HPPD inhibitor herbicide resistance related gene, and encoded protein and use thereof

Abstract

Provided are an HPPD inhibitor herbicide resistance gene BPR1, and an encoded protein and a use thereof. The loss of function of the protein encoded by this gene confers broad-spectrum resistance to HPPD inhibitor herbicides in rice. Evolutionary analysis of the BPR1 gene finds that homologous genes thereof exist in gramineous food crops such as rice, maize, wheat, millet, sorghum, barley, oat and millet, as well as in crops such as tobacco, cotton, grape, soybean and rapeseed. Technologies such as gene editing and RNAi can be used to reduce the expression level of the BPR1 gene and homologous genes thereof in different species, or disrupt coding reading frames thereof, leading to the loss of function of the BPR1 protein and the homologous proteins thereof in different species, thereby making the corresponding plants broad-spectrum resistant to HPPD inhibitor herbicides. The present invention has broad application prospects in the field of cultivating crops resistant to HPPD inhibitor herbicides.

Description

HPPD inhibitor herbicide resistance-related genes and their encoding proteins and applications Technical Field

[0001] The present invention belongs to the field of biotechnology and mainly relates to HPPD inhibitor herbicide resistance-related genes and encoded proteins and their application in cultivating plants resistant to broad-spectrum HPPD inhibitor herbicides. Background Art

[0002] During their growth and development, crops inevitably experience interference from various biotic and abiotic stresses. Weeds, one of the most common biotic stressors, not only compete with crops for resources like sunlight, water, nutrients, and growing space, but also serve as hosts for pests and diseases, spreading them and severely impacting crop growth, leading to yield losses. Chemical herbicides are currently one of the most cost-effective methods for weed control and have made significant contributions to ensuring stable crop yields and increasing incomes over the past decades.

[0003] 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a vital enzyme in living organisms. It participates in tyrosine metabolism and is present in nearly all aerobic organisms. Within organisms, tyrosine is converted to p-hydroxyphenylpyruvic acid (HPPA) under the catalysis of tyrosine aminotransferase (TAT). HPPD, a ferrous-dependent, non-heme oxygenase, further catalyzes HPPA in the presence of oxygen to 2,5-dihydroxyphenylacetate (HGA), a key precursor of plastoquinone and tocopherols. Plastoquinone and tocopherols are crucial components of plant defense against oxidative stress and in promoting photosynthesis. Disturbances in their metabolism can lead to a deficiency of carotenoids in plants, impaired photosynthesis, and ultimately, plant death due to albinism.

[0004] Since HPPD was first identified as a herbicide target in the 1990s, HPPD inhibitors have gradually emerged due to their advantages such as high efficiency, low toxicity, and low resistance to cross-resistance. Common HPPD inhibitors include, but are not limited to, bipyrazone (CAS No. 1622908-18-2), mesotrione (CAS No. 104206-82-8), tembotrione (CAS No. 335104-84-2), and topramezone (CAS No. 210631-68-8).

[0005] However, with the extensive and repeated use of specific herbicides in recent years, resistant weeds have also emerged. Therefore, the development of new herbicides and the cultivation of new herbicide-resistant crops are of great significance and have broad application prospects.

[0006] Currently, the cultivation of crops resistant to HPPD inhibitor herbicides primarily involves increasing the expression levels of homologous or heterologous HPPD genes through transgenic methods, or by modifying the plant's endogenous HPPD gene through gene editing techniques to reduce the binding of HPPD herbicide binding sites to HPPD inhibitor herbicides, thereby reducing sensitivity to herbicides and thereby improving plant resistance to HPPD inhibitor herbicides. Few other HPPD inhibitor herbicide resistance genes have been reported, and to date, only HIS1 (HPPD INHIBITOR SENSITIVE 1) has been reported to confer significant resistance to triketone herbicides. Therefore, identifying genes associated with HPPD inhibitor herbicide resistance is of great significance for the development of resistant crops.

[0007] Summary of the Invention

[0008] In view of the above problems, the present invention provides a BPR1 gene and its encoded protein, and its use in cultivating plants resistant to HPPD inhibitor herbicides. After the BPR1 gene or the protein encoded by its homologous gene in the plant loses or weakens its function, the plant has significantly enhanced resistance to HPPD inhibitor herbicides.

[0009] Specifically, the present invention provides the following technical solutions.

[0010] In one aspect, the present invention provides a use of an HPPD inhibitor herbicide-related resistance protein in cultivating or breeding plants having enhanced HPPD inhibitor herbicide resistance compared to wild-type plants, wherein the amino acid sequence of the protein is as follows:

[0011] 1) the amino acid sequence shown in SEQ ID NO: 2; or

[0012] 2) An amino acid sequence of a derivative protein that has undergone substitution, deletion and / or addition of one or more amino acid residues compared to the amino acid sequence shown in SEQ ID NO: 2 and has the same activity as the amino acid sequence shown in SEQ ID NO: 2.

[0013] In some embodiments, the amino acid sequence of the derivative protein has at least 60%, 65%, for example 67%-99% (such as 68%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) homology with the amino acid sequence shown in SEQ ID NO: 2. Preferably, the amino acid sequence of the derivative protein is an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 26.

[0014] In some embodiments, the nucleotide sequence of the gene encoding the protein is as follows:

[0015] 1) the DNA sequence shown in SEQ ID NO: 1;

[0016] 2) a DNA sequence that has a conservative substitution, deletion, or addition of one or more bases compared to the DNA sequence shown in SEQ ID NO: 1 and has the same function as the DNA sequence shown in SEQ ID NO: 1; or

[0017] 3) A nucleotide sequence that can hybridize to the DNA sequence shown in SEQ ID NO: 1 under highly stringent conditions.

[0018] In some embodiments, the DNA sequence having the same function as the DNA sequence shown in SEQ ID NO: 1 has at least 60% identity with the DNA sequence shown in SEQ ID NO: 1, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0019] In another aspect, the present invention provides a method for cultivating plants with broad-spectrum resistance to HPPD inhibitor herbicides, the method comprising completely losing or weakening the function of an HPPD inhibitor herbicide-related resistance protein in wild-type plants or plant cells, thereby achieving the purpose of enhancing the plant's broad-spectrum resistance to HPPD inhibitor herbicides, wherein the amino acid sequence of the protein is as follows:

[0020] 1) the amino acid sequence shown in SEQ ID NO: 2; or

[0021] 2) An amino acid sequence of a derivative protein that has undergone substitution, deletion and / or addition of one or more amino acid residues compared to the amino acid sequence shown in SEQ ID NO: 2 and has the same activity as the amino acid sequence shown in SEQ ID NO: 2.

[0022] In some embodiments, the amino acid sequence of the derivative protein has at least 60%, 65%, for example 67%-99% (such as 68%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) homology with the amino acid sequence shown in SEQ ID NO: 2. Preferably, the amino acid sequence of the derivative protein is an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 26.

[0023] In some embodiments, the complete loss or attenuation of the function of the HPPD inhibitor herbicide-related resistance protein is achieved by:

[0024] 1) The amino acid sequence of the protein is replaced, deleted and / or added by molecular means (e.g., gene editing, RNAi interference), resulting in complete loss or weakening of the function of the protein;

[0025] 2) knocking out the gene encoding the protein in the plant by gene editing (e.g., CRISPR / CAS gene editing), resulting in the inability to translate the biologically active protein in the plant; or

[0026] 3) Through protein modification (e.g., ubiquitination, acetylation, proteolysis), the amino acid sequence of the protein is modified, thereby causing the complete loss or weakening of the function of the protein.

[0027] In another aspect, the present invention provides a method for preparing a plant with broad-spectrum resistance to HPPD inhibitor herbicides, comprising partially or completely deleting a gene encoding a resistance protein associated with the HPPD inhibitor herbicide in a wild-type plant or plant cell, or replacing, introducing and / or deleting one or more bases therein, culturing the plant or plant cell, and screening for plants having broad-spectrum resistance to HPPD inhibitor herbicides compared to the wild-type plant, wherein the nucleotide sequence of the gene is as follows:

[0028] 1) the DNA sequence shown in SEQ ID NO: 1;

[0029] 2) a DNA sequence that has a conservative substitution, deletion, or addition of one or more bases compared to the DNA sequence shown in SEQ ID NO: 1 and has the same function as the DNA sequence shown in SEQ ID NO: 1; or

[0030] 3) A nucleotide sequence that can hybridize to the DNA sequence shown in SEQ ID NO: 1 under highly stringent conditions.

[0031] In some embodiments, the DNA sequence having the same function as the DNA sequence shown in SEQ ID NO: 1 has at least 60% identity with the DNA sequence shown in SEQ ID NO: 1, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0032] In some embodiments, the plant is a dicotyledonous or monocotyledonous plant, preferably rice, soybean, corn, wheat, sorghum, barley, millet, oat, Arabidopsis, tobacco, cotton, grape, or rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1. Figure 1A is a schematic diagram of the continuous soaking and germination of LG31 and bpr86-1 mutant seeds for resistance to dimethoate; Figure 1B is a schematic diagram of the BPR1 gene structure and the BPR1 gene mutation sites in the bpr1-1, bpr48-3, bpr51-1 and bpr86-1 mutants.

[0034] Figure 2. Figure 2A shows the growth of LG31 and bpr86-1 mutant seeds after being soaked for two days in different concentrations of bispyribac (0, 25, 50, 75, 100, and 200 mg / L) and then accelerated. Figure 2B shows the survival rate 21 days after sowing under treatment with different concentrations of bispyribac. The difference between a and b in Figure 2B indicates a significant difference in the survival rate between the control group and the mutant after drug application.

[0035] Figure 3. Figure 3A shows the growth of LG31 and bpr86-1 mutant seedlings after 14 days of soil growth and subsequent spraying with different concentrations of bispyribac-1 (0, 0.1, 0.5, 1.0, 2.0, and 4.0 g / L). Figure 3B shows the survival rate 40 days after spraying with different concentrations of bispyribac-1.

[0036] Figure 4. LG31 and bpr86-1 mutant seedlings grown in soil for 14 days before spraying (A) and after spraying with 405g aiha -1 Mesotrione (B), 324g aiha -1 Tembotrione (C) and 81g aiha -1 Topramezone (D), and the growth diagram and survival rate after 21 days of continuous growth (E).

[0037] Figure 5. Schematic diagram of the growth of 14-day-old seedlings of ZH11, bpr1-ko and BPR1-OX strains 21 days after spraying with 100 mg / L (A) and 400 mg / L (B) of bispyribac herbicide, respectively; (C) (AB) Survival rate statistics.

[0038] Figure 6. Growth diagram (A) and survival rate statistics (B) of 14-day-old soybean wild-type and loss-of-function mutant seedlings sprayed with 50 mg / L bispyribac herbicide 10 days later.

[0039] Figure 7. Amino acid sequence similarity and evolutionary relationships between different species and rice BPR1. Figure 7A shows an alignment of the amino acid sequences of proteins encoded by BPR1 genes from different species, and a schematic diagram of the BPR1 gene mutation sites in the bpr1-1, bpr48-3, bpr51-1, and bpr86-1 mutants. Red indicates that the amino acid residue at that site in the BPR1 protein is identical in all species, while blue indicates that the amino acid residue at that site is shared across most species. Figure 7B shows the evolutionary relationship. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0041] Unless otherwise specified, the experimental materials and reagents used in the following examples are all commercially available. The following examples are primarily used to illustrate the present invention, but the scope of the present invention is not limited to the examples. Unless otherwise specified, the examples are based on conventional experimental methods. The experimental equipment and reagents used in the following examples are all commercially available and can be purchased on the market.

[0042] Example 1 Obtaining the Bifenazolin-Resistant Rice Mutant bpr86-1 and Mapping the Related Genes

[0043] 8 kg of wild-type seeds of the rice variety Longjing 31 (LG31) (provided by Pan Guojun, Rice Research Institute, Heilongjiang Academy of Agricultural Sciences) were induced by soaking them in 0.5% ethyl methanesulfonate (EMS, CAS: 62-50-0) at room temperature (25°C) for 16 hours. The EMS-induced seeds were then subjected to conventional seedling germination, transplanting, and harvesting, followed by selfing to create an M2 generation rice mutant library. The seeds were then soaked in 0.1 mg / L bispyribac (Qingyuan Agro-Guan, CAS: 1622908-18-2) for two weeks to accelerate germination and screen for candidate resistant mutants. The specific procedure involved soaking approximately one million M2 generation seeds in a 0.1 mg / L bipyrazone solution for two weeks. Viable, green seedlings were selected as candidate resistant mutants (see Figure 1). These seedlings were then transplanted to the field and allowed to grow normally until flowering and seed setting. M3 generation mutant seeds were harvested and soaked in 0.1 mg / L bipyrazone (Qingyuan Agro-Crown, CAS No. 1622908-18-2) for two weeks to accelerate germination. These candidate resistant mutants were then screened for repeated bipyrazone resistance screening. A further bipyrazone-resistant mutant, designated bipyrazone resistance 86-1 (abbreviated as bpr86-1), was isolated through the M3 generation screening.

[0044] The BC1F1 progeny were obtained by hybridizing bpr86-1 with the wild type Longjing 31, and the BC1F1 progeny were further selfed to construct a BC1F2 population. The BC1F1 and BC1F2 progeny were screened for herbicide resistance using the above-mentioned herbicide resistance screening method. The results showed that all 20 BC1F1 progenies were albino (susceptible), while the ratio of albino seedlings (susceptible) to green seedlings (tolerant) in the BC1F2 progeny was 150:48 [χ 2 =0.11<χ 2 (P<0.05)=3.84], a segregation ratio close to 3:1, indicating that the gene associated with bispyribac-resistance in bpr86-1 is a single recessive gene. Whole-genome resequencing technology identified the bispyribac-associated gene in bpr86-1 as BPR1, also known as DNR1, with the gene ID LOC_Os01g08270 and the gene sequence as SEQ ID No: 1. First-generation sequencing confirmed that the mutation site in the bpr86-1 mutant gene is a guanine-to-adenine (G1085A) mutation at position 1085 in the gene coding region, resulting in a glycine-to-arginine (G362Q) mutation at amino acid position 362 in the gene-encoded protein. In addition, using the same isolation method as bpr86-1, we also isolated three other bispyribac-resistant mutants, bpr1-1, bpr48-3, and bpr51-1. The results of first-generation sequencing verification showed that they all had mutations in the BPR1 gene coding region, with the nucleotide mutation sites being G523A, G896T, and G1054A, respectively. They also caused amino acid mutations in the BPR1 gene-encoded protein, with the mutation sites being G175R, R299L, and G352R, respectively, as shown in Figure 1B.

[0045] Example 2: Resistance mutants have resistance to bispyribac during seed germination (taking bpr86-1 as an example)

[0046] M4-generation seeds of LG31 and the bpr86-1 mutant were soaked in solutions containing different concentrations of mefenacet (0, 25, 50, 75, 100, and 200 mg / L) for two consecutive days. Sixteen seeds were placed in each pot, with three replicates, and the herbicide solution was changed daily. The seeds were then germinated for 2-3 days and sown in PINDSTRUP soil at room temperature (25-28°C, 50%-75% humidity in a greenhouse) and grown for 7, 14, and 21 days. The wild-type Longjing 31 (LG31) control showed a 90% survival rate (seedlings exhibited early albinism and subsequent death) when treated with 25 mg / L, and did not recover after 21 days. Treatment with 50 mg / L and above resulted in complete albinism, which did not recover after 21 days. In contrast, the bpr86-1 mutant showed zero albinism after 21 days of growth when treated with 200 mg / L. The experimental results showed that the resistance of the bpr86-1 mutant to the bispyribac-ethyl solution was increased by more than 4 times compared with the wild-type control under the condition of continuous seed soaking for 2 days, as shown in Figure 2.

[0047] Example 3: Resistant mutants are resistant to bispyribac in the seedling stage (taking bpr86-1 as an example)

[0048] LG31 and bpr86-1 mutant M4 seeds were soaked in tap water for germination and then sown in triplicate, with 16 seeds per pot. After 14 days of growth, the seeds were sprayed with different concentrations of bispyribac-1 (0, 0.1, 0.5, 1.0, 2.0, and 4.0 g / L) at a spraying dose of 40 ml per square meter. Photos were taken before, 20 days after, and 40 days after spraying. The results showed that the Longjing 31 wild-type control died 20 days after spraying at concentrations of 0.5 g / L and above. In contrast, bpr86-1 exhibited a clear bispyribac-1 resistance phenotype, surviving at a concentration of 2 g / L. This indicates that bpr86-1 exhibited more than four times the resistance of the control, as shown in Figure 3.

[0049] Example 4: Resistant mutants have broad-spectrum resistance to HPPD inhibitor herbicides (taking bpr86-1 as an example)

[0050] The M4 generation seeds of LG31 and bpr86-1 mutants were soaked in tap water for germination, sown and grown for 14 days, and then sprayed with different HPPD inhibitor herbicides, including 324g aiha -1 Tembotrione (commercially available under the brand name "Yucaojue", pesticide product standard certificate number Q / JYN78-2021, recommended effective concentration is 90-126g aiha -1 ), 405g aiha -1 Mesotrione (commercially available under the brand name "Yuzhongbang", pesticide product standard certificate number is GB / T28155-2011, and the recommended effective concentration is 112-146g aiha -1 ) and 81g aiha -1 Topramezone (brand name "BASF", pesticide product standard certificate number is WQ / 49BASFCN107, recommended effective concentration is 22-27g aiha -1 ), and the results were observed after 21 days. The experimental results showed that bpr86-1 had more than three times the resistance to all three herbicides compared to the control Longjing 31 wild-type (LG31). This indicates that bpr86-1 possesses a broad-spectrum resistance phenotype to HPPD inhibitor herbicides, as shown in Figures 4A-E.

[0051] Example 5 Knockout of the BPR1 gene in Zhonghua 11 to enhance resistance to bispyribac

[0052] To further verify that the herbicide-resistant phenotype of the bpr1-1, bpr48-3, bpr51-1, and bpr86-1 mutants is caused by the loss of function of the rice BPR1 gene, we obtained wild-type seeds in the Zhonghua 11 (ZH11) background and BPR1 (DNR1) gene knockout (bpr1-ko) and gene overexpression (BPR1-OX) seeds in the ZH11 background. All of the above seeds were donated by Professor Li Shan of Nanjing Agricultural University (see Zhang, S., et al. (2021). Natural allelic variation in a modulator of auxin homeostasis improves grain yield and nitrogen use efficiency in rice. Plant Cell 33:566-580.10.1093 / plcell / koaa037). The bpr1-ko gene knockout mutant causes premature termination of gene translation due to base deletion.

[0053] Subsequently, ZH11, bpr1-ko, and BPR1-OX seeds were germinated and grown in soil for two weeks until the three-leaf stage. They were then sprayed with two different concentrations of bispyribac-1 solutions: 100 mg / L and 400 mg / L (these two concentrations are, respectively, the recommended field use dose for commercial bispyribac-1 and four times the recommended dose, which play an important role in guiding agricultural growth). Twenty-one days after spraying, bpr1-ko exhibited a significant bispyribac-1 herbicide resistance phenotype at both 100 mg / L and 400 mg / L concentrations. In contrast, the BPR1-OX line was more sensitive to bispyribac-1, further demonstrating that loss of BPR1 gene function enhances bispyribac-1 herbicide resistance in rice, as shown in Figures 5A-B.

[0054] The results suggest that the herbicidal activity of HPPD inhibitors may depend on the transaminase function of BPR1. Therefore, functional loss of the BPR1 gene and its homologous genes may enhance crop resistance to HPPD inhibitors.

[0055] Example 6 Knockout of soybean BPR1 homologous gene to enhance resistance to bispyribac

[0056] To further investigate whether BPR1 homologs in other crops also contribute to mesotrione resistance, we obtained wild-type seeds from the Williams 82 (WT) background and three soybean BPR1 homolog GLYMA_13G361500 mutant seeds (KO-1, KO-2, and KO-3) from this background. (All seeds were kindly provided by Professor Song Qingxin of Nanjing Agricultural University. The mutants are from the EMS-induced mutant library developed by Professor Song's research group; for details, see http: / / isoybean.org / .) These mutants all harbor missense mutations, resulting in the V94I, A320V, and D192N amino acid substitutions in the soybean BPR1 protein sequence, respectively. Subsequently, the wild-type seeds from the Williams 82 (WT) background and the mutant seeds (KO-1, KO-2, and KO-3) were grown in soil for two weeks. Ten seeds per seed were grown in triplicate and then sprayed with a 50 mg / L mesotrione solution. Ten days after spraying, the mutant seedlings showed significant resistance to mesotrione compared to wild-type controls, further demonstrating that loss of function in the soybean BPR1 homologous gene, GLYMA_13G361500, enhances mesotrione resistance, as shown in Figures 6A-B. This also suggests that loss of function in BPR1 homologous genes in other plants can similarly enhance plant resistance to mesotrione.

[0057] Example 7 Evolutionary Analysis of the BPR1 Gene and Identification of Its Homologous Genes

[0058] Evolutionary analysis of the protein encoded by the BPR1 gene revealed that this gene has very few homologous genes in gramineous crops such as rice, maize, wheat, millet, sorghum, barley, and oats, as well as crops such as tobacco, cotton, grapes, soybeans, and rapeseed, and the amino acid sequence similarity ranges from 67% to 91%, indicating a high degree of conservation (Figure 7A). The mutation sites in the four rice-resistant mutants (bpr1-1, bpr48-3, bpr51-1, and bpr86-1) and the three mutation sites (V94I, A320V, and D192N) in the soybean GLYMA_13G361500 gene mutant are all primarily located at highly conserved amino acid sites in the BPR1 protein amino acid sequence ( FIG7A ). Furthermore, both the rice mutants and the soybean GLYMA_13G361500 gene mutant in Example 6 disrupted the function of the BPR1 protein, thereby producing a mesotrione-resistant phenotype. This indicates that BPR1 is functionally conserved across species, and disrupting the function of the BPR1 protein can induce plants to develop a broad-spectrum HPPD inhibitor herbicide resistance phenotype. The BPR1 (bipyrazone resistance 1) gene is a gene found in plants. For example, the BPR1 gene in rice (Oryza sativa L.) is also called LOC_Os01g08270 or Os01g0178000, and the amino acid sequence of the protein encoded is SEQ ID NO: 2; the BPR1 homologous genes in corn (Zea mays L.) are Zm00001eb336900 and Zm00001eb122460, and the amino acid sequences of the proteins encoded are SEQ ID NO: 3 and SEQ ID NO: 4, respectively; the BPR1 homologous genes in wheat (Triticum aestivum L.) are TraesCS3B02G141600, TraesCS3A02G122400, and TraesCS3D02G124300, and the amino acid sequences of the proteins encoded are SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively. NO:7, with a 90-91% similarity to BPR1; the BPR1 homologous gene in barley (Hordeum vulgare L.) is HORVU.MOREX.r3.3HG0240080, encoding a protein with an amino acid sequence of SEQ ID NO:8; the BPR1 homologous gene in millet (Setaria italica) is SETIT_001761mg, encoding a protein with an amino acid sequence of SEQ ID NO:9; the BPR1 homologous gene in sorghum (Sorghum bicolor) is SORBI_3003G045600, encoding a protein with an amino acid sequence of SEQ ID NO:10; and the BPR1 homologous gene in oats (Avena sativa L.) is AVESA.00001b.r3.3Ag0000581, AVESA.00001b.r3.3Dg0000054 and AVESA.00001b.r3.3Cg0000528, encoding protein amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively; the BPR1 homologous genes in soybean (Glycine max L.) are GLYMA_15G012300 and GLYMA_13G361500, encoding protein amino acid sequences of SEQ ID NO: 14 and SEQ ID NO: 15, respectively, with a similarity of 74% to BPR1. Example 6 has preliminarily demonstrated that knocking out the soybean BPR1 homologous gene can cause bifenazolidone resistance; the BPR1 homologous gene in cotton (Gossypium hirsutum L.) is B456_009G132000, encoding protein amino acid sequence of SEQ ID NO:16; the BPR1 homologous gene in tobacco (Nicotiana attenuata) is PAT_0-1, and the amino acid sequence of the protein encoded is SEQ ID NO:17; the BPR1 homologous genes in grape (Vitis vinifera) are Vitvi09g00444 and Vitvi04g00367, and the amino acid sequences of the proteins encoded are SEQ ID NO:18 and SEQ ID NO:19, respectively, with a similarity of 67% to BPR1; the BPR1 homologous genes in rapeseed (Brassica napus) are BnaC02g23160D, BnaA02g19790D, BnaA07g38380D, BnaC06g19110D, BnaA07g35690D, and BnaC06g40630D, and the amino acid sequences of the proteins encoded are SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25; the BPR1 homologous gene in Arabidopsis thaliana is AT1G80360 (VAS1), and the amino acid sequence of the encoded protein is SEQ ID NO:26. Phylogenetic analysis is shown in Figure 7B.

[0059] Combined with the above examples, it can be seen that, first, the functional loss of BPR1 in different rice varieties leads to resistance to HPPD inhibitor herbicides; second, missense mutations in the BPR1 gene in soybeans also produce a phenotype of resistance to HPPD inhibitor herbicides; third, based on evolutionary analysis, the amino acid sequences of BPR1 proteins in different plant species are highly similar.

[0060] Therefore, in the context of this application, the BPR1 gene includes BPR1 genes of different varieties of rice and BPR1 genes in other plants (such as soybean), which are homologous to the rice BPR1 gene and have the same or similar functions in plants involved in resistance to HPPD inhibitor herbicides.

[0061] Sequence Listing

[0062] SEQ ID NO: 1 BPR1-CDS in rice (Oryza sativa L.)

[0063] SEQ ID NO:2 Amino acid sequence of BPR1 protein in rice (Oryza sativa L.) - Protein

[0064] SEQ ID NO:3 Amino acid sequence of protein encoded by BPR1 homologous gene Zm00001eb336900 in maize (Zea mays L.) - Protein

[0065] SEQ ID NO:4 Amino acid sequence of protein encoded by BPR1 homologous gene Zm00001eb122460 in maize (Zea mays L.) - Protein

[0066] SEQ ID NO:5 Amino acid sequence of protein encoded by BPR1 homologous gene TraesCS3B02G141600 in wheat (Triticum aestivum L.) - Protein

[0067] SEQ ID NO:6 Amino acid sequence of protein encoded by BPR1 homologous gene TraesCS3A02G122400 in wheat (Triticum aestivum L.) - Protein

[0068] SEQ ID NO:7 Amino acid sequence of protein encoded by BPR1 homologous gene TraesCS3D02G124300 in wheat (Triticum aestivum L.) - Protein

[0069] SEQ ID NO:8 Amino acid sequence of protein encoded by the BPR1 homologous gene HORVU.MOREX.r3.3HG0240080 in barley (Hordeum vulgare L.) - Protein

[0070] SEQ ID NO:9 Amino acid sequence of protein encoded by BPR1 homologous gene SETIT_001761mg in millet (Setaria italica (L.) Beauv.)

[0071] SEQ ID NO:10 Amino acid sequence of protein encoded by the BPR1 homologous gene SORBI_3003G045600 in sorghum (Sorghum bicolor (L.) Moench)

[0072] SEQ ID NO:11 Amino acid sequence of protein encoded by BPR1 homologous gene AVESA.00001b.r3.3Ag0000581 in oat (Avena sativa L.) - Protein

[0073] SEQ ID NO:12 Amino acid sequence of protein encoded by BPR1 homologous gene AVESA.00001b.r3.3Dg0000054 in oat (Avena sativa L.) - Protein

[0074] SEQ ID NO:13 Amino acid sequence of protein encoded by BPR1 homologous gene AVESA.00001b.r3.3Cg0000528 in oat (Avena sativa L.) - Protein

[0075] SEQ ID NO:14 Amino acid sequence of protein encoded by BPR1 homologous gene GLYMA_15G012300 in soybean (Glycine max L.) - Protein

[0076] SEQ ID NO:15 Amino acid sequence of protein encoded by BPR1 homologous gene GLYMA_13G361500 in soybean (Glycine max L.) - Protein

[0077] SEQ ID NO:16 Amino acid sequence of protein encoded by BPR1 homologous gene B456_009G132000 in cotton (Gossypium hirsutum L.)

[0078] SEQ ID NO:17 Amino acid sequence of protein encoded by BPR1 homologous gene PAT_0-1 in tobacco (Nicotiana attenuata) - Protein

[0079] SEQ ID NO:18 Amino acid sequence of protein encoded by BPR1 homologous gene Vitvi09g00444 in grape (Vitis vinifera) - Protein

[0080] SEQ ID NO:19 Amino acid sequence of protein encoded by BPR1 homologous gene Vitvi04g00367 in grape (Vitis vinifera) - Protein

[0081] SEQ ID NO:20 Amino acid sequence of protein encoded by BnaC02g23160D, a homologous gene of BPR1 in Brassica napus

[0082] SEQ ID NO: 21 Amino acid sequence of the protein encoded by the BPR1 homologous gene BnaA02g19790D in Brassica napus

[0083] SEQ ID NO:22 Amino acid sequence of protein encoded by BnaA07g38380D, a homologous gene of BPR1 in Brassica napus

[0084] SEQ ID NO:23 Amino acid sequence of protein encoded by BPR1 homologous gene BnaC06g19110D in Brassica napus - Protein

[0085] SEQ ID NO:24 Amino acid sequence of protein encoded by BPR1 homologous gene BnaA07g35690D in Brassica napus - Protein

[0086] SEQ ID NO:25 Amino acid sequence of protein encoded by BnaC06g40630D, a homologous gene of BPR1 in Brassica napus

[0087] SEQ ID NO:26 Amino acid sequence of protein encoded by BnaC06g40630D, a BPR1 homologous gene in Arabidopsis thaliana L.

[0088] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a HPPD inhibitor herbicide-related resistance protein in cultivating or breeding a plant with enhanced resistance to HPPD inhibitor herbicides compared to a wild-type plant, wherein the amino acid sequence of the protein is as follows: 1) The amino acid sequence shown in SEQ ID NO:2; or 2) The amino acid sequence of a derivative protein that has substitutions, deletions, and / or additions of one or several amino acid residues compared to the amino acid sequence shown in SEQ ID NO:2 and has the same activity as the amino acid sequence shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that The amino acid sequence of the derivative protein has at least 60%, 65%, for example, 67%-99% (such as 68%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) homology with the amino acid sequence shown in SEQ ID NO:

2. Preferably, the amino acid sequence of the derivative protein is the amino acid sequence shown in any one of SEQ ID NO:3 to 26.

3. The use according to claim 1, wherein the nucleotide sequence of the gene encoding the protein is as follows:. 1) The DNA sequence shown in SEQ ID NO:1; 2) A DNA sequence that has conservative substitutions, deletions, or addition of more than one base compared to the DNA sequence shown in SEQ ID NO:1 and has the same function as the DNA sequence shown in SEQ ID NO:1; or 3) A nucleotide sequence that can hybridize with the DNA sequence shown in SEQ ID NO:1 under high stringency conditions.

4. The application according to claim 3, wherein The DNA sequence that has the same function as the DNA sequence shown in SEQ ID NO:1 has at least 60% identity with the DNA sequence shown in SEQ ID NO:1, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

5. Method for cultivating plants with broad-spectrum resistance to HPPD inhibitor herbicides, said method comprising completely or partially inactivating the function of the HPPD inhibitor herbicide-related resistance protein in wild-type plants or plant cells, so as to achieve the purpose of enhancing the broad-spectrum resistance of plants to HPPD inhibitor herbicides, wherein, The amino acid sequence of the protein is as follows: 1) The amino acid sequence shown in SEQ ID NO:2; or 2) The amino acid sequence of a derivative protein that has substitutions, deletions, and / or additions of one or several amino acid residues compared to the amino acid sequence shown in SEQ ID NO:2 and has the same activity as the amino acid sequence shown in SEQ ID NO:

2.

6. The method according to claim 1, wherein The amino acid sequence of the derivative protein has at least 60%, 65%, for example, 67%-99% (such as 68%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) homology with the amino acid sequence shown in SEQ ID NO:

2. Preferably, the amino acid sequence of the derivative protein is the amino acid sequence shown in any one of SEQ ID NO:3 to 26.

7. The method according to claim 7, characterized in that, The complete loss or weakening of the function of the HPPD inhibitor herbicide-related resistance protein is achieved by the following methods: 1) By molecular means (e.g., gene editing, RNAi interference), the amino acid sequence of the protein is subjected to substitution, deletion, and / or addition of one or more amino acid residues, resulting in complete loss or weakening of the protein function; 2) By gene editing (e.g., CRISPR / CAS gene editing), the gene encoding the protein in the plant is knocked out, resulting in the inability to translate the biologically active protein in the plant; Or 3) By protein modification (e.g., ubiquitination, acetylation, proteolysis), the amino acid sequence of the protein is protein-modified, thereby resulting in complete loss or weakening of the protein function.

8. A method for preparing a plant with broad-spectrum resistance to HPPD inhibitor herbicides, which comprises partially or completely deleting the gene encoding the HPPD inhibitor herbicide-related resistance protein existing in a wild-type plant or plant cell, or substituting, introducing and / or deleting one or more bases therein, culturing the plant or plant cell and screening out a plant with broad-spectrum resistance to HPPD inhibitor herbicides compared with the wild-type plant, wherein, The nucleotide sequence of the gene is as follows: 1) The DNA sequence shown in SEQ ID NO:1; 2) A DNA sequence that has conservative substitutions, deletions, or addition of more than one base compared to the DNA sequence shown in SEQ ID NO:1 and has the same function as the DNA sequence shown in SEQ ID NO:1; or 3) A nucleotide sequence that can hybridize with the DNA sequence shown in SEQ ID NO:1 under high stringency conditions.

9. The method according to claim 8, wherein The DNA sequence having the same function as the DNA sequence shown in SEQ ID NO:1 has at least 60% identity with the DNA sequence shown in SEQ ID NO:1, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

10. The application according to any one of claims 1-4, the method according to any one of claims 5-9, wherein the plant is a dicotyledonous or monocotyledonous plant, preferably rice, soybean, corn, wheat, sorghum, barley, millet, oats, Arabidopsis thaliana, tobacco, cotton, grape, rapeseed.

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