CN117987385A - HPPD protein, gene, vector, cell, composition and application thereof, and method for improving crop herbicide resistance - Google Patents

Abstract

The invention relates to the field of genetic engineering, discloses HPPD proteins, genes, vectors, cells, compositions and application thereof, and a method for improving crop herbicide resistance, in particular to a p-hydroxyphenylpyruvate dioxygenase protein, a gene for encoding the p-hydroxyphenylpyruvate dioxygenase protein, a recombinant vector, a transgenic cell, a composition and application thereof in improving crop herbicide resistance, and also discloses a method for improving crop herbicide resistance. The HPPD protein and the coding gene thereof provided by the invention can be used for improving the herbicide resistance of crops on the premise that the catalytic activity of the enzyme is not affected basically.

Description

HPPD protein, gene, vector, cell, composition and its application, method for improving crop herbicide resistance

This invention is a divisional application with a filing date of January 21, 2022, a patent application number of 202210070645.7, and an invention name of "HPPD protein, gene, vector, cell, composition and its application, and method for improving crop herbicide resistance".

Technical Field

The present invention relates to the field of genetic engineering, and in particular to an HPPD protein, a gene, a vector, a cell, a composition and application thereof, and a method for improving crop herbicide resistance.

Background Art

HPPD is a member of the non-heme iron, α-ketoacid-dependent oxygenase family and a key enzyme in the tyrosine metabolic pathway. It can further catalyze the catalytic product of tyrosine aminotransferase, p-hydroxyphenylpyruvate, to produce homogentisate.

In plants, homogentisate is a precursor for the biosynthesis of plastoquinone and tocopherol. Plastoquinone is not only a key cofactor of phytoene desaturase, but also a carrier of electron transfer, directly participating in photosynthesis. Tocopherol is one of the structural components of the cell membrane and an antioxidant of the cell membrane. Once the activity of HPPD is inhibited, the synthesis of plastoquinone will be reduced, which will in turn lead to the obstruction of the catalytic process of phytoene desaturase, thereby affecting the biosynthesis of carotenoids, and ultimately causing the plant to show symptoms of albinism and die. Therefore, HPPD is an important target of herbicides.

At present, there are more than 16 commercial HPPD inhibitor herbicides. Due to the characteristics of broad spectrum, high efficiency, safe use, and slow growth of resistance, HPPD herbicides have attracted more and more attention from pesticide chemistry researchers, and their annual sales are increasing year by year.

Herbicide-resistant crops have brought great benefits to farmers and the environment in the past 20 years, such as glyphosate-resistant corn and soybean crops. Therefore, it is necessary to develop new herbicide-resistant crops, such as crops resistant to HPPD inhibitor herbicides.

There have been some studies on HPPD herbicide resistance genes in the field. Most of these studies are based on HPPD from various bacteria, looking for mutation sites that are resistant to herbicides, and then introducing the exogenous mutant genes into cash crops to enhance their tolerance to herbicides. However, there are few reports on studies that use HPPD proteins from cash crops themselves as research objects to enhance their resistance to herbicides by mutating amino acid residues at individual sites without changing their normal catalytic function. Therefore, it is still necessary to develop and improve tolerance systems to HPPD inhibitors.

Summary of the invention

The purpose of the present invention is to overcome the above-mentioned defects of the prior art and provide an HPPD protein resistant to HPPD inhibitors and a gene encoding the same and applications thereof.

In order to achieve the above object, the present invention provides a para-hydroxyphenylpyruvate dioxygenase protein in a first aspect, wherein the protein has an amino acid sequence selected from at least one of the following:

(1) a first amino acid sequence derived from a mutation in at least one of positions 418, 423 and 432 of the amino acid sequence as shown in SEQ ID NO: 1;

(2) a protein derived from the first amino acid sequence in which the first amino acid sequence is substituted, deleted or added with one or more amino acids and the enzyme activity remains unchanged, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the first amino acid sequence;

(3) a second amino acid sequence derived from a mutation at position 422 of the amino acid sequence shown in SEQ ID NO: 2;

(4) a protein derived from the second amino acid sequence in which the second amino acid sequence is substituted, deleted or added with one or more amino acids and the enzyme activity remains unchanged, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the second amino acid sequence;

(5) a third amino acid sequence derived from a mutation at position 426 in the amino acid sequence as shown in SEQ ID NO: 3;

(6) a protein derived from the third amino acid sequence in which one or more amino acids are substituted, deleted or added and the enzyme activity remains unchanged, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the third amino acid sequence;

(7) a fourth amino acid sequence derived from a mutation at position 418 in the amino acid sequence as shown in SEQ ID NO: 4;

(8) a protein derived from the fourth amino acid sequence in which one or more amino acids are substituted, deleted or added and the enzyme activity remains unchanged, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the fourth amino acid sequence;

(9) the fifth amino acid sequence derived from a mutation at position 426 in the amino acid sequence as shown in SEQ ID NO:5;

(10) A protein derived from the fifth amino acid sequence in which one or more amino acids are substituted, deleted or added and the enzyme activity remains unchanged, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the fifth amino acid sequence.

The second aspect of the present invention provides a gene encoding a p-hydroxyphenylpyruvate dioxygenase protein, wherein the nucleotide sequence of the gene is a nucleotide sequence capable of encoding the amino acid sequence of the p-hydroxyphenylpyruvate dioxygenase protein described in the first aspect.

The third aspect of the present invention provides a recombinant vector, which contains the gene described in the second aspect.

The fourth aspect of the present invention provides a transgenic cell, which contains the gene described in the second aspect.

The fifth aspect of the present invention provides a composition comprising the p-hydroxyphenylpyruvate dioxygenase protein described in the first aspect.

The sixth aspect of the present invention provides the use of at least one of the p-hydroxyphenylpyruvate dioxygenase protein described in the first aspect, the gene described in the second aspect, the recombinant vector described in the third aspect, and the transgenic cell described in the fourth aspect in improving crop herbicide resistance.

The seventh aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector described in the third aspect into a target plant, so that the target plant expresses the para-hydroxyphenylpyruvate dioxygenase protein described in the first aspect to obtain resistance to the herbicide.

The eighth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector in a mutant crop containing the recombinant vector described in the third aspect into a target plant through hybridization, breeding or backcrossing, so that the target plant expresses the para-hydroxyphenylpyruvate dioxygenase protein described in the first aspect to obtain resistance to the herbicide.

The ninth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: modifying the HPPD gene of a target plant by a CRISPR/Cas gene editing method so that the target plant expresses the para-hydroxyphenylpyruvate dioxygenase protein described in the first aspect to obtain resistance to the herbicide.

Compared with the prior art, the HPPD protein and its encoding gene resistant to HPPD inhibitors provided by the present invention have at least the following advantages:

By transferring the gene encoding the enzyme provided by the present invention into the target plant, the target plant can acquire resistance to HPPD inhibitors (herbicides), but the catalytic activity of the enzyme itself is basically unaffected. Therefore, the HPPD protein and its encoding gene provided by the present invention can be used to cultivate plants with herbicide resistance.

Other features and advantages of the present invention will be described in detail in the following detailed description.

DETAILED DESCRIPTION

The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article.

In the present invention, unless otherwise specified, the term "enzyme activity" used refers to the amount of enzyme content, which is expressed in enzyme activity units, namely enzyme units (U).

As mentioned above, the first aspect of the present invention provides a para-hydroxyphenylpyruvate dioxygenase protein having an amino acid sequence selected from at least one of the following:

(1) a first amino acid sequence derived from a mutation in at least one of positions 418, 423 and 432 of the amino acid sequence as shown in SEQ ID NO: 1;

(2) a protein derived from the first amino acid sequence in which the first amino acid sequence is substituted, deleted or added with one or more amino acids and the enzyme activity remains unchanged, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the first amino acid sequence;

(3) a second amino acid sequence derived from a mutation at position 422 of the amino acid sequence shown in SEQ ID NO: 2;

(4) a protein derived from the second amino acid sequence in which the second amino acid sequence is substituted, deleted or added with one or more amino acids and the enzyme activity remains unchanged, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the second amino acid sequence;

(5) a third amino acid sequence derived from a mutation at position 426 in the amino acid sequence as shown in SEQ ID NO: 3;

(6) a protein derived from the third amino acid sequence in which one or more amino acids are substituted, deleted or added and the enzyme activity remains unchanged, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the third amino acid sequence;

(7) a fourth amino acid sequence derived from a mutation at position 418 in the amino acid sequence as shown in SEQ ID NO: 4;

(8) a protein derived from the fourth amino acid sequence in which one or more amino acids are substituted, deleted or added and the enzyme activity remains unchanged, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the fourth amino acid sequence;

(9) the fifth amino acid sequence derived from a mutation at position 426 in the amino acid sequence as shown in SEQ ID NO:5;

(10) A protein derived from the fifth amino acid sequence in which one or more amino acids are substituted, deleted or added and the enzyme activity remains unchanged, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and/or carboxyl terminus of the fifth amino acid sequence.

In the present invention, unchanged enzyme activity means that under the same assay conditions, the percentage (relative activity) between the enzyme activity of the proteins derived from the first amino acid sequence, the second amino acid sequence, the third amino acid sequence, the fourth amino acid sequence, and the fifth amino acid sequence and the enzyme activity of the wild type is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%).

Preferably, the first amino acid sequence is an amino acid sequence selected from at least one of the following:

(1a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 wherein lysine at position 418 is mutated to aspartic acid;

(1b) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 1 wherein the glutamic acid at position 423 is mutated to methionine;

(1c) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 1 wherein the glutamic acid at position 423 is mutated to glutamine;

(1d) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 wherein the glutamic acid at position 432 is mutated to methionine;

(1e) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 1 wherein the glutamic acid at position 432 is mutated to isoleucine.

Preferably, the first amino acid sequence is at least one of the amino acid sequences shown by K418D, E423M, E423Q, E432M, and E432I.

According to a specific embodiment of the present invention, the amino acid sequence of the protein obtained by mutating position 418 in the amino acid sequence shown in SEQ ID NO: 1 to aspartic acid is called K418D.

According to a specific embodiment of the present invention, the amino acid sequence of the protein obtained by mutating the 423 position in the amino acid sequence shown in SEQ ID NO: 1 to methionine is called E423M.

According to a specific embodiment of the present invention, the amino acid sequence of the protein obtained by mutating the 423 position in the amino acid sequence shown in SEQ ID NO: 1 to glutamine is called E423Q.

According to a specific embodiment of the present invention, the amino acid sequence of the protein obtained by mutating position 432 of the amino acid sequence shown in SEQ ID NO: 1 to methionine is called E432M.

According to a specific embodiment of the present invention, the amino acid sequence of the protein obtained by mutating position 432 in the amino acid sequence shown in SEQ ID NO: 1 to isoleucine is called E432I.

Preferably, the second amino acid sequence is the following amino acid sequence:

(2a) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 wherein the glutamic acid at position 422 is mutated to methionine.

Preferably, the second amino acid sequence is the amino acid sequence shown by E422M.

According to a specific embodiment of the present invention, the amino acid sequence of the protein obtained by mutating position 422 in the amino acid sequence shown in SEQ ID NO: 2 to methionine is called E422M.

Preferably, the third amino acid sequence is the following amino acid sequence:

(3a) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 3 wherein the glutamic acid at position 426 is mutated to methionine.

Preferably, the third amino acid sequence is the amino acid sequence shown by E426M.

According to a specific embodiment of the present invention, the amino acid sequence of the protein obtained by mutating position 426 in the amino acid sequence shown in SEQ ID NO: 3 to methionine is called E426M.

Preferably, the fourth amino acid sequence is the following amino acid sequence:

(4a) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 4 wherein the glutamic acid at position 418 is mutated to methionine.

Preferably, the fourth amino acid sequence is the amino acid sequence shown by Q418M.

According to a specific embodiment of the present invention, the amino acid sequence of the protein obtained by mutating position 418 in the amino acid sequence shown in SEQ ID NO: 4 to methionine is called Q418M.

Preferably, the fifth amino acid sequence is the following amino acid sequence:

(5a) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 5 wherein the glutamic acid at position 426 is mutated to glutamine.

Preferably, the fifth amino acid sequence is the amino acid sequence shown by E426Q.

According to a specific embodiment of the present invention, the amino acid sequence of the protein obtained by mutating position 426 in the amino acid sequence shown in SEQ ID NO: 5 to glutamine is called E426Q.

In the present invention, the method for obtaining the above-mentioned protein is well known to those skilled in the art. For example, when the amino acid sequence of the protein is known, it can be directly obtained by chemical synthesis, or by first obtaining a gene encoding the protein and then obtaining the protein by biological expression.

The protein provided by the present invention can also be modified. Modifications (usually without changing the primary structure, i.e., without changing the amino acid sequence) include: chemical derivatization forms of proteins in vivo or in vitro such as acetylation or hydroxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the protein or in further processing steps, which can be completed by exposing the protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine).

In the present invention, in order to facilitate purification, the protein can also be modified by using common tags in the art. For example, it can be obtained by connecting the common purification tags shown in Table 1 (such as at least one of GST, Poly-His, FLAG, His-SUMO and c-myc) to the amino terminal and/or carboxyl terminal of the protein. The tag will not affect the activity of the protein provided by the present invention. In actual application, it can be selected whether to add a tag according to needs.

Table 1

As mentioned above, the second aspect of the present invention provides a gene encoding a p-hydroxyphenylpyruvate dioxygenase protein, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence of the p-hydroxyphenylpyruvate dioxygenase protein described in the first aspect.

Preferably, the nucleotide sequence of the gene is at least one nucleotide sequence selected from the following:

(1) the nucleotide sequence shown in SEQ ID NO:6;

(2) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 6;

(3) the nucleotide sequence shown in SEQ ID NO:7;

(4) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:7;

(5) the nucleotide sequence shown in SEQ ID NO: 8;

(6) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 8;

(7) the nucleotide sequence shown in SEQ ID NO:9;

(8) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 9, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical;

(9) the nucleotide sequence shown in SEQ ID NO: 10;

(10) A nucleotide sequence that is at least 90% identical to SEQ ID NO:10, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical.

In the present invention, it is well known in the art that, among the 20 different amino acids constituting proteins, except for Met (ATG) or Trp (TGG) which are respectively encoded by a single codon, the other 18 amino acids are respectively encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there are usually more than one codon to determine an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the composition of the amino acids, so the nucleotide sequences of genes encoding the same protein can be different.

Those skilled in the art can fully deduce the nucleotide sequences of genes encoding the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and the specific mutation patterns of the proteins provided by the present invention based on the known codon table, and obtain the nucleotide sequences by biological methods (such as PCR method, mutation method) or chemical synthesis method, so this part of the nucleotide sequences should all be included in the scope of the present invention.

Similarly, using the nucleotide sequences SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and the specific mutation mode of the protein provided by the present invention, it is also possible to obtain the amino acid sequence provided by the present invention by modifying SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 by methods well known in the art, such as the method of Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989).

In the present invention, a person skilled in the art can easily obtain the gene encoding the protein provided by the present invention based on the above-mentioned protein and nucleotide sequences SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Exemplarily, site-directed mutagenesis can be performed on the basis of SEQ ID NO: 6, and the site-directed mutagenesis method includes but is not limited to ZFN site-directed mutagenesis method, TALEN site-directed mutagenesis method, and/or genome site-directed mutagenesis methods such as CRISPR-Cas9.

In the present invention, as described in the first aspect, accordingly, the 5' end and/or 3' end of the nucleotide sequence provided by the present invention may also be connected to the coding sequence of the common purification tag shown in Table 1.

The nucleotide sequence provided by the present invention can usually be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Exemplarily, those skilled in the art can easily obtain templates and primers based on the nucleotide sequence provided by the present invention, and amplify the nucleotide sequence using PCR. After obtaining the nucleotide sequence, the amino acid sequence can be obtained in large quantities by recombination. The obtained nucleotide sequence is usually cloned into a vector, then transferred into genetically engineered bacteria, and then the nucleotide sequence is separated from the host cell after the proliferation by conventional methods. In addition, the nucleotide sequence can also be synthesized by artificial chemical synthesis methods known in the art.

As mentioned above, the third aspect of the present invention provides a recombinant vector, which contains the gene described in the second aspect.

In the present invention, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as various commercially available plasmids, cosmids, phages and retroviruses, etc., which can be selected according to specific circumstances, and can be exemplified by pGWC, pB2GW7.0 or pET-28a, etc. The recombinant vector construction can be obtained by enzyme digestion with various nucleases that have a cleavage site in the vector multiple cloning site to obtain a linear plasmid, and then connected with a gene fragment cut with the same nuclease to obtain a recombinant plasmid.

As mentioned above, the fourth aspect of the present invention provides a transgenic cell, which contains the gene described in the second aspect.

Preferably, the transgenic cell is a prokaryotic cell.

In the present invention, the recombinant vector can be transformed, transduced or transfected into a host cell by methods known in the art, such as calcium chloride chemical transformation, high voltage electroporation transformation, preferably electroporation transformation. The host cell can be a prokaryotic cell or a eukaryotic cell, which can be selected according to actual conditions. The cell can be a DH5α strain, an Agrobacterium strain GV3101, etc.

As mentioned above, the fifth aspect of the present invention provides a composition, which contains the p-hydroxyphenylpyruvate dioxygenase protein described in the first aspect.

The composition provided by the present invention contains the p-hydroxyphenylpyruvate dioxygenase protein described in the first aspect of the present invention as an active ingredient, and the content of the protein is 50-90% by weight based on the total weight of the composition. The composition may also contain a solvent known to those skilled in the art (such as glycerol, sugars and protein protectants such as protease inhibitors).

As mentioned above, the sixth aspect of the present invention provides the use of at least one of the p-hydroxyphenylpyruvate dioxygenase protein described in the first aspect, the gene described in the second aspect, the recombinant vector described in the third aspect, and the transgenic cell described in the fourth aspect in improving crop herbicide resistance.

Preferably, the herbicide is a HPPD inhibitor.

More preferably, the herbicide is at least one of triketone compounds, pyrazole compounds, isoxazole compounds, diketonitrile compounds and heterocyclic amide compounds.

Further preferably, the herbicide is at least one of mesotrione, quintrione, Y13287, Y18024 and benzylpyrazone, wherein Y13287 (CN104557739A) and Y18024 (CN110669016A) are herbicides made by this laboratory, and the molecular structural formulas of the above compounds are as follows:

As mentioned above, the seventh aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector described in the third aspect into a target plant, so that the target plant expresses the para-hydroxyphenylpyruvate dioxygenase protein described in the first aspect to obtain resistance to the herbicide.

As described above, the eighth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector in a mutant crop containing the recombinant vector described in the third aspect into a target plant through hybridization, breeding or backcrossing, so that the target plant expresses the para-hydroxyphenylpyruvate dioxygenase protein described in the first aspect to obtain resistance to the herbicide.

As mentioned above, the ninth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: modifying the HPPD gene of the target plant by the CRISPR/Cas gene editing method, so that the target plant expresses the para-hydroxyphenylpyruvate dioxygenase protein described in the first aspect above to obtain resistance to the herbicide.

In the present invention, transferring the recombinant vector into the target plant specifically means: transferring the nucleotide sequence of the protein with herbicide resistance provided by the present invention into the target plant by transgenic method, so that the target plant acquires resistance to HPPD inhibitor herbicides; the nucleotide sequence of the protein provided by the present invention can also be transferred into the target plant by hybridization, breeding, backcrossing and other methods, so that the target plant acquires resistance to HPPD inhibitor herbicides; in addition, the HPPD gene of the target plant can be directly modified by gene editing technology such as CRISPR/Cas, so that the target plant acquires resistance to HPPD inhibitor herbicides. More specifically, the protein can be used as a parent material, hybridized with other excellent plant varieties and further backcrossed, and the herbicide resistance trait can be further bred into other target plant varieties.

The transgenic method used in the present invention is well known to those skilled in the art, and includes direct or indirect transformation methods. Direct transformation methods include chemical induction, liposome method, gene gun method, electroporation method and microinjection method.

In the present invention, the term "plant" has the broadest meaning. Examples of plants include, but are not limited to, vascular plants, vegetables, grains, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi and algae, as well as clones and plant parts used for asexual reproduction (e.g., cuttings, layering, shoots, rhizomes, underground stems, clumps, root collars, bulbs, corms, tubers, rhizomes, plants/tissues produced in tissue culture, etc.). The term "plant" further encompasses whole plants, plant parents and offspring, and plant parts, including seeds, branches, stems, leaves, roots (including tubers), flowers, florets, fruits, fleshy stems, peduncles, stamens, anthers, stigmas, styles, ovaries, petals, sepals, carpels, root tips, root caps, root hairs, leaf hairs, seed hairs, pollen grains, microspores, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organs, tissues and cells and tissues and organs of plants. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristem zones, gametophytes, sporophytes, pollen and microspores. Wherein, all of the above-mentioned include target gene/nucleic acid provided by the present invention.

Plants particularly suitable for use in the methods of the invention include all plants belonging to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants, including food crops, fodder or forage legumes, ornamental plants, trees or shrubs.

According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include rice, wheat, corn, sorghum, soybean, sunflower, rape, alfalfa, cotton, tomato, potato or tobacco. More preferably, the crop plant is a cereal, such as rice, wheat, corn, sorghum, barley, millet, rye or oats.

The beneficial effects achieved by the present invention are to obtain proteins, genes, recombinant vectors, transgenic cells, compositions, and plants that can replace the prior art and make plants resistant to herbicides, to obtain applications and methods of plants with herbicide resistance, and to obtain plant varieties with herbicide resistance through transgenic or non-transgenic methods.

The present invention will be described in detail below by way of examples.

In the following examples, unless otherwise specified, room temperature refers to 25±2°C.

In the following examples, unless otherwise specified, the experimental methods used are conventional methods.

In the following examples, unless otherwise specified, the materials and reagents involved can be obtained from commercial sources.

Agarose gel recovery kit was purchased from TIANGEN Company;

Restriction enzymes and supporting solutions were purchased from Takara;

The ligation kit was purchased from New England BioLabs.

Example 1

Synthesis of coding genes and nucleotide sequences

The following coding genes and nucleotide sequences were synthesized by Wuhan Jinkairui Bioengineering Co., Ltd. based on the nucleotide sequence provided by the present invention.

(1) SEQ ID NO: 6

According to the nucleotide sequence shown in SEQ ID NO: 6, the coding gene of the protein shown in Table 2 and the nucleotide sequence shown in SEQ ID NO: 6 were synthesized.

Table 2

protein Coding genes Mutation site codon SEQ ID NO: 1 WT (wild type) / K418D K418D GAT E423M E423M ATG E423Q E423Q CAG E432M E432M ATG E432I E432I ATT

(2) SEQ ID NO: 7

According to the nucleotide sequence shown in SEQ ID NO: 7, the coding gene of the protein shown in Table 3 and the nucleotide sequence shown in SEQ ID NO: 7 were synthesized.

Table 3

protein Coding genes Mutation site codon SEQ ID NO: 2 WT (wild type) / E422M E422M ATG

(3) SEQ ID NO: 8

According to the nucleotide sequence shown in SEQ ID NO: 8, the coding gene of the protein shown in Table 4 and the nucleotide sequence shown in SEQ ID NO: 8 were synthesized.

Table 4

protein Coding genes Mutation site codon SEQ ID NO: 3 WT (wild type) / E426M E426M ATG

(4) SEQ ID NO: 9

According to the nucleotide sequence shown in SEQ ID NO:9, the coding gene of the protein shown in Table 5 and the nucleotide sequence shown in SEQ ID NO:9 were synthesized.

Table 5

protein Coding genes Mutation site codon SEQ ID NO: 4 WT (wild type) / Q418M Q418M ATG

(5) SEQ ID NO: 10

According to the nucleotide sequence shown in SEQ ID NO: 10, the coding gene of the protein shown in Table 6 and the nucleotide sequence shown in SEQ ID NO: 10 were synthesized.

Table 6

protein Coding genes Mutation site codon SEQ ID NO: 5 WT (wild type) / E426Q E426Q CAG

Example 2

Construction of expression vector

(1) Amplification of target gene

Using the nucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 as templates, PCR reactions were performed to obtain wild-type and mutant gene sequences of HPPD as shown in Tables 2, 3, 4, 5, and 6, and ligated into corresponding vectors.

The PCR reaction system and PCR program settings are as follows:

Table 7

PCR reaction system composition Addition amount (volume) 10× PCR buffer 5μL 5×dNTP(10mM) 1μL stencil 1μL Forward primer 1.0μL Direction Primer 1.0μL PfuDNA polymerase 0.5μL dd H2O To a total volume of 50 μL

Table 8

step Temperature(℃) Time(s) 1 95 240 2 95 45 3 55 45 4 72 400 5 72 600 6 4 save

The extension time at 72°C depends on the specific fragment length and the efficiency of the enzyme used. Steps 2 to 4 in the above procedure are repeated for 25 cycles.

(2) Agarose gel electrophoresis of nucleic acids and recovery of PCR products

a. Weigh 1g agarose into a conical flask, add 100mL of 1×TAE solution (prepare a 50-fold stock solution, preparation method: weigh 242g Tris, 18.6g EDTA into a 1L beaker; add about 800mL deionized water and stir; add 57.1ml glacial acetic acid and fully dissolve; adjust the pH to 8.3 with NaOH, add deionized water to 1L, and store at room temperature, the same below), put it in a microwave oven and heat until the agarose particles are completely dissolved, cool it until it is not hot, add 5μL of ethidium bromide solution, mix well, pour it into the gel tank with the comb in place, and let it cool and solidify;

b. Remove the comb before use, put the gel into the electrophoresis tank, add an appropriate amount of 1×TAE solution; add the sample to the well, cover the lid and start electrophoresis, and stop electrophoresis when the bromophenol blue moves to 1/3 of the bottom;

c. Cut the target band, put it into a clean 1.5mL EP tube and recover the gel; the gel recovery kit was purchased from TIANGEN (Cat. No. DP209); the amount of gel recovery was 300μL of sol solution PN (provided by the kit) for 100mg of gel, and incubated in a 50℃ water bath, constantly inverting and mixing to help dissolve the gel;

d. Add the solution in c to the pretreated adsorption column (provided in the kit), let it stand at room temperature for 5 minutes to allow it to fully bind, and then centrifuge at 12000 rpm for 1 minute;

e. Pour out the liquid in the collection tube and repeat step d once to increase the recovery rate of the product;

f. Add 500 μL of the washing solution PW (provided by the kit) to the purification column and centrifuge at 12000 rpm for 1 min; repeat this operation once;

g. Centrifuge the adsorption column at 13000 rpm for 2 min to remove the PW solution as much as possible;

h. Place the purification column in a new EP tube and heat it at 65°C for 10 minutes to evaporate the PW solution;

i. Add 50 μL of elution solution (provided by the kit) to the middle of the adsorption column, place it on a 65°C heating table for 2 minutes, and centrifuge it at 13,000 rpm for 2 minutes to elute the DNA; repeat this operation once;

j. Finally, the recovered PCR products were stored at -20°C.

(3) Enzyme digestion reaction

After the DNA with the correct size fragment is verified by electrophoresis, the vector and PCR product are double-digested (the digestion solution is purchased together with the restriction endonuclease). The digestion system is shown in Table 9:

Table 9

Components Volume (μL) PCR product/vector 43 10×Cutsmart solution 5 B H 1 XOt 1

After incubation at 37°C for 3 h, electrophoresis was performed and the fragments were recovered using a gel recovery kit. To improve the efficiency of vector cutting, 1.5 μL of restriction endonuclease was added and the enzyme cutting time was extended by 5 h.

(4) Ligation reaction

The recovered gene fragments were ligated according to the following system. The ligation reagent Solution 1 (including T4 DNA ligase and matching buffer) was purchased from New England BioLabs (Cat. No. M0202S), and then incubated at 16° C. for more than 5 hours.

The connection system is shown in Table 10:

Table 10

Element Volume (μL) Solution 1 8 Purpose fragment 7 Carrier 1

(5) Conversion

After the ligation is completed, the ligation product is transformed into Escherichia coli JM109 competent cells (purchased from Promega, product number ST1105) and cultured. The specific steps are as follows:

a. Take out a tube of competent cells and place it on ice to thaw for 10 minutes;

b. Add the ligation product to the competent cells, mix well, and place on ice for 30 minutes;

c. Heat shock at 42°C for 90 seconds, then place on ice for 2 minutes;

d. Add 200 μL of Luria-Bertani (LB) medium (without antibiotics) to the tube and culture at 37°C and 220 rpm for 30 min; preheat the solid LB plate with the corresponding resistance to room temperature;

e. Take out the bacterial solution and spread it evenly on the plate, and culture it in a 37℃ incubator for 16 hours.

(6) Sequencing

Select some single clones from the plate cultured for 16 hours and transfer them to a clean plate, culture them at 37°C for 5 hours, and then send them to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing; after the sequencing results are fed back, sequence comparison is performed to confirm the correct clone.

Example 3

Expression and purification of target protein

(1) Expression

First, extract the protein in small quantities to explore the best expression conditions and purification strategies, and then extract in large quantities after the conditions are determined. Protein expression is carried out in Escherichia coli BL21 (DE3) cells (purchased from Kangti Life Science Technology Co., Ltd., product number KTSM109), and the process is as follows:

a. Pick a single clone from the plate after 16 hours of culture and add it to 100 mL of LB medium containing the required antibiotic resistance. Culture it at 37°C and 220 rpm for 5 hours until obvious turbidity is observed.

b. Expand the vial of bacteria in step a into a large bottle of LB medium (containing antibiotics) at a volume ratio of 1:100, and culture at 37°C and 220rpm for 3h; when the _OD600 value reaches 0.7h, lower the temperature to 20°C and add 0.2mM isopropyl-β-D-thiogalactoside (IPTG) for induction, and the induction time is 14h;

c. Centrifuge at 4°C, 4000 rpm for 10 min, collect the bacteria and use them for protein purification.

(2) Purification

a. Resuspend the collected E. coli with cell lysis solution, and the volume ratio of the cell lysis solution to the LB culture medium of the collected E. coli is 30mL:1L; stir on a magnetic stirrer for 20 minutes to mix the cells, during which 1mM of serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF), 40μg/mL of lysozyme for lysing bacterial cell walls, 1μg/mL of deoxyribonuclease I for decomposing nucleic acids, and 10mM of cofactor MgCl ₂ are added; then ultrasonic disruption is performed;

b. After the sonication, centrifuge at 13000 rpm and 4°C for 1 hour, collect the supernatant for affinity chromatography; protein purification was carried out in a constant temperature room at 4°C;

c. First, pour the supernatant after centrifugation into the treated Ni column for full binding; then rinse with a solution containing different concentrations (10mM, 30mM, 50mM) of imidazole to remove impurities; finally, elute the target protein with a high concentration (250mM) of imidazole solution; and then analyze the results by SDS-PAGE;

d. The protein after affinity chromatography was diluted and loaded onto an ion exchange column. After loading, a linear gradient (NaCl from 0 to 500 mM) elution was performed with a NaCl buffer solution (the buffer solution was Tris, pH 7.0). This step was completed with the aid of a protein purifier. SDS-PAGE was run to analyze the results, and proteins with better properties and purity were combined and concentrated for the next step of molecular sieve chromatography.

e. Before loading the sample on molecular sieve chromatography, centrifuge at 13000rpm for 5min to remove some precipitated proteins and impurities, and then use a syringe to transfer the sample to the loading loop; then use elution buffer (containing 100mM NaCl, Tris, pH 7.0) to elute the protein, collecting 0.5mL of the sample in each tube; then use SDS-PAGE to detect and analyze the protein, combine the proteins that need to be preserved, and then divide them into small portions for freezing; for proteins used for activity testing, add glycerol at a final concentration of 30% by volume for preservation.

Test Example 1

Study on the inhibition kinetics of herbicides

The coupled enzyme activity test method was used to determine the inhibition kinetics of the enzyme at different concentrations of inhibitors under saturated substrate concentration.

The specific test method steps are as follows:

a. Preparation of buffer solution, substrate and cofactor:

HEPES buffer for activity measurement: Prepare a 1 M stock solution, adjust its pH to 7.0 with sodium hydroxide, dilute to 20 mM before use, and filter with a 0.22 μm filter membrane;

Preparation of the substrate HPPA (p-hydroxyphenylpyruvic acid): first prepare a stock solution with a concentration of 100 mM using DMSO, and dilute it with activity measurement buffer before use;

Preparation of sodium ascorbate: Prepare to a concentration of 20 mM with deionized water. Store at -80°C;

Preparation of ferrous sulfate: Prepare to a concentration of 1mM with deionized water. Store at -80℃;

Preparation of inhibitors: Prepare a 10 mM stock solution with DMSO and dilute it to different concentrations with activity assay buffer before use to ensure that the volume of inhibitor added to each well is the same;

b. Preparation of coupling enzyme HGD (homogeneous dioxygenase): The construction of the expression vector of HGD is consistent with the method of "Construction of expression vector in Example 2", and human HGD is used here; the expression method of HGD protein is consistent with the method of "Expression and purification of target protein in Example 3", but the HGD used for testing is not subjected to subsequent purification, but directly uses the supernatant of cell lysate;

c. Before starting the activity test, the dosage of HGD needs to be explored. When HGD is insufficient, the reaction cannot be well coupled, and the measured reaction rate cannot truly reflect the activity of HPPD. Therefore, it is necessary to do an HGD concentration-dependent experiment. When the reaction rate no longer increases with the increase in the amount of HGD, it is considered that the HGD dosage is saturated at this time; and the saturated dosage is doubled to ensure sufficient coupling of the reaction.

d. During the test, first mix the activity buffer (20mM), substrate (50μM), sodium ascorbate (2mM), ferrous sulfate (100μM), and coupled enzyme HGD, and incubate at 30℃ for 5min, then add them to the 96-well ELISA plate (3 parallel wells), and then add different concentrations of inhibitors to the above wells. Among them, the wells without inhibitors are filled with activity buffer and 1μL DMSO (as a control); finally, HPPD is added to start the reaction, and HPPD needs to be incubated at 30℃ for 5min in advance;

e. First, place the ELISA plate in the instrument and shake it for 15 seconds, then read the UV absorbance at 318nm, read it every 30 seconds, and monitor for a total of 10 minutes. Repeat three times.

In this test example, two types of representative commercial herbicides were tested: triketides (mesotrione, quintrione, Y18024 and Y13287) and pyrazoles (benpyraclostrobin), and the inhibition kinetics (IC ₅₀ ) of HPPD protein in rice, wheat, sorghum, corn and Arabidopsis were tested, as shown in Table 11, Table 12, Table 13, Table 14 and Table 15, respectively.

Control: refers to adding substrate and 1 μL of DMSO (organic solvent for dissolving inhibitors, total system is 200 μL) to the enzyme reaction system, which is regarded as the full activity of the enzyme in the absence of inhibitors.

Sample: Add inhibitors of different concentrations to the enzyme reaction system and observe the effect of the inhibitors on enzyme activity.

Data processing:

The _IC50 value was obtained according to the following formula:

In the above formula:

y——the percentage of the residual activity in the presence of the corresponding concentration of inhibitor to the activity without inhibitor;

max, min——maximum and minimum values of relative activity;

x——corresponding inhibitor concentration;

IC ₅₀ - the concentration of inhibitor at which the enzyme activity remains 50%.

In order to compare the resistance multiples of the wild type and each mutant to different compounds, the protein concentrations of the wild type and mutants of the same genus were adjusted to be consistent for testing. For example, in Table 11, the concentration of each protein (including the wild type and mutants K418D, E423M, E423Q, E432M, E432I) is 22.5nM. In Table 12, the concentration of each protein (including the wild type and mutant E422M) is 22.5nM. In Table 13, the concentration of each protein (including the wild type and mutant E426M) is 26.2nM. In Table 14, the concentration of each protein (including the wild type and mutant Q418M) is 18.0nM. In Table 15, the concentration of each protein (including the wild type and mutant E426Q) is 36.0nM.

To obtain the relative resistance multiple, the IC ₅₀ value of each compound against the wild type was used as a benchmark, and the relative resistance multiple was set to 1.000. The resistance multiple of the mutant relative to the wild type is: the IC ₅₀ value of the compound against the mutant divided by the IC ₅₀ value of the wild type. For example, in Table 11, the IC ₅₀ value of mesotrione against wild-type rice HPPD is 0.236 μM, and its relative resistance multiple is set to 1.000; the IC ₅₀ value of mesotrione against rice HPPD mutant K418D is 0.501 μM, so the resistance multiple of the mutant relative to the wild type is: 0.501/0.236=2.12 times; and so on for other compounds, mutants, and different species.

Table 11 Inhibition kinetics (IC ₅₀ ) results and resistance folds of rice HPPD mutants

In the above table, K418D refers to the mutant at position 418 of rice HPPD, which mutated from lysine to aspartic acid; E423M refers to the mutant at position 423 of rice HPPD, which mutated from glutamate to methionine; E423Q refers to the mutant at position 423 of rice HPPD, which mutated from glutamate to glutamine; E432M refers to the mutant at position 432 of rice HPPD, which mutated from glutamate to methionine; E432I refers to the mutant at position 432 of rice HPPD, which mutated from glutamate to isoleucine.

Table 12 Inhibition kinetics (IC ₅₀ ) results and resistance folds of wheat HPPD mutants

In the table above, E422M refers to a mutant at position 422 of wheat HPPD, which mutates from glutamate to methionine.

Table 13 Inhibition kinetics (IC ₅₀ ) results and resistance folds of sorghum HPPD mutants

In the table above, E426M refers to the mutant at position 426 of sorghum HPPD, which mutated from glutamate to methionine.

Table 14 Inhibition kinetics (IC ₅₀ ) results and resistance folds of corn HPPD mutants

In the table above, Q418M refers to the mutant at position 418 of HPPD in maize, which mutates from glutamine to methionine.

Table 15 Inhibition kinetics (IC ₅₀ ) results and resistance folds of Arabidopsis HPPD mutants

In the table above, E426Q refers to a mutant at position 426 of the Arabidopsis HPPD gene, which mutated from glutamate to glutamine.

From the test results, it can be seen that the mutation at site 418 in the amino acid sequence shown in SEQ ID NO: 1 (K418D) has a resistance to each inhibitor of 1.6 to 2.6 times (relative to the wild type); the mutation at site 423 in the amino acid sequence shown in SEQ ID NO: 1 (E423M, E423Q) has a resistance to each inhibitor of 1.4 to 2.1 times (relative to the wild type); the mutation at site 432 in the amino acid sequence shown in SEQ ID NO: 1 (E432M, E432I) has a resistance to each inhibitor of 1.5 to 2.9 times (relative to the wild type); the mutation at site 422 in the amino acid sequence shown in SEQ ID NO: 2 (E422M) has a resistance to each inhibitor of 2.6 to 4.0 times (relative to the wild type); The mutation at position 426 (E426M) in the amino acid sequence shown in NO:3 has a resistance multiple of 1.0 to 1.5 times (relative to the wild type), and "none" means no resistance, that is, the IC ₅₀ value of the inhibitor to the mutant is less than the IC ₅₀ value of the wild type; the mutation at position 418 (Q418M) in the amino acid sequence shown in SEQ ID NO:4 has a resistance multiple of 1.4 to 2.1 times (relative to the wild type); the mutation at position 426 (E426Q) in the amino acid sequence shown in SEQ ID NO:5 has a resistance multiple of 1.2 to 1.9 times (relative to the wild type). The inhibitory activity of the HPPD protein provided by the present invention is weakened, that is, the HPPD protein provided by the present invention is insensitive to herbicides. Therefore, the HPPD protein provided by the present invention can be used to improve the herbicide resistance of crops.

The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims (12)

1. A p-hydroxyphenylpyruvate dioxygenase protein, characterized in that the amino acid sequence of the protein is shown as a third amino acid sequence; the third amino acid sequence is the amino acid sequence shown in SEQ ID NO.3 after mutation of glutamic acid at position 426 into methionine.

2. A gene encoding a p-hydroxyphenylpyruvate dioxygenase protein, characterized in that the nucleotide sequence of the gene is a nucleotide sequence which is capable of encoding the amino acid sequence of the p-hydroxyphenylpyruvate dioxygenase protein of claim 1.

3. A recombinant vector comprising the gene according to claim 2.

4. A transgenic cell comprising the gene of claim 2; the transgenic cells are prokaryotic cells.

5. A composition comprising the p-hydroxyphenylpyruvate dioxygenase protein of claim 1.

6. Use of at least one of the p-hydroxyphenylpyruvate dioxygenase protein of claim 1, the gene of claim 2, the recombinant vector of claim 3, the transgenic cell of claim 4 for increasing herbicide resistance in crops.

7. The use according to claim 6, wherein the herbicide is an HPPD inhibitor.

8. The use according to claim 6 or 7, wherein the herbicide is at least one of a triketone compound and a pyrazole compound.

9. The use according to claim 8, wherein the herbicide is at least one of mesotrione, quinclorac, Y13287, Y18024 and topramezone.

10. A method of increasing herbicide resistance in a crop, the method comprising: transferring the recombinant vector of claim 3 into a target plant, such that the target plant expresses the p-hydroxyphenylpyruvate dioxygenase protein of claim 1 to obtain resistance to herbicides.

11. A method of increasing herbicide resistance in a crop, the method comprising: transferring the recombinant vector in the mutant crop containing the recombinant vector of claim 3 into a target plant by crossing, transferring or backcrossing, so that the target plant expresses the p-hydroxyphenylpyruvate dioxygenase protein of claim 1 to obtain resistance to herbicides.

12. A method of increasing herbicide resistance in a crop, the method comprising: the HPPD gene of the target plant is engineered by CRISPR/Cas gene editing methods such that the target plant expresses the p-hydroxyphenylpyruvate dioxygenase protein of claim 1 to obtain resistance to herbicides.