CN114645028A

CN114645028A - ALS mutant protein and application thereof

Info

Publication number: CN114645028A
Application number: CN202210201414.5A
Authority: CN
Inventors: 罗祖勇; 李莹莹; 李亮; 谭旭光; B·刘; 张李; 刘政; 张云会
Original assignee: Keji Dalong Beijing Biotechnology Co ltd
Current assignee: Keji Dalong Beijing Biotechnology Co ltd
Priority date: 2021-11-04
Filing date: 2022-03-02
Publication date: 2022-06-21
Also published as: CN113717957A

Abstract

The application belongs to the technical field of plant proteins, and particularly discloses an ALS mutant protein and application thereof, wherein the ALS mutant protein has an S629N mutation. The application has at least one of the following beneficial effects: the ALS mutant protein provided by the application is positioned on the 6BL chromosome of the wheat B genome, and the 629 th serine of the ALS mutant protein is changed into asparagine, so that the wheat has herbicide resistance activity and can tolerate the mebendazole nicotinic acid with 8-fold concentration (720 gr ae/ha). Therefore, the mutant protein can be used for cultivating herbicide-resistant plants, such as wheat, tobacco or arabidopsis thaliana and the like, especially crops, so as to reduce the phytotoxicity of the crops and widen the application range of the herbicide.

Description

ALS mutant protein and application thereof

Technical Field

The application belongs to the technical field of plant proteins, and more particularly relates to ALS mutant proteins, nucleic acids, expression cassettes, recombinant vectors, cells and application.

Background

The farmland weeds are one of the main causes of crop yield reduction, and compared with the traditional cultivation measures, artificial weeding, mechanical weeding and other methods, the application of the chemical herbicide is the most efficient, simple and economical method for preventing and killing the farmland weeds.

Acetolactate synthase (ALS) (also known as acetohydroxyacid synthase, AHAS; Ec 4.1.3.18) is the first enzyme to catalyze the synthesis of the branched-chain amino acids valine, leucine and isoleucine. Five classes of herbicides developed and developed with ALS as a target exist, namely Sulfonylureas (SU), imidazolinones (IMI), sulfonylamino-carbonyl triazolinones (SCT), pyrimidine salicylates (PTB), and Triazolopyrimidines (TP). In this context, herbicide resistance refers to tolerance to herbicides including sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidine salicylates. ALS inhibitor herbicides have the advantages of strong selectivity, wide weed control spectrum, low toxicity, high efficiency, low toxicity to mammals and the like, and are widely popularized and used at present.

The action mechanism of the herbicide developed by taking ALS as a target is to kill plants by inhibiting the activity of acetolactate synthase (ALS), destroying protein synthesis, interfering DNA synthesis and cell division and growth and finally causing plant death. The resistance of plants to such herbicides results from one or more amino acid mutations in the ALS gene that result in structural changes in the enzyme.

Disclosure of Invention

It has now been found that ALS muteins are mutated at the 122, 155, 197, 205, 574, 653, 654 alleles of homologous proteins from different plants, including maize, wheat, rice, oilseed rape and sunflower, to render the corresponding plants resistant to herbicides such as imidazolinones, sulfonylureas, triazolopyrimidines and pyrimidinylsalicylates (Tan S, Evzns R, Dahmerm L, et al.1 midazonone-tolerant crops: History, current status and future.pest Management science 2005, 61: 246-. However, there is no report that amino acid mutation from S to N at 629 site in ALS gene of triticum aestivum makes triticum aestivum herbicide resistant.

Through long-term and arduous research and practice, the inventor finally obtains a wheat strain insensitive to ALS inhibitor herbicides from a series of mutant plants by performing EMS chemical mutagenesis treatment and herbicide screening on a large number of seeds of the rye 247, so that the wheat has imidazolinone herbicide resistance (tolerance), and the wheat can keep the normal plant physiological functions of the original protein under the condition of spraying the herbicides. The ALS protein in the wheat can be used for cultivating plants with herbicide-resistant activity, especially crops.

The application is realized by the following scheme:

the present application provides an ALS mutein comprising a S629N mutation.

In the present application, the ALS mutein has a herbicide resistant activity because serine at position 629 is changed to asparagine, and thus, there has been no report.

In a specific embodiment of the present application, the amino acid sequence of the ALS mutein is set forth in SEQ ID NO 2. The protein mutation enables the plants to have the activity of resisting imidazolinone herbicides.

In one embodiment of the present application, the imidazolinone herbicide is imazapyr.

In one embodiment of the present application, the ALS mutein is derived from triticum aestivum: (a)Triticum aestivum L.）。

In one embodiment of the present application, the wheat is rye 247.

In a specific embodiment of the present application, the ALS mutein is capable of conferring tolerance to 8 times the concentration (720 gr ae/ha) of imazapic in wheat.

In another aspect, the present application provides a nucleic acid encoding the ALS mutein described above.

In a specific embodiment of the application, nucleotide G at nucleotide 1886 of the nucleic acid sequence is mutated.

In a specific embodiment of the present application, the nucleotide at position 1886 of the nucleic acid sequence is mutated from guanine G to adenine A.

In a specific embodiment of the present application, the ALS mutein is located on the 6BL chromosome of the wheat B genome.

In one embodiment of the present application, the nucleic acid has the sequence shown in SEQ ID NO 1.

In another aspect of the present application, there is provided an expression cassette comprising the nucleic acid described above.

In another aspect, the present application provides a recombinant vector comprising the above-described nucleic acid.

In another aspect, the present application provides a cell comprising the nucleic acid described above.

In another aspect, the present application provides a use of the ALS mutein as defined above, or the nucleic acid as defined above, or the expression cassette as defined above, or the recombinant vector as defined above, or the cell as defined above, for combating herbicides in plants.

In one embodiment herein, the herbicide is an imidazolinone herbicide.

In one embodiment herein, the herbicide includes, but is not limited to, one or more of imazamox, imazapic, imazapyr, imazaquin, and derivatives thereof.

In one embodiment herein, the herbicide is imazapic.

In one embodiment of the present application, the plant is wheat, tobacco, arabidopsis, rice, maize or sorghum, or the like.

In another aspect, the present application provides a method for obtaining a plant having herbicide resistance, comprising the steps of:

1) allowing the plant to comprise the nucleic acid described above; and/or the presence of a gas in the gas,

2) allowing the plant to express the ALS mutant protein.

In one embodiment of the present application, the method of obtaining a herbicide resistant plant comprises the steps of transgenosis, crossing, backcrossing or asexual propagation.

The ALS mutant protein provided by the application has at least one of the following beneficial effects:

the serine at position 629 of the ALS mutein provided by the application is changed into asparagine, so that the mutant wheat has herbicide resistance activity and can tolerate imazapic acid with 8 times concentration (720 gr ae/ha).

Drawings

FIG. 1 is a photograph of the J401 mutants screened as provided in the examples of the present application.

FIG. 2 is a diagram showing the results of agarose gel electrophoresis experiments on the J401 mutant provided in the examples of the present application, wherein 6A-ALS is the A genome PCR product of wild-type plants; J6A-ALS is the A genome PCR product of J401 mutant plant; 6B-ALS is a B genome PCR product of a wild plant; J6B-ALS is the B genome PCR product of J401 mutant plants; 6D-ALS is a D genome PCR product of a wild type plant; J6D-ALS is the D genome PCR product of J401 mutant plants; m is DNA marker.

Fig. 3 is a graph of experimental results of the resistance of progeny of the J401 mutant to imazapic in the field, provided in the examples of the present application, wherein plants in

rows

1 and 2 are the J401 mutant, and plants in

rows

3 and 4 are the linkman 247 control.

FIG. 4 is a graph of experimental results of the tolerance of progeny of the J401 mutant to different concentrations of imazapic when cultured in the greenhouse for 2 weeks, wherein "ck 0" is wild type wheat without imazapic as a control; "ck 2 x" is the control of 2 times concentration of imazethapyr sprayed on wild wheat; 0 x is that no imazapyr is sprayed on the wheat in the experimental group; and 2x refers to spraying imazapic with the concentration 2 times of that of the wheat in the experimental group, and the like.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The technical solutions of the present application will be described clearly and completely in conjunction with the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

Example 1 construction of wheat mutagenized population and screening of herbicide-resistant wheat

In 10 months 2018, 120kg of boat wheat 247 seeds (approval number: Country wheat 2016029, purchased from Shuxin International species Co., Ltd. of Beijing) were separately packaged in 12 gauze bags, each 10kg of which was washed clean with tap water. Soaking in clear water at room temperature for 12 hr, taking out the seeds, and draining.

10mM PBS (Shanghai Producer, product No. B040100-0005, powder, pH 7.0) solution was prepared, and 0.4% (w/w) EMS (product No. M0880. CAS No. 62-50-0. Sigma. Ethyl methanesulfonate, concentration: 1.206 g/ml) solution was prepared using PBS solution as a mother liquor.

Seeds were soaked with 0.4% EMS solution (w/v =1:2 seed to EMS solution ratio) and the seeds were tumbled or the solution bucket shaken every 1 hour. After 12 hours, EMS solution is discarded, tap water is changed for soaking the seeds for 5 minutes each time, and the soaking is repeated for 5 times. And finally, taking out the seeds, washing the seeds for 2 hours by using running water, turning over the seeds in the washing process, and washing the EMS completely. And then draining, slightly airing, transporting to a field for mechanical sowing, and performing conventional fertilizer and water management. In 6 months of 2019, the seeds are harvested in different regions and dried, and the seeds are of M1 generation.

Wheat seeds (M1 generation) are sown in furrows in the early 9 th month of 2019, the width of a compartment surface is 80cm, the width of a furrow is 40cm, when the wheat grows to one leaf and one core, 240g/L imazapyr (Imazamox, CAS number 114311-32-9, 2- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -5-methylnicotinic acid, an aqueous solution) is sprayed by a fan-shaped spray head, and the spraying dose is 50 ml/mu (2 times of field use concentration, 180gr ae/ha). After 2 weeks, the seedlings were examined for withering and death, and green surviving seedlings were found (see FIG. 1), and the rest of the wheat were dead. The green seedlings were transplanted into nursery pots, and after vernalization for 30 days outdoors, the seedlings were transferred to a greenhouse for management and harvest, and the number of seeds was M2.

Example 2 mutation site analysis of herbicide-resistant wheat

The herbicide-resistant wheat mutant plants obtained in example 1 above were selected for molecular testing. The method comprises the following specific steps:

taking 20mg of leaves of the mutant and the wild plants, putting the leaves into a centrifuge tube, adding steel balls, putting the centrifuge tube into liquid nitrogen for quick cooling, fully vibrating and grinding the leaves into powder by a tissue grinder, and extracting sample DNA by a CTAB method for genome sequencing.

Common wheat is hexaploid, contains A, B, D genomes, and has 3 ALS genes, namely 6A-ALS, 6B-ALS and 6D-ALS. Specific primers are respectively designed according to reference sequences for amplification, and specific primer sequences are shown in Table 1.

TABLE 1 specific primers

(1) The gene 6A-ALS was amplified using KOD FX (available from Toyobo, Shanghai, Biotech, Inc.) in the following reaction scheme:

2X PCR Buffer for KOD FX 25.0 ul; 2mM dNTP 10 ul; KOD FX 1 ul; 10 μ M forward primer 1.5 ul; 10 μ M reverse primer 1.5 ul; wheat genome DNA of 30 ng/microliter is 5.0 ul; sterile water was added to the total volume of 50 ul.

The PCR amplification reaction procedure was as follows: pre-denaturation: 2min at 98 ℃; 35 cycles: denaturation at 98 ℃ for 30 sec; annealing at 58 ℃ for 30 sec; extending at 68 ℃ for 2.5 min; and (3) heat preservation: at 68 ℃ for 3 min. The amplified PCR product was subjected to agarose gel electrophoresis detection, and the experimental results are shown in FIG. 2, using wild-type PCR product as a control.

(2) The genes 6B-ALS and 6D-ALS were amplified using Q5 High-Fidelity 2X master mix (purchased from BioLabs) in the following reaction system:

q5 High-Fidelity 2X master mix 25.0 ul; 10 μ M forward primer 1.0 ul; 10 μ M reverse primer 1.0 ul; wheat genome DNA of 30 ng/microliter is 5.0 ul; sterile water was added to the total volume of 50 ul.

The PCR amplification reaction procedure was as follows: pre-denaturation: 2min at 98 ℃; 35 cycles: denaturation at 98 ℃ for 20 sec; annealing at 57 ℃ for 20 sec; extension 72 ℃ 90 sec; and (3) heat preservation: 72 ℃ for 2 min. The amplified PCR product was subjected to agarose gel electrophoresis detection, and the experimental results are shown in FIG. 2, using wild-type PCR product as a control.

And sending the residual PCR products to Beijing Optimalaceae new biotechnology limited company for sequencing to obtain the 6A-ALS, 6B-ALS and 6D-ALS gene sequences of the wheat mutant J401.

Sequencing comparison shows that, compared with the wild type rye 247, the mutant strain with the number J401 has no base change of the acetolactate synthase gene in A, D genome, the acetolactate synthase gene has mutation at the 1886 th nucleic acid in B genome, G is changed into A, the 629 th amino acid of the corresponding coded protein is changed into asparagine from serine, and the genotype is homozygous. The nucleotide sequence of J401 is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

Example 3: testing of herbicide resistant plants for imidazolinone herbicide resistance in the field

In 10 months of 2020, planting in the field at row spacing of 30 × 10cm and row length of 1.5m, and using wild type as control. When the wheat grows to one leaf and one heart, the imazapic is sprayed according to the dosage of 1 time of the field use concentration (90 gr ae/ha). After 14 days of spraying, the withered condition of the plants is investigated, and the wild type control is found to stop growing after the imazapic is sprayed, the whole plants wither, while the screened herbicide-resistant plant J401 is completely tolerant to the imazapic, the growth and development of the herbicide-resistant plant are not influenced and are consistent with those of the wild type plants without spraying the pesticide, and the test result is shown in figure 3. Normal tillering, jointing, flower raising and fructification at the later stage. This indicates that the herbicide-resistant mutants we screened can stably inherit the imidazolinone herbicide resistance.

Example 4: testing of herbicide resistant plants for resistance to imidazolinone herbicides in the greenhouse

In 2021, 4 months, the control Hordeum vulgare 247 and the J401 mutant were sown in seedling pots. The control group was set without imazapic (0 ×) and with 2 × imazapic, the test groups were set with 0 ×, 2 ×, 4 ×, 8 ×, 10 ×, 12 × imazapic, for a total of 6 concentration gradients, 4 pots were sown for each concentration, with 3 replicates per treatment. Before the imazethapyr is sprayed, abnormal seedlings are removed, and no less than 10 seedlings are left after each treatment. The wheat was sprayed with herbicide at the two-leaf one-heart stage, and the phytotoxicity symptoms, the number of damaged plants, the plant height, and the experimental results after 2 weeks were investigated and recorded at 1 week and 2 weeks after the application of the herbicide, respectively, as shown in fig. 4 and table 2.

For wheat herbicide phytotoxicity, a five-level visual method is adopted, and the classification standard is as follows:

level 0: the crops grow normally without any damage symptoms;

level 1: slight phytotoxicity of crops is less than 10%;

and 2, stage: the medium phytotoxicity of crops can be recovered later without influencing the yield;

and 3, level: the crop has serious phytotoxicity and is difficult to recover, thereby causing the reduction of yield;

4, level: the crop has serious phytotoxicity and can not be recovered, thereby causing obvious yield reduction or no yield.

The formula for calculating the phytotoxicity is as follows:

victim rate = (number of victim strain/total number of test strain) × 100%

The phytotoxicity index = ∑ (phytotoxicity grade obtained by classification visual method × number of phytotoxicity strain of corresponding grade) × 100/(total strain number × highest grade of classification visual method), wherein the highest grade of 5-grade visual method is 4.

Table 2 experimental results of different treatments

Note: "ck 0" is wild type wheat not sprayed with imazaquin as control; "ck 2 x" is the control of 2 times concentration of imazethapyr sprayed on wild wheat; 0 x is that no imazapyr is sprayed on the wheat in the experimental group; and 2x is that the wheat in the experimental group is sprayed with imazamox with the concentration 2 times, and the like.

As can be seen from FIG. 4 and Table 2, after spraying for 2 weeks, mutant J401, which was sprayed with imazapic at a concentration of 8 times or less, did not cause phytotoxicity, and grew normally, and plant height was not affected, and thus, this ALS mutein was able to tolerate at least 8 times (720 gr ae/ha) imazapic in wheat.

In conclusion, the serine at the 629 th position of the ALS mutant protein provided by the application is changed into the asparagine, so that the ALS mutant protein has herbicide-resistant activity, and the ALS mutant protein can make wheat tolerate imazapic acid with 8-fold concentration (720 gr ae/ha) (the concentration is far higher than the normal use concentration in the field), so that herbicide-resistant plants, such as wheat, tobacco or arabidopsis thaliana, and the like, especially crops can be cultivated by using the ALS mutant protein, so that the phytotoxicity of the crops is reduced, and the application range of the herbicide is widened.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Sequence listing

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Claims

ALS mutein, characterized in that the ALS mutein carries the mutation S629N, preferably the amino acid sequence of the ALS mutein is shown in SEQ ID NO 2.
2. The ALS mutein of claim 1, wherein the ALS mutein is capable of conferring herbicide resistance to a plant, the herbicide being an imidazolinone herbicide.
3. The ALS mutein of claim 2, wherein the herbicide is imazapic.
4. The ALS mutein according to claim 1, wherein the ALS mutein is derived from wheat, preferably wherein the wheat is rye 247.
5. A nucleic acid encoding the protein of any one of claims 1 to 4.
6. The nucleic acid according to claim 5, wherein the nucleotide at position 1886 of the nucleic acid sequence is mutated from guanine G to adenine A, preferably the nucleic acid sequence is shown in SEQ ID NO. 1.
7. An expression cassette, recombinant vector or cell comprising the nucleic acid of any one of claims 5 to 6.
8. Use of the ALS mutein of claims 1-4 or the nucleic acid of any one of claims 5-6 or the expression cassette, recombinant vector or cell of claim 7 for herbicide resistance in a plant, preferably in wheat, tobacco, Arabidopsis, rice, maize or sorghum.
9. A method for obtaining a plant with herbicide resistance comprising the steps of:

allowing a plant to comprise the nucleic acid of any one of claims 4-6; and/or the presence of a gas in the gas,

expressing the ALS mutein according to any one of claims 1 to 3 in a plant.
10. The method according to claim 9, characterized in that it comprises a transgenic, crossing, backcrossing or asexual propagation step.