WO2019233349A1 - 突变型对羟苯基丙酮酸双氧化酶、其编码核酸以及应用 - Google Patents

突变型对羟苯基丙酮酸双氧化酶、其编码核酸以及应用 Download PDF

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WO2019233349A1
WO2019233349A1 PCT/CN2019/089512 CN2019089512W WO2019233349A1 WO 2019233349 A1 WO2019233349 A1 WO 2019233349A1 CN 2019089512 W CN2019089512 W CN 2019089512W WO 2019233349 A1 WO2019233349 A1 WO 2019233349A1
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seq
idno
hppd
protein
plant
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PCT/CN2019/089512
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French (fr)
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连磊
莫苏东
李华荣
苑广迪
李振国
张俊杰
丁德辉
陈波
刘桂智
宋超
王蕾
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青岛清原化合物有限公司
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Priority claimed from CN201910077823.7A external-priority patent/CN110616203B/zh
Application filed by 青岛清原化合物有限公司 filed Critical 青岛清原化合物有限公司
Priority to MX2020013127A priority Critical patent/MX2020013127A/es
Priority to BR112020024811-8A priority patent/BR112020024811A2/pt
Priority to JP2021517887A priority patent/JP2021526849A/ja
Priority to CA3103851A priority patent/CA3103851A1/en
Priority to US15/734,623 priority patent/US20210230563A1/en
Priority to EP19814351.3A priority patent/EP3805377A4/en
Priority to AU2019280480A priority patent/AU2019280480A1/en
Publication of WO2019233349A1 publication Critical patent/WO2019233349A1/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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of agricultural genetic engineering, and particularly relates to a novel mutant p-hydroxyphenylpyruvate dioxidase (HPPD), which imparts resistance or tolerance to an HPPD inhibitory herbicide to a plant, a nucleic acid encoded by the same and application thereof.
  • HPPD p-hydroxyphenylpyruvate dioxidase
  • HPPD P-Hydroxyphenylpyruvate dioxidase
  • HPP hydroxyphenylpyruvate
  • Herbicides that work by inhibiting HPPD are well known and include many types, such as isoxazoles, diketononitriles, triketones, and pyrazoline salts.
  • Inhibition of HPPD blocked biosynthesis of plastid quinone (PQ) from tyrosine.
  • PQ is an essential cofactor in the biosynthesis of carotenoid pigments, and these carotenoid pigments are necessary for photoprotection of photosynthetic centers.
  • Herbicides that inhibit HPPD are bleaches that move through the bast, and they cause new meristems and leaves exposed to light to appear white. In the absence of carotenoids, chlorophyll is photodestructive and becomes a photolytic lysate by itself through the photosensitivity of singlet oxygen.
  • HPPD-inhibiting herbicides are also known, and include over-expressing the HPPD enzyme to produce large amounts of HPPD enzymes in plants. These HPPD enzymes are associated with a given herbicide. It is sufficiently related to have enough functional enzymes available (despite its inhibitors), or to mutate the target HPPD enzyme into a functional HPPD that is more insensitive to herbicides.
  • HPPD inhibitory herbicides are a large class that cover many different types. Although a given mutant HPPD enzyme can provide a useful level of tolerance to one or some HPPD inhibitory herbicides, the same or single mutant HPPD may not be sufficient to provide resistance to another or another Commercial levels of tolerance for different, more desirable HPPD inhibitory herbicides (see, for example, U.S.
  • CRISPR / Cas9 is a new gene-targeted editing technology that has appeared since 2012 (Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, JA, and Charpentier, E. 2012.
  • the CRISPR / Cas 9 system recognizes edited targets based on complementary base pairing between nucleic acid molecules, and can edit any 20bp target sequence immediately following PAM (NGG).
  • NGS PAM
  • the CRISPR / Cas 9 system is simple to operate, and only needs to replace the 20-30bp target nucleotide sequence on the original vector for each target, which is suitable for high-throughput operations. Multiple sites of the same gene and multiple different genes can be edited simultaneously.
  • this technology has shown great application prospects in biomedicine, functional genomics, improvement of animal and plant traits, and creation of new traits. It is revolutionizing the promotion of animal and plant breeding (Hui Zhang, Jinshan Zhang, Zhaobo Lang, Jose Ramon Botellad, and Jian-Kang ZhuZhu. 2017. Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement, Critical Reviews, Science, Reviews, 36: 4, 291-309, 072.1080.
  • CRISPR / Cas 9 mainly implements site-specific editing in three ways.
  • the first is a site-directed knockout of the gene to obtain a mutant.
  • Cas 9 recognizes and cleaves targets under the guidance of targeted RNA (gRNA) to generate double-stranded DNA breaks; broken DNA is usually repaired with non-homologous end junctions (NHEJ); it is easy to generate frameshifts during repair Mutation to destroy this gene. Targeted knockouts are more efficient.
  • the second is to replace the target sequence with a homologous substitution of the target or to insert the site.
  • Single base editing is a gene editing method that uses the CRISPR / Cas9 system to target deaminase to specific sites in the genome to modify specific bases. This method has been successfully applied in rice.
  • CRISPR / Cpf1 can also be used for gene editing (Zetsche, B., Gootenberg, JS, Abudayyeh, OO, Slaymaker, IM, Makarova, KS, Essletzbichler, P., Volz, SE, Joung, J., Oost , J., Regev, A., Koonin, EV, and Zhang, F. 2015.
  • Cpf1 is a single RNA-guided class of Endonuclease of Class 2 CRISPR Cas System.Cell.163: 759–771; Endo, A., Masafumi, M., Kaya, H., and Toki, S. 2016a.
  • CRISPR / Cpf1 has two main components: the Cpf1 enzyme and crRNA, which determines system specificity. Although the CRISPR / Cpf1 and CRISPR / Cas9 systems are similar, there are some important differences (Hui Zhang, Jinshan Zhang, Zhaobo Lang, Jose Ramón Botella & Jian-Kang Zhu (2017) Genome Editing—Principles, and Applications for functional Functional GenomicsResearch , Critical Reviews in Science, 36: 4,291-309, DOI: 10.1080 / 07352689.2017.1402989).
  • the CRISPR / Cpf1 system does not require trans-activated crRNA (tracrRNA), but CRISP / Cas9 does. Therefore, it is relatively short, only 42-44 nucleotides, including a 19-nucleotide repeat and a 23-25 nucleotide long spacer.
  • the target sequence of Cpf1 is cut 23bp downstream of the PAM sequence, and the non-target single strand is 18bp downstream of the PAM sequence. A 5bp overhanging sticky end was generated.
  • the resulting sticky ends can increase the efficiency of HDR-mediated insertion of donor DNA into the Cpf1 splice site.
  • the CRISPR / Cpf1 system only needs one promoter to drive multiple small crRNAs arrays when editing multiple targets or genes, which is very suitable for multi-target editing.
  • the CRISPR / Cas9 system requires a G (5'-NGG-3 ')-rich PAM sequence at the 3' end of the target sequence, and the CRISPR / Cpf1 system requires a T (5'-rich) T (5 ') at the 5' end of the target sequence.
  • -TTTN-3'or 5'-TTN-3 ') PAM sequence suitable for editing multi-A / T DNA or gene.
  • Cpf1 Three modified CRISPR / Cpf1 systems have been developed, including FnCpf1 from Francisella novicida, AsCpf1 from Acidaminococcus sp. And LbCpf1 from Lachnospiraceae bacterium.
  • Three Cpf1 systems have been used for plant genome editing on the following species: rice, Arabidopsis, tobacco, and soybean (Endo, A., Masafumi, M., Kaya, H., and Toki, S. 2016a. Efficient target targeted mutagenesis ofrice and tobaccogenomes using Cpf1 from Francisella novelicida.Sci.Rep.
  • the present application provides a mutant p-hydroxyphenylpyruvate dioxidase (HPPD) that imparts resistance or tolerance to HPPD inhibitory herbicide to plants, said mutant HPPD retains or enhances its catalysis
  • HPPD p-hydroxyphenylpyruvate dioxidase
  • HPP p-hydroxyphenylpyruvate
  • the present invention also relates to a biologically active fragment of mutant p-hydroxyphenylpyruvate dioxidase, a polynucleotide encoding the protein or fragment, and an application thereof.
  • an aspect of the present invention provides a mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein, which corresponds to wild-type rice p-hydroxyphenylpyruvate dioxidase corresponding to SEQ ID ID NO: 2.
  • One or more positions at positions 419, 420, 430, and 431 have one or more mutations selected from the group consisting of: 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226H, 276W, 277N , 336D, 337A, 338D, 338S, 338Y, 342D, 346C, 346D, 346H, 346S, 346Y, 370N, 377C, 386T, 390I, 392L, 403G, 410I, 418P, 419F, 419L, 419V, 420S, 420T, 430G And 431L.
  • the amino acid sequence of the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein further has at least 80%, at least 85%, at least 90%, at least 95% of the amino acid sequence shown in SEQ ID NO: 2. %, At least 96%, at least 97%, at least 98%, at least 99% sequence identity. More preferably, the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein has the amino acid sequence shown in SEQ ID NO: 2, and the difference is only in the wild-type rice corresponding to SEQ ID ID NO: 2.
  • Another aspect of the present invention provides a biologically active fragment of a mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein, which has one or more deletions at the N and / or C terminus of the protein (for example, 1- 50, 1-25, 1-10, or 1-5, such as 1, 2, 3, 4 or 5) amino acid residues, but still retain the required biological activity of the full-length protein That is, its property of catalyzing the conversion of p-hydroxyphenylpyruvate (HPP) to urinic acid is retained or enhanced, and its sensitivity to HPPD inhibitory herbicides is significantly lower than that of wild-type HPPD or its corresponding biologically active fragment.
  • HPPD p-hydroxyphenylpyruvate dioxidase
  • the present invention further relates to a fusion protein comprising the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein of the present invention or a biologically active fragment thereof and other components fused thereto, such as a peptide or a polypeptide Components.
  • the component imparts the desired properties to the fusion protein, such as facilitating its isolation and purification, improving its stability, extending its half-life, providing additional biological activity, and guiding the fused HPPD protein into the target region.
  • plastids such as chloroplasts. The selection of the respective components is well known to those skilled in the art.
  • Another aspect of the present invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein or a biologically active fragment or fusion protein thereof.
  • HPPD p-hydroxyphenylpyruvate dioxidase
  • the invention also provides a nucleic acid construct comprising the polynucleotide and a regulatory element operably linked thereto.
  • the present invention provides an expression vector comprising the polynucleotide and an expression control element operably linked thereto.
  • the invention provides a host cell comprising the polynucleotide, a nucleic acid construct, or an expression vector.
  • the present invention also provides a method for producing a plant having increased resistance or tolerance to an HPPD inhibitory herbicide.
  • the invention further relates to plants produced by the method described above.
  • the present invention also provides a method for increasing the resistance or tolerance of a plant to an HPPD-inhibiting herbicide, which comprises expressing in the plant the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) Protein or biologically active fragment or fusion protein thereof.
  • HPPD p-hydroxyphenylpyruvate dioxidase
  • the present invention further provides a method for increasing plant resistance or tolerance to an HPPD-inhibiting herbicide, which comprises expressing the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein of the present invention or an organism thereof The plant of the active fragment or fusion protein is crossed with another plant.
  • HPPD p-hydroxyphenylpyruvate dioxidase
  • the present invention further provides a method for increasing plant resistance or tolerance to an HPPD-inhibiting herbicide, which includes gene editing of the endogenous HPPD protein of the plant cell, plant tissue, plant part, or plant.
  • the invention further relates to the use of the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein or a biologically active fragment or fusion protein thereof according to the invention for improving the resistance or tolerance of a plant's HPPD-inhibiting herbicide.
  • HPPD p-hydroxyphenylpyruvate dioxidase
  • the present invention further relates to a method for controlling weeds in a plant site, which comprises applying to the site containing a plant or seed of the present invention an effective amount of one or more HPPD-inhibiting herbicides for controlling weeds without significantly affecting the plant site. Mentioned plant.
  • FIG. 1 shows the color response of a recombinant E. coli culture medium transformed with wild-type or mutant rice HPPD genes cultured in a 96-well plate.
  • Recombinant E. coli expresses one of wild-type rice HPPD (WT) or single-point mutant rice HPPD, and they contain different concentrations of the herbicide cyclosulfonone (left) or the metabolite of flufenazone ( Right, its structural formula is: ) Under the conditions of culture, showing different degrees of color change. The darker the color of the herbicide wells with the same concentration, the higher the resistance / tolerance to this herbicide.
  • FIG. 2 shows the color response of a recombinant E. coli culture medium transformed with wild-type or mutant rice HPPD genes cultured in a 96-well plate.
  • Recombinant E. coli expresses wild-type rice HPPD (WT) and each unit of point-mutant rice HPPD, and they contain different concentrations of the herbicides cicofazone (left) or oxaflutole (right) in the culture medium. Incubation under conditions showed varying degrees of color change. The darker the color of the herbicide wells with the same concentration, the higher the resistance / tolerance to this herbicide.
  • Figure 3 shows the color response of a recombinant E. coli culture medium transformed with wild-type or mutant rice HPPD genes cultured in a 96-well plate.
  • recombinant E. coli expressed wild-type rice HPPD (WT) and various single-point mutant rice HPPD, and they were cultured in the medium containing different concentrations of the herbicide mesotrione, showing different degrees of color response. The darker the color of the herbicide wells with the same concentration, the higher the resistance / tolerance to this herbicide.
  • FIG 4 shows the color response of a recombinant E. coli culture medium transformed with wild-type or mutant rice HPPD genes cultured in a 96-well plate.
  • Recombinant E. coli expresses wild-type rice HPPD (WT) or one of the point mutations H141R, G342D, D370N or a combination thereof, and metabolizes them in different concentrations of the herbicide sulfazone (top) or flubendicarb
  • the after-products (bottom) were cultured under different conditions and showed varying degrees of color change. The darker the color of the herbicide wells with the same concentration, the higher the resistance / tolerance to this herbicide.
  • Figure 5 shows the color response of recombinant coliforms cultured in wild-type or mutant rice HPPD genes cultured in 96-well plates.
  • recombinant E. coli expresses wild-type rice HPPD (WT) or single point mutant H141R, G342D, D370N or a combination thereof (141 + 342 means H141R / G342D; 141 + 370 means H141R / D370N; 342 + 370 means G342D / D370N; 141 + 342 + 370 means H141R / G342D / D370N), and they were cultured under the conditions containing different concentrations of the herbicides cicoflavone (top) or oxaflutole (bottom), showing different degrees of color change. The darker the color of the herbicide wells with the same concentration, the higher the resistance / tolerance to this herbicide.
  • FIG. 6 shows the color response of a recombinant E. coli culture medium transformed with wild-type or mutant rice HPPD genes cultured in a 96-well plate.
  • the recombinant E. coli culture solution expresses wild-type rice HPPD (WT) or single point mutant H141R, G342D, D370N or a combination thereof, and they are cultured under conditions containing different concentrations of the herbicide mesotrione, showing different degrees of color Variety. The darker the color of the herbicide wells with the same concentration, the higher the resistance / tolerance to this herbicide.
  • Figure 7 shows all amino acid mutations marked on the rice HPPD wild-type enzyme protein.
  • Figure 8 shows the color response of recombinant E. coli culture medium transformed with mutant rice HPPD gene cultured in a 96-well plate.
  • the recombinant E. coli culture medium expressed similar mutation points in various combinations of 336-338-342-346 and 141R + 342D + 370N (336D, 338D, 338S, 338Y, 342D, 346C, 346H, 346S respectively represent P336D, N338D, N338S , N338Y, G342D, R346C, R346H, R346S; 141R + 342D + 370N means H141R / G342D / D370N), they are contained in different concentrations of the herbicide diazolozone metabolite (code 101, its structural formula is: ) Under different conditions, showing different degrees of color change. The darker the color of the herbicide wells with the same concentration, the higher the resistance / tolerance to this herbicide.
  • Figure 9 shows the color response of recombinant E. coli culture medium transformed with mutant rice HPPD gene cultured in a 96-well plate.
  • the recombinant E. coli culture medium expressed three or four mutation points in each combination (141R, 336D, 338D, 338S, 338Y, 342D, 346C, 346S, 346H, 370N, 418P, and 419F respectively representing H141R, P336D, N338D, N338S, N338Y , G342D, R346C, R346S, R346H, D370N, K418P, G419F), and they were cultured under the condition of containing metabolites of the herbicide diazolozone, showing different degrees of color change. The darker the color of the herbicide wells with the same concentration, the higher the resistance / tolerance to this herbicide.
  • Figure 10 shows the inhibition curves of the metabolites of oxadiazon for OsHPPD, WT, and each mutant.
  • the abscissa represents the concentration of compound 101, and the ordinate represents the reaction rate at 100% when the inhibitor concentration is 0, and the enzymes at different concentrations of 101. Residual activity, the numbers in the figure represent each mutation site. It can be intuitively seen from the figure that the wild-type WT is extremely sensitive to 101, and the activity is completely inhibited at a concentration of about 60 uM of 101, while each mutant shows a strong resistance increase. Based on this result, 101 pairs of mutants could be calculated to inhibit the IC50 value of the mutants.
  • each mutant showed significantly improved resistance to wild-type OsHPPD (of which 141R, 338D, 342D, 346C, 346H, 370N, 386T , 418P, 419F, and 420S represent H141R, N338D, G342D, R346C, R346H, D370N, P386T, K418P, G419F, N420S).
  • Figure 11 shows the sensitivity of transgenic rice (Zhonghua 11) to the HPPD inhibitor herbicide sulfazone.
  • the mutant rice OsHPPD3M H141R / G342D / D370N
  • CK negative control
  • Figure 12 shows the tolerance of the transgenic rice (Zhonghua 11) to the HPPD inhibitor herbicide diazolozone.
  • Plants expressing rice OsHPPD3M in the T0 generation plants can tolerate 8-16 g of the active ingredient diazoxynone / mu, but the non-transgenic control (CK) died soon after severe albinism (A, B); plants in the T1 generation expressing rice OsHPPD3M The plant can tolerate 32-64 g of the active ingredient, azoxystrozone / mu, but the non-transgenic control died quickly after severe albinism (C, D).
  • Figure 13 shows a rice HPPD single base editing vector.
  • Figure 14 shows the sequence analysis of a single base-edited rice seedling and its target H141R (CAC> CGC).
  • Figure 15 shows the structure of the rice hppd gene (Oshppd> NC029257.1), showing two exons, one intron, three mutation sites (141, 342, 370) and the designed Target cleavage sites (gRNA1-2, gRNA2-1).
  • Figure 16 shows the structure of the template DNA.
  • the length of the core substitution region of the three mutant amino acids 141-324-370 is 1056bp, and the left and right homology arms are 350bp each.
  • the left and right ends are left 6bp, and the total length of the template is 1768bp.
  • the PCR product was quickly genotyped, and the NcoI digestion site was removed.
  • PAM NGG at the original cutting site on the template was also removed.
  • Figure 17 shows three mutation sites (H141R-G342D-D370N) of homologous substitutions of rice HPPD gene.
  • an "HPPD-inhibiting herbicide” is a substance that has herbicidal activity per se or a substance that is used in combination with other herbicides and / or additives that can change its effect, and can work by inhibiting HPPD.
  • Substances capable of acting as herbicides by inhibiting HPPD are well known in the art, including many types, 1) triketones, for example, sulcotrione (CAS number: 99105-77-8); mesotrione Mesotrione (CAS number: 104206-82-8); Bicyclopyrone (CAS number: 352010-68-5); Tetramone (CAS number: 335104-84-2); Sulfuran Ketone (tefuryltrione, CAS number: 473278-76-1); biscyclotrione (CAS number: 156963-66-5); 2) diketonitriles, for example, 2-cyano-3-cyclopropyl -1- (2-methylsulfonyl-4-trifluoromethylphenyl) propan-1,3-dione (CAS number: 143701-75-1); 2-cyano-3-cyclopropyl- 1- (2-methylsulfonyl-3,4-dichlorophenyl) propan-1,3-dione (CAS number: 2128
  • a plant "increased tolerance to an HPPD-inhibiting herbicide” or “increased resistance to an HPPD-inhibiting herbicide” refers to a plant that has tolerance or resistance to the HPPD-inhibiting herbicide. Compared to plants containing the wild-type HPPD gene.
  • HPPD enzymes with "increased tolerance to HPPD-inhibiting herbicides” or “increased resistance to HPPD-inhibiting herbicides” refer to HPPD enzymes that are known to inhibit the activity of the corresponding wild-type HPPD enzyme protein At herbicide concentrations, it exhibits an enzyme activity that is at least 10% higher, preferably at least 15% higher, and more preferably at least 20% higher than the wild-type HPPD enzyme.
  • the terms "HPPD inhibitory herbicide tolerance” and "HPPD inhibitory herbicide resistance” are used interchangeably, and both refer to the tolerance to HPPD inhibitory herbicides and to HPPD Resistance to inhibitory herbicides.
  • wild type refers to a nucleic acid molecule or protein that can be found in nature.
  • protein protein
  • polypeptide and “peptide” are used interchangeably in the present invention and refer to polymers of amino acid residues, including the polymerization of chemical analogues in which one or more amino acid residues are natural amino acid residues Thing.
  • the proteins and polypeptides of the present invention can be produced recombinantly or chemically.
  • mutant protein or “mutant protein” refers to a protein that has one or more amino acid residue substitutions, insertions, deletions, and / or additions compared to the amino acid sequence of a wild-type protein.
  • polynucleotide and “nucleic acid” are used interchangeably and include DNA, RNA, or hybrids thereof, which may be double-stranded or single-stranded.
  • a "host organism” is to be understood as any unicellular or multicellular organism that can introduce mutant HPPD protein-encoding nucleic acids, including, for example, bacteria such as E. coli, fungi such as yeast (such as Saccharomyces cerevisiae), molds (such as Aspergillus ), Plant cells and plants.
  • plant should be understood as any differentiated multicellular organism capable of photosynthesis, especially monocotyledonous or dicotyledonous plants, for example: (1) food crops: Oryza spp., Such as rice Oryzasativa, Oryza latifolia, Oryzasativa, Oryza glaberrima; Triticum spp., Such as Triticum triestumum, T. Turgidumssp .durum); Hordeum (e.g.
  • avena such as oat (Avena sativa), wild oat (Avenafatua), Avena byzantina, Avenafatuavar.sativa, Hybrid oats (Avenahybrida); Echinochloa spp., For example, Pennisetum (Sorghum), Sorghum (two-colored sorghum) bicolor), sorghum (Sorghum vulgare)), triticale, maize or corn, millet, rice, millet, millet, Sorghum bicolor, gardenia, Fagopyrum spp., Panicum mi (liaceum), millet (Setaria) italica, Zizania (palustris), Ethiopian thrush (Eragrostis tef), cricket (Panicum miliaceum
  • Lycopersicon spp. e.g. tomato (Lycopersicon esculentum), tomato (Lycopersicon lycopersicum), pear-shaped tomato (Lycopersicon pyriforme)), sclerotium (Macrotyloma spp.), kale, angular loofah (Luffa acutangula), Lentil, okra, onion, potato, artichoke, asparagus, broccoli, Brussels sprouts, cabbage ), Carrot, cauliflower, celery, collard greens, squash, benasahispida, Asparagus officinalis, Apium apigraveolens, Amaranthus spp., Allium spp., Abelmoschus spp., Cichorium endivia, Cucurbita spp., Coriandrum
  • Tree crops Pinus spp., Salix spp., Acer spp., Hibiscus spp.
  • Pistachio (Pistacia vera), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), green root, macadamia, pistachio, badan and nut-producing plants; (17) Other: Arabidopsis, Brachiaria, Tribulus, Large Dogtail, Goosegrass, Cadaba farinosa, algae, Carex elata, ornamental plants, Carissa macrocropa, Cynara spp., Wild carrot (Daucus carota), Dioscorea spp.), Erianthus sp., Festuca arundinacea, Hemerocallis fulva, Lotus spp., Luzula sylvatica, Medicago sativa, Melilotus Genus (Melilotus spp.), Morus nigra, Nicotiana spp., Olea spp., Ornithopus spp., Pastinaca
  • plant tissue or “plant part” includes plant cells, protoplasts, plant tissue cultures, plant callus, plant pieces, and plant embryos, pollen, ovules, seeds, leaves, stems, flowers, Branches, seedlings, fruits, nuclei, ears, roots, root tips, anthers, etc.
  • a "plant cell” is to be understood as any cell derived from or found in a plant that is capable of forming, for example, undifferentiated tissue such as callus tissue, differentiated tissue such as embryos, plant components, plants or seeds.
  • the first letter represents a naturally occurring amino acid at a certain position in a specific sequence
  • the following number represents a position relative to SEQ ID NO: 2
  • the second letter represents a substitution of the natural amino acid Of different amino acids.
  • A103S indicates that, relative to the amino acid sequence of SEQ ID NO: 2, the alanine at position 103 is replaced by serine.
  • an amino acid substitution that does not exist in the first letter it refers to the amino acid sequence of its wild-type protein.
  • the natural amino acid is represented by a letter after the number. Amino acid substitution. For double or multiple mutations, each mutation is separated by "/".
  • H141R / G342D / D370N means that with respect to the amino acid sequence of SEQ ID NO: 2, histidine at position 141 is replaced by arginine, glycine at position 342 is replaced by aspartic acid, and position 370 Aspartic acid is replaced by asparagine, and all three mutations are present in the specific mutant HPPD protein.
  • the present invention discloses a mutant HPPD protein or a biologically active fragment thereof which retains the catalytic conversion of hydroxyphenylpyruvate (HPP) to urine black compared to wild-type p-hydroxyphenylpyruvate dioxidase protein. Acid activity, while increasing resistance or tolerance to HPPD inhibitory herbicides.
  • the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein of the present invention corresponds to the amino acid sequence of the wild-type rice p-hydroxyphenylpyruvate dioxidase protein corresponding to SEQ ID ID NO: 2.
  • One or more positions at position 431 have one or more mutations selected from: 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226H, 276W, 277N, 336D, 337A, 338D , 338S, 338Y, 342D, 346C, 346D, 346H, 346S, 346Y, 370N, 377C, 386T, 390I, 392L, 403G, 410I, 418P, 419F, 419L, 419V, 420S, 420T, 430G, and 431L.
  • the amino acid sequence of the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein further has at least 80%, at least 85%, at least 90%, at least 95% of the amino acid sequence shown in SEQ ID NO: 2. %, At least 96%, at least 97%, at least 98%, at least 99% sequence identity. More preferably, the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein has the amino acid sequence shown in SEQ ID NO: 2, and the difference is only in the wild-type rice corresponding to SEQ ID ID NO: 2.
  • One or more mutations at positions 392, 403, 410, 418, 419, 420, 430, and 431 have one or more mutations selected from the following: 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226H, 276W, 277N, 336D, 337A, 338D, 338S, 338Y, 342D, 346C, 346D, 346H, 346S, 346Y, 370N, 377C, 386T, 390I, 392L, 403G, 410I, 418P, 419F, 419L, 419V, 420S, 420T, 430G
  • the amino acid sequence of the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein of the present invention and the wild-type rice p-hydroxyphenylpyruvate dioxidase protein amino acid shown in SEQ ID NO: 2 The sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, and corresponds to the amino acid shown in SEQ ID NO: 2 93, 103, 141, 165, 191, 220, 226, 276, 277, 336, 337, 338, 342, 346, 370, 377, 386, 390, 392, 403, 410, 418, 419,
  • One or more mutations at positions 420, 430, and 431 have one or more mutations selected from the group consisting of 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226H
  • the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein of the present invention has the amino acid sequence shown in SEQ ID NO: 2, and the difference is only in the wild-type rice corresponding to SEQ ID ID NO: 2.
  • the specific amino acid position (number) in the protein of the present invention is determined by comparing the amino acid sequence of the target protein with SEQ ID NO: 2 using a standard sequence alignment tool, such as using the Smith-Waterman algorithm or CLUSTALW2
  • the algorithm aligns two sequences, where the sequences are considered aligned when the alignment score is highest.
  • the comparison score can be calculated according to the method described in Wilbur, W.J. and Lipman, D.J. (1983) Rapid similarity searches of nuclear acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80: 726-730.
  • the AlignX program (part of the vectorNTI group) is preferably used to align the amino acid sequence of the protein with SEQ ID NO: 2 with the default parameters suitable for multiple alignments (gap opening penalty: 10og gap extension penalty 0.05). The location of specific amino acids within the protein of the invention is determined.
  • amino acid sequence identity can be determined by conventional methods, for example, Smith and Waterman, 1981, Adv. Appl. Math. 2: 482, Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, Thompson et al., The teachings of 1994, Nucleic Acids Res 22: 467380, etc., were determined by computerized operating algorithms (GAP, BESTFIT, FASTA, and TFASTA, Genetics Computer Group in the Wisconsin Genetics software package). The BLAST algorithm available from the National Center for Biotechnology Information www.ncbi.nlm.nih.gov/ (Altschul et al., 1990, Mol. Biol. 215: 403-10 can also be used ), Determined using default parameters.
  • the mutant p-hydroxyphenylpyruvate dioxidase protein of the present invention has SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO : 12, SEQ IDNO: 14, SEQ IDNO: 16, SEQ IDNO: 18, SEQ IDNO: 20, SEQ IDNO: 22, SEQ IDNO: 32, SEQ IDNO: 34, SEQ IDNO: 36 , SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ IDNO: 54, SEQ IDNO: 56, SEQ IDNO: 58, SEQ IDNO: 60, SEQ IDNO: 62, SEQ IDNO: 64, SEQ IDNO: 66, SEQ IDNO: 68, SEQ IDNO : 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 4, SEQ
  • the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein of the present invention has the following amino acid mutations in its amino acid sequence: H141R / G342D, H141R / D370N, G342D / D370N, H141R / N338D, H141R / G342D, N338D / G342D, K418P / G419F, G419F / N420S, G342D / R346C, G342D / R346H, H141R / N420S, G338D / K418P, P277N / N338D, L276W / P277N, H141R / G342D / D370N, H141R / N420S, H141R / N338S / N420S, P336D / N338D / G342D, P336D / N338D / G
  • the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein of the present invention has SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30 , SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO : 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134 ,
  • the wild-type p-hydroxyphenylpyruvate dioxidase protein may be derived from any plant, especially the aforementioned monocotyledonous or dicotyledonous plant.
  • the wild-type p-hydroxyphenylpyruvate dioxidase protein of the present invention is derived from Oryza, especially rice. More preferably, the wild-type p-hydroxyphenylpyruvate dioxidase protein has the amino acid sequence shown in SEQ ID NO: 2 or has at least 80%, at least 85%, Amino acid sequences with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the structure of a protein can be changed without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions can be introduced into the protein amino acid sequence without affecting the activity of the protein molecule And / or three-dimensional configuration. Examples and embodiments of conservative amino acid substitutions will be apparent to those skilled in the art.
  • the amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, that is, a non-polar amino acid residue is used to replace another non-polar amino acid residue, and the polarity is uncharged.
  • mutant HPPD protein of the present invention may include one or more other mutations such as conservative substitutions in the amino acid sequence in addition to the above-mentioned mutations.
  • the invention also encompasses mutant HPPD proteins that also contain one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the proteins of the invention.
  • the invention also relates to the deletion of one or more amino acid residues from the N and / or C terminus of a mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein while retaining its required Functionally active fragments, which are also within the scope of the present invention, are called biologically active fragments.
  • biologically active fragment refers to a part of the mutant HPPD protein of the present invention, which retains the biological activity of the mutant HPPD protein of the present invention, and at the same time is resistant or resistant to HPPD inhibitors.
  • a biologically active fragment of a mutant HPPD protein may be one or more deleted at the N and / or C terminus of the protein (eg, 1-50, 1-25, 1-10, or 1-5). (E.g., 1, 2, 3, 4 or 5) amino acid residues, but it still retains the biological activity of the full-length protein.
  • the present invention also provides a fusion protein comprising the mutant HPPD protein of the present invention or a biologically active fragment thereof, and other components fused thereto.
  • the other component is a plastid-directing peptide, such as a peptide directed into the chloroplast, which targets the mutant HPPD protein to the chloroplast.
  • the other component is a tag peptide, such as 6 ⁇ His.
  • the other component is a peptide, such as a NusA peptide, that helps increase the solubility of the mutant HPPD protein.
  • the present invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein or a biologically active fragment thereof, or a complementary sequence thereof.
  • isolated polynucleotide refers to a polynucleotide that contains substantially no components that normally accompany it in a naturally occurring environment.
  • the amino acid sequence of the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein has at least 80%, at least 85%, at least 90%, and the amino acid sequence shown in SEQ ID NO: 2, An amino acid sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, and further in a wild-type rice p-hydroxyphenylpyruvate bis shown in SEQ ID NO: 2 93, 103, 141, 165, 191, 220, 226, 276, 277, 336, 337, 338, 342, 346, 370, 377, 386, 390, 392, 403, 410, oxidase protein amino acid sequence, One or more mutations at positions 418, 419, 420, 430, and 431 have one or more mutations selected from the following: 93S, 103S, 141R, 141K, 141T, 165V, 191I, 220K, 226
  • the mutation is one or more mutations selected from the group consisting of R93S, A103S, H141R, H141K, H141T, A165V, V191I, R220K, G226H, L276W, P277N, P336D, P337A, N338D, N338S, N338Y , G342D, R346C, R346D, R346H, R346S, R346Y, D370N, I377C, P386T, L390I, M392L, E403G, K410I, K418P, G419F, G419L, G419V, N420S, N420T, E430G and Y431L. More preferably, the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein or a biologically active fragment thereof is derived from rice HPPD protein and has one or more amino acid substitutions selected from the above.
  • HPPD p
  • nucleic acid sequences that can encode the amino acid sequences disclosed herein. It is within the ability of one of ordinary skill in the art to generate other nucleic acid sequences that encode the same protein, and therefore the present invention encompasses nucleic acid sequences that encode the same amino acid sequence due to the degeneracy of the genetic codon.
  • the gene in order to achieve high expression of a heterologous gene in a target host organism, such as a plant, the gene can be optimized using codons preferred by the host organism to make it better expressed.
  • a polynucleotide of the invention has a nucleic acid sequence selected from:
  • SEQ ID NO: 4 Code SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 ,, SEQ IDNO: 44, SEQ IDNO: 46, SEQ IDNO: 48, SEQ IDNO: 50, SEQ IDNO: 52, SEQ IDNO: 54, SEQ IDNO: 56, SEQ IDNO: 58, SEQ ID NO: 60, SEQ IDNO: 62, SEQ IDNO: 64, SEQ IDNO: 66, SEQ IDNO: 68, SEQ IDNO: 70, SEQ IDNO: 72, SEQ IDNO: 74, SEQ IDNO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID
  • SEQ ID NO: 3 SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 , SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO : 51, SEQ IDNO: 53, SEQ IDNO: 55, SEQ IDNO: 57, SEQ IDNO: 59, SEQ IDNO: 61, SEQ IDNO: 63, SEQ IDNO: 65, SEQ IDNO: 67 , SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO:
  • the polynucleotide has a member selected from SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ IDNO: 19, SEQ IDNO: 21, SEQ IDNO: 23, SEQ IDNO: 25, SEQ IDNO: 27, SEQ IDNO: 29, SEQ IDNO: 31.SEQ IDNO: 33, SEQ IDNO: 35, SEQ IDNO: 37, SEQ IDNO: 39, SEQ IDNO: 41, SEQ IDNO: 43, SEQ IDNO: 45, SEQ IDNO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ IDNO: 67, SEQ IDNO: 69, SEQ IDNO: 71
  • the stringent conditions may refer to the conditions of 6M urea, 0.4% SDS, 0.5 ⁇ SSC or equivalent hybridization conditions, or may refer to the conditions of more stringent conditions, such as 6M urea, 0.4% SDS, 0.1 ⁇ SSC or Equivalent hybridization conditions.
  • the temperature may be about 40 ° C or more. If more stringent conditions are required, the temperature may be about 50 ° C, for example, and further about 65 ° C.
  • the wild-type and mutant codons corresponding to the amino acid mutation site are as follows:
  • the invention also provides a nucleic acid construct comprising a nucleic acid sequence encoding the mutant p-hydroxyphenylpyruvate dioxidase protein or a biologically active fragment or fusion protein of the invention and one or more operatively linked thereto Regulatory elements.
  • regulatory element refers in the present invention to a nucleic acid sequence capable of regulating the transcription and / or translation of a nucleic acid to which it is operatively linked.
  • Regulatory elements may be appropriate promoter sequences that are recognized by host cells for expression of a nucleic acid sequence encoding a protein of the invention.
  • the promoter sequence contains transcriptional regulatory sequences that mediate protein expression. Promoters can be any nucleotide sequence that exhibits transcriptional activity in a selected host cell, including mutated, truncated, and heterozygous promoters, and may be obtained from a coding extracellular homologous or heterologous host cell. Or intracellular polypeptide genes.
  • a promoter expressed in a plant cell or a plant a native promoter of p-hydroxyphenylpyruvate dioxidase or a heterologous promoter which is active in a plant can be used.
  • the promoter may be constitutively expressed, or may be inducibly expressed. Examples of the promoter include, for example, a histone promoter, a rice actin promoter, a plant virus promoter such as a cauliflower mosaic virus promoter, and the like.
  • the regulatory element may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3 'end of the nucleic acid sequence encoding the protein of the present invention. Any terminator that functions in a selected host cell can be used in the present invention.
  • leader sequences ie untranslated regions of mRNA that are important for translation by the host cell.
  • the leader sequence is operably linked to the 5 'end of a nucleic acid sequence encoding a protein of the invention. Any leader sequence that functions in a selected host cell can be used in the present invention.
  • the regulatory element may also be a polyadenylation sequence, that is, a sequence that is operably linked to the 3 'end of a nucleic acid sequence and is recognized by the host cell as a signal that adds a polyadenylation residue to the transcribed mRNA .
  • a polyadenylation sequence that functions in a selected host cell can be used in the present invention.
  • the regulatory element may also be a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a protein and directs the encoded protein into the cell's secretory pathway.
  • the 5 'end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region that is naturally linked within the translation reading frame to a portion of the coding region encoding the secreted polypeptide.
  • the 5 'end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. Where the coding sequence naturally does not contain a signal peptide coding region, an exogenous signal peptide coding region may be required.
  • an exogenous signal peptide coding region may simply replace the natural signal peptide coding region in order to promote the secretion of the polypeptide.
  • any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the selected host cell, ie, into the culture medium, may be used in the present invention.
  • Regulatory sequences that allow the expression of the polypeptide to be regulated relative to the growth of the host cell may also be appropriately added.
  • Regulatory systems include, for example, regulatory systems that enable gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds, such as lac, tec, and tip manipulation subsystems, ADH2 systems, or GAL1 systems.
  • regulatory compounds such as lac, tec, and tip manipulation subsystems, ADH2 systems, or GAL1 systems.
  • regulatory sequences are those that allow gene amplification. In eukaryotic systems, these include dihydrofolate reductase genes that are amplified in the presence of methotrexate, and metallothionein genes that are amplified due to heavy metals.
  • the nucleotide sequence encoding the polypeptide will be operably linked to the regulatory sequence.
  • the regulatory element may also be a transcriptional activator, that is, an enhancer, such as the tobacco mosaic virus translation activator described in WO87 / 07644, or an intron, such as the adh1 intron of corn, the corn bronze 1 Gene (maize) intron or rice actin intron 1. They can enhance the expression of the mutant HPPD protein, its biologically active fragment or fusion protein of the present invention in transgenic plants.
  • an enhancer such as the tobacco mosaic virus translation activator described in WO87 / 07644
  • an intron such as the adh1 intron of corn, the corn bronze 1 Gene (maize) intron or rice actin intron 1.
  • the present invention also provides an expression vector, which contains a nucleic acid sequence encoding the mutant p-hydroxyphenylpyruvate dioxidase protein or a biologically active fragment thereof or a fusion protein of the present invention, and an expression control element operatively connected thereto. .
  • the expression vector also contains at least one origin of replication to achieve self-replication. The choice of the vector usually depends on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • a vector may be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity. Its replication does not depend on the replication of chromosomes, such as plasmids, extrachromosomal elements, minichromosomes, or artificial chromosomes.
  • the vector may contain any element that guarantees self-replication.
  • the vector may be a vector that is integrated into the genome when it is introduced into a host cell and replicates with the chromosome (s) into which it is integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the host cell genome, or transposons can be used.
  • the vector may be a vector for gene editing of an endogenous HPPD gene in a host cell.
  • Vectors can be, for example, plasmids, viruses, cosmids, phages, and the like, which are well known to those skilled in the art and are described in the art.
  • the expression vector in the present invention is a plasmid.
  • the expression vector may include a promoter, a ribosome binding site for translation initiation, a polyadenylation site, a transcription terminator, an enhancer, and the like.
  • the expression vector may also contain one or more selectable marker genes for use in selecting a host cell containing the vector. Such selectable markers include genes encoding dihydrofolate reductase, genes conferring resistance to neomycin, genes conferring resistance to tetracycline or ampicillin, and the like.
  • the vector of the present invention may contain elements that allow the vector to integrate into the host cell genome or to replicate autonomously within the cell independent of the genome.
  • the vector may rely on a polynucleotide sequence encoding a polypeptide or any other element of a vector suitable for integration into the genome by homologous or non-homologous recombination.
  • the vector may contain additional nucleotide sequences for directing integration into the host cell genome by homologous recombination at the exact location of the chromosome.
  • the integration element should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and more preferably 800 to 10,000 base pairs. Has a high degree of identity to the corresponding target sequence to increase the probability of homologous recombination.
  • An integration element may be any sequence that is homologous to a target sequence in the host cell genome.
  • integration elements may be non-coding or coding nucleotide sequences.
  • the vector may be integrated into the host cell's genome through non-homologous recombination.
  • the vector may further include an origin of replication that enables the vector to autonomously replicate within the host cell.
  • the origin of replication may be any plasmid replicon that functions intracellularly to mediate autonomous replication.
  • the term "origin of replication" or "plasmid replicon” is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.
  • More than one copy of the polynucleotide of the present invention can be inserted into a host cell to increase the yield of a gene product.
  • An increase in the number of polynucleotide copies may be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, in the latter case Cells containing an amplified copy of a selectable marker gene and an additional copy of a polynucleotide therefrom can be selected by artificially culturing the cells in the presence of a suitable alternative formulation.
  • nucleic acid sequence of the present invention can be inserted into a vector by various methods, for example, by ligating the insert and the vector with an appropriate restriction endonuclease.
  • a variety of cloning techniques are known in the art, and these are all within the knowledge of those skilled in the art.
  • Vectors suitable for use in the present invention include commercially available plasmids such as, but not limited to, pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, WI, USA) pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174, pBluescript II, KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8, pCM7, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia) and the like.
  • the invention also provides a host cell comprising a nucleic acid sequence, a nucleic acid construct or an expression vector of the invention.
  • a vector encoding the present invention into a host cell allows the vector to exist as part of a chromosomal integrant or as a self-replicating extrachromosomal vector as described earlier, or the vector can gene-edit the HPPD gene endogenous to the host cell.
  • the host cell may be any host cell familiar to those skilled in the art, including prokaryotic cells and eukaryotic food cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells or plant cells, examples of which are E. coli ( E.
  • the term "host cell” also encompasses any parent cell progeny that is not identical to the parent cell due to mutations that occur during replication.
  • nucleic acid sequences, nucleic acid constructs or expression vectors of the present invention can be introduced into host cells by a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun or Ti-plasmid-mediated gene delivery, and calcium phosphate transduction. Staining, DEAE-dextran-mediated transfection, lipofection, or electroporation (see Davis, L., Dibner, M., Batty, I., Basic Methods in Molecular Biology, 1986).
  • the mutant HPPD protein of the present invention can be targeted to plastids, such as chloroplasts, in plants. This can be achieved by concatenating the nucleic acid sequence encoding the mutant HPPD protein of the present invention with the reading frame of a nucleic acid sequence encoding a plastid guide peptide such as a chloroplast transit peptide.
  • the polynucleotide, nucleic acid construct or expression vector of the present invention can be directly transformed into the chloroplast genome of a plant cell. Those skilled in the art will know vectors and methods that can be used to transform plant cell chloroplast genomes.
  • the nucleic acid sequence encoding the mutant HPPD protein of the present invention can be integrated by bombarding the leaves of a target plant with DNA-coated ions and by homologous recombination or non-homologous recombination.
  • the transformed host cells can be cultured in conventional nutrient media. After transforming a suitable host cell and culturing the host cell to an appropriate cell density, the selected promoter can be induced by a suitable method such as temperature change or chemical induction, and the cell can be cultured for a period of time to produce the A mutant HPPD protein or a biologically active fragment or fusion protein thereof.
  • the present invention also relates to a method for producing the mutant HPPD protein or the biologically active fragment or fusion protein of the present invention, comprising: (a) a method for assisting the production of the mutant HPPD protein or the biologically active fragment or fusion protein thereof; Culturing the host cell under conditions; and (b) recovering the mutant HPPD protein or a biologically active fragment or fusion protein thereof.
  • the cells are cultured on a nutrient medium suitable for the production of the polypeptide by a method well known in the art.
  • a nutrient medium suitable for the production of the polypeptide for example, in a laboratory or industrial fermentor with a suitable culture medium and under conditions that permit the expression and / or isolation of the polypeptide by shake flask culture and small-scale or large-scale fermentation (including continuous, batch, (Batch feed or solid state fermentation).
  • the culture is performed on a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media are available from suppliers or formulated according to published compositions (for example, on the catalog of the American Type Culture Collection). If the polypeptide is secreted into a nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the culture medium, it can be recovered from the cell lysate.
  • the polypeptide can be detected by methods known in the art that are specific to the polypeptide. These detection methods may include the use of specific antibodies, formation of enzyme products, or disappearance of enzyme substrates.
  • the produced polypeptide can be recovered by methods known in the art. For example, cells can be harvested by centrifugation, broken physically or chemically, and the resulting crude extract can be retained for further purification.
  • Transformed host cells expressing the mutant HPPD protein or its biologically active fragment or fusion protein of the invention can be lysed by any convenient method, including freeze-thaw cycles, ultrasound, mechanical disruption, or the use of cytolytic agents. These methods are well known to those skilled in the art.
  • the mutant HPPD protein or a biologically active fragment thereof of the present invention can be recovered and purified from a culture of transformed host cells by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, and phosphate cellulose chromatography. , Hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and phytohemagglutinin chromatography, etc.
  • the present invention also relates to a method for preparing a host organism, particularly a plant cell, plant tissue, plant part or plant, which is tolerant or resistant to an HPPD-inhibiting herbicide, which comprises using the mutant p-hydroxybenzene comprising the present invention.
  • the pyruvate dioxidase protein or a biologically active fragment thereof encodes a nucleic acid sequence, a nucleic acid construct or an expression vector comprising the nucleic acid sequence to transform the host organism.
  • Methods for transforming host cells such as plant cells, are known in the art and include, for example, protoplast transformation, fusion, injection, electroporation, PEG-mediated transformation, ion bombardment, viral transformation, Agrobacterium-mediated transformation, electroporation Perforation or bombardment.
  • a series of such transformation methods are described in the prior art, for example, a soybean transformation technology is described in EP1186666, and a suitable technology for transformation of a monocotyledonous plant, particularly rice, is described in WO 92/09696. It may also be advantageous to cultivate plant explants with Agrobacterium tumefaciens or Agrobacterium rhizogenes to transfer DNA into plant cells.
  • Intact plants can then be regenerated from infected plant material parts (such as leaf fragments, stem segments, roots, and protoplasts or suspension cultured cells) in a suitable culture medium that can contain antibiotics for selection or kill Insecticide.
  • Transformed cells grow in plants in the usual manner, they can form germ cells and transfer transformed traits to the progeny plants. Such plants can be grown in a normal manner and crossed with plants having the same transformed genetic factor or other genetic factors. The obtained heterozygous individuals have corresponding phenotypic characteristics.
  • the invention also provides a method for preparing a host organism, especially a plant cell, plant tissue, plant part or plant, which is tolerant or resistant to an HPPD-inhibiting herbicide, which comprises the mutant p-hydroxybenzene of the invention
  • a nucleic acid encoding a pyruvate dioxidase protein or a biologically active fragment thereof is integrated into the host organism's genome and expressed.
  • Suitable vectors and selectable markers are well known to those skilled in the art, for example, a method for integration into the tobacco genome is described in WO06 / 108830, the disclosure of which is incorporated herein by reference.
  • the gene of interest is preferably expressed in a plant cell by a constitutive or inducible promoter.
  • the gene encoding a protein expressed in a plant cell may be under the control of a constitutive promoter, a tissue-specific promoter, or an inducible promoter.
  • bacterial-derived promoters such as the octopine synthase promoter, nopaline synthase promoter, and mannoline synthase promoter
  • viral-derived promoters such as cauliflower mosaic virus (35S and 19S), 35T ( For a further modified 35S promoter, see US Patent No. 6,166,302, particularly Example 7E) and the like.
  • Plant promoter regulatory elements can also be used, including but not limited to the ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), ⁇ -conglycinin promoter, ⁇ - Phaseolin promoter, ADH promoter, heat shock promoter, and tissue-specific promoter.
  • Constitutive promoter regulatory elements can also be used to direct continuous gene expression (eg actin, ubiquitin, CaMV35S, etc.) in all cell types and at all times.
  • Tissue-specific promoter regulatory elements are also applicable in the present invention and are responsible for gene expression (e.g., zein, oleosin, napin, ACP, globulin) in a specific cell or tissue type (e.g., leaf or seed) Wait).
  • promoter regulatory elements that are active (or inactive) at certain stages of plant development can also be used.
  • these promoter regulatory elements include, but are not limited to, pollen specific, embryo specific, corn ear silk specific, cotton fiber specific, root specific, seed endosperm specific or asexual reproduction specific promoter regulatory elements, and the like.
  • inducible promoter regulatory elements that are responsible for responding to specific signals such as physical stimuli (heat shock genes), light (RUBP carboxylase), hormones (Em), metabolites, chemicals ( Tetracycline response) and stress) gene expression.
  • Other required transcription and translation elements that function in plants can be used.
  • the invention also provides a method for increasing the tolerance or resistance of HPPD-inhibiting herbicides in plant cells, plant tissues, plant parts or plants, which comprises dioxidizing with a mutant p-hydroxyphenylpyruvate comprising the invention
  • a nucleic acid molecule encoding a nucleic acid sequence encoding an enzyme protein or a biologically active fragment thereof or a fusion protein transforms the plant or a part thereof and expresses it.
  • the nucleic acid molecule can be expressed as an extrachromosomal entity, or it can be integrated into the genome of a plant cell to achieve expression, especially by homologous recombination integrated into the endogenous gene position of the plant cell to achieve expression.
  • the present invention also provides a method for increasing the tolerance or resistance of a plant or part thereof to an HPPD-inhibiting herbicide, which comprises expressing the mutant p-hydroxyphenylpyruvate dioxidase (HPPD) protein of the present invention or A plant whose biologically active fragment or fusion protein is crossed with another plant, and plants or parts thereof having improved resistance or tolerance to HPPD-inhibiting herbicides are screened.
  • HPPD p-hydroxyphenylpyruvate dioxidase
  • the invention also provides a method for increasing the tolerance or resistance of HPPD-inhibiting herbicides in plant cells, plant tissues, plant parts or plants, which comprises The endogenous HPPD protein is subjected to gene editing to achieve expression of the mutant p-hydroxyphenylpyruvate dioxidase protein of the present invention or a biologically active fragment or fusion protein thereof.
  • the present invention also relates to a method for preparing a herbicide-tolerant or resistance-improving plant by traditional breeding techniques, which comprises using the mutant p-hydroxyphenylpyruvate dioxidase protein of the present invention or a biological activity thereof integrated in the genome
  • the plants of the fragment encoding nucleic acid sequences are selfed or crossed, and progeny that are heterozygous or homozygous for containing the encoding nucleic acid sequence are screened.
  • the invention further relates to plant cells, plant tissues, plant parts and plants obtained by the method described above, and their progeny.
  • plant cells, plant tissues or plant parts transformed with a polynucleotide of the invention can be regenerated into whole plants.
  • the invention includes cell cultures, including tissue cell cultures, liquid cultures, and solid plate cultures. Seeds produced by and / or used to regenerate the plants of the invention are also included within the scope of the invention. Other plant tissues and parts are also included in the present invention.
  • the invention also includes methods for producing plants or cells containing a nucleic acid molecule of the invention. A preferred method of producing such plants is by planting the seeds of the invention. Plants transformed in this way can gain resistance to a variety of herbicides with different modes of action.
  • explants can be mixed with the transformed Agrobacterium and incubated for a sufficient time to allow their transformation. After transformation, Agrobacterium is killed by selection with an appropriate antibiotic, and plant cells are cultured with an appropriate selection medium. Once callus is formed, bud formation can be promoted by using appropriate plant hormones according to methods well known in the fields of plant tissue culture and plant regeneration. However, intermediate callus is not always necessary. After bud formation, the plant cells can be transferred to a medium that promotes root formation, thereby completing plant regeneration. The plant can then be cultivated to produce seeds that can be used to establish future generations.
  • the gene encoding a bacterial protein into a gene transfer vector, and to incorporate the plant promoter regulatory element and a 3 'untranslated transcription termination region (such as Nos, etc.) into the vector to make the transfer vector suitable for The gene is expressed in plant cells.
  • a 3 'untranslated transcription termination region such as Nos, etc.
  • the present invention also provides a method for controlling weeds in a plant site, which comprises applying to the site containing a plant or seed of the present invention an effective amount of one or more HPPD-inhibiting herbicides for controlling weeds.
  • the term "site” includes a site where the plant of the present invention is cultivated, such as soil, and also includes, for example, plant seeds, plant seedlings, and grown plants.
  • an effective amount for controlling weeds refers to an amount of the herbicide sufficient to affect the growth or development of the target weed, for example, to prevent or inhibit the growth or development of the target weed, or to kill the weed.
  • the effective amount of weed control does not significantly affect the growth and / or development of the plant seed, plant seedling or plant of the invention. Those skilled in the art can determine such an effective amount for controlling weeds through routine experiments.
  • the present invention also provides a mutant pair that retains or enhances the property of catalyzing the conversion of p-hydroxyphenylpyruvate (HPP) to urnic acid and is significantly less sensitive to HPPD-inhibiting herbicides than wild-type HPPD.
  • HPP p-hydroxyphenylpyruvate
  • HPPD hydroxyphenylpyruvate dioxidase
  • the component for increasing solubility is NusA, which constitutes a fusion protein with the mutant HPPD protein of the present invention.
  • the expression vector is a pET-44a vector.
  • the host cell may be a bacterial cell, a fungal cell, or a plant cell.
  • the rice (Oryza sativa Japonica Group) p-hydroxyphenylpyruvate dioxygenase (HPPD) gene is located at the second chromosome Os02g0168100 locus. According to its cDNA sequence (NCBI number XP_015626163.1), its coding region DNA (OsHPPD) (general organism, China, Anhui, Luzhou) was directly used as a PCR template.
  • primers NusOsF: acg, gat, gac, gac, ag, ATGCCTCCCACTCCCACCCC, and NusOsR: tccacgagctcccggggccTAACTAGGATCCTTGAA, CTGTAG were designed and synthesized.
  • These primers, a synthetic template and Q5 DNA polymerase were used for PCR amplification.
  • Amplification conditions were: 98 ° C for 2 minutes; then 98 ° C for 20 seconds, 65 ° C for 30 seconds, and 72 ° C for 60 seconds, repeated 35 times; finally, 72 ° C for 2 minutes.
  • the amplified fragment showed 1.3Kb in agarose gel electrophoresis, and its DNA concentration was determined by ultraviolet absorption after recovery.
  • the pET-44a (Novagen) plasmid was digested with BoxI (Thermo Fisher Scientific, Shanghai, China) at 37 ° C for 1 hour and then heated to 65 ° C to inactivate BoxI. Take an equal amount of the OsHPPD DNA fragment and BoxI linearized pET-44a vector, add an equal volume of 2 ⁇ Gibson Assembly Master Mix (Hanheng Bio, China, Shanghai), mix it, incubate at 50 ° C for one hour, and take 5ul of ligation The product was used to transform E. coli DH5a competent cells. The bacterial solution was spread on the surface of a LB solid medium plate containing 100 ppm ampicillin and cultured at 37 ° C overnight.
  • the rice full-length OsHPPD enzyme has 446 amino acids, and its amino acid sequence is shown in SEQ ID NO: 2. Among them, amino acids 1-50 are considered to constitute signal peptides and are responsible for directing them into the chloroplast (Siehl et al. Plant Physiol. 2014 Nov; 166 (3): 1162-1176.). So, starting from amino acid position 51 up to amino acid position 446, saturated random mutations were made for each amino acid. This is achieved by performing a bypass PCR with a primer containing the amino acid code to be mutated to NNK and another appropriate conventional primer. In NNK, N stands for A / T / G / C and K stands for G / T.
  • the NNK codon can encode any of the 20 amino acids and terminators, so this is a saturation mutation.
  • the NNK codon can encode any of the 20 amino acids and terminators, so this is a saturation mutation.
  • Kille Acevedo-Rocha, CG, Parra, LP, Zhang, ZG, Opperman, DJ, Reetz, MT, Acevedo, JP (2013) Reducing Codon, Redundancy, and Screening, Effectiveness of combinatorial, Protein, Libraries, created, Bysaturation, Mutagenesis, ACS, Synthesis -92; Directed Evolution: Library: Creations and protocols 2nd edited. Edited by Elizabeth MJGillam, Janine N. Copp and David F. Ackerley New York, NY United States: Springer, 2014. doi: 10.1007 / 978-1-4939- 1053-3.
  • the mutant was cloned into a linearized pET-44a vector, and then transformed into E. coli.
  • a 96-well plate was used in the presence of HPPD inhibitor herbicides (such as cyclosulfonone, 1-2 ⁇ M) and substrate tyrosine (1 G / l) in 2 ⁇ YT medium, cultured at 28 ° C, 150 rpm / min on a shaker for 24 hours, expressed, and then quickly screened these mutants based on their browning.
  • HPPD inhibitor herbicides such as cyclosulfonone, 1-2 ⁇ M
  • substrate tyrosine (1 G / l
  • the method of this patent improves the solubility of HPPD expression in bacteria by fusing NusA with rice HPPD protein, so that the protein can be expressed at the same time at 28 degrees Celsius and the enzymatic reaction can be performed, greatly saving screening time and steps.
  • H141R mutant The production and screening of H141R mutant is taken as an example to illustrate the whole process.
  • TGCGCG was used as a primer, and a synthetic full-length OsHPPD template and Q5 DNA polymerase (NEB, New England Biolabs, Boston, USA) were subjected to PCR amplification to obtain the latter DNA fragment.
  • the previous fragment and the next fragment have 19 bases in the middle (OsHPPD-H141R-F and OsHPPD-H141R-R). Therefore, the two fragments were each mixed in equal molar numbers, and an equal volume of 2 ⁇ Glodstar MasterMix (Kangwei Century Biotechnology Co., Ltd., Beijing) was added, and then 10 pmol NusOsF and NusOsR primers were added to perform a bypass PCR reaction.
  • the amplification conditions were: 96 ° C for 2 minutes; 96 ° C for 20 seconds, 65 ° C for 30 seconds, 72 ° C for 60 seconds, and repeated 30 times; finally, 72 ° C for 5 minutes. After detection by agarose gel electrophoresis, a band with a size of 1.3 Kb was recovered, and the concentration was determined by ultraviolet absorption method.
  • HPPD inhibitory herbicides inhibit HPPD enzyme activity.
  • HPPD 4-hydroxyphenylpyruvate
  • HPPD 4-hydroxyphenylpyruvate
  • HGA 4-hydroxyphenylpyruvate
  • Uric acid is dark brown. Therefore, if an HPPD mutant is resistant or herbicide-resistant, it can oxidize 4-hydroxyphenylpyruvate to urosuric acid and appear dark brown after expression in E. coli. Therefore, we used a 96-well plate to culture E. coli in a 2 ⁇ YT broth with HPPD inhibitor herbicide and substrate tyrosine, and express HPPD, and then quickly screened these mutants based on their color changes.
  • the 96-well plate was cultured on a shaker at 150 rpm at 28 ° C for 24 hours. Visually or detect the light absorption of the culture at 400 nM, use the inoculation loop to pick out clones that produce melanin obvious, and further culture, extract its plasmid DNA for sequencing and further research, such as OsHPPD protein expression, purification and enzyme activity test.
  • the present invention also tested H141R, G342D, D370N and their complex site-mutated HPPD proteins on five HPPD inhibitory herbicides, sulfazone, metabolite of flubendione, and cyclamate, on different 96-well plates at different times.
  • the test results are shown in Figs. 4, 5, and 6.
  • Table 3 shows the approximate resistance / tolerance of these mutants to the corresponding herbicides based on their color shades. It can be seen from Table 3 that the two-site mutant and the three-site mutant also exhibit high drug resistance.
  • amino acid mutations in rice HPPD (OsHPPD) proteins can exist in combination, which can also achieve more High HPPD inhibitory herbicide resistance / tolerance.
  • Example 4 Saturation mutation was performed on the basis of the 3-point mutation H141R-G342D-D370N.
  • the mutation sites are combined based on the following three principles: similar sites facilitate homologous substitutions during gene editing and high editing efficiency; base changes are also A ⁇ G / T ⁇ C or C ⁇ T / G ⁇ A Base editing; and minimal resistance sites to facilitate editing and avoid possible negative effects. Design combinations, corresponding primers, and construct prokaryotic expression vectors according to the above principles, and then perform color reaction screening to find combinations suitable for editing and high resistance for gene editing.
  • the resistance of the single point is 10-20uM; the resistance of the two points is about 20-120um, which is stronger than the single point, and the color is lighter at 100uM; H141R / N338D / G342D, H141R / G342D / K418P , H141R / G342D / G419F, 338D / 342D / 346C / H, H141R / G342D / N420S, H141R / N338D / N420S and other three-site combination sorting resistance is good, can still have a pale color to 1000uM; H141R / N338D / G342D / K418P, H141R / G342D / K418P / G419F, H141R / N338D / G342D / R346C, H141R / N338D / G342D / R346H, H141R
  • Rice OsHPPD protein and homogenisate oxidase HGD are heterologously expressed in E. coli. Genes are inserted into the pET-15b expression vector, and expressed using BL21 (DE3) expression strains. -NTA obtained by purification.
  • the positive cloned HPPD open reading frame (ORF) was cloned into pET-15b vector to form a 6His-HPPD expression vector, and transformed into BL21 (DE3) cells.
  • the expression strain was inoculated into 10 mL of 2 ⁇ YT broth, and cultured at 37 ° C. on a shaker at 200 rpm overnight. 10mL of the culture was inoculated into 1L 2 ⁇ YT medium, cultured until the OD 600 reached 0.6-0.8, cooled to 16 ° C, 0.2mM IPTG (isopropylthiogalactoside) induced expression overnight, 2800xg centrifuged to harvest .
  • IPTG isopropylthiogalactoside
  • buffer A 50mM Tris pH 8.0, 500mM NaCl, 20mM imidazole
  • PMSF benzylsulfonyl fluoride
  • protease inhibitor inhibitor a variety of proteases Inhibitor mixture
  • Cells were sonicated in an ice bath (40% total power, 3 sec operation / 6 sec gap, 2x30 minutes (Ningbo Xinzhi Technology Co., Ltd., Ningbo, China)); at 12000 rpm, 4 ° C, 30 minutes, Centrifuge, take the supernatant, and filter through a 0.22uM filter.
  • Ni-NTA column purification After combining the above supernatant with Ni-NTAresin, washing with buffer A containing 50 mM imidazole and finally eluting with an elution buffer containing 400 mM imidazole.
  • HPPD activity is through the detection of HPPD enzyme to catalyze the conversion of 4-HPP (4-hydroxyphenylpyruvate) to HGA (urnic acid).
  • HGA uronic acid
  • MAA methacrylate acid
  • Maleic acetoacetate maleic acetoacetate has the maximum absorption at 318 nm, and the absorption constant is 14.7 OD M -1 cm -1 .
  • reaction substrate 4-HPP Add 6ul 50-fold substrate 4-HPP to the enzyme-labeled plate, and add 294uL at a final concentration of 25mM HEPES (hydroxyethylpiperazine ethanesulfuric acid), pH 7, 2mM vitamin C, 10mM FeSO4, 50nM homogentisate dioxygenase and 5 to 240nM HPPD enzyme.
  • the final concentration of the reaction substrate 4-HPP is generally 1 to 100 uM.
  • V max is the maximum catalytic reaction rate that can be achieved when the enzyme catalyzes.
  • the Mie constant K m is the concentration of the substrate required when the enzyme catalyzed reaction reaches half the maximum rate (V max ).
  • the value of K m is a constant that is equal to the enzyme concentration. It has nothing to do with the type of substrate, temperature, pH and ionic strength of the reaction.
  • K cat is the catalytic constant of the enzyme, which indicates how many substrates an enzyme molecule or enzyme active center can catalyze each second.
  • K cat / K m represents the catalytic efficiency of the enzyme. As shown in Table 10, these enzyme parameters of rice HPPD wild type (WT) and various mutants were determined. From the data in the table, it can be seen that the catalytic efficiency of most mutant HPPD has been enhanced.
  • Enzyme fitness is an indicator of the adaptability of the enzyme to the inhibitor. A larger value indicates that the enzyme is more resistant to the inhibitor. Since the substrate concentration during the reaction is much larger than the K m value, and different OsHPPD Mutant reaction conditions are the same, so K cat can be replaced by the rate of V max when catalyzed by the same concentration of enzyme (500 nM).
  • N338D / G342D / R346H shortest, suitable for homologous substitution HDR
  • H141R / N338D / G342D shorter, also suitable for homologous substitution
  • H141R / N338D / N420S suitable for single base editing, resistance (Strong)
  • Primers are designed to amplify three-point mutant HPPD (H141R / G342D / D370N) (OsHPPD3M) based on the selected restriction site and the nucleotide sequence of the gene itself. The designed primers were synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd .: HPPD–F, GATAGCCGGTACGGGTTCGA GCCACC ATGCCTCCCACT CCCACCC, HPPD–R, GATCTTTGTAATCGGGGTAC CTAGGATCCTTGAACTGTAGGGGC.
  • PCR amplification Amplify the gene of interest using synthetic primers and Q5 DNA polymerase (NEB, New England Biolabs, Boston, USA). The amplified product was detected by agarose gel electrophoresis, and the product was recovered according to the instructions of TIANquick Midi Purification Kit. After the recovery was completed, the concentration of the extracted DNA was detected by Nanodrop.
  • Agrobacterium culture Pick the transformed Agrobacterium clones, shake culture at 28 ° C in YEB liquid culture medium (Kalamycin + Rifampin) to OD600 of 0.5, collect colonies at 3500 rpm, equal amount of AAM (1 ml AAM + 1 ⁇ l 1000 ⁇ AS). The callus was infected after the liquid medium was diluted.
  • rice callus Before preparing Agrobacterium, rice callus must be prepared. Strip the rice seeds, and wash the seeds with sterile water until the water becomes clear after washing. Unlimited times, disinfect with 70% alcohol for 30 seconds, and then place the 5% sodium hypochlorite on a horizontal shaker for 20 minutes. Sterile water after disinfection with sodium hypochlorite It was washed 5 times, placed on sterile absorbent paper, air-dried on the surface of the seeds, inoculated on an induction medium, and cultured at 28 ° C. to callus.
  • Agrobacterium-infected rice callus Huaidao No. 5 callus with a diameter of 3 mm was selected for subculture for 10 days, and the callus was collected into a 50 ml centrifuge tube; the adjusted concentration of the Agrobacterium bacterial solution Pour into a centrifuge tube containing callus and infect it for 20 minutes in a shaker at 200 rpm at 28 ° C; after the infection is complete, discard the bacterial solution, and place the callus on a sterile filter paper to air dry for about 20 minutes. , Placed on a common culture plate for co-cultivation.
  • the plate was covered with a piece of sterile filter paper soaked in AAM (1ml AAM + 30 ⁇ l 1000 ⁇ AS) liquid medium; after 3 days of infection, the Agrobacterium was washed away to remove It was washed 5 times with water, and then washed with 500 mg / L cephalosporin antibiotic for 20 minutes), and then placed on a 50 mg / L hygromycin selection medium for screening and culture.
  • Screening, differentiation and rooting of resistant callus transfer the co-cultured callus to the screening medium for the first round of screening (2 weeks); after the first round of screening, the newly grown callus is transferred Go to the screening medium (containing 50mg / L hygromycin) for the second round of screening (2 weeks); after the screening is completed, pick the yellow and white callus with good growth status to differentiate, and add 1uM-5uM to the differentiation medium.
  • Sulfazone is screened for herbicide resistance, and about 1 cm seedlings can be obtained after 3 to 4 weeks; the differentiated seedlings are transferred to the rooting medium for rooting culture; the rooted seedlings are subjected to seedling treatment, and then transferred to the equipment. Soil pots were placed in the greenhouse for cultivation; OsHPPD3M 55 seedlings or events were obtained.
  • the transgenic seedling (T0 generation) herbicide resistance is tested again:
  • the T0 generation transgenic seedlings are transplanted into large plastic barrels in the greenhouse and cultivated to obtain T1 generation seeds.
  • two events were randomly selected from the over-expressed mutant events, and a group of non-transgenic Zhonghua 11 rice seedlings of the same growth period were added to determine the herbicide resistance.
  • the herbicide used is oxadiazon, and its field dose is usually 4 grams of active ingredient per acre (4 g, a.i./mu).
  • the doses of oxadiazon in this experiment were 8 and 16 g / mu.
  • the three-point mutant OsHPPD3M (H141R / G342D / D370N) overexpression all enhanced the resistance of transgenic rice to HPPD inhibitory herbicides, and the resistance multiple was at least 4 times. From the preliminary observation of the growth and development and flowering of the transgenic seedlings of the T0 and T1 generations, most of the event plants were normal.
  • Hygromycin resistance gene The GC content of rice hppd (Oshppd) gene is high, which affects the efficiency of PCR amplification. In addition, there is a copy of hppd endogenous to rice. Therefore, we selected the hygromycin resistance gene hyg as the foreign gene and the sucrose phosphate synthase (SPS) gene as the endogenous reference gene for copy number estimation.
  • the sucrose phosphate synthase (SPS) gene is a rice-specific gene and is a single copy and can be used as an endogenous reference gene for rice (Ding Jiayu, Jia Junwei, Yang Li Tao Tao et al.
  • Genomic DNA Extraction and purification of genomic DNA from rice leaves using a plant genomic DNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd., and detection of DNA content and purity using a nanodrop nucleic acid protein analyzer (nanodrop)
  • the ratio of OD260 / OD280 is in the range of 1.8-2.0. When the ratio of OD260 / OD230 is around 2.0, the purity is considered to be better.
  • Primers Design two pairs of primers: Hyg-F: 5'-GTACACAAATCGCCCGCAG-3 ', Hyg-R: 5'-TCTATTTCTTTGCCCTCGGAC-3', amplify a 111 bp fragment of hygromycin resistance gene; Sps-F: 5 ' -GTACACAAATCGCCCGCAG-3 ', Sps-R: 5'-TCTATTTCTTTGCCCTCGGAC-3', and a 170 bp fragment of the sucrose phosphate synthase (SPS) gene was amplified.
  • SPS sucrose phosphate synthase
  • Quantitative PCR reaction system Prepare a reaction solution (20 ⁇ L) according to the SYBR Premix ExTaq II system and perform real-time fluorescent quantitative PCR.
  • Procedure for PCR amplification pre-denaturation at 95 °C / 30S, and then 40 cycles of 95 °C / 5S ⁇ 55 °C / 30S ⁇ 72 °C / 30S.
  • Preparation of standard curve 400 bp sequences of SPS gene and HYG gene were selected respectively.
  • the selected sequences should contain the fragments amplified by quantitative PCR and ligated together by homologous recombination, and then ligated into the pClone007 vector.
  • the constructed standard quality granules containing the HYG gene and the SPS gene were digested with restriction enzyme Psha I to linearize the DNA, and the concentration was measured with a nucleic acid protein detector. The concentration was diluted with ddH 2 O to 10 6 copies / ⁇ L, 10 5 copies / ⁇ L, 10 4 copies / ⁇ L, 10 3 copies / ⁇ L, 10 2 copies / ⁇ L.
  • Real-time quantitative PCR The qRT-PCR method was used to analyze the expression levels of transgenic rice-related genes to verify the efficiency of gene overexpression.
  • Rice UBQ5 gene was selected as the internal reference gene.
  • the qRT-PCR amplification program was set as follows: pre-denaturation at 95 ° C for 30s; denaturation at 95 ° C for 5s; annealing at 60 ° C for 30s; extension at 65 ° C for 5min, for a total of 40 cycles.
  • the data obtained from the experiment was exported by software and analyzed by Excel.
  • the relative expression of genes was calculated using ⁇ CT. All samples were set up with 3 independent biological replicates.
  • the rice reference gene Sps is a homozygous diploid, and the transgenic contemporary exogenous target gene is homozygous, the data obtained by dividing the number of HYG starting templates by the number of SPS starting templates is multiplied by 2. The number of copies of the gene of interest in the rice genome.
  • the results in Table 13 show that among the 54 transgenic lines, 36 had a copy number of 1, and 13 had a copy number of 2, and the copy number There were 4 strains with 3, 1 strain with 4 copy number, and 0 copies of the negative control detected.
  • HYG 0 and SPS 0 represent the number of starting templates for the Hyg and Sps genes in the PCR reaction, respectively.
  • Single base editing is a gene editing method that uses the CRISPR / Cas9 system to target deaminase to specific sites in the genome to modify specific bases.
  • This method has been successfully applied in rice.
  • the amino acid of the rice HPPD gene located at the 141st, 342th, and 370th positions of the second chromosome Os02g0168100 was edited. Histidine (Histidine; Codon) of the 141 amino acid residue of HPPD gene in rice Edited to arginine (Arginine; codon by single base editing method) Will be the original Becomes ). Similarly, the glycine (Glycine; codon) at amino acid residue 342 ) Edited as aspartic acid; Will be the original becomes ); The 370th amino acid residue aspartic acid (Aspartic acid; codon ) Edited asparagine (Asparagine; codon Will be the original becomes ).
  • the mutant protein xCas9 (3.7) -ABE with the broader PAM Cas9 protein was selected as the editing tool (Hu, JHet al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature http://dx.doi.org/ 10.1038 / nature26155 (2016)).
  • a target site for sgRNA was designed: GGTGC a CGCCGTGGCGCTGC-GCG, where a is the action site of ABE to achieve the editing of A bases to G bases.
  • the PAM of this sgRNA is GCG conforming to the requirements of xCas9 (3.7).
  • a sgRNA target site is designed: GCACG c CGTCGTAGTAGTTG GGC, where c is the action site of CBE to achieve C-base to T-base editing.
  • the PAM of this sgRNA is GGC and meets the requirements of xCas9 (3.7).
  • the DNA sequence near the 370th amino acid of rice HPPD gene was analyzed, and the target site of sgRNA was designed: CCTGGT c ATCCCTGTCCACG AGC, where c is the action site of CBE to realize the editing of C bases to T bases.
  • the PAM of this sgRNA is GGC and also meets the requirements of xCas9 (3.7).
  • aaacGCAGCGCCACGGCGtGCAC 342GE-F: ggcgCACGcCGTCGTAGTAGTTG and 342GE-R: aaacCAACTACTACGACGgCGTG; 370GE-F: ggcg CCTGGTcATCCCTGTCCACG and 370GE-R: aaacCGTGGACAGGGATgACCAGG. Dilute with ultrapure water to 10uM, mix in equal amounts and place in a boiling water bath. Allow it to cool to room temperature before using.
  • pQY000140 vector 1ug pQY000140 vector was cut with BsaI enzyme at 37 ° C for one hour, and after detection by agarose gel electrophoresis, the target fragment was recovered, and the concentration was determined by ultraviolet absorption method. It was mixed with the beacon fragment 1:10 and ligated with T4 DNA ligase (NEB, New England Biolabs, Boston, USA) at 16 ° C for 2 hours. Trans5a competent cells (full-form genes, Beijing) were transformed and cultured overnight at 37 ° C. Pick the single clone to correct the sequence of the single base editing vector by Sanger sequencing. The construction vector pQY000141 is shown in FIG. 13. The plasmids extracted from the correctly sequenced E. coli clones were transformed into Agrobacterium tumefaciens EH105 (Weidi Biological, Shanghai).
  • Huaidao 5 callus (at least 3000 callus) was transformed.
  • the infected callus was transferred to a 50 mg / L hyg screening medium for selection and culture.
  • yellow-white callus with good growth status was picked and differentiated on a differentiation medium, and 0.2uM sulfonone was added during the differentiation process for screening for 3 to 4 weeks to obtain about 1 cm.
  • the rice hppd gene has two exons and one intron.
  • the three target sites H141, G342, and D370 are all located in the first exon.
  • gRNA design design at least one gRNA upstream of H141 and downstream of D370, and cut each knife one by one, and then replace the three sites at the same time by the method of homologous substitution. Enter the exon 1 sequence into http://crispor.tefor.net/ to evaluate all possible gRNAs. According to the specific score value greater than 90 (Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Crack TJ, Marraffini LA, Bao G, Zhang F. Nat Biotechnol.
  • the template donor DNA design is shown in Figure 16: According to Zhao Yunde Laboratories (Sun Y, Zhang X, Wu C, He Y, Ma Y, Hou H, Guo X, Du W, Zhao Y, Xia L. Engineering Herbicide- Resistant Rice Through Plants CRISPR / Cas9-Mediated Homologous Recombination Acetolactate Synthase.Mol Plant.
  • the homology arm is designed at 350bp; in order to increase the possibility of homology substitution, two versions of the template donor are designed for each editing vector: the template is directly connected to the editing vector, so that gRNA, Cas9 and the template enter the same simultaneously Cells, once Cas9 and gRNA cut the genomic target DNA of the cell, the template donor DNA can be repaired in time; another version is the free template donor DNA generated by PCR amplification.
  • additional repair templates will be 20: 1 (free repair template: editing vector, molar ratio) is mixed with the editing vector, and then bombarded with a gene gun.
  • the length of the core substitution region of the three mutated amino acids 141-342-370 is determined by the two selected target RNA cleavage positions (1056bp), and the left and right homology arms are 350bp each. 6bp was left at each end, and the total length of the template was 1768bp.
  • the NcoI restriction site was removed; and to avoid re-cutting after replacement, the The PAM (NGG) at the original cleavage site was also removed.
  • Editing vectors The rice U3 promoter was used to express gRNA1-2 and gRNA2-1, respectively. Therefore, the two gRNA expression boxes were ligated with the template and sent to Kingsray Biotechnology (Nanjing) for synthesis. The synthesized DNA fragment was then ligated to the backbone vector pCXUN-Cas9 at KpnI using seamless cloning technology (from Huazhong Agricultural University and Dr. Yu Bing, Mol Plant. 2016 Apr 4; 9 (4): 628-31.doi: 10.1016 / j.molp. 2016.01.001. Epub 2016 Jan 6.).
  • Gene gun transformation, screening, differentiation, rooting and soil culture After editing and multi-enzyme digestion, the edited vector constructed above was combined with free template donor DNA generated by PCR amplification, and the ratio was 20: 1 (free repair).
  • primer pairs are 290-F: AGATACAGACGTACCTGGACCACCA and 1553-R: GCCGGCAAAAAGGAACTGGG (342-370 mutation site region), 90-F: AGATACAGACGTACCTGGACCACCA and donor-out-R: AGTGATTGTACCATCATTTGTC (342-370 region + part of the downstream genomic DNA sequence) , And 54-F: TTCCACCACGTCGAGCTC and 356-R: GGTGAACCCGGAGATGTACG (141 single points).
  • the introduction of the gene of the present invention into model plants such as Arabidopsis thaliana and Brachypodium spp. Produced a corresponding increase in drug resistance.
  • the CRISPR / Cpf1 system is also used to edit the above mutation sites and combinations. It can be known that the transgene or gene is edited into other aforementioned plants, such as food crops, legumes, oil crops, fiber crops, fruit crops, rhizome crops, vegetable crops, flower crops, medicinal crops, raw materials Crops, forage crops, sugar crops, beverage crops, lawn plants, tree crops, nut crops, etc. will also produce corresponding resistance traits, which has good industrial value.

Abstract

本发明公开了突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白、其生物活性片段以及包含编码所述蛋白或片段的核酸序列的分离的多核苷酸，其中所述突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段保留或加强了其催化对羟基苯基丙酮酸(HPP)转化为尿黑酸的性质，且对HPPD抑制剂的敏感性低于野生型HPPD。本发明还公开了包含所述多核苷酸的核酸构建体、表达载体和宿主细胞，以及生产具有催化对羟基苯基丙酮酸(HPP)转化为尿黑酸的性质、同时对HPPD抑制性除草剂的敏感性降低的植物的方法。

Description

突变型对羟苯基丙酮酸双氧化酶、其编码核酸以及应用 技术领域
本发明属于农业基因工程领域，具体涉及向植物赋予HPPD抑制性除草剂抗性或耐受性的新型突变型对羟苯基丙酮酸双氧化酶(HPPD)、其编码核酸以及应用。
背景技术
对羟苯基丙酮酸双氧化酶(HPPD)是催化羟基苯丙酮酸(HPP)转化成尿黑酸盐的反应的酶。这个反应在酶结合的铁和氧的存在下发生。通过抑制HPPD而起作用的除草剂是熟知的，包括多种类型，比如异噁唑类、二酮腈类、三酮类以及吡唑啉盐类等。对HPPD的抑制作用阻断了从酪氨酸生物合成质体醌(PQ)。PQ在类胡萝卜素色素的生物合成中是一种必需的辅因子，这些类胡萝卜素色素对于光合中心的光防护是必需的。抑制HPPD的除草剂是通过韧皮部可移动的漂白剂，它们引起暴露于光的新的分生组织和叶子显现出白色。在缺乏类胡萝卜素下，叶绿素是光破坏性的并且通过单线态氧的光敏作用而自身变成一种光化裂解剂。
用于提供耐受HPPD抑制性除草剂的植物的技术路线与方法同样是已知的，包括将HPPD酶过量表达从而在植物中产生大量的HPPD酶，这些HPPD酶与一种给定的除草剂是充分相关的从而具有足够的可供使用的功能性酶(尽管存在它的抑制剂)，或者是将目标HPPD酶突变成一种功能性的、但是对于除草剂更加不敏感的HPPD。HPPD抑制性除草剂是一个大类，它涵盖了许多不同类型。虽然一种给定的突变体HPPD酶可以提供对于一种或一些HPPD抑制性除草剂的一个有用水平的耐受性，但同一或单一突变体HPPD可能不足以提供对于另一种或另一类不同的、更希望的HPPD抑制性除草剂的商业水平的耐受性(参见例如美国申请公开号2004/0058427；以及PCT申请公开号WO98/20144和WO02/46387；还参见美国申请公开号2005/0246800，它涉及作为相对耐受HPPD的大豆品种的鉴定和标记)。而且，不同HPPD抑制性除草剂可以在它们控制的杂草范围、使用的作物对象、制造成本以及环境益处方面是不同的。因此，本领域中仍然需要用于向不同的作物和作物品种赋予对HPPD抑制性除草剂的抗性/耐受性的新型突变HPPD。
在创制除草剂耐受性作物和作物品种方面，转基因技术已广泛应用。但是，转基因作物的应用一直受到登记成本高的限制。以CRISPR/Cas 9为代表的基因编辑技术进步可以改变这一现状。CRISPR/Cas 9是2012年以来出现的新基因定点编辑技术(Jinek,M.,Chylinski,K.,Fonfara,I.,Hauer,M.,Doudna,J.A.,and Charpentier,E.2012.A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity.Science.337:816–821.； Cong,L.,Ran,F.A.,Cox,D.,Lin,S.,Barretto,R.,Habib,N.,Hsu,P.D.,Wu,X.,Jiang,W.,Marraffini,L.A.,and Zhang,F.2013.Multiplex genome engineering using CRISPR/Cas systems.Science.339:819–823；Li,J.F.,Norville,J.E.,Aach,J.,Mccormack,M.,Zhang,D.,Bush,J.,Church,G.M.,and Sheen,J.2013.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.Nat.Biotechnol.31:688.；Mali,P.,Yang,L.,Esvelt,K.M.,Aach,J.,Guell,M.,Dicarlo,J.E.,Norville,J.E.,and Church,G.M.2013.RNA-guided human genome engineering via Cas9.Science.339:823.)。CRISPR/Cas 9系统对编辑靶点的识别是依靠核酸分子之间的碱基互补配对，可对任何紧跟PAM(NGG)的20bp的靶点序列进行编辑。另外，CRISPR/Cas 9系统操作简单，每次打靶只需替换原有载体上的20-30bp的靶向核苷酸序列，适宜高通量操作。可以同时编辑同一基因的多个位点以及多个不同的基因。目前，这项技术在生物医学、功能基因组学、动植物性状改良和新性状的创制等方面已显现出巨大的应用前景，正在对动植物育种产生革命性的促进作用(Hui Zhang，Jinshan Zhang,Zhaobo Lang,Jose Ramon Botellad,and Jian-Kang Zhu.2017.Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement,Critical Reviews in Plant Sciences,36:4,291-309,DOI:10.1080/07352689.2017.1402989).
CRISPR/Cas 9作为第三代基因编辑工具，主要是通过三种方式来实现定点编辑的。第一种是基因的定点敲除获得突变体。具体讲，Cas 9在靶向RNA(gRNA)的指导下识别和切割靶点，产生双链DNA断裂；断裂的DNA通常以非同源末端连接(NHEJ)来修复；在修复时容易产生移码突变以破坏这个基因。定点敲除的效率都较高。第二种是对靶标进行同源置换来更换靶标序列或者定点插入。在产生双链DNA断裂时，如果在附近存在同源修复模板，这时可能发生同源置换或定点插入。同源置换的效率较低，并随着要置换的序列的长度增长而变得更低。第三种是单碱基编辑(Komor AC,Kim YB,Packer MS,Zuris JA,Liu DR.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature.2016 May 19；533(7603):420-4.doi:10.1038/nature17946；Gaudelli NM,Komor AC,Rees HA,Packer MS,Badran AH,Bryson DI,Liu DR.Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage.Nature.2017 Nov 23；551(7681):464-471.doi:10.1038/nature 24644.Epub 2017 Oct 25.Erratum in:Nature.2018 May 2)。单碱基编辑是利用CRISPR/Cas 9系统将脱氨酶靶向基因组中特定的位点从而对特定碱基进行修饰的基因编辑方法。此种方法已经在水稻中成功运用。如：Yan F.,Kuang Y.,Ren B.,Wang J.,Zhang D.,Lin H.,Yang B.,Zhou X.,and Zhou H.(2018).High-efficient A·T to G·C base  editing by Cas9n-guided tRNA adenosine deaminase in rice.Mol.Plant.doi:10.1016/j.molp.2018.02.008。
另外，CRISPR/Cpf1也同样可以用来做基因编辑(Zetsche,B.,Gootenberg,J.S.,Abudayyeh,O.O.,Slaymaker,I.M.,Makarova,K.S.,Essletzbichler,P.,Volz,S.E.,Joung,J.,Oost,J.,Regev,A.,Koonin,E.V.,and Zhang,F.2015.Cpf1is a single RNA-guided endonuclease of a Class 2 CRISPR Cas system.Cell.163:759–771；Endo,A.,Masafumi,M.,Kaya,H.,and Toki,S.2016a.Efficient targeted mutagenesis of rice and tobacco genomes usingCpf1 from Francisella novicida.Sci.Rep.6:38169.)。CRISPR/Cpf1有两个主要组成部分：Cpf1酶和决定系统特异性的crRNA。尽管CRISPR/Cpf1和CRISPR/Cas9系统相似，但还是有一些重要的差异(Hui Zhang,Jinshan Zhang,Zhaobo Lang,José Ramón Botella&Jian-Kang Zhu(2017)Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement,Critical Reviews in Plant Sciences,36:4,291-309,DOI:10.1080/07352689.2017.1402989)。首先，CRISPR/Cpf1系统不需要反式激活crRNA(tracrRNA)，但CRISP/Cas9却必须要。因此，它比较短小，只有42-44核苷酸，包括一个19核苷酸重复序列和1个23-25核苷酸长的间隔区。第三，不像Cas9在相同位置(PAM上游3-4bp)剪切DNA双链产生平头末端，Cpf1的目标序列剪切位置在PAM序列下游23bp处，非目标单链为PAM序列下游18bp处，产生一个5bp突出的粘性末端。产生的粘性末端可以增加HDR介导的供体DNA插入到Cpf1剪切位点的效率。第四，CRISPR/Cpf1系统编辑多个目标或基因时只需要一个启动子来驱动多个小crRNAs阵列，很适合多靶点编辑。第五，CRISPR/Cas9系统在目标序列的3’端需要一个富含G(5’-NGG-3’)的PAM序列，CRISPR/Cpf1系统需要一个位于目标序列5’端富含T(5’-TTTN-3’or 5’-TTN-3’)的PAM序列，适合于编辑多A/T的DNA或基因。目前已经开发出三种改造过的CRISPR/Cpf1系统，包括来自Francisella novicida(弗朗西斯氏菌)的FnCpf1，来自Acidaminococcus sp.(氨基酸球菌)的AsCpf1和来自Lachnospiraceae bacterium(毛螺菌)的LbCpf1。三种Cpf1系统已经在以下物种上用作植物基因组编辑：水稻、拟南芥、烟草和大豆(Endo,A.,Masafumi,M.,Kaya,H.,and Toki,S.2016a.Efficient targeted mutagenesis of rice and tobacco genomes usingCpf1 from Francisella novicida.Sci.Rep.6:38169；Kim,H.,Kim,S.T.,Ryu,J.,Kang,B.C.,Kim,J.S.,and Kim,S.G.2017.CRISPR/Cpf1-mediated DNA-free plant genome editing.Nat.Commun.8:14406.；Tang,X.,Lowder,L.G.,Zhang,T.,Malzahn,A.A.,Zheng,X.,Voytas,D.F.,Zhong,Z.,Chen,Y.,Ren,Q.,and Li,Q.2017.A CRISPR-Cpf1system for efficient genome editing and transcriptional repression in plants.Nat.Plants.3:17018.；Wang,M.,Mao,Y.,Lu,Y.,Tao,X.,and Zhu,J.K.2017a.Multiplex gene editing in rice using the CRISPR-Cpf1 system.Mol.Plant.10:1011–1013)。
通过基因编辑介导的同源置换、定点修饰或单碱基编辑提高重要农作物对除草剂的耐受性是当前基因编辑研究领域的热点之一，现已报道几个成功实例，但都集中在抗乙酰乳酸合成酶(ALS)抑制性除草剂(Yongwei Sun，Xin Zhang，Chuanyin Wu，ubing He，Youzhi Ma,Han Hou，Xiuping Guo,Wenming Du，Yunde Zhao and Lanqin Xia.2016.Engineering Herbicide-Resistant Rice Plants through CRISPR/Cas9-Mediated Homologous Recombination of Acetolactate Synthase.Molecular Plant 9,628-631 doi.org/10.1016/j.molp.2016.01.001；Yiyu Chen,Zhiping Wang,Hanwen Ni,Yong Xu,Qijun Chen,Linjian Jiang.2017.CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis.Sci China Life Sci 60.doi:10.1007/s11427-017-9021-5)和草甘膦除草剂(WO2017028768A1)。这就需要科学家们不断研究进而开发出新的提高农作物对不同类型除草剂耐受性的方法。
发明内容
基于此，本申请提供了一种向植物赋予HPPD抑制性除草剂抗性或耐受性的突变型对羟苯基丙酮酸双氧化酶(HPPD)，所述突变型HPPD保留或加强了其催化对羟基苯基丙酮酸(HPP)转化为尿黑酸的性质，同时对HPPD抑制性除草剂的敏感性明显低于野生型HPPD。本发明还涉及突变型对羟苯基丙酮酸双氧化酶的生物学活性片段，编码所述蛋白或片段的多核苷酸及其应用。
因此，本发明一方面提供了一种突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白，其在对应于SEQ ID NO:2所示的野生型水稻对羟苯基丙酮酸双氧化酶蛋白氨基酸序列中的第93、103、141、165、191、220、226、276、277、336、337、338、342、346、370、377、386、390、392、403、410、418、419、420、430和431位中的一个或多个位置处具有选自下述中的一个或多个突变：93S、103S、141R、141K、141T、165V、191I、220K、226H、276W、277N、336D、337A、338D、338S、338Y、342D、346C、346D、346H、346S、346Y、370N、377C、386T、390I、392L、403G、410I、418P、419F、419L、419V、420S、420T、430G和431L。优选地，所述突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白的氨基酸序列进一步与SEQ ID NO:2所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性。更优选地，所述突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白具有SEQ ID NO:2所示的氨基酸序列，区别仅在于在对应于SEQ ID NO:2所示的野生型水稻对羟苯基丙酮酸双氧化酶蛋白氨基酸序列中的第93、103、141、165、191、220、226、276、277、336、337、338、342、346、370、377、386、390、392、403、410、418、419、420、430和431位中的一个或多个位置处具有选自下述中的一个或多个突变：93S、103S、141R、141K、141T、 165V、191I、220K、226H、276W、277N、336D、337A、338D、338S、338Y、342D、346C、346D、346H、346S、346Y、370N、377C、386T、390I、392L、403G、410I、418P、419F、419L、419V、420S、420T、430G和431L。
本发明另一方面提供了突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白的生物学活性片段，其在所述蛋白的N和/或C末端缺失了一个或多个(例如1－50个、1－25个、1－10个或1－5个，例如1、2、3、4或5个)氨基酸残基的部分，但仍然保留了该全长蛋白的所需生物学活性，即保留或加强了其催化对羟基苯基丙酮酸(HPP)转化为尿黑酸的性质，同时对HPPD抑制性除草剂的敏感性明显低于野生型HPPD或其相应的生物学活性片段。
本发明另外涉及一种融合蛋白，其中包含有本发明的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物学活性片段以及与之相融合的其它组分，例如肽或多肽组分。优选地，所述组分赋予该融合蛋白以希望的性质，例如便于其分离、纯化，提高其稳定性，延长其半寿期，提供另外的生物学活性，引导所融合的HPPD蛋白进入目标区域例如质体如叶绿体等。相应组分的选择是本领域技术人员熟知的。
本发明另一方面提供了包含编码所述突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段或者融合蛋白的核酸序列的分离的多核苷酸。
本发明还提供了包含所述多核苷酸以及与之可操作连接的调控元件的核酸构建体。
在进一步的方面，本发明提供了一种表达载体，其中包含所述多核苷酸以及与之可操作连接的表达调控元件。
在又一个方面，本发明提供了包含有所述多核苷酸、核酸构建体或表达载体的宿主细胞。
本发明还提供了一种生产对于HPPD抑制性除草剂的抗性或耐受性提高的植物的方法。
本发明另外涉及通过上述方法所生产的植物。
本发明还提供了一种提高植物对HPPD抑制性除草剂的抗性或耐受性的方法，其中包括在所述植物中表达本发明的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段或者融合蛋白。
本发明另外提供了一种提高植物对HPPD抑制性除草剂的抗性或耐受性的方法，其中包括将表达本发明的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段或者融合蛋白的植物与另一植物杂交。
本发明另外提供了一种提高植物对HPPD抑制性除草剂的抗性或耐受性的方法，其 中包括对所述植物细胞、植物组织、植物部分或植物的内源性HPPD蛋白进行基因编辑。
本发明另外涉及本发明的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段或者融合蛋白用于提高植物的HPPD抑制性除草剂抗性或耐受性的用途。
本发明另外涉及一种控制植物场所的杂草的方法，其中包括对包含本发明的植物或种子的场所施用控制杂草有效量的一种或多种HPPD抑制性除草剂，并且不显著影响所述植物。
附图说明
图1显示了在96-孔板中培养的转化了野生型或突变型水稻HPPD基因的重组大肠杆菌培养液的颜色反应。其中重组大肠杆菌表达野生型水稻HPPD(WT)或者单位点突变型水稻HPPD之一，将它们在培养液中含有不同浓度的除草剂环磺酮(左)或苯唑氟草酮代谢后产物(右，其结构式为：
Figure PCTCN2019089512-appb-000001
)的条件下培养，显示有不同程度的颜色变化。在有相同浓度的除草剂孔中，颜色越深代表对此除草剂的抗性/耐受性越高。
图2显示了在96-孔板中培养的转化了野生型或突变型水稻HPPD基因的重组大肠杆菌培养液的颜色反应。其中重组大肠杆菌表达野生型水稻HPPD(WT)和各个单位点突变型水稻HPPD，将它们在培养液中含有不同浓度的除草剂环吡氟草酮(左)或苯唑草酮(右)的条件下培养，显示有不同程度的颜色变化。在有相同浓度的除草剂孔中，颜色越深代表对此除草剂的抗性/耐受性越高。
图3显示了在96-孔板中培养的转化了野生型或突变型水稻HPPD基因的重组大肠杆菌培养液的颜色反应。其中重组大肠杆菌表达野生型水稻HPPD(WT)和各个单位点突变型水稻HPPD，将它们在培养液中含有不同浓度的除草剂硝磺草酮的条件下培养，显示有不同程度的颜色反应。在有相同浓度的除草剂孔中，颜色越深代表对此除草剂的抗性/耐受性越高。
图4显示了在96-孔板中培养的转化了野生型或突变型水稻HPPD基因的重组大肠杆菌培养液的颜色反应。其中重组大肠杆菌表达野生型水稻HPPD(WT)或者单位点突变型H141R、G342D、D370N之一或其组合，将它们在含有不同浓度的除草剂环磺酮(上)或苯唑氟草酮代谢后产物(下)的条件下培养，显示有不同程度的颜色变化。在有相同浓度的除草剂孔中，颜色越深代表对此除草剂的抗性/耐受性越高。
图5显示了在96-孔板中培养的转化了野生型或突变型水稻HPPD基因的重组大肠杆 菌培养液的颜色反应。其中重组大肠杆菌表达野生型水稻HPPD(WT)或者单位点突变型H141R、G342D、D370N或其组合(141+342表示H141R/G342D；141+370表示H141R/D370N；342+370表示G342D/D370N；141+342+370表示H141R/G342D/D370N)，将它们在含有不同浓度除草剂环吡氟草酮(上)或苯唑草酮(下)的条件下培养，显示有不同程度的颜色变化。在有相同浓度的除草剂孔中，颜色越深代表对此除草剂的抗性/耐受性越高。
图6显示了在96-孔板中培养的转化了野生型或突变型水稻HPPD基因的重组大肠杆菌培养液的颜色反应。其中重组大肠杆菌培养液表达野生型水稻HPPD(WT)或者单位点突变型H141R、G342D、D370N或其组合，将它们在含有不同浓度除草剂硝磺草酮的条件下培养，显示不同程度的颜色变化。在有相同浓度的除草剂孔中，颜色越深代表对此除草剂的抗性/耐受性越高。
图7显示了水稻HPPD野生型酶蛋白上标注的所有的氨基酸突变。
图8显示了在96-孔板中培养的转化了突变型水稻HPPD基因的重组大肠杆菌培养液的颜色反应。其中重组大肠杆菌培养液表达相近突变点336-338-342-346各种组合与141R+342D+370N组合(336D、338D、338S、338Y、342D、346C、346H、346S分别表示P336D、N338D、N338S、N338Y、G342D、R346C、R346H、R346S；141R+342D+370N表示H141R/G342D/D370N)，将它们在含有不同浓度除草剂双唑草酮代谢产物(代号为101，其结构式为：
Figure PCTCN2019089512-appb-000002
)的条件下培养，显示不同程度的颜色变化。在有相同浓度的除草剂孔中，颜色越深代表对此除草剂的抗性/耐受性越高。
图9显示了在96-孔板中培养的转化了突变型水稻HPPD基因的重组大肠杆菌培养液的颜色反应。其中重组大肠杆菌培养液表达三、四个突变点各组合(141R、336D、338D、338S、338Y、342D、346C、346S、346H、370N、418P、419F分别表示H141R、P336D、N338D、N338S、N338Y、G342D、R346C、R346S、R346H、D370N、K418P、G419F)，将它们在含有不同浓度除草剂双唑草酮代谢产物的条件下培养，显示不同程度的颜色变化。在有相同浓度的除草剂孔中，颜色越深代表对此除草剂的抗性/耐受性越高。
图10显示了双唑草酮代谢产物对OsHPPD WT以及各突变体的抑制曲线，横坐标表示化合物101浓度，纵坐标以抑制剂浓度为0时反应速率为100％，表示在不同浓度101时酶的活性残余活性，图中数字代表各突变位点。从图中可以很直观的看出，野生型WT对101极为敏感，在101浓度约为60uM时活性被完全抑制，而各突变体则表现出了较强的抗性提高。 根据此结果，可计算出101对各突变体活性抑制IC50值，同样证明了各突变体对野生型OsHPPD表现出明显提高的抗性(其中，141R、338D、342D、346C、346H、370N、386T、418P、419F、420S分别表示H141R、N338D、G342D、R346C、R346H、D370N、P386T、K418P、G419F、N420S)。
图11显示了转基因水稻(中花11)对HPPD抑制剂除草剂环磺酮的敏感性。表达突变型水稻OsHPPD3M(H141R/G342D/D370N)能在含3uM环磺酮培养基中保持绿色，但负对照(CK)表达mCherry的苗子在1.0uM的环磺酮的培养基也严重白化(药害)。
图12显示了转基因水稻(中花11)对HPPD抑制剂除草剂双唑草酮的耐受性。T0代植株表达水稻OsHPPD3M的植株能耐受8-16克有效成分双唑草酮/亩，但非转基因对照(CK)严重白化后很快死亡(A，B)；T1代植株表达水稻OsHPPD3M的植株能耐受32-64克有效成分双唑草酮/亩，但非转基因对照严重白化后很快死亡(C，D)。
图13显示了水稻HPPD单碱基编辑载体。
图14显示了单碱基编辑水稻苗及其靶点H141R(CAC>CGC)序列分析。
A：单碱基编辑苗：在0.4uM环磺酮存在的培养基里，编辑不成功者白化(药害)，编辑成功者保持绿色；
B：单碱基编辑靶点的序列：水稻HPPD第141位氨基酸野生型为组氨酸His，密码子为CAC(上图)，编辑后为精氨酸Arg，密码子为CGC(本例为杂合体，出现双峰)。
图15显示了水稻hppd基因(Oshppd>NC029257.1)的结构，展示两个外显子(exon)，一个内含子(intron)，三个突变位点(141，342，370)和设计的靶向切割位置(gRNA1-2,gRNA2-1)。
图16显示了模板DNA的结构。三个突变氨基酸141-342-370的核心置换区域的长度1056bp，左右同源臂各350bp，再加上从载体上切割后在左右两端各留下6bp，模板总长1768bp；为便于PCR扩增后对其PCR产物进行快速基因型鉴定，去掉了其中的NcoI酶切位点；同时为避免置换后又被重新切割，模板上的原切割位点处的PAM(NGG)也被去掉。
图17显示了水稻HPPD基因同源置换三突变点(H141R-G342D-D370N)。
A：水稻HPPD基因编辑苗：在0.4uM环磺酮存在的培养基里，未成功编辑的(野生型WT)白化(药害)，编辑成功的2株苗(AW2，AW3)保持绿色；
B：同源置换后，氨基酸342和370位点的密码子变化，GGC变为GAC和GAC变为AAC(杂合体；导致部分G342D和D370N)；H141R(CAC>CGC)也成功编辑(图中未列序列)。
发明详述
本说明书中使用的一些术语定义如下。
在本发明中，“HPPD抑制性除草剂”是本身有除草活性的物质或者与能改变其效果的其他除草剂和/或添加剂合用的物质，其能够通过抑制HPPD而起作用。本身能够通过抑制HPPD而起除草作用的物质在本领域中是熟知的，包括许多类型，1)三酮类，例如，磺草酮(Sulcotrione，CAS号：99105-77-8)；硝磺草酮(Mesotrione，CAS号：104206-82-8)；氟吡草酮(bicyclopyrone，CAS号：352010-68-5)；环磺酮(tembotrione，CAS号：335104-84-2)；呋喃磺草酮(tefuryltrione，CAS号：473278-76-1)；双环磺草酮(Benzobicyclon，CAS号：156963-66-5)；2)二酮腈类，例如，2-氰基-3-环丙基-1-(2-甲基磺酰基-4-三氟甲基苯基)丙-1,3-二酮(CAS号：143701-75-1)；2-氰基-3-环丙基-1-(2-甲基磺酰基-3,4-二氯苯基)丙-1,3-二酮(CAS号：212829-55-5)；2-氰基-1-[4-(甲基磺酰基)-2-三氟甲基苯基]-3-(1-甲基环丙基)丙-1,3-二酮(CAS号：143659-52-3)；3)异噁唑类，例如，异噁氟草(isoxaflutole，CAS号：141112-29-0)；异噁氯草酮(isoxachlortole，CAS号：141112-06-3)异恶草酮(clomazone，CAS号：81777-89-1)；4)吡唑类，例如，苯唑草酮(topramezone，CAS号：210631-68-8)；磺酰草吡唑(pyrasulfotole，CAS号：365400-11-9)；苄草唑(pyrazoxyfen，CAS号：71561-11-0)；吡唑特(pyrazolate，CAS号：58011-68-0)；吡草酮(benzofenap，CAS号：82692-44-2)；双唑草酮(CAS号：1622908-18-2)；Tolpyralate(CAS号：1101132-67-5)；苯唑氟草酮(CAS号：1992017-55-6)；环吡氟草酮(CAS号：1855929-45-1)；三唑磺草酮(CAS号：1911613-97-2)；5)二苯酮类；6)其他类：lancotrione(CAS号：1486617-21-3)；fenquinotrione(CAS号：1342891-70-6)。优选地，所述除草剂是环磺酮、苯唑氟草酮、环吡氟草酮、苯唑草酮、硝磺草酮、双唑草酮或其任意组合等。
“对HPPD抑制性除草剂的耐受性增强”或“对HPPD抑制性除草剂的抗性增强”的植物是指这样的植物，其对所述HPPD抑制性除草剂的耐受性或抗性与含有野生型HPPD基因的植物相比提高。“对HPPD抑制性除草剂的耐受性增强”或“对HPPD抑制性除草剂的抗性增强”的HPPD酶是指这样的HPPD酶，其在已知会抑制相应野生型HPPD酶蛋白活性的除草剂浓度下，表现出比野生型HPPD酶高至少10％、优选高至少15％、更优选高至少20％的酶活性。在本发明中，“HPPD抑制性除草剂耐受性”和“HPPD抑制性除草剂抗性”两个术语可以互换使用，均指的是对HPPD抑制性除草剂的耐受性和对HPPD抑制性除草剂的抗性。
术语“野生型”指的是可以在自然界中被发现存在的核酸分子或蛋白质。
术语“蛋白”、“多肽”和“肽”在本发明中可以互换使用，指的是氨基酸残基聚合物，包括其中一个或多个氨基酸残基是天然氨基酸残基的化学类似物的聚合物。本发明的蛋白 和多肽可以重组产生，也可以通过化学合成。术语“突变蛋白”或“突变型蛋白”指的是这样的蛋白质，其与野生型蛋白质的氨基酸序列相比，具有一个或多个氨基酸残基的取代、插入、缺失和/或添加。
术语“多核苷酸”和“核酸”可以互换使用，包括DNA、RNA或者其杂交体，可以是双链或单链的。
在本发明中，“宿主生物”应理解为可以引入突变型HPPD蛋白编码核酸的任何单细胞或多细胞生物，包括例如细菌如大肠杆菌，真菌如酵母(例如酿酒酵母)、霉菌(例如曲霉菌)，植物细胞和植物等。
在本发明中，“植物”应理解为能够进行光合作用的任何分化的多细胞生物，特别是单子叶或双子叶植物，例如：(1)粮食作物：稻属(Oryza spp.)，例如稻(Oryza sativa)、阔叶稻(Oryza latifolia)、水稻(Oryza sativa)、光稃稻(Oryza glaberrima)；小麦属(Triticum spp.)，例如普通小麦(Triticum aestivum)、硬粒小麦(T.Turgidumssp.durum)；大麦属(Hordeum spp.)，例如大麦(Hordeum vulgare)、亚利桑那大麦(Hordeum arizonicum)；黑麦(Secale cereale)；燕麦属(Avena spp.)，例如燕麦(Avena sativa)、野燕麦(Avena fatua)、比赞燕麦(Avena byzantina)、Avena fatua var.sativa、杂种燕麦(Avena hybrida)；稗属(Echinochloa spp.)，例如，珍珠粟(Pennisetum glaucum)、高粱(两色高粱(Sorghum bicolor)、高粱(Sorghum vulgare))、黑小麦、玉蜀黍或玉米、粟、稻(rice)、谷子、糜子、两色蜀黍(Sorghum bicolor)、黍子、荞麦属(Fagopyrum spp.)、黍(Panicum miliaceum)、小米(Setaria italica)、沼生菰(Zizania palustris)、埃塞俄比亚画眉草(Eragrostis tef)、稷(Panicum miliaceum)、龙爪稷(Eleusine coracana)；(2)豆类作物：大豆属(Glycine spp.)，例如大豆(Glycine max)、黄豆(Soja hispida)、Soja max)、野豌豆属(Vicia spp.)、豇豆属(Vigna spp.)、豌豆属(Pisum spp.)、芸豆(field bean)、羽扇豆属(Lupinus spp.)、蚕豆属(Vicia)、酸豆(Tamarindus indica)、兵豆(Lens culinaris)、山黧豆属(Lathyrus spp.)、扁豆属(Lablab)、蚕豆、绿豆、红豆、鹰嘴豆；(3)油料作物：花生(Arachis hypogaea)、落花生属(Arachis spp)、胡麻属(Sesamum spp.)、向日葵属(Helianthus spp.)(例如向日葵(Helianthus annuus))、油棕属(Elaeis)(例如油棕(Eiaeis guineensis)、美洲油棕(Elaeis oleifera))、大豆(soybean)、油菜(Brassicanapus)、芸苔、芝麻、芥菜(Brassicajuncea)、油菜籽油菜(oilseedrape)、油茶、油棕、油橄榄、蓖麻、欧洲油菜(Brassica napus L.)、卡诺拉油菜(canola)；(4)纤维作物：剑麻(Agave sisalana)、棉属(棉花、海岛棉(Gossypium barbadense)、陆地棉(Gossypium hirsutum))、红麻、剑麻、蕉麻、亚麻(Linum usitatissimum)、黄麻、苎麻、大麻(Cannabis sativa)、火麻；(5)水果类作物：枣属(Ziziphus spp.)、香瓜属(Cucumis spp.)、 鸡蛋果(Passiflora edulis)、葡萄属(Vitis spp.)、越桔属(Vaccinium spp.)、西洋梨(Pyrus communis)、李属(Prunus spp.)、番石榴属(Psidium spp.)、石榴(Punica granatum)、苹果属(Malus spp.)、西瓜(Citrullus lanatus)、柑桔属(Citrus spp.)、无花果(Ficus carica)、金桔属(Fortunella spp.)、草莓属(Fragaria spp.)、山楂属(Crataegus spp.)、柿树属(Diospyros spp.)、红仔果(Eugenia unifora)、枇杷(Eriobotrya japonica)、龙眼(Dimocarpus longan)、番木瓜(Carica papaya)、椰子属(Cocos spp.)、阳桃(Averrhoa carambola)、狲猴桃属(Actinidia spp.)、扁桃(Prunus amygdalus)、芭蕉属(Musa spp.)(香蕉)、鳄梨属(Persea spp.)(鳄梨(Persea americana))、番石榴(Psidium guajava)、曼密苹果(Mammea americana)、芒果(Mangifera indica)、橄榄(油橄榄(Oleaeuropaea))、番木瓜(Caricapapaya)、椰子(Cocos nucifera)、凹缘金虎尾(Malpighia emarginata)、人心果(Manilkara zapota)、菠萝(Ananas comosus)、番荔枝属(Annona spp.)、柑桔树(柑桔属物种(Citrus spp.))、波罗蜜属(Artocarpus spp.)、荔枝(Litchi chinensis)、茶藨子属(Ribes spp.)、悬钩子属(Rubus spp.)、梨、桃、杏、梅、杨梅、柠檬、金橘、榴莲、橙、草莓(straw berry)、蓝莓、哈密瓜、甜瓜、椰枣、胡桃树、樱桃树；(6)根茎类作物：木薯属(Manihot spp.)、甘薯(Ipomoea batatas)、芋(Colocasia esculenta)、榨菜、洋葱、荸荠、油莎草、山药；(7)蔬菜类作物：菠菜属(Spinacia spp.)、菜豆属(Phaseolus spp.)、莴苣(Lactuca sativa)、苦瓜属(Momordica spp)、欧芹(Petroselinum crispum)、辣椒属(Capsicum spp.)、茄属(Solanum spp.)(例如马铃薯(Solanum tuberosum)、红茄(Solanum integrifolium)或蕃茄(Solanum lycopersicum))、蕃茄属(Lycopersicon spp.)(例如西红柿(Lycopersicon esculentum)、蕃茄(Lycopersicon lycopersicum)、梨形蕃茄(Lycopersicon pyriforme))、硬皮豆属(Macrotyloma spp.)、无头甘蓝(kale)、棱角丝瓜(Luffa acutangula)、小扁豆(lentil)、秋葵(okra)、洋葱(onion)、马铃薯(potato)、洋蓟(artichoke)、芦笋(asparagus)、西兰花(broccoli)、球芽甘蓝(Brussels sprouts)、卷心菜(cabbage)、胡萝卜(carrot)、花椰菜(cauliflower)、芹菜(celery)、羽衣甘蓝(collard greens)、西葫芦(squash)、冬瓜(Benincasa hispida)、石刁柏(Asparagus officinalis)、旱芹(Apium graveolens)、苋属(Amaranthus spp.)、葱属(Allium spp.)、秋葵属(Abelmoschus spp.)、苦苣(Cichorium endivia)、南瓜属(Cucurbita spp.)、芫荽(Coriandrum sativum)、埃塞俄比亚芥(B.carinata)、萝卜(Rapbanus sativus)、芸苔属(Brassica)物种(例如例如欧洲油菜(Brassica napus)、芜菁亚种(Brassica rapa ssp.)、卡诺拉油菜(canola)、油籽油菜(oilseed rape)、芜菁油菜(turnip rape)、芥菜、甘蓝、黑芥、油菜籽油菜)、孢子甘蓝、茄科植物(茄子)、甜椒、黄瓜、丝瓜、白菜、油菜、甘蓝、葫芦、韭菜、莲、藕、生菜；(8)花卉作物：小金莲花(Tropaeolum minus)、金莲花(Tropaeolum majus)、美人蕉(Canna indica)、 仙人掌属(Opuntia spp.)、万寿菊属(Tagetes spp.)、兰花、文殊兰、君子兰、朱顶红、玫瑰、月季、茉莉花、郁金香、樱花、牵牛花、金盏花、荷花、雏菊、康乃馨、矮牵牛花、郁金香、百合、梅花、水仙、迎春、报春、瑞香、山茶、白玉兰、紫玉兰、琼花、君子兰、海棠、牡丹、芍药、丁香、杜鹃、西洋杜鹃、含笑、紫荆、棣棠、锦带花、连翘、云南黄馨、金雀花、仙客来、蝴蝶兰、石斛、风信子、鸢尾、马蹄莲、金盏菊、百枝莲、四季海棠、吊钟海棠、竹节海棠、天竺葵；(9)药用作物：红花(Carthamus tinctorius)、薄荷属(Mentha spp.)、波叶大黄(Rheum rhabarbarum)、番红花(Crocus sativus)、枸杞、玉竹、黄精、知母、麦冬、川贝、郁金、砂仁、何首乌、大黄、甘草、黄芪、人参、三七、五加、当归、川芎、北柴胡、曼佗罗、洋金花、薄荷、益母草、藿香、黄芩、夏枯草、除虫菊、银杏、金鸡纳树、天然橡胶树、苜蓿、胡椒；(10)原料作物：橡胶、蓖麻(Ricinus communis)、油桐、桑、忽布、桦、桤木、漆树；(11)牧草作物：冰草属(Agropyron spp.)、车轴草属(Trifolium spp.)、芒(Miscanthus sinensis)、狼尾草属(Pennisetum sp.)、虉草(Phalaris arundinacea)、柳枝稷(Panicum virgatum)、草原草(prairiegrasses)、印度草(Indiangrass)、大须芒草(Big bluestem grass)、梯牧草(Phleum pratense)、草皮草(turf)、莎草科(高山嵩草、脚苔草(Carex pediformis)、低苔草)、苜蓿、梯牧草、紫花苜蓿、草木犀、紫云英、柽麻、田菁、红萍、水葫芦、紫穗槐、羽扇豆、三叶草、沙打旺、水浮莲、水花生、黑麦草；(12)糖料作物：甘蔗(甘蔗属物种(Saccharum spp.))、甜菜(Beta vulgaris)；(13)饮料作物：大叶茶(Camellia sinensis)、茶(Camellia Sinensis)、茶树(tea)、咖啡(咖啡属物种(Coffea spp.))、可可树(Theobroma cacao)、蛇麻花(啤酒花)；(14)草坪植物：固沙草(Ammophila arenaria)、早熟禾属(Poa spp.)(草地早熟禾(Poa pratensis)(蓝草))、剪股颖属物种(Agrostis spp.)(剪股颖、匍匐剪股颖(Agrostis palustris))、黑麦草属物种(Lolium spp.)(黑麦草)、羊茅属物种(Festuca spp.)(羊茅)、结缕草属物种(Zoysia spp.)(结缕草(Zoysiajaponica))、狗牙根属物种(Cynodon spp.)(百慕大草、狗牙根)、侧钝叶草(Stenotaphrum secunda tum)(圣奥古斯丁草)、雀稗属物种(Paspalum spp.)(巴哈草)、假俭草(Eremochloa ophiuroides)(百足草)、地毯草属物种(Axonopus spp.)(地毯草)、指形垂穗草(Bouteloua dactyloides)(野牛草)、垂穗草属变种物种(Bouteloua var.spp.)(格兰马草)、马唐(Digitariasanguinalis)、香附子(Cyperusrotundus)、短叶水蜈蚣(Kyllingabrevifolia)、阿穆尔莎草(Cyperusamuricus)、加拿大飞蓬(Erigeroncanadensis)、天胡荽(Hydrocotylesibthorpioides)、鸡眼草(Kummerowiastriata)、地锦(Euphorbiahumifusa)、耕地堇菜(Violaarvensis)、白颖苔草、异穗苔草、草皮草(turf)；(15)树木作物：松属(Pinus spp.)、柳属(Salix sp.)、槭树属(Acer spp.)、木槿属(Hibiscus spp.)、桉属 (Eucalyptus sp.)、银杏(Ginkgo biloba)、箣竹属(Bambusa sp.)、杨属(Populus spp.)、牧豆树属(Prosopis spp.)、栎属(Quercus spp.)、刺葵属(Phoenix spp.)、山毛榉属(Fagus spp.)、吉贝(Ceiba pentandra)、樟属(Cinnamomum spp.)、黄麻属(Corchorus sp.)、南方芦苇(Phragmites australis)、酸浆属(Physalis spp.)、山蚂蝗属(Desmodium spp.)、杨、常春藤、白杨、珊瑚树、银杏、栎类、臭椿、木荷、冬青、悬铃木、女贞、大叶黄扬、落叶松、黑荆树、马尾松、思茅松，云南松、南亚松、油松、红松、黑胡桃、柠檬、悬铃木、蒲桃、珙桐、木棉、爪哇木棉、洋紫荆、羊蹄甲、雨树、合欢、龙牙花、刺桐、广玉兰、苏铁、紫薇、针叶树、乔木、灌木；(16)坚果作物：巴西栗(Bertholletia excelsea)、栗属(Castanea spp.)、榛属(Corylus spp.)、山核桃属(Carya spp.)、核桃属(Juglans spp.)、阿月浑子(Pistacia vera)、腰果(Anacardium occidentale)、澳洲坚果(全缘叶澳洲坚果(Macadamia integrifolia))、碧根果、夏威夷果、开心果、巴旦木以及产生坚果的植物；(17)其他：拟南芥、臂形草、蒺藜草、大狗尾草、牛筋草、Cadaba farinosa、藻类(algae)、Carex elata、观赏植物、大果假虎刺(Carissa macrocarpa)、菜蓟属(Cynara spp.)、野胡萝卜(Daucus carota)、薯蓣属(Dioscorea spp.)、蔗茅属(Erianthus sp.)、苇状羊茅(Festuca arundinacea)、萱草(Hemerocallis fulva)、百脉根属(Lotus spp.)、Luzula sylvatica、紫苜蓿(Medicago sativa)、草木樨属(Melilotus spp.)、黑桑(Morus nigra)、烟草属(Nicotiana spp.)、木犀榄属(Olea spp.)、鸟足豆属(Ornithopus spp.)、欧防风(Pastinaca sativa)、接骨木属(Sambucus spp.)、白芥属(Sinapis sp.)、蒲桃属(Syzygium spp.)、鸭茅状摩擦禾(Tripsacum dactyloides)、Triticosecale rimpaui、香堇(Viola odorata)等。
在本发明中，术语“植物组织”或“植物部分”包括植物细胞、原生质体、植物组织培养物、植物愈伤组织、植物块以及植物胚、花粉、胚珠、种子、叶、茎、花、枝、幼苗、果实、核、穗、根、根尖、花药等。
在本发明中，“植物细胞”应理解为来自或发现于植物的任何细胞，其能够形成例如：未分化组织如愈伤组织，分化组织如胚胎，植物的组成部分，植物或种子。
对于说明书中所用的有关氨基酸取代的术语，第一个字母代表特定序列某一位置上天然存在的氨基酸，后面的数字代表相对于SEQ ID NO:2的位置，第二个字母代表取代该天然氨基酸的不同氨基酸。譬如A103S表示相对于SEQ ID NO:2的氨基酸序列而言，第103位的丙氨酸被丝氨酸取代。对于第一个字母不存在的氨基酸取代，其指的是相对于其野生型蛋白的氨基酸序列而言，在对应于SEQ ID NO:2的所述位置处，天然氨基酸被数字后的字母代表的氨基酸所取代。对于双重或多重突变，各突变之间以“/”隔开。例如，H141R/G342D/D370N表示相对于SEQ ID NO:2的氨基酸序列而言，第141位的组 氨酸被精氨酸取代，第342位的甘氨酸被天冬氨酸取代，以及第370位的天冬氨酸被天冬酰胺取代，全部三个突变均存在于所述具体的突变型HPPD蛋白内。
在一个方面，本发明公开了一种突变型HPPD蛋白或其生物活性片段，其与野生型对羟苯基丙酮酸双氧化酶蛋白相比保留了催化羟基苯丙酮酸(HPP)转变成尿黑酸的活性，同时对HPPD抑制性除草剂的抗性或耐受性有所提高。特别地，本发明的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白在对应于SEQ ID NO:2所示的野生型水稻对羟苯基丙酮酸双氧化酶蛋白氨基酸序列中的第93、103、141、165、191、220、226、276、277、336、337、338、342、346、370、377、386、390、392、403、410、418、419、420、430和431位中的一个或多个位置处具有选自下述中的一个或多个突变：93S、103S、141R、141K、141T、165V、191I、220K、226H、276W、277N、336D、337A、338D、338S、338Y、342D、346C、346D、346H、346S、346Y、370N、377C、386T、390I、392L、403G、410I、418P、419F、419L、419V、420S、420T、430G和431L。优选地，所述突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白的氨基酸序列进一步与SEQ ID NO:2所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性。更优选地，所述突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白具有SEQ ID NO:2所示的氨基酸序列，区别仅在于在对应于SEQ ID NO:2所示的野生型水稻对羟苯基丙酮酸双氧化酶蛋白氨基酸序列中的第93、103、141、165、191、220、226、276、277、336、337、338、342、346、370、377、386、390、392、403、410、418、419、420、430和431位中的一个或多个位置处具有选自下述中的一个或多个突变：93S、103S、141R、141K、141T、165V、191I、220K、226H、276W、277N、336D、337A、338D、338S、338Y、342D、346C、346D、346H、346S、346Y、370N、377C、386T、390I、392L、403G、410I、418P、419F、419L、419V、420S、420T、430G和431L。
在一个实施方案中，本发明的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白的氨基酸序列与SEQ ID NO:2所示的野生型水稻对羟苯基丙酮酸双氧化酶蛋白氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性，并且在对应于SEQ ID NO:2所示的氨基酸序列中的第93、103、141、165、191、220、226、276、277、336、337、338、342、346、370、377、386、390、392、403、410、418、419、420、430和431位中的一个或多个位置处具有选自下述中的一个或多个突变：93S、103S、141R、141K、141T、165V、191I、220K、226H、276W、277N、336D、337A、338D、338S、338Y、342D、346C、346D、346H、346S、346Y、370N、377C、386T、390I、392L、403G、410I、418P、419F、419L、419V、420S、420T、430G 和431L。优选地，本发明的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白具有SEQ ID NO:2所示的氨基酸序列，区别仅在于在对应于SEQ ID NO:2所示的野生型水稻对羟苯基丙酮酸双氧化酶蛋白氨基酸序列中的第93、103、141、165、191、220、226、276、277、336、337、338、342、346、370、377、386、390、392、403、410、418、419、420、430和431位中的一个或多个位置处具有选自下述中的一个或多个突变R93S、A103S、H141R、H141K、H141T、A165V、V191I、R220K、G226H、L276W、P277N、P336D、P337A、N338D、N338S、N338Y、G342D、R346C、R346D、R346H、R346S、R346Y、D370N、I377C、P386T、L390I、M392L、E403G、K410I、K418P、G419F、G419L、G419V、N420S、N420T、E430G和Y431L。
本发明所述蛋白质内的特定氨基酸位置(编号)是利用标准序列比对工具通过将目标蛋白质的氨基酸序列与SEQ ID NO:2进行比对而确定的，譬如用Smith-Waterman运算法则或用CLUSTALW2运算法则比对两个序列，其中当比对得分最高时认为所述序列是对准的。比对得分可依照Wilbur,W.J.and Lipman,D.J.(1983)Rapid similarity searches of nucleic acid and protein data banks.Proc.Natl.Acad.Sci.USA,80:726-730中所述的方法进行计算。在ClustalW2(1.82)运算法则中优选使用默认参数：蛋白质缺口开放罚分＝10.0；蛋白质缺口延伸罚分＝0.2；蛋白质矩阵＝Gonnet；蛋白质/DNA端隙＝-1；蛋白质/DNA GAPDIST＝4。
优选采用AlignX程序(vectorNTI组中的一部分)，以适于多重比对的默认参数(缺口开放罚分:10og缺口延伸罚分0.05)通过将蛋白质的氨基酸序列与SEQ ID NO:2进行比对来确定本发明所述蛋白质内特定氨基酸的位置。
氨基酸序列的同一性可以通过常规方法，参考例如Smith and Waterman,1981,Adv.Appl.Math.2:482，Pearson&Lipman,1988,Proc.Natl.Acad.Sci.USA 85:2444，Thompson et al.,1994,Nucleic Acids Res 22:467380等的教导，通过计算机化运行运算法则(Wisconsin Genetics软件包中的GAP,BESTFIT,FASTA,和TFASTA，Genetics Computer Group)来确定。也可使用可从美国国立生物技术信息中心(National Center for Biotechnology Information  www.ncbi.nlm.nih.gov/)获得的BLAST运算法则(Altschul et al.,1990,Mol.Biol.215:403-10)，使用默认参数确定。
在进一步的实施方案中，本发明的突变型对羟苯基丙酮酸双氧化酶蛋白具有SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO: 44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82或SEQ ID NO:84所示的氨基酸序列。
在进一步的实施方案中，本发明的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白在其氨基酸序列具有如下氨基酸突变：H141R/G342D、H141R/D370N、G342D/D370N、H141R/N338D、H141R/G342D、N338D/G342D、K418P/G419F、G419F/N420S、G342D/R346C、G342D/R346H、H141R/N420S、G338D/K418P、P277N/N338D、L276W/P277N、H141R/G342D/D370N、H141R/N338D/N420S、H141R/N338S/N420S、P336D/N338D/G342D、P336D/N338S/G342D、P336D/N338Y/G342D、N338D/G342D/R346C、N338D/G342D/R346H、N338D/G342D/R346S、N338S/G342D/R346C、N338S/G342D/R346H、N338S/G342D/R346S、N338Y/G342D/R346C、N338Y/G342D/R346H、N338Y/G342D/R346S、P336D/G342D/R346C、P336D/G342D/R346H、P336D/G342D/R346S、P336D/N338D/R346C、P336D/N338D/R346H、P336D/N338D/R346S、P336D/N338S/R346C、P336D/N338S/R346H、P336D/N338S/R346S、P336D/N338Y/R346C、P336D/N338Y/R346H、P336D/N338Y/R346S、H141R/N338D/G342D、H141R/G342D/K418P、H141R/G342D/G419F、H141R/G342D/P386T、K418P/G419F/N420T、K418T/G419F/N420T、H141R/G342D/R346C、H141R/G342D/R346H、H141R/G342D/N420S、H141R/G342D/P277N、H141R/G342D/P336D、H141R/G342D/L276W、H141R/G342D/R346S、H141R/G342D/L390I、H141R/G342D/I377C、H141R/G342D/M392L、H141R/P337A/G342D、H141R/N338S/G342D、H141R/N338Y/G342D、P277N/N338D/G342D、P277N/G342D/R346C、P277N/N338D/N420S、N338D/G342D/K418P、H141R/N338D/G342D/K418P、H141R/N338D/G342D/G419F、H141R/N338D/G342D/P386T、H141R/N338D/G342D/R346C、H141R/N338D/G342D/R346H、H141R/G342D/K418P/G419F、H141R/G342D/L276W/P277N、P336D/N338D/G342D/R346C、P336D/N338D/G342D/R346H、P336D/N338D/G342D/R346S、P336D/N338S/G342D/R346C、P336D/N338S/G342D/R346H、P336D/N338S/G342D/R346S、P336D/N338Y/G342D/R346C、P336D/N338Y/G342D/R346H、P336D/N338Y/G342D/R346S、P277N/P336D/N338D/G342D、P277N/N338D/G342D/R346C、P277N/N338D/K418P/G419F、H141R/N338D/G342D/K418P/G419F、H141R/N338D/G342D/G419F/N420S、H141R/G336D/G342D/K418P/ G419F/N420S、H141R/N338D/G342D/K418P/G419F/N420S、H141R/N338D/G342D/K418P/G419F/N420T、H141R/N338D/G342D/R346C/K418P/G419F/N420S、H141R/N338D/G342D/R346H/K418P/G419F/N420S、H141R/P277N/N338D/G342D/K418P/G419F/N420S、H141R/P277N/P336D/N338D/G342D/K418P/G419F/N420S。
在更进一步的实施方案中，本发明的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白具有SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:116、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:122、SEQ ID NO:124、SEQ ID NO:126、SEQ ID NO:128、SEQ ID NO:130、SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138、SEQ ID NO:140、SEQ ID NO:142、SEQ ID NO:144、SEQ ID NO:146、SEQ ID NO:148、SEQ ID NO:150、SEQ ID NO:152、SEQ ID NO:154、SEQ ID NO:156、SEQ ID NO:158、SEQ ID NO:160、SEQ ID NO:162、SEQ ID NO:164、SEQ ID NO:166、SEQ ID NO:168、SEQ ID NO:170、SEQ ID NO:172、SEQ ID NO:174、SEQ ID NO:176、SEQ ID NO:178、SEQ ID NO:180、SEQ ID NO:182、SEQ ID NO:184、SEQ ID NO:186、SEQ ID NO:188、SEQ ID NO:190、SEQ ID NO:192、SEQ ID NO:194、SEQ ID NO:196、SEQ ID NO:198、SEQ ID NO:200、SEQ ID NO:202、SEQ ID NO:204、SEQ ID NO:206、SEQ ID NO:208、SEQ ID NO:210、SEQ ID NO:212、SEQ ID NO:214、SEQ ID NO:216、SEQ ID NO:218、SEQ ID NO:220、SEQ ID NO:222、SEQ ID NO:224、SEQ ID NO:226、SEQ ID NO:228、SEQ ID NO:230、SEQ ID NO:232、SEQ ID NO:234、SEQ ID NO:236、SEQ ID NO:238、SEQ ID NO:240、SEQ ID NO:242、SEQ ID NO:244、SEQ ID NO:246、SEQ ID NO:248、SEQ ID NO:250、SEQ ID NO:252、SEQ ID NO:254、SEQ ID NO:256、SEQ ID NO:258或SEQ ID NO:260所示的氨基酸序列。
在本发明中，野生型对羟苯基丙酮酸双氧化酶蛋白可以来源于任何植物，特别是前述单子叶或双子叶植物。现有技术文献中已经公开了一些来源的野生型对羟苯基丙酮酸双氧化酶蛋白序列以及编码序列，这些现有技术文献在此引入本文作为参考。
优选地，本发明的野生型对羟苯基丙酮酸双氧化酶蛋白来源于稻属，特别是水稻。更优选地，所述野生型对羟苯基丙酮酸双氧化酶蛋白具有SEQ ID NO:2所示的氨基酸序列，或者与SEQ ID NO:2所示氨基酸序列有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列。
本领域技术人员还清楚的是，可以改变蛋白质的结构而不对其活性和功能性产生不利影响，例如可以在蛋白质氨基酸序列中引入一个或多个保守性氨基酸取代，而不会对蛋白质分子的活性和/或三维构型产生不利影响。本领域技术人员清楚保守性氨基酸取代的实例以及实施方式。具体的说，可以用与待取代位点属于相同组的另一氨基酸残基取代该氨基酸残基，即用非极性氨基酸残基取代另一非极性氨基酸残基，用极性不带电荷的氨基酸残基取代另一极性不带电荷的氨基酸残基，用碱性氨基酸残基取代另一碱性氨基酸残基，和用酸性氨基酸残基取代另一酸性氨基酸残基。只要取代不损害蛋白质的生物活性，则一种氨基酸被属于同组的其他氨基酸替换的保守取代落在本发明的范围内。
因此，本发明的突变型HPPD蛋白除了包含上述突变之外，还可以在氨基酸序列中包含一个或多个其他突变例如保守性取代。另外，本发明也涵盖还包含一个或多个其他非保守取代的突变型HPPD蛋白，只要该非保守取代不显著影响本发明的蛋白质的所需功能和生物活性即可。
如本领域中所熟知的，可以从蛋白质的N和/或C末端缺失一或多个氨基酸残基而仍保留其功能活性。因此，在另一方面，本发明还涉及从突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白的N和/或C末端缺失了一或多个氨基酸残基、同时保留了其所需功能活性的片段，它们也在本发明的范围内，被称为生物活性片段。在本发明中，“生物活性片段”是指本发明的突变型HPPD蛋白的一部分，其保留了本发明的突变型HPPD蛋白的生物学活性、同时对HPPD抑制剂的耐受性或抗性相比于不具有所述突变的HPPD片段有所提高。例如，突变型HPPD蛋白的生物学活性片段可以是在所述蛋白质的N和/或C末端缺失了一个或多个(例如1－50个、1－25个、1－10个或1－5个，例如1、2、3、4或5个)氨基酸残基的部分，但其仍然保留了全长蛋白的生物学活性。
本发明还提供了一种融合蛋白，其中包含本发明的突变型HPPD蛋白或其生物活性片段，以及与之融合的其它组分。在一个优选的实施方案中，所述其它组分是质体引导肽，例如引导到叶绿体内的肽，其将突变的HPPD蛋白靶向叶绿体。在另一个实施方案中，所述其它组分是标签肽，例如6×His。在又一个实施方案中，所述其它组分是有助于提高所述突变型HPPD蛋白的溶解性的肽，例如NusA肽。
在又一方面，本发明提供了包含编码上述突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物学活性片段的核酸序列或者其互补序列的分离的多核苷酸。术语“分离的”多核苷酸是指该多核苷酸基本上不包含在天然存在环境中通常伴随着其的成分。在一个实施方案中，所述突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白的氨基酸序列与SEQ ID NO:2所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、 至少97％、至少98％、至少99％序列同一性的氨基酸序列，并且进一步地在对应于SEQ ID NO:2所示的野生型水稻对羟苯基丙酮酸双氧化酶蛋白氨基酸序列中的第93、103、141、165、191、220、226、276、277、336、337、338、342、346、370、377、386、390、392、403、410、418、419、420、430和431位中的一个或多个位置处具有选自下述中的一个或多个突变：93S、103S、141R、141K、141T、165V、191I、220K、226H、276W、277N、336D、337A、338D、338S、338Y、342D、346C、346D、346H、346S、346Y、370N、377C、386T、390I、392L、403G、410I、418P、419F、419L、419V、420S、420T、430G和431L。优选地，所述突变是选自下述中的一个或多个突变：R93S、A103S、H141R、H141K、H141T、A165V、V191I、R220K、G226H、L276W、P277N、P336D、P337A、N338D、N338S、N338Y、G342D、R346C、R346D、R346H、R346S、R346Y、D370N、I377C、P386T、L390I、M392L、E403G、K410I、K418P、G419F、G419L、G419V、N420S、N420T、E430G和Y431L。更优选地，所述突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段衍生自水稻HPPD蛋白，并且具有选自上述的一个或多个氨基酸取代。
本领域技术人员十分清楚，由于遗传密码的简并性，有多种不同的核酸序列可以编码本文公开的氨基酸序列。产生编码相同蛋白质的其他核酸序列在本领域普通技术人员的能力范围内，因此本发明涵盖因遗传密码子的简并性而编码相同氨基酸序列的核酸序列。例如，为了在目标宿主生物例如植物中实现异源基因的高表达，可以对所述基因采用宿主生物偏好的密码子进行优化，以使其更好地表达。
因此，在一些实施方案中，本发明的多核苷酸具有选自下述的核酸序列：
(1)编码SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:116、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:122、SEQ ID NO:124、 SEQ ID NO:126、SEQ ID NO:128、SEQ ID NO:130、SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138、SEQ ID NO:140、SEQ ID NO:142、SEQ ID NO:144、SEQ ID NO:146、SEQ ID NO:148、SEQ ID NO:150、SEQ ID NO:152、SEQ ID NO:154、SEQ ID NO:156、SEQ ID NO:158、SEQ ID NO:160、SEQ ID NO:162、SEQ ID NO:164、SEQ ID NO:166、SEQ ID NO:168、SEQ ID NO:170、SEQ ID NO:172、SEQ ID NO:174、SEQ ID NO:176、SEQ ID NO:178、SEQ ID NO:180、SEQ ID NO:182、SEQ ID NO:184、SEQ ID NO:186、SEQ ID NO:188、SEQ ID NO:190、SEQ ID NO:192、SEQ ID NO:194、SEQ ID NO:196、SEQ ID NO:198、SEQ ID NO:200、SEQ ID NO:202、SEQ ID NO:204、SEQ ID NO:206、SEQ ID NO:208、SEQ ID NO:210、SEQ ID NO:212、SEQ ID NO:214、SEQ ID NO:216、SEQ ID NO:218、SEQ ID NO:220、SEQ ID NO:222、SEQ ID NO:224、SEQ ID NO:226、SEQ ID NO:228、SEQ ID NO:230、SEQ ID NO:232、SEQ ID NO:234、SEQ ID NO:236、SEQ ID NO:238、SEQ ID NO:240、SEQ ID NO:242、SEQ ID NO:244、SEQ ID NO:246、SEQ ID NO:248、SEQ ID NO:250、SEQ ID NO:252、SEQ ID NO:254、SEQ ID NO:256、SEQ ID NO:258或SEQ ID NO:260所示氨基酸序列的核酸序列或其互补序列；
(2)SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:115、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:121、SEQ ID NO:123、SEQ ID NO:125、SEQ ID NO:127、SEQ ID NO:129、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137、SEQ ID NO:139、SEQ ID NO:141、SEQ ID NO:143、SEQ ID NO:145、SEQ ID NO:147、SEQ ID NO:149、SEQ ID NO:151、SEQ ID NO:153、SEQ ID NO:155、SEQ ID NO:157、SEQ ID NO:159、SEQ ID NO:161、SEQ ID NO:163、SEQ ID NO:165、SEQ ID NO: 167、SEQ ID NO:169、SEQ ID NO:171、SEQ ID NO:173、SEQ ID NO:175、SEQ ID NO:177、SEQ ID NO:179、SEQ ID NO:181、SEQ ID NO:183、SEQ ID NO:185、SEQ ID NO:187、SEQ ID NO:189、SEQ ID NO:191、SEQ ID NO:193、SEQ ID NO:195、SEQ ID NO:197、SEQ ID NO:199、SEQ ID NO:201、SEQ ID NO:203、SEQ ID NO:205、SEQ ID NO:207、SEQ ID NO:209、SEQ ID NO:211、SEQ ID NO:213、SEQ ID NO:215、SEQ ID NO:217、SEQ ID NO:219、SEQ ID NO:221、SEQ ID NO:223、SEQ ID NO:225、SEQ ID NO:227、SEQ ID NO:229、SEQ ID NO:231、SEQ ID NO:233、SEQ ID NO:235、SEQ ID NO:237、SEQ ID NO:239、SEQ ID NO:241、SEQ ID NO:243、SEQ ID NO:245、SEQ ID NO:247、SEQ ID NO:249、SEQ ID NO:251、SEQ ID NO:253、SEQ ID NO:255、SEQ ID NO:257或SEQ ID NO:259所示的核酸序列或其互补序列；
(3)在严谨条件下与(1)或(2)所示序列杂交的核酸序列；和
(4)因遗传密码的简并性而与(1)或(2)所示序列编码相同氨基酸序列的核酸序列，或其互补序列。
进一步优选地，所述多核苷酸具有选自SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:115、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:121、SEQ ID NO:123、SEQ ID NO:125、SEQ ID NO:127、SEQ ID NO:129、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137、SEQ ID NO:139、SEQ ID NO:141、SEQ ID NO:143、SEQ ID NO:145、SEQ ID NO:147、SEQ ID NO:149、SEQ ID NO:151、SEQ ID NO:153、SEQ ID NO:155、SEQ ID NO:157、SEQ ID NO:159、SEQ ID NO:161、SEQ ID NO:163、SEQ ID NO:165、SEQ ID NO:167、SEQ ID NO:169、SEQ ID NO:171、SEQ ID NO:173、SEQ ID NO:175、SEQ ID NO:177、SEQ ID NO:179、SEQ ID NO:181、SEQ ID  NO:183、SEQ ID NO:185、SEQ ID NO:187、SEQ ID NO:189、SEQ ID NO:191、SEQ ID NO:193、SEQ ID NO:195、SEQ ID NO:197、SEQ ID NO:199、SEQ ID NO:201、SEQ ID NO:203、SEQ ID NO:205、SEQ ID NO:207、SEQ ID NO:209、SEQ ID NO:211、SEQ ID NO:213、SEQ ID NO:215、SEQ ID NO:217、SEQ ID NO:219、SEQ ID NO:221、SEQ ID NO:223、SEQ ID NO:225、SEQ ID NO:227、SEQ ID NO:229、SEQ ID NO:231、SEQ ID NO:233、SEQ ID NO:235、SEQ ID NO:237、SEQ ID NO:239、SEQ ID NO:241、SEQ ID NO:243、SEQ ID NO:245、SEQ ID NO:247、SEQ ID NO:249、SEQ ID NO:251、SEQ ID NO:253、SEQ ID NO:255、SEQ ID NO:257或SEQ ID NO:259所示核酸序列、其简并序列或者它们的互补序列的核酸序列。
优选地，所述严谨条件可以指6M尿素、0.4％SDS、0.5×SSC的条件或与其同等的杂交条件，也可以指严谨性更高的条件，例如6M尿素、0.4％SDS、0.1×SSC或与其同等的杂交条件。在各种条件中，温度可约为40℃以上，如需要严谨性更高的条件时，温度例如可约为50℃，进一步可约为65℃。
更进一步优选地，所述氨基酸突变位点所对应的野生型和突变型密码子如下：
氨基酸突变位点 野生型密码子 突变型密码子
A103S GCC TCG；TCA；TCC；TCT；AGC；AGT
H141R CAC CCG；CGA；CGC；CGT；AGG；AGA
H141K CAC AAG；AAA
H141T CAC ACC；ACT；ACA；ACG
A165V GCG GTG；GTA；GTC；GTT
V191I GTC ATA；ATC；ATT
R220K CGG AAG；AAA
G342D GGC GAC；GAT
D370N GAC AAC；AAT
K410I AAG ATA；ATC；ATT
R93S CGC AGC；AGT；TCA；TCC；TCG；TCT
G226H GGC CAC；CAT
L276W CTG TGG
P277N CCG AAC；AAT
P336D CCG GAT；GAC
P337A CCC GCA；GCC；GCG；CGT
N338D AAC GAC；GAT
N338S AAC AGC；TCG；TCA；TCC；TCT；AGT
N338Y AAC TAC；TAT
G342D GGC GAC；GAT
R346C CGC TGC；TGT
R346H CGC CAC；CAT
R346S CGC AGC；TCG；TCA；TCC；TCT；AGT
R346D CGC GAC；GAT
R346Y CGC TAC；TAT
I377C ATC TGC；TGT
P386T CCA ACA；ACC；ACG；ACT
L390I TTG ATA；ATC；ATT
M392L ATG CTA；CTC；CTG；CTT；TTA；TTG
E403G GAG GGA；GGC；GGG；GGT
P418P AAG CCA；CCC；CCG；CCT
G419F GGC TTC；TTT
G419L GGC CTA；CTC；CTG；CTT；TTA；TTG
G419V GGC GTA；GTC；GTG；GTT
N420S AAC AGC；TCG；TCA；TCC；TCT；AGT
N420T AAC ACA；ACC；ACG；ACT
E430G GAG GGA；GGC；GGG；GGT
Y431L TAT CTA；CTC；CTG；CTT；TTA；TTG
本发明还提供了一种核酸构建体，其中包含编码本发明的突变型对羟苯基丙酮酸双氧化酶蛋白或其生物活性片段或者融合蛋白的核酸序列以及与之可操作连接的一个或多个调控元件。术语“调控元件”在本发明中指的是能够调节与之可操作连接的核酸的转录和/或翻译的核酸序列。
调控元件可能是适当的启动子序列，被宿主细胞识别用于表达编码本发明蛋白质的核酸序列。启动子序列包含介导蛋白质表达的转录调控序列。启动子可以是在选定宿主细胞内显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合的启动子，并且可 能获自与宿主细胞同源或异源的编码细胞外或细胞内多肽的基因。作为在植物细胞或植物中表达的启动子，可使用对羟苯基丙酮酸双氧化酶天然的启动子，或者在植物中具有活性的异源启动子。所述启动子可以是组成型表达的，或者可以是诱导型表达的。启动子的实例包括例如组蛋白启动子，水稻肌动蛋白启动子，植物病毒启动子例如花椰菜花叶病毒启动子等。
调控元件也可以是合适的转录终止子序列，被宿主细胞识别来终止转录的序列。终止子序列与编码本发明蛋白质的核酸序列的3'末端可操作性的连接。在选定宿主细胞内起作用的任何终止子均可用于本发明中。
调控元件也可能是合适的前导序列，即对于宿主细胞的翻译而言非常重要的mRNA非翻译区。前导序列与编码本发明蛋白质的核酸序列的5'末端可操作性连接。在选定宿主细胞内起作用的任何前导序列均可用于本发明中。
调控元件还可以是多聚腺苷酸化序列，即是与核酸序列的3'末端可操作性连接并且在转录时被宿主细胞识别为添加多聚腺苷酸残基至被转录mRNA的信号的序列。在选定宿主细胞内起作用的任何多聚腺苷酸化序列均可用于本发明中。
调控元件也可以是编码与蛋白质的氨基末端连接的氨基酸序列并指导所编码蛋白质进入细胞分泌途径的信号肽编码区。核酸序列的编码序列的5'末端可能固有的包含在翻译读码框架内与编码所述分泌多肽的编码区部分天然连接的信号肽编码区。或者，编码序列的5'末端可能包含对于编码序列而言是外来的信号肽编码区。在编码序列天然不包含信号肽编码区的情况下可能需要外源的信号肽编码区。或者，外源的信号肽编码区可能简单地替代天然信号肽编码区以便促进所述多肽的分泌。无论如何，指导被表达多肽进入选定宿主细胞之分泌途径即分泌入培养基中的任何信号肽编码区均可能用于本发明中。
还可适当的添加允许所述多肽表达相对于宿主细胞的生长进行调节的调节序列。调节系统有例如使得基因表达响应化学或物理刺激物(包括调节化合物的存在)而开启或关闭的调节系统，例如lac,tec,和tip操纵子系统，ADH2系统或GAL1系统等。其它的调控序列的例子有允许进行基因扩增的那些调控序列。在真核体系内，这些包括在氨甲喋呤存在下被扩增的二氢叶酸还原酶基因，由于重金属而被扩增的金属硫蛋白基因。在这些案例中，编码多肽的核苷酸序列将可操作性的与所述调控序列连接。
在本发明中，调控元件也可以是转录激活剂即增强子，例如WO87/07644中描述的烟草花叶病毒翻译激活剂，或内含子等，例如玉米的adh1内含子、玉米青铜色1基因(maize bronze 1 gene)内含子或水稻肌动蛋白内含子1。它们可以增强本发明的突变型HPPD蛋 白、其生物活性片段或者融合蛋白在转基因植物中的表达。
本发明还提供了一种表达载体，其中包含有编码本发明的突变型对羟苯基丙酮酸双氧化酶蛋白或其生物活性片段或者融合蛋白的核酸序列以及与之可操作连接的表达调控元件。表达载体中还至少含有一个复制起点，以实现自我复制。载体的选择通常取决于载体与该载体待引入之宿主细胞的相容性。载体可能是自主复制载体，即作为染色体外实体存在的载体，它的复制不依赖于染色体的复制，例如质粒、染色体外元件、微型染色体或人工染色体。该载体可能包含保证自我复制的任何元件。或者，所述载体可能是当引入宿主细胞时被整合入基因组中并与其所整合入的染色体一起复制的载体。此外，可使用单个载体或质粒或者一起包含待引入宿主细胞基因组之总DNA的两个或更多个载体或质粒，或者转座子。或者，所述载体也可以是对宿主细胞内源性的HPPD基因进行基因编辑的载体。
载体可以是例如质粒、病毒、粘粒、噬菌体等类型，它们是本领域技术人员所熟知的，在本领域中众多描述。优选地，本发明中的表达载体是质粒。表达载体可包含启动子、翻译起始的核糖体结合位点、聚腺苷酸化位点、转录终止子、增强子等。表达载体中也可以含有一个或多个可选择标记基因以便用于选择包含载体的宿主细胞。这种可选择的标记包括编码二氢叶酸还原酶的基因，或赋予新霉素耐受性的基因，赋予对四环素或氨苄青霉素耐受性的基因等。
本发明的载体可以包含允许载体整合入宿主细胞基因组或在细胞内不依赖于基因组而自主复制的元件。对于整合进入宿主细胞基因组的方面，所述载体可依靠编码多肽的多核苷酸序列或适于通过同源或非同源重组整合入基因组的载体的任何其它元件。或者，载体可包含用于指导在染色体的准确位置通过同源重组整合入宿主细胞基因组的附加的核苷酸序列。为了提高在准确位置处整合的可能性，整合元件应优选包含足够数目的核酸，譬如100至10,000个碱基对，优选400至10,000个碱基对，更优选800至10,000个碱基对，它们与相应的靶序列具有高度的同一性以提高同源重组的概率。整合元件可能是与宿主细胞基因组内靶序列同源的任何序列。此外，整合元件可能是非编码的或编码的核苷酸序列。另一方面，载体可能通过非同源重组整合入宿主细胞的基因组内。对于自主复制而言，载体可能进一步包含能使载体在所述宿主细胞内自主复制的复制起点。复制起点可能是在细胞内发挥作用的介导自主复制的任何质粒复制子。术语"复制起点"或"质粒复制子"在此定义为能使质粒或载体在体内进行复制的核苷酸序列。
可将一拷贝以上的本发明之多核苷酸插入宿主细胞中以提高基因产物的产量。可通过将至少一个额外拷贝的序列整合入宿主细胞基因组中或者通过将可扩增的可选择标记 基因与所述多核苷酸包含在一起来达到多核苷酸拷贝数目的增加，在后一情形下，包含扩增拷贝的选择标记基因以及由此而来的附加拷贝的多核苷酸的细胞可通过在适当的可选择制剂存在的条件下人工培养所述细胞进行选择。
本发明的核酸序列可通过多种方法插入载体中，例如通过用适当的限制性核酸内切酶消化插入物和载体后进行连接。多种克隆技术在本领域中是已知的，这些均在本领域技术人员的知识范围内。
本发明中适用的载体包括可从商业渠道获得的质粒，例如但不限于：pBR322(ATCC 37017)，pKK223-3(Pharmacia Fine Chemicals，Uppsala，Sweden)，GEM1(Promega Biotec，Madison，WI，USA)pQE70，pQE60，pQE-9(Qiagen)，pD10，psiX174 pBluescript II KS，pNH8A，pNH16a，pNH18A，pNH46A(Stratagene)，ptrc99a，pKK223-3，pKK233-3，pDR540，pRIT5(Pharmacia)，pKK232-8，pCM7，pSV2CAT，pOG44，pXT1，pSG(Stratagene)，pSVK3，pBPV，pMSG，和pSVL(Pharmacia)等。
本发明还提供了包含本发明核酸序列、核酸构建体或表达载体的宿主细胞。将包含编码本发明的载体引入宿主细胞中使得载体作为染色体整合体的一部分存在或如早先所述作为自我复制的染色体外载体存在，或者载体可以对宿主细胞内源性的HPPD基因进行基因编辑。宿主细胞可以是本领域技术人员熟悉的任何宿主细胞，包括原核生物细胞和真核食物细胞，例如细菌细胞、真菌细胞、酵母细胞、哺乳动物细胞、昆虫细胞或植物细胞，其实例有大肠杆菌(E.coli)、链霉菌(Streptomyces)、枯草芽孢杆菌(Bacillus subtilis)、鼠伤寒沙门氏菌(Salmonella typhimurium)、假单胞菌属(Pseudomonas)、链霉菌属(Streptomyces)、葡萄球菌属(Staphylococcus)、Spodoptera Sf9、CHO、COS等。适当宿主细胞的选择在本领域技术人员的能力范围内。
在本发明中，术语“宿主细胞”还涵盖由于复制期间发生的突变而与亲本细胞不完全相同的任何亲本细胞后代。
本发明的核酸序列、核酸构建体或表达载体可以通过多种技术导入宿主细胞，包括转化、转染、转导、病毒感染、基因枪或Ti-质粒介导的基因传递，以及钙磷酸盐转染、DEAE-葡聚糖介导的转染、脂转染或电穿孔等(参考Davis，L.，Dibner，M.，Battey，I.，Basic Methods in Molecular Biology,1986)。
在一个具体实施方案中，本发明的突变型HPPD蛋白可以被靶向植物内的质体，例如叶绿体。这可以通过将编码本发明的突变型HPPD蛋白的核酸序列与编码质体引导肽例如叶绿体转运肽的核酸序列阅读框一致地连接来实现。或者，可以将本发明的多核苷酸、核酸构建体或表达载体直接转化植物细胞的叶绿体基因组来实现。本领域技术人员 清楚可用于转化植物细胞叶绿体基因组的载体和方法。例如，可以通过将DNA包被的离子轰击目标植物的树叶，并通过同源重组或非同源重组而整合编码本发明突变型HPPD蛋白的核酸序列。
在适当的情况下，转化的宿主细胞可以在常规的营养培养基中培养。在转化适合的宿主细胞并培养宿主细胞至适当的细胞密度后，可以用适当的方法例如温度改变或化学诱导等诱导所选择的启动子，并可另外培养细胞一段时间以使其产生本发明的突变型HPPD蛋白或其生物活性片段或融合蛋白。
因此，本发明还涉及产生本发明的突变型HPPD蛋白或其生物活性片段或融合蛋白的方法，包括：(a)在有助于所述突变型HPPD蛋白或其生物活性片段或融合蛋白生产的条件下培养上述宿主细胞；和(b)回收所述突变型HPPD蛋白或其生物活性片段或融合蛋白。
在本发明的生产方法中，用本领域众所周知的方法将所述细胞培养于适于所述多肽产生的营养培养基上。例如，在实验室或工业发酵罐中以合适的培养基并且在允许所述多肽表达和/或分离的条件下通过摇瓶培养和小规模或大规模发酵(包括连续的、分批的、分批投料或固态发酵)培养细胞。用本领域已知的步骤在包含碳和氮源以及无机盐的合适营养培养基上进行培养。合适的培养基可购自供应商或依照已发表的组成配制(例如，在美国典型培养物保藏(American Type Culture Collection)的目录上)。若所述多肽被分泌入营养培养基中，则可直接从培养基中回收该多肽。若所述多肽不分泌到培养基中，则可从细胞裂解物中回收它。
可用本领域已知特异于所述多肽的方法检测该多肽。这些检测方法可包括使用特异抗体、形成酶产物或酶底物的消失。
产生的多肽可用本领域已知的方法回收。例如，可以通过离心收获细胞，用物理的或化学的方法使之破碎，并保留得到的粗提取液以进一步纯化。可以用任何方便的方法裂解表达本发明的突变型HPPD蛋白或其生物活性片段或融合蛋白的转化宿主细胞，包括冻融循环、超声波、机械破碎或使用细胞溶解剂。这些方法是本领域技术人员熟知的。可以从转化宿主细胞的培养物中回收和纯化本发明的突变型HPPD蛋白或其生物活性片段，采用的方法包括硫酸铵或乙醇沉淀、酸提取、阴离子或阳离子交换层析、磷酸纤维素层析、疏水作用层析、亲合层析、羟磷灰石层析和植物血凝素层析等等。
本发明还涉及一种制备对HPPD抑制型除草剂具有耐受性或抗性的宿主生物特别是植物细胞、植物组织、植物部分或植物的方法，其中包括用包含本发明的突变型对羟苯基丙酮酸双氧化酶蛋白或其生物活性片段的编码核酸序列、包含所述核酸序列的核酸构 建体或表达载体对所述宿主生物进行转化。宿主细胞例如植物细胞的转化方法是现有技术中已知的，包括例如原生质体转化、融合、注射、电穿孔、PEG介导的转化、离子轰击、病毒转化、农杆菌介导的转化、电穿孔或轰击等。现有技术中描述了一系列这样的转化方法，例如EP1186666中描述了大豆转化的技术，WO 92/09696中描述了单子叶植物特别是水稻转化的合适技术等。还可以有利地用根癌农杆菌或发根农杆菌培养植物外植体，以将DNA转移进植物细胞。然后可以在合适的培养基中从感染的植物材料部分(如叶碎片、茎节段、根以及原生质体或悬浮培养的细胞)再生完整植物，所述培养基可以含有用于选择的抗生素或杀虫剂。转化细胞以通常的方式在植物中生长，它们可以形成生殖细胞并将转化的性状传递到子代植物。这样的植物能以正常方式培养并与具有相同转化遗传因子或其他遗传因子的植物杂交。得到的杂合个体具有相应的表型特性。
本发明还提供了一种制备对HPPD抑制型除草剂具有耐受性或抗性的宿主生物特别是植物细胞、植物组织、植物部分或植物的方法，其中包括将本发明的突变型对羟苯基丙酮酸双氧化酶蛋白或其生物活性片段的编码核酸整合到宿主生物的基因组中并使之表达。合适的载体和选择标记是本领域技术人员所熟知的，例如WO06/108830中描述了一种整合入烟草基因组中的方法，其公开内容通过引用并入本文中作为参考。目的基因在植物细胞中优选由组成型或诱导型启动子表达。mRNA一旦表达之后，就被翻译成蛋白质，从而将目的氨基酸掺入蛋白质中。在植物细胞中表达的编码蛋白质的基因可以在组成型启动子、组织特异性启动子或诱导型启动子的控制下。例如可以使用细菌来源的启动子，如章鱼碱合酶启动子、胭脂碱合酶启动子、甘露碱合酶启动子；病毒来源的启动子，如花椰菜花叶病毒(35S和19S)、35T(再改造的35S启动子，参阅美国专利No.6,166,302，特别是实施例7E)等。也可以使用植物启动子调节元件，包括但不仅限于核酮醣-1，6-二磷酸(RUBP)羧化酶小亚基(ssu)、β-伴大豆球蛋白(conglycinin)启动子、β-菜豆素启动子、ADH启动子、热休克启动子和组织特异性启动子。还可以使用组成型启动子调节元件从而指导在所有细胞类型和所有时间的持续基因表达(如肌动蛋白、泛素、CaMV35S等)。组织特异性启动子调节元件在本发明中也是适用的，其负责在特定细胞或组织类型(如叶或种子)中的基因表达(如玉米醇溶蛋白、油质蛋白、napin、ACP、球蛋白等)。同样的，还可以使用在植物发育的某些阶段有活性(或无活性)的启动子调节元件。这些启动子调节元件的实例包括但不仅限于花粉特异性、胚胎特异性、玉米穗丝特异性、棉花纤维特异性、根特异性、种子胚乳特异性或无性繁殖期特异性启动子调节元件等。在某些情况下可能需要使用诱导型启动子调节元件，其负责应答于特定信号(如物理刺激(热休克基因)、光(RUBP羧化酶)、激素(Em)、代谢物、化学品(四环素应答)和胁迫)的基 因表达。可以使用在植物中发挥功能的其他所需的转录和翻译元件。
本发明还提供了一种提高植物细胞、植物组织、植物部分或植物的HPPD抑制性除草剂耐受性或抗性的方法，其中包括用包含本发明的突变型对羟苯基丙酮酸双氧化酶蛋白或其生物活性片段或者融合蛋白的编码核酸序列的核酸分子转化所述植物或其部分，并使之表达。所述核酸分子可以作为染色体外实体存在而进行表达，或者可以整合到植物细胞的基因组中实现表达，特别是通过同源重组整合到植物细胞的内源基因位置处实现表达。这些实施方式均在本发明的范围之内。
本发明还提供了一种提高植物或其部分的HPPD抑制性除草剂耐受性或抗性的方法，其中包括将表达本发明的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段或者融合蛋白的植物与另一植物杂交，以及筛选具有提高的HPPD抑制性除草剂抗性或耐受性的植物或其部分。
本发明还提供了一种提高植物细胞、植物组织、植物部分或植物中的HPPD抑制性除草剂耐受性或抗性的方法，其中包括对所述植物细胞、植物组织、植物部分或植物的内源性HPPD蛋白进行基因编辑，以实现在其中表达本发明的突变型对羟苯基丙酮酸双氧化酶蛋白或其生物活性片段或者融合蛋白。
本发明还涉及通过传统育种技术制备除草剂耐受性或抗性提高的植物的方法，其中包括用在基因组中整合了本发明的突变型对羟苯基丙酮酸双氧化酶蛋白或其生物活性片段的编码核酸序列的植物自交或者杂交，并筛选杂合或纯合地包含了所述编码核酸序列的后代。
本发明进一步涉及通过上述方法获得的植物细胞、植物组织、植物部分和植物，及其后代。
优选地，可以将转化了本发明多核苷酸的植物细胞、植物组织或植物部分再生为整个植株。本发明包括细胞培养物，包括组织细胞培养物、液体培养物和固体平板培养物。由本发明植物所产生和/或用于再生本发明植物的种子也包括在本发明范围内。其他植物组织和部分也包括在本发明中。本发明同样包括产生含有本发明核酸分子的植物或细胞的方法。产生这类植物的一种优选方法为通过种植本发明的种子。以这种方式转化的植物可以获得对多种具有不同作用模式的除草剂的抗性。
例如，对于用农杆菌转化植物细胞，可以将外植体与转化的农杆菌混合并孵育足够的时间以允许其转化。转化后，通过用适当抗生素的选择杀死农杆菌，并用适当的选择培养基培养植物细胞。一但形成愈伤组织，可以根据植物组织培养和植物再生领域熟知的方法通过使用适当的植物激素促进芽形成。然而，愈伤组织中间期并不总是必要的。 芽形成以后，可以将所述植物细胞转移到促进根形成的培养基，从而完成植物再生。然后可以培养植物产生种子，所述种子可以用于建立将来的世代。不论转化技术如何，优选将编码细菌蛋白质的基因整合进基因转移载体中，通过在载体中纳入植物启动子调节元件以及3’非翻译转录终止区(如Nos等)使所述转移载体适于在植物细胞中表达该基因。
本发明还提供了一种控制植物场所的杂草的方法，其中包括对包含本发明的植物或种子的场所施用控制杂草有效量的一种或多种HPPD抑制性除草剂。
在本发明中，术语“场所”包括栽培本发明植物的场地例如土壤，也包括例如植物种子、植物苗以及长成的植物。术语“控制杂草有效量”指的是除草剂的量足以影响目标杂草的生长或发育，例如阻止或抑制目标杂草的生长或发育，或者杀灭所述杂草。有利地，所述控制杂草有效量不会显著影响本发明植物种子、植物苗或植物的生长和/或发育。本领域技术人员可以通过常规实验确定这样的控制杂草有效量。
本发明还提供了一种制备保留或加强了催化对羟基苯基丙酮酸(HPP)转化为尿黑酸的性质、且对HPPD抑制型除草剂的敏感性明显低于野生型HPPD的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白的方法，其中包括对编码野生型HPPD的核酸进行突变，将突变核酸在表达载体中与编码提高溶解度的组分的核酸序列读框一致地融合和连接形成融合蛋白编码序列，将所获得的重组表达载体转化到宿主细胞中，在含有所述HPPD抑制型除草剂和HPPD酶促底物的合适的条件下表达所述融合蛋白以及筛选保留或加强了催化对羟基苯基丙酮酸(HPP)转化为尿黑酸的性质、且对HPPD抑制型除草剂的敏感性明显降低的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白的步骤。优选地，所述提高溶解度的组分是NusA，其与本发明的突变型HPPD蛋白构成融合蛋白。更优选地，所述表达载体是pET-44a载体。所述宿主细胞可以是细菌细胞、真菌细胞或植物细胞。
除非具体说明或暗示，如本文中所用，术语“一”、“一个/一种”和“所述”表示“至少一个”。本文提到或引用的所有专利、专利申请和出版物整体引入本文作为参考，其引用程度如同单独地个别引用一样。
具体实施方式
以下结合实施例对本发明做进一步说明。在这些实施例中描述的所有方法和操作是以举例方式提供的，其不应被理解为限制性的。有关DNA操作的方法可参考Current Protocols in Molecular Biology，第1和2卷，Ausubel F.M.,Greene Publishing Associates and Wiley Interscience,1989,Molecular Cloning,T.Maniatis et al.,1982，或者Sambrook J.and Russell D.,2001，Molecular Cloning:a laboratory manual,version 3。
实施例
实施例1 水稻HPPD(OsHPPD)基因的克隆
水稻(Oryza sativa Japonica Group)对羟苯基丙酮酸双氧化酶(4-hydroxyphenylpyruvate dioxygenase，HPPD)基因位于第二染色体Os02g0168100位点。根据其cDNA序列(NCBI编号XP_015626163.1)直接合成其编码区域DNA(OsHPPD)(通用生物，中国，安徽，滁州)当作PCR模板。根据载体pET-44a(Novagen)和XP_015626163.1序列，设计和合成引物NusOsF:acg att gat gac gac gac aag ATGCCTCCCACTCCCACCCC和NusOsR:tccacgagctcccggactcTTA CTAGGATCCTTGAA CTGTAG。用这些引物，合成的模板和Q5 DNA聚合酶(NEB,New England Biolabs,Boston,USA)进行PCR扩增。扩增条件为：98℃ 2分钟；然后98℃ 20秒，65℃ 30秒和72℃ 60秒，重复35次；最后72℃ 2分钟。扩增片段在琼脂糖凝胶电泳中显示为1.3Kb，回收后通过紫外吸收法确定其DNA浓度。
pET-44a(Novagen)质粒经BoxI(Thermo Fisher Scientific，中国，上海)在37℃酶切1小时后，加热至65℃失活BoxI。取等量OsHPPD DNA片段与BoxI线性化的pET-44a载体混合，加入等体积2×Gibson Assembly Master Mix(汉恒生物，中国，上海)，混匀后，于50℃孵育一个小时，取5ul连接产物来转化E.coli DH5a感受态，将菌液涂布至含100ppm氨苄青霉素的LB固体培养基平板表面，在37℃过夜培养。第二天，经单菌落PCR确定正确克隆后，在37℃下过夜培养三个正确克隆，提取足够质粒DNA送到擎科生物技术公司(中国，北京)用sanger法测序。测序结果证明得到了正确的全长水稻HPPD编码区DNA。
实施例2 水稻HPPD(OsHPPD)蛋白中每一氨基酸的饱和随机突变
水稻全长OsHPPD酶有446个氨基酸，其氨基酸序列示于SEQ ID NO:2中。其中氨基酸1－50被认为构成信号肽，负责指引到叶绿体中(Siehl et al.Plant Physiol.2014 Nov；166(3):1162-1176.)。所以，从第51位氨基酸开始直到第446位氨基酸，对每一个氨基酸都做了饱和随机突变。这是通过用一个含有将需要突变的氨基酸编码子改为NNK的引物与另一适当的常规引物做搭桥PCR来实现的。在NNK中，N代表A/T/G/C，K代表G/T，因此NNK密码子可以编码20种氨基酸以及终止子中的任何一个，所以这是饱和突变。参考Kille S,Acevedo-Rocha CG,Parra LP,Zhang ZG,Opperman DJ,Reetz MT,Acevedo JP(2013)Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis.ACS Synth Biol 2(2):83-92；Directed Evolution Library Creation:methods and protocols 2nd ed.Edited by Elizabeth M.J.Gillam,Janine N.Copp and David F.Ackerley New York,NY United States:Springer,2014. doi:10.1007/978-1-4939-1053-3。这将产生大量的突变体。将突变体克隆到线性化pET-44a载体内，再转化到大肠杆菌里，用96-孔板在有HPPD抑制剂除草剂(比如环磺酮，1-2μM)和底物酪氨酸(1克/升)的2×YT培养液里，28℃150转/分钟摇床上培养24小时，表达，再根据其颜色变褐快速筛选这些突变体。我们用这样的方法，筛选到10个单氨基酸突变体，它们是A103S，H141R，H141K，H141T，A165V，V191I，R220K，G342D，D370N和K410I。这些抗性突变体相对于野生型对环磺酮和苯唑氟草酮代谢后产物的颜色反应如图1所示，其中，H141R和G342D颜色变化最为显著，明显深于野生型。
本专利的方法通过将NusA与水稻HPPD蛋白融合的方式提高了HPPD在细菌中表达的溶解度，从而可以在28摄氏度同时表达蛋白和进行酶促反应，大大节约筛选时间和步骤。
同样地，测试了这些突变体对另外三种HPPD抑制剂除草剂环吡氟草酮、苯唑草酮和硝磺草酮的颜色反应，参考图2和图3。根据其颜色深浅，对各个突变体针对这5种除草剂的抗性/耐受性进行了粗约估计，结果示于表1中。加号“+”越多，相对于野生型的颜色越深，抗性/耐受性就越高。
表1.水稻HPPD单位点突变体及其对5种HPPD抑制剂除草剂相对抗性
Figure PCTCN2019089512-appb-000003
以下以H141R突变体的产生和筛选为例说明其整个过程。
1、以NusOsF和OsHPPD-H141R-R：CACCGCGAGGCCGTGGTCC为引物，合成的全长OsHPPD模板和Q5 DNA聚合酶(NEB，New England Biolabs,Boston,USA)进行PCR扩增，得到前一个片段DNA。扩增条件为：98℃/2分；98℃/20秒→65℃/30秒→72℃/60秒(重复35次)；72℃/2秒。通过琼脂糖凝胶电泳检测后，回收大小正确的条带。并通过紫外吸收法确定浓度。同样地，以NusOsR和OsHPPD-H141R-F：
Figure PCTCN2019089512-appb-000004
TGCGCG为引物，合成的全长OsHPPD模板和Q5DNA聚合酶(NEB，New England Biolabs,Boston,USA)进行PCR扩增，得到后一个DNA片段。
2、前一个片段和后一个片段在中间(OsHPPD-H141R-F与OsHPPD-H141R-R)有19个碱基重叠。因此，将这两个片段各取等摩尔数混合，加入等体积的2×Glodstar MasterMix(康为世纪生物科技有限公司，北京)，再加入10pmol NusOsF和NusOsR引物，做搭桥PCR反应。扩增条件为：96℃2分；96℃20秒，65℃30秒，72℃60秒，重复30次；最后72℃5分。通过琼脂糖凝胶电泳检测后，回收1.3Kb大小正确的条带，并通过紫外吸收法确定浓度。
3、取等量OsHPPDMut DNA片段与上述线性化pET-44a载体混合，加入等体积2×Gibson Assembly Master Mix(汉恒生物)，于50℃孵育一个小时后，取5ul连接产物转化E.coli DH5a感受态，将菌液涂布到含100ppm氨苄青霉素的LB固体培养基平板表面，在37℃过夜培养。将平板上所有克隆(菌落)刮下，提取质粒，并用紫外吸收法定量DNA，取100ng质粒转化至BL21(DE3)感受态中，涂布平板，37℃过夜培养，暂时保存这些转化后的大肠杆菌平板在4℃下供突变体筛选用。
4、通过颜色反应筛选HPPD抑制剂除草剂抗性突变体
HPPD抑制性除草剂抑制HPPD酶活性，当酪氨酸在酪氨酸转氨酶作用下生成4-羟基苯丙酮酸(HPP)时，失活的HPPD无法将4-羟基苯丙酮酸氧化为尿黑酸(HGA)。尿黑酸呈黑褐色。所以，如果一个HPPD突变体抗或耐除草剂，在大肠杆菌里表达后，还能氧化4-羟基苯丙酮酸为尿黑酸而显现黑褐色。因此，我们用96-孔板在有HPPD抑制剂除草剂和底物酪氨酸的2×YT培养液里，培养大肠杆菌，表达HPPD，再根据其颜色变化快速筛选这些突变体。
(1)配制2×YT培养基(加入1g/L L-Tyr、0.1mM IPTG、0.01mM MnCl 2和氨苄青霉素100mg/L)。
(2)在96-孔板中每孔加入上述培养基0.1mL，从上述转化获得的大肠杆菌平板上，挑取OsHPPD野生型或突变体(OsHPPD Mut)表达克隆进行96-孔板液体培养。根据筛选药物的不同加入终浓度1uM-20uM的HPPD抑制剂，例如：环磺酮终浓度为1.7uM、硝磺草酮终浓度为10uM等。当所有组分加好后，盖上强透气封板膜(索来保生物制剂公司，中国，北京)。
(3)将96-孔板置于28℃150转/分钟摇床上培养24小时。用目视或检测培养物在400nM的光吸收，用接种环挑取产生黑色素明显的克隆，进一步培养，提取其质粒DNA进行测序和进一步研究，比如OsHPPD蛋白表达，纯化和酶活性测试。
用同样的方法，用表2所述引物我们分别得到了A103S、A165V、V191I、R220K、D370N、K410I单位点突变体和H141R/G342D/D370N三位点突变体。
表2 用于制备HPPD突变体的引物
引物名称 引物序列(5'-3')
NusOsF acg att gat gac gac gac aag ATGCCTCCCACTCCCACCCC
NusOsR tccacgagctcccggactcTTA CTAGGATCCTTGAACTGTAG
OsHPPD-H141R-F CACGGCCTCGCGGTGNNKGCCGTGGCGCTGCGCG
OsHPPD-H141R-R CACCGCGAGGCCGTGGTCC
OsHPPD-A103S-F GCGTTCCTCTTCACCNNKCCCTACGGCGGCGACCACG
OsHPPD-A103S-R GGTGAAGAGGAACGCGACGGAGGC
OsHPPD-A165V-F TCGCGGCCGGTGCGCGCCCGNNKTTCCAGCCCG
OsHPPD-A165V-R CGGGCGCGCACCGGCCGCGAC
OsHPPD-V191I-F GTCGTGCTCCGCTTCNNKAGCCACCCGG
OsHPPD-V191I-R GAAGCGGAGCACGACGTCGCC
OsHPPD-R220K-F CGCCGTGGACTACGGCCTCCGCNNKTTCGACCACG
OsHPPD-R220K-R GCGGAGGCCGTAGTCCACGGCGC
OsHPPD-D370N-F CTCGTGGACAGGGATNNKCAGGGGGTGTTGCTCCAGATCT
OsHPPD-D370N-R ATCCCTGTCCACGAGCACCCC
OsHPPD-K410I-F TGGGCAGGAGTACCAGNNKGGCGGCTGCGGCG
OsHPPD-K410I-R CTGGTACTCCTGCCCACTCTCATCC
OsHPPD-H141R/G342D/D370N-F GGCCTCAACTCGGTGGTGCTCG
OsHPPD-H141R/G342D/D370N-R GCGAGCACCACCGAGTTGAGGC
另外，又通过类似桥式PCR的方式制备了单突变组合成的一些双突变体，比如H141R/G342D，H141R/D370N和G342D/D370N。
实施例3 水稻HPPD(OsHPPD)蛋白多位点突变组合
本发明在不同时间不同96-孔板上还测试了H141R、G342D、D370N及其复合位点突变型HPPD蛋白对五种HPPD抑制性除草剂环磺酮、苯唑氟草酮代谢后产物、环吡氟草酮、苯唑草酮和硝磺草酮的颜色反应。测试结果示于图4、图5和图6。表3中显示了根据其颜色深浅粗约估计的这些突变体对相应除草剂的抗性/耐受性。从表3中可以看出，双位点突变体以及三位点突变体也展现出较高的耐药性，可见，水稻HPPD(OsHPPD)蛋白中的氨基酸突变可以组合存在，其同样能实现更高的HPPD抑制性除草剂抗性/耐受性。
表3.单位点及多位点突变型水稻HPPD对5种除草剂的抗性
Figure PCTCN2019089512-appb-000005
实施例4.在3点突变H141R-G342D-D370N基础上再次进行饱和突变。
1、在大肠杆菌表达3点突变OsHPPD H141R-G342D-D370N的颜色反应中，用双唑草酮代谢产物(代号101)来抑制时，需要120uM的浓度才无明显颜色反应。因此，我们用120uM的101做初始筛选。用前述的技术路线，针对氨基酸51-446的每一个氨基酸 (141R，342D，370N位点除外)分别设计一对引物，其中一个引物在要饱和突变的氨基酸位点用NNK表示，PCR扩增，产生一系列的突变体，将这些突变体在大肠杆菌BL21(DE3)里表达，表达后用120uM的化合物101进行反复的筛选。完成所有单点突变筛选后得到18个新的突变位点。这些新位点是R93S，G226H，L276W，P277N，P336D，P337A，N338D，N338S，N338Y，R346C，R346D，R346H，R346S，R346Y，I377C，P386T，L390I，M392L，E403G，K418P，G419F，G419L，G419V，N420S，N420T，E430G，Y431L。这些新突变点的氨基酸变化和核苷酸(碱基)以及产生这些新突变点所用的引物列在表4中，以及引物序列在表5中。综上，如图7所示，总共筛选得到26个突变点，其中，有的位点可以从原来的氨基酸变为多个不同氨基酸，比如H141R,K,T；N338D,S,Y；R346C,D,H,S,Y；G419F,L,V和N420S,T。
表4.新筛选出的所有突变位点氨基酸、碱基变化及所用引物
Figure PCTCN2019089512-appb-000006
Figure PCTCN2019089512-appb-000007
表5.新筛选出的所有突变位点位置及所用引物序列
Figure PCTCN2019089512-appb-000008
实施例5.突变位点的组合
基于以下三个原则对突变位点进行组合：位点相近便于基因编辑时的同源置换，编辑效率高；碱基改变同为A→G/T→C或C→T/G→A便于单碱基编辑；以及抗性位点尽量少便于编辑和避免可能的负面影响。分别按上述原则来设计组合、相应的引物和构建原核表达载体，再进行颜色反应筛选，找出适合于编辑和抗性高的组合来实施基因编辑。
(1)按相近距离原则设计了33个组合，其中24个组合有3个突变位点，9个组合有4个突变位点。表6展示了这些组合及其所用引物序列。
表6 相近原则设计的突变位点组合(三和四个突变位点)
Figure PCTCN2019089512-appb-000009
Figure PCTCN2019089512-appb-000010
Figure PCTCN2019089512-appb-000011
如图8所示，这些相近突变位点的各种组合，经克隆、表达和颜色反应比较后发现，其中，三个组合N338D/G342D/R346C、N338D/G342D/R346H和N338S/G342D/R346C的抗性最高，在高达1000-1500uM的双唑草酮代谢产物101存在下还有明显颜色反应；其次为两个各有四个相互接近的突变位点的组合P336D/N338D/G342D/R346C和P336D/N338D/G342D/R346H。(2)按有利于单碱基编辑原则，设计了6个组合：同为A→G/T→C的H141R/N338D/N420S，H141R/N338S/N420S，H141R/N338D和H141R/N420S，G342D/R346C和同为C→T/G→A的G342D/R346H。表7列出了产生这些组合的引物及序列。
表7.有利于单碱基编辑原则设计的突变位点组合
Figure PCTCN2019089512-appb-000012
经测试发现，上述6个组合的颜色反应也都较强，除草剂101浓度在600-1000uM的大肠杆菌培养液还能辨别有颜色反应。
(3)其它组合如表8所示。
表8.两、三、四个等突变位点的组合
Figure PCTCN2019089512-appb-000013
Figure PCTCN2019089512-appb-000014
Figure PCTCN2019089512-appb-000015
Figure PCTCN2019089512-appb-000016
经测试发现，肉眼可辨上述两点组合的颜色反应较弱，都在100uM的101以内；三点、四点及更多位点的组合中，H141R/G342D/N338D/R346C、H141R/G342D/N338D/R346H、H141R/G342D/N338D/K418P、H141R/G342D/N338D/G419F和H141R/G342D/N338D/G419F/N420S的颜色反应都很强，即使在1000-2000uM的101浓度下都有颜色(如图9所示)。
综上所述，单位点的抗性均在10-20uM；两位点抗性在20-120um左右，均比单位点强，100uM时颜色较淡；H141R/N338D/G342D、H141R/G342D/K418P、H141R/G342D/G419F、338D/342D/346C/H、H141R/G342D/N420S、H141R/N338D/N420S等三位点组合排序抗性较好，可到1000uM仍有淡的颜色；H141R/N338D/G342D/K418P、H141R/G342D/K418P/G419F、H141R/N338D/G342D/R346C、H141R/N338D/G342D/R346H、H141R/N338D/G342D/K418P/G419F、H141R/N338D/G342D/G419F/N420S、H141R/N338D/G342D/K418P/G419F/N420S等四或更多位组合的抗性较高，在高达2500uM的101时颜色仍很明显。
实施例6.OsHPPD酶蛋白的表达、分离和纯化
水稻OsHPPD蛋白和尿黑酸氧化酶HGD(homogentisate 1,2-dioxygenase)是由大肠杆菌异源表达，将基因插入到pET-15b表达载体中，利用BL21(DE3)表达菌株进行表达，再经过Ni-NTA resin纯化获得。
(1)将阳性克隆的HPPD开放读码框(ORF)再克隆至pET-15b载体中形成6His-HPPD表达载体，并转化至BL21(DE3)细胞中。接种表达菌株至10mL 2×YT培养液，37℃，200转/分钟摇床上培养过夜。将10mL培养物接种至1L 2×YT培养液中，培养到OD 600达到0.6-0.8时，降温到16℃，0.2mM IPTG(异丙基硫代半乳糖苷)诱导表达过夜，2800xg离心收菌。
(2)将收集好的菌体利用buffer A(50mM Tris pH 8.0,500mM NaCl,20mM咪唑)重悬，加入终浓度为1mM PMSF(苯甲基磺酰氟)，250ul protease inhibitor cocktail(多种蛋白酶抑制剂混合物)，混匀。在冰浴里用超声破碎细胞(40％总功率，3秒工作/6秒间隙，2x30分钟(宁波新芝科技有限公司，中国，宁波))；在12000转/分钟，4℃，30分钟，离心，取上清液，用0.22uM滤膜过滤。
(3)Ni-NTA柱纯化，将上述上清液与Ni-NTA resin结合后，用含有50mM咪唑的buffer A洗杂，最后用含400mM咪唑的洗脱缓冲液洗脱。
(4)SDS-PAGE分析目标蛋白的大致纯度，将含有目的蛋白的洗脱液合并后用超滤管(10kDa molecular weight cut-off,Amicon Ultra)浓缩。并用20mM Tris-HCl，pH 7.5溶液至少3次更换溶液和脱盐。用BCA法确定总蛋白浓度。所有表达、纯化的水稻HPPD野生型和各种突变体如表9所示。
表9.水稻HPPD野生型和各突变位点组合
Figure PCTCN2019089512-appb-000017
(5)透析或过脱盐柱，将缓冲液换为贮存液50mM Tris pH 8.0,500mM NaCl。BCA法测定浓度，分装后用液氮速冻后，保存于-80℃冰箱。
实施例7.化合物对OsHPPD蛋白各种酶学参数的测定
1、HPPD活性是通过检测HPPD酶催化4-HPP(4-羟基苯丙酮酸)转化为HGA(尿黑酸)，HGA(尿黑酸)在尿黑酸氧化酶的催化下转化为MAA(马来酰乙酰乙酸)，马来酰乙酰乙酸在318nm有最大吸收，吸收常数为14.7OD M -1cm -1。
2、在酶标板中加入6ul 50倍底物终浓度的4-HPP，通过一次加入294uL终浓度为25mM HEPES(羟乙基哌嗪乙硫磺酸)，pH 7，2mM维生素C，10mM FeSO4，50nM尿黑酸氧化酶(homogentisate dioxygenase)和5至240nM HPPD酶。反应底物4-HPP的终浓度一般在1至100uM。
3、用紫外酶标检测仪(ReadMax1900型光吸收型全波长酶标仪)(上海)连续监测反应孔在318nm的吸光度变化。
4、OsHPPD K m、K cat和K cat/K m测定：
V max是酶催化时所能达到的最大催化反应速率，米氏常数K m是酶催化反应达到最大速率(V max)一半时所需底物的浓度，K m值是一个常数，与酶浓度无关，随测定的底物种类、反应的温度、pH及离子强度而改变。K cat是酶的催化常数，表示每秒钟内一个酶分子或者酶活性中心能催化多少个底物发生反应。K cat/K m表示酶的催化效率。如表10所示，测定了水 稻HPPD野生型(WT)和各种突变体的这些酶学参数。从表中数据可以看出，绝大多数突变体HPPD的催化效率都有所增强。
表10 最大反应速率V max及米氏常数K m值测定
Figure PCTCN2019089512-appb-000018
5、除草剂101和环吡氟草酮对OsHPPD蛋白抑制能力IC50测定
通过对双唑草酮代谢产物101和环吡氟草酮对水稻野生型(WT)和各种突变型OsHPPD蛋白的抑制作用测定，如图10和表11所示，各突变体对101和环吡氟草酮除草剂化合物的IC50值相对于野生型都有明显提高，例如，H141R/N338D/G342D/K418P/G419F/N420S突变体对101除草剂化合物的IC50值相对于野生型提高至13.7倍，对环吡氟草酮的IC50值提高至1.8倍。IC50值的增加表明这些突变体对HPPD除草剂的耐受性增加，因101和环吡氟草酮各自的独特性质等，其增加程度在这两种HPPD除草剂之间并非完全一致。
表11.抑制剂101和环吡氟草酮对野生型WT、各突变型OsHPPDIC50值
Figure PCTCN2019089512-appb-000019
Figure PCTCN2019089512-appb-000020
6、OsHPPD蛋白对抑制剂适合度(Enzyme Fitness)测定
酶适合度(Enzyme Fitness)是酶对抑制剂适应性强弱的一个指标，值越大代表该酶对抑制剂抗性越强，由于反应时底物浓度远远大于K m值，且不同OsHPPD突变体反应条件相同，所以K cat可用相同浓度酶(500nM)催化时的速率V max值代替。Enzyme Fitness＝K cat*K m -1*Ki，Ki＝IC50/(1+S/K m)(抑制常数)。我们测试了除草剂化合物101和环吡氟草酮对水稻HPPD野生型(WT)和各种突变体的抑制常数和酶适合度，来进一步评估这些突变体对除草剂的耐受性。如表12所述结果表明，所有突变体的Enzyme Fitness与野生型相比都有不同程度的增强，确证其耐受性增强。
表12.OsHPPD对不同化合物的酶适合度(Enzyme Fitness)测定
Figure PCTCN2019089512-appb-000021
综上所述，通过一系列酶学测试证明，水稻OsHPPD各突变体耐药性相对野生型WT均有提高。其中，在颜色反应中显示出最强抗性的四突变体H141R/N338D/G342D/K418P，在体外酶活实验中也有较高的抗性，且结果显示带有K418P位点突变的突变体，抗性提高都比较好，但是底物亲和性(K m)有所降低；而突变体H141R/G342D/P277N与H141R/N338D/G342D/K418P抗性相当，并且没有底物亲和性(K m)变低的问题；另外，三突变体H141R/N338D/G342D和四突变体H141R/N338D/G342D/P386T也具有较强抗性，且K m没有降低；最短三突变体N338D/G342D/R346H，同样具有较好抗性，且易于编辑。
因此，优先考虑N338D/G342D/R346H(最短，适合同源置换HDR)、H141R/N338D/G342D(较短，也适合做同源置换)以及H141R/N338D/N420S(适合单碱基编辑，抗性较强)进行下一步试验，做转基因和基因编辑植物后，再测试去耐受性变化。
实施例8.转基因水稻过表达三点突变体OsHPPD3M
1、过表达载体的构建
1)引物：根据选取的酶切位点以及基因本身的核苷酸序列，设计引物用于扩增三点突变型HPPD(H141R/G342D/D370N)(OsHPPD3M)。设计的引物由北京擎科新业生物技术有限公司合成：HPPD–F， GATAGCCGGTACGGGTTCGAGCCACC ATGCCTCCCACT CCCACCC，HPPD–R， GATCTTTGTAATCGGGGTACCTAGGATCCTTGAACTGTAGGGGC。
2)PCR扩增：利用合成的引物和Q5DNA聚合酶(NEB,New England Biolabs,Boston,USA)扩增目的基因。扩增产物经琼脂糖凝胶电泳检测，按照TIANquick Midi Purification kit操作说明，回收产物。回收完成后，用Nanodrop检测提取的DNA浓度。
3)水稻过表达载体构建：将经KpnI和HindIII酶切后回收的HPPD片段和质粒pCAMBIA1301，采用汉恒生物科技(上海)有限公司的HB-in fusion TM无缝克隆试剂盒构建水稻过表达载体pCAMBIA1301-OsHPPD3M。经转化到感受态的E.coli DH5α，得到阳性克隆，再经测序验证和限制性内切酶酶切验证后，转化农杆菌。
2、水稻愈伤的农杆菌转化和转基因事件产生：
1)水稻过表达载体pCAMBIA1301-OsHPPD3M以及空载只表达mCherry(荧光蛋白标记基因)的pCAMBIA1301，各1ug加入到农杆菌EH105感受态中，放在冰上5分钟，浸入液态氮中速冻5分钟，捞出放在37℃下5分钟，最后放置在冰上5分钟。加入500μl YEB液体培养基(无抗生素)，置于28℃、200转/分摇床中培养2～3h；用3500转/分离心收集菌体，将收集的菌体涂布于YEB(卡拉霉素+利福平)平板上，28℃培养箱培养2天；挑取单克隆至液体培养基培养，-80℃保菌。
2)农杆菌的培养：挑取转化后的农杆菌单克隆，YEB液体培养基(卡拉霉素+利福平)中28℃振荡培养至OD600为0.5，3500转/分收集菌落，等量AAM(1ml AAM+1μl 1000×AS)液体培养基稀释后侵染愈伤组织。
3)诱导水稻中花11愈伤组织：在准备农杆菌之前，先要准备水稻愈伤。剥取水稻种子，无菌水清洗种子，直至洗后的水变清澈，不限次数，70％酒精消毒30秒，之后5％次氯酸钠置于水平摇床摇晃培养20分，次氯酸钠消毒后无菌水清洗5次，置于无菌吸水纸，风干种子表面水分，接种于诱导培养基上在28℃下培养愈伤。
4)农杆菌侵染水稻愈伤：选取继代培养10天，直径为3mm的淮稻5号愈伤组织，将愈伤组织收集至50ml的离心管中；将已调制浓度的农杆菌菌液倒入含有愈伤组织的离心管中，置于28℃、200转/分摇床中侵染20分；侵染完毕，倒掉菌液，将愈伤组织放置于无菌滤纸上风干20min左右，置于共同培养基平板上共同培养，平板上铺有一张AAM(1ml AAM+30μl  1000×AS)液体培养基浸湿的无菌滤纸；侵染3天后，清洗去除农杆菌(先用无菌水洗5遍，再用500mg/L的头孢抗生素清洗20分钟)，置于50mg/L潮霉素筛选培养基上筛选培养。
5)抗性愈伤的筛选、分化和生根：将共培养后的愈伤组织移至筛选培养基进行第一轮筛选(2周)；第一轮筛选完毕后将新长出的愈伤移至筛选培养基(含50mg/L潮霉素)进行第二轮筛选(2周)；筛选完成后，挑取生长状态良好的黄白色愈伤组织进行分化，分化培养基中添加1uM-5uM的环磺酮进行除草剂抗性筛选，3～4周后可以获得1cm左右的幼苗；将分化出的幼苗移至生根培养基进行生根培养；将生根完成的幼苗进行炼苗处理后，移至装有土壤的花盆中置温室进行培养；获得OsHPPD3M 55株幼苗或事件。
3、转基因苗(T0代)除草剂抗性初步检测：在分化培养基中添加1-5uM的环磺酮，结果显示空载体对照对环磺酮没有耐受性，过表达3点突变HPPD(H141R/G342D/D370N)的转化苗可以耐受3uM的环磺酮，如图11所示。
4、转基因苗(T0代)除草剂抗性再次检测：T0代转基因苗移栽到温室的大的塑料桶里培育以求得到T1代种子。在拔节期从过表达突变型的事件中，随机各选出2个事件，加一组非转基因的中花11相同生育期的稻苗，进行除草剂抗性测定。所用除草剂是双唑草酮，其田间剂量通常是每亩4克有效成分(4g，a.i./mu)。本次实验双唑草酮的剂量是8和16克/亩。
抗性检测结果：喷施后第5天(16克/亩)、7天(8克/亩)时，非转基因水稻苗开始显现白化症状，但转基因的过表达突变型的Event 1、Event 2、Event 3和Event 4都保持正常绿色。直到32天后，喷过药的非转基因苗接近死亡，但转基因过表达突变型的，不论8克/亩，还是16克/亩处理，都继续保持绿色，生长也正常，并开始抽穗(如图12A、12B所示)。
5、转基因苗(T1代)除草剂抗性再次检测：
a)转基因过表达突变型HPPD的三个事件Event 20、Event 28、Event 37，加上非转基因野生型的淮稻5号(淮稻5号对HPPD双唑草酮的天然耐受性高于中花11，因此不能以淮稻5号做基数计算抗性倍数，实际抗性应该更高)。
b)所用双唑草酮的剂量为0、4、8、16、32到64克/亩，由于冬季温室的温度较低，光照弱，药害症状显现慢。到喷药后第14天，在32和64克/亩处理下的非转基因淮稻5号显现症状，但转基因的事件都没有症状，仍为绿色(如图12C、12D所示)。
综上，过表达三点突变型OsHPPD3M(H141R/G342D/D370N)都增强了转基因水稻对HPPD抑制性除草剂的抗性，抗性倍数至少是4倍。从T0代和T1代的转基因苗生长发育、开花结实的初步观察，大部分事件植株都很正常。
实施例9 转基因水稻过表达HPPD拷贝数测定
潮霉素抗性基因：水稻hppd(Oshppd)基因的GC含量很高，影响PCR扩增效率，另外 水稻内源也有一个拷贝的hppd。因此，我们选择筛选标记基因潮霉素抗性基因hyg作为外源基因，蔗糖磷酸合成酶基因(sucrose phosphate synthase,SPS)作为内源参照基因进行拷贝数估算。蔗糖磷酸合成酶(SPS)基因是水稻特有的基因，并且为单拷贝，可以作为水稻的内源参照基因(Ding Jiayu，Jia Junwei,Yang Li tao et al.Validation of a rice specific gene,sucrose phosphate synthase,used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes[J].J.Agric.Food Chem.,2004,52:3372-7)。可以通过测定转基因水稻筛选标记的潮霉素基因(hyg)的拷贝数来间接评估过表达hppd的拷贝数。
基因组DNA：利用天根生化科技(北京)公司的植物基因组DNA提取试剂盒，对水稻叶片基因组DNA进行提取和纯化，利用纳米滴核酸蛋白测定仪(nanodrop)对DNA的含量和纯度进行检测，当OD260/OD280的比值在1.8-2.0范围内，OD260/OD230的比值在2.0左右时认为纯度较好。
引物：设计两对引物：Hyg-F：5’-GTACACAAATCGCCCGCAG-3’，Hyg-R:5’-TCTATTTCTTTGCCCTCGGAC-3’，扩增潮霉素抗性基因111bp长的片段；Sps-F:5’-GTACACAAATCGCCCGCAG-3’，Sps-R:5’-TCTATTTCTTTGCCCTCGGAC-3’，扩增蔗糖磷酸合成酶(SPS)基因170bp的片段。
定量PCR反应体系：按SYBR Premix ExTaq II体系配制反应液(20μL)，进行实时荧光定量PCR。PCR扩增程序：预变性95℃/30S，再经95℃/5S→55℃/30S→72℃/30S，40个循环。
标准曲线的制作：分别选取SPS基因和HYG基因的400bp的序列，所选取的序列需包含定量PCR扩增的片段，并通过同源重组的方式将其连接在一起，然后将其连接进入pClone007载体内。将构建好的含有HYG基因和SPS基因的标准品质粒用限制性内切酶PshaⅠ酶切为线性化DNA，用核酸蛋白检测仪测定浓度，用ddH 2O梯度稀释为10 6拷贝/μL、10 5拷贝/μL、10 4拷贝/μL、10 3拷贝/μL、10 2拷贝/μL。将5个不同稀释度的标准品与空白对照同时进行扩增，每个样品设置三个技术重复。然后按照上述方法进行PCR扩增。浓度与拷贝数换算公式：拷贝数(拷贝/mL)＝(6.02×1023拷贝/mol)×(DNA浓度g/ml)/(MW g/mol)。平均分子量(MW g/mol)：dsDNA＝(碱基数)×(660dalton/bp)。
转基因拷贝数的计算：每一个被检测样本在达到阈值时都有一个循环数Ct，将Ct值代入标准曲线中得到该样本的起始模板量，目的基因与内源基因起始模板量的比值即是目的基因的拷贝数。实验所得数据经过软件输出后采用Excel进行数据分析。
实时荧光定量PCR：采用qRT-PCR方法，对转基因水稻相关基因表达量进行分析，验证基因过表达的效率。选用水稻的UBQ5基因作为内参基因。按配制反应液，进行实时荧光定 量PCR。按SYBR Premix ExTaq II体系配制反应液(20μL)。qRT-PCR扩增程序设定为：95℃预变性，30s；95℃变性，5s；60℃退火，30s；65℃延伸5min，共进行40次循环。实验所得数据经过软件输出后采用Excel进行数据分析，运用△△CT对基因的相对表达量进行计算。所有样品均设置3次独立生物学重复。
在本实验中，选取54株PCR检测为阳性的植株和4株非转基因对照植株，利用植物基因组DNA提取试剂盒提取基因组DNA，每个样品做3个重复，进行定量PCR反应，获得扩增曲线，荧光域值的设定与制作基因标准曲线时相同，获得待测样品的Ct值，并由方程：HYG 0＝10 (-0.260CT+10.442)计算出该样品HYG基因的起始模板数，由方程：SPS 0＝10 (-0.260CT+10.172)计算出该样品Sps基因的起始模板数。由于水稻内参基因Sps是纯合二倍体，而转基因当代的外源目的基因是纯合体的几率极小，因此HYG起始模板数除以SPS起始模板数所获得的数据，乘以2就是目的基因在水稻基因组中的拷贝数。通过目的基因Hyg和水稻内源参照基因Sps起始模板数的比较，由表13结果显示：在54株转基因株系中，有36株拷贝数为1，有13株拷贝数为2，拷贝数为3的有4株，拷贝数为4的有1株，而所检测阴性对照拷贝数为0。
表13 转基因植株目的基因拷贝数的估算
Figure PCTCN2019089512-appb-000022
Figure PCTCN2019089512-appb-000023
注：HYG 0和SPS 0分别表示Hyg和Sps基因在PCR反应中的起始模板数。
实施例10.水稻基因编辑抗HPPD抑制性除草剂
在上述突变、筛选水稻HPPD基因，得到三点突变141、342、370及其组合，在离体测定中能耐受HPPD抑制性除草剂的基础上，先用转基因水稻过表达一个突变体组合OsHPPD3M(H141R/G342D/D370N)确证其对HPPD抑制性除草剂的高耐受性，稍后又对HPPD开展了基因编辑以得到非转基因的耐受HPPD抑制性除草剂的水稻品种。首先针对氨基酸141、342、370三位点分别进行单碱基编辑和141-342-370三位点的同源置换，其基因编辑过程及结果如下：
(1)单碱基编辑是利用CRISPR/Cas9系统将脱氨酶靶向基因组中特定的位点从而对特定碱基进行修饰的基因编辑方法。此种方法已经在水稻中成功运用。如：Yan F.,Kuang Y.,Ren B.,Wang J.,Zhang D.,Lin H.,Yang B.,Zhou X.,and Zhou H.(2018).High-efficient A·T to G·C base editing by Cas9n-guided tRNA adenosine deaminase in rice.Mol.Plant.doi: 10.1016/j.molp.2018.02.008。
在本实施例中，对水稻HPPD基因位于第二染色体Os02g0168100位点中第141、342和370位的氨基酸分别进行编辑。水稻对HPPD基因141位氨基酸残基的组氨酸(Histidine；密码子
Figure PCTCN2019089512-appb-000024
)通过单碱基编辑的方法编辑为精氨酸(Arginine；密码子
Figure PCTCN2019089512-appb-000025
，将原来的
Figure PCTCN2019089512-appb-000026
变为
Figure PCTCN2019089512-appb-000027
)。同样地，将第342位氨基酸残基的甘氨酸(Glycine；密码子
Figure PCTCN2019089512-appb-000028
)编辑为天冬氨酸(Aspartic acid；密码子
Figure PCTCN2019089512-appb-000029
，将原来的
Figure PCTCN2019089512-appb-000030
变为
Figure PCTCN2019089512-appb-000031
)；将第370位氨基酸残基天冬氨酸(Aspartic acid；密码子
Figure PCTCN2019089512-appb-000032
)编辑为天冬酰胺(Asparagine；密码子
Figure PCTCN2019089512-appb-000033
，将原来的
Figure PCTCN2019089512-appb-000034
变为
Figure PCTCN2019089512-appb-000035
)。选择具有更广泛PAM的Cas9蛋白的突变型蛋白xCas9(3.7)-ABE作为编辑工具(Hu,J.H.et al.Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.Nature http://dx.doi.org/10.1038/nature26155(2018))。
根据水稻HPPD基因第141位氨基酸附近的DNA序列，设计了sgRNA的靶位点：GGTGC aCGCCGTGGCGCTGC-GCG,其中 a为ABE的作用位点，以实现A碱基至G碱基的编辑。此sgRNA的PAM为GCG符合xCas9(3.7)的要求。
同理，根据水稻HPPD基因第342位氨基酸附近的DNA序列，设计了sgRNA的靶位点：GCACG cCGTCGTAGTAGTTG GGC,其中 c为CBE的作用位点，以实现C碱基至T碱基的编辑。此sgRNA的PAM为GGC符合xCas9(3.7)的要求。
分析水稻HPPD基因第370位氨基酸附近的的DNA序列，设计了sgRNA的靶位点：CCTGGT cATCCCTGTCCACG AGC,其中 c为CBE的作用位点，以实现C碱基至T碱基的编辑。此sgRNA的PAM为GGC也符合xCas9(3.7)的要求。
因此，我们合成三对引物141GE-F:ggcgGTGCaCGCCGTGGCGCTGC和141GE-R：
aaacGCAGCGCCACGGCGtGCAC；342GE-F：ggcgCACGcCGTCGTAGTAGTTG和342GE-R:aaacCAACTACTACGACGgCGTG；370GE-F：ggcg CCTGGTcATCCCTGTCCACG和370GE-R:aaacCGTGGACAGGGATgACCAGG。以超纯水稀释至10uM后，等量混合后置于沸水浴中，待其自然冷却至室温后备用。将1ug pQY000140载体经过BsaI酶37℃切割一小时，通过琼脂糖凝胶电泳检测后，回收目的片段，并通过紫外吸收法确定浓度。与煺火片段1：10混合，以T4DNA连接酶(NEB，New England Biolabs,Boston,USA)于16℃连接2小时。转化Trans5a感受态细胞(全式基因，北京)，置于37℃培养过夜。挑取单克隆通过sanger测序单碱基编辑载体序列是否正确。构建载体pQY000141如图13所示。将测序正确的大肠杆菌克隆提取质粒，转化至EH105农杆菌(唯地生物，上海)。
按前述水稻愈伤农杆菌转化方法，对淮稻5号愈伤(至少3000愈伤)进行转化。农杆菌侵染后，将侵染后的愈伤转到50mg/L hyg筛选培养基上筛选培养。三轮(15天x3)筛选后， 挑取生长状态良好的黄白色愈伤组织在分化培养基上进行分化，在分化过程中加入0.2uM环磺酮进行筛选3～4周后获得1cm左右的幼苗约1500株。在这约1500株分化苗中，绝大多数已白化，只有4株任是正常绿色。将这4株绿色苗移植到含有0.4uM的环磺酮的生根培养基中继续培养2周，其中两株也白化，只有2株还保持绿色(图14A)。取少量叶片以CTAB法提取基因组DNA。以引物oshppd54F：TTCCACCACGTCGAGCTC和Oshppd356R：GGTGAACCCGGAGATGTACG进行PCR。扩增产物以1％琼脂糖电泳检测后，进行sanger测序。
测序结果表明，这2株绿色苗(QY000141-1和QY000141-2)在氨基酸141位点都有成功编辑(图14B)而白化苗是野生型。
(2)CRISPR/cas9介导的水稻HPPD突变体同源置换以获得除草剂抗性
在获得转基因事件过表达3点突变体H141R/G342D/D370N后，我们也对这3突变点组合进行同源置换，以期获得抗除草剂的非转基因水稻。
水稻hppd基因有两个外显子(exon)，一个内含子(intron)。三个靶标位点H141、G342和D370都位于第一外显子。
gRNA设计：在H141的上游和D370的下游至少各设计一个gRNA，拟各切一刀，再用同源置换的方法把三个位点同时置换。把外显子1序列输入到 http://crispor.tefor.net/评估所有可能的gRNA.根据特异性分数值大于90(Hsu PD,Scott DA,Weinstein JA,Ran FA,Konermann S,Agarwala V,Li Y,Fine EJ,Wu X,Shalem O,Cradick TJ,Marraffini LA,Bao G,Zhang F.Nat Biotechnol.2013Sep；31(9):827-32.doi:10.1038/nbt.2647.Epub 2013Jul 21)，尽量避免脱靶效应和缩短其长度等原则，选择了下列2个gRNA：OshppdgRNA-PAM1-2:5’-GGAACGCGAGCGCCTGGAAC
Figure PCTCN2019089512-appb-000036
-3’(GC＝70％)(bottom strand)；
OshppdgRNA-PAM2-1(GC＝39％):5’-CACCTCTTTCATGATGAAAA
Figure PCTCN2019089512-appb-000037
-3’(top strand)；
有下划线的为PAM序列，加粗表示的
Figure PCTCN2019089512-appb-000038
是指设计置换模板DNA时，在置换模板上这个
Figure PCTCN2019089512-appb-000039
将被变为其它碱基，以破坏这些PAM，避免置换后又被重新切开。
这两个gRNA1-2和gRNA2-1在水稻基因组DNA上的分布如图15所示。
模板供体DNA设计如图16所示：根据赵云德实验室(Sun Y,Zhang X,Wu C,He Y,Ma Y,Hou H,Guo X,Du W,Zhao Y,Xia L.Engineering Herbicide-Resistant Rice Plants through CRISPR/Cas9-Mediated Homologous Recombination of Acetolactate Synthase.Mol Plant.2016 Apr 4；9(4):628-31.doi:10.1016/j.molp.2016.01.001.Epub 2016Jan 6)，我们先将同源臂设计在350bp；为增加同源置换可能性，为每个编辑载体都设计了两个版本的模板供体：将模板直接 连在编辑载体上，这样gRNA、Cas9和模板同时进入同一细胞，一旦Cas9和gRNA把细胞基因组靶标DNA切割后，模板供体DNA能及时修复；另一版本是通过PCR扩增而产生的自由的模板供体DNA，这些额外的修复模板，将以20：1(自由修复模板：编辑载体，摩尔比)与编辑载体混合，再用基因枪轰击。三个突变氨基酸141-342-370的核心置换区域的长度是由两个选定的靶向RNA切割位置所确定(1056bp)，左右同源臂各350bp，再加上从载体上切割后在左右两端各留下6bp，模板总长1768bp；为便于PCR扩增后对其PCR产物进行快速基因型鉴定，去掉了其中的NcoI酶切位点；同时为避免置换后又被重新切割，模板上的原切割位点处的PAM(NGG)也被去掉。
编辑载体：用水稻U3启动子分别表达gRNA1-2和gRNA2-1。因此，将这两个gRNA表达框与模板连接在一起后送到金斯瑞生物科技公司(南京)合成。合成后DNA片段再用无缝克隆技术在KpnI处连接到骨架载体pCXUN-Cas9(来自华中农业大学和玉兵博士,Mol Plant.2016 Apr 4；9(4):628-31.doi:10.1016/j.molp.2016.01.001.Epub 2016 Jan 6.)产生编辑载体。
基因枪转化、筛选、分化、生根和土培苗：上述构建的编辑载体经测序和多酶切验证后，与通过PCR扩增而产生的自由的模板供体DNA，以20：1(自由修复模板：编辑载体，摩尔比)与编辑载体混合，用基因枪转化淮稻5号愈伤。约3000愈伤被转化后，转到50mg/L hyg筛选培养基上筛选培养以期先得到转基因植株。三轮(15天x3)筛选后，挑取生长状态良好的黄白色愈伤组织在分化培养基上进行分化，在分化过程中加入0.2uM环磺酮进行筛选3～4周后获得1cm左右的幼苗约1000株。在这约1000株分化苗中，绝大多数已白化，但有21株还保持绿色。将这21株绿色苗转移到含0.4uM环磺酮的生根培养基中继续培养，促进根系生长。两周后，又有19株白化，将剩下的2株绿苗(编号AW2和AW3)移栽到花盆中，置温室培养。移栽前拍摄的绿色苗如图17A所示。
编辑苗的hppd基因型鉴定：为鉴定其基因型，我们设计了三对PCR引物来分别扩增342-370突变位点区域、342-370区域+部分下游基因组DNA序列和141单点。这些引物对是290-F：AGATACAGACGTACCTGGACCACCA与1553-R：GCCGGCAAAAAGGAACTGGG(342-370突变位点区域)、90-F：AGATACAGACGTACCTGGACCACCA与donor-out-R：AGTGATTGTACCATCATTTGTC(342-370区域+部分下游基因组DNA序列)，以及54-F：TTCCACCACGTCGAGCTC与356-R：GGTGAACCCGGAGATGTACG(141单点)。
鉴定结果显示：这2株绿苗确有成功编辑(图17B)。PCR产物用NcoI酶切后产生的条带符合预期；测序结果也显示在141点由野生型的组氨酸His变为精氨酸Arg(密码子从
Figure PCTCN2019089512-appb-000040
变为
Figure PCTCN2019089512-appb-000041
)、在342点由野生型的甘氨酸Gly变为天冬氨酸Asp(密码子从
Figure PCTCN2019089512-appb-000042
变为
Figure PCTCN2019089512-appb-000043
)，在370点由野生型天冬氨酸Asp变为天冬酰胺Asn(密码子从
Figure PCTCN2019089512-appb-000044
变为
Figure PCTCN2019089512-appb-000045
)。
同时经过很多测试发现，将本发明所述基因导入拟南芥、二穗短柄草等模式植物中，都产生了相应水平的耐药性提升。另外，经CRISPR/Cpf1系统来编辑上述突变位点及组合也在应用中。由此可知，将其转基因或者基因编辑到其他前述的植物，如粮食作物、豆类作物、油料作物、纤维作物、水果类作物、根茎类作物、蔬菜类作物、花卉作物、药用作物、原料作物、牧草作物、糖料作物、饮料作物、草坪植物、树木作物、坚果作物等，也会产生相应的抗性性状，具有良好的产业价值。
说明书中提及的所有出版物和专利申请均通过引用并入本文，如同每篇出版物或专利申请被单独、特别地通过引用并入本文一样。
尽管为清楚理解起见，前述发明已通过举例说明和实施例的方式较为详细地进行了描述，但显而易见的是，可以在所附权利要求书的范围内实施某些改变和修改，这样的改变和修改均在本发明的范围之内。

Claims (21)

  1. 一种突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段，其中所述突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白的氨基酸序列与野生型水稻对羟苯基丙酮酸双氧化酶蛋白的氨基酸序列相比，在对应于SEQ ID NO:2所示的野生型水稻对羟苯基丙酮酸双氧化酶蛋白氨基酸序列中的第93、103、141、165、191、220、226、276、277、336、337、338、342、346、370、377、386、390、392、403、410、418、419、420、430和431位中的一个或多个位置处具有选自下述中的一个或多个突变：93S、103S、141R、141K、141T、165V、191I、220K、226H、276W、277N、336D、337A、338D、338S、338Y、342D、346C、346D、346H、346S、346Y、370N、377C、386T、390I、392L、403G、410I、418P、419F、419L、419V、420S、420T、430G和431L。
  2. 权利要求1所述的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段，其中所述突变型对羟苯基丙酮酸双氧化酶蛋白的氨基酸序列进一步与SEQ ID NO:2所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性的氨基酸序列。
  3. 权利要求1所述的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段，其中所述突变型对羟苯基丙酮酸双氧化酶蛋白具有SEQ ID NO:2所示的氨基酸序列，区别在于具有权利要求1中所定义的一个或多个氨基酸突变。
  4. 权利要求1－3中任一项所述的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段，其中所述突变型对羟苯基丙酮酸双氧化酶蛋白的氨基酸序列在对应于SEQ ID NO:2所示的野生型水稻对羟苯基丙酮酸双氧化酶蛋白氨基酸序列中的第93、103、141、165、191、220、226、276、277、336、337、338、342、346、370、377、386、390、392、403、410、418、419、420、430和431位中的一个或多个位置处具有选自下述中的一个或多个突变：R93S、A103S、H141R、H141K、H141T、A165V、V191I、R220K、G226H、L276W、P277N、P336D、P337A、N338D、N338S、N338Y、G342D、R346C、R346D、R346H、R346S、R346Y、D370N、I377C、P386T、L390I、M392L、E403G、K410I、K418P、G419F、G419L、G419V、N420S、N420T、E430G和Y431L。
  5. 权利要求4所述的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段，其中所述突变型对羟苯基丙酮酸双氧化酶蛋白具有SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ  ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82或SEQ ID NO:84所示的氨基酸序列。
  6. 权利要求4所述的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段，其中所述突变型对羟苯基丙酮酸双氧化酶蛋白的氨基酸序列具有如下氨基酸突变：H141R/G342D、H141R/D370N、G342D/D370N、H141R/N338D、H141R/G342D、N338D/G342D、K418P/G419F、G419F/N420S、G342D/R346C、G342D/R346H、H141R/N420S、G338D/K418P、P277N/N338D、L276W/P277N、H141R/G342D/D370N、H141R/N338D/N420S、H141R/N338S/N420S、P336D/N338D/G342D、P336D/N338S/G342D、P336D/N338Y/G342D、N338D/G342D/R346C、N338D/G342D/R346H、N338D/G342D/R346S、N338S/G342D/R346C、N338S/G342D/R346H、N338S/G342D/R346S、N338Y/G342D/R346C、N338Y/G342D/R346H、N338Y/G342D/R346S、P336D/G342D/R346C、P336D/G342D/R346H、P336D/G342D/R346S、P336D/N338D/R346C、P336D/N338D/R346H、P336D/N338D/R346S、P336D/N338S/R346C、P336D/N338S/R346H、P336D/N338S/R346S、P336D/N338Y/R346C、P336D/N338Y/R346H、P336D/N338Y/R346S、H141R/N338D/G342D、H141R/G342D/K418P、H141R/G342D/G419F、H141R/G342D/P386T、K418P/G419F/N420T、K418T/G419F/N420T、H141R/G342D/R346C、H141R/G342D/R346H、H141R/G342D/N420S、H141R/G342D/P277N、H141R/G342D/P336D、H141R/G342D/L276W、H141R/G342D/R346S、H141R/G342D/L390I、H141R/G342D/I377C、H141R/G342D/M392L、H141R/P337A/G342D、H141R/N338S/G342D、H141R/N338Y/G342D、P277N/N338D/G342D、P277N/G342D/R346C、P277N/N338D/N420S、N338D/G342D/K418P、H141R/N338D/G342D/K418P、H141R/N338D/G342D/G419F、H141R/N338D/G342D/P386T、H141R/N338D/G342D/R346C、H141R/N338D/G342D/R346H、H141R/G342D/K418P/G419F、H141R/G342D/L276W/P277N、P336D/N338D/G342D/R346C、P336D/N338D/G342D/R346H、P336D/N338D/G342D/R346S、P336D/N338S/G342D/R346C、P336D/N338S/G342D/R346H、P336D/N338S/G342D/R346S、P336D/N338Y/G342D/R346C、P336D/N338Y/G342D/R346H、P336D/N338Y/G342D/R346S、P277N/P336D/N338D/G342D、P277N/N338D/G342D/R346C、P277N/N338D/K418P/G419F、H141R/N338D/G342D/K418P/G419F、H141R/N338D/G342D/G419F/N420S、H141R/G336D/G342D/K418P/G419F/N420S、H141R/N338D/G342D/K418P/G419F/N420S、H141R/N338D/G342D/K418P/G419F/N420T、H141R/N338D/G342D/R346C/K418P/G419F/N420S、H141R/N338D/G342D/ R346H/K418P/G419F/N420S、H141R/P277N/N338D/G342D/K418P/G419F/N420S、H141R/P277N/P336D/N338D/G342D/K418P/G419F/N420S。
  7. 权利要求6所述的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段，其中所述突变型对羟苯基丙酮酸双氧化酶蛋白具有SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:116、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:122、SEQ ID NO:124、SEQ ID NO:126、SEQ ID NO:128、SEQ ID NO:130、SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138、SEQ ID NO:140、SEQ ID NO:142、SEQ ID NO:144、SEQ ID NO:146、SEQ ID NO:148、SEQ ID NO:150、SEQ ID NO:152、SEQ ID NO:154、SEQ ID NO:156、SEQ ID NO:158、SEQ ID NO:160、SEQ ID NO:162、SEQ ID NO:164、SEQ ID NO:166、SEQ ID NO:168、SEQ ID NO:170、SEQ ID NO:172、SEQ ID NO:174、SEQ ID NO:176、SEQ ID NO:178、SEQ ID NO:180、SEQ ID NO:182、SEQ ID NO:184、SEQ ID NO:186、SEQ ID NO:188、SEQ ID NO:190、SEQ ID NO:192、SEQ ID NO:194、SEQ ID NO:196、SEQ ID NO:198、SEQ ID NO:200、SEQ ID NO:202、SEQ ID NO:204、SEQ ID NO:206、SEQ ID NO:208、SEQ ID NO:210、SEQ ID NO:212、SEQ ID NO:214、SEQ ID NO:216、SEQ ID NO:218、SEQ ID NO:220、SEQ ID NO:222、SEQ ID NO:224、SEQ ID NO:226、SEQ ID NO:228、SEQ ID NO:230、SEQ ID NO:232、SEQ ID NO:234、SEQ ID NO:236、SEQ ID NO:238、SEQ ID NO:240、SEQ ID NO:242、SEQ ID NO:244、SEQ ID NO:246、SEQ ID NO:248、SEQ ID NO:250、SEQ ID NO:252、SEQ ID NO:254、SEQ ID NO:256、SEQ ID NO:258或SEQ ID NO:260所示的氨基酸序列。
  8. 一种融合蛋白，其中包含权利要求1-7中任一项所述的突变型HPPD蛋白或其生物活性片段，以及与之融合的其它组分，例如标签肽如6×His，或者质体引导肽例如引导到叶绿体内的肽。
  9. 一种分离的多核苷酸，其中包含编码权利要求1-7中任一项的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段或者权利要求8中所述的融合蛋白的核酸序列或者其互补序列；所述的多核苷酸优选是DNA、RNA或者其杂合体；所述的多核苷酸优选是单链的或双链的。
  10. 权利要求9所述的多核苷酸，其具有选自下述的核酸序列：
    (1)编码SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:116、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:122、SEQ ID NO:124、SEQ ID NO:126、SEQ ID NO:128、SEQ ID NO:130、SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138、SEQ ID NO:140、SEQ ID NO:142、SEQ ID NO:144、SEQ ID NO:146、SEQ ID NO:148、SEQ ID NO:150、SEQ ID NO:152、SEQ ID NO:154、SEQ ID NO:156、SEQ ID NO:158、SEQ ID NO:160、SEQ ID NO:162、SEQ ID NO:164、SEQ ID NO:166、SEQ ID NO:168、SEQ ID NO:170、SEQ ID NO:172、SEQ ID NO:174、SEQ ID NO:176、SEQ ID NO:178、SEQ ID NO:180、SEQ ID NO:182、SEQ ID NO:184、SEQ ID NO:186、SEQ ID NO:188、SEQ ID NO:190、SEQ ID NO:192、SEQ ID NO:194、SEQ ID NO:196、SEQ ID NO:198、SEQ ID NO:200、SEQ ID NO:202、SEQ ID NO:204、SEQ ID NO:206、SEQ ID NO:208、SEQ ID NO:210、SEQ ID NO:212、SEQ ID NO:214、SEQ ID NO:216、SEQ ID NO:218、SEQ ID NO:220、SEQ ID NO:222、SEQ ID NO:224、SEQ ID NO:226、SEQ ID NO:228、SEQ ID NO:230、SEQ ID NO:232、SEQ ID NO:234、SEQ ID NO:236、SEQ ID NO:238、SEQ ID NO:240、SEQ ID NO:242、SEQ ID NO:244、SEQ ID NO:246、SEQ ID NO:248、SEQ ID NO:250、SEQ ID NO:252、SEQ ID NO:254、SEQ ID NO:256、SEQ ID NO:258或SEQ ID NO:260所示氨基酸序列的核酸序列或其互补序列；
    (2)SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ  ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:115、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:121、SEQ ID NO:123、SEQ ID NO:125、SEQ ID NO:127、SEQ ID NO:129、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137、SEQ ID NO:139、SEQ ID NO:141、SEQ ID NO:143、SEQ ID NO:145、SEQ ID NO:147、SEQ ID NO:149、SEQ ID NO:151、SEQ ID NO:153、SEQ ID NO:155、SEQ ID NO:157、SEQ ID NO:159、SEQ ID NO:161、SEQ ID NO:163、SEQ ID NO:165、SEQ ID NO:167、SEQ ID NO:169、SEQ ID NO:171、SEQ ID NO:173、SEQ ID NO:175、SEQ ID NO:177、SEQ ID NO:179、SEQ ID NO:181、SEQ ID NO:183、SEQ ID NO:185、SEQ ID NO:187、SEQ ID NO:189、SEQ ID NO:191、SEQ ID NO:193、SEQ ID NO:195、SEQ ID NO:197、SEQ ID NO:199、SEQ ID NO:201、SEQ ID NO:203、SEQ ID NO:205、SEQ ID NO:207、SEQ ID NO:209、SEQ ID NO:211、SEQ ID NO:213、SEQ ID NO:215、SEQ ID NO:217、SEQ ID NO:219、SEQ ID NO:221、SEQ ID NO:223、SEQ ID NO:225、SEQ ID NO:227、SEQ ID NO:229、SEQ ID NO:231、SEQ ID NO:233、SEQ ID NO:235、SEQ ID NO:237、SEQ ID NO:239、SEQ ID NO:241、SEQ ID NO:243、SEQ ID NO:245、SEQ ID NO:247、SEQ ID NO:249、SEQ ID NO:251、SEQ ID NO:253、SEQ ID NO:255、SEQ ID NO:257或SEQ ID NO:259所示的核酸序列或其互补序列；
    (3)在严谨条件下与(1)或(2)所示序列杂交的核酸序列；和
    (4)因遗传密码的简并性而与(1)或(2)所示序列编码相同氨基酸序列的核酸序列，或其互补序列。
  11. 权利要求10所述的多核苷酸，所述核酸序列被优化用于在植物细胞中表达。
  12. 一种核酸构建体，其中包含权利要求9-11中任一项所述的多核苷酸以及与之可操作连接的调控元件。
  13. 一种表达载体，其中包含有权利要求9-11中任一项所述的多核苷酸以及与之可操作连接的表达调控元件。
  14. 一种宿主细胞，其中包含有权利要求9-11中任一项所述的多核苷酸、权利要求12所述的核酸构建体或权利要求13所述的表达载体，优选地，所述的宿主细胞是植物 细胞。
  15. 一种生产对于除草剂的抗性或耐受性提高的植物的方法，其中包括将权利要求14所述的植物细胞再生成植物。
  16. 通过权利要求15所述的方法所生产的植物。
  17. 一种提高植物细胞、植物组织、植物部分或植物的HPPD抑制性除草剂抗性或耐受性的方法，其中包括在所述植物细胞、植物组织、植物部分或植物中表达权利要求1-7中任一项所述的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段或者权利要求8中所述的融合蛋白；
    或者，其中包括将表达权利要求1-7中任一项所述的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段或者权利要求8所述的融合蛋白的植物与另一植物杂交，以及筛选具有提高的HPPD抑制性除草剂抗性或耐受性的植物或其部分；
    或者，其中包括对所述植物细胞、植物组织、植物部分或植物的内源性HPPD蛋白进行基因编辑，以实现在其中表达权利要求1-7中任一项所述的突变型对羟苯基丙酮酸双氧化酶蛋白或其生物活性片段或者权利要求8所述的融合蛋白。
  18. 权利要求1-7中任一项所述的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白或其生物活性片段、权利要求8中所述的融合蛋白或者权利要求9-11中任一项所述的多核苷酸用于提高宿主细胞或者植物细胞、植物组织、植物部分或植物对HPPD抑制性除草剂的抗性或耐受性的用途，优选地，所述宿主细胞是细菌细胞或真菌细胞。
  19. 一种控制植物栽培场所的杂草的方法，其中所述植物包括权利要求16所述的植物或者通过权利要求15和17中任一项的方法制备的植物，所述方法包括对所述栽培场所施用控制杂草有效量的一种或多种HPPD抑制性除草剂；优选地，所述HPPD抑制性除草剂包含以下有效成分中的至少一种：1)三酮类：磺草酮、硝磺草酮、氟吡草酮、环磺酮、呋喃磺草酮、双环磺草酮；2)二酮腈类：2-氰基-3-环丙基-1-(2-甲基磺酰基-4-三氟甲基苯基)丙-1,3-二酮、2-氰基-3-环丙基-1-(2-甲基磺酰基-3,4-二氯苯基)丙-1,3-二酮、2-氰基-1-[4-(甲基磺酰基)2-三氟甲基苯基]-3-(1-甲基环丙基)丙1,3-二酮；3)异噁唑类：异噁氟草、异噁氯草酮、异恶草酮；4)吡唑类：苯唑草酮、磺酰草吡唑、苄草唑、吡唑特、吡草酮、双唑草酮、Tolpyralate、苯唑氟草酮、环吡氟草酮、三唑磺草酮；5)二苯酮类；6)其他类：lancotrione、fenquinotrione；更优选地，所述HPPD抑制性除草剂包含以下有效成分中的至少一种：环磺酮、苯唑氟草酮、环吡氟草酮、苯唑草酮、硝磺草酮、双唑草酮。
  20. 权利要求16所述的植物、权利要求17和19中任一项所述的方法或者权利要求 18所述的用途，其中所述植物是双子叶植物或单子叶植物，例如粮食作物、豆类作物、油料作物、纤维作物、水果类作物、根茎类作物、蔬菜类作物、花卉作物、药用作物、原料作物、牧草作物、糖料作物、饮料作物、草坪植物、树木作物、坚果作物等。
  21. 一种制备保留或加强了催化对羟基苯基丙酮酸(HPP)转化为尿黑酸的性质、且对HPPD抑制型除草剂的敏感性明显低于野生型HPPD的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白的方法，其中包括对编码野生型HPPD的核酸进行突变，将突变核酸在表达载体中与编码提高溶解度的组分的核酸序列读框一致地融合和连接形成融合蛋白编码序列，将所获得的重组表达载体转化到宿主细胞中，在含有所述HPPD抑制型除草剂和HPPD酶促底物的合适的条件下表达所述融合蛋白以及筛选保留或加强了催化对羟基苯基丙酮酸(HPP)转化为尿黑酸的性质、且对HPPD抑制型除草剂的敏感性明显降低的突变型对羟苯基丙酮酸双氧化酶(HPPD)蛋白的步骤；所述提高溶解度的组分优选NusA，其与本发明的突变型HPPD蛋白构成融合蛋白；所述表达载体优选pET-44a载体；所述宿主细胞优选细菌细胞、真菌细胞或植物细胞。
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CN113061602A (zh) * 2021-02-26 2021-07-02 未米生物科技(江苏)有限公司 一种高通量启动子变异创制方法
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CN113249343A (zh) * 2020-02-07 2021-08-13 山东舜丰生物科技有限公司 具有抗除草剂特性的多肽、核酸及其应用
CN113249345A (zh) * 2020-02-07 2021-08-13 山东舜丰生物科技有限公司 抗除草剂多肽、核酸及其应用
CN113637701A (zh) * 2021-09-10 2021-11-12 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) 一种菰米遗传转化体系建立的方法
CN114364793A (zh) * 2021-01-15 2022-04-15 江苏省农业科学院 具有除草剂抗性的hppd突变型蛋白及其应用
CN116064430A (zh) * 2021-09-14 2023-05-05 山东舜丰生物科技有限公司 突变的hppd多肽及其应用
CN113249343B (zh) * 2020-02-07 2024-04-26 山东舜丰生物科技有限公司 具有抗除草剂特性的多肽、核酸及其应用

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CN113249344B (zh) * 2020-02-07 2023-05-30 山东舜丰生物科技有限公司 抗除草剂突变蛋白、核酸及其应用
CN113249343B (zh) * 2020-02-07 2024-04-26 山东舜丰生物科技有限公司 具有抗除草剂特性的多肽、核酸及其应用
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