半含有外源基因, 含外源基因的花粉失活 (即失去授精 能力), 所以外源基因仅通过雌配子传递至下一代, 不会通过花粉漂移到环境中; (2) 转化 体自交可结实, 所结可育种子 (含有抗除草剂基因) 与不育种子 (不含有抗除草剂基因)
的比例为 1 : 1, 通过除草剂筛选, 可以将两类种子区分开来, 其中可育株(带有外源基因) 抗除草剂筛选存活下来用作保持系, 可以通过与不育系杂交授粉方便地、 源源不断地繁殖 不育系, 另一方面, 可育株也可以通过自交方便地、 源源不断地生产保持系, 而通过保持 系与不育系杂交授粉繁殖的不育株(不含转基因成分)在生产上用作杂交制种的亲本; (3 ) 因为不育系不含转基因, 因此用其生产的杂交种子不含转基因, 用此杂交种生产的商品粮 食更不含转基因, 从而消除了转基因生物安全的隐患。 该新型杂交育种体系为充分利用植 物杂种优势提供了切实可行的技术新突破。
在本发明中所使用的术语 "核酸"可以是任何包含脱氧核糖核苷酸或者核糖核苷酸的 聚合物, 包括但不限于经过修饰的或者未经修饰的 DNA、 RNA, 其长度不受任何特别限制。 对于用于构建重组细胞的载体, 优选核酸为 DNA, 因为 DNA相对于 RNA而言, 其更稳定, 并 且易于操作。
油菜为四倍体, 含有 AA基因组和 CC基因组, 油菜细胞核隐性核不育通常由两对基因 控制, 如隐性核不育材料 S45AB的育性受两个隐性基因 (BnMSl和 BnMS2 ) 调控, 当两个基 因都为隐性纯合时表现为不育。 隐性核不育材料 9012AB 的不育性状受双基因 (BnMs3, BnMs4)控制。 根据本发明的实施例, 油菜雄性不育恢复基因的类型并不受特别限制。 在本 发明的一个实施例中, 所述油菜雄性不育恢复基因编码具有如 SEQ ID NO: 1所示氨基酸序 列的蛋白质, 可以采用的油菜雄性不育恢复基因为 BrMS2, 可以将其作为油菜受体 brms2和 brmsl纯合突变体(完全雄性不育) 的野生型育性恢复基因。 据本发明的具体实施例, 在本 发明的一个实施例中,所述油菜雄性不育恢复基因具有如 SEQ ID NO: 2所示的核苷酸序列, 与油菜内源的雄性不育基因 BnMS2 (其核苷酸序列如 SEQ ID NO: 3所示) 相比, 本发 明载体中的雄性不育恢复基因有 8个单碱基突变, 其中 6个是同义突变, 2个是错义 突变, 导致两处(第 179位和第 297位)编码的氨基酸发生改变。 该序列能够有效地使油 菜 brms2/brms2和 brmsl/brmsl不育受体植株的育性得到恢复。在本发明的一个实施例中, 所述油菜转化载体的第一表达盒还可以进一步包括: 第一启动子, 所述第一启动子与所述 第一核酸分子可操作地相连; 以及第一终止子, 所述第一终止子与所述第一核酸分子可操 作地相连。 根据本发明的实施例, 第一启动子和第一终止子的类型并不受特别限制。 根据 本发明的一个实施例, 对于 BrMS2基因, 可以采用 BrMS2的内源启动子、 0RF区及终止区的 序列, 均为野生油菜基因组序列。在本发明的一个实施例中, 所述第一启动子具有如 SEQ ID NO: 4所示的核苷酸序列。 在本发明的一个实施例中, 所述第一终止子具有如 SEQ ID NO: 5所示的核苷酸序列。 发明人惊奇地发现, 利用该启动子和终止子的组合, 能够进一步显著
地提高表达相应蛋白的效率, 进而能够提高利用构建体构建保持系的效率,并且能够更有效 地使油菜 brms2和 brmsl不育受体植株的育性得到恢复。
根据本发明的实施例, 油菜花粉失活基因的类型并不受特别限制。 已知的花粉失活基 因可以编码碳水化合物降解或修饰酶、淀粉酶、脱支酶和果胶酶, 茁长素(auxin)、 rol B, 细胞毒素、白喉毒素、 DAM甲基化酶、亲和素,或者可选自原核调控***。举例而言, Mariani, et al. , Nature Vol. 347 ; pp. 737 ; (1990)表明, Aspergi l lus oryzae RNase_Tl或 Baci l lus amylol iquefaciens的 RNase (被命名为 "barnase ")在绒毡层中的表达诱导了绒毡层细胞 的破裂, 导致雄性不能生育。 Quaas, et al. , Eur. J. Biochem. Vol. 173 : pp. 617 (1988) 描述了 RNase-Tl的化学合成, 而 barnase基因的核苷酸序列由 Hartley, J. Molec. Biol.; Vol. 202 : pp. 913 (1988)公开。 Agrobacterium rhizogenes的 rolB基因编码通过从吲 哚酚 -β -甙释放游离吲哚从而干扰茁长素代谢的酶。 Estruch, et al. , ΕΜΒΟ J. Vol. 11 : pp. 3125 (1991)和 Spena, et al. , Theor. Appl. Genet.; Vol. 84 : pp. 520 (1992)表 明, 烟草中 rolB基因的花粉囊 -特异性表达产生了花粉囊枯萎的植株, 其中, 花粉的产生 大大减少, rolB基因是可用于控制花粉产生的基因的例子。 Sl ightom, et al. , J. Biol. Chem. Vol. 261 : pp. 108 (1985)公开了 rolB基因的核苷酸序列。 编码白喉毒素基因的 DNA分子 可从 American Type Culture Col lection (Rockvi l le, MD), ATCC No. 39359或 ATCC No. 67011获得, 关于例子和使用方法, 见 Fabijanski, et al. , Ε. P. Appl. No. 90902754. 2, "Molecular Methods of Hybrid Seed Production" 。 DAM 甲基化酶基因用于在美国专利 No. 5, 689, 049 和 PCT/US95/15229 Cigan, A. M. and Albertsen, M. C. , "Reversible Nuclear Genetic System for Male Steri l ity in Transgenic Plants. "讨论的方法中导 致不育。 还见 Albertsen et al的美国专禾 lj No. 5, 962, 769 " Induction of Male Steri l ity in Plants by Expression of High Levels of Avidin"对亲禾口素基因导致不育的讨论。
根据本发明的实施例, 所述花粉失活基因 BrSZl编码具有如 SEQ ID NO: 6所示氨基酸 序列的蛋白质, 由此, 可以编码 Barnase 蛋白。 根据本发明的实施例, 所述花粉失活基因 BrSZl具有如 SEQ ID N0: 7所示的核苷酸序列。 由此, 可以进一步提高表达相应蛋白的效 率。 根据本发明的实施例, 第二表达盒进一步包括: 第二启动子, 所述第二启动子与所述 第二核酸分子可操作地相连, 所述第二启动子为花粉特异性启动子; 以及第二终止子, 所 述第二终止子与所述第二核酸分子可操作地相连。 由此, 可以更有效的提高相应基因的表 达效率。 另外, 根据本发明的实施例, 在第二表达盒以外, 构建体中还可以进一步包括编 码 Pr2蛋白的表达盒, 所述 Pr2编码 SEQ ID N0: 8所示氨基酸序列的蛋白质, 由此, 可以
编码 Barstar蛋白,该表达盒的启动子为 P2和终止子为 T2序列分别如 SEQ ID NO: 13和 SEQ ID NO: 14所示, 构成 P2 : : Pr2 : : T2表达盒。 由此, 第二表达盒可以有效地编码花粉失活蛋 白, 可以通过花粉特异性启动子使得目的基因 (花粉失活基因) 能够被靶向定位到特定的 细胞中, 而 Pr2表达框编码的 Pr2蛋白, 则可以在花粉以外的组织器官抑制 BrSZl的泄露 表达,最终可以使含有转基因的花粉失活,但是又不造成植物其它组织器官的非预期表型。 该设计使得所有含有此基因的转基因花粉失活, 不能授精, 还能严格防止基因漂移等生物 安全问题, 失活的花粉不能与周围其它植株或杂草授粉, 因而转基因不能通过花粉漂移到 环境中。
另外, 根据本发明的实施例, 油菜转化的构建体还可以进一步包括: 第三表达盒, 所 述第三表达盒包含第三核酸分子, 所述第三核酸分子编码抗除草剂基因, 所述抗除草剂基 因的类型并不受特别限制。 由此, 便于通过抗除草剂基因的表达来确定植物是否含有构建 体所引入的基因。 根据本发明的实施例, 可以采用选自抗草甘膦、 抗草丁膦、 抗百草枯、 抗咪唑啉酮基因的至少一种作为筛选基因。 在本发明的一个实施例中, 可以采用 Pr4 蛋白 作为抗除草剂基因, 跟野生型油菜植株的基因相比, 该突变基因 319位点核苷酸 G变为 A, 导致所编码的氨基酸位点由 Ala变为 Thr, 含有该突变基因的油菜具有除草剂抗性, 过表达 该突变基因可以使转基因植物具有咪唑啉酮类除草剂抗性。 在本发明的一个实施例中, 所 述第三表达盒进一步包括: 第三启动子, 所述第三启动子与所述第三核酸分子可操作地相 连, 所述第三启动子为组成型启动子; 第三终止子, 所述第三终止子与所述第三核酸分子 可操作地相连。 在本发明的一个实施例中, 所述第三启动子 P4具有如 SEQ ID NO: 16所示 的核苷酸序列。 在本发明的一个实施例中, 所述第三终止子 T3具有如 SEQ ID NO: 11所示 的核苷酸序列。 由此, 含有该表达框的水稻种子在除草剂筛选下, 可以呈现抗与不抗除草 剂两类种子, 因此该表达框在本发明中用于辨认和分选保持系种子。
由此, 根据本发明的实施例, 可以利用根据本发明的实施例的构建体, 以非转基因隐 性核雄性不育油菜(brms2/brms2和 brmsl/brmsl)作为转化的受体, 进行遗传转化, 得到整 合含有以下紧密连锁的四个外源基因 Pr4, BrMS2, BrSZl , Pr2的油菜保持系。 外源基因的 ***与内源雄性不育位点 (brms2/brms2和 brmsl/brmsl ) 是非连锁的, 因此得到的转基因 油菜保持系含有独立的纯合的 brms2和 brmsl隐性不育位点及杂合的外源基因整合位点。
由此, 可以通过常规技术, 例如农杆菌介导法, 将前述构建体引入到油菜的细胞、 组 织或器官中, 以便得到可以后续用于研究、 杂交的样本。 因而, 在本发明的第二方面, 本 发明提出了一种油菜细胞、 组织或器官。 根据本发明的实施例, 该油菜细胞、 组织或器官
中含有前面所述的构建体。 在本发明的一个实施例中, 所述油菜细胞、 组织或器官来自油 菜纯合隐性雄性不育植株。 在本发明的一个实施例中, 所述油菜纯合隐性雄性不育植株包 含 BrMS2 基因的纯合隐性等位基因。 由此, 本发明的油菜细胞、 组织或器官, 可以有效地 用于构建保持系并繁殖雄性不育系。 前面关于构建体所描述的特征和优点, 也适用于该油 菜细胞、 组织或器官, 不再赘述。
根据本发明的实施例, 水稻雄性不育恢复基因的类型并不受特别限制。 在本发明的一 个实施例中, 所述水稻雄性不育恢复基因编码具有如 SEQ ID NO: 18所示氨基酸序列的蛋 白质。即,可以采用的水稻雄性不育恢复基因为 0sFG2, 由此,可以将其作为水稻受体 osfg2 纯合突变体(完全雄性不育) 的野生型育性恢复基因。 0sFG2基因编码的蛋白, 在花药发育 阶段特异表达。 根据本发明的具体实施例, 在本发明的一个实施例中, 所述水稻雄性不育 恢复基因具有如 SEQ ID NO: 19所示的核苷酸序列, 该序列能够有效地使水稻 osfg2/OSfg2 不育受体植株的育性得到恢复。 水稻受体 osfg2纯合突变体是经过 EMS诱变黄华占品种所 得, 在黄华占不育突变体中, 与野生型黄华占相比, 该基因编码区的第 1688位点由 G突变 为 A, 导致对应的其编码的蛋白序列的第 563位点的氨基酸由甘氨酸 (G) 变为天门冬氨酸 (D) 。 在本发明的一个实施例中, 所述水稻转化载体的第一表达盒还可以进一步包括: 第 一启动子, 所述第一启动子与所述第一核酸分子可操作地相连, 所述第一启动子为雄配子 特异性启动子; 以及第一终止子, 所述第一终止子与所述第一核酸分子可操作地相连。 根 据本发明的实施例, 第一启动子和第一终止子的类型并不受特别限制。 根据本发明的一个 实施例, 对于 0sFG2基因, 可以采用 0sFG2的内源启动子、 0RF区及终止区的序列, 均为野 生水稻基因组序列。 在本发明的一个实施例中, 所述第一启动子具有如 SEQ ID NO: 20所 示的核苷酸序列。 在本发明的一个实施例中, 所述第一终止子具有如 SEQ ID NO: 21 所示 的核苷酸序列。 发明人惊奇地发现, 利用该启动子和终止子的组合, 能够进一步显著地提 高表达相应蛋白的效率, 进而能够提高利用构建体构建保持系的效率,并且能够更有效地使 水稻 osfg2不育受体植株的育性得到恢复。
根据本发明的实施例, 水稻花粉失活基因的类型并不受特别限制。 根据本发明的实施 例, 所述花粉失活基因编码具有如 SEQ ID NO: 22 所示氨基酸序列的蛋白质。 由此, 可以 编码 Zm-AAl所编码的 α -淀粉酶。根据本发明的实施例,所述花粉失活基因具有如 SEQ ID NO: 23所示的核苷酸序列。由此,可以进一步提高表达相应蛋白的效率。根据本发明的实施例, 第二表达盒进一步包括:第二启动子,所述第二启动子与所述第二核酸分子可操作地相连, 所述第二启动子为花粉特异性启动子; 以及第二终止子, 所述第二终止子与所述第二核酸
分子可操作地相连。 由此, 可以更有效的提高相应基因的表达效率。 另外, 根据本发明的 实施例, 在第二表达盒中还可以进一步包括编码导肽的序列, 由此, 第二表达盒可以有效 地编码具有导肽的花粉失活蛋白, 由此, 可以使得目的基因 (花粉失活基因) 能够被靶向 定位到特定的细胞器中。 例如, 根据本发明的实施例, 编码导肽的序列具有如 SEQ ID N0: 24所示的核苷酸序列 (来自于玉米的 brittle-Ι基因的编码导肽(TP) 的序列)。 由此, 可 以有效地将所表达的蛋白靶向淀粉体, 分解花粉中的淀粉, 从而使花粉失去活力, 丧失授 精能力, 造成转基因花粉失活。 进而, 根据本发明的具体实施例, 该基因在玉米花粉特异 性启动子 PG47驱动下, 与来自于玉米的 brittle-Ι基因的编码导肽 (TP) 的序列及终止子 IN2-1组成表达框, 可在发育后期的成熟花粉中特异性表达淀粉酶, 并靶向淀粉体, 分解花 粉中的淀粉, 从而使花粉失去活力, 丧失授精能力, 造成转基因花粉失活。 该设计使得所 有含有此基因的转基因花粉失活, 不能授精还能严格防止基因漂移等生物安全问题, 失活 的花粉不能与周围其它植株或杂草授粉, 因而转基因不能通过花粉漂移到环境中。
另外, 根据本发明的实施例, 水稻转化的构建体还可以进一步包括: 第三表达盒, 所 述第三表达盒包含第三核酸分子, 所述第三核酸分子编码抗除草剂基因, 所述抗除草剂基 因的类型并不受特别限制。 由此, 便于通过抗除草剂基因的表达来确定植物是否含有构建 体所引入的基因。 根据本发明的实施例, 可以采用选自抗草甘膦、 抗草丁膦、 抗百草枯、 抗咪唑啉酮基因的至少一种作为筛选基因。 在本发明的一个实施例中, 可以采用 Pr5 蛋白 作为抗除草剂基因, 该基因是 OsALS 548 (核苷酸 1642、 1643位由 TG变为 AT, 导致 548 位氨基酸由 Trp变为 Met)位点突变后的具有除草剂抗性的水稻基因, 过表达该突变的水稻 基因可以使转基因植物具有咪唑啉酮类除草剂抗性。 在本发明的一个实施例中, 所述第三 表达盒进一步包括: 第三启动子, 所述第三启动子与所述第三核酸分子可操作地相连, 所 述第三启动子为组成型启动子; 第三终止子, 所述第三终止子与所述第三核酸分子可操作 地相连。 在本发明的一个实施例中, 所述第三启动子具有如 SEQ ID NO: 25所示的核苷酸 序列。 在本发明的一个实施例中, 所述第三终止子具有如 SEQ ID NO: 11 所示的核苷酸序 列。 由此, 含有该表达框的水稻种子在除草剂筛选下, 可以呈现抗与不抗除草剂两类种子, 因此该表达框在本发明中用于辨认和分选保持系种子。
由此, 根据本发明的实施例, 可以利用根据本发明的实施例的构建体, 以非转基因隐 性核雄性不育水稻 (osfg2/OSfg2)作为转化的受体, 进行遗传转化, 得到整合含有以下紧密 连锁的三个外源基因 Pr5, 0sFG2, Zm-AAl的水稻保持系。 外源基因的***与内源雄性不育 位点 (OSfg2/OSfg2) 是非连锁的, 因此得到的转基因水稻保持系含有独立的纯合的 osfg2
隐性不育位点及杂合的外源基因 (包括 0SFG2基因) 整合位点。
由此, 可以通过常规技术, 例如农杆菌介导法, 将前述构建体引入到水稻的细胞、 组 织或器官中, 以便得到可以后续用于研究、 杂交的样本。 因而, 在本发明的第二方面, 本 发明提出了一种水稻细胞、 组织或器官。 根据本发明的实施例, 该水稻细胞、 组织或器官 中含有前面所述的构建体。 在本发明的一个实施例中, 所述水稻细胞、 组织或器官来自水 稻纯合隐性雄性不育植株。 在本发明的一个实施例中, 所述水稻纯合隐性雄性不育植株包 含 0sFG2 基因的纯合隐性等位基因。 由此, 本发明的水稻细胞、 组织或器官, 可以有效地 用于构建保持系并繁殖雄性不育系。 前面关于构建体所描述的特征和优点, 也适用于该水 稻细胞、 组织或器官, 不再赘述。
由此, 在本发明的第 2 方面, 本发明提出了一种构建油菜或水稻雄性不育系的方法。 根据本发明的实施例, 参考图 7, 该方法包括: 将前面所述的油菜或水稻构建体引入到第一 油菜或水稻纯合隐性雄性不育植株中, 以便获得携带外源基因的第二油菜或水稻植株, 所 述第二油菜或水稻植株能够产生可育雄性配子, 并且第二油菜或水稻植株中的外源基因处 于杂合状态, 因此第二油菜或水稻植株中有一半花粉不含外源基因, 一半含有外源基因, 含外源基因的花粉失活 (即失去授精能力)。 进而培育所得到的第二油菜或水稻植株, 通过 第二油菜或水稻植株即转化体与其相应的不育植株杂交授精, 可以使不育植株恢复结实得 到不携带外源基因的种子,从而使油菜或水稻雄性不育系得以繁殖。根据本发明的实施例, 第一油菜或水稻纯合隐性雄性不育植株包含 BrMSl/BrMS2 或 0sFG2基因的纯合隐性等位基 因。 另外, 根据本发明的实施例, 可以通过除草剂筛选进行分拣的步骤, 即通过检测油菜 或水稻种子是否抗除草剂来进行分拣, 区分其是否携带外源基因。 前面关于构建体所描述 的特征和优点, 也适用于该方法, 不再赘述。
在本发明的第 3 方面, 本发明提出了一种恢复油菜或水稻不育植株雄性育性的方法。 根据本发明的实施例, 该方法包括: 将前面所述的油菜或水稻构建体引入到油菜或水稻纯 合隐性雄性不育植株中。 在本发明的一个实施例中, 所述油菜或水稻纯合隐性雄性不育植 株本别包含 BrMSl/BrMS2或 0sFG2基因的纯合隐性等位基因。 前面关于构建体所描述的特 征和优点, 也适用于该方法, 不再赘述。
在本发明的第 4 方面, 本发明提出了一种制备油菜或水稻种子的方法。 根据本发明的 实施例, 该方法包括以下步骤: 将前面所述的油菜或水稻构建体分别引入到油菜或水稻植 株中; 以及将所述油菜或水稻植株自体受精, 以获得含有前面所述的构建体的种子。 在本 发明的一个实施例中, 所述油菜或水稻植株为油菜或水稻纯合隐性雄性不育植株。 在本发
明的一个实施例中, 所述油菜或水稻纯合隐性雄性不育植株包含 BrMSl/BrMS2或 0sFG2基 因的纯合隐性等位基因。
在本发明的第 5 方面, 本发明提出了一种用于制备杂交油菜或水稻的方法。 根据本发 明的实施例, 该方法采用油菜或水稻雄性不育系, 该油菜或水稻雄性不育系是通过前面构 建油菜或水稻雄性不育系的方法构建的。 由此, 可以进一步利用本发明的油菜或水稻雄性 不育系进行杂交, 提高油菜或水稻杂交的效率。 前面关于构建体所描述的特征和优点, 也 适用于该方法, 不再赘述。
在本发明的第 6方面,本发明提出了油菜或水稻雄性不育系在制备杂交种子中的用途。 根据本发明的实施例, 所述油菜或水稻雄性不育系是通过前面构建油菜或水稻雄性不育系 的方法构建的。由此,可以进一步利用本发明的油菜或水稻雄性不育系与恢复系进行杂交, 筛选优良的杂交组合, 提高杂交制种的效率。 前面关于构建体所描述的特征和优点, 也适 用于该用途, 不再赘述。 需要说明的是, 根据本发明实施例的构建体及其用途是本申请的发明人经过艰苦的创 造性劳动和优化工作才完成的。 具体实施方式述中, 除非另有说明, "多个" 的含义是两个 或两个以上。
具体实 ¾fc¾r式
下面根据具体的实施例对本发明进行说明。 需要说明的是, 这些实施例仅仅是为了说 明本发明, 而不能以任何方式解释为对本发明的限制。 另外, 除非特别说明, 在下面的实 施例中所涉及的方法为常规方法, 可以参照 《分子克隆实验指南》 第三版或者相关产品进 行。 所用试剂或仪器未注明生产厂商者, 均为可以通过市购获得的常规产品。
实施例 1: 油菜表达载体 pBnSI的构建:
在构建油菜的植物表达载体之前, 发明人首先分别对表达载体内的每个表达盒单独进 行了油菜转化, 并进一步对各个表达盒的功能进行了验证。 结果表明各个表达盒单独转化 油菜时, 都能够工作良好, 达到预期的设计效果。 进一步发明人构建了以下表达载体。 其 中抗除草剂基因参考中国专利 201310116228. 2, 本发明通过引用并入本专利。
通过装配 BrSZl、 Pr2、 BrMS2和 Pr4表达框的各个元件, 来构建如图 1所示的 pBnSI 载体。 该载体含有 4个表达框, 其中, 第一表达框为 barnase的表达框, 组成为来自拟南 芥基因组的花粉特意表达启动子 Pl, 如序列表中 SEQ ID NO: 4所示, 细菌来源的 barnase 基因编码区 BrSZl , 如需列表中 SEQ ID NO: 7 所示, 以及来自于拟南芥 Rbcs基因的终止
子 Tl, 如序列表中 SEQ ID NO: 5所示, 构成第一表达框 PI : : BrSZl : : T1 ; 第二表达框为 barstar表达框, 组成为来自于烟草花叶病毒的组成型启动子 35S启动子 P2, 如序列表中 SEQ ID NO: 13所示, 人工合成的来自于细菌的 barstar基因 Pr2, 如序列表中 SEQ ID NO: 8所示,以及 35S终止子 T2,如序列表中 SEQ ID NO: 14所示,构成第二表达框 P2:: Pr2 :: T2; 第三表达框为油菜育性恢复基因表达框 BrMS2,该表达框包括来自油菜基因组的 BrMS2启动 子, 其核苷酸序列如序列表中 SEQ ID NO: 26所示, 和来自油菜基因组的 BrMS2基因编码 区, 其核苷酸序列如序列表中 SEQ ID NO: 2所示, 以及人工合成的 rbcs 3A终止子 T3, 如 序列表中 SEQ ID N0: 15所示,构成第三表达框 BrMS2;第四表达框为抗除草剂基因表达框, 组成为 N0S启动子 P4, 如序列表中 SEQ ID NO: 16所示, 抗除草剂基因 Pr4, 如序列表中 SEQ ID NO: 17 所示, Nos 终止子 T4, 如序列表中 SEQ ID NO: 11 所示, 构成第四表达框 P4 :: Pr4 :: T4。
将上述各表达框的元件测序验证后, 在 PCAMBIA1301 载体中依次连入上述片段, 最终 得到植物表达载体 pBnSI , 如图 1所示。
实施例 2: 油¾ ^化
利用电激法将质粒 PBnSI转入农杆菌 EHA105菌株, 利用农杆菌介导的蘸花法对含有纯 合的 brms2和 brmsl隐性不育位点的油菜进行遗传转化,得到 22株单拷贝转基因植株材料。 所述的含有纯合的 brms2和 brmsl隐性不育位点的油菜转化受体材料为甘蓝型油菜。
实施例 3: 转基因油菜植株的花粉育性检测
对实施例 2所得到的 22株单拷贝转基因油菜 (含有纯合的 brms2和 brmsl隐性不育位 点) 植株进行分析发现, 转基因植株和非转基因对照植株之间没有明显的形态差异, 但是 花粉育性明显不同。
对 pBnSI 构建体转化油菜得到的转基因植株材料, 进行花粉可染率检测, 同时对野生 型油菜进行花粉可染率检测 (图 2)。
采用的方法为: 在油菜开花期, 从转基因油菜植株、 及其野生型对照植株各随机抽取 单株, 各株取一朵花, 每朵花取 1个花药, 置于载玻片中央, 滴加一滴 1%的 Alexander溶 液, 用镊子和解剖针释放花粉后, 盖上盖玻片, 在显微镜下观察、 计数可染色花粉数和花 粉总数, 染成橘红色的为可育花粉, 绿色的为败育花粉 (图 2 显示了染色后的可育花粉粒 和不可育花粉粒)。
分析转基因油菜植株的花粉可染率, 结果显示对照植株的橘红色花粉占 98%〜100%; 而 多个随机抽取的转基因植株中, 正常花粉与败育花粉比例接近 1 : 1, 表明所构建的保持系
可以产生等量的携带外源基因的花粉和不携带外源基因的花粉, 即构建体 pBnSI使转基 因株系花粉的 50%失活。该结果表明本发明所提供的载体能够达到预期的花粉失活功能。 BP : 一方面 barnase基因的表达由花粉特异性启动子驱动, 另一方面由 35S组成型启动子驱动 barstar基因, 这样, barnase在花粉中特异性高表达, 可以致死雄配子, 从而达到花粉败 育的目的, 同时, barnase在花粉以外的其它植物组织中的表达泄露, 就可以完全被组成型 表达的 barstar所彻底抑制, 因此植株的其他表型完全正常。
实施例 4: 转基因油菜植株的抗除草剂种子与不抗除草剂种子分离分析
对实施例 2所得到的 22株单拷贝转基因油菜植株 (含有纯合的 brms2和 brmsl隐性不 育位点)所结 T1代种子进行抗除草剂性分离比例调查, 结果表明这些种子均显示 1 : 1分离 比(图 3), 即携带外源基因的抗除草剂种子和不携带外源基因的不抗除草剂种子表现为 1 : 1 分离, 表明本发明所提供的载体各元件作为整体表达达到预期的花粉失活功能和种子 筛选标记功能。
实施例 5: 水稻表达载体 pOsSI的构建:
在构建水稻的植物表达载体之前, 发明人首先分别对表达载体内的每个表达盒单独进 行了水稻转化, 并进一步对各个表达盒的功能进行了验证。 结果表明各个表达盒单独转化 水稻时, 都能够工作良好, 达到预期的设计效果。进一步发明人构建了以下水稻表达载体。 其中抗除草剂基因参考中国专利 201210037789. 9, 本发明通过引用并入本专利。
通过装配 Zm-AA1、 0sFG2和 Pr5表达框各个元件, 来构建图 4所示的 pOsSI载体。 该 载体含有 3个表达框, 其中, 第一表达框为 Zm-AAl的表达框, 组成为来自玉米基因组的花 粉特异表达启动子 PG47, 如序列表中 SEQ ID NO: 28所示, 融合了 brittle-1导肽序列的 玉米来源的 Zm-AAl基因编码区, 如序列表中 SEQ ID NO: 29所示, 以及来自于玉米基因组 的终止子 In2-1, 如序列表中 SEQ ID NO: 30 所示, 构成第一表达框 PG47 :: TP :: Zm-AAl:: In2-1 ; 第二表达框为水稻育性恢复基因 0sFG2表达框, 组成为来自于水稻基 因组的启动子 P5, 如序列表中 SEQ ID NO: 31所示, 来自水稻基因组的 0sFG2基因编码区, 如序列表中 SEQ ID NO: 32所示, 以及来源的水稻基因组的终止子 T5, 如序列表中 SEQ ID NO: 33所示, 构成第二表达框 P5 : : 0sFG2 : : T5; 第三表达框为抗除草剂基因表达框, 组成 为来自玉米基因组的组成型表达启动子 Ubi, 如序列表中 SEQ ID NO: 25所示, 抗除草剂基 因 Pr5, 如序列表中 SEQ ID NO: 27所示, 以及人工合成的终止子 T3, 如序列表中 SEQ ID NO: 15所示, 构成第四表达框 P6 : : Pr5 : : T6。
将上述各表达框的元件测序验证后, 在 PCAMBIA1301 载体中依次连入上述片段, 最终
得到植物表达载体 pOsSI , 如图 4所示。
实施例 6: 水稻转化
利用电激法将质粒 pOsSI转入农杆菌 AglO菌株,利用农杆菌介导法对含有纯合的 osfg2 隐性不育位点的水稻进行遗传转化, 得到 32株单拷贝转基因植株材料。 具体的转化受体材 料为水稻黄华占品种。
实施例 7: 转基因水稻植株的花粉育性检测
对实施例 6所得到的 32株单拷贝转基因水稻 (含有纯合的 osfg2隐性不育位点)植株 进行分析发现, 转基因植株和非转基因对照植株之间没有明显的形态差异, 但是花粉育性 明显不同。
对 pOsSI 构建体转化水稻得到的转基因植株材料, 进行花粉可染率检测, 同时对野生 型水稻进行花粉可染率检测 (图 5)。
采用的方法为: 在水稻开花期, 从转基因水稻植株、 及其野生型对照植株各随机抽取 单株, 各株取一朵花, 每朵花取 1个花药, 置于载玻片中央, 滴加一滴 1%的 I2-KI溶液, 用镊子和解剖针释放花粉后, 盖上盖玻片, 在显微镜下观察、 计数可染色花粉数和花粉总 数, 可以着色为深蓝色的为可育花粉, 而不能够着色的为败育花粉 (图 5 显示了染色后的 可育花粉粒和不可育花粉粒)。 分析转基因水稻植株的花粉可染率。
结果显示对照植株的可着色的花粉占 98%〜100%; 而多个随机抽取的转基因植株中, 正 常花粉 (可着色) 与败育花粉 (不能着色) 比例接近 1 : 1, 表明所构建的保持系可以产生 等量的携带外源基因的花粉和不携带外源基因的花粉, 即构建体 pOsSI使转基因株系花 粉的 50%失活。 该结果表明本发明所提供的载体能够达到预期的花粉失活功能。
实施例 8: 转基因水稻植株的抗除草剂种子与不抗除草剂种子分离分析
对实施例 6所得到的 32株单拷贝转基因水稻植株 (含有纯合的 osfg2隐性不育位点) 所结 T1代种子进行抗除草剂性分离比例调查,结果表明这些种子均显示 1: 1分离比(图 6), 即携带外源基因的抗除草剂种子和不携带外源基因的不抗除草剂种子表现为 1 : 1分离, 表明本发明所提供的载体各元件作为整体表达达到预期的花粉失活功能和种子筛选标记功 能。
实施例 9: 创制保持系、 繁殖^ 系及生产杂交种的技术路线
通过以上在油菜和水稻中经过一系列实施例创制新型保持系和不育系,发明人提出 以下创制保持系、繁殖不育系及生产杂交种的技术路线(图 7): 该技术涉及三个连锁的功 能元件: 花粉致死基因, 抗除草剂基因, 育性恢复基因。 其中所选用的花粉致死基因在
花粉中表达的时间、水平及致死效率都直接关系到该技术能否成功应用, 因此本发明利 用花粉发育后期特异性启动子驱动花粉失活基因在花粉成熟期特异表达,实现了特异性 花粉致死的效果;利用植物内源启动子控制的雄性不育恢复核基因转化对应的植物雄性 不育植株, 使转基因植株恢复育性; 利用抗除草剂基因可以方便地将转基因植株自交后 产生的两类种子分开:不携带转基因的不育种子,没有除草剂抗性,可通过除草剂筛除, 携带转基因的可育种子,具有除草剂抗性,可以作为保持系存活下来,由此可以方便地、 源源不断地得到保持系; 保持系进一步与不育系授粉杂交, 使不育系恢复结实, 得到不 含有转基因的不育系, 使不育系得到繁殖; 由此得到的不育系既可以与任意恢复系父本 杂交, 用于杂交种子的生产。利用本发明构建的新型育性调控载体转化植物雄性不育植 株, 得到的转基因植物植株恢复育性, 其花粉育性和种子的除草剂抗性均表现预期的结 果。
Claims
1. 一种繁殖不育系的方法, 所述方法包括:
(a)提供第一植株, 所述第一植株为不育系, 所述不育系含有一个纯合隐性的等位不育 基因;
(b) 向第二植株中引入下述构建体, 所述第二植株含有一个纯合隐性的等位不育基因, 所述构建体包含:
( i )第一核苷酸序列, 当其在所述第一或第二植株中表达时, 能功能性互补所述植株的 纯合隐性植物不育性状;
( ϋ ) 第二核苷酸序列, 其表达抑制所述第二植株中雄性配子的形成或功能, 从而使得 在含有所述隐性等位基因的所述第二植株中产生的雄配子不含所述构建体;
( iii )第三核苷酸序列,该序列编码产物的表达能用于选择具有所述构建体的植物细胞; 以及
( c )用所述第二植株的所述雄性配子使所述第一植株受精, 以繁殖保持了所述第一 植株纯合隐性等位状态的后代; 其特征在于所述的第三核苷酸序列选自抗草甘膦、抗草丁膦、抗百草枯、抗草铵膦、 抗莠去津、 抗溴苯腈、 抗 2, 4-D、 抗咪唑啉酮或抗磺酰脲类等除草剂的基因构成的 组。
2. 权利要求 1所述的方法, 其中所述第一核苷酸序列为育性恢复基因, 选自 0sFG2、 BrMS2 MSP1、 PAIR1、 PAIR2、 ZEP1、 MELL, PSS1、 TDR、 UDT1、 GAMYB4、 PTC1、 API5、 WDA1、 CYP704B2、 MS26、 MS22、 DPW、 MADS3、 0SC6、 RIP1、 CSA或 AID1构成的组。
3. 权利要求 2所述的方法,其中所述的第一核苷酸序列与第四核苷酸序列可操作地相连, 所述第四核苷酸序列指导偏好于雄性植物细胞的表达。
4. 权利要求 3所述的方法,其中所述的第四核苷酸序列仅在存在诱导物质或诱导条件时 才具有功能。
5. 权利要求 3所述的方法,其中所述的第四核苷酸序列选自 0sFG2、BrMS2、MSPl、PAIRl、 PAIR2、 ZEP1、 MELL, PSS1、 TDR、 UDT1、 GAMYB4、 PTC1、 API5、 WDA1、 CYP704B2, MS26、 MS22、 DPW、 MADS3、 0SC6、 RIP1、 CSA、 AID1、 5126、 Ms26、 Ms22或 Ms45的雄 性组织调控序列构成的组。
6. 权利要求 1所述的方法, 其中第二核苷酸序列选自 DAM甲基化酶基因、 Zea mays a 淀 粉酶基因、 细胞毒素编码基因、 或 Barnase与 Barstar的组合序列构成的组。
权 利 要 求 书
7. 权利要求 6所述的方法,其中所述的第二核苷酸序列与第五核苷酸序列可操作地相连, 所述第五核苷酸序列指导偏好于雄性配子的表达。
8. 权利要求 7所述的方法, 其中所述的第五核苷酸序列选自聚半乳糖醛酸酶 47基因、 Zml3基因、 果胶甲基酯酶基因、 钙调蛋白结合蛋白基因、 肌动蛋白解聚因子基因、 prol fi l in基因和硫酸化五肽 phytosulphokine基因的调控区域构成的组。
9. 权利要求 1所述的方法, 其中所述的第三核苷酸序列具体为具有除草剂抗性的 ALS基 因突变体。
10. 权利要求 9所述的方法,其中所述的 ALS基因突变体在水稻中的突变为 Trp548、 Ala96 和 /或 Ser627突变, 优选带有 Trp548Cys、 Trp548Met Ala96Val、 Ala96Thr和 /或 Ser627Asn 突变。
11. 权利要求 9所述的方法,其中所述的 ALS基因突变体在油菜中的突变为 Alal07、Alal90、 Trp559和 /或 Ser638突变。
12. 权利要求 9所述的方法,其中所述的第三核苷酸序列与第六核苷酸序列可操作地连接, 所述第六核苷酸序列为组成型表达启动子。
13. 权利要求 12所述的方法, 其中所述的第六核苷酸序列为 35s启动子。
14. 一种方法, 用于从具有雌性配子和雄性配子的植株产生种子,所述方法包括:
( a) 向第一植株中引入下述构建体, 所述第一植株含有一个纯合隐性的等位不育基因, 所述构建体包含:
( i )第一核苷酸序列, 当其在所述第一植株中表达时, 能功能性互补所述植株的纯合隐 性植物不育性状;
( ϋ ) 第二核苷酸序列, 其表达抑制所述第一植株中雄性配子的形成或功能, 从而使得 在含有所述隐性等位基因的所述第一植株中产生的雄配子不含所述构建体;
( i i i )第三核苷酸序列,该序列编码产物的表达能用于选择具有所述构建体的植物细胞;
(b ) 使所述植株自体受精; 以及
( c ) 产生含有所述构建体的种子;
其特征在于所述的第三核苷酸序列选自抗草甘膦、抗草丁膦、抗百草枯、抗草铵膦、 抗莠去津、 抗溴苯腈、 抗 2, 4-D、 抗咪唑啉酮或抗磺酰脲类等除草剂的基因构成的 组。
15. 权利要求 14所述的方法, 其中所述第一核苷酸序列为育性恢复基因, 选自 0sFG2、
权 利 要 求 书
BrMS2 MSP1、 PAIR1、 PAIR2、 ZEP1、 MELL, PSS1、 TDR、 UDT1、 GAMYB4、 PTC1、 API5、 WDA1、 CYP704B2、 MS26、 MS22、 DPW、 MADS3、 0SC6、 RIP1、 CSA或 AID1构成的组。
16. 权利要求 15所述的方法,其中所述的第一核苷酸序列与第四核苷酸序列可操作地相 连, 所述第四核苷酸序列指导偏好于雄性植物细胞的表达。
17. 权利要求 16所述的方法,其中所述的第四核苷酸序列仅在存在诱导物质或诱导条件 时才具有功能。
18. 权利要求 16所述的方法, 其中所述的第四核苷酸序列选自 0sFG2、 BrMS2、 MSP1、 PAIR1、 PAIR2、 ZEPU MELL, PSS1、 TDR、 UDT1、 GAMYB4、 PTC1、 API5、 WDA1、 CYP704B2, MS26、 MS22、 DPW、 MADS3、 0SC6、 RIP1、 CSA、 AID1、 5126、 Ms26、 Ms22或 Ms45的雄 性组织调控序列构成的组。
19. 权利要求 14所述的方法, 其中第二核苷酸序列选自 DAM甲基化酶基因、 Zea mays a 淀粉酶基因、 细胞毒素编码基因、 或 Barnase与 Barstar的组合序列构成的组。
20. 权利要求 19所述的方法,其中所述的第二核苷酸序列与第五核苷酸序列可操作地相 连, 所述第五核苷酸序列指导偏好于雄性配子的表达。
21. 权利要求 20所述的方法, 其中所述的第五核苷酸序列选自聚半乳糖醛酸酶 47基因、 Zml3基因、 果胶甲基酯酶基因、 钙调蛋白结合蛋白基因、 肌动蛋白解聚因子基因、 prolfi l in基因和硫酸化五肽 phytosulphokine基因的调控区域构成的组。
22. 权利要求 14所述的方法, 其中所述的第三核苷酸序列具体为具有除草剂抗性的 ALS 基因突变体。
23. 权利要求 22所述的方法,其中所述的 ALS基因突变体在水稻中的突变为 Trp548、 Ala96 和 /或 Ser627突变, 优选带有 Trp548Cys、 Trp548Met Ala96Val、 Ala96Thr和 /或 Ser627Asn 突变。
24. 权利要求 22所述的方法, 其中所述的 ALS基因突变体在油菜中的突变为 Alal07、 Alal90 Trp559和 /或 Ser638突变。
25. 权利要求 22所述的方法,其中所述的第三核苷酸序列与第六核苷酸序列可操作地连接, 所述第六核苷酸序列为组成型表达启动子。
26. 权利要求 25所述的方法, 其中所述的第六核苷酸序列为 35s启动子。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993025695A1 (en) * | 1992-06-12 | 1993-12-23 | Plant Genetic Systems N.V. | Maintenance of male-sterile plants |
WO1999042598A2 (en) * | 1998-02-20 | 1999-08-26 | Zeneca Limited | Hybrid seed production |
US6172279B1 (en) * | 1989-01-26 | 2001-01-09 | Zeneca Limited | Plant gene construct encoding a protein capable of disrupting the biogenesis of viable pollen |
CN101248183A (zh) * | 2005-06-24 | 2008-08-20 | 先锋高级育种国际公司 | 介导植物雄性生育力的核苷酸序列以及使用其的方法 |
CN102586215A (zh) * | 2011-09-06 | 2012-07-18 | 深圳兴旺生物种业有限公司 | 水稻抗除草剂蛋白及其在植物育种中的应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999042587A1 (en) * | 1998-02-20 | 1999-08-26 | Zeneca Limited | Pollen specific promoter |
-
2014
- 2014-05-21 CN CN201480003077.2A patent/CN104837334B/zh active Active
- 2014-05-21 WO PCT/CN2014/077967 patent/WO2014187312A1/zh active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6172279B1 (en) * | 1989-01-26 | 2001-01-09 | Zeneca Limited | Plant gene construct encoding a protein capable of disrupting the biogenesis of viable pollen |
WO1993025695A1 (en) * | 1992-06-12 | 1993-12-23 | Plant Genetic Systems N.V. | Maintenance of male-sterile plants |
WO1999042598A2 (en) * | 1998-02-20 | 1999-08-26 | Zeneca Limited | Hybrid seed production |
CN101248183A (zh) * | 2005-06-24 | 2008-08-20 | 先锋高级育种国际公司 | 介导植物雄性生育力的核苷酸序列以及使用其的方法 |
CN102586215A (zh) * | 2011-09-06 | 2012-07-18 | 深圳兴旺生物种业有限公司 | 水稻抗除草剂蛋白及其在植物育种中的应用 |
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2918681A4 (en) * | 2012-11-09 | 2016-10-12 | Shenzhen Inst Of Molecular Crop Design | FERTILITY GENE AND USES THEREOF |
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