WO2021104220A1 - Régulation de l'expression de gènes de compétitivité de pollen stk1 ; 2 et application associée dans l'amélioration de l'efficacité de la lignée mâle-stérile de noyau végétal de propagation - Google Patents

Régulation de l'expression de gènes de compétitivité de pollen stk1 ; 2 et application associée dans l'amélioration de l'efficacité de la lignée mâle-stérile de noyau végétal de propagation Download PDF

Info

Publication number
WO2021104220A1
WO2021104220A1 PCT/CN2020/130966 CN2020130966W WO2021104220A1 WO 2021104220 A1 WO2021104220 A1 WO 2021104220A1 CN 2020130966 W CN2020130966 W CN 2020130966W WO 2021104220 A1 WO2021104220 A1 WO 2021104220A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
pollen
male sterile
male
competitiveness
Prior art date
Application number
PCT/CN2020/130966
Other languages
English (en)
Chinese (zh)
Inventor
赖锦盛
宋伟彬
赵海铭
Original Assignee
中国农业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国农业大学 filed Critical 中国农业大学
Publication of WO2021104220A1 publication Critical patent/WO2021104220A1/fr

Links

Images

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the invention belongs to the field of plant genetic breeding and seed production, and specifically relates to the regulation and control of the expression of pollen fertilization competitiveness genes STK1 and STK2 and the application in improving the efficiency of multiplication of nuclear male sterile lines of plants.
  • hybrids Due to the existence of heterosis, the biomass, disease and insect resistance, and stress tolerance (drought, high temperature, low temperature, saline-alkali, etc.) of hybrids are considerably improved compared to their parents, such as hybrid corn and hybrid rice. Higher than homozygous parents.
  • the method usually used to produce hybrids is: planting the female parent and the male parent together, removing the tassels of the female parent, and retaining the tassels of the male parent. The seeds harvested by the female parent are the hybrids.
  • Self-pollination refers to the phenomenon that the pollen of a plant pollinates the pistil of the same individual. In plants with bisexual flowers, it can be divided into same-flower pollination (Phaseolus spp.) for pollination between the stamens and pistils of the same flower, and adjacent-flower pollination, which pollinates between different flowers in an inflorescence (individual), and the same plant as different Cross-pollination of the same plant for pollination between flowers. Some plants do not grow stamens and pistils in the same flower, or even on the same plant, and cannot self-pollinate. Their pistils can only get pollen from other flowers. This is called cross-pollination. A category of crops with a natural hybridization rate higher than 50% and self-recession is classified as often cross-pollinated crops, such as corn.
  • Maize is a monoecious plant, and male and female flowers are located on different parts of the plant. Maize can reproduce offspring through self-pollination or cross-pollination. Under natural conditions, when the wind blows the pollen from the tassel to the filaments of the female ear That completes natural pollination.
  • a homozygous maize inbred line should be developed first, and then the two inbred lines should be crossed, and the yield and stress resistance of the hybrid offspring should be evaluated to determine whether it has commercial potential.
  • Each inbred line may have one or more excellent traits that the other inbred line lacks, or supplement one or more undesirable traits of the other inbred line.
  • the first generation of seeds crossed between two inbred lines is F1 generation seeds.
  • F1 generation plants are obtained after the F1 generation seeds germinate. Compared with the two inbred line parents (parents), the F1 generation plants are more vigorous and resistant to stress. Both have strong advantages and at the same time have more biomass.
  • Hybrids can be produced by artificially detasseling the female parent, that is, the unpolished female parent (which can be sown in the field with the male parent, such as sowing 5 rows of female parent, one row of male parent) tassels, and retain the male parent tassels . Subsequently, as long as the foreign corn pollen is isolated, the female ears of the female parent can only receive the pollen of the male parent, and the resulting seed is a hybrid (F1), which can be used for agricultural production.
  • F1 hybrid
  • Machines can also be used to emasculate the female parent.
  • the reliability of mechanical emasculation and manual emasculation are basically the same, but faster and at lower cost.
  • most emasculation machines will cause more damage to the plant. Therefore, there is no completely satisfactory emasculation method at present, and people are still looking for a cheaper and more thorough replacement method for emasculation. .
  • a stable male sterility system provides a simple and efficient method.
  • CMS inbred lines some genotypes can avoid heavy detasseling work.
  • the method includes three main materials, namely sterile lines: male sterile materials, maintainer lines: can provide pollen for the sterile lines, so that the offspring of the sterile lines remain sterile lines, restore lines: can restore the sterile lines Fertility.
  • the sterile line and the restorer line are crossed to produce F1, which is a hybrid for agricultural production. More specifically, the nucleo-cytoplasmic interactive sterile type is manifested as nucleo-cytoplasmic interaction heredity.
  • cytoplasmic gene is fertile N, no matter whether the nuclear gene is fertile (RfRf) or sterile (rfrf), it is male fertile.
  • rfrf homozygous sterility gene
  • This male sterile line formed by nucleoplasmic interaction has a genetic composition of S(rfrf), which cannot produce normal pollen, but it can be used as a hybrid female parent. Since the maintainer line N(rfrf) can be found [using it to cross with the sterile line, the resulting F1 can still maintain male sterility, namely: S(rfrf)( ⁇ ) ⁇ N(rfrf) ⁇ S(rfrf)(not Breeding)] and can accept restorer lines S(RfRf) or N(RfRf) [using them to cross with sterile lines, the resulting F1 are all fertile, namely: S(rfrf)( ⁇ ) ⁇ S(RfRf) ⁇ S(Rfrf)(F1)(fertile), or S(rfrf)( ⁇ ) ⁇ N(RfRf) ⁇ S(Rfrf)(F1)(fertile)] pollen to restore F1 to male fertility, F1 plants produce F2 by selfing
  • Male sterile lines can avoid manual detasseling, save manpower, reduce seed costs, and ensure seed purity.
  • rice, corn, sorghum, onion, castor, sugar beet and rapeseed have used nuclear-cytoplasmic male sterility to produce hybrid seeds; the nuclear-plasma male sterile lines of other crops are also being widely used. Research.
  • CMS also has its shortcomings. One is that it is observed that individual CMS materials are susceptible to disease, and the other is that recovery lines are more difficult to find. These problems hinder the wide application of the CMS system in seed production.
  • Fabijanski, et al. developed methods to make plants male sterile (EPO 89/3010153.8 and PCT/CA90/00037). It is mainly through the following two ways to inhibit the male flower fertility of the plant.
  • One method is to link the promoter specifically expressed by the male tissue to the cytotoxic gene and transfer it into the plant so that the male flower cannot disperse normally without affecting other traits; the other is
  • gene interference means that the cloned gene that controls the male fertility of the plant is interfered by transgenic means, so that it cannot function normally.
  • transgenic means so that it cannot function normally.
  • transgenic methods have also been used to maintain the sterility of male sterile plants (US6743968).
  • the pollen lethal gene and the male fertility restoration gene are constructed in a vector and introduced into the male sterile plant.
  • the transgenic offspring are fertile, but only pollen without the restoration gene can be produced.
  • the homozygous recessive state of the recessive sterile plant is maintained. It first constructs a transgenic vector, which contains a pollen cell lethal gene, and at the same time, the vector also contains a dominant gene to restore plant fertility. The vector is transferred into male sterile plants, and the vector exists in a heterozygous state in the transgenic plants.
  • the plant is fertile due to the existence of the restorer fertility gene. When it is crossed with the male sterile plant, the pollen contains the restorer gene. (Msms) also contains lethal genes, which make pollen abortion. Therefore, only pollen (ms) that does not contain restorer genes can cross with the female gametes (ms) of male sterile plants, and the offspring are recessive homozygous individuals (msms).
  • male sterility system As mentioned above, an important issue in many work using male sterility system is how to use male sterility genes and how to distinguish male sterile seeds, plants and fertile seeds and plants, and also need to consider how to make sterile seeds. The infertility of the individual is maintained.
  • the purpose of the present invention is to improve the production efficiency of sterile lines.
  • the method provided by the present invention is used to maintain the homozygous recessive state of male sterile plants, and the method includes:
  • the construct in the example, a transgenic element Ms45-RNAi
  • the construct exists in a heterozygous state in the second plant (the heterozygous Ms45-RNAi/- insert is only present in one chromatid, and its sister chromatid does not contain transgenic elements)
  • the construct contain:
  • the first nucleotide sequence which when expressed in the first plant will restore the male fertility of the first plant
  • the second nucleotide sequence when it exists in a heterozygous state, can affect the competitiveness of pollen fertilization
  • the first nucleotide sequence in the example, the Ms45 gene expression element shown in sequence 1
  • the second nucleotide sequence in the example, the interference fragments of STK1 and STK2 genes
  • the above method is a method of multiplying male sterile lines of plants
  • the plant, the first plant and the second plant are all dicotyledonous plants or monocotyledonous plants;
  • the plant, the first plant and the second plant can be not only corn (Zea mays), but also rice (Oryza sativa), sorghum (Sorghum bicolor), wheat (Triticumaestivum), soybean (Glycine max), cotton (Gossypiumhirsutum), sunflower (Helianthus annuus) and other crops.
  • the first nucleotide sequence includes a gene for controlling male fertility, such as the wild-type allele Ms45 of ms45 in Table 1.
  • This gene for controlling male fertility is not limited to the genes listed in Table 1. , Genes that control male fertility in maize or other species can also achieve the purpose of the present invention, and therefore are also within the protection scope of the present invention.
  • the first plant is the maize male sterile mutant ms45, specifically the ms45 homozygous recessive inbred line Zheng 58 ( ⁇ 58(ms45ms45)), which is derived from ms45 Homozygous recessive mutant (Maize Genetics Cooperation Stock Center, 905I; the mutation site is recorded in Table 1 ms45) and the backcross offspring of Zheng 58 (zheng58).
  • the first nucleotide sequence is the Ms45 gene expression element in the embodiment of the present invention, and the Ms45 gene expression element expresses the protein Ms45.
  • the protein Ms45 is as follows a) or b):
  • the Ms45 gene expression elements include Ms45 gene promoter, Ms45 gene 5'UTR, Ms45 gene exon, Ms45 gene intron, Ms45 gene 3'UTR and Ms45 gene terminator;
  • the Ms45 gene expression element is a DNA molecule shown in 1) or 2) or 3) as follows:
  • the coding region includes a DNA molecule with sequence 1 or the coding region includes a DNA molecule with the reverse complementary sequence of sequence 3 from positions 4762-8265 at the 5'end;
  • the second nucleotide sequence when the second nucleotide sequence exists in a heterozygous state, it reduces pollen fertilization competitiveness, but does not affect pollen fertility.
  • the second nucleotide sequence includes a functional fragment; the functional fragment inhibits the expression of a specific gene; the specific gene is a gene capable of regulating pollen fertilization competitiveness; the expression of the specific gene is reduced It will lead to reduced competitiveness of pollen fertilization, but does not affect pollen fertility (different from pollen lethality). Pollen can develop normally, but the pollen that changes the expression of the transgene is significantly different from wild-type pollen in terms of fertilization competitiveness, which is different from wild-type pollen. Under the condition of mixed pollen, the competitiveness of genetically modified pollen to participate in fertilization is greatly reduced. However, the transgenic pollen can complete normal pollination and fertilization. When all pollen is transgenic (no competition), it can be pollinated, and it can produce normal fruit and achieve a high seed setting rate.
  • the specific genes are STK1 and STK2 genes.
  • the functional fragments are interference fragments of STK1 and STK2 genes.
  • the reverse complementary sequence of the interference fragment is shown at positions 11282-11875 from the 5'end in sequence 3 of the sequence table.
  • the second nucleotide sequence that is, the reverse complementary sequence of the complete expression element of the interference fragment, is shown at positions 10982-13578 from the 5'end in the sequence 3 of the sequence listing.
  • the present invention relates to nucleotide sequences for controlling male fertility and pollen competitiveness of plants, and a method for multiplying male sterility of plants developed by using these nucleotide sequences and transgenic technology.
  • Another object of the present invention is to provide a DNA construct.
  • the construct provided by the present invention is the construct described in the above method.
  • the above-mentioned DNA construct can restore the fertility of the male sterile mutant while changing the pollen competitiveness of the plant.
  • the third object of the present invention is to provide any of the following substances.
  • the above-mentioned second plant can maintain the sterility of the male sterile plant.
  • the key of the present invention is to construct the male restorer gene Ms45 and the elements for controlling plant pollen competitiveness in a vector, which can restore the fertility of the male sterile mutant ms45, and meanwhile, the pollen competitiveness of plants containing the transgenic elements decreases. Therefore, in the process of cultivating sterile lines, the proportion of plants (fertile individuals) containing transgenic elements decreases, and the proportion of sterile individuals (sterile lines) increases, thereby realizing the expansion of sterile individuals (sterile lines).
  • the inventors first constructed a plant transformation vector, which contains the expression elements of the gene to restore male fertility and the interference fragments of the maize pollen fertilization competitiveness regulatory genes STK1 and STK2 genes.
  • a plant transformation vector which contains the expression elements of the gene to restore male fertility and the interference fragments of the maize pollen fertilization competitiveness regulatory genes STK1 and STK2 genes.
  • Into HiIIA ⁇ HiIIB maize hybrids and then use male sterile plants to backcross the obtained transgenic plants, so as to introduce the interference fragments of the Ms45 restorer gene and the maize pollen fertility competitiveness regulating gene STK1 and STK2 genes into the control plant male fertility
  • Male sterile plants are in ms45. Due to the presence of the restorer gene Ms45, the transgenic plants appear to be fertile.
  • transgenic heterozygous plant Msmsms
  • male sterile plant msms
  • sterile line genotype msms
  • the sterile line can be restored to fertility by any wild-type plant and can be used as a sterile line in the seed production process; the other is a fertile grain containing transgenic elements (maintainer line, genotype Msmsms), the maintainer line It is recessively homozygous at the site of male fertility control. Because it contains complementary transgene sequences, the plant is fertile.
  • Figure 1 shows the male flower phenotypes of male fertility mutant ms45 (A) and wild-type Ms45 (B).
  • Nuclear male sterility is the result of mutations, suppression or other effects of key genes in the process of microspore formation. These genes are collectively referred to as male sterility genes.
  • the pollen development pathway is controlled by many genes, so many gene mutations will eventually lead to male sterility.
  • a large number of male sterility mutants have been identified in maize (as shown in Table 1), and each male sterility gene is There is a specific restorer gene, that is, each male sterile mutant can only be restored by its wild-type allele.
  • the present invention takes a male sterile mutant in Table 1 as an example.
  • ms45 the male flower of the mutant cannot be powdered normally (as shown in Figure 1, A is a male sterile mutant and B is a wild type), and the mutant can be sporty. It was restored by the Ms45 gene in the wild-type plant.
  • the wild-type restorer gene Ms45 in the present invention is derived from the inbred line B73, and its sequence is shown in sequence 1.
  • the sequence contains the complete expression elements of the Ms45 gene, that is, the promoter, 5'UTR, exon, intron, and 3'UTR And terminator. After the nucleotide shown in sequence 1 was transferred into the ms45 male sterile mutant, the mutant plant became fertile.
  • the Ms45 gene expression element shown in sequence 1 encodes the Ms45 protein shown in sequence 2.
  • the present invention introduces functional elements in the process of expanding plant nuclear male sterile lines, and the functional elements include promoters, interference fragments of maize pollen fertilization competitiveness regulatory genes STK1 and STK2 genes, and terminator.
  • the expression of functional elements can lead to a decline in the competitiveness of pollen pollination.
  • accession number of STK1 gene in MaizeGDB is Zm00001d045056.
  • accession number of STK2 gene in MaizeGDB is Zm00001d003377.
  • Example 1 Construction of a plant transformation vector containing the male fertility gene Ms45, functional elements and selectable marker genes
  • the present invention takes the ms45 male sterility mutant in Table 1 as an example, and specifically describes the embodiment.
  • the above-mentioned complete expression elements and functional elements of the Ms45 gene are constructed into the vector to obtain the transformation vector constructed by the method according to the invention (the circular plasmid shown in sequence 3 of the sequence table).
  • the transformation vector includes the male fertility gene Ms45, functional elements and the selection marker gene bar.
  • positions 4762-8265 from the 5'end are the reverse complementary sequence of the complete expression element of the Ms45 gene (when constructing the vector and amplifying, the sequence 3 has a mutation from the 5'th position 5664, which is consistent with the sequence 1.
  • a difference of 1 base is produced, but it does not result in a change in the encoded amino acid sequence, does not affect the function of the protein, and the remaining bases are unchanged).
  • Positions 10982-13578 are the reverse complementary sequence of the functional element (including corn pollen fertilization competition)
  • the reverse complement sequence of the gene is shown in positions 10986-11238)
  • positions 13899-15353 are the complete expression elements of the selectable marker gene bar (wherein, positions 13899-14576 encode promoters, and positions 14621-15172 encode selectable marker genes). bar, coded terminator at positions 15179-15353).
  • the male fertility gene Ms45, functional elements and selectable marker gene bar are located on the same T-DNA.
  • the present invention obtains transgenic plants through the method of Agrobacterium infecting maize immature embryos.
  • the plant transformation vector of Example 1 was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium, which was then used to infect maize immature embryos, as follows:
  • Example 1 The plant transformation vector of Example 1 was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium.
  • the recipient used in the transgenic process in the laboratory is the hybrid F1 generation of inbred lines HiIIA and HiIIB.
  • Corn inbred lines HiIIA and HiIIB Armstrong C L, Green C E and Phillips R L. Development and availability of germplasm with high Type II culture formation response. Maize Genetics Cooperation News Letter, 1991, 65: 92-93).
  • the plant transformation vector of Example 1 was introduced into the immature embryos of recipient plants by the Agrobacterium tumefaciens EHA105 infection method, and transgenic plants were obtained after screening with the herbicide dialaphos.
  • the specific method is:
  • Recombinant Agrobacterium should be cultured on YEP (containing Kana33mg/L and Str100mg/L antibiotics) medium one week in advance, and stored in a refrigerator at 4 degrees for about one month. Long-term storage should be stored at -80 degrees glycerol. 2. Recombinant Agrobacterium To cultivate on YEP medium at 19°C for 3 days, add Kana (33mg/L) and Str (50mg/L) at the same time.
  • the selection medium contains 1.5 mg of bialaphos/ L. After two weeks, the concentration of bialaphos can be increased to 3mg/L for subculture.
  • the cells containing the transformants will grow into visible type II callus for about 5 weeks after being infected.
  • regeneration medium I the solute is shown in Table 2, the solvent is water
  • the solute is shown in Table 2, the solvent is water
  • MS salt was purchased from Phyto Technology Laboratories, and the product number is M524.
  • bar gene detection method use the following primers Bar669F and Bar669R: PCR amplification of the hybrid progeny, if it contains the target fragment of 669bp, the hybrid progeny contains the transgenic element Ms45- If the hybrid offspring of RNAi does not contain the 669bp target fragment, the hybrid offspring is the hybrid offspring that does not contain the transgenic element Ms45-RNAi.
  • Bar669F 5'TCTCGGTGACGGGCAGGAC 3'(sequence 4);
  • Bar669R 5'TGACGCACAATCCCACTATCCTT 3'(sequence 5);
  • Table 3 shows the proportion of grains containing expression elements on the hybrid ears of some hybrid progeny.
  • STK-68, STK-111 and STK-125 were used in subsequent experiments.
  • transgenic plants prepared in the examples can be used to multiply the ms45 male sterile inbred line, as follows:
  • ms45 homozygous recessive mutant (Maize Genetics Cooperation Stock Center, 905I, as the female parent, crossed with an inbred line (such as Zheng 58 (zheng58)), the obtained F1 continued to be crossed with the maize inbred line Zheng 58 (Henan City Agricultural The Institute of Grain Crops, Academy of Sciences) backcrossing, genotype analysis of the obtained BC1 population, screening of plants heterozygous for Ms45 and continuing backcrossing with Zheng58, after such backcrossing for 5-6 generations, using molecular markers to screen Ms45 The point is heterozygous, and the individual plants with agronomic phenotypes close to Zheng 58 are selfed to obtain ms45 homozygous recessive inbred line Zheng 58 ( ⁇ 58(ms45ms45)), which can be used as a sterile line , Call it the first plant;
  • the method for screening the genotype of the Ms45 locus is as follows: PCR amplification is performed with the following primers Ms45F and Ms45R, the size of the Ms45 target fragment: 859 bp, and the target fragment size of ms45: 811 bp. If both 859bp and 811bp target fragments are obtained, the Ms45 position is heterozygous Ms45/ms45, if there is no 811bp fragment, the Ms45 position is dominantly homozygous for Ms45/Ms45; if there is no 859bp target fragment, it is ms45/ms45 Recessive homozygous.
  • Ms45R 5'-TGTTGTTTCTTGGCAAAGGTCAG-3' (sequence 7).
  • This inbred line is the second plant that is heterozygous for Ms45-RNAi and homozygous for ms45, also known as Zheng 58 (Ms45-Lc heterozygous and ms45 homozygous recessive).
  • the inbred line Zheng 58 (Zheng 58 (ms45ms45)) was crossed, and the grains containing transgenic elements were selected from the hybrid offspring (the bar gene was used to determine whether the transgenic elements were transgenic elements, the same below), and then the T0 generation in Example 2 was transformed into the field.
  • Ms45-RNAi corn (male parent) and the ms45 obtained from (1) were recessively sprayed with 200 mM bialaphos, and the surviving plants continued to be backcrossed with Zheng 58 (ms45ms45).
  • the grains or plants containing transgenic elements contain transgenic sites (Ms45-RNAi), so the resulting offspring contains transgenic grains or plants that contain transgenic elements.
  • the loci (Ms45-RNAi) are all heterozygous. Pollinate the first plant with the grains or plant pollen containing the transgenic elements, and if the obtained grains or normal plants without transgenic elements are sterile, the transgenic site Ms45-RNAi of the pollen-providing plant is heterozygous and ms45 The point is invisible homozygous, which is the second plant.
  • the second plant of Zheng 58 (Ms45-RNAi heterozygous and ms45 homozygous) obtained in (2) was used as the male parent, and the MS45 homozygous recessive inbred line Zheng 58 ( ⁇ 58(ms45ms45)) obtained in (1) was used as the male parent.
  • This) hybridization produced not only Zheng 58 (ms45ms45), but also Zheng 58 (Ms45-RNAi heterozygous ms45ms45), a maintainer line of male sterile Zheng 58.
  • the grains of Zheng 58 (ms45ms45) do not contain transgenic elements, while the grains of Zheng 58 (Ms45-RNAi heterozygous ms45ms45) contain transgenic elements.
  • the first plant male sterile line Zheng 58 (ms45ms45) obtained in (1) and the male sterile maintainer line Zheng 58 (Ms45-RNAi heterozygous ms45ms45) obtained in (3) were sown to In the field, the two materials are sown separately, and each row of the maintainer line is sown with 5 rows of sterile lines to ensure that no other corn is sown within 300 meters of the breeding, so that the sterile line and the maintainer line are naturally pollinated in the field.
  • the maintainer line can only accept its own pollen, and the offspring produced cannot distinguish between the homozygous genetically modified grains and heterozygous grains.
  • the sterile line material receives the pollen of the maintainer line, and the offspring that does not contain the transgenic element is the sterile line, and the transgenic element is the maintainer line. All the maintainer lines are used for the expansion of sterile lines and maintainers in the next year, most of the sterile lines are used for the production of commercial seeds, and the remaining small part is used for the expansion of sterile lines and maintainers in the next year.
  • maintainer lines account for about 20%, sterile lines account for about 80%), thereby achieving sterility
  • the expansion coefficient of the department is improved, and the cost is reduced.
  • Example 4 Using the male sterile line in Example 3 to produce hybrids on a large scale
  • the sterile line produced in Example 3 is a recessive homozygous sterile line controlled by the nucleus, and the sterile line can be restored to fertility by any wild-type plant (Ms45Ms45).
  • an inbred line with male sterile such as male sterile Zheng 58 (ms45ms45) with high combining ability, such as Chang 7-2
  • hybrids with excellent agronomic traits can be produced.
  • the male sterile inbred line and the wild-type inbred line are sown alternately in the field to ensure that no other corn is sown within 300 meters of the breeding.
  • the ears of the sterile line can only accept the pollen of the wild-type inbred line.
  • wild-type inbred lines can only be selfed. In this way, the seeds produced on the ears of the sterile line are hybrids.
  • the present invention Compared with the existing methods of expanding plant nuclear male sterile lines (for example, the patent "morphological marking method to expand plant nuclear male sterile lines" applied by the inventor of this patent), the present invention has an outstanding advantage, that is, the sterility obtained The ratio of lines is significantly increased (the ratio of sterility obtained by the present invention is increased to about 80%, and the ratio of sterility obtained by the existing method is only about 50%).
  • the present invention reduces the expression level of pollen fertilization competitiveness regulatory genes STK1 and STK2 (serine threoninekinase 1; 2) at the RNA level, reduces the competitiveness of corn pollen to participate in fertilization, and combines them with transgenic technology to expand the plant male sterile line technology system Combine to improve the production efficiency of sterile lines.
  • STK1 and STK2 serine threoninekinase 1; 2

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Botany (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'invention concerne la régulation de l'expression de gènes de compétitivité de pollen STK1 ; 2 et une application associée dans l'amélioration de l'efficacité de propagation d'une lignée mâle-stérile de noyau végétal. En réduisant le niveau d'expression de gènes régulateurs de compétitivité de fertilisation de pollen STK1 et STK2 (sérine thréonine kinase 1 ; 2) au niveau de l'ARN, la compétitivité du pollen de maïs à participer à la fécondation est réduite, cette dernière étant combinée avec le système technologique de propagation d'une lignée mâle-stérile végétale conjointement avec une technologie transgénique de façon à améliorer l'efficacité de production de lignées stériles.
PCT/CN2020/130966 2019-11-25 2020-11-24 Régulation de l'expression de gènes de compétitivité de pollen stk1 ; 2 et application associée dans l'amélioration de l'efficacité de la lignée mâle-stérile de noyau végétal de propagation WO2021104220A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911164326.7 2019-11-25
CN201911164326.7A CN110714022B (zh) 2019-11-25 2019-11-25 对花粉竞争力基因stk1；2的表达调控及在提高扩繁植物核雄性不育系效率中的应用

Publications (1)

Publication Number Publication Date
WO2021104220A1 true WO2021104220A1 (fr) 2021-06-03

Family

ID=69215548

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/130966 WO2021104220A1 (fr) 2019-11-25 2020-11-24 Régulation de l'expression de gènes de compétitivité de pollen stk1 ; 2 et application associée dans l'amélioration de l'efficacité de la lignée mâle-stérile de noyau végétal de propagation

Country Status (2)

Country Link
CN (1) CN110714022B (fr)
WO (1) WO2021104220A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114591967A (zh) * 2022-03-02 2022-06-07 华南农业大学 玉米tcp基因在杂交育种中的应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110714022B (zh) * 2019-11-25 2023-01-24 中国农业大学 对花粉竞争力基因stk1；2的表达调控及在提高扩繁植物核雄性不育系效率中的应用
CN112094332A (zh) * 2020-09-25 2020-12-18 信阳师范学院 一种糖转运蛋白及其在调控植物雄性不育中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060288440A1 (en) * 2000-09-26 2006-12-21 Pioneer Hi-Bred International, Inc. Nucleotide sequences mediating male fertility and method of using same
CN104837334A (zh) * 2013-05-23 2015-08-12 深圳市作物分子设计育种研究院 植物新型保持系及不育系建立及其用途
CN110178721A (zh) * 2019-06-10 2019-08-30 中国农业大学 形态标记法扩繁植物核雄性不育系
CN110714022A (zh) * 2019-11-25 2020-01-21 中国农业大学 对花粉竞争力基因stk1;2的表达调控及在提高扩繁植物核雄性不育系效率中的应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7517975B2 (en) * 2000-09-26 2009-04-14 Pioneer Hi-Bred International, Inc. Nucleotide sequences mediating male fertility and method of using same
US8492623B1 (en) * 2010-04-22 2013-07-23 Agrigenetics, Inc. Hybrid corn variety 570076
CN103237441B (zh) * 2011-11-02 2014-12-03 未名兴旺系统作物设计前沿实验室（北京）有限公司 一种小麦细胞核雄性不育系的保持方法
CN104611364A (zh) * 2015-02-15 2015-05-13 浙江新安化工集团股份有限公司 转基因元件及其应用、区分雄性不育系和可育保持系的方法、扩繁玉米雄性不育系的方法
CN107304428B (zh) * 2016-07-25 2021-04-02 未名兴旺系统作物设计前沿实验室（北京）有限公司 小麦育性恢复基因及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060288440A1 (en) * 2000-09-26 2006-12-21 Pioneer Hi-Bred International, Inc. Nucleotide sequences mediating male fertility and method of using same
CN104837334A (zh) * 2013-05-23 2015-08-12 深圳市作物分子设计育种研究院 植物新型保持系及不育系建立及其用途
CN110178721A (zh) * 2019-06-10 2019-08-30 中国农业大学 形态标记法扩繁植物核雄性不育系
CN110714022A (zh) * 2019-11-25 2020-01-21 中国农业大学 对花粉竞争力基因stk1;2的表达调控及在提高扩繁植物核雄性不育系效率中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JUN T HUANG, QINGHUA WANG, WONKEUN PARK, YAPING FENG, DIBYENDU KUMAR, ROBERT MEELEY, HUGO K DOONER: "Competitive Ability of Maize Pollen Grains Requires Paralogous Serine Threonine Protein Kinases STK1 and STK2", GENETICS, vol. 207, no. 4, 6 October 2017 (2017-10-06), pages 1361 - 1370, XP055815798, ISSN: 0016-6731, DOI: 10.1534/genetics.117.300358 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114591967A (zh) * 2022-03-02 2022-06-07 华南农业大学 玉米tcp基因在杂交育种中的应用
CN114591967B (zh) * 2022-03-02 2023-05-23 华南农业大学 玉米tcp基因在杂交育种中的应用

Also Published As

Publication number Publication date
CN110714022A (zh) 2020-01-21
CN110714022B (zh) 2023-01-24

Similar Documents

Publication Publication Date Title
US20230227836A1 (en) Simultaneous gene editing and haploid induction
WO2021104220A1 (fr) Régulation de l'expression de gènes de compétitivité de pollen stk1 ; 2 et application associée dans l'amélioration de l'efficacité de la lignée mâle-stérile de noyau végétal de propagation
CN110178721B (zh) 形态标记法扩繁植物核雄性不育系
WO2021223727A1 (fr) Système de production de semences et de sélection génétique intelligent pour sélection de croisement de cultures et production de semences hybrides et application associée
US20220106607A1 (en) Gene for parthenogenesis
WO2016054236A1 (fr) Sauvetage d'embryons in situ et récupération d'hybrides non génétiquement modifiés à partir de croisements intergénétiques
CN102960234B (zh) 扩繁植物雄性不育系的高效种子标记方法
CN113544290A (zh) 同时基因编辑和单倍体诱导
CN102965391B (zh) 扩繁植物雄性不育系的高效种子标记方法
JP2023547548A (ja) 単為結果性スイカ植物体
Zhou et al. Rapid generation of a tomato male sterility system and its feasible application in hybrid seed production
CN116724886B (zh) 颜色标记扩繁玉米细胞核雄性不育系的方法
US20050120416A1 (en) Novel method for the production of hybrid maize seeds
Zubko et al. Self-fertile cybrids Nicotiana tabacum (+ Hyoscyamus aureus) with a nucleo-plastome incompatibility
US11572573B2 (en) Method for propagating sterile male plant line
US20230183737A1 (en) Tomato plants having suppressed meiotic recombination
Kumar Male sterility in vegetables
US10724044B2 (en) Reversible genic male sterility in compositae
Vannozzi et al. technology based on genome editing applications
Tomlekova et al. hybrids Chapter 1–Trends and achievements in F
CN118652929A (zh) 种子形态标记扩繁玉米细胞核雄性不育系的方法
Van der Veken Tools for hybrid breeding in industrial chicory (Cichorium intybus L.)
WO2022038536A1 (fr) Procédés d'augmentation des taux de croisement de gramineae
CN118086366A (zh) 种子形态标记扩繁玉米细胞核雄性不育系的方法
JP2024501117A (ja) クロロシス抵抗性細胞質雄性不稔ブラシカ属植物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20892200

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20892200

Country of ref document: EP

Kind code of ref document: A1