WO2014131342A1 - 一种小麦新型育性调控构建体及其应用 - Google Patents

一种小麦新型育性调控构建体及其应用 Download PDF

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WO2014131342A1
WO2014131342A1 PCT/CN2014/072428 CN2014072428W WO2014131342A1 WO 2014131342 A1 WO2014131342 A1 WO 2014131342A1 CN 2014072428 W CN2014072428 W CN 2014072428W WO 2014131342 A1 WO2014131342 A1 WO 2014131342A1
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gene
wheat
promoter
mutant
pollen
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PCT/CN2014/072428
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English (en)
French (fr)
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邓兴旺
马力耕
李健
周宽基
王峥
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未名兴旺系统作物设计前沿实验室（北京）有限公司
兴旺投资有限公司
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Priority to CN201480001231.2A priority Critical patent/CN104379751A/zh
Publication of WO2014131342A1 publication Critical patent/WO2014131342A1/zh

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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
    • 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
    • C12N15/8289—Male sterility

Definitions

  • the present invention relates to the field of plant genetic engineering and plant breeding technology, and particularly relates to a new method for effectively utilizing recessive nuclear male sterility mutants and their additional lines for hybrid seed production. method.
  • Heterosis is a common phenomenon in the biological world, and the use of heterosis can significantly increase crop yield, quality and resistance. Compared with crops such as corn and rice, the research on the utilization of wheat heterosis is relatively lagging. The core problem to be solved is the establishment of a technical system for efficient production of wheat hybrids. Based on the research progress in the past 50 years, the research on the utilization of wheat heterosis mainly focuses on the use of nuclear infertility male sterility ("three-line method”), the use of chemical killing technology (“slaughter method”) and light temperature sensitivity Use of nuclear male sterility ("two-line method”).
  • the three-line method has not been widely used in production because of the difficulty in breeding of the sterile line, narrow recovery sources, and cytoplasmic side effects.
  • the killing method avoids the relationship between recovery and maintenance. It has been considered as a promising new technology for wheat hybrid seed production, but due to its poor stability during seed production, high seed production costs and environmental pollution. For many reasons, it is difficult to promote and utilize it in actual production.
  • the two-line method based on light temperature sensitivity has the advantages of low seed production cost, wide recovery source, and easy to obtain superior combination, it also faces two key problems: the influence of environmental factors instability on the fertility of sterile lines.
  • the wheat sterile lines selected for use with light temperature sensitivity characteristics are very limited.
  • Nuclear sterility is used for the utilization of crop heterosis, and its sterility has the characteristics of being easily recovered and not easily maintained. According to the conventional method, a large amount of efficient production of the pure sterility line is not achieved. Therefore, aiming at the current situation of wheat heterosis utilization, the establishment of a new system for efficient male sterile breeding in wheat is one of the most critical factors for the successful application of hybrid wheat.
  • the core purpose is to achieve efficient maintenance of nuclear sterility.
  • the core technical measures adopted are to build a complete and perfect function. Special artificial recombination of exogenous chromosomes (arms) or transgenic exogenous chromosomes (arms).
  • the above-mentioned hybrid wheat production system has certain defects, mainly due to the inability to produce a high-purity male sterile line, or because the marker-based sterile line retention procedure is too complicated, or due to Maintaining the line is genetically unstable, or because the maintenance of the maintainer line and the production of the sterile line are too laborious, resulting in too high a production cost of the hybrid. Therefore, these systems have not been applied on a large scale in production.
  • Cigu nuclear sterile wheat mutant is a single gene-controlled dominant mutant that exhibits complete male sterility and is not affected by genetic background and environmental conditions, and can accept other varieties, strains or males. Pollen of fertile sister strains.
  • Liu Binghua et al. combined the Taigu nuclear sterility gene Ms2 with the Dwarf No. 1 wheat RhtlO dominant dwarf gene to produce dwarf wheat, and the sterility gene and the dwarf gene are inherited, which can identify the fertility early. To the wheat to get up and joint.
  • "Taigu nuclear sterile wheat” and other dominant genic male sterile mutants have not yet made breakthroughs in the application of hybrid wheat breeding.
  • the core of the 4E-ms system is its light blue grain retention system, which is a 4E monomeric addition line of mslg/mslg homozygous sterile wheat type, which also has a white kernel.
  • the exogenous univalent body 4E carried by the light blue granules can transmit at the same time through the female and male gametes at a rate of about 20% during the selfing process, thereby producing about 4% of 4E
  • the inbreds of the deep blue granules only produced the deep blue granules themselves, but did not directly isolate the offspring of the white sterility lines, thus causing the light blue granules to self-bred white nucleus sterility.
  • the efficiency of the system function is reduced; at the same time, although the photoelectric color sorter can achieve complete color selection between blue and white particles, the color selection between the deep and light blue particles is incomplete (grain photoelectric color sorter is dark blue and shallow The color selection accuracy between the blue particles is only about 50%), and it will occur in the production of F1 seedlings with the generation of white sterile seeds in the production of light blue granules. The accumulation of dark blue granules affects the production efficiency of light blue granules self-propagating white sterility lines.
  • the object of the present invention is to provide a means for effectively constructing a novel stable wheat male sterile line and fully utilizing wheat germplasm resources for cross breeding and improving the purity of hybrids, at least to some extent to solve the above technical problems or At least provide a useful business option.
  • the present invention was completed based on the following findings of the inventors:
  • the inventors used a homozygous recessive nuclear male sterile wheat mutant as a female parent to hybridize with a heterologous line of wheat to construct a homozygous recessive nuclear male sterile wheat mutant.
  • the heterochromosomal addition line wherein the additional chromosomes are capable of restoring the phenotype of the wheat mutant male sterility.
  • the heterologous addition line of the homozygous recessive nuclear male sterile wheat mutant is used as a transforming receptor material, and the target gene is integrated into the additional chromosome by transforming the closely linked two target genes into the heterologous additional receptor plants. Transgenic plants on.
  • pollen-inactivated genes can inactivate pollen containing foreign genes, ie, lose fertility
  • screening genes can be used for sorting of transgenic seeds and non-GM seeds
  • sorted non-GM seeds are used as The breeding line produces hybrids
  • the transgenic seeds are used as a source of maintenance to continuously and stably produce sterile lines.
  • the wheat recessive nuclear male sterility mutant mslg can be used as a female parent to hybridize with the wheat 4E dimer extracellular line 81529 to construct a wheat mslg mutant 4E heterologous line.
  • the wheat mslg mutant 4E heterologous line was used as the transgenic receptor material, and the two closely related target genes were transformed into the wheat mslg mutant 4E heterologous line, and the transgenic plants with the target gene integrated into the additional 4E chromosome were screened.
  • the fertility restorer gene on the 4E chromosome restores the sterile phenotype of the wheat mslg mutant.
  • the pollen inactivating gene ZmBT1 TP-AA1 containing the leader sequence can inactivate the pollen containing the foreign gene, ie lose fertilization.
  • the present invention proposes a wheat heterochromosomal addition line.
  • the additional line comprises one or two chromosomes of wheat relatives, such as the 4E chromosome of Elymus sibiricum, which contains a fertility restorer gene of the wheat recessive nuclear male sterility mutant mslg. , can restore the sterile phenotype of wheat mslg mutant, the plant appears to be fertile.
  • the invention also proposes a construct.
  • the construct comprises: a first expression cassette,
  • the first expression cassette contains a first nucleic acid molecule encoding a pollen inactivating gene, including but not limited to an enzyme or a modified enzyme that encodes a carbohydrate-producing enzyme, an amylase, and a debranching Enzymes and pectinase, more specifically such as maize a-amylase gene, auxin, rot B, cytotoxin gene, diphtheria toxin, DAM methylase, avidin, or may be selected from prokaryotic regulatory systems, Also a dominant male sterility gene; and a second expression cassette, the second expression cassette comprising a second nucleic acid molecule, the second nucleic acid molecule encoding a fluorescent color selection gene, the fluorescent color selection gene including but not It is limited to genes such as red fluorescent gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, and
  • the wheat pollen inactivating gene and the fluorescent color selection gene can be effectively introduced into the heterologous additional line plant of the homozygous recessive nuclear male sterile wheat mutant, thereby obtaining the foreign chromosome and the transgene.
  • the breeding plant can also be used as a female parent to carry out cross-breeding as a female parent, so that the sterile line and the maintainer can be easily produced through the self-crossing of the maintainer line.
  • the maintainer line carrying the foreign chromosome and the transgene and the sterile line not carrying the foreign chromosome and the transgene can be distinguished by the fluorescent color selection of the seed, and the seed of the maintainer line is fluorescently labeled, and the seed of the sterile line does not have a fluorescent label.
  • the first expression cassette further comprises a first promoter operably linked to the first nucleic acid molecule encoding the pollen inactivating gene, the first promoter It is a pollen-specific promoter, including but not limited to the ZmAPB1 promoter, the ZmABP promoter, the ZmC5 promoter, the MPCBP promoter, and the ZmPG47 promoter, etc., which prefer to specifically express the gene linked to it during the late stage of plant pollen development.
  • the nucleotide sequence of the first promoter is as shown in SEQ ID NO: 2.
  • the nucleotide sequence of the peptide and the pollen inactivating gene as shown in SEQ ID NO: 1, encoding a corn alpha amylase having a peptide.
  • the maize alpha amylase gene AA1 is driven by the maize pollen-specific promoter ZmPG47, and the sequence and terminator encoding the leader peptide (TP) from the brittle- ⁇ gene derived from maize.
  • the IN2-1 constitutes an expression cassette in which the nucleotide sequence encoding the leader peptide (TP) is as shown in SEQ ID NO: 14, and the nucleotide sequence encoding the maize alpha amylase gene AA1 is shown in SEQ ID NO: 15.
  • the pollen-inactivated gene expression cassette can specifically express amylase in the mature pollen in the late development stage, and target the amyloplast to decompose the starch in the pollen, thereby depriving the pollen, losing the ability to insemination, and inactivating the transgenic pollen.
  • This design inactivates all transgenic pollen containing this gene. Insemination can also prevent biosafety issues such as gene drift. Inactivated pollen cannot be pollinated with other plants or weeds around it, so transgenes cannot drift through the pollen into the environment.
  • the second expression cassette further comprises a second promoter, the second promoter being seed specific Sex promoters include, but are not limited to, the END2 promoter, the LTP2 promoter.
  • 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, a DNA fragment containing the above expression cassette, and a virus.
  • the construct (sometimes referred to as an expression vector, genetic vector or vector) is in the form of a plasmid.
  • the plasmid has the characteristics of simple operation, can carry large fragments, 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, i.e., either single-stranded or double-stranded. Those skilled in the art can make selections as needed.
  • a plurality of DNA fragments can be manipulated to provide a DNA sequence in the proper orientation or in the correct reading frame.
  • DNA fragments can be ligated using adaptors or adaptors, or further procedures can be included to provide convenient restriction sites and the like.
  • the invention also encompasses a wheat cell, tissue or organ.
  • the wheat cell, tissue or organ contains the construct described above.
  • the construct is introduced into cells, tissues or organs of rice by conventional techniques, such as Agrobacterium-mediated transformation, to obtain samples which can be subsequently used for research and hybridization.
  • the invention also proposes a method of constructing a male sterile line of wheat.
  • the method comprises: introducing the wheat relative chromosomes described above into a wheat homozygous recessive nuclear male sterile plant to obtain a wheat concealed with a foreign chromosome A homozygous plant with a male sterile male mutation site becomes a heterologous line of a wheat homozygous recessive nuclear male sterility mutant, and the additional chromosome can restore the phenotype of the male mutant male sterility.
  • the invention provides a method for restoring male fertility of a recessive nuclear male sterility mutant of wheat.
  • the method comprises: introducing a chromosome of a wheat relative species into a wheat homozygous recessive nuclear male sterile mutant plant to obtain a recessive nuclear male sterility carrying the foreign chromosome Plants homozygous for the mutation site become heterologous lines of wheat homozygous recessive nuclear male sterility mutants, and additional chromosomes can restore the phenotype of male mutant male sterility, which produces fertile male gametes
  • the introduced construct is introduced into the heterologous line of the wheat homozygous recessive nuclear male sterility mutant to obtain a transgenic plant carrying the foreign gene and the foreign gene is located on the foreign chromosome;
  • the transgenic plants are bred, and the transgenic plants are capable of self-fertilization, thereby obtaining seeds (maintaining lines) carrying foreign genes and foreign chromosomes
  • Seeds carrying foreign genes and exogenous chromosomes can continuously produce sterile lines and maintainer lines by self-intersection, and seeds that do not carry foreign genes and foreign chromosomes can be used as hybrid wheat seedlings for hybrid seed production. Parents. Thus, it can be effectively used for hybrid seed production of wheat.
  • the invention also proposes a method of preparing wheat seeds.
  • the method comprises introducing the construct described above into a heterologous chromosome of a wheat recessive nuclear male sterility mutant, the wheat comprising the construct of the invention on an additional chromosome
  • the recessive nuclear male sterile mutant plants are self-fertilized to obtain seeds containing the constructs described above.
  • the invention provides the use of a wheat male sterile line in the preparation of hybrid wheat.
  • the wheat male sterile line is obtained by the method of constructing a wheat male sterile line in the foregoing.
  • Figure 1 is a comparison of the phenotype of wheat mslg mutants with wild type.
  • 1A-C are wheat wild-type, wheat mslg mutants and wheat mslg mutant 4E monomeric additional lineages in the flowering stage;
  • Figure 1D-F is wheat wild type, wheat mslg mutant and wheat mslg mutant 4E, respectively The stamens and pistils of the heterozygous cultivar in the flowering stage;
  • Fig. 1G-I are the grain of wheat wild type, wheat mslg mutant and wheat mslg mutant 4E monomer different addition line respectively;
  • 1J-L is wheat wild I 2 -KI staining of type, wheat mslg mutant and wheat mslg mutant 4E monomeric additional lineage;
  • Fig. 1M-0 is wheat wild type, wheat mslg mutant and wheat mslg mutant 4E monomer addition line Mature seeds.
  • Figure 2 is a schematic diagram showing the construction of a wheat mslg mutant 4E heterologous line.
  • ® indicates self-intersection, ms indicates mslg; 4E' and 4E" represent one and two 4E chromosomes, respectively.
  • Figure 3 is a cytogenetic identification of the wheat mslg mutant 4E heterologous line.
  • Figure 3A shows the root tip cells of the mslg male sterile mutant
  • Figure 3B shows the root tip cells of the mslg mutant 4E monomeric addition line
  • Figure 3C shows the root tip cells of the mslg mutant 4E dimerism.
  • the blue is the DAPI-stained wheat chromosome
  • the red is the rhodamine-labeled long-eared buckwheat chromosome.
  • Figure 4 is a schematic view showing the structure of the plant expression vector pl300-130.
  • Figure 5 is a schematic diagram showing the construction of the plant expression vector pl300-130.
  • Fig. 6 is a schematic diagram showing that a wheat mslg mutant 4E heterologous line is used as a transforming receptor material, and a transformant obtained by transgene is selfed to obtain a sterile line.
  • Fig. 7 shows the results of staining of fertile pollen grains and sterile pollen grains I 2 -KI. detailed description
  • the methods used in the following examples are conventional methods unless otherwise specified.
  • the primers used are synthesized by Shanghai Yingjun Biotechnology Co., Ltd., and the sequencing is completed by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd., during the construction of PCR kits and vectors.
  • the endonuclease was purchased from Bao Bioengineering Co., Ltd.
  • the pEASY-Blunt-simple cloning kit was purchased from Beijing Quanjin Biotechnology Co., Ltd.
  • the T4 DNA ligase was purchased from NEB.
  • the methods were all carried out according to the method provided by the kit.
  • the vector pCAMBIA 1300 used in the experiment was from CAMBIA.
  • Fig. 1A and Fig. 1B are the wheat ears of the flowering stage, in which the ears of the wheat ms mutant (Fig. 3B) are more fluffy than the wild type ears (Fig. 3A);
  • Fig. 1D and 1E are the stamens and pistils of the flowering stage, in which the wild The anthers of the type have been cracked, and the stigma of the pistil has been covered with yellow pollen grains (Fig. 1D). The anthers of the mutants are not cracked, and there are no pollen on the stigma of the pistil (Fig. 1E); Figs.
  • 1J and 1K are pollen I 2 -KI staining, in which the wild type pollen is round and round, all stained blue-black, indicating strong vigor (Fig. 1J), while the pollen shape of the ms mutant is shrunken, all yellow-brown, indicating that the pollen has no vigor ( Fig. 1K);
  • Fig. 3G and 3H are seeds at the grain filling stage, in which the wild type is fertilized successfully, the seeds are full (Fig. 1G), and the ms mutant is not finished fertilized, and the seeds are not developed (Fig. m).
  • the ms mutant was crossed with 9 conventional wheat lines including Chinese spring, and the F1 generation was selfed.
  • the separation ratio between the F2 generation sterile line and the fertile line was observed, which all met the ratio of 1:3, indicating the mutation.
  • the body is a typical single-gene recessive nuclear male-sterile mutant (Table 1).
  • the ⁇ mutant was homologously analyzed with the wheat male sterile mutants mslc and ms5.
  • the specific method was to hybridize the F1 plants of Chinese spring with the mslc and ms5 male sterile mutants respectively, and analyze the sterile plants in the hybrid progeny.
  • the ratio to the fertile strain, the ratio of the sterile and fertile plants in the hybrid progeny of the mslc male sterile mutant is 1:1, and there is no sterile strain in the hybrid progeny of the ms5 male sterile mutant (Table) 2), the description ⁇ mutant is an allelic mutant of msl, named mslg. Table 2 Allelic analysis of mutants and fflWc mutants and mutants
  • the expected separation ratio of the sterile plants of the sterile plants is ⁇ 2 ⁇
  • the mslg mutant was used as the female parent, and the blue granule 81529 was used as the male parent.
  • the hybrid progeny was selected and identified, and the 4E chromosome monomer was successfully added to the homozygous genic male sterile glytype mslg/mslg.
  • a self-crossing fertile mslg/mslg genotype wheat 4E monomer-additional line with a blue color was obtained.
  • Fig. 1C shows the spikes of the wheat msg mutant 4E monomer-added line in the flowering stage, and the bulkiness is restored to the wild type; Fig.
  • FIG. 1F is the wheat stamen and pistil of the wheat mslg mutant 4E monomer-added line. The same type, the anther has been cracked, the stigma of the pistil has been covered with yellow pollen grains;
  • Figure 1L is the I 2 -KI staining of the wheat mslg mutant 4E monomeric additional line pollen, the same as the wild type, the pollen shape is round, all It is dyed blue-black, indicating strong vigor;
  • Figure 31 is the seed of wheat mslg mutant 4 ⁇ monomer addition line. It is the same as wild type, fertilization is successful, and seeds are full.
  • Figure 3M-0 is a mature seed of wheat wild type, wheat mslg mutant and wheat mslg mutant 4E monomer addition line, wherein the seeds of wheat wild type and wheat mslg mutant are normal seed color, hereinafter referred to as white
  • the seeds of the wheat mslg mutant 4E monomeric addition line are blue. This indicates that 4E carries not only a part of the homologous male fertile gene, but also a blue particle marker gene.
  • the fertility gene on the wheat chromosome is wild type. (MS/MS). Take the mslg mutant as the female parent and the blue grain 81529 as the male parent.
  • the fertility gene MS is heterozygous (MS/ms) and the F1 plant is male fertile.
  • the 4E chromosome of Elytrigia sinensis the F2 generation seeds are light blue granules; the F3 generation seeds do not have blue and white separation, indicating that the F2 generation genome is supplemented with the 4E chromosome of two long-eared buckwheat grasses, and the F2 generation seeds are dark blue grains.
  • the F2 generation seeds were light blue granules, and the F3 generation seeds obtained by the single plant were blue and white, and the F3 generation blue granules and white granules were called an F3 pedigree.
  • the F3 family blue and white granules were planted separately to check the isolation of the male sterile plants in the white granules: If the male sterile plants in the F3 generation family were not isolated, it indicated that the pedigree did not carry the male sterility gene (MS).
  • Rhododendron chinense L. Rhodamine
  • the roots of wheat mslg mutant, wheat mslg mutant 4E monomer addition line and wheat mslg mutant 4E two-body addition line were used by GISH method.
  • the apical cells were analyzed by cytogenetics. The results are shown in Figure 3.
  • the blue color is the DAPI-stained wheat chromosome, the red is the rhodamine-labeled long-eared buckwheat chromosome, and the other is the root tip cell of the wheat mslg mutant.
  • FIG. 3B is the root tip cell of the wheat 4lg mutant 4E monomer addition line, in addition to the wheat chromosome, there is a chromosome of the long-eared buckwheat grass
  • Figure 3C is the wheat mslg mutant 4E two-body addition Root tip cells, except wheat chromosome
  • the cytogenetic analysis results of the root tip cells of the above three materials completely corresponded with the traits such as plant fertility and seed color.
  • the blue grain 81529 is a 4E additional line blue-grain spring wheat material obtained by systematically breeding blue-grain wheat and Gansu spring wheat material cultivated by Academician Li Zhensheng, and is a 4E two-body different addition line.
  • Example 3 Using wheat mslg mutant 4E heterologous system to create a new generation of wheat hybrid breeding system
  • the heterologous addition line of the homozygous recessive nuclear male sterile wheat mutant can be used as a transforming receptor material, through the tightly linked two target genes. Transformation into a heterologous lineage recipient plant, screening for transgenic plants in which the target gene is integrated into an additional chromosome. In transgenic plants, pollen-inactivated genes can inactivate pollen containing foreign genes, ie, lose fertility, screening genes can be used for sorting of transgenic seeds and non-GM seeds, and sorted non-GM seeds are used as The breeding line produces hybrids, and the transgenic seeds are used as a source of maintenance to continuously and stably produce sterile lines.
  • a wheat mslg mutant 4E heterologous line is used as a transgenic acceptor material, and two closely related target genes are transformed into a wheat mslg mutant 4E heterologous line, and the target gene is integrated into the target gene.
  • Additional transgenic plants on the 4E chromosome The fertility restorer gene on the 4E chromosome restores the sterile phenotype of the wheat mslg mutant, and the pollen inactivating gene ZmBT1 TP-AA1 inactivates pollen containing the foreign gene.
  • the fluorescent color selection gene mCherryW is used for sorting of transgenic seeds and non-transgenic seeds, and the sorted non-transgenic seeds are used as hybrid lines for producing sterile lines, and the transgenic seeds are used as the source of the maintenance system continuously and stably. Production of sterile lines. Because the technology uses biotechnology to produce non-GM products, it solves the bottleneck problem in the process of hybrid wheat 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.
  • Example 4 Construction of plant expression vector
  • a plant expression vector designated pl300-130 shown in Fig. 4 was constructed by assembling the following DNA element on the pCAMBIA1300 vector.
  • Notl-EcoRI three restriction sites (aagctt gcggccgc gaattc) and B Bglll restriction sites (agatct)
  • the synthesized IN2-1 terminator was ligated to the pEASY-Blunt-simple vector and verified by sequencing.
  • the ZmBT1 TP-ZmAAl gene sequence was obtained by artificial synthesis. During the synthesis, the Notl cleavage site (gcggccgc) and the EcoRI cleavage site (gaattc) were added to the 5' and 3' of ZmBT1 TP-ZmAAl, respectively. The synthesized ZmBT1 TP The -ZmAAl gene was ligated to the pEASY-T3 vector and verified by sequencing.
  • the pEASY-Blunt-simple plasmid ligated with ZmBT1 TP-ZmAAl gene was digested with the restriction enzyme sites Notl and EcoRI at both ends of the gene to obtain ZmBT1 with correct sequence and corresponding restriction sites at both ends. TP-ZmAAl gene fragment.
  • the pEASY-Blunt-simple vector ligated with the IN2-1 terminator was cleaved with restriction endonucleases Notl and EcoRI, and ligated into the ZmBT1 TP-ZmAAl gene, and the ZmBT1 TP-ZmAAl gene was located upstream of the IN2-1 terminator.
  • the ZmPG47 promoter was amplified from the maize genome, and the primers for amplifying ZmPG47 were:
  • Fl aagctt TGCACCGGACACTGTCTG (SEQ ID NO: 8), aaggcc is a Hindlll restriction site, Rl: gcggccgc TGTCGTGATCGATGCTTTATTC (SEQ ID NO: 9), and gcggccgc is a Notl restriction site;
  • the pEASY-Blunt- simple plasmid ligated with the ZmPG47 promoter was doubled with the cleavage sites Hindlll and Notl flanked by the ZmPG47 promoter. After digestion, the ZmPG47 promoter fragment with the correct sequence and the corresponding restriction sites at both ends was obtained.
  • the pEASY-Blunt-simple vector ligated with the IN2-1 terminator and the ZmBT1 TP-ZmAAl gene was digested with restriction endonucleases Hindlll and Notl and ligated into the ZmPG47 promoter, and the ligated ZmPG47 promoter was located at ZmBT1 TP-ZmAAl. Upstream of the gene. This was constructed as an intermediate vector A with a gene expression cassette ZmPG47: ZmBTl TP-ZmAAl: IN2-1.
  • the terminator was obtained by artificial synthesis. During the synthesis, BamHI-EcoRI-Xhol-Sall- four restriction sites (ggatcc gaattc ctcgag gtcgac) and Kpnl restriction sites (ggtacc) were added to the 5' and 3', respectively.
  • the synthetic PINII terminator was ligated to the pEASY-Blunt-simple vector and verified by sequencing.
  • the mCherryW gene sequence was obtained by artificial synthesis. During the synthesis, the Xhol cleavage site (ctcgag) and the Sail restriction site (gtcgac) were added to the 5' and 3' of the mCherryW gene, respectively.
  • the synthesized mCherryW gene was ligated into pEASY-Blunt. -simple vector and verified by sequencing.
  • the pEASY-Blunt-simple plasmid harboring the mCherryW gene was digested with the restriction sites Xhol and Sail at both ends of the gene to obtain the mCherryW gene fragment with the correct sequence and the corresponding restriction sites at both ends.
  • the Ltp2 promoter was amplified from the genome of barley, and the primers for amplifying Ltp2 were:
  • gaattc AACCGTCTCTTCGTGAGAATAACC (SEQ ID NO: 10), gaattc is an EcoRI restriction site,
  • R2 ctcgag TACTCGGCTACACTCACACGC (SEQ ID NO: 11), ctcgag is a Xhol cleavage site;
  • the pEASY-Blunt-simple plasmid ligated with the Ltp2 promoter was doubled with the restriction sites EcoRI and Xhol on both sides of the Ltp2 promoter. After digestion, the Ltp2 promoter fragment with the correct sequence and the corresponding restriction sites at both ends was obtained.
  • the pEASY-Blunt-simple vector ligated with the scorpion terminator and the mCherryW gene was digested with restriction endonucleases EcoRI and Xhol, ligated into the Ltp2 promoter, and the ligated Ltp2 promoter was located upstream of the mCherryW gene.
  • the CaMV35S enhancer sequence was obtained by artificial synthesis. During the synthesis, the BamHI restriction site (ggatcc) and the EcoRI restriction site (gaattc) were added to the 5' and 3' of the CaMV35S enhancer, respectively. The synthesized CaMV35S enhancer was ligated. The pEASY-Blunt-simple vector was verified by sequencing. The pEASY-Blunt- simple plasmid harboring the CaMV35S enhancer was digested with the cleavage sites BamHI and EcoRI at both ends of the gene to obtain the correct sequence of CaMV35S enhancer with corresponding restriction sites at both ends. Fragment.
  • the pEASY-Blunt-simple vector harboring the scorpion terminator, mCherryW gene and Ltp2 promoter was digested with restriction endonuclease BamHI and EcoRI, and ligated into the CaMV35S enhancer, which was located upstream of the Ltp2 promoter.
  • This is constructed as an intermediate vector B with the gene expression cassette CaMV35S-Ltp2: mCherryW: ⁇ .
  • the gene expression cassette ZmPG47: ZmBT1 TP-ZmAAl: IN2-1 was excised from the intermediate vector A with restriction endonucleases Bglll and Hindlll, and the gene expression cassette CaMV35S-Ltp2 was cloned with restriction endonucleases Kpnl and BamHI.
  • mCherryW: PINII was cleaved from the intermediate vector B, and lig300-130 was obtained by ligating the pCAMBIA 1300 vector cut with restriction endonucleases Kpnl and Hindlll.
  • Figure 5 The above construction process is shown in Figure 5.
  • the wheat was co-transformed by Agrobacterium method and gene gun method.
  • the specific transforming receptor material was wheat 4lg mutant 4E heterologous line (see Example 2 for the construction process), which may be the wheat 4lg mutant 4E monomer
  • the addition line may also be a wheat mslg mutant 4E dimerism addition line.
  • a wheat mslg mutant 4E monomer addition line is used. It should be noted that all the heterochromosomal addition lines capable of fully recovering the male sterility phenotype of the wheat msl mutant can be used as the transforming receptor material of the present invention, except for the 4E chromosome derived from E. longissima in this case.
  • 2R and 4R of rye can also restore the male sterility phenotype of wheat msl mutants, but not limited to from rye 2R and 4R.
  • all of the heterologous lines capable of restoring the wheat recessive nuclear male-sterile mutant (not limited to the msl mutant) using the additional chromosome of the related species can be used as the receptor of the present invention.
  • the primers were designed to identify the transgenic wheat plants by PCR.
  • R3 5'- TGGCCTTGTAGGTGGTCTTC -3 ' ( SEQ ID NO: 13 )
  • the reaction conditions were: pre-denaturation at 94 °C for 5 minutes; denaturation at 94 °C for 30 seconds; annealing at 55 °C for 30 seconds; extension at 72 °C for 40 seconds; 35 cycles; extension at 72 °C for 10 minutes.
  • Amplified is a partial fragment of mCherryW, which is 359 bp in length.
  • positive transgenic plants were selected. Southernem-blot was used to identify the integrity and copy number of the exogenous transgene in the transgenic positive plants. The results showed that the exogenous genes in the transgenic positive plants were intact and integrated in a single copy.
  • Example 7 Pollen activity detection of transgenic plant material
  • Example 5 Analysis of the transgenic plants described in Example 5 revealed that no significant morphological differences were observed between the transgenic plants and the non-transgenic control plants, but the male fertility was different.
  • the wheat mslg mutant (infertility, hereinafter referred to as CK1) and the non-transgenic wheat mslg mutant 4E heterologous line (fertile, hereinafter referred to as CK2) were used as controls for pollen activity detection.
  • the ratio of the fluorescence of the transformant described in Example 6 to the non-fluorescent seed was investigated. The results showed that the fluorescent seed accounted for about 20% of the total seed, and the non-fluorescent seed accounted for about 80% of the total seed, with wheat.
  • Ffl ⁇ Mutant 4E The ratio of 4E chromosome to the progeny through the female gametophyte in the heterologous addition line was approximately 20%, further demonstrating that T-DNA is integrated on the 4E chromosome.
  • the results of Examples 6 and 7 indicate that the expression vector elements provided in the present invention are well expressed as a whole.

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Abstract

提供了一种调控小麦育性的方法及其应用。该方法包括：以纯合隐性核雄性不育小麦突变体作为母本，与小麦的异附加系杂交，构建纯合隐性核雄性不育小麦突变体的异染色体附加系，其中附加的染色体能够恢复小麦突变体雄性不育的表型，再以纯合隐性核雄性不育小麦突变体的异染色体附加系为转化受体材料，通过将紧密连锁的2个目标基因转化至异附加系受体植株中，筛选目标基因整合到附加的染色体上的转基因植株。

Description

一种小麦新型育性调控构建体及其应用 技术领域 本发明属于植物基因工程和植物育种技术领域， 具体涉及一项有效利用隐性细胞核雄 性不育突变体及其附加系进行杂交制种的新方法。
背景技术
杂种优势是生物界的一种普遍现象， 利用杂种优势可以显著提高作物产量、 品质和抗 性。 与玉米、 水稻等作物相比， 小麦杂种优势利用的研究相对滞后， 亟待解决的核心问题 是高效生产小麦杂交种的技术体系的建立。 综合近 50年来的研究进展， 小麦杂种优势利用 研究主要集中于： 核质互作雄性不育的利用（"三系法")、化学杀雄技术的利用（"化杀法"） 和光温敏核雄性不育的利用 （"两系法"）。 三系法由于不育系难以繁殖、 恢复源较窄、 细胞 质副效应等原因， 未能在生产上大面积应用。 化杀法避开了恢复与保持间的相互关系， 曾 被认为是一种很有希望的小麦杂交制种新技术， 但由于其在制种过程中稳定性差、 制种成 本高及环境污染等多方面原因， 在实际生产上也难以推广利用。 基于光温敏的两系法虽然 具有制种成本低、 恢复源广泛， 较易获得优势组合等优点， 但也面临着两大关键问题一 环境因素的不稳定性对不育系育性的影响和利用光温敏特性所选育的小麦不育系十分有限。
细胞核不育用于作物杂种优势利用， 其不育性具有易被恢复而不易被保持的特性， 按 照常规方法无法实现纯合核不育系种子的大量有效生产。 因此， 针对小麦杂种优势利用的 现状， 建立高效小麦雄性不育繁育新体系， 是杂交小麦获得成功应用的最关键因素之一。
1972年澳大利亚学者 Driscoll提出了著名的 XYZ杂交小麦生产体系， 即在雄性不育系 (Z系， Cornerstone mslc 小麦突变体） 中， 增加一条或一对携带同源等位显性可育基因 Ms的外源染色体 （来自黑麦的 2R， 其携带的显性标记性状是 "穗下节有绒毛" ）， 分别获 得 Y系和 X系， 外源染色体不能与小麦染色体配对及不能发生同源等位基因交换， 在 Y系 中外源染色体通过花粉的传递率低， 用 Y系 （保持系） 作父本和 Z系杂交生产 Z系， 其后 代绝大部分将表现雄性不育； 用 Z系和 X系杂交生产 Y系。 XYZ体系没有取得生产应用的 成功， 其主要原因有： 1 ) 标记性状是植株阶段性状， 生产应用中不可能在开花期有限的时 间内， 人工拔除所有有绒毛株或无绒毛株。 2) 2R单价染色体能够通过雄配子途径传递， 造 成不育系和杂交种生产纯度不高的问题。 此后， 许多学者致力于 XYZ体系的改良， 核心目 的是实现细胞核不育性的高效保持， 采取的核心技术措施是构建一条功能较为齐全、 完美 的特殊人工重组外源染色体 （臂） 或转基因外源染色体 （臂）。 但是， 针对高效保持亲本系 的要求， 上述杂交小麦生产体系都存在一定的缺陷， 主要是由于不能产生高纯度的雄性不 育系、或由于基于标记基因的不育系保持程序太复杂、或由于保持系在遗传上尚不稳定、 或 由于保持系的繁殖和用其生产不育系太费力、 导致杂交种的生产成本太高。 因此， 这些体 系并没有在生产上得到大规模应用。
我国的 "太谷核不育小麦"突变体（Ms2)是一个单基因控制的显性突变体， 表现完全 雄性不育， 且不受遗传背景和环境条件影响， 可接受其它品种、 品系或雄性可育姐妹株的 花粉。 刘秉华等将太谷核不育基因 Ms2与矮变 1号小麦 RhtlO显性矮杆基因结合于一体， 培育出矮败小麦，其不育基因与矮秆基因呈连锁遗传， 可将育性识别提早到小麦起身拔节期。 但由于实现其有效标记系统的研制工作困难较大， "太谷核不育小麦"及其它显性核不育 突变体至今未取得杂交小麦育种应用的研究突破。
此外，黄寿松等（1991 )报导的 "蓝标型小麦核不育、保持系" 、周宽基报导的 "4E-ms 杂交小麦生产体系" （周宽基等 1996， 1998， 2006), 都是将携带同源显性可育基因 MsE 和显性蓝色糊粉层标记基因 Ba的外源染色体 4E， 附加到各自的核不育小麦上 （msms), 分 别实现了不育系种子 （正常小麦粒色） 和保持系种子 （蓝粒） 的细胞遗传学途径的粒色标 记， 与 XYZ体系相比， 取得了标记性状方面的重要技术改进， 实现了小麦隐性核不育的有 效保持——生产大量整倍体核不育系种子， 开辟了小麦核不育杂种优势利用的新途径、 方 法。
周宽基等 （1996,1998,2006) 4E-ms体系的核心是其浅蓝粒保持系， 它是 mslg/mslg纯 合不育基因型小麦的 4E单体异附加系， 它同时具备白粒核不育系的 "自交"繁育和浅蓝粒 保持系自身繁殖的双重功能—— "一系两用" 。 然而， 浅蓝粒系所携带的外源单价体 4E， 在其自交过程中以约 20%的传递率， 能够同时通过雌、 雄配子两个途径传递， 由此产生约 4%的 4E二体异附加系（深蓝粒系）后代， 深蓝粒系自交后代只产生其深蓝粒自身， 而不直 接分离出白粒不育系后代， 从而造成浅蓝粒系自交繁育白粒核不育系功能的效率降低； 同 时， 虽然光电色选机可实现蓝、 白粒间的完全色选， 但是对深、 浅蓝粒间的色选是不完全 的 （谷物光电色选机对深蓝和浅蓝粒间的色选精度只有 50%左右）， 加之当生产上随着逐年 逐代的白粒不育系种子的分出用于 F1制种， 在浅蓝粒系自交繁殖群体中将发生深蓝粒的累 加， 从而影响浅蓝粒自交繁殖白粒不育系种子的生产效率。
利用常规的育种方法来选育不育系存在周期长， 见效慢， 不育基因单一， 对环境因素 影响敏感等问题， 远不能满足生产发展的需要。 近年来， 通过基因工程创造植物雄性不育 系及其恢复系已在一些作物上获得了成功， 为作物杂种优势的利用开创了新的前景。 目前 利用基因工程创造雄性不育的策略主要是利用花粉发育的特异启动子与外源基因嵌合， 构 建表达载体， 转化植物来阻断花粉发育的过程从而达到雄性不育的目的。 发明内容
本发明的目的在于提出一种能够有效构建新型稳定的小麦雄性不育系和充分利用小麦 种质资源用于杂交育种、 提高杂交种纯度的手段， 至少在一定程度上解决上述技术问题之 一或至少提供一种有用的商业选择。
本发明是基于发明人的下列发现而完成的： 发明人以纯合隐性核雄性不育小麦突变体 作为母本， 与小麦的异附加系杂交， 构建纯合隐性核雄性不育小麦突变体的异染色体附加 系， 其中， 附加的染色体能够恢复小麦突变体雄性不育的表型。 再以纯合隐性核雄性不育 小麦突变体的异染色体附加系为转化受体材料， 通过将紧密连锁的 2个目标基因转化至异附 加系受体植株中， 筛选目标基因整合到附加的染色体上的转基因植株。 在转基因植株中， 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， 筛选基因可以用于转基因 种子和非转基因种子的分拣， 分拣出的非转基因种子用作不育系生产杂交种， 转基因种子 用作保持系来源源不断地、 稳定地生产不育系。
根据本发明的实施例， 可以以小麦隐性核雄性不育突变体 mslg为母本， 与小麦 4E二体 异附加系 81529杂交，构建小麦 mslg突变体 4E异附加系。再以小麦 mslg突变体 4E异附加系作 为转基因受体材料， 将紧密连锁的 2个目标基因转化至小麦 mslg突变体 4E异附加系中， 并筛 选目标基因整合到附加的 4E染色体上的转基因植株： 4E染色体上带有的育性恢复基因可以 恢复小麦 mslg突变体的不育表型， 包含导肽序列的花粉失活基因 ZmBTl TP-AA1 可使含有 外源基因的花粉失活， 即失去授精能力， 荧光色选基因 mCherryW用于转基因种子和非转基 因种子的分拣， 分拣出的非转基因种子用作不育系生产杂交种， 转基因种子用作保持系来 源源不断地稳定地生产不育系。 由于该技术利用生物技术生产非转基因产品， 解决了小麦 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。
本发明提出了一种小麦异染色体附加系。 根据本发明的实施例， 该附加系包含一条或 两条小麦近缘种的染色体， 如长穗偃麦草的 4E染色体， 该染色体上含有小麦隐性核雄性不 育突变体 mslg的育性恢复基因， 能够恢复小麦 mslg突变体的不育表型， 植株表现为可育。
本发明还提出了一种构建体。 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花粉失活基因， 所述花粉失活基 因包括但不限于可编码促使碳水化合物降解的酶或修饰酶、 淀粉酶、 脱支酶和果胶酶， 更 具体的如玉米 a淀粉酶基因、 生长素 (auxin) , rot B、 细胞毒素基因、 白喉毒素、 DAM甲基 化酶、 亲和素， 或者可选自原核调控系统， 还可以是显性的雄性不育基因； 以及第二表达 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荧光色选基因， 所述荧光 色选基因包括但不限于红色荧光基因、 青色荧光蛋白基因、 黄色荧光蛋白基因、 荧光素酶 基因、 绿色荧光蛋白基因、 花青甙 pi等基因。 利用该构建体， 能够有效地将小麦花粉失活 基因和荧光色选基因引入到纯合隐性核雄性不育小麦突变体的异染色体附加系植株中， 从 而得到携带外源染色体和转基因的可育株作为保持系， 同时得到不携带外源染色体和转基 因的不育株作为母本进行杂交制种， 从而可以方便地通过保持系的自交源源不断地生产不 育系和保持系， 另外， 携带外源染色体和转基因的保持系与不携带外源染色体和转基因的 不育系可以通过种子的荧光色选加以区分， 保持系种子带有荧光标记， 不育系种子不带有 荧光标记。
本发明所述的构建体中， 第一表达盒中还包含第一启动子， 所述第一启动子与编码花 粉失活基因的第一核酸分子可操作性的相连， 所述第一启动子为花粉特异性启动子， 包括 但不限于 ZmAPBl启动子、 ZmABP启动子、 ZmC5启动子、 MPCBP启动子、 以及 ZmPG47启动子 等， 其偏好于指导其连接的基因在植物花粉发育后期特异性表达。 根据本发明的一个实施 例， 所述第一启动子的核苷酸序列如 SEQ ID N0 : 2所示。 在第一表达盒中还可以进一步包 括一个导肽序列， 所述导肽与花粉失活基因构成的核苷酸序列如 SEQ ID NO: 1所示， 可以 编码具有导肽的玉米 α淀粉酶， 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。
更具体的， 根据本发明的具体实施例， 玉米 α淀粉酶基因 AA1在玉米花粉特异性启动 子 ZmPG47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， 其中编码导肽 （TP) 的核苷酸序列如 SEQ ID NO : 14所示， 编码玉米 α淀粉酶 基因 AA1的核苷酸序列如 SEQ ID NO : 15所示。 上述花粉失活基因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。
本发明所述的构建体中， 第二表达盒还包括第二启动子， 所述第二启动子为种子特异 性的启动子， 包括但不限于 END2启动子、 LTP2启动子。
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)、 包含上述表达盒的 DNA片段、 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。
在制备表达盒的过程中， 可对多种 DNA片段加以操作， 以提供处于合适方向， 或是处 于正确读码框中的 DNA序列。 为达到此目的， 可使用衔接子或接头， 将 DNA片段连起来， 或者进一步包括其它操作， 以提供方便的限制性酶切位点等。
本发明还包括一种小麦细胞、 组织或器官。 根据本发明的实施例， 该小麦细胞、 组织 或器官中含有前面所述的构建体。 该构建体是通过常规技术， 例如农杆菌介导法， 导入到 水稻的细胞、 组织或器官中的， 以获得可以后续用于研究、 杂交的样本。
本发明还提出了一种构建小麦雄性不育系的方法。 根据本发明的实施例， 参考图 6， 该 方法包括： 将前面所述的小麦近缘种染色体引入到小麦纯合隐性核雄性不育植株中， 以便 获得携带有外源染色体的、 小麦隐性核雄性不育突变位点纯合的植株， 成为小麦纯合隐性 核雄性不育突变体的异附加系， 附加的染色体能够恢复小麦突变体雄性不育的表型， 所述 异附加系产生可育雄性配子； 再将前面所述的构建体引入小麦纯合隐性核雄性不育突变体 的异附加系中， 以获得携带外源基因且外源基因位于外源染色体上的转基因植株； 培育所 述转基因植株， 所述转基因植株能够进行自体受精， 从而得到携带外源基因和外源染色体 的种子 （保持系） 和不携带外源基因和外源染色体的种子 （不育系）。 携带外源基因和外源 染色体的种子可以通过自交源源不断的生产不育系和保持系， 而不携带外源基因和外源染 色体的种子可以作为小麦雄性不育系用于杂交制种的亲本。 由此， 可以有效用于小麦的杂 交制种。
本发明提出了一种恢复小麦隐性核雄性不育突变体雄性育性的方法。 根据本发明的实 施例， 该方法包括： 将小麦近缘种的染色体引入到小麦纯合隐性核雄性不育突变体植株中, 以便获得携带有外源染色体的、 小麦隐性核雄性不育突变位点纯合的植株， 成为小麦纯合 隐性核雄性不育突变体的异附加系， 附加的染色体能够恢复小麦突变体雄性不育的表型， 所述异附加系产生可育雄性配子； 再将前面所述的构建体引入小麦纯合隐性核雄性不育突 变体的异附加系中， 以获得携带外源基因且外源基因位于外源染色体上的转基因植株； 培 育所述转基因植株， 所述转基因植株能够进行自体受精， 从而得到携带外源基因和外源染 色体的种子 （保持系） 和不携带外源基因和外源染色体的种子 （不育系）。 携带外源基因和 外源染色体的种子可以通过自交源源不断的生产不育系和保持系， 而不携带外源基因和外 源染色体的种子可以作为小麦雄性不育系用于杂交制种的亲本。 由此， 可以有效用于小麦 的杂交制种。
本发明还提出了一种制备小麦种子的方法。 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小麦隐性核雄性不育突变体的异附加染色体中， 使所述在附加染色体 上含有本发明所述构建体的小麦隐性核雄性不育突变体植株自体受精， 以获得含有前面所 述的构建体的种子。
本发明提出了小麦雄性不育系在制备杂交小麦中的用途。 根据本发明的实施例， 所述 小麦雄性不育系是通过前面构建小麦雄性不育系的方法获得的。 附图说明
图 1是小麦 mslg突变体与野生型的表型比较。图 1A-C分别是小麦野生型、小麦 mslg 突变体和小麦 mslg突变体 4E单体异附加系扬花期的穗子； 图 1D-F分别是小麦野生型、小 麦 mslg突变体和小麦 mslg突变体 4E单体异附加系扬花期的雄蕊和雌蕊； 图 1G-I分别是 小麦野生型、小麦 mslg突变体和小麦 mslg突变体 4E单体异附加系灌浆期的籽粒；图 1J-L 分别是小麦野生型、小麦 mslg突变体和小麦 mslg突变体 4E单体异附加系花粉的 I2-KI染 色； 图 1M-0分别是小麦野生型、小麦 mslg突变体和小麦 mslg突变体 4E单体异附加系成 熟的种子。
图 2是小麦 mslg突变体 4E异附加系的构建示意图。 ®表示自交， ms表示 mslg; 4E' 和 4E"分别表示一条和两条 4E染色体。
图 3是小麦 mslg突变体 4E异附加系的细胞遗传学鉴定。 图 3A是 mslg雄性不育突变 体的根尖细胞， 图 3B是 mslg突变体 4E单体异附加系的根尖细胞， 图 3C是 mslg突变体 4E二体异附加系的根尖细胞。 蓝色的是经 DAPI染色的小麦染色体， 红色的是罗丹明标记 的长穗偃麦草染色体。
图 4是植物表达载体 pl300-130的结构示意图。
图 5是植物表达载体 pl300-130的构建示意图。
图 6是小麦 mslg突变体 4E异附加系作为转化受体材料， 通过转基因所得到的转化体 自交得到不育系的示意图。 图 7是可育花粉粒和不育花粉粒 I2-KI的染色结果。 具体实施方式
下述实施例中所用方法如无特别说明均为常规方法， 所用引物均由上海英骏生物技术 公司合成， 测序由北京三博远志生物技术有限责任公司完成， PCR试剂盒、 载体构建过程 中的核酸内切酶购自宝生物工程有限公司， pEASY-Blunt-simple cloning kit购自北京全式金 生物技术公司， T4 DNA连接酶购自 NEB公司， 方法均参照试剂盒提供的方法进行。 实验 中所用的载体 pCAMBIA 1300来自于 CAMBIA公司。 实施例 1. 小麦 mslg突变体
小麦 ms突变体是在春小麦品种间杂交组合的杂种 F4代群体间发现的， 是一个自发突 变的雄性不育突变体， 雄性不育度为 100%。 图 1A和 1B是扬花期的小麦穗子， 其中小麦 ms突变体的穗子 （图 3B) 较野生型的穗子 （图 3A) 颖壳蓬松； 图 1D和 1E是扬花期的小 麦雄蕊和雌蕊，其中野生型的花药已经开裂，雌蕊的柱头上已经布满黄色的花粉粒（图 1D)， ^突变体的花药不开裂，雌蕊的柱头上没有花粉（图 1E);图 1J和 1K是花粉的 I2-KI染色， 其中野生型的花粉外形浑圆， 全部被染成蓝黑色， 表明具有较强的活力 （图 1J)， 而 ms突 变体的花粉外形皱縮， 全部呈黄褐色， 表明花粉没有活力 （图 1K); 图 3G和 3H是灌浆期 的种子， 其中野生型受精成功， 种子饱满 （图 1G)， 而 ms突变体未完成受精， 种子没有发 育 （图 m)。
将 ms突变体与包括中国春在内的 9个小麦常规品系杂交， 得到的 F1代自交， 观察 F2 代不育株与可育株的分离比， 均符合 1:3的比例， 表明该突变体是典型的单基因隐性核雄性 不育突变体 （表 1 )。
表 1 突变体与 9个小麦株系杂交 F2不育株与可育株的分离比
组合 F2代不育株 F2代可育株 预期分离比 X 2 P 中国春 299 809 1： 3 2 2250 0. 20-0. 10
ms/阿勃 319 963 1： 3 0 0042 >0. 90 甘麦 8号 210 678 1： 3 0 7943 0. 50-0. 30
陇春 7号 241 747 1： 3 0 1633 0. 70-0. 50
陇春 11号 199 604 1： 3 0 0104 >0. 90 ms/宁春 4号 283 893 1： 3 0 5000 0. 5-0. 3
¾s/宁春 16号 227 694 1： 3 0 0438 0. 90-0. 80 KUZA 203 649 1： 3 0 5649 0. 50-0. 30
ms/宁农 1376 213 689 1： 3 0 8514 0. 50-0. 30 将 ^突变体与小麦雄性不育突变体 mslc和 ms5进行等位分析，具体的做法是将 中 国春的 F1代植物分别与 mslc和 ms5雄性不育突变体进行杂交， 分析杂交后代中不育株与 可育株的比例， 与 mslc雄性不育突变体的杂交后代中不育株与可育株的比例为 1:1， 而与 ms5雄性不育突变体的杂交后代中没有不育株 (表 2)，说明 ^突变体是 msl的等位突变体， 命名为 mslg。 表 2 突变体与 fflWc突变体和 突变体的等位分析
组合 不育株 可育株 预期分离比 χ 2 Ρ
中国春 14 22 1 : 1 1. 778 0. 20-0. 10
ms5 //ffis/中国春 0 34 1 : 1 1 1 实施例 2. 小麦 mslg突变体 4Ε异附加系的构建
以 mslg突变体为母本， 以蓝粒 81529为父本杂交， 并对杂交后代进行了有目标的鉴定 选择， 成功地将 4E染色体单体异附加到具有纯合核不育基因型 mslg/mslg背景的普通小麦 上， 获得了籽粒为蓝色的自交可育的 mslg/mslg基因型小麦 4E单体异附加系。 图 1C是小 麦 mslg突变体 4E单体异附加系扬花期的穗子， 蓬松度恢复成野生型的状态； 图 1F是小麦 mslg突变体 4E单体异附加系扬花期的小麦雄蕊和雌蕊， 与野生型相同， 花药已经开裂， 雌蕊的柱头上已经布满黄色的花粉粒； 图 1L是小麦 mslg突变体 4E单体异附加系花粉的 I2-KI染色， 与野生型相同， 花粉外形浑圆， 全部被染成蓝黑色， 表明具有较强的活力； 图 31是小麦 mslg突变体 4Ε单体异附加系灌浆期的种子， 与野生型相同， 受精成功， 种子饱 满。 图 3M-0分别是小麦野生型、 小麦 mslg突变体和小麦 mslg突变体 4E单体异附加系 成熟的种子，其中小麦野生型和小麦 mslg突变体的种子为正常的种子颜色，后面称为白色; 小麦 mslg突变体 4E单体异附加系的种子为蓝色。由此说明 4E不但携带部分同源雄性可育 基因，而且携带蓝粒标记基因。具体的育种选育方法和过程如图 2所示 (图中用 ms表示 mslg; 和 4E"分别表示一条和两条 4E染色体； 浅蓝粒是在二倍体组织细胞中附加一条 4E染色 体， 深蓝粒是在二倍体组织细胞中附加两条 4E染色体)。
小麦 mslg突变体的基因型为 2n = 42 (ms/ms) = 42, 42条染色体全部是小麦的染色体， 且有一个育性基因突变 （ms/ms ) ; 4E 二体异附加系蓝粒小麦 81529 的基因型为 2n = 42 (MS/MS) + 4E" = 44, 除小麦的 42条染色体外， 还有两条来自长穗偃麦草的 4E染色体， 小 麦染色体上的育性基因为野生型 （MS/MS)。 以 mslg突变体为母本， 以蓝粒 81529为父本， 进行杂交， F1的种子为浅蓝粒， 其基因型为2n = 42 (MS/ms) + 4E = 43， 除小麦的 42条染 色体外，还有一条长穗偃麦草的 4E染色体，小麦染色体上的育性基因 MS为杂合（MS/ms)， F1植株表现为雄性可育。
F1植株自交获得的 F2代种子， 共有 9种基因型， 其中白粒的基因型 3种， 全部是没有 附加长穗偃麦草 4E染色体的； 深蓝粒和浅蓝粒的基因型各 3种， 全部为雄性可育， 其中只 有 2η = 42 (ms/ms) + 4E' = 43是下一步选育的目标基因型。 由于深蓝粒和浅蓝粒很难区分， 因此将 F2的蓝粒种子全部种下， 自交后单株收种为 F3代， F3代种子存在蓝白分离的说明 F2代基因组中只附加一条长穗偃麦草的 4E染色体， F2代种子为浅蓝粒； F3代种子不存在 蓝白分离的说明 F2代基因组中附加了两条长穗偃麦草的 4E染色体， F2代种子为深蓝粒。
将 F2代种子为浅蓝粒，单株收获的 F3代种子存在蓝白分离的 F3代蓝粒和白粒称为一 个 F3家系。 将 F3家系蓝白粒分别种植， 检查白粒行中的雄性不育株分离情况： 若 F3代家 系白粒行中无雄性不育株分离出来， 则说明该家系不携带雄性不育基因 （MS/MS), 淘汰该 家系； 若 F3代家系白粒行中出现雄性不育株系和可育株系的分离， 则说明该家系中的雄性 不育基因是杂合的 (MS/ms)， 也不是目标基因型； 若 F3代家系白粒行中出现 100%雄性不育 株率， 则说明该家系的雄性不育基因已经纯合（ms/ms)，其基因型为2n = 42 (ms/ms) + 4E = 43， 是目标基因型， 该家系相应的蓝粒行收获后下代继续分株系分蓝白成对种植， 以便对 其农艺性状进行选择。 以此类推， 对杂种后代中进行上述鉴定选择， 在 F12代最终选育出 农艺性状优良的小麦 mslg突变体 4E单体异附加系，其基因型为 2n = 42(ms/ms) + 4E' = 43， 浅蓝粒， 雄性可育。
由于 4E单价体的雌雄配子体向后代传递的效率分别为 19.0-25.8%和 11.1-18.4%,因此， 其自交后代可分离出 64.3%的 mslg雄性不育突变体 （白粒， 2n = 42 (ms/ms) = 42)、 32.1% 的 mslg突变体 4E单体异附加系 （浅蓝粒， 2n = 42 (ms/ms) + 4E' = 43) 禾 B 3.6%的 mslg突 变体 4E二体异附加系 （深蓝粒， 2n = 42 (ms/ms) + 4E"= 44)， mslg突变体 4E单体异附加 系和 mslg突变体 4E二体异附加系统称为 mslg突变体 4E异附加系。
以长穗偃麦草的基因组 DNA (罗丹明标记） 做探针， 利用 GISH 的方法对小麦 mslg 突变体、 小麦 mslg突变体 4E单体异附加系和小麦 mslg突变体 4E二体异附加系的根尖细 胞进行细胞遗传学分析， 结果如图 3所示， 蓝色的是经 DAPI染色的小麦染色体， 红色的是 罗丹明标记的长穗偃麦草染色体， 图 3A是小麦 mslg突变体的根尖细胞， 只有小麦的染色 体； 图 3B是小麦 mslg突变体 4E单体异附加系的根尖细胞， 除小麦染色体外还有一条长穗 偃麦草的染色体； 图 3C是小麦 mslg突变体 4E二体异附加系的根尖细胞， 除小麦染色体外 还有两条长穗偃麦草的染色体。 对上述三种材料根尖细胞的细胞遗传学分析结果与植株育 性、 种子颜色等性状完全对应。
所述蓝粒 81529是李振声院士培育的蓝粒小麦与甘肃春小麦材料杂交后经系统选育获 得的 4E附加系蓝粒春小麦材料， 为 4E二体异附加系。 实施例 3.利用小麦 mslg突变体 4E异附加系创建新一代小麦杂交育种体系
如图 6所示， 在没有克隆到育性恢复基因的情况下， 可以以纯合隐性核雄性不育小麦突 变体的异染色体附加系为转化受体材料， 通过将紧密连锁的 2个目标基因转化至异附加系受 体植株中， 筛选目标基因整合到附加的染色体上的转基因植株。 在转基因植株中， 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， 筛选基因可以用于转基因种子和 非转基因种子的分拣， 分拣出的非转基因种子用作不育系生产杂交种， 转基因种子用作保 持系来源源不断地、 稳定地生产不育系。 例如根据本发明的后续实施例， 以小麦 mslg突变 体 4E异附加系作为转基因受体材料， 将紧密连锁的 2个目标基因转化至小麦 mslg突变体 4E 异附加系中， 并筛选目标基因整合到附加的 4E染色体上的转基因植株： 4E染色体上带有的 育性恢复基因可以恢复小麦 mslg突变体的不育表型， 花粉失活基因 ZmBTl TP-AA1 可使含 有外源基因的花粉失活， 即失去授精能力， 荧光色选基因 mCherryW用于转基因种子和非转 基因种子的分拣， 分拣出的非转基因种子用作不育系生产杂交种， 转基因种子用作保持系 来源源不断地稳定地生产不育系。 由于该技术利用生物技术生产非转基因产品， 解决了小 麦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。 实施例 4.植物表达载体的构建
通过在 pCAMBIA1300载体上装配下述 DNA元件， 构建图 4所示的被称为 pl300-130 的植物表达载体。
( 1 )基因表达盒 ZmPG47: ZmBTl TP-ZmAAl : IN2-1 , 目标基因 ZmBTl TP-ZmAAl (SEQ ID NO: 1 ) 的开放读码框连接于启动子 ZmPG47 (SEQ ID NO: 2) 的下游， 终止子 IN2-1
(SEQ ID NO: 3) 的上游。
(2)基因表达盒 CaMV35S-Ltp2: mCherryW: PINII, mCherryW基因（SEQ ID NO: 4) 的开放读码框连接于 CaMV35S增强子 （SEQ ID NO: 5) -Ltp2启动子 （SEQ ID NO: 6) 禾口 PINII终止子 （SEQ ID NO: 7) 之间。 具体地， 参见图 5 的载体 pl300-130 的构建流程示意图， 具体描述如下： 首先， 终止子 IN2-1 由人工合成获得， 合成过程中在 IN2-1 的 5' 端和 3' 分别加入 Hindlll-Notl-EcoRI三个酶切位点 （aagctt gcggccgc gaattc)禾 B Bglll酶切位点 （agatct), 合成 的 IN2-1终止子连在 pEASY-Blunt-simple载体上并经测序验证正确。
ZmBTl TP-ZmAAl基因序列由人工合成获得，合成过程中在 ZmBTl TP-ZmAAl的 5'端 和 3' 分别加入 Notl酶切位点 （gcggccgc) 和 EcoRI酶切位点 （gaattc), 合成的 ZmBTl TP-ZmAAl 基因连在 pEASY-T3 载体上并经测序验证正确。 用基因两端所带有的酶切位点 Notl和 EcoRI对连有 ZmBTl TP-ZmAAl基因的 pEASY-Blunt-simple质粒进行双酶切， 得到 序列正确、 两端带有相应酶切位点的 ZmBTl TP-ZmAAl基因片段。 将连有 IN2-1终止子的 pEASY-Blunt-simple载体用限制性内切酶 Notl和 EcoRI切开，连入 ZmBTl TP-ZmAAl基因， ZmBTl TP-ZmAAl基因位于 IN2-1终止子的上游。
从玉米的基因组中扩增 ZmPG47启动子， 扩增 ZmPG47的引物为：
Fl : aagctt TGCACCGGACACTGTCTG (SEQ ID NO: 8)， aaggcc为 Hindlll酶切位点， Rl : gcggccgc TGTCGTGATCGATGCTTTATTC (SEQ ID NO: 9)， gcggccgc为 Notl酶 切位点；
将 PCR产物连在 pEASY-Blunt-simple载体上并经测序验证正确后， 用 ZmPG47启动子 两侧所带的酶切位点 Hindlll和 Notl对连有 ZmPG47启动子的 pEASY-Blunt- simple质粒进 行双酶切， 得到序列正确、 两端带有相应酶切位点的 ZmPG47 启动子片段。 将连有 IN2-1 终止子和 ZmBTl TP-ZmAAl基因的 pEASY-Blunt- simple载体用限制性内切酶 Hindlll和 Notl 切开， 连入 ZmPG47启动子， 连入的 ZmPG47启动子位于 ZmBTl TP-ZmAAl基因的上游。 这样就构建成了中间载体 A， 上面连有基因表达盒 ZmPG47: ZmBTl TP-ZmAAl : IN2-1。
接下来，
终止子 ΡΙΝΠ由人工合成获得，合成过程中在 ΡΙΝΠ的 5'端和 3'分别加入 BamHI- EcoRI - Xhol-Sall-四个酶切位点 （ggatcc gaattc ctcgag gtcgac) 和 Kpnl酶切位点 （ggtacc), 合成的 PINII终止子连在 pEASY-Blunt-simple载体上并经测序验证正确。
mCherryW基因序列由人工合成获得， 合成过程中在 mCherryW基因的 5' 端和 3' 分 别加入 Xhol酶切位点 （ctcgag) 和 Sail酶切位点 （gtcgac), 合成的 mCherryW基因连在 pEASY-Blunt-simple载体上并经测序验证正确。 用基因两端所带有的酶切位点 Xhol和 Sail 对连有 mCherryW基因的 pEASY-Blunt-simple质粒进行双酶切，得到序列正确、两端带有相 应酶切位点的 mCherryW基因片段。将连有 ΡΙΝΠ终止子的 pEASY-Blunt-simple载体用限制 性内切酶 Xhol和 Sail切开， 连入 mCherryW基因， mCherryW基因位于 ΡΙΝΠ终止子的上 游。
从大麦的基因组中扩增 Ltp2启动子， 扩增 Ltp2的引物为：
F2: gaattc AACCGTCTCTTCGTGAGAATAACC (SEQ ID NO: 10)， gaattc为 EcoRI 酶切位点，
R2: ctcgag TACTCGGCTACACTCACACGC (SEQ ID NO: 11 )， ctcgag为 Xhol酶切位 点；
将 PCR产物连在 pEASY-Blunt-simple载体上并经测序验证正确后，用 Ltp2启动子两侧 所带的酶切位点 EcoRI和 Xhol对连有 Ltp2启动子的 pEASY-Blunt-simple质粒进行双酶切， 得到序列正确、两端带有相应酶切位点的 Ltp2启动子片段。将连有 ΡΙΝΠ终止子和 mCherryW 基因的 pEASY-Blunt-simple载体用限制性内切酶 EcoRI和 Xhol切开， 连入 Ltp2启动子， 连入的 Ltp2启动子位于 mCherryW基因的上游。
CaMV35S增强子序列由人工合成获得， 合成过程中在 CaMV35S增强子的 5' 端和 3' 分别加入 BamHI酶切位点 （ggatcc)和 EcoRI酶切位点 （gaattc), 合成的 CaMV35S增强子 连在 pEASY-Blunt-simple载体上并经测序验证正确。 用基因两端所带有的酶切位点 BamHI 和 EcoRI对连有 CaMV35S增强子的 pEASY-Blunt- simple质粒进行双酶切， 得到序列正确、 两端带有相应酶切位点的 CaMV35S增强子片段。 将连有 ΡΙΝΠ终止子、 mCherryW基因和 Ltp2 启动子的 pEASY-Blunt-simple 载体用限制性内切酶 BamHI 禾卩 EcoRI 切开， 连入 CaMV35S增强子， CaMV35S增强子位于 Ltp2启动子的上游。这样就构建成了中间载体 B， 上面连有基因表达盒 CaMV35S-Ltp2: mCherryW: ΡΙΝΠ。
接下来， 用限制性内切酶 Bglll和 Hindlll将基因表达盒 ZmPG47: ZmBTl TP-ZmAAl： IN2- 1从中间载体 A上切下来，用限制性内切酶 Kpnl和 BamHI将基因表达盒 CaMV35S-Ltp2： mCherryW: PINII从中间载体 B 上切下来， 同时连入用限制性内切酶 Kpnl和 Hindlll切开 的 pCAMBIA 1300载体， 即得到 pl300-130。 上述构建过程如图 5所示。
实施例 5. 小麦的遗传转化
利用农杆菌法和基因枪法两种方法对小麦进行共转化， 具体的转化受体材料为小麦 mslg突变体 4E异附加系 （构建过程见实施例 2)， 可以是小麦 mslg突变体 4E单体异附加 系， 也可以是小麦 mslg突变体 4E二体异附加系， 本实施例采用的是小麦 mslg突变体 4E 单体异附加系。 需要说明的是， 所有能够完全恢复小麦 msl 突变体雄性不育表型的异染色 体附加系都可以作为本发明的转化受体材料， 除本案的来自长穗偃麦草的 4E染色体外， 例 如黑麦的 2R和 4R也可以恢复小麦 msl突变体的雄性不育表型，还有但不限于来自黑麦 2R 和 4R。 进一步， 所有能够利用近缘种的附加染色体恢复小麦隐性核雄性不育突变体 （不限 于 msl突变体） 的异附加系， 都可以作为本发明的受体。
实施例 6.转化事件的分子鉴定
设计引物对转基因小麦植株进行 PCR 鉴定。
F3: 5'- ATTCATGTACGGCTCCAAGG-3 ' ( SEQ ID NO: 12)
R3: 5'- TGGCCTTGTAGGTGGTCTTC -3 ' ( SEQ ID NO: 13 )
反应条件为： 94 °C预变性 5分钟； 94 °C变性 30秒； 55 °C退火 30秒； 72°C延伸 40秒； 35个循环； 72°C延伸 10分钟。 扩增的是 mCherryW的部分片段， 长度为 359 bp。 根据 PCR 鉴定结果， 选出阳性的转基因植株。 采用 Southem-blot对转基因阳性植株中外源转基因的 完整性和拷贝数进行鉴定， 结果表明转基因阳性植株中外源基因完整且为单拷贝整合。 实施例 7.转基因植株材料的花粉活性检测
对实施例 5 所述的转基因植株进行分析发现， 转基因植株和非转基因对照植株之间没 有观察到明显的形态上的不同， 但是其雄性生育能力不同。
以小麦 mslg突变体（不育， 下面简称为 CK1 )和非转基因小麦 mslg突变体 4E异附加 系 （可育， 下面简称为 CK2) 为对照， 进行花粉活性检测。
在小麦盛花期， 从外源基因整合在受体小麦 mslg突变体 4E异附加系中附加的长穗偃 麦草 4E染色体上的转基因植株、 CK1 及 CK2 中各随机抽取单株， 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、 计数不育花粉数和花粉总数 （图 7 显示了染色后的可育花 粉粒和不可育花粉粒）， 不育花粉占花粉总数的约 20%， 与小麦 mslg突变体 4Ε单体异附加 系中 4E染色体通过雄配子体向后代传递的比例为约 20%相符，说明了 T-DNA整合在 4E染 色体上， 且 T-DNA上带有的花粉致死基因能够导致转基因花粉失活。
实施例 8.荧光种子与非荧光种子分离比例
对实施例 6所述的转化体所结实的荧光与非荧光种子的分离比例进行了调查， 结果表明 荧光种子占总种子数的约 20%， 非荧光种子占种子总数的约 80%， 与小麦 ffl^ 突变体 4E 单体异附加系中 4E染色体通过雌配子体向后代传递的比例为约 20%相符， 进一步也证明 了 T-DNA整合在 4E染色体上。 实施例 6和 7的结果表明本发明中所提供的表达载体各元 件作为整体表达良好。

Claims

权利要求
1. 一种调控小麦育性的方法， 所述方法包括： 在纯合隐性核雄性不育小麦突变 体的异染色体附加系材料中， 引入下述构建体， 所述构建体包含花粉失活基 因和筛选基因， 其特征在于： 所述纯合隐性核雄性不育小麦突变体的异染色 体附加系材料中含有一个能恢复其纯合隐性核雄性不育的异附加染色体， 所 述构建体在转入所述小麦材料后， 其整合在该异附加染色体上。
2. 权利要求 1所述的方法，其中所述的异附加染色体来自长穗偃麦草的 4E染色 体、 或黑麦的 2R或 4R。
3. 权利要求 1或 2所述的方法， 其中所述的花粉失活基因包括但不限于可编码 促使碳水化合物降解的酶， 如玉米 a淀粉酶基因、 生长素 (auxin) , rot B、 细胞毒素基因、 白喉毒素、 DAM甲基化酶、 或显性的雄性不育基因。
4. 权利要求 3所述的方法，其中所述的玉米 a淀粉酶基因的核苷酸序列如 SEQ ID NO : 15所示。
5. 权利要求 4所述的方法， 其中所述的玉米 a淀粉酶基因的 5 ' 端连有一个导 肽， 所述导肽的核苷酸序列如 SEQ ID NO : 14所示。
6. 权利要求 5所述的方法， 其中所述的玉米 a淀粉酶基因由一个花粉发育晚期 特异表达的启动子驱动表达， 所述花粉发育晚期特异表达的启动子包括但不 限于 ZmAPBl启动子、 ZmABP启动子、 ZmC5启动子、 MPCBP启动子、或 ZmPG47 启动子。
7. 权利要求 1或 2所述的方法， 其中所述的筛选基因为一个荧光筛选基因， 包 括但不限于红色荧光基因、 青色荧光蛋白基因、 黄色荧光蛋白基因、 荧光素 酶基因、 绿色荧光蛋白基因、 花青甙 pl、 mCherryW等基因。
8. 权利要求 7所述的方法， 其中所述的荧光筛选基因由一个种子特异表达的启 动子驱动表达，所述种子特异表达的启动子包括但不限于 END2启动子、 LtP2 启动子等。
9. 一种表达盒， 包含一个花粉失活基因和一个筛选基因， 其特征在于所述花粉 失活基因包括但不限于可编码促使碳水化合物降解的酶， 如玉米 a淀粉酶基 因、 生长素 (auxin) , rot B、 细胞毒素基因、 白喉毒素、 DAM甲基化酶、 或 显性的雄性不育基因； 所述筛选基因为一个荧光筛选基因， 包括但不限于红 色荧光基因、 青色荧光蛋白基因、 黄色荧光蛋白基因、 荧光素酶基因、 绿色 荧光蛋白基因、 花青甙 pl、 mCherryW等基因。
权利要求 9所述的表达盒， 其中所述的其中所述的花粉失活基因的 5 ' 端连 有一个导肽， 所述导肽的核苷酸序列如 SEQ ID NO : 14所示。
权利要求 9或 10所述的表达盒，其中所述的花粉失活基因由一个花粉发育晚 期特异表达的启动子驱动表达， 所述花粉发育晚期特异表达的启动子的核苷 酸序列如 SEQ ID NO : 2所示。
权利要求 11所述的表达盒，其中所述的花粉失活基因还连有一个终止子，所 述终止子的核苷酸序列如 SEQ ID N0 : 3所示。
权利要求 11所述的表达盒，其中所述的筛选基因由一个种子特异表达的启动 子驱动表达， 所述种子特异表达的启动子包括但不限于 EN D2启动子、 LtP2 启动子等。
权利要求 13所述的表达盒， 其中所述的启动子的 5 ' 端还连有一个增强子序 列， 所述增强子的核苷酸序列如 SEQ ID N0 : 5所示。
权利要求 14所述的表达盒， 其中所述的荧光筛选基因的 3 ' 端连有一个终止 子， 所述终止子的核苷酸序列如 SEQ ID N0 : 7所示。
一种小麦细胞、 组织、 器官或种子， 其特征在于所述小麦细胞、 组织、 器官 或种子来自一种纯合隐性核雄性不育小麦突变体的异染色体附加系材料， 所 述纯合隐性核雄性不育小麦突变体的异染色体附加系材料中含有一个能恢复 其纯合隐性核雄性不育的异附加染色体， 在该异附加染色体上外源转入了一 个构建体， 所述构建体包含权利要求 9-15之任一所述的表达盒。
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