A herbicide-resistant hppd mutant protein

Abstract

The present application relates to a herbicide-resistant HPPD mutant protein. At least three excellent mutants are obtained by gene editing technology, which express different HPPD mutant proteins and can confer herbicide resistance or tolerance to plants.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to a herbicide-resistant HPPD mutant protein. Background Technology

[0002] 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an important enzyme in the metabolism of tyrosine in organisms. Inhibition of HPPD will lead to photosynthetic uncoupling, lack of auxiliary light-harvesting pigments, and destruction of chlorophyll in plant cells. As a result, the photosynthetic tissues of plants will produce whitening symptoms, growth will be inhibited, and eventually death will occur.

[0003] Herbicides that work by inhibiting HPPD include various types, such as isoxazoles, diketonitrs, triketones, benzophenones, and pyrazolides. These herbicides have broad-spectrum herbicidal activity, but while killing weeds indiscriminately, they also cause some damage to crops. Therefore, obtaining herbicide-tolerant crops is particularly important.

[0004] One current strategy is to mutate HPPD to obtain target enzymes with higher tolerance to HPPD inhibitors without affecting normal plant growth. While single-point or multi-point mutations in the HPPD-encoding gene may screen for suitable HPPD mutations, such screening is extremely labor-intensive and highly random, making it difficult to predict the effect of each mutation. The inventors, combining CRI SPR technology, have successfully screened three new HPPD mutants in rice that can grow normally under herbicides, aiming to improve plant tolerance to HPPD inhibitors. Summary of the Invention

[0005] This application provides a mutant HPPD protein, characterized in that the amino acid sequence of the HPPD protein is as shown in SEQ ID NO.2, or as shown in SEQ ID NO.3, or as shown in SEQ ID NO.4.

[0006] This application also provides biological materials related to the above-mentioned HPPD protein, which are any one of A1) to A8) below:

[0007] A1) The nucleic acid molecule encoding the HPPD protein described above;

[0008] A2) An expression cassette containing the nucleic acid molecules described in A1);

[0009] A3) A recombinant vector containing the nucleic acid molecules described in A1);

[0010] A4) A recombinant vector containing the expression cassette described in A2);

[0011] A5) Plant cell lines containing the nucleic acid molecules described in A1);

[0012] A6) Plant cell lines containing the expression cassette described in A2);

[0013] A7) Plant cell lines containing the recombinant vector described in A3);

[0014] A8) Plant cell lines containing the recombinant vector described in A4).

[0015] In some embodiments, the plant cell lines described above do not develop into whole plants or complete plants. This application also provides the use of the aforementioned HPPD protein or biomaterial in plant herbicide resistance.

[0016] This application also provides a method for obtaining herbicide-resistant plant cells, plant tissues, plant parts, or plants, comprising:

[0017] 1) To induce the expression of the aforementioned mutated HPPD protein in plant cells, plant tissues, plant parts, or the plant itself; or

[0018] 2) To make plant cells, plant tissues, plant parts, or plants contain the aforementioned biological materials; or

[0019] 3) Mutate or edit the endogenous HPPD gene in plant cells, plant tissues, plant parts or plants to achieve the expression of the above-mentioned mutated HPPD protein therein.

[0020] In some embodiments, the methods for obtaining herbicide-resistant plant cells, plant tissues, plant parts or plants further include steps of hybridization, backcrossing or asexual reproduction.

[0021] This application also provides a method for identifying herbicide-resistant plants, comprising the following steps:

[0022] 1) Determine whether the plant expresses the aforementioned HPPD protein; or

[0023] 2) Determine whether the plant contains the aforementioned biological material.

[0024] This application also provides a method for controlling weeds around crops, characterized by comprising: applying an effective dose of herbicide to a field where crops are grown, wherein the crops express the aforementioned mutated HPPD protein or contain the aforementioned biological material.

[0025] This application also provides a method for protecting plants from damage caused by herbicides, comprising: 1) expressing the aforementioned mutated HPPD protein in plant cells, plant tissues, plant parts, or the plant; or

[0026] 2) To make plant cells, plant tissues, plant parts, or plants contain the aforementioned biological materials; or

[0027] 3) Mutate or edit the endogenous HPPD gene in plant cells, plant tissues, plant parts or plants to achieve the expression of the above-mentioned mutated HPPD protein therein.

[0028] In some implementations, the proteins are expressed in the roots, stems, leaves, flowers, fruits, or seeds of a plant or crop.

[0029] In another preferred embodiment, the plants include monocotyledons and dicotyledons.

[0030] In another preferred embodiment, the plants include herbaceous plants and woody plants.

[0031] In another preferred embodiment, the plants include Arabidopsis thaliana, tobacco, rice, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rapeseed, cabbage, strawberry, etc.

[0032] In one embodiment, the herbicide is, for example, an HPPD inhibitor herbicide (or HPPD-suppressing herbicide) including triketones, diketonitriles, isoxazoles, pyrazoles, benzophenones, quinazoline diketones, or combinations thereof. Triketone herbicides are preferably one or more of bicyclosulfuron, mesosulfuron, methylsulfuron, cyclosulfuron, terfurazolidone, or flupyrflupyr; isoxazole herbicides are preferably one or more of isoxaflutole, isoxaflutole-chlorpyrifos, or isoxaflutole; pyrazole herbicides are preferably one or more of bensulfuron, pyrazosulfuron, pyrazosulfuron-methyl, sulfosulfuron-methyl, or benzoxazolone; quinazoline diketone herbicides are preferably quinazon, methyl quinazon, etc. Attached Figure Description

[0033] Figure 1 Comparison of growth status of mutants C6-14, C7-20, C3-10, and C6-21 with wild type 15 days after herbicide application.

[0034] Figure 2 Comparison of growth status of mutants C6-14, C7-20, C3-10, and C6-21 with wild type 45 days after herbicide application.

[0035] Figure 3 Comparison of growth status of mutants P6-17 429, P6-17 381, P6-11, and P6-54 with wild type 15 days after herbicide application.

[0036] Figure 4 Comparison of growth status of mutants P6-17 429, P6-17 381, P6-11, and P6-54 with wild type 45 days after herbicide application.

[0037] Figure 5 Comparison of growth status of mutants A2-36 and A2-40 with wild type 15 days after herbicide application.

[0038] Figure 6 Comparison of growth status of mutants A2-36 and A2-40 with wild type 45 days after herbicide application. Detailed Implementation

[0039] To illustrate the universal design concept of this application, specific experimental parameters are used as examples below, but this should not be used as a reason to limit the scope of protection of this application.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] Example 1: Constructing an Editing Carrier

[0042] Three editing vectors were constructed using CRISPR technology. The first vector, pHK-dCas9-CBE, was constructed to replace C bases with T bases in the target sequence, thereby altering the encoded amino acids. The pHK-dCas9-CBE plasmid was 17648 bp in length. After digestion with BsaI, a 22 bp fragment was removed, yielding a 17626 bp linear plasmid. T4 DNA ligase was used to ligate the sticky ends and the target primer. The ligation product was transformed into competent cells (e.g., DH5α), and single clones were selected for sequencing verification. Plasmids were extracted from sequencing-positive clones and transformed into Agrobacterium EV105 for transformation into rice callus tissue.

[0043] The second method involves constructing the pH-A3A-PBE editing vector, which enables more efficient single-base editing of DNA (CT) than CBE. The third method involves constructing the pHK-dCas9-ABE editing vector, which converts adenine (A) in DNA to guanine (G). The construction methods for the second and third types of editing vectors are the same as those for the first method.

[0044] Example 2: Transformation of Rice Callus

[0045] 2.1 Preparation of Rice Callus Tissue Select clean, mold-free, and plump Nanjing 46 rice seeds. Remove the seed coat without damaging the embryo. Immerse the dehulled rice seeds in 75% anhydrous ethanol for 1 min, then disinfect with 30% NaClO for 20 min, shaking continuously during the process to ensure thorough disinfection. After disinfection, rinse 5-6 times with sterile water, then blot dry with sterile filter paper. Transfer the disinfected seeds to induction medium, placing 25-30 seeds per plate, and incubate at 25-27℃ for approximately two weeks.

[0046] 2.2 Agrobacterium infection and co-culture

[0047] Agrobacterium strains carrying the target gene site were activated and cultured on Yep solid medium containing Kan and Rif antibiotics. The culture was incubated upside down at 28°C for 48 hours. All colonies were then scraped onto AAM+AS (20mg) medium and incubated at 28°C for 1-2 hours, adjusting the OD600 of the Agrobacterium suspension to 0.4-0.6. The Agrobacterium suspension was poured into 50ml sterile centrifuge tubes, and pre-cultured callus was added and soaked for 2 minutes with constant shaking. Two layers of sterile filter paper were pre-placed on an empty dish, and the infected callus was placed on top and aerated for about 30 minutes. Three layers of sterile filter paper were pre-placed on an empty dish, with 2ml of AAM+AS (20mg) culture medium added evenly beforehand. The dried callus was then inoculated into the dish containing the three layers of sterile filter paper and incubated in the dark at 25°C for 48-72 hours.

[0048] 2.3 Screening, Differentiation, and Rooting of Resistant Callus

[0049] After co-culturing, the callus tissue was transferred to a selection medium containing antibiotics and cultured at 25–27°C for approximately 30 days. The surviving callus tissue after selection was inoculated onto a differentiation medium and cultured at 25–27°C for about one week. Callus tissue that began to turn green and sprout seedlings was then transferred back to a new differentiation medium and cultured at 25–27°C for about two weeks. For rooting culture, once the rice seedlings reached approximately 5 cm in height, all the original roots were removed, and the seedlings were transplanted onto a rooting medium and cultured under light. When the seedlings reached the same height as the cap, sterile water was added for hardening off for 4–6 days. The growth status of the seedlings in the rooting medium was observed. Afterward, the edited seedlings were transplanted to the field for planting and cultivation, and the edited seedlings were tested.

[0050] Example 3 Herbicide Resistance Test

[0051] T0 plants were generated and cultured to obtain T1 generation seeds. T1 generation seeds were germinated and cultured to the three-leaf stage. Then, the plants were foliar-sprayed with a herbicide (isoxamethonium, pesticide registration number: PD20183297, concentration 2.5 mg / L, 1 / 8X) using a pneumatic sprayer, ensuring even distribution on the leaf surface. Wild-type rice plants were used as a control and cultured in a 28℃ incubator. Plant growth was observed at 15 and 45 days. Representative independent lines were photographed. Since not all lines could be displayed side-by-side in a single image, the results are shown separately on [image list missing]. Figure 1-6 And Table 1.

[0052] Table 1

[0053]

[0054] It is evident that the effects of single-point or multi-point mutations in HPPD vary considerably among the mutants. C6-14 and C3-10 showed the best results, followed by P6-17 429.

[0055] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application.

Claims

1. A mutated HPPD protein, characterized in that, The amino acid sequence of the HPPD protein is shown in SEQ ID NO.3 or SEQ ID NO.

4.

2. A biomaterial related to the HPPD protein of claim 1, characterized by any one of the following A1) to A4): A1) A nucleic acid molecule encoding the HPPD protein of claim 1; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecules described in A1); A4) A recombinant vector containing the expression cassette described in A2).

3. The use of the HPPD protein of claim 1 or the biomaterial of claim 2 in plant herbicide resistance; wherein the herbicide is an HPPD-inhibiting herbicide.

4. A method for obtaining herbicide-resistant plant cells, plant tissues, plant parts, or plants, characterized in that, include: 1) To induce plant cells, plant tissues, plant parts, or plants to express the mutated HPPD protein of claim 1; or 2) To make plant cells, plant tissues, plant parts or plants contain the biological material of claim 2; The herbicide is an HPPD inhibitory herbicide.

5. The method according to claim 4, characterized in that, It further includes steps such as hybridization, backcrossing, or asexual reproduction.

6. A method for controlling weeds around crops, characterized in that, include: An effective dose of herbicide is applied to a field where crops are grown, wherein the crops express the mutated HPPD protein of claim 1 or contain the biological material of claim 2; the herbicide is an HPPD-inhibiting herbicide.

7. A method for protecting plants from damage caused by herbicides, characterized in that, include: 1) To induce plant cells, plant tissues, plant parts, or plants to express the mutated HPPD protein of claim 1; or 2) To make plant cells, plant tissues, plant parts or plants contain the biological material of claim 2; The herbicide is an HPPD inhibitory herbicide.

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