Hppd mutant proteins and uses thereof

Abstract

The application relates to an HPPD mutant protein and application thereof. At least three excellent mutants are obtained through a gene editing technology, the mutants express different HPPD mutant proteins, and can endow plants with herbicide resistance or tolerance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a HPPD mutant protein and application thereof. BACKGROUND

[0002] 4-Hydroxyphenyl pyruvate dioxygenase (HPPD) is an important enzyme in the tyrosine metabolic process of organisms. Inhibition of HPPD will cause decoupling of photosynthesis in plant cells, lack of auxiliary light-harvesting pigments, and destruction of chlorophyll. As a result, plants will show albino symptoms in photosynthetic tissues, growth will be inhibited, and even death will occur.

[0003] Herbicides that act by inhibiting HPPD include various types, such as isoxazoles, dinitroanilines, triketones, benzophenones, and pyrazoles. Such herbicides have broad-spectrum herbicidal activity, but also cause certain damage to crops when they kill weeds indiscriminately. Therefore, it is particularly important to obtain crops that are resistant to herbicides.

[0004] One current strategy is to mutate HPPD to obtain a target enzyme with higher resistance to HPPD inhibitors while not affecting the normal growth of plants. Although single-point or multi-point mutations in the coding gene of HPPD can screen for a suitable HPPD mutant, such screening is extremely labor-intensive and random, and it is difficult to predict the effect of each mutation. The inventors combined CRI SPR 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

[0005] The present 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] The present application also provides biological materials related to the above-mentioned HPPD protein, which are any one of the following A1) to A8):

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

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

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

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

[0011] A5) a plant cell line comprising the nucleic acid molecule of A1);

[0012] A6) a plant cell line comprising the expression cassette of A2);

[0013] A7) a plant cell line comprising the recombinant vector of A3);

[0014] A8) a plant cell line comprising the recombinant vector of A4).

[0015] In some embodiments, the above plant cell line does not develop into a whole plant or a complete plant. The present application also provides the use of the above 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 with herbicide resistance, comprising:

[0017] 1) causing the plant cell, plant tissue, plant part or plant to express the above mutated HPPD protein; or

[0018] 2) causing the plant cell, plant tissue, plant part or plant to comprise the above biological material; or

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

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

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

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

[0023] 2) determining whether the plant comprises the above biological material.

[0024] The present application also provides a method for controlling weeds around crops, characterized in that it comprises: applying an effective dose of herbicide to a field where crops are planted, the crops expressing the above mutated HPPD protein or comprising the above biological material.

[0025] The present application also provides a method for protecting plants from damage caused by herbicides, comprising: 1) causing the plant cell, plant tissue, plant part or plant to express the above mutated HPPD protein; or

[0026] 2) causing a plant cell, plant tissue, plant part, or plant to comprise the above-mentioned biological material; or

[0027] 3) mutating or editing an endogenous HPPD gene of a plant cell, plant tissue, plant part, or plant to express the above-mentioned mutated HPPD protein therein.

[0028] In some embodiments, the above-mentioned protein is expressed in the roots, stems, leaves, flowers, fruits, or seeds of the plant or crop.

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

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

[0031] In another preferred embodiment, the plant comprises Arabidopsis thaliana, tobacco, rice, maize, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, canola, Chinese cabbage, strawberry, and the like.

[0032] In one embodiment, the herbicide is, for example, an HPPD inhibitor herbicide (or HPPD-inhibiting herbicide) including a triketone, dinitroaniline, isoxazole, pyrazole, diphthalimide, quinazolinone, or a combination thereof. The triketone herbicide is preferably one or any of benzobicyclon, mesotrione, sulcotrione, tefuryltrione, or pyrazolynate; the isoxazole herbicide is preferably one or any of isoxaflutole, isoxachlortole, or clomazone; the pyrazole herbicide is preferably one or any of benzofluor, norflurazon, pyrazolate, pyrazolynate, or norflurazon; and the quinazolinone herbicide is preferably one or any of quinoclamine, and the like. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

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

[0037] Figure 5 Comparison of growth status of mutant A2-36, A2-40 and wild type after spraying herbicide for 15 days.

[0038] Figure 6 Comparison of growth status of mutant A2-36, A2-40 and wild type after spraying herbicide for 45 days. DETAILED DESCRIPTION

[0039] In order to specifically illustrate the design idea of the present application, the following specific experimental parameters are taken as examples for demonstration, but should not be taken as a reason to limit the protection scope 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 can be obtained from commercial channels unless otherwise specified.

[0041] Example 1 Construction of editing vector

[0042] Three kinds of editing vectors were constructed using CRISPR technology. First, the pHK-dCas9-CBE editing vector was constructed, which was expected to replace the C base in the target sequence with a T base, thereby realizing the change of the encoded amino acid. The pHK-dCas9-CBE plasmid is 17648 bp long, which is cut with BsaI enzyme to remove a 22 bp fragment, and a linear plasmid of 17626 bp is recovered. The sticky ends and target primers were connected using T4 DNA ligase, and the enzyme-cut ligation product was transformed into competent cells (such as DH5a), and single colonies were picked for sequencing verification. The positive clones were extracted for plasmid, transformed into Agrobacterium EV105, and prepared for rice callus transformation.

[0043] Second, the pH-A3A-PBE editing vector was constructed, which could achieve more efficient C-T single base editing than CBE. Third, the pHK-dCas9-ABE editing vector was constructed, which could convert adenine (A) in DNA to guanine (G). The construction methods of the second and third editing vectors refer to the method of the first.

[0044] Example 2 Rice callus transformation

[0045] 2.1 Preparation of rice callus Select the rice seeds of Nangjing 46 with clean seed coat, no mold spot, and full grain. Shell the rice seeds without damaging the embryo. Infect the shelled rice seeds with 75% anhydrous ethanol for 1 min, and sterilize the rice seeds with 30% NaClO for 20 min, with constant shaking to ensure sufficient sterilization. After sterilization, rinse the rice seeds with sterile water for 5-6 times, dry the rice seeds with sterile filter paper, and move the sterilized rice seeds to the induction medium. Place 25-30 rice seeds on each plate, and culture the rice seeds at 25-27°C for about two weeks.

[0046] 2.2 Agrobacterium infection and co-culture

[0047] Activate the Agrobacterium strain carrying the target site of the target gene on the Yep solid medium containing Kan and Rif antibiotics, and culture the Agrobacterium strain in an inverted incubator at 28°C for 48 h. Scrape all the colonies on the plate to the AAM+AS (20 mg) medium with a spreader, and culture the Agrobacterium strain at 28°C for 1-2 h. Adjust the OD600 of the Agrobacterium suspension to be within the range of 0.4-0.6. Pour the suspended Agrobacterium infection solution into a 50 ml sterilized centrifuge tube, pour the pre-cultured callus into the centrifuge tube, and soak the callus for 2 min with constant shaking. Pad two layers of sterile filter paper on the empty dish in advance, place the infected callus on the filter paper, and dry the callus for about 30 min. Pad three layers of sterile filter paper on the empty dish in advance, and evenly add 2 ml of AAM+AS (20 mg) medium to the filter paper. Inoculate the dried callus into the dish containing the three layers of sterile filter paper, and culture the callus at 25°C in the dark for 48-72 h.

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

[0049] Move the callus after co-culture to the screening medium containing antibiotics, and culture the callus at 25-27°C for about 30 days. Inoculate the surviving callus after screening to the differentiation medium, and culture the callus at 25-27°C for about one week. Move the callus that starts to turn green and start to grow seedlings to the new differentiation medium, and culture the callus at 25-27°C for about two weeks. Root the rice seedlings when the seedlings grow to about 5 cm, remove all the original roots, and move the seedlings to the rooting medium. Culture the seedlings under light, and add sterile water to the seedlings for 4-6 days when the seedlings grow to the height of the cover. Observe the growth state of the seedlings in the rooting medium, and then move the edited seedlings to the field for planting culture, and detect the edited seedlings.

[0050] Example 3 Herbicide resistance detection

[0051] The edited T0 plants were generated, and the T1 generation plant seeds were obtained by seed culture. The T1 generation seeds were germinated and cultured to the three-leaf-one-heart stage, and then the plants were treated with herbicide (isoxaflutole, pesticide registration number: PD20183297, concentration 2.5 mg / L, 1 / 8X) by using an air pressure sprayer to make the herbicide evenly distributed on the surface of the leaves, and wild-type rice plants were used as controls and continued to be cultured in a 28°C culture room. The plant growth was observed at 15 days and 45 days, respectively, and representative independent lines were photographed. Since all the plants cannot be displayed side by side in one figure, the results are shown in Table 1 and FIGS. 1-3, respectively. Figures 1-6 Table 1

[0052] Table 1

[0053]

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

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

Claims

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

2.

2. A biological material 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 molecule of A1); A3) a recombinant vector containing the nucleic acid molecule of A1); A4) a recombinant vector containing the expression cassette of A2).

3. Use of the HPPD protein of claim 1 or the biological material of claim 2 in plant resistance to herbicides; the herbicides being HPPD-inhibiting herbicides.

4. A method of obtaining a plant cell, plant tissue, plant part or plant having herbicide resistance, characterized in that, comprising: 1) causing a plant cell, plant tissue, plant part, or plant to express the mutated HPPD protein of claim 1; or 2) causing a plant cell, plant tissue, plant part, or plant to comprise the biological material of claim 2; the herbicides being HPPD-inhibiting herbicides.

5. The method of claim 4, wherein, It further comprises the steps of hybridization, backcrossing, or vegetative reproduction.

6. A method of controlling weeds around a crop, characterized by, comprising: applying an effective dose of herbicides to a field where crops are planted, the crops expressing the mutated HPPD protein of claim 1 or comprising the biological material of claim 2; the herbicides being HPPD-inhibiting herbicides.

7. A method for protecting a plant from damage caused by a herbicide, characterized in that, comprising: 1) causing a plant cell, plant tissue, plant part, or plant to express the mutated HPPD protein of claim 1; or 2) causing a plant cell, plant tissue, plant part, or plant to comprise the biological material of claim 2; the herbicides being HPPD-inhibiting herbicides.

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