Herbicide-resistant HPPD mutant protein

Abstract

The invention relates to a herbicide-resistant HPPD mutant protein. Through a gene editing technology, at least three excellent mutants are obtained, different HPPD mutant proteins are expressed, and herbicide resistance or tolerance can be given to plants.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology, and specifically relates to a herbicide-resistant HPPD mutant protein. Background Art

[0002] 4-Hydroxypheny l pyruvate D ioxygenase (HPPD) is an important enzyme in the tyrosine metabolism process in organisms. The inhibition of HPPD will lead to the uncoupling of photosynthesis in plant cells, the lack of auxiliary light-harvesting pigments, and the destruction of chlorophyll, resulting in albinism symptoms in the photosynthetic tissues of plants, growth inhibition, and even death.

[0003] There are many types of herbicides that work by inhibiting HPPD, such as isoxazoles, diketonitriles, triketones, benzophenones, and pyrazols, etc. These herbicides have broad-spectrum herbicidal activity, but they also cause certain damage to crops while killing weeds indiscriminately, so it is particularly important to obtain crops that are tolerant to herbicides.

[0004] One current strategy is to mutate HPPD to obtain a target enzyme with higher tolerance to HPPD inhibitors without affecting the normal growth of plants. Although single-point or multi-point mutations of the HPPD encoding gene may screen for a suitable HPPD mutation, such screening is extremely labor-intensive and highly random, and it is difficult to predict the effect of each mutation. The inventors combined CRISPR technology and fortunately screened three new HPPD mutants in rice that can grow normally under herbicides, in order to improve the tolerance of plants to HPPD inhibitors. Summary of the invention

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

[0006] The present application also provides a biological material related to the above-mentioned HPPD protein, which is any one of the following A1) to A8):

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

[0008] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0009] A3) a recombinant vector containing the nucleic acid molecule described in A1);

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

[0011] A5) a plant cell line containing the nucleic acid molecule described in A1);

[0012] A6) a plant cell line containing the expression cassette described in A2);

[0013] A7) a plant cell line containing the recombinant vector described in A3);

[0014] A8) A plant cell line containing the recombinant vector described in A4).

[0015] In some embodiments, the plant cell line does not develop into a whole plant or a complete plant. The present application also provides the use of the HPPD protein or biological material in plant resistance to herbicides.

[0016] The present application also provides a method for obtaining a plant cell, plant tissue, plant part or plant having herbicide resistance, comprising:

[0017] 1) causing plant cells, plant tissues, plant parts or plants to express the mutated HPPD protein; or

[0018] 2) causing plant cells, plant tissues, plant parts or plants to contain the above-mentioned biological materials; or

[0019] 3) Mutating or editing the endogenous HPPD gene of a plant cell, plant tissue, plant part or plant to achieve expression of the mutated HPPD protein therein.

[0020] In some embodiments, the above method for obtaining a plant cell, plant tissue, plant part or plant having herbicide resistance further comprises the steps of hybridization, backcrossing or asexual reproduction.

[0021] The present application also provides a method for identifying a herbicide-resistant plant, comprising the following steps:

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

[0023] 2) Determining whether the plant contains the above-mentioned biological material.

[0024] The present application also provides a method for controlling weeds around crops, characterized in that it includes: applying an effective dose of herbicide to a field where crops are planted, wherein the crops express the above-mentioned mutant HPPD protein or contain the above-mentioned biological material.

[0025] The present application also provides a method for protecting plants from damage caused by herbicides, comprising: 1) causing plant cells, plant tissues, plant parts or plants to express the above-mentioned mutant HPPD protein; or

[0026] 2) causing plant cells, plant tissues, plant parts or plants to contain the above-mentioned biological materials; or

[0027] 3) Mutating or editing the endogenous HPPD gene of a plant cell, plant tissue, plant part or plant to achieve expression of the mutated HPPD protein therein.

[0028] In some embodiments, the protein is expressed in roots, stems, leaves, flowers, fruits or seeds of plants or crops.

[0029] In another preferred embodiment, the plants include monocotyledonous plants and dicotyledonous plants.

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

[0031] In another preferred embodiment, the plants include Arabidopsis, 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 referred to as an HPPD-inhibiting herbicide) including triketones, diketonitriles, isoxazoles, pyrazoles, benzophenones, quinazolinediones or combinations thereof. Triketone herbicides are preferably one or any of bicyclonazole, mesotrione, mesotrione, cyproconazole, teflonone or flupyralidone; isoxazole herbicides are preferably one or any of isoxaflutole, isoxaflutole, isoxaflutole; pyrazole herbicides are preferably one or any of benzylpyraclostrobin, pyrazolone, pyrazolamide, sulfonylurea or benzylpyraclostrobin; quinazolinedione herbicides are preferably quinazoline, methylquinazoline, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Comparison of the growth status of mutants C6-14, C7-20, C3-10, C6-21 and the wild type 15 days after spraying herbicides.

[0034] Figure 2 : Comparison of the growth conditions of mutants C6-14, C7-20, C3-10, C6-21 and the wild type 45 days after spraying herbicides.

[0035] Figure 3 : Comparison of the growth status of mutants P6-17 429, P6-17 381, P6-11, P6-54 and the wild type 15 days after spraying herbicides.

[0036] Figure 4 : Comparison of the growth status of mutants P6-17 429, P6-17 381, P6-11, P6-54 and the wild type 45 days after spraying herbicides.

[0037] Figure 5 : Comparison of the growth status of mutants A2-36, A2-40 and the wild type 15 days after spraying herbicides.

[0038] Figure 6 : Comparison of the growth status of mutants A2-36, A2-40 and the wild type 45 days after spraying herbicides. DETAILED DESCRIPTION

[0039] In order to specifically illustrate the universal design concept of the present application, specific experimental parameters are used as an example below, but this should not be used as a reason to limit the scope of protection of the present application.

[0040] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0041] Example 1 Construction of editing vector

[0042] Three editing vectors were constructed using CRISPR technology. The first one was to construct a pHK-dCas9-CBE editing vector, hoping to replace the C base in the target sequence with the T base, thereby achieving a change in the encoded amino acid. The total length of the pHK-dCas9-CBE plasmid is 17648bp. The plasmid was cut with BsaI, a 22bp fragment was removed, and a 17626bp long linear plasmid was recovered. T4 DNA ligase was used to connect the sticky ends and the target primers, and the enzyme-cut ligation product was transformed into competent cells (such as DH5ɑ), and a single clone was picked for sequencing verification. The plasmid was extracted from the sequencing-positive clone and transformed into Agrobacterium EV105 to prepare for the transformation of rice callus.

[0043] The second method is to construct a pH-A3A-PBE editing vector, which can achieve more efficient CT single-base editing than CBE. The third method is to construct a pHK-dCas9-ABE editing vector, which converts adenine (A) in DNA into guanine (G). The construction methods of the second and third editing vectors refer to the first method.

[0044] Example 2 Rice Callus Transformation

[0045] 2.1 Preparation of rice callus Select rice seeds of Nanjing 46 with clean seed coat, no mildew spots and full grains, dehull them without damaging the embryo, infect the dehulled rice seeds with 75% anhydrous ethanol for 1 min, and disinfect them with 30% NaClO for 20 min, shaking them continuously during the period to ensure sufficient disinfection. After disinfection, rinse them with sterile water for 5-6 times, rinse them clean and dry them with sterile filter paper. Move the disinfected seeds to the induction medium, put 25-30 rice seeds per plate of medium, and culture them at 25-27℃ for about two weeks.

[0046] 2.2 Agrobacterium infection and co-cultivation

[0047] The Agrobacterium strain carrying the target gene target site was activated and cultured on Yep solid medium containing Kan and Rif antibiotics, inverted and cultured in a 28°C incubator for 48 hours, and all colonies were scraped into AAM+AS (20 mg) medium with a spreader, cultured at 28°C for 1-2 hours, and the OD600 of the Agrobacterium suspension was adjusted to be within the range of 0.4-0.6. The suspended Agrobacterium infection solution was poured into a 50ml sterilized centrifuge tube, and the pre-cultured callus tissue was poured in, soaked for 2 minutes, and shaken continuously during the period. Two layers of sterile filter paper were pre-padded on the empty dish, and the infected callus tissue was placed on it and blown for about 30 minutes. Three layers of sterile filter paper were pre-padded on the empty dish, and 2mIAAM+AS (20 mg) culture solution was evenly added to the filter paper in advance. The dried callus tissue was inoculated into the dish containing three layers of sterile filter paper, and cultured in the dark at 25°C for 48-72 hours.

[0048] 2.3 Screening, differentiation and rooting of resistant calli

[0049] Transfer the co-cultivated callus to a screening medium containing antibiotics and culture at 25-27°C for about 30 days. Inoculate the surviving callus after screening onto a differentiation medium and culture at 25-27°C for about a week. Transfer the callus that begins to turn green and grow seedlings back to a new differentiation medium and culture at 25-27°C for about two weeks. Rooting culture When the rice seedlings grow to about 5 cm, pull out all the original roots, transplant the seedlings to the rooting medium, culture under light, and when the seedlings grow to the same height as the lid, add sterile water to refine the seedlings for 4-6 days. Observe the growth status of the seedlings in the rooting medium, then transplant the edited seedlings into the field for planting and culture, and test the edited seedlings.

[0050] Example 3 Herbicide resistance detection

[0051] The edited T0 plants were produced and cultured to obtain T1 generation plant seeds. The T1 generation seeds were germinated and cultured to the three-leaf and one-heart stage, and the herbicide (isoxamethasone, pesticide registration number: PD20183297, concentration of 2.5 mg / L, 1 / 8X) was sprayed on the leaves using a pressure sprayer to evenly distribute it on the surface of the leaves. The wild-type rice plants were used as controls and continued to be cultured in a 28°C culture room. The growth of the plants was observed at 15 days and 45 days, and representative independent strains were selected for photography. Since all of them cannot be displayed side by side in one picture, the results are shown in Figure 1-6 And Table 1.

[0052] Table 1

[0053]

[0054] It can be seen that after single-point or multi-point mutation of HPPD, the effects of the mutants are quite different. Among them, C6-14 and C3-10 have the best effects, and P6-17 429 has the second best effect.

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

Claims

1. A mutant 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. The biomaterial related to the HPPD protein according to claim 1, characterized by the following A1) to A8) Any of: A1) a nucleic acid molecule encoding the HPPD protein according to claim 1; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1); A4) a recombinant vector containing the expression cassette described in A2); A5) a plant cell line containing the nucleic acid molecule described in A1); A6) a plant cell line containing the expression cassette described in A2); A7) a plant cell line containing the recombinant vector described in A3); A8) A plant cell line containing the recombinant vector described in A4).

3. Use of the HPPD protein according to claim 1 or the biomaterial according to claim 2 in plant resistance to herbicides.

4. A method for obtaining a plant cell, plant tissue, plant part or plant having herbicide resistance, characterized in that: include: 1) causing plant cells, plant tissues, plant parts or plants to express the mutated HPPD protein of claim 1; or 2) causing plant cells, plant tissues, plant parts or plants to contain the biological material of claim 2; or 3) Mutating or editing the endogenous HPPD gene of a plant cell, plant tissue, plant part or plant to achieve expression of the mutated HPPD protein of claim 1 therein.

5. The method according to claim 4, characterized in that It may further include the steps of hybridization, backcrossing or asexual reproduction.

6. A method for identifying herbicide-resistant plants, characterized in that: The following steps are involved: 1) determining whether the plant expresses the HPPD protein of claim 1; or 2) Determining whether the plant contains the biological material of claim 2.

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

8. A method for protecting plants from damage caused by herbicides, characterized in that include: 1) causing plant cells, plant tissues, plant parts or plants to express the mutated HPPD protein of claim 1; or 2) causing plant cells, plant tissues, plant parts or plants to contain the biological material of claim 2; or 3) Mutating or editing the endogenous HPPD gene of a plant cell, plant tissue, plant part or plant to achieve expression of the mutated HPPD protein of claim 1 therein.

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