Corn HPPD mutant and application thereof in improving tolerance of plant herbicide

Abstract

The invention discloses a corn HPPD mutant and application of the corn HPPD mutant in improvement of plant herbicide tolerance. The invention specifically discloses a mutant protein, and the amino acid sequence of the mutant protein comprises mutation at at least one of the following positions relative to the amino acid sequence as shown in SEQ ID NO: 1: 18th, 20th and 26th sites. The invention also discloses an application of the mutant protein in improving the tolerance of plants to HPPD inhibitor herbicides (such as mesotrione). Experiments show that when the coding gene of the mutant protein is used for transforming rice, the mesotrione tolerance of a transgenic plant can be remarkably improved, and the mesotrione tolerance is improved by four times compared with that of a wild plant. Good gene resources are provided for herbicide-resistant crop breeding, HPPD inhibitor herbicide resistance genes are enriched, a foundation is laid for cultivating mesotrione-resistant new germplasm, and potential market value and wide application prospects are achieved in the agricultural field.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a corn HPPD mutant and an application thereof in improving plant herbicide tolerance. Background Art

[0002] The development of herbicides has played a significant role in improving weed control efficiency in crop fields, reducing agricultural production costs, and increasing crop yields. HPPD inhibitors are a newly developed class of selective, contact herbicides with advantages such as high efficiency, low toxicity, high crop safety, and safety for subsequent crops. The main HPPD inhibitors include mesotrione, sulcotrione, and bicyclonimide, which are widely used to control broadleaf and grass weeds in crops such as corn and soybeans. In plants, HPPD inhibitors are absorbed through young roots, inhibiting the synthesis of homogentisate (HGA), hindering the biosynthesis of plastoquinone (PQ) and tocopherols, and thus affecting carotene biosynthesis. Reduced carotenoid content leads to light and organ instability in plants. Excessive light energy causes chlorophyll degradation, ultimately leading to leaf bleaching and plant death. HPPD inhibitor herbicides are safe, broad-spectrum, and have a low risk of resistance, sparking interest in developing transgenic crops tolerant to HPPD inhibitors. HPPD genes from both microbial and plant sources have been screened and cloned, but their tolerance to the herbicides has been limited, hindering their practical application. Therefore, creating HPPD mutant plants with significantly enhanced resistance to HPPD inhibitor herbicides is of great practical significance.

[0003] The idea of ​​directed evolution can be traced back to the 1960s. In 1967, Sol Spiegelman conducted in vitro evolution of Qβ phage RNA, demonstrating Darwin's theory of evolution at the molecular level for the first time and proving the potential of directed evolution. In the 21st century, with the development of PCR-driven random mutagenesis and phage display technology, new strategies have been provided for the directed evolution of proteins and enzymes. In 2021, the research team of Michael, Romas and Yi Xiao at the University of Minnesota designed a protein complex called a targeted artificial DNA replisome (TADR) that can perform error-prone replication of one strand of a plasmid in cells. This system uses a three-protein complex of the viral nickase cisA, the bacterial Rep helicase, and the error-prone T5 DNA polymerase to perform targeted mutagenesis in vivo to replicate one strand of the target plasmid with errors while preserving the integrity of the rest of the genome. Compared with traditional directed evolution methods, the TADR mutation rate is approximately 230,000 times higher. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to improve the tolerance of plants to HPPD inhibitor herbicides. The technical problem to be solved is not limited to the technical subject matter described herein. Those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0005] To solve the above technical problems, the present invention first provides a mutant protein, the amino acid sequence of which comprises a mutation at at least one of the following positions relative to the amino acid sequence shown in SEQ ID NO: 1: positions 18, 20 and 26.

[0006] The amino acid sequence shown in SEQ ID NO: 1 may be the amino acid sequence of wild-type maize p-hydroxyphenylpyruvate dioxygenase ZmHPPD1.

[0007] Furthermore, the mutant protein comprises a mutation as shown in any one of the following A1)-A6):

[0008] A1)S18L;

[0009] A2)A20V;

[0010] A3)R26H;

[0011] A4)S18L+A20V;

[0012] A5)S18L+R26H;

[0013] A6)A20V+R26H.

[0014] Furthermore, the amino acid sequence of the mutant protein has only the following mutations relative to the amino acid sequence shown in SEQ ID NO: 1: S18L, A20V, R26H, S18L+A20V, S18L+R26H or A20V+R26H.

[0015] The mutant protein may be any of the following:

[0016] (1) mutant protein ZmHPPD1(S18L), whose amino acid sequence is obtained by mutating Ser at position 18 of SEQ ID NO:1 to Leu, while keeping the other amino acid residues unchanged;

[0017] (2) mutant protein ZmHPPD1(A20V), whose amino acid sequence is obtained by mutating Ala at position 20 of SEQ ID NO:1 to Val, while keeping the other amino acid residues unchanged;

[0018] (3) mutant protein ZmHPPD1(R26H), whose amino acid sequence is obtained by mutating Arg at position 26 of SEQ ID NO:1 to His, while keeping the other amino acid residues unchanged;

[0019] (4) mutant protein ZmHPPD1 (S18L+A20V), whose amino acid sequence is obtained by mutating Ser at position 18 to Leu and Ala at position 20 to Val, while keeping the other amino acid residues unchanged;

[0020] (5) mutant protein ZmHPPD1 (S18L+R26H), whose amino acid sequence is obtained by mutating Ser at position 18 to Leu and Arg at position 26 to His in SEQ ID NO: 1, while keeping the other amino acid residues unchanged;

[0021] (6) Mutant protein ZmHPPD1 (A20V+R26H), whose amino acid sequence is obtained by mutating Ala at position 20 to Val and Arg at position 26 to His in SEQ ID NO: 1, while keeping the other amino acid residues unchanged.

[0022] The present invention also provides a biomaterial, which may be any of the following:

[0023] B1) a nucleic acid molecule encoding the mutant protein;

[0024] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0025] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0026] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0027] B5) A recombinant host cell containing the nucleic acid molecule of B1), or a recombinant host cell containing the expression cassette of B2), or a recombinant host cell containing the recombinant vector of B3).

[0028] Furthermore, the biological materials can all express the nucleic acid molecule described in B1).

[0029] In the above biological materials, the recombinant vector can be a cloning vector or an expression vector.

[0030] The recombinant vector can be constructed using an expression vector. The structure of the expression vector is well known to those skilled in the art. The expression vector generally contains elements required for target gene expression such as promoters, multiple cloning sites, terminators, ribosome binding sites, etc., and may also contain screening marker genes (such as kanamycin resistance gene kanr, neomycin resistance gene neo, hygromycin resistance gene hyg, chloramphenicol resistance gene cat, streptomycin resistance gene str, bleomycin resistance gene ble, etc.). The expression vector can be constructed using any method known in the art (such as recombinant technology, synthetic technology, etc.) or can be purchased commercially. For example, in one or more embodiments of the present invention, the expression vector includes pET28, pBAD, and BGV005.

[0031] Furthermore, the recombinant vector can be a recombinant expression vector obtained by cloning the gene encoding the mutant protein described herein into an expression vector (such as a prokaryotic expression vector, a eukaryotic expression vector, and a viral expression vector). Although the expression vectors used in the embodiments provided herein are pET28, pBAD, and BGV005, the present invention is not limited to these specific vectors. Those skilled in the art may use other suitable expression vectors, as long as the expression vector is capable of expressing the mutant protein described herein.

[0032] The gene encoding the mutant protein can be any gene capable of encoding the mutant protein described herein. For example, the nucleotide sequence of the gene encoding the mutant protein can be selected from SEQ ID NO: 3 to SEQ ID NO: 8.

[0033] The prokaryotic expression vector may be selected from Escherichia coli expression vectors (e.g., pET series vectors, pGEX series vectors, pMAL series vectors, etc.). The eukaryotic expression vector may be selected from yeast expression vectors (e.g., pYES2, pPICZaA, pUG6, etc.), plant expression vectors (e.g., pBI series vectors, pCAMBIA series vectors, etc.). The viral expression vector may be selected from tobacco mosaic virus (TMV) vectors, cowpea mosaic virus (CPMV) vectors, potato virus X (PVX) vectors, cauliflower mosaic virus (CaMV) vectors, etc.

[0034] Available existing plant expression vector construction contains the recombinant expression vector of the coding gene of mutant protein described herein.Described plant expression vector includes but is not limited to binary expression vector (as pBI series vector (as pBI121), pBIN series vector (as pBin19), pCAMBIA series vector (as pCAMBIA1300 vector), pPZP series vector, pGreen series vector, pBIBAC series vector, pSKI015 vector, pSKI074 vector, pRI101-AN vector etc.) and co-integration vector (can be by inserting the section homologous to Ti plasmid or its section in intermediate vector with homologous recombination or cloning mode to construct).Described plant expression vector contains the required element such as promoter, multiple cloning site, terminator, ribosome binding site etc. of foreign gene expression.Described plant expression vector can also comprise the 3 ' end non-translated region of foreign gene, i.e. comprise polyadenylic acid signal and any other DNA fragment participating in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as, but not limited to, the non-translated region transcribed at the 3' end of Agrobacterium crown gall induction (Ti) plasmid genes (such as the rouge synthase Nos gene) and plant genes (such as soybean storage protein genes) all have similar functions. When the encoding gene of the mutant protein described herein is introduced by Agrobacterium tumefaciens, it is preferred to use an expression vector suitable for Agrobacterium tumefaciens, such as a binary vector or a modified vector thereof. Examples of these plant expression vectors include pBI121, pBIN19, pSMAB704, pCAMBIA series vectors, and pGreen series vectors.

[0035] In the above biological materials, the nucleic acid molecule in B1) includes a DNA molecule having a coding sequence as shown in any one of SEQ ID NOs: 3-8.

[0036] B1) The nucleic acid molecule may also include a nucleic acid molecule obtained by modifying the codon preference based on the nucleotide sequence shown in any one of SEQ ID NOs: 3-8.

[0037] The DNA molecule shown in SEQ ID NO: 3 can encode the mutant protein ZmHPPD1 (S18L) described herein;

[0038] The DNA molecule shown in SEQ ID NO: 4 can encode the mutant protein ZmHPPD1 (A20V) described herein;

[0039] The DNA molecule shown in SEQ ID NO: 5 can encode the mutant protein ZmHPPD1 (R26H) described herein;

[0040] The DNA molecule shown in SEQ ID NO: 6 can encode the mutant protein ZmHPPD1 (S18L+A20V) described herein;

[0041] The DNA molecule shown in SEQ ID NO: 7 can encode the mutant protein ZmHPPD1 (S18L+R26H) described herein;

[0042] The DNA molecule shown in SEQ ID NO: 8 can encode the mutant protein ZmHPPD1 (A20V+R26H) described herein.

[0043] The present invention also provides the use of the mutant protein or the biomaterial in any of the following:

[0044] C1) Use in improving plant tolerance to HPPD inhibitor herbicides;

[0045] C2) Use in the preparation of plants tolerant to HPPD inhibitor herbicides;

[0046] C3) Use in breeding plants with enhanced tolerance to HPPD inhibitor herbicides.

[0047] In the above application, the HPPD inhibitor herbicides include mesotrione, sulcotrione, cypermethrin, cyclazone, cypermethrin and isoxaflutole, but are not limited thereto.

[0048] The present invention also provides a method for improving the tolerance of a plant to an HPPD inhibitor herbicide, the method comprising allowing the plant to express the mutant protein.

[0049] In the above method, causing the plant to express the mutant protein can be achieved by any of the following methods:

[0050] D1) expressing or overexpressing the gene encoding the mutant protein in the plant;

[0051] D2) mutating the protein with the amino acid sequence of SEQ ID NO: 1 contained in the plant, wherein the mutation is any one of A1) to A6) herein.

[0052] Furthermore, the D1) can be achieved by introducing the gene encoding the mutant protein into the plant.

[0053] Methods for introducing the gene encoding the mutant protein into plants are well known to those skilled in the art. For example, the target gene can be transferred into plant recipient cells by physical or chemical methods, such as gene guns (also known as microprojectile bombardment or biomissiles), chemical stimulation, electric shock, liposome-mediated methods, microinjection, laser microbeams, pollen tube channels, ultrasound, air guns, and eddy current methods. The target gene can also be transferred into plant recipient cells using vectors as a medium, such as Agrobacterium-mediated methods (Agrobacterium-mediated methods) mediated by Agrobacterium Ti plasmid vectors (including Ti plasmid-derived vectors such as co-integration vector systems and binary vector systems), plant virus vector-mediated transformation methods, and the like.

[0054] Furthermore, the mutation method described in D2) is well known to those skilled in the art, such as PCR-mediated site-directed mutagenesis, oligonucleotide primer-mediated site-directed mutagenesis, cassette mutagenesis, gene editing technology or homologous recombination technology.

[0055] In the above method, the nucleotide sequence of the gene encoding the mutant protein may be SEQ ID NO: 3, 4, 5, 6, 7 or 8.

[0056] Furthermore, the method for increasing plant tolerance to HPPD inhibitor herbicides described herein may include the following steps:

[0057] (1) constructing a recombinant expression vector comprising a gene encoding any of the mutant proteins described herein;

[0058] (2) introducing the recombinant expression vector constructed in step (1) into the plant;

[0059] (3) Transgenic plants with enhanced tolerance to HPPD inhibitor herbicides were obtained through screening and identification.

[0060] Furthermore, the nucleotide sequence of the gene encoding the mutant protein may be SEQ ID NO: 3, 4, 5, 6, 7 or 8.

[0061] Furthermore, the introduction method described in step (2) includes but is not limited to: Agrobacterium-mediated method, plant virus vector-mediated transformation method, gene gun method (also known as microparticle bombardment method or biological missile method), chemical stimulation method, electric shock method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasonic method, air gun method and vortex method, etc.

[0062] Furthermore, the introduction method may be Agrobacterium-mediated method.

[0063] Furthermore, the Agrobacterium-mediated method may include the following steps: introducing the recombinant expression vector constructed in step (1) into Agrobacterium (such as a Ca ion-induced transformation method, a polyethylene glycol-mediated transformation method, a metal cation-mediated transformation method, an electroporation transformation method, a phage transduction method, etc.) to obtain recombinant Agrobacterium, and infecting the callus tissue or explant of the plant with the recombinant Agrobacterium; and inducing and culturing the positive callus tissue or explant obtained after identification to obtain a regenerated plant.

[0064] The explants include but are not limited to seeds, roots, leaves, petioles, cotyledons, cotyledon petioles, hypocotyls, stem segments, stem apical meristems, epidermal parenchyma cells, tubers, stolon segments, embryonic suspension cells and protoplasts.

[0065] The screening and identification methods are known to those skilled in the art, and for example, transformed transgenic plants (including transgenic offspring materials) can be identified by techniques such as PCR detection, Southern hybridization, immunoblotting, Northern hybridization, enzyme-linked immunosorbent assay (ELISA), functional identification (testing the presence of selection marker genes and target genes) and / or in situ hybridization.

[0066] Herein, the plant may be any of the following:

[0067] E1) Monocotyledonous or dicotyledonous plants;

[0068] E2) Grasses;

[0069] E3) Oryza plants.

[0070] As used herein, the transgenic plants (plants with enhanced tolerance to HPPD inhibitor herbicides) are understood to include not only first-generation transgenic plants obtained by introducing a gene encoding any of the mutant proteins described herein, but also their progeny. These transgenic plants include seeds, callus tissue, whole plants, and cells.

[0071] When referring to a mutant protein herein, its amino acid sequence is determined by reference to the amino acid sequence of wild-type maize p-hydroxyphenylpyruvate dioxygenase ZmHPPD1 (SEQ ID NO: 1). When referring to a mutation, the mutation is described using the following format: "single-letter abbreviation of the amino acid before mutation, mutation position, and single-letter abbreviation of the amino acid after mutation." For example, "S18L" indicates that at position 18 of the amino acid sequence set forth in SEQ ID NO: 1, the amino acid has mutated from Ser to Leu. The first methionine (Met) in the amino acid sequence set forth in SEQ ID NO: 1 is at position 1.

[0072] Multiple mutations can also be separated by a plus sign ("+"), for example, "S18L+A20V"; "S18L+A20V" indicates that at positions 18 and 20 of the amino acid sequence shown in SEQ ID NO: 1, the amino acids are mutated from Ser and Ala to Leu and Val, respectively.

[0073] The present invention constructed a TADR system for directed evolution of the maize HPPD gene. Using the constructed efficient mutant screening system for Escherichia coli, mutant strains with strong tolerance to mesotrione were identified through prokaryotic expression. The mutation sites were then verified and bioinformatics analyzed. Finally, single-site mutations and combined mutations at Ser18, Ala20, and Arg26 were screened, resulting in six mutant proteins: ZmHPPD1(S18L), ZmHPPD1(A20V), ZmHPPD1(R26H), ZmHPPD1(S18L+A20V), ZmHPPD1(S18L+R26H), and ZmHPPD1(A20V+R26H). Experimental results showed that both single-site mutations and combined mutations significantly improved the tolerance of the recipient bacteria to mesotrione. Recipient bacteria containing single-point mutations (S18L, A20V, R26H) can tolerate 800 μM mesotrione, among which the recipient bacteria containing a single-point mutation (R26H) can tolerate 1000 μM mesotrione. Combination mutant strains (S18L+A20V, S18L+R26H, A20V+R26H) can also tolerate 800 μM mesotrione. In addition, transforming rice with the gene encoding the mutant protein can significantly improve the tolerance of transgenic plants to mesotrione. Plants overexpressing ZmHPPD1(S18L), ZmHPPD1(A20V) and ZmHPPD1(R26H) can tolerate 420 g ai ha in field experiments. -1 concentration of mesotrione, while the wild-type plant's tolerance to mesotrione was only 105 g ai ha -1 The results showed that the expression of the HPPD locus mutation in rice significantly enhanced herbicide resistance.

[0074] By directing the modification of wild-type maize HPPD1 (ZmHPPD1), the present invention has generated ZmHPPD1 mutants (i.e., the mutant proteins described herein) that significantly improve plant tolerance to HPPD inhibitor herbicides. These mutant proteins have been used to successfully create rice with enhanced tolerance to HPPD inhibitor herbicides. This invention provides a valuable genetic resource for breeding herbicide-resistant crops, enriches the pool of HPPD inhibitor herbicide resistance genes, and lays a foundation for the development of new mesotrione-resistant germplasm. The invention has potential market value and broad application prospects in the agricultural sector.

[0075] Definition of terms

[0076] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0077] The term "expression cassette" generally refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a gene of interest. A typical expression cassette comprises a promoter, an MCS (multiple cloning site) and / or a terminator. An expression cassette may also include a gene of interest, a marker gene (such as a TK gene, a DHFR gene, a CAT gene, and a NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly (A) signal sequence, and / or an mRNA splicing signal sequence. The elements in the expression cassette may be directly connected or indirectly connected via a linker.

[0078] The term "vector" generally refers to a vehicle capable of transporting exogenous DNA or a gene of interest into host cells for amplification and / or expression. Such a vector can be a cloning vector or an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be amplified and / or expressed in the host cell. Those skilled in the art can select an appropriate vector based on the purpose of the genetic engineering project and the properties of the recipient cell. The vector includes, but is not limited to, a plasmid, a phage (e.g., lambda phage or M13 phage), a cosmid (i.e., cosmid), a phagemid, a shuttle vector (e.g., a yeast expression vector), a Ti plasmid, an artificial chromosome (e.g., a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a P1 artificial chromosome (PAC), or a Ti plasmid artificial chromosome (TAC)), a viral vector (e.g., a baculovirus vector, a retrovirus (including a lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papillomavirus (e.g., SV40), or a herpes virus (e.g., herpes simplex virus)). A vector may contain a variety of elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may also contain a replication initiation site.

[0079] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, etc. For example, the bacteria can be from the genus Corynebacterium sp. (such as Corynebacterium glutamicum, Corynebacterium pekinensis, Corynebacterium crenulate, etc.), the genus Brevibacterium sp. (such as Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium ammoniaphagoides, etc.), the genus Escherichia sp. (such as Escherichia coli), the genus Erwinia sp., the genus Agrobacterium sp. (such as Agrobacterium tumefaciens), the genus Flavobacterium sp., the genus Alcaligenes sp., the genus Pseudomonas sp. and the genus Bacillus sp. (such as Bacillus sp.), etc. The virus may include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (such as SV40) and herpes virus (such as herpes simplex virus), etc. The fungus may be from the genus Saccharomyces (such as Saccharomyces cerevisiae, Candida, Methanol yeast, Pichia pastoris), Fusarium (Fusarium sp.), Rhizoctonia (Rhizoctonia sp.), Verticillium (Verticillium sp.), Penicillium (Penicillium sp.), Aspergillus (Aspergillus sp.) and Cephalosporium (Cephalosporium sp.), etc. The actinomycete may be from the genus Streptomyces (Streptomyces sp.) (such as Streptomyces). The algae may be from Cyanophyta (such as cyanobacteria), Fucus sp., Achnanthes sp., Amphiprora sp., Amphoras sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., and the like.

[0080] The term "host cell," also referred to as a recipient cell, generally refers to any type of cell into which a vector can be introduced, such as plant cells and animal cells. The term "host cell" is understood to refer not only to a specific recipient cell but also to the progeny of such a cell. Due to natural, accidental, or intentional mutations and / or changes, such progeny may not necessarily be completely identical to the original parent cell, but are still included within the scope of host cells. Suitable host cells are known in the art, wherein: the plant cell can be, but is not limited to, plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum); the animal cell can be a mammalian cell (e.g., Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell substrain (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells, or NK cells), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., African clawed frog (Xenopus laevis) cells, or giant salamander (Andrias davidianus) cells). davidianus) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells, Sf-9 cells or Hi-5 cells), etc., but are not limited thereto.

[0081] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0082] The term "recombinant microorganism" generally refers to a microorganism whose genes have been manipulated and modified to produce a functionally altered recombinant microorganism. This can be achieved by introducing an exogenous gene of interest or a recombinant vector into the microorganism, or by directly editing the endogenous genes of the microorganism.

[0083] The term "recombinant host cell" generally refers to a recombinant host cell whose genes have been manipulated and modified to produce functionally altered recombinant host cells. This can include introducing an exogenous gene of interest or a recombinant vector into a host cell, or directly editing the endogenous genes of the host cell.

[0084] The term "mutation" generally refers to changes in amino acid sequences or nucleotide sequences, which may include changes in the base pair composition or arrangement order of the gene structure, such as point mutations caused by single base changes, or deletions, duplications and insertions of multiple bases, etc. It may also include replacement, deletion and insertion (addition) of one or more amino acid residues in a protein.

[0085] The term "site-directed mutagenesis" generally refers to the alteration of one or more bases in a gene through site-directed mutagenesis. This includes base additions, deletions, and point mutations, resulting in changes in the amino acid composition of the corresponding protein. Site-directed mutagenesis methods include oligonucleotide primer-mediated mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis.

[0086] The term "gene editing technology" generally refers to technologies that can alter specific gene sequences within cells, causing base deletions, duplications, insertions, frameshift mutations, replacements, and knockouts of target genes. These technologies can achieve genomic sequence replacements, deletions, splicing, and single-base changes, effectively "editing" the genome or the sequence of a specific gene. Gene editing includes zinc finger nuclease knockout technology, TALEN gene editing technology, and CRISPR gene editing technology.

[0087] The term "homologous recombination technology" generally refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. For example, a site-directed mutagenesis strategy based on homologous recombination can be achieved by ligating the ends of a gene encoding a mutant protein of the present invention (e.g., a DNA molecule represented by SEQ ID NO: 3, 4, 5, 6, 7, or 8) to homologous arms of the wild-type ZmHPPD1 gene, followed by introduction into a recipient plant to replace the wild-type gene via homologous recombination, thereby achieving site-directed mutagenesis.

[0088] The term "explant" generally refers to a portion of a plant used as in vitro culture material in plant tissue culture. After appropriate treatment and under suitable conditions, it can be regenerated into a whole plant. In practice, those skilled in the art will select the appropriate explant for transformation based on the specific plant.

[0089] The term "callus" generally refers to the newly formed tissue that forms on the surface of a wound after a local injury to the original plant. It is composed of living parenchyma cells and can originate from living cells in any tissue within the plant's organs. In plant tissue culture, it refers to a mass of unorganized, actively dividing parenchyma cells formed from an explant. Cultivating callus on an appropriate culture medium can induce it to form a whole plant.

[0090] The term "comprising" is not intended to be limiting, but rather inclusive and means that there may be additional elements other than the listed elements, and can be interpreted as "including, but not limited to." The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of." The terms "comprising" and "including" are used interchangeably herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 HPPD is a catalytic reaction and metabolite in Escherichia coli and plants. Tyrosine is converted to para-hydroxyphenylpyruvate (4-HPP) by the enzyme tyrosine aminotransferase (TAT). HPPD then catalyzes the conversion of 4-HPP into homogentisate (HGA) in the presence of oxygen. In E. coli, HGA cannot be further metabolized and undergoes oxidative polymerization to produce a brown substance. In plants, HGA can be further converted into plastoquinone (PQ) and tocopherol (a-tocopherol).

[0092] Figure 2 Construction of an E. coli expression system for the maize HPPD gene. a. Prokaryotic expression of the maize HPPD gene in E. coli produces a brown homogentisate product. 1: pET28 control group; 2: pET28-ZmHPPD1 experimental group; 3: pBAD control group; 4: pBAD-ZmHPPD1 experimental group; 5: pET28 control group; 6: pET28-ZmHPPD2 experimental group; 7: pBAD control group; 8: pBAD-ZmHPPD2 experimental group. b. Expression levels of the maize HPPD protein in the pellet (undissolved components after cell disruption) and supernatant of the pET28 prokaryotic expression system. 1: pET28 supernatant; 2: pET28-ZmHPPD1 supernatant; 3: pET28-ZmHPPD1 precipitate; 4: pET28 precipitate; 5: pET28 supernatant; 6: pET28-ZmHPPD2 supernatant; 7: pET28-ZmHPPD2 precipitate; 8: pET28 precipitate; M: protein marker. c. Growth of the pET28-ZmHPPD1 strain in LB medium containing different concentrations of mesotrione.

[0093] Figure 3 Construction of a directed evolution system for maize HPPD. a. Schematic diagram of the TADR-ZmHPPD1 system. b. Flowchart of the ZmHPPD1 directed evolution process. The flowchart was created using the CNSknowall website (https: / / cnsknowall.com). c. Growth of the mutant strain T-4-36 on LB agar plates containing 6000 μM mesotrione and in LB liquid medium containing 800 μM mesotrione.

[0094] Figure 4Identification of the mutation site in the ZmHPPD1 mutant strain T-4-36. a Information on the 17 mutation sites in the T-4-36 strain. b Bioinformatics analysis of the secondary structure associated with the mutation sites in the T-4-36 strain. c Prokaryotic expression analysis of the mutation sites in the T-4-36 strain.

[0095] Figure 5 Single-site and combined-site mutations in ZmHPPD1 confer strong tolerance to mesotrione. The figure shows the inhibitory effect of mesotrione on wild-type ZmHPPD1 and its mutants in LB liquid culture. Darker colors indicate strains with greater mesotrione tolerance.

[0096] Figure 6 The enzyme activities and half inhibitory concentrations (IC 50 ) analysis. a. Enzyme activity analysis of the wild type and six mutants. b. IC values ​​of mesotrione on the wild type and six mutants 50 All data were statistically analyzed using one-way analysis of variance combined with honestly significant difference test (P<0.05, n≥3).

[0097] Figure 7 Single-site mutations in ZmHPPD1 significantly enhance rice tolerance to mesotrione. a. Phenotype of Nipponbare rice 14 days after spraying with mesotrione. b. Phenotype of rice plants overexpressing wild-type ZmHPPD1 14 days after spraying with mesotrione. c. Phenotype of rice plants overexpressing the ZmHPPD1(S18L) mutant 14 days after spraying with mesotrione. d. Phenotype of rice plants overexpressing the ZmHPPD1(A20V) mutant 14 days after spraying with mesotrione. e. Phenotype of rice plants overexpressing the ZmHPPD1(R26H) mutant 14 days after spraying with mesotrione. 1–9: mesotrione concentrations of 0, 52.5, 105, 157.5, 210, 262.5, 315, 367.5, and 420 g ai ha -1 ). DETAILED DESCRIPTION

[0098] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0099] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0100] The vector pET28 in the following examples was purchased from EMD Life Sciences, a subsidiary of the Merck Group in Germany, with the product number 69864-3.

[0101] The vector pBAD in the following examples was purchased from Shanghai Zeye Biotechnology Co., Ltd. with the product number ZY6201Z.

[0102] The vector BGV005 in the following examples is the vector pCAMBIA1301, which was purchased from Shanghai Zeye Biotechnology Co., Ltd. with the product number ZY61464Z.

[0103] In the following examples, HPPD is 4-hydroxyphenylpyruvatedioxygenase, also known as 4-hydroxyphenylpyruvatedioxygenase.

[0104] The sequences of the proteins and mutant proteins involved in the following examples are:

[0105] The amino acid sequence of wild-type maize HPPD1 (ZmHPPD1) is SEQ ID NO: 1, and the nucleotide sequence of its encoding gene is SEQ ID NO: 2.

[0106] The mutant protein ZmHPPD1 (S18L) has an amino acid sequence obtained by mutating Ser at position 18 of SEQ ID NO: 1 to Leu while keeping other amino acid residues unchanged. The nucleotide sequence of the encoding gene is SEQ ID NO: 3.

[0107] The mutant protein ZmHPPD1 (A20V) has an amino acid sequence obtained by mutating Ala at position 20 of SEQ ID NO: 1 to Val, while keeping other amino acid residues unchanged. The nucleotide sequence of the encoding gene is SEQ ID NO: 4.

[0108] The mutant protein ZmHPPD1 (R26H) has an amino acid sequence obtained by mutating Arg at position 26 of SEQ ID NO: 1 to His, while keeping other amino acid residues unchanged. The nucleotide sequence of the encoding gene is SEQ ID NO: 5.

[0109] The mutant protein ZmHPPD1 (S18L+A20V) has an amino acid sequence obtained by mutating Ser at position 18 of SEQ ID NO: 1 to Leu and Ala at position 20 to Val, while keeping other amino acid residues unchanged. The nucleotide sequence of its encoding gene is SEQ ID NO: 6.

[0110] The mutant protein ZmHPPD1 (S18L+R26H) has an amino acid sequence obtained by mutating Ser at position 18 of SEQ ID NO: 1 to Leu, and Arg at position 26 to His, while keeping other amino acid residues unchanged. The nucleotide sequence of its encoding gene is SEQ ID NO: 7.

[0111] The mutant protein ZmHPPD1 (A20V+R26H) has an amino acid sequence obtained by mutating Ala at position 20 to Val and Arg at position 26 to His in SEQ ID NO: 1, while keeping the other amino acid residues unchanged. The nucleotide sequence of the encoding gene is SEQ ID NO: 8.

[0112] Example 1: Cloning of ZmHPPD gene and prokaryotic expression activity analysis

[0113] Reference sequences for ZmHPPD1 (Zm00001d015356) and ZmHPPD2 (Zm00001d019365) were obtained from the NCBI gene database. The ZmHPPD1 gene is located on maize chromosome 5 and encodes 444 amino acid residues with a molecular weight of 46.9 kDa. The ZmHPPD2 gene is located on maize chromosome 7 and encodes 445 amino acid residues with a molecular weight of 47.3 kDa. Protein sequence alignment revealed a low similarity of only 65% ​​between the two HPPD genes (Table 1).

[0114] Table 1. Basic information of two maize HPPD genes

[0115]

[0116]

[0117] Genomic DNA and RNA were extracted from young leaves of the maize inbred line B73 using a plant DNA / RNA extraction kit (purchased from Tiangen Biochemical). cDNA was obtained by reverse transcription of the plant RNA using a reverse transcription kit (purchased from Takara). The full genomic and coding sequences (CDS) of ZmHPPD1 and ZmHPPD2 were amplified using the high-fidelity DNA polymerase KOD-Plus-Neo (purchased from Toyobo) using specific primers (ZmHPPD1-F / R and ZmHPPD2-F / R; primer sequences are shown in Table 2) using genomic DNA and cDNA as templates, respectively. Sequences were confirmed by Sanger sequencing.

[0118] The pET28 and pBAD vectors were selected as backbone vectors, and specific primers (pET28-ZmHPPD1-ss-F / R; pET28-ZmHPPD2-ss-F / R; pBAD-ZmHPPD1-ss-F / R; pBAD-ZmHPPD2-ss-F / R) were designed (primer sequences are shown in Table 2). Using genomic DNA from the maize inbred line B73 as a template, the CDS regions of ZmHPPD1 and ZmHPPD2 (with vector homology arms at both ends) were amplified using the above-designed specific primers. The pET28 plasmid vector was double-digested with BamHI and HindIII restriction endonucleases (purchased from NEB), while the pBAD plasmid vector was double-digested with BglII and EcoRI restriction endonucleases (purchased from NEB). The amplified CDS fragments were then ligated into the digested vector using SoSoo recombinase (purchased from Qingke Biotechnology). The final constructed plasmid was transformed into BL21(DE3) competent cells (purchased from Qingke Biotechnology), thus forming the prokaryotic expression vectors pET28-ZmHPPD1, pET28-ZmHPPD2, pBAD-ZmHPPD1, and pBAD-ZmHPPD2. The constructed vectors were prokaryotically expressed in Escherichia coli BL21(DE3) strains. HPPD was used to catalyze the production of homogentisate metabolites in E. coli. The activity of HPPD can be determined by observing the level of pigment accumulation ( Figure 1 ).

[0119] Table 2. Sequence list

[0120]

[0121]

[0122] In the prokaryotic expression experiment, using 0.4 mM IPTG as an inducer, it was found that the host bacteria containing pET28-ZmHPPD1 and pBAD-ZmHPPD1 both formed brown homogentisate metabolites, while the two host bacteria containing pET28-ZmHPPD2 and pBAD-ZmHPPD2 did not form homogentisate, indicating that the metabolic pathway of ZmHPPD1 can function normally in microorganisms ( Figure 2 (a)

[0123] The host bacteria were lysed using an ultrasonic cell disruptor. The cell suspension was kept on ice during the disruption process. The ultrasonic probe was turned on for 30 seconds, then paused for 1-1.5 minutes, and this process was repeated 5 times. SDS-PAGE protein gel electrophoresis was used to separate the two HPPD proteins (ZmHPPD1 and ZmHPPD2) in maize. The results showed that both HPPD1 and HPPD2 proteins were expressed normally. The molecular weight of the two ZmHPPD proteins was approximately 47 kDa, which is consistent with the reported molecular size ( Figure 2 (b)

[0124] ZmHPPD1 was used as the research object. To further verify the tolerance level of ZmHPPD1 to mesotrione, the strain containing pET28-ZmHPPD1 was spread on LB agar plates containing 0μM, 2000μM, 4000μM, 6000μM, and 8000μM mesotrione, respectively. 0.4mM IPTG was used as an inducer. It was found that the strain could grow on the 0μM, 2000μM, and 4000μM plates, but grew slowly in the presence of mesotrione. The strain did not grow on the 6000μM and 8000μM mesotrione plates, indicating that mesotrione has the effect of inhibiting the growth of Escherichia coli, and the growth of the strain was inhibited at a concentration of 6000μM mesotrione ( Figure 2 (c)

[0125] Example 2: Directed evolution and mutant screening of ZmHPPD1

[0126] In the construction of the TADR system, CRISPR / Cas9 technology was used to knock out the rep helicase of the Escherichia coli strain BL21, and the phage CisA gene was knocked in, and the expression on the E. coli chromosome was initiated by the pLAC promoter; an auxiliary plasmid was constructed to initiate the error-prone T5 DNA polymerase and Rep helicase protein complex with the pBAD promoter; the target sequence (Target-up / down) recognized by CisA was added to the N-terminus and C-terminus of the ZmHPPD1 gene (sequence see Table 2), respectively, and it was initiated by the pTAC promoter. The modified E. coli strain and the auxiliary plasmid were combined to form a complete TADR-ZmHPPD1 system. The function of the system was verified by biochemical and genetic experiments to ensure that the directed evolution process could proceed as expected ( Figure 3 (a)

[0127] Transform the modified TADR-ZmHPPD1 vector into competent BL21 cells and culture on 2×YT solid medium containing arabinose at 37°C for 12-14 hours. Select a single colony and incubate in 3 ml of 2×YT liquid medium containing arabinose at 37°C at 200 rpm for 12 hours. Transfer 30 μL of the incubated culture to a fresh 3 ml of 2×YT liquid medium containing arabinose for subculture. Extract plasmids from the remaining culture for storage or sequencing. Plasmids must be extracted from each generation to preserve the mutation. Transform the resulting mutant plasmid into BL21 cells and plate onto LB solid medium containing 6000 μM mesotrione and 0.4 mM IPTG. Incubate at 37°C for 12 hours and observe colony growth. Count the number of colonies growing on the 6000 μM mesotrione-resistant plate, take a single colony and continue to culture in LB medium for 12 h (this step is for the propagation of the mutant strain, and IPTG is not added). Take 30 μL of the propagation solution and add it to 3 ml of LB liquid medium containing tyrosine-containing gradient concentrations of mesotrione (0, 600, 800, 1000 μM), and culture at 37°C and 200 rpm until the OD 600 =0.6, add IPTG, continue to culture for 24 hours, and observe the color and growth of the cells. Screening of mesotrione-tolerant strains that can grow in a high concentration of mesotrione environment and have darker cell color ( Figure 3 (b)

[0128] After multiple generations of screening, a mutant strain capable of tolerating 800 μM mesotrione was obtained and named T-4-36 (where "4" indicates the fourth generation of subculture and "36" represents the strain number within that generation). This mutant strain was able to grow on LB agar plates containing 6000 μM mesotrione and exhibited darker cell color and higher homogentisate metabolite accumulation than the control strain in LB liquid medium containing 800 μM mesotrione. Figure 3 (c)

[0129] Example 3: Verification of mutation sites in ZmHPPD1 mutants

[0130] The plasmid of the T-4-36 strain was extracted and sequenced for the first generation. The sequencing results showed that it contained 39 mutation sites (Table 3). Structural analysis of the mutation sites showed that Q389 mutated into a stop codon, and the amino acid sequence of the entire ZmHPPD1 was reduced by 55 amino acids. Sequencing showed that it was a homozygote, which indicated that the amino acid sequence before position 389 was the main structure and contained the active center of the protein (His219, His301 and Glu387). Therefore, the structure after position 389 was not analyzed. There were 17 effective mutation sites involving changes in amino acid codons ( Figure 4 (a)

[0131] Table 3. Mutation site information of T-4-36 strain

[0132]

[0133]

[0134] Note: The 39 mutation sites (Position) in Table 3 are positions relative to the amino acid sequence of wild-type ZmHPPD1 (SEQ ID NO: 1). For example, the first row (Number 1) in Table 3 indicates that at position 18 of the amino acid sequence shown in SEQ ID NO: 1, the amino acid has mutated from Ser to Leu.

[0135] Further analysis of the protein structure changes at the 17 mutation sites revealed that nine mutation sites, including S18L, A20V, R26H, G190S, G238E, E248K, P276L, R282C, and Q389*, were involved in changes in protein structure or properties. Among them, the mutations of S18L, A20V, G190S, and E248K were located inside the α-helix and β-sheet, and did not significantly change the secondary structure; the mutation of R26H was a basic amino acid mutation, located inside the hinge region, and did not form an extra structure; G238E was located at the second amino acid of the random coil, and the hydrophilic neutral amino acid mutated to an acidic amino acid, and the amino acid properties changed; the mutation of P276L caused the Cα-N bond of proline to be unable to rotate, and the mutation to leucine directly led to a structural change; R282 was located at the third amino acid of the random coil, and the mutation caused the basic amino acid to mutate to a neutral amino acid, and the amino acid properties changed ( Figure 4 (b)

[0136] The PCR-mediated site-directed mutagenesis method was used with pET28 as the backbone vector to construct prokaryotic expression vectors of the above 9 mutation sites, which were transformed into Escherichia coli BL21 (DE3) strain to obtain recombinant bacteria, which were incubated in LB medium for 12 h. The recombinant strains were incubated in LB liquid medium containing 800 μM mesotrione, 0.4 mM IPTG and 1 g / L tyrosine for 24 h and the bacterial color and pigment production level were observed. The results showed that the bacterial color of pET28-ZmHPPD1(S18L), pET28-ZmHPPD1(A20V) and pET28-ZmHPPD1(R26H) was darker than that of other mutation sites and was comparable to the level of T-4-36. Therefore, it can be determined that the mutations at the S18L and A20V sites can confer tolerance to 800 μM mesotrione on ZmHPPD1, while the R26H mutation can confer tolerance to 1000 μM mesotrione on ZmHPPD1. Figure 4 (c)

[0137] The recombinant vector pET28-ZmHPPD1 is a recombinant expression vector obtained by cloning the DNA molecule shown in SEQ ID NO: 2 into the pET28 vector. The recombinant vector pET28-ZmHPPD1 expresses the ZmHPPD1 protein (SEQ ID NO: 1).

[0138] The recombinant vector pET28-ZmHPPD1(S18L) is a recombinant expression vector obtained by cloning the DNA molecule shown in SEQ ID NO: 3 into the pET28 vector. The recombinant vector pET28-ZmHPPD1(S18L) expresses the mutant protein ZmHPPD1(S18L).

[0139] The recombinant vector pET28-ZmHPPD1(A20V) is a recombinant expression vector obtained by cloning the DNA molecule shown in SEQ ID NO: 4 into the pET28 vector. The recombinant vector pET28-ZmHPPD1(A20V) expresses the mutant protein ZmHPPD1(A20V).

[0140] The recombinant vector pET28-ZmHPPD1(R26H) is a recombinant expression vector obtained by cloning the DNA molecule shown in SEQ ID NO: 5 into the pET28 vector. The recombinant vector pET28-ZmHPPD1(R26H) expresses the mutant protein ZmHPPD1(R26H).

[0141] Example 4: Single-site mutations and combined mutations improve ZmHPPD1 tolerance to mesotrione

[0142] Using pET28 as the backbone vector, prokaryotic expression vectors containing paired combinations of the three sites, S18L, A20V, and R26H, were constructed: pET28-ZmHPPD1(S18L+A20V), pET28-ZmHPPD1(S18L+R26H), and pET28-ZmHPPD1(A20V+R26H). The vectors harboring the three single-site mutations and the three combined mutations were transformed into Escherichia coli BL21 strains. Cell color and pigment production were observed after 24 h of culture in LB liquid medium (containing 0.4 mM IPTG and 1 g / L tyrosine) containing a gradient of mesotrione concentrations (0, 200, 400, 600, 800, and 1000 μM). The results showed that at the same mesotrione concentration, all mutant types exhibited higher cytochrome accumulation than the wild-type, indicating that the HPPD enzyme activity of the six single-site mutations and the combined mutations was stronger than that of the wild-type HPPD. At a concentration of 600 μM mesotrione, homogentisate levels were comparable in single-site and combined mutations. Furthermore, the S18L, A20V, S18L+A20V, S18L+R26H, and A20V+R26H strains were able to produce homogentisate at a concentration of 800 μM mesotrione, and R26H could even produce homogentisate at a concentration of 1000 μM mesotrione. Figure 5 ).

[0143] The recombinant vector pET28-ZmHPPD1(S18L+A20V) is a recombinant expression vector obtained by cloning the DNA molecule shown in SEQ ID NO: 6 into the pET28 vector. The recombinant vector pET28-ZmHPPD1(S18L+A20V) expresses the mutant protein ZmHPPD1(S18L+A20V).

[0144] The recombinant vector pET28-ZmHPPD1(S18L+R26H) is a recombinant expression vector obtained by cloning the DNA molecule shown in SEQ ID NO:7 into the pET28 vector. The recombinant vector pET28-ZmHPPD1(S18L+R26H) expresses the mutant protein ZmHPPD1(S18L+R26H).

[0145] The recombinant vector pET28-ZmHPPD1(A20V+R26H) is a recombinant expression vector obtained by cloning the DNA molecule shown in SEQ ID NO: 8 into the pET28 vector. The recombinant vector pET28-ZmHPPD1(A20V+R26H) expresses the mutant protein ZmHPPD1(A20V+R26H).

[0146] Example 5. Enzyme activity and IC of ZmHPPD1 mutants 50 Determination

[0147] 50 μL of BL21 (DE3) strains carrying pET28-ZmHPPD1 and pET28-ZmHPPD1(S18L), pET28-ZmHPPD1(A20V), pET28-ZmHPPD1(R26H), pET28-ZmHPPD1(S18L+A20V), pET28-ZmHPPD1(S18L+R26H), and pET28-ZmHPPD1(A20V+R26H) were added to kanamycin-resistant LB medium for overnight culture, and 30 μL was transferred to 3 mL LB medium containing 0, 200, 400, 600, 800, or 1000 μM mesotrione and continued to culture for 3-4 h. When OD 600 When the value reached 0.6, 0.4 mM IPTG was added and the culture was continued for 24 h.

[0148] Observe the bacterial growth and pigment accumulation of each treatment group, take 500 μL of bacterial solution and centrifuge it in a 1.5 ml centrifuge tube, transfer 200 μL of supernatant obtained by centrifugation to a microplate, and use a microplate reader to measure the absorbance at 420 nm and 450 nm. OD 420 Used to measure the pigment content in bacterial suspension, OD 450 Used to calculate IC 50 value, IC 50 The values ​​were calculated using an online calculation tool (https: / / www.aatbio.com / tools / ic50-calculator).

[0149] To further compare the tolerance of different HPPD mutants to mesotrione, we measured the OD of wild type and mutants. 420 The results showed that the pigment accumulation of wild-type HPPD and its mutants gradually decreased with the increase of mesotrione concentration. R26H showed higher OD values ​​than wild-type and other mutants at all concentrations. 420 values, indicating its excellent enzyme activity and metabolic capacity ( Figure 6 (a) IC values ​​of different concentrations of mesotrione against the wild type and six HPPD mutants 50 The results showed that the IC values ​​of the six HPPD mutants were 50 The values ​​were 1.5 to 2 times higher than those of the wild type, and R26H showed IC 50 The values ​​were significantly higher than those of other mutants ( Figure 6 (middle b) These results indicate that the HPPD mutant has significantly enhanced tolerance to mesotrione concentrations.

[0150] Example 6: ZmHPPD1 single-site mutation significantly improves mesotrione resistance in rice

[0151] The CDS sequences of the wild type and mutant ZmHPPD1 in the recombinant vectors pET28-ZmHPPD1, pET28-ZmHPPD1(S18L), pET28-ZmHPPD1(A20V) and pET28-ZmHPPD1(R26H) were amplified using recombination primers (BGV-ZmHPPD1-ss-F / R) (sequences shown in Table 2); the BGV005 vector was double-digested with KpnI and BamHI restriction endonucleases (purchased from NEB), and the recombinant ligation was used to transform DH5α competent cells to construct plant overexpression vectors BGV005-ZmHPPD1, BGV005-ZmHPPD1(S18L), BGV005-ZmHPPD1(A20V) and BGV005-ZmHPPD1(R26H).

[0152] The recombinant vector BGV005-ZmHPPD1 is a recombinant expression vector obtained by replacing the fragment (small fragment) between the KpnI and BamHI recognition sites of the BGV005 vector with the DNA fragment with the nucleotide sequence set forth in SEQ ID NO: 2, while maintaining the remaining nucleotide sequence of the BGV005 vector unchanged. The recombinant vector BGV005-ZmHPPD1 expresses the ZmHPPD1 protein with the amino acid sequence set forth in SEQ ID NO: 1.

[0153] The recombinant vector BGV005-ZmHPPD1(S18L) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the KpnI and BamHI recognition sites of the BGV005 vector with the DNA fragment with the nucleotide sequence set forth in SEQ ID NO: 3, while maintaining the remaining nucleotide sequences of the BGV005 vector. The recombinant vector BGV005-ZmHPPD1(S18L) expresses the mutant protein ZmHPPD1(S18L).

[0154] The recombinant vector BGV005-ZmHPPD1(A20V) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the KpnI and BamHI recognition sites of the BGV005 vector with the DNA fragment with the nucleotide sequence set forth in SEQ ID NO:4, while maintaining the remaining nucleotide sequences of the BGV005 vector. The recombinant vector BGV005-ZmHPPD1(A20V) expresses the mutant protein ZmHPPD1(A20V).

[0155] The recombinant vector BGV005-ZmHPPD1(R26H) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the KpnI and BamHI recognition sites of the BGV005 vector with the DNA fragment with the nucleotide sequence set forth in SEQ ID NO: 5, while maintaining the remaining nucleotide sequences of the BGV005 vector. The recombinant vector BGV005-ZmHPPD1(R26H) expresses the mutant protein ZmHPPD1(R26H).

[0156] The above-mentioned recombinant vectors BGV005-ZmHPPD1, BGV005-ZmHPPD1(S18L), BGV005-ZmHPPD1(A20V), and BGV005-ZmHPPD1(R26H) were transformed into EHA105 Agrobacterium using the heat shock method to obtain recombinant Agrobacterium.

[0157] The rice Nipponbare was transformed by Agrobacterium-mediated method. The recombinant Agrobacterium was used to infect the mature rice embryos, and the transgenic plants were obtained through screening and regeneration. Figure 7 a) and the obtained transgenic plants: OE-ZmHPPD1 ( Figure 7 b), OE-ZmHPPD1(S18L) ( Figure 7 c), OE-ZmHPPD1(A20V)( Figure 7 d), OE-ZmHPPD1(R26H)( Figure 7 (e) Spray mesotrione at a gradient concentration. Active, undamaged transgenic and wild-type rice seeds of the T2 generation were germinated in a dark incubator at 28-30°C for 3 days. Seeds with uniform germination were sown in a greenhouse at a temperature of 25-30°C. When the seeds reached the tillering stage (5-6 leaves), mesotrione was sprayed at a gradient concentration. At 52.5 g ai ha -1 As the initial concentration (g ai ha -1 g ai / ha), and seven higher concentration gradients were set (105, 157.5, 210, 262.5, 315, 367.5 and 420 g ai ha -1 ). Select mesotrione with an active ingredient content of 15% and prepare the working solution at the following concentrations: 52.5g ai ha -1 (778μL / L) (add 778μL of mesotrione with an active ingredient content of 15% to 1L of water, the same below), 105g ai ha -1 (1556μL / L), 157.5g ai ha -1 (2334μL / L), 210g ai ha -1 (3112μL / L), 262.5g ai ha -1(3890μL / L), 315g ai ha -1 (4668μL / L), 367.5g ai ha -1 (5446μL / L), 420g ai ha -1 (6224μL / L). During the spraying process, all leaves should be evenly covered. After spraying, regular observation of plant changes should be conducted to calculate the degree of damage.

[0158] After 14 days of treatment, wild-type Nipponbare plants grew at 105 g ai ha -1 In contrast, OE-ZmHPPD1 overexpressing plants showed leaf bleaching symptoms at 262.5 g aiha -1 The OE-ZmHPPD1(S18L), OE-ZmHPPD1(A20V) and OE-ZmHPPD1(R26H) overexpressing plants showed a certain growth inhibition under high dose conditions, but even at 420 g ai ha -1 No bleaching symptoms occurred even at concentrations below 20%.

[0159] In summary, the herbicide resistance identification experiment of transgenic rice further proved that mutations in Ser18, Ala20 and / or Arg26 amino acids of corn HPPD1 can effectively improve its tolerance to mesotrione.

[0160] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A mutant protein, characterized in that The amino acid sequence of the mutant protein comprises a mutation at at least one of the following positions relative to the amino acid sequence shown in SEQ ID NO: 1: positions 18, 20 and 26.

2. The mutant protein according to claim 1, characterized in that The mutant protein comprises a mutation as shown in any one of the following A1)-A6): A1)S18L; A2)A20V; A3)R26H; A4)S18L+A20V; A5)S18L+R26H; A6)A20V+R26H.

3. Biomaterial, characterized in that The biological material is any one of the following: B1) a nucleic acid molecule encoding the mutant protein according to claim 1 or 2; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A recombinant host cell containing the nucleic acid molecule of B1), or a recombinant host cell containing the expression cassette of B2), or a recombinant host cell containing the recombinant vector of B3).

4. The biomaterial according to claim 3, characterized in that B1) The nucleic acid molecule includes a DNA molecule whose coding sequence is shown in any one of SEQ ID NOs: 3-8.

5. Use of the mutant protein according to claim 1 or 2, or the biomaterial according to claim 3 or 4, in any of the following: C1) Use in improving plant tolerance to HPPD inhibitor herbicides; C2) Use in the preparation of plants tolerant to HPPD inhibitor herbicides; C3) Use in breeding plants with enhanced tolerance to HPPD inhibitor herbicides.

6. The use according to claim 5, characterized in that The HPPD inhibitor herbicides include mesotrione, sulcotrione, cypermethrin, betacyclone, cyproconazole and isoxaflutole.

7. A method for increasing plant tolerance to HPPD inhibitor herbicides, characterized in that: The method comprises causing a plant to express the mutant protein according to claim 1 or 2.

8. The method according to claim 7, characterized in that The plant is caused to express the mutant protein according to claim 1 or 2 by any one of the following methods: D1) expressing or overexpressing the gene encoding the mutant protein according to claim 1 or 2 in a plant; D2) mutating the protein with the amino acid sequence of SEQ ID NO: 1 contained in the plant, wherein the mutation is the mutation shown in any one of A1) to A6) of claim 2.

9. The method according to claim 8, characterized in that The nucleotide sequence of the gene encoding the mutant protein is SEQ ID NO: 3, 4, 5, 6, 7 or 8.

10. The method according to any one of claims 7 to 9, characterized in that: The plant is any one of the following: E1) Monocotyledonous or dicotyledonous plants; E2) Grasses; E3) Oryza plants.

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