WO2022205974A1 - Mutant de hir ayant une résistance aux herbicides à base de tricétone et son application dans la sélection des plantes - Google Patents

Mutant de hir ayant une résistance aux herbicides à base de tricétone et son application dans la sélection des plantes Download PDF

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WO2022205974A1
WO2022205974A1 PCT/CN2021/133986 CN2021133986W WO2022205974A1 WO 2022205974 A1 WO2022205974 A1 WO 2022205974A1 CN 2021133986 W CN2021133986 W CN 2021133986W WO 2022205974 A1 WO2022205974 A1 WO 2022205974A1
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hir
wild
type
reference sequence
plant
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Chinese (zh)
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韦叶娜
陈容
候青江
邓龙群
张震
胡江博
唐海峰
冯小容
胥南飞
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四川天豫兴禾生物科技有限公司
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    • G01N2333/90222—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3) in general
    • G01N2333/90225—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3) in general with a definite EC number (1.10.3.-)
    • G01N2333/90235—Ascorbate oxidase (1.10.3.3)

Definitions

  • the present disclosure relates to the field of plant proteins, in particular, to a HIR mutant with resistance to triketone herbicides and its application in plant breeding.
  • Triketones herbicides are a class of albino (belonging to cyclohexanone) herbicides newly developed by Syngenta, which are used in various (such as soybean, cotton, rapeseed, fruit and beet, etc.) broad-spectrum herbicides.
  • Leaf crop fields control monocotyledonous weeds, sensitive weeds absorb and conduct through young roots, competitively inhibit 4-Hydroxyphenylpyruvate dioxygenase (HPPD), inhibit the synthesis of homogentisic acid, make plastoquinone and The biosynthesis of tocopherols is blocked, causing the plant to develop symptoms such as bleaching, stunted growth, and ultimately killing the plant.
  • Triketone herbicides have a broad herbicidal spectrum and can control a variety of annual grass and broad-leaved weeds, with an efficacy of 3 to 5 days, and are environmentally friendly.
  • mesotrione and cyclosulfonone are the main herbicides in corn fields, which can effectively control 1-year-old broad-leaved weeds in corn fields, as well as some grass weeds in corn fields, such as barnyardgrass, foxtail, horsetail Tang et al.
  • Mesotrione has been well promoted in domestic corn fields.
  • triketone herbicides in other crops is very limited. Because it is harmful to weeds and crops. Even in maize, resistance to triketone herbicides varies among cultivars, and care must be taken when applying them.
  • the purpose of the present disclosure is to provide a HIR mutant with resistance to triketone herbicides and its application in plant breeding.
  • the HIR mutants provided by the present disclosure are capable of degrading triketone herbicides, thereby rendering plants resistant to triketone herbicides.
  • the present disclosure provides an HIR mutant with resistance to triketone herbicides, wherein the HIR mutant is obtained by mutating a plant-derived wild-type HIR, wherein the mutation is as follows (1)-( 9) any one or a combination of several:
  • amino acid sequence of the reference sequence is shown in SEQ ID NO.1.
  • HIR Iron/ascorbate-dependent oxidoreductases
  • Fe(II)/2-oxoglutarate(2OG)-dependent oxygenases HIR
  • the HIR mutant and its encoding nucleic acid can be used not only for the cultivation of transgenic crops, but also for the cultivation of non-transgenic plants with resistance to triketone herbicides, and has broad application prospects.
  • the reference sequence shown in SEQ ID NO.1 is the wild-type HIR of rice Nipponbare, and the HIR obtained by any plant-derived wild-type HIR through any one or combination of mutations in the above-mentioned mutation modes (1)-(9)
  • the mutants were all resistant to triketone herbicides.
  • the above-mentioned plants include but are not limited to rice, wheat, corn, barley, oats, sorghum, buckwheat, millet, mung bean, broad bean, pea, lentil, sweet potato, potato, cotton, soybean, rapeseed, sesame, peanut, sunflower, radish, Carrots, turnips, beets, cabbage, mustard greens, cabbage, cauliflower, kale, cucumber, zucchini, pumpkin, winter melon, bitter gourd, loofah, vegetable melon, watermelon, melon, tomato, eggplant, pepper, kidney bean, cowpea, edamame, leek, Green onions, onions, leeks, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grapes, strawberries, beets, sugar cane, tobacco, alfalfa, pasture, lawn grass, tea and cassava, etc.
  • the alignment method used in the protein sequence alignment involved in the present disclosure is Clustal online alignment, and its website address is: http://www.ebi.ac.uk/Tools/msa/clustalo/.
  • sequence alignment tools such as DNAMAN, the relevant parameter settings are set by default
  • an amino acid sequence refers to the type and arrangement of amino acid residues constituting a protein or polypeptide, usually the three-letter format commonly used in the art is used to denote amino acid residues, and the single-letter format commonly used in the art can also be used to denote amino acid residues Basically, those skilled in the art should be able to convert the three-letter amino acid sequence to the one-letter amino acid sequence. It should be clear that no matter which way is used in the present disclosure, those skilled in the art can understand and convert.
  • alanine a single letter is A, three letters are Ala; arginine, a single letter is R, three letters are Arg; aspartic acid, a single letter is D, three letters are Asp; cysteine, Single letter is C, three letters are Cys; glutamine, single letter is Q, three letters are Gln; glutamic acid, single letter is E, three letters are Glu; histidine, single letter is H, three letters are His; Isoleucine, one letter is I, three letters are Ile; Glycine, one letter is G, three letters are Gly; Asparagine, one letter is N, three letters are Asn; Leucine, one letter is L, three letters are Leu; lysine, one letter is K, three letters are Lys; methionine, one letter is M, three letters are Met; phenylalanine, one letter is F, three letters are Phe ; Proline, single letter is P, three letters are Pro; Serine, single letter is S, three letters are Ser;
  • the amino acid sequence of the wild-type HIR is selected from any one of SEQ ID NOs. 1-2.
  • SEQ ID NO.1 is the wild-type HIR of rice (Oryza sativa) Nipponbare, which is both a reference sequence and a wild-type HIR, and the mutant mutated in the above-mentioned mutation mode has resistance to triketone herbicides.
  • SEQ ID NO.2 is the wild-type HIR of maize, and the mutant after it is mutated according to the above-mentioned mutation mode has resistance to triketone herbicides.
  • positions 5, 75, 80, 89, 111, 145, 218, 283 and 322 on the reference sequence are referred to as reference sites
  • the sites on the wild-type HIR or HIR mutants corresponding to the above reference sites are referred to as mutation sites.
  • the positions of the wild-type HIRs of different plant sources (take rice and maize as examples) corresponding to the mutation sites of the above-mentioned reference sites on their wild-type HIR sequences are shown in the following table:
  • position 5 of the maize wild-type HIR corresponds to position 5 of the reference sequence
  • position 76 corresponds to position 75 of the reference sequence
  • position 81 corresponds to position 80 of the reference sequence.
  • bit 90 corresponds to bit 89 of the reference sequence
  • bit 112 corresponds to bit 111 of the reference sequence
  • bit 146 corresponds to bit 145 of the reference sequence
  • bit 220 corresponds to bit 218 of the reference sequence
  • bit 220 corresponds to bit 218 of the reference sequence.
  • Bit 285 corresponds to bit 283 of the reference sequence
  • bit 324 corresponds to bit 322 of the reference sequence.
  • the maize wild-type HIR is aligned with the reference sequence, and the amino acid of the maize wild-type HIR corresponding to the 5th position of the reference sequence (that is, the 5th position of the maize wild-type HIR) is mutated. is P;
  • the maize wild-type HIR is aligned with the reference sequence, and the maize wild-type HIR corresponds to the amino acid at position 75 of the reference sequence (ie, the 76th position of the maize wild-type HIR). mutated to L.
  • the triketone herbicide is selected from the group consisting of mesotrione, sulfenolone, bicyclosulfuron, sulcotrione, sulcotrione, diflufenazone, and Any one of bispyrazone.
  • the resistance to triketone herbicides refers to the resistance to herbicides including mesotrione, cyclosulfazone, bicyclosulfuron, furoscotrione, sulcotrione, fenoxadiazone, and diazotrione, etc.
  • the triketone herbicides within are tolerant.
  • the present disclosure provides an isolated nucleic acid molecule encoding a HIR mutant having triketone herbicide resistance as described in any of the above.
  • nucleic acid may be DNA or RNA, preferably DNA.
  • Nucleic acids of HIR mutant proteins Once the nucleic acid is obtained, it can be cloned into a vector, transformed or transfected into corresponding cells, and then propagated by conventional host cells, from which the nucleic acid can be isolated in large quantities.
  • the present disclosure provides a recombinant vector containing the nucleic acid molecule as described above.
  • vectors refer to bacterial plasmids, cosmids, phagemids, yeast plasmids, plant cell viruses, animal cell viruses and various other viral vectors commonly used in the art.
  • Vectors can be divided into cloning vectors, expression vectors and transformation vectors according to the purpose of application, which means that the purpose of use is to clone and verify genes, express corresponding genes and transform corresponding genes respectively.
  • Vectors useful in the present disclosure include, but are not limited to, vectors for expression in bacteria (prokaryotic expression vectors), vectors for expression in yeast (eg, Pichia vectors), baculovirus vectors for expression in insect cells, Vectors for expression in mammalian cells (retroviral vectors, adenovirus vectors, etc.), plant viral vectors for expression in plants, and various vectors for expression in mammalian mammary glands.
  • the present disclosure provides a recombinant bacteria or recombinant cell containing the nucleic acid molecule as described above or the recombinant vector as described above.
  • Recombinant bacteria can be cocci, bacilli such as Escherichia coli or spirochete; it can also be autoxic bacteria or heterooxygen bacteria and the like.
  • Cells can be prokaryotic or eukaryotic.
  • Eukaryotic cells can be animal cells or plant cells.
  • Another preferred cell is a plant cell, preferably a rice cell, more preferably a Nipponbare cell.
  • the above-mentioned nucleic acid molecules contained in the plant cells can be introduced into plant cells by transgenic technology, including introduction into the nucleus, chloroplast and/or plastid of plant cells, or by mutation techniques (such as chemical mutagenesis) conventional in the art. Such as ethyl methanesulfonate (EMS) mutagenesis or radiation mutagenesis, etc.) so that the above-mentioned nucleic acid molecules exist in plant cells.
  • EMS ethyl methanesulfonate
  • the present disclosure provides the HIR mutant as described in any one of the above, the nucleic acid molecule as described above, the recombinant vector as described above, or the recombinant bacteria or recombinant cell as described above, after obtaining a herbicide having triketones Applications in resistant plant varieties.
  • the means of obtaining plant varieties with resistance to triketone herbicides in the present disclosure include preparing, breeding, producing or otherwise producing, for example, including obtaining by transgenic breeding, such as transforming the above-mentioned nucleic acid molecules into plants and making them Expression of the above-mentioned HIR mutants; also including those obtained by non-transgenic breeding, such as by crossing, backcrossing, selfing or asexual propagation and sorting plant varieties comprising the above-mentioned nucleic acid molecules and expressing the above-mentioned HIR mutants.
  • transgenic breeding such as transforming the above-mentioned nucleic acid molecules into plants and making them Expression of the above-mentioned HIR mutants
  • non-transgenic breeding such as by crossing, backcrossing, selfing or asexual propagation and sorting plant varieties comprising the above-mentioned nucleic acid molecules and expressing the above-mentioned HIR mutants.
  • the application comprises: modifying the endogenous HIR gene of the plant of interest to encode the HIR mutant.
  • the application comprises: mutagenizing, screening plant cells, tissues, individuals or populations to encode the HIR mutant.
  • Plant varieties described in the present disclosure include, but are not limited to, individual plants, groups of plants or their propagation material, plant events, plant progeny, plant seeds, or other reproducible parts of plants.
  • the plant progeny itself is the plant, including the plant progeny produced by transgenic technology, the plant progeny produced by crossing with other plant varieties, and the plant progeny produced by backcrossing or self-crossing.
  • the plant species described in the present disclosure can be dicotyledonous plants or monocotyledonous plants, including but not limited to rice, wheat, corn, barley, oats, sorghum, buckwheat, millet, mung beans, broad beans, peas, lentils, sweet potatoes , potato, cotton, soybean, rapeseed, sesame, peanut, sunflower, radish, carrot, turnip, beet, cabbage, mustard greens, cabbage, cauliflower, kale, cucumber, zucchini, pumpkin, winter squash, bitter gourd, loofah, vegetable melon, watermelon , melon, tomato, eggplant, pepper, kidney bean, cowpea, edamame, leek, green onion, onion, leek, spinach, celery, amaranth, lettuce, chrysanthemum, daylily, grape, strawberry, beet, sugar cane, tobacco, alfalfa, pasture , lawn grass, tea and cassava, etc.
  • the triketone herbicide is selected from the group consisting of mesotrione, sulfenolone, bicyclosulfuron, sulcotrione, sulcotrione, diflufenazone, and Any one of bispyrazone.
  • a plant refers to a single plant, group of plants, or reproductive material thereof, including plants, plant varieties, plants, plant events, plant progeny, plant seeds, or other reproductive parts of plants.
  • the plant progeny itself is the plant, including the plant progeny produced by transgenic technology, the plant progeny produced by crossing with other plant varieties, and the plant progeny produced by backcrossing or self-crossing.
  • the applying comprises the steps of:
  • the target plant is made to contain the above-mentioned nucleic acid molecule, or the target plant is made to express the above-mentioned HIR mutant, thereby obtaining a plant variety having resistance to triketone herbicides.
  • Those skilled in the art can use gene editing technology, breeding technology, transgenic technology, etc. well-known in the art to make the target plant contain the above-mentioned nucleic acid molecule and make the target plant express the above-mentioned HIR mutant, and these methods include but are not limited to the following methods:
  • the present disclosure provides a method for breeding a plant having resistance to a triketone herbicide, comprising: sexually or vegetatively a plant variety obtained by the application as described above.
  • the methods of sexual reproduction include crossbreeding, backcrossing and selfing.
  • the present disclosure provides a method for identifying a plant with resistance to triketone herbicides, comprising: determining whether the plant to be tested expresses the HIR mutant described in any one of the above; and/or determining the plant to be tested Whether it contains nucleic acid molecules as described above.
  • Figure 1 shows the growth of rice seedlings on different days after spraying mesotrione.
  • Fig. 2 shows the growth of rice seedlings after spraying of bisoxazone.
  • Figure 3 shows the growth of rice seedlings after spraying with cyclopentanone.
  • Figure 4 shows the amino acid sequence alignment results of wild-type rice OsHIR (OsWT) and OsB, OsC, OsD, OsE, OsI and OsJ.
  • Figure 5 shows the growth of rice mutant seeds at different seeding densities.
  • Figure 6 shows the growth of Kasalath seeds 10 days after sowing.
  • Figure 7 shows the growth of Kasalath seeds 20 days after sowing.
  • Figure 8 shows the alignment results of the amino acid sequences of wild-type rice OsHIR (OsWT) and wild-type maize ZmHIR (ZmWT).
  • Figure 9 is a map of the pPAH vector.
  • Figure 10 shows the color reaction results of rice HIR mutants.
  • Figure 11 shows the color response results of the maize ZmHIR mutant.
  • Sterilize the seeds of the rice (Oryza sativa) variety F1 of the seed industry company take the mature seeds, manually dehull them, select the seeds with full sterile spots, put them into a 50ml sterile centrifuge tube, and add 70% alcohol for sterilization for 30 seconds. Pour off the alcohol and wash it once with sterile water; add an appropriate amount of 2.6% sodium hypochlorite solution, soak and disinfect for 15 minutes. Pour off the sodium hypochlorite solution, soak and wash with sterile water 6-7 times, 3 minutes each time.
  • Induction and subculture The seeds were blotted dry on sterile filter paper, placed in induction medium, 12 per dish; after the operation, the petri dish was sealed with parafilm, and cultured at 30°C in the dark for 21-28 days, the more The wounded tissue was transferred to fresh medium and cultured for about 7 days, and the spherical callus of 1-2 mm in size was taken as the culture material.
  • Induction medium NB+hydrolyzed casein 0.3g/L+L-proline 2.8g/L+sucrose 30g/L+2,4-D 4mg/L+agar 8g/L, pH5.8. Autoclave.
  • S0 medium NB+L-proline 2.8g/L+sucrose 30g/L+2,4-D 2mg/L, pH5.8, autoclaved.
  • the callus of suspension culture was blotted dry on sterile filter paper, irradiated with UV light for 5 minutes at the same time, transferred to differentiation medium (containing 0.1-5.0 mg/L mesotrione), 30°C, light, and cultured 21 days or so. Subculture 1 time. Select the new young green shoots and move them to a new differentiation medium at 30°C, light, and continue to culture for about 21 days.
  • differentiation medium containing 0.1-5.0 mg/L mesotrione
  • the new seedlings grow to about 2cm, they are transferred to the rooting medium, illuminated at 30°C, and cultivated for 3 to 4 weeks. medium, and moved to soil.
  • Rooting medium 1/2MS+inositol 0.1g/L+sucrose 20g/L, pH 5.8, autoclaved, plus NAA 0.2mg/L+ mesotrione 0.1-5mg/L.
  • the transplanted rice seedlings were evenly arranged in the same experimental area (to avoid overlapping leaves). Calculate the area occupied by the experimental group and the control group, and according to the area, 105 grams per hectare (10.5 mg per square meter) of the active ingredient measured by 1 time of mesotrione was sprayed with mesotrione. Mesotrione 6 times the dose is 630 g/ha. The actual spraying is according to 40ml per square meter, and the drug content is 6 times of mesotrione. Photographs were taken 4 days, 10 days and 21 days after spraying. Some representative plants were photographed, and the results are shown in Figure 1.
  • the OsHIR gene and OsHIR protein of F1 mutant plant 1 to F1 mutant plant 6 each have The mutations are shown in Table 1 below:
  • This example provides HIR mutants derived from maize, and the HIR mutants are obtained by the following mutation methods:
  • the wild-type ZmHIR protein (SEQ ID NO.2) encoded by the ZmHIR gene shown in SEQ ID NO.4 of wild-type maize (Zea mays) Zheng 58 is compared with the reference sequence SEQ ID NO.1, the result is shown in the figure 8, the wild-type ZmHIR protein corresponds to one or more of the reference sites (positions 111, 75, 218, 322, 5, 80, and 89) of the reference sequence. 1 site (as indicated by the arrow in Figure 8), the mutation site is mutated, and the mutant obtained after the mutation and the mutation it has are shown in Table 4 below:
  • genes encoding the maize ZmHIR mutants and the maize ZmHIR mutants provided in the embodiments of the present disclosure can be obtained by chemical synthesis.
  • PCR primers were designed, and enzyme cleavage sites (Pac1 and Sbf1) were introduced at both ends of the gene.
  • DH5 ⁇ Escherichia coli is transformed into DH5 ⁇ E. coli.
  • Tryptone 10g NaCl 10g Yeast Extract 5g ddH 2 O Dilute to 250ml and sterilize for later use.
  • LTM25 LTM0 + 25 ⁇ M mesotrione
  • LTM50 LTM0 + 50 ⁇ M mesotrione
  • LTM100 LTM0 + 100 ⁇ M mesotrione.
  • the mutations S5P, F75L, H80R, S89G, L111S, E145G, D218N, H283R and/or R322G mutations can confer or enhance mesotrione resistance in rice OsHIR mutants.
  • Example 4 With reference to the detection method of Example 4, the mesotrione resistance of the maize ZmHIR mutants ZmC, ZmE, ZmI and ZmJ provided in Example 3 was verified. The results are shown in Figure 11. Among them, wild-type maize ZmHIR was used as control CK3.
  • the mutations S5P, F75L, H80R, D89G, L111S, D218N and/or R322G mutations can confer or enhance the mesotrione resistance of maize ZmHIR mutants.
  • the mutants obtained by mutating wild-type HIR derived from plants can degrade triketone herbicides and have resistance to triketone herbicides. Plants expressing such mutants also have For resistance to triketone herbicides, the aforementioned mutation mode can be any one or a combination of the following (1)-(9):

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Abstract

L'invention concerne un mutant de l'oxydoréductase dépendante du fer/ascorbate (HIR) ayant une résistance aux herbicides à base de tricétone et son application dans la sélection des plantes. Le mutant de HIR est obtenu par mutation du HIR de type sauvage dérivé d'une plante, et peut dégrader les herbicides à base de tricétone, afin que les plantes exprimant le mutant soient résistantes aux herbicides à base de tricétone.
PCT/CN2021/133986 2021-03-31 2021-11-29 Mutant de hir ayant une résistance aux herbicides à base de tricétone et son application dans la sélection des plantes WO2022205974A1 (fr)

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