Use of the wat1 gene in modulating the uptake and accumulation of plants to sulfonylurea herbicides

Abstract

This invention discloses the application of the WAT1 gene in regulating the absorption and accumulation of sulfonylurea herbicides in plants. The invention found that knocking out the OsWAT1 gene in rice reduces the absorption and accumulation of bensulfuron-methyl, lowers the bensulfuron-methyl content in rice, and increases resistance to bensulfuron-methyl. Rice with the OsWAT1 gene knocked out also developed resistance to ethoxysulfuron and sulfadiazine. Furthermore, knocking out the NtWAT1 homolog of flue-cured tobacco K326, indoor resistance verification experiments showed that the tobacco mutant also acquired bensulfuron-methyl resistance, also achieved by reducing herbicide absorption and accumulation. This indicates that the WAT1 gene plays a role in regulating the absorption and accumulation of sulfonylurea herbicides in plants and can be used to create new plant varieties with resistance to sulfonylurea herbicides.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to the application of the WAT1 gene in regulating the uptake and accumulation of bensulfuron-methyl in plants. Background Technology

[0002] Weeds in direct-seeded rice paddies are a key issue limiting the mechanized direct-seeding of rice. Sulfonylurea herbicides are highly effective in rice paddies, but improper use can inhibit rice seed germination and seedling growth, and they tend to remain in the soil, affecting subsequent crops. Therefore, developing sulfonylurea-resistant crop varieties to simplify weed control in rice paddies and promoting crop rotation are of great significance.

[0003] Sulfonylurea herbicides are widely used worldwide, primarily for controlling weeds in crops. These herbicides are selectively systemic and translocated. They are absorbed into the plant through the roots and leaves and translocated through the xylem and phloem. They inhibit the biosynthesis of branched-chain amino acids in plant cells, such as valine, leucine, and isoleucine. Simultaneously, the accumulation of butanone in the plant hinders DNA synthesis during cell division, preventing normal cell growth and ultimately achieving weed control. Most sulfonylurea herbicides are readily hydrolyzed in acidic environments. In alkaline soils, the degradation rate is slowest; 1%-20% of the applied herbicide can remain in the soil for a long time, damaging subsequent sensitive crops. The residual effects can persist for 2-3 years after application. Most sulfonylurea herbicides ionize and hydrolyze in both water and soil, with a half-life of up to 8 weeks. Sulfonylurea herbicides have low volatility and do not exhibit significant photodegradability; however, some, such as chlorsulfuron and metsulfuron-methyl, can remain in the soil for extended periods.

[0004] With the promotion and popularization of direct-seeding rice technology, weeds in paddy fields have become one of the main obstacles to high and stable rice yields. Sulfonylurea herbicides, as widely used herbicides in paddy fields, have good control effects on broadleaf weeds and sedges. Bensulfuron-methyl (BSM) is a highly effective ALS inhibitor herbicide, usually used as a soil preservative. However, because it inhibits rice germination, it is not suitable for direct-seeding rice fields, and different crops have different sensitivities to bensulfuron-methyl. Therefore, if crop varieties resistant to sulfonylurea herbicides can be cultivated, the phytotoxicity of sulfonylurea herbicides on rice can be avoided while reducing their residues, further mitigating the threat to subsequent sensitive crops such as tobacco, and providing a guarantee for paddy and dryland planting systems.

[0005] The Walls Are Thin 1 (WAT1) gene is a novel auxin transporter gene in plants that integrates auxin signaling in the formation of secondary walls in Arabidopsis fibers. Studies by Ranocha et al. found that WAT1 is expressed in all tissues and organs of Arabidopsis, with the highest expression levels in the stem and hypocotyl, and high expression in organs with a high proportion of cells containing secondary walls. Genes encoding the secondary wall-associated NAC domain protein SND1 and the NAC secondary wall thickening promoter NST1 play important regulatory roles in the formation of secondary walls in plant fibers. Silencing these two genes using RNAi leads to a significant reduction in the thickness of the secondary wall. In Arabidopsis WAT1 mutants, the expression of SND1 and NST1, as well as their target genes KNAT7, MYB46, and MYB103, is significantly reduced, indicating that WAT1 may be a regulator of SND1 and NST1, playing a crucial role in the formation of secondary walls in plant fibers. The Arabidopsis WAT1 mutant exhibits reduced lignin content, decreased secondary cell wall thickness in stem fibers, and dwarfing, along with significantly reduced auxin content and impaired auxin transport in the stem. Furthermore, research indicates that WAT1 acts as a transporter for auxin via local vacuolar membranes, playing a crucial role in plant morphogenesis. Ju et al. found that in the upland cotton variety 'L28', with its longer internodes and looser plant structure, WAT1 gene expression was higher than in the shorter, more compact variety 'XLZ77'. Moreover, in 'L28', WAT1 gene expression increased with the growth of fruiting branch internodes, suggesting that the WAT1 gene plays a role in upland cotton internode development. Recent studies have also revealed that WAT1, in addition to regulating plant growth and development, also plays a role in plant disease resistance responses. Ye et al. found that silencing GhWATs significantly increased resistance to Verticillium wilt in cotton. Denancé et al. found that the Arabidopsis WAT1 mutant exhibited enhanced resistance to bacterial wilt, Verticillium wilt, and some vascular pathogens. They also pointed out that WAT1 can influence Arabidopsis disease resistance by regulating salicylic acid (SA) and tryptophan metabolism. SA is a key hormone in plant stress defense responses, while tryptophan metabolism can produce precursors for auxin synthesis. This indicates that WAT1 plays a crucial role in plant growth, development, and stress defense responses by regulating the metabolism of hormones such as auxin and SA. However, there are currently no reports on the role of the WAT1 gene in the uptake and accumulation of sulfonylurea herbicides in plants. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of the WAT1 gene in regulating the absorption and accumulation of sulfonylurea herbicides in plants.

[0007] A second objective of this invention is to provide the application of the WAT1 gene in the creation of germplasm resources resistant to sulfonylurea herbicides.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention reveals that knocking out the OsWAT1 gene in rice can reduce the absorption and accumulation of BSM, decrease the BSM content in rice, and increase resistance to BSM. Rice with the OsWAT1 gene knocked out also developed resistance to ethoxysulfuron and ethersulfuron. After knocking out the homologous gene NtWAT1 in flue-cured tobacco K326, indoor resistance verification experiments showed that the tobacco mutant also acquired BSM resistance, which was also achieved by reducing the absorption and accumulation of herbicides. This indicates that the WAT1 gene has the function of regulating the absorption and accumulation of sulfonylurea herbicides in plants.

[0010] Specifically, this invention uses BSM to screen a yeast-enriched library of rice transporter proteins, obtains the OsWAT1 gene through sequencing and alignment, and uses a transient expression system in tobacco (tobacco leaf disc absorption and tobacco translocation experiments) to find that the absorption and translocation amounts of tobacco expressing this gene are significantly different from those of the unexpressed vector. Subsequently, root absorption experiments show that rice with the OsWAT1 gene knocked out and tobacco with the NtWAT1 gene knocked out absorbed less BSM compared to their wild-type varieties. This invention first verified that OsWAT1 mutant rice has higher resistance to herbicides under culture medium conditions, and then combined this with herbicide seed coating technology to coat rice seeds. It was found that even after coating, OsWAT1 mutant rice still exhibited higher herbicide resistance and relatively good control efficacy against broadleaf weeds. Simulating field pre-emergence herbicides, it was found that the OsWAT1 rice mutant also exhibits a certain degree of resistance. Experiments using tobacco seed germination rate determination, hydroponics, and spraying verified that the NtWAT1 tobacco mutant also exhibits BSM resistance. The results of this invention indicate that the WAT1 gene in plants plays a role in regulating the uptake and accumulation of sulfonylurea herbicides, and can be used to create new plant varieties resistant to sulfonylurea herbicides.

[0011] Therefore, this invention seeks protection for the following novel applications relating to the WAT1 gene or WAT1 protein in plants:

[0012] Application of the WAT1 gene or WAT1 protein in regulating the uptake and accumulation of sulfonylurea herbicides in plants.

[0013] Application of the WAT1 gene or WAT1 protein in the breeding of plant varieties resistant to sulfonylurea herbicides.

[0014] Furthermore, in order to obtain plant varieties resistant to sulfonylurea herbicides by inhibiting the expression of the WAT1 gene in plants, or inhibiting the expression level and / or activity of the WAT1 protein.

[0015] Furthermore, the inhibition of WAT1 gene expression in plants, or the inhibition of WAT1 protein expression level and / or activity, is achieved through gene saturation mutation, gene site editing, RNA interference, homologous recombination, or gene knockout.

[0016] Furthermore, the gene knockout specifically involves constructing a CRISPR-Cas9 system for the WAT1 gene and transforming plants to obtain plants with WAT1 gene mutations, thereby obtaining plant varieties resistant to sulfonylurea herbicides; the CRISPR-Cas9 system contains sgRNA that recognizes the WAT1 gene.

[0017] The present invention also provides the application of reagents for knocking out the WAT1 gene in inhibiting the uptake and accumulation of sulfonylurea herbicides in plants or in the breeding of plant varieties resistant to sulfonylurea herbicides.

[0018] Preferably, the WAT1 gene is the rice OsWAT1 gene, whose nucleotide sequence is as shown in SEQ ID No. 1; or has at least 50% homology with the nucleotide sequence shown in SEQ ID No. 1 and encodes the amino acid shown in SEQ ID No. 2; or hybridizes with the nucleotide shown in SEQ ID No. 1 under stringent conditions and encodes the amino acid shown in SEQ ID No. 2; the amino acid sequence of the OsWAT1 protein is as shown in SEQ ID No. 2.

[0019] Furthermore, by constructing a CRISPR-Cas9 system for the rice OsWAT1 gene and transforming plants, plants with OsWAT1 gene mutations were obtained, thus yielding plant varieties resistant to sulfonylurea herbicides; the CRISPR-Cas9 system contains sgRNA that recognizes the OsWAT1 gene, and its target sequence is shown in SEQ ID No. 5.

[0020] Preferably, the WAT1 gene is the tobacco NtWAT1 gene, whose nucleotide sequence is as shown in SEQ ID No. 3; or has at least 50% homology with the nucleotide sequence shown in SEQ ID No. 3 and encodes the amino acid shown in SEQ ID No. 4; or hybridizes with the nucleotide shown in SEQ ID No. 3 under stringent conditions and encodes the amino acid shown in SEQ ID No. 4; the amino acid sequence of the NtWAT1 protein is as shown in SEQ ID No. 4.

[0021] Furthermore, by constructing a CRISPR-Cas9 system for the tobacco NtWAT1 gene and transforming plants, plants with NtWAT1 gene mutations were obtained, i.e., plant varieties resistant to sulfonylurea herbicides were obtained; the CRISPR-Cas9 system contains sgRNA that recognizes the NtWAT1 gene, and its target sequence is shown in SEQ ID NO.6.

[0022] Furthermore, the plant in question is a crop.

[0023] Preferably, the crop is rice or tobacco.

[0024] Preferably, the sulfonylurea herbicides include, but are not limited to, bensulfuron-methyl, ethoxysulfuron-methyl, or ethersulfuron-methyl.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention provides the application of the WAT1 gene in regulating the uptake and accumulation of sulfonylurea herbicides in plants. This invention found that knocking out the OsWAT1 gene in rice reduces the uptake and accumulation of BSM, lowers the BSM content in rice, and increases resistance to BSM. Rice with the OsWAT1 gene knocked out also developed resistance to ethoxysulfuron and ethersulfuron-methyl. Furthermore, knocking out the NtWAT1 homolog of flue-cured tobacco K326, in indoor resistance verification experiments, showed that the tobacco mutant also acquired herbicide resistance, also achieved by reducing herbicide uptake and accumulation. This indicates that the WAT1 gene in plants plays a role in regulating the uptake and accumulation of sulfonylurea herbicides and can be used to create new plant varieties resistant to sulfonylurea herbicides. This invention provides a good option for the research of herbicide-resistant crops by combining the uptake and accumulation of sulfonylurea herbicides with the resistance mechanism. Attached Figure Description

[0027] Figure 1 The sensitivity and growth curves of the OsWAT1 overexpressing yeast strain under BSM treatment. A: Sensitivity test results; B: Growth curve.

[0028] Figure 2 Figure 1 shows the absorption and translocation of BSM in tobacco leaf discs to illustrate the effects of OsWAT1 overexpression. A: Results of bensulfuron-methyl absorption experiments in tobacco leaf discs; BC: Results of bensulfuron-methyl translocation experiments in tobacco.

[0029] Figure 3 The sequence alignment results of mutants of the Zhonghua 11 and OsWAT1 genes are shown in the figure.

[0030] Figure 4 This is a sequence alignment diagram of wild-type tobacco and mutants of the NtWAT1 gene.

[0031] Figure 5 Figure 1 shows the germination results of different genotypes of OsWAT1 rice after adding BSM to the culture medium. A: Phenotype of rice seeds of different genotypes after 7 days of germination; B: Root length of germinating rice seeds of different genotypes; C: Aboveground length of germinating rice seeds of different genotypes.

[0032] Figure 6 Figure 1 shows the growth of different genotypes of OsWAT1 rice seedlings after adding BSM to the culture medium. A: Photographs of seedling establishment of different genotypes of rice seedlings; B: Seedling establishment rate of different genotypes of rice seedlings; C: Root length of different genotypes of rice seedlings; D: Aboveground length of different genotypes of rice seedlings.

[0033] Figure 7 Figures showing the results of BSM coating verification of the resistance and herbicidal activity of the OsWAT1 mutant. A: Phenotypic photos of rice in different treatment groups; B: Plant height and fresh weight of rice in different treatment groups on the 14th, 21st and 28th days of growth; C: Survival status of weeds (grass sedge and sedge) in the paddy field.

[0034] Figure 8 Figure 1 shows the results of validating the resistance of the OsWAT1 mutant under BSM blocking treatment. A: Phenotypic photos of rice in different treatment groups; B and D: Plant height and fresh weight of rice in different treatment groups on day 21 and day 35 of growth.

[0035] Figure 9 Figure 1 shows the results of BSM uptake in the roots of different OsWAT1 rice genotypes. A: Root content of different genotypes of rice after 21 days of growth, cultured for 6 hours in buffer solutions with different concentrations of BSM; B: Stem content of different genotypes of rice after 21 days of growth, cultured for 6 hours in buffer solutions with different concentrations of BSM; C: Whole plant content of different genotypes of rice after 21 days of growth, cultured for 6 hours in buffer solutions with different concentrations of BSM; D: BSM uptake kinetic curves of different genotypes of rice.

[0036] Figure 10 The images show the results of the petri dish bioassay to verify the resistance of tobacco mutants. A: Photographs of the germination of tobacco seeds of different genotypes in different treatment groups; B: Germination rate of tobacco seeds of different genotypes in different treatment groups; C: Germination inhibition rate of tobacco seeds of different genotypes in different treatment groups.

[0037] Figure 11 Figure 1 shows the results of verifying the resistance of tobacco mutants by adding BSM to hydroponics. A: Photographs of the growth of tobacco seedlings of different genotypes in different treatment groups; B: Chlorophyll a content; C: Chlorophyll b content; D: Carotene content; E: Root length; F: Plant height; G: Leaf length; H: Leaf width; I: Fresh weight.

[0038] Figure 12 Figure 1 shows the BSM uptake results of different tobacco genotypes in hydroponics. A: Benzylsulfuron content in roots; B: Benzylsulfuron content in stems.

[0039] Figure 13 Figure 1 shows the results of verifying the resistance of tobacco mutants by foliar spraying with BSM. A: Photographs of the growth of tobacco seedlings of different genotypes in different treatment groups; B: Plant height of tobacco seedlings of different genotypes in different treatment groups; C: Fresh weight of tobacco seedlings of different genotypes in different treatment groups.

[0040] Figure 14 Figure showing the germination results of different OsWAT1 genotypes of rice after adding ethersulfuron-methyl to the culture medium.

[0041] Figure 15 Figure 1 shows the growth of OsWAT1 rice seedlings of different genotypes after adding ethersulfuron-methyl to the culture medium. A: Germination phenotype of rice seeds of different genotypes; B: Root length of rice seeds of different genotypes after germination; C: Stem length of rice seeds of different genotypes after germination.

[0042] Figure 16 Figure 1 shows the germination results of different genotypes of OsWAT1 rice after adding ethoxysulfuron to the culture medium. A: Germination phenotype of rice seeds of different genotypes; B: Germination rate of rice seeds of different genotypes; C: Root length of rice seeds of different genotypes; D: Stem length of rice seeds of different genotypes.

[0043] Figure 17 Figure 1 shows the growth of different genotypes of OsWAT1 rice seedlings after adding ethoxysulfuron to the culture medium. A: Photographs of seedling establishment of different genotypes of rice seedlings; B: Seedling establishment rate of different genotypes of rice seedlings; C: Root length of different genotypes of rice seedlings; D: Stem length of different genotypes of rice seedlings. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0045] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0046] In this example, *Escherichia coli* DH5α and *Agrobacterium* EHA105 are commonly used strains and are commercially available; the rice variety is wild-type Zhonghua 11 (a publicly used rice variety, commercially available), and the tobacco variety is wild-type K326 (a publicly used tobacco variety). The primers used in this example were synthesized by BGI Genomics Co., Ltd. in Shenzhen, and sequencing was performed at BGI Genomics Co., Ltd. in Shenzhen.

[0047] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0048] Example 1: Screening of genes related to bensulfuron-methyl transport using a yeast enrichment library of rice transport proteins.

[0049] 1. Yeast Library Screening: A yeast library containing 1452 rice transporter proteins was used to screen for yeast strains sensitive to bensulfuron-methyl. Yeast strains stored at -80℃ in 96-well plates were copied to SD-gal solid medium using a 96-pin replicator. After incubation at 30℃ for 2-3 days, the strains were copied again to 96-well plates containing SD-gal liquid medium and incubated overnight. 20 μL of the overnight cultured yeast was added to 1 mL of fresh SD-gal liquid medium. After incubation at 30℃ and 180 rpm for 1 hour, the OD value at 595 nm was measured using a microplate reader. The OD value of the bacterial culture in each well was adjusted to 0.01 with sterile water. Liquid medium was used as a blank control. The OD-adjusted yeast was copied to solid medium containing 20 g / L galactose and 2.5 μM and 5 μM solutions. After drying, the culture was sealed with adhesive tape and incubated at 30℃ for 2-3 days. The results were observed and photographed daily.

[0050] 2. Sequence identification of the rice bensulfuron-methyl transporter gene: Based on the above screening results, corresponding strains were selected from the preserved yeast library. After culturing in SD-glu liquid medium at 30℃ and 180rpm for 1-2 days, yeast plasmids were extracted and transformed into E. coli. Single colonies were picked and sequenced using pYES2 vector-specific primers. The sequencing results were compared with the database http: / / rice.plantbiology.msu.edu / to determine the target transporter gene sequence and functional annotation, showing that the transporter gene is rice OsWAT1, its nucleotide sequence is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2. The results showed that after overexpression of OsWAT1, the yeast strain was more sensitive to BSM (Bacillus subtilis). Figure 1 A).

[0051] 3. Yeast Growth Inhibition Curve: The selected yeast strains were cultured overnight at 30℃ and 200 rpm in SD-glu liquid medium. OD values ​​were measured using a spectrophotometer. Different concentrations of BSM-containing liquid medium were prepared, and 100 μL of each concentration was added to 96-well plates, with three replicates per concentration. A certain amount of yeast culture was added to 100 μL of medium to achieve an initial OD value of 0.01. The OD value was then measured every 1 hour using an automated growth curve analyzer. The control group (without BSM) was used, and the average of the three measurements was calculated. The results showed that after overexpression of OsWAT1, the corresponding yeast strains grew more slowly in BSM-containing medium and were more sensitive to BSM. Figure 1 B).

[0052] The culture medium used in the above experiment is as follows:

[0053]

[0054] Example 2: Verification of the function of rice bensulfuron-methyl transporter using a transient expression system in tobacco.

[0055] 1. Construction of pEAQ recombinant vector

[0056] The OsWAT1 gene, sequenced from a yeast strain, was used as a template for PCR amplification. The plasmid concentration was 5 ng / μL. The following reaction mixture was used: 10 μL 5× Prime Star Max DNA polymerase; 0.8 μL of each primer (10 μM); 1 μL DNA (5 ng); and H2O added to a final volume of 20 μL. The specific reaction program was as follows: enzyme activation at 98℃ for 1 min, one cycle; denaturation at 98℃ for 30 s, annealing at 60℃ for 30 s, and extension at 72℃ for 1 min 30 s, for a total of 32 cycles.

[0057] The primer sequences for OsWAT1 amplification are as follows:

[0058] F1:5'-CAAATTCGCGACCGGTGTCGACGCTGTAATACGACTCACTATAGG GAATATTAAGCTT-3'

[0059] R1:5'-AGTTAAAGGCCTCGAGGTCGACGCATGCTCGAGCGGCCGCCAG TGTGATGGATATCTG-3'

[0060] Agarose gel electrophoresis (1.0%) was used for identification, and the PCR products obtained sequentially were used... The Gel Extraction Kit (OMEGA) is used for gel recovery and purification.

[0061] Establish the enzyme digestion reaction system: 6 μL SaII (Takara), 8 μL 10xH buffer (Takara); 3 μg intact pEAQ plasmid; deionized H2O to a final volume of 100 μL. Digest at 37℃ for 3 h, and purify using an OMEGA gel extraction kit.

[0062] Follow the instructions in the ClonExpress II One Step Cloning Kit (vazyme) to connect. The connection system is as follows:

[0063]

[0064] Colony PCR was performed to screen for correct clones, which were named pEAQ-OsWAT1. Positive transformants were selected, and plasmids were extracted and sequenced. The correctly sequenced plasmids were then transformed into Agrobacterium and the bacterial culture was preserved for subsequent experiments.

[0065] 2. Tobacco injection expression protein

[0066] The preserved Agrobacterium culture was activated on LB solid medium (50 mg / L Kan + 50 mg / L Rif) plates, and then inoculated into 1 mL of LB (50 mg / L Kan + 50 mg / L Rif) liquid medium. The culture was incubated at 28°C for 24 h at 180 rpm. 50 μL of the culture was then added to 50 mL of LB (50 mg / L Kan + 50 mg / L Rif) liquid medium and incubated at 28°C for 15 h at 180 rpm. The culture was then aliquoted into 10 mL centrifuge tubes, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the cells were washed with 7 mL of sterile water. The cells were then resuspended in 7 mL of resuspension solution and incubated in the dark for 3 h. The OD of the culture was then calculated. 600 Adjust the concentration to 0.3 for injection inoculation. Select 2-3 larger leaves from the middle of the tobacco plant for injection, injecting the pEAQ vector and Agrobacterium tumefaciens solution onto both sides of the tobacco leaf, respectively. Use a syringe to push the bacterial injection solution into the tobacco leaf until the bacterial solution can no longer spread. After the tobacco is injected, use a marker to mark the area where the bacterial solution has spread, wipe the Agrobacterium tumefaciens off the tobacco surface to prevent the surface bacterial solution from causing the leaves to wilt, and incubate in the dark for 1 day, then incubate under normal conditions for 1 day.

[0067] 3. Tobacco leaf disc absorption of bensulfuron-methyl experiment

[0068] Two days after transient expression of pEAQ-OsWAT1 in tobacco, a small portion of tobacco leaves injected with pEAQ-GFP was observed for luminescence. Tobacco leaves with normal and uniform luminescence were selected, and leaf discs were created using a 1.4 cm diameter punch. After pre-culturing in leaf disc absorption medium (containing BSM) at pH 5.8 for 30 min, six leaf discs constituted one replicate, with three replicates for each concentration. The drug absorption time was 2 h. The tobacco leaves were washed three times with 0.5 mM CaCl2 to ensure complete removal of the drug adhering to the tobacco surface. After absorption, the tobacco leaves were ground in liquid nitrogen, and 5 mL of acetonitrile was added. The tissue homogenate was collected and placed in a 10 mL centrifuge tube. After vortexing for 3 min, sonication for 30 min, followed by vortexing for another 2 min, and centrifugation at 4000 rpm for 5 min was performed for extraction. 1 mL of the supernatant was collected and impurities were removed using a 0.22 μm organic filter membrane. The filtrate was then analyzed by LCMS-MS. The results are as follows: Figure 2As shown in Figure A, the BSM content absorbed by tobacco leaf discs expressing pEAQ-OsWAT1 was higher than that of tobacco leaf discs expressing pEAQ without BSM.

[0069] 4. Tobacco Transport Experiment with Benzylsulfuron

[0070] Two days after transient expression of pEAQ-OsWAT1 in tobacco, 100 μL of 200 μM BSM was evenly applied to the area of ​​the same leaf that was not injected with the pEAQ vector and gene using a pipette. After 24 hours, the luminescence of the tobacco parts injected with pEAQ-GFP was observed. Leaves with normal and uniform luminescence were selected. Two leaf discs were punched from each tobacco leaf at the site of pEAQ vector and gene injection using a 1.4 cm diameter punch. The tobacco leaves were washed three times with 0.5 mM CaCl2 to ensure complete removal of BSM adhering to the tobacco surface. The tobacco leaves were then ground using a grinder, vortexed for 5 min, sonicated for 30 min, and centrifuged at 14000 rpm for 5 min. 1 mL of the supernatant was collected, filtered through a 0.22 μm microporous membrane, and analyzed by LCMS-MS.

[0071] The results of tobacco leaf disc absorption and translocation experiments showed that, compared with the empty control, OsWAT1 improved the absorption and translocation of BSM in tobacco leaves. Figure 2 B, 2C).

[0072] Example 3: CRISPR knockout construction of WAT1 rice and tobacco mutant plants

[0073] 1. Constructing OsWAT1 rice mutant plants by CRISPR knockout

[0074] (1) Using a simple and efficient CRISPR / Cas9 system, a specific target sequence was selected based on the OsWAT1 exon sequence. The target sequence is 5'-GTGCATCCTCGCCGGATACG-3' (SEQ ID NO.5). The target sequence targets the OsWAT1 gene and specifically inactivates the OsWAT1 protein.

[0075] (2) Constructing a pCRISPR / Cas9 recombinant vector containing the above target sequence fragments.

[0076] 1) Design adapter primers with sticky ends based on the target sequence.

[0077] The designed target sequence was fitted with a specific sticky end adapter for the pCRISPR / Cas9 system, and the complete adapter primer was synthesized.

[0078] Rice-OsWAT1-F2:5'-TGTGTGCGCCGAGCAGGCCGTGAGC-3';

[0079] Rice-OsWAT1-R2:5'-AAACCGTATCCGGCGAGGATGCACA-3';

[0080] 2) Annealing the adapter primers with sticky ends to form complementary double-stranded fragments with sticky ends.

[0081] Dilute the F2 and R2 primers to a concentration of 10 μM, take 10 μL of each and mix well. Perform an annealing reaction in a PCR instrument, reducing the temperature from 98℃ to 22℃, so that the F2 and R2 primers complement each other to form a double-stranded fragment with sticky ends.

[0082] 3) Enzymatic digestion of the original vector pOs-sgRNA containing sg-RNA (TAKARACat#632640)

[0083] The original vector pOs-sgRNA containing sg-RNA was digested with the restriction endonuclease BsaⅠ to generate sticky ends that are complementary to the sticky ends of the target sequence. The digestion system of the original pOs-sgRNA vector with BsaⅠ was as follows: 2 μL of 10× buffer, 1 μL of BsaⅠ enzyme, 4 μg of pOs-sgRNA vector, and ddH2O to a final volume of 20 μL. Digestion was carried out at 37℃ for 12 h. After verifying the band size by 1% agarose gel electrophoresis, the digested product was purified by column chromatography using a kit (OMEGACat#D2500-02) to obtain the digested pOs-sgRNA vector. The digested product was dissolved in sterile ddH2O, and the concentration was determined before use.

[0084] 4) The double-stranded fragment with sticky ends is ligated into the enzyme-digested pOs-sgRNA vector to form a recombinant vector containing the target sequence and sg-RNA.

[0085] The double-stranded fragment from step 2) and the digested pOs-sgRNA vector from step 3) were ligated using T4 ligase to form a complete recombinant vector containing the target sequence for OsWAT1 protein and sg-RNA. The 15 μL ligation system consisted of: 1.5 μL 10×T4 ligation buffer, 4 μL double-stranded fragment, 3 μL digested pOs-sgRNA vector, 1 μL T4 DNA ligase, and ddH2O to a final volume of 15 μL. Ligation was performed at 16°C for 12 hours. The ligation product was transformed into *E. coli* DH5α and cultured overnight on kanamycin-resistant LB agar plates (containing 10 mg / L kanamycin). Positive strains were selected for sequencing to obtain correctly sequenced recombinant vectors containing the target sequence and sg-RNA.

[0086] 5) Using LR mix, the recombinant vector containing the target sequence and sg-RNA is recombined with the vector pH-Ubicas9-7 containing Cas9 to form a complete recombinant vector containing the target sequence-sg-RNA+Cas9.

[0087] 6) The recombinant vector obtained in step 4) and the Cas9-containing vector pH-Ubi-cas9-7 (provided by Baige Gene Technology Co., Ltd.) were recombined using LR mix (Shanghai Beinuo Biotechnology Co., Ltd.). The LR reaction system consisted of: 25-50 ng of the recombinant vector containing the target sequence and sg-RNA, 75 ng of pH-Ubi-cas9-7 vector, 1 μL of 5×LR Clonase™ buffer, TE buffer (pH 8.0) to a final volume of 4.5 μL, and 0.5 μL of LR Clonase™. The system was incubated at 25°C for 2 h. After the reaction, 2 μL of 2 μg / μL Proteinase K was added, and the mixture was treated at 37°C for 10 min. Then, 2 μL of the reaction product was transferred into *E. coli* DH5α and cultured overnight at 37°C on gentamicin-resistant LB plates. Positive strains were selected for sequencing to obtain the correctly sequenced complete pCRISPR / Cas9-OsWAT1 recombinant expression vector containing the OsWAT1 protein target sequence-sg-RNA+Cas9.

[0088] (3) The obtained complete recombinant vector containing the OsWAT1 protein target sequence-sg-RNA+Cas9 was introduced into rice callus to obtain transgenic plants.

[0089] 1) The recombinant expression vector pCRISPR / Cas9-OsWAT1 obtained in step (2) was electroporated into Agrobacterium EHA105 (Olivia CD, 2019) to obtain recombinant bacteria AGL1 / pCRISPR / Cas9-OsWAT1.

[0090] 2) The recombinant strain AGL1 / pCRISPR / Cas9-OsWAT1 was transformed into Zhonghua 11 rice callus using Agrobacterium-mediated transformation, as detailed below:

[0091] 3) Pick a single colony of AGL1 / pCRISPR / Cas9-OsWAT1 and inoculate it into 10 mL of Agrobacterium tumefaciens medium (containing 50 mg / L kanamycin and 50 mg / L rifampin). Incubate at 28°C and 180 rpm for 2-3 days. Take 4 mL of the bacterial suspension, centrifuge at 4000 rpm for 3 min, discard the supernatant, add a small amount of AAM medium to resuspend the cells, and then add 20 mL of AAM medium (containing 0.1 mM acetylsyleugenol As). Incubate at 28°C and 150 rpm in the dark for 1-2 h until OD reaches the target value. 600= Approximately 0.4. Select healthy, granular callus tissue from Zhonghua 11 rice (hereinafter also known as wild-type rice). Immerse the callus tissue in Agrobacterium culture medium (YEP without agar), shake at 28℃ and 150-200 rpm for 20 minutes. Decant the callus tissue, blot off excess bacterial solution with sterile filter paper, and spread the callus tissue evenly on a sterile Petri dish containing multiple layers of filter paper. Dry the callus tissue on a laminar flow hood (the callus should be dispersed and not clump). Then transfer the callus tissue to a co-culture medium and incubate in the dark for 2-3 days. Transfer the callus tissue to NB basal medium containing 100 mg / L hygromycin and 400 mg / L cephalosporin for 3-4 weeks (first screening). Transfer the surviving callus tissue to a second screening medium (NB basal medium containing 100 mg / L hygromycin and 200 mg / L cephalosporin) for 3 weeks. The resistant callus was transferred to a differentiation medium (containing 100 mg / L hygromycin) for differentiation. After the regenerated plants rooted on a seedling strengthening medium containing 100 mg / L hygromycin (about 3-4 weeks), they were transferred to a greenhouse. Transgenic plants with completely inactivated OsWAT1 protein could be obtained from the T0 generation plants.

[0092] The culture media used in the above transformation are as follows:

[0093] Co-culture medium (Beijing Huayueyang Biotechnology Co., Ltd.): callus induction and subculture medium + As (0.1 mmol / L) + glucose (10 g / L), pH 5.2.

[0094] Agrobacterium-infected rice callus culture medium (AAM medium, Beijing Huayueyang Biotechnology Co., Ltd.): AA macro-elements + AA micro-elements + AA amino acids + MS vitamins + hydrolyzed casein (500 mg / L) + sucrose (68.5 g / L) + glucose (36 g / L) + As (0.1 mM), pH 5.2.

[0095] NB basic culture medium (Beijing Huayueyang Biotechnology Co., Ltd.): N6 macroelements + B5 microelements + B5 organic components + iron salts + hydrolyzed casein (300mg / L) + proline (500mg / L) + sucrose (30g / L) + agar (8g / L), pH 5.8.

[0096] Callus induction and subculture medium: NB basal medium + 2,4-D (2 mg / L).

[0097] Differentiation medium: NB basal medium + 6-BA (3 mg / L) + NAA (1 mg / L).

[0098] Seedling growth medium: 1 / 2 MS medium + NAA (0.5 mg / L) + MET (0.25 mg / L).

[0099] Agrobacterium tumefaciens medium (YEP): 10 g / L tryptone + 10 g / L yeast extract + 5 g / L sodium chloride + 15 g / L agar.

[0100] (4) Screening for transgenic positive plants from transgenic plants

[0101] DNA (OMEGACat#D3485-02) was extracted from the transgenic plants (T0 generation) transplanted in step (3) and the target sequence sites were detected. A total of 15 positive plants were detected.

[0102] Mutant plants were obtained from transgenic positive plants.

[0103] 1) Identification of mutation sites

[0104] DNA (OMEGACat#D3485-02) was extracted from the positive plants in step 4 of the transplantation. Specific primers F3 and R3 were designed to amplify the DNA fragment containing the target site within 500 bp. The 283 bp PCR product obtained was purified and sent to the company for sequencing. The sequencing results were compared with the wild-type plant sequence to screen out mutant plants.

[0105] F3:5'-GTAATGGTGGCCGGAGTACC-3';

[0106] R3:5'-GCACGCAAAGAAAGGAAGGA-3'.

[0107] 2) The mutant plants were propagated, and seeds were collected from individual plants lacking transgenic elements such as hygromycin and Cas9 in the T1 generation transgenic segregating population to obtain loss-of-function mutants, named oswat1-1 and oswat1-2, respectively. The mutation analysis results of the loss-of-function mutants and wild-type plants are as follows: Figure 3 As shown, oswat1-1 has a 2bp deletion mutation, and oswat1-2 has a 1bp insertion mutation.

[0108] 2. Constructing NtWAT1 tobacco mutant plants by CRISPR knockout

[0109] Construction of mutant plants of the rice OsWAT1 gene based on the tobacco homology NtWAT1 (its nucleotide sequence is shown in SEQ ID NO.3, and its encoding amino acid sequence is shown in SEQ ID NO.4).

[0110] (1) Using the CRISPR / Cas9 system, target sequences were selected based on the exon sequences of NtWAT1.

[0111] Using a simple and efficient CRISPR / Cas9 system, a specific target sequence was selected based on the NtWAT1 exon sequence. The target sequence is 5'-CAAACTTACTTCACCTGCAATGG-3' (SEQ ID No. 6). The target sequence targets the NtWAT1 gene and specifically inactivates the NtWAT1 protein.

[0112] (2) Constructing a pCRISPR / Cas9 recombinant vector containing the above target sequence fragments.

[0113] 1) Design adapter primers with sticky ends based on the target sequence.

[0114] The designed target sequence was fitted with a specific sticky end adapter for the pCRISPR / Cas9 system, and the complete adapter primer was synthesized.

[0115] Tobacco-NtWAT1-F5:5'-TGTGTGCGCCGAGCAGGCCGTGAGC-3';

[0116] Tobacco-NtWAT1-R5:5'-AAACCGTATCCGGCGAGGATGCACA-3';

[0117] Other experimental procedures were performed using the OsWAT1 rice mutant plants described above. The three functional mutants obtained were named Ntwat1-1, Ntwat1-2, and Ntwat1-3, respectively. The mutant analysis results are shown below. Figure 4 Ntwat1-1 has a 1bp insertion mutation, Ntwat1-2 has a 1bp deletion mutation, and Ntwat1-3 has a 2bp deletion mutation.

[0118] Example 4: Rice Resistance Test to Sulfonylurea Herbicides

[0119] To test the resistance of the loss-of-function mutant rice obtained in Example 3 to BSM, resistance was verified by culture medium sensitivity test, indoor coated pot culture and indoor closed treatment test.

[0120] 1. Germination rate of rice seeds of different genotypes was determined by adding bensulfuron-methyl to the culture medium.

[0121] OsWAT1 gene mutant germination rate experiment

[0122] Seeds of rice mutants of the Zhonghua 11 and OsWAT1 genes were sterilized in a workbench using 75% anhydrous ethanol and 30% sodium hypochlorite solutions (1-2 drops of Tween could be added to the solutions). First, the seeds were washed with 75% anhydrous ethanol for 3 minutes, repeated 3 times. Then, they were washed with 30% sodium hypochlorite solution for 3 minutes, then rinsed again with 30% sodium hypochlorite solution for 15-20 minutes. During this time, the seeds could be placed in a shaker at 150 rpm. Finally, the seeds were rinsed thoroughly with sterile water, and then air-dried on sterilized filter paper. They were then sown in MS medium containing 0, 1.0, and 1.5 μM BSM, respectively, and cultured in a 28℃ climate incubator for 7 days. Each treatment was performed in triplicate, with 30 seeds tested in each replicate. Germination was recorded daily for 1-5 days after sowing, and the rice phenotype was photographed and statistically analyzed after 7 days. The germination rate of rice seeds of different genotypes and the inhibition rate of germination by BSM were calculated. The results showed that the final germination rate of mutant seeds was not significantly different from that of wild-type seeds. Figure 5 On the fifth day of germination, the two concentrations of BSM showed the lowest inhibition rates against mutants OsWAT1-1 and OsWAT1-2 (Table 1).

[0123] Table 1. Inhibition rate (%) of bensulfuron-methyl on seed germination of different genotypes

[0124]

[0125] The same planting method was used to verify the efficacy of the mutants OsWAT1-1 and OsWAT1-2 against ethersulfuron (Table 2). Figure 14 ) and ethoxysulfuron (Table 3, Figure 16 The resistance of rice to germination was investigated, and the results showed that the germination inhibition rate of rice seeds in each line was as follows: WT > OsWAT1-1 > OsWAT1-2.

[0126] Table 2. Inhibition rate of ethersulfuron-methyl on seed germination of various genotypes

[0127]

[0128]

[0129] Table 3. Inhibition rate of ethoxysulfuron on seed germination of various genotypes

[0130]

[0131] 2. Verification of herbicide resistance in rice seedlings by adding sulfonylurea herbicides to the culture medium.

[0132] OsWAT1 gene mutant seedlings are resistant

[0133] The rice seed disinfection method was the same as described above. After drying, the seeds were sown in MS medium containing 0, 0.5, 1.5, and 2.5 μM BSM, respectively, and cultured in an artificial climate incubator for 14 days. After 14 days, root length, stem length, and the number of seedlings were measured and photographed. oswat1-1 and oswat1-2 showed higher BSM tolerance than the wild type, with higher root length, aboveground part length, and seedling rate than both the wild type and the overexpression lines. Figure 6 After being cultured for 14 days using the same planting method in a medium containing ethersulfuron-methyl and ethoxysulfuron-methyl, the root length and aboveground length of the mutants OsWAT1-1 and OsWAT1-2 were greater than those of the wild-type lines. Figure 15 , Figure 17 ).

[0134] 3. Verification of rice resistance and herbicidal activity through bensulfuron-methyl seed coating

[0135] (1) OsWAT1 gene mutant coating

[0136] A seed pelleting powder (2:1, w / w) was prepared using kaolin and attapulgite. A 5 g / L bensulfuron-methyl suspension was prepared from the technical grade bensulfuron-methyl, and a binder was prepared using 1.5% (w / w) polyvinyl alcohol. Seeds were then placed in a coating machine with the powder, suspension, and binder to produce pelleted seeds with a drug-to-seed ratio of 1:2500. Water coating was used as a control.

[0137] The experiment was conducted in a plant climate chamber using field soil. The collected field soil was dried for 3-4 days before use, then mixed with an appropriate amount of water to form a slurry, which was then placed in a non-porous plastic basin and leveled. Plump rice seeds of Zhonghua 11, oswat1-1, and oswat1-2 were selected for BSM seed coating at a ratio of 1:2500. Water-coated seeds served as a control. The experiment was conducted in plastic basins of varying sizes, with three replicates per treatment group. At sowing, the area was divided into three sections from left to right, and Zhonghua 11, oswat1-1, and oswat1-2 rice were planted sequentially. Each rice line was planted in eight holes, with two seeds per hole. To verify whether BSM seed coating had herbicidal activity against broadleaf weeds, two common paddy field weeds (sedge and sedge) were selected for testing, with equal amounts of weed seeds sown in each plastic basin. After sowing, keep the soil moist. Seven days later, add water to the pots, maintaining a water level 2 cm above the soil surface. Apply rice fertilizer every seven days thereafter. On days 14, 21, and 28 of growth, photographs were taken to record the rice phenotype. On the last day, weed survival was also recorded. BSM coating inhibited seedling growth to varying degrees, with the wild-type Zhonghua 11 showing the most severe inhibition, exhibiting shorter plant height and reduced fresh weight. The oswat1 mutant line had significantly higher plant height and fresh weight than Zhonghua 11. Figure 7 )

[0138] 4. Verification of rice resistance by bensulfuron-methyl pretreatment

[0139] The experiment was conducted in a plant climate chamber, with a blank control group and a 25 μM BSM treatment group, each treatment having 3 replicates. To ensure the results closely resembled field soil conditions, field soil was used in this experiment. The collected field soil was dried for 3-4 days before use, then mixed with an appropriate amount of water to form a slurry, which was then placed in a non-porous plastic basin and leveled. After breaking dormancy, the seeds of Zhonghua 11, oswat1-1, and oswat1-2 rice were soaked and germinated at 37℃ for 1 day. Rice seedlings with consistent germination trends were selected and sown in the basins. The basins were divided into 3 areas from left to right, and Zhonghua 11, oswat1-1, and oswat1-2 rice were planted sequentially, with 8 holes for each rice line and 2 seeds per hole. After sowing, the soil in the basins was kept moist, but the water level did not exceed the soil surface. A sealing treatment was performed 1 day after sowing. A solution containing 0.1% Tween 80 was mixed with water and 25 μM BSM solution and sprayed evenly into pots. The amount of pesticide used per pot was 25 mL. After application, the soil was kept moist, but the water level should not exceed the soil surface. After 7 days, water was added to the pots, maintaining a water level 2 cm above the soil surface. Fertilizer was then applied to the rice every 7 days. The rice phenotype was photographed and statistically analyzed on days 21 and 35 of growth. After BSM treatment, the growth of Zhonghua 11 was severely inhibited, with a significant decrease in plant height and fresh weight. The mutant showed significantly better growth than Zhonghua 11. Figure 8 ).

[0140] Example 5: Absorption of different rice genotypes by rice

[0141] After removing the rice seeds from the oven, they were first washed with 75% anhydrous ethanol for 3 minutes, then disinfected with 30% sodium hypochlorite solution for 20 minutes, and finally rinsed 5-6 times with clean water. After disinfection, the rice seeds were soaked in a 37℃ incubator to promote germination. Once the seeds showed signs of germination, they were hydroponically cultured using a modified Hoagland rice nutrient solution and then transferred to a plant climate chamber for further cultivation. During cultivation in the plant climate chamber, the hydroponic nutrient solution was changed every 4-5 days. The rice was used for BSM root absorption experiments after 21 days of growth.

[0142] (1) OsWAT1 gene mutant root absorption

[0143] 0.5 mM CaCl2 buffer solution (pH 5.8) was used as the absorption culture medium. Five concentration gradient experiments were set up: 2.5, 5, 12.5, 25, and 50 μM. A control group without the drug was also included. Three seedlings constituted one replicate, for a total of three replicates. Samples were collected after 6 hours of culture. During collection, the parts that had come into contact with the drug solution were repeatedly washed with buffer solution to ensure minimal BSM residue. The samples were blotted dry with filter paper, weighed, and placed in sample bags for later testing. Roots and stems of rice samples were collected, weighed, and recorded. Samples were cut short and placed in 5 mL sample grinding tubes, with one grinding bead in each tube. 2 mL of acetonitrile was added to the root samples for extraction, and 3 mL to the aerial samples. The tubes were then sealed and ground in an automated rapid sample grinder at 70 Hz for 60 seconds. After removal, the samples were vortexed for 3 minutes, then sonicated for 20 minutes, and centrifuged at 4000 rpm for 10 minutes. Take 1 mL of the supernatant after centrifugation and add purification reagents; the root purification reagent formula is: 150 mg anhydrous magnesium sulfate, 25 mg PSA; the aerial part formula is: 150 mg anhydrous magnesium sulfate, 25 mg PSA and 5 mg GCB. Vortex for 2 min, centrifuge at 14000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane for analysis. The BSM content was determined using high performance liquid chromatography-mass spectrometry (HPLC-MS). The absorption results showed that the absorbance of oswat1-1 and oswat1-2 was lower than that of the wild type, and their affinity for BSM was reduced. Figure 9 ).

[0144] Example 6: Verification of tobacco resistance to bensulfuron-methyl

[0145] 1. Determination of tobacco germination rate using petri dish bioassay

[0146] Fifty plump, healthy tobacco seeds of each of the four genotypes were selected and scattered on petri dishes lined with filter paper. 5 mL of 1 / 1000 DMSO aqueous solution, 10 μM BSM solution, and 100 μM BSM solution were added respectively, with three replicates for each treatment. The plants were incubated at 30℃, with 2 mL of BSM aqueous solution added every two days. Germination rate was recorded after six days and statistically analyzed. Results showed that after BSM treatment, the germination rate of Nt wat1-1 was not significantly different from that of the wild type, while the germination rates of Nt wat1-2 and Nt wat1-3 were higher than those of the wild type. Figure 10 ).

[0147] 2. Hydroponic testing to verify tobacco resistance

[0148] Select a number of plump and healthy tobacco seeds, sow them on nutrient soil, cover with plastic wrap, and poke several small holes with a toothpick. Cultivate at 30℃. After 5 days, uncover the plastic wrap and water appropriately daily. After 12 days, select tobacco seedlings with uniform growth and transplant them into hydroponic boxes, with 6 replicates per group. Add 800mL of nutrient solution to each group and cultivate at 26℃, changing the nutrient solution every 3 days. Use 1 / 4 Hoagland hydroponic nutrient solution for tobacco hydroponics. After 15 days, treat the tobacco with BSM, preparing 10μM and 100μM BSM solutions, with 6 replicates for each concentration. Continue cultivation at 26℃, changing the culture medium every 3 days. After 7 days, collect samples and measure root length, plant height, leaf length, leaf width, fresh weight, etc., for statistical analysis. After weighing and recording the samples, the BSM content was detected: After weighing the tobacco samples, the same pretreatment method as the rice samples was used for sample pretreatment, and the samples were detected using high performance liquid chromatography-mass spectrometry.

[0149] The results showed that Ntwat1-1 and Ntwat1-2 were significantly higher than the wild type, while Ntwat1-3 showed no significant difference compared to the wild type. The plant height and fresh weight of all three mutant lines were significantly higher than the wild type. Figure 11 The absorption results showed that the aboveground BSM uptake was significantly lower than that of the wild type, decreasing by 76.00%, 68.16%, and 72.28%, respectively. Figure 12 ).

[0150] 3. Verification of tobacco resistance by bensulfuron-methyl spray treatment

[0151] Mix nutrient soil and vermiculite in a 3:1 ratio. Select tobacco seedlings of uniform growth and transplant them onto the nutrient soil. Cultivate them at 30℃. After 14 days, spray the tobacco seedlings. Prepare a 1mM bensulfuron-methyl stock solution. Take 150μL and add it to 150mL of deionized water to make a 1μM BSM solution. Then add 150μL of surfactant Silwet L-77. Use dimethyl sulfoxide as a control. Take 30mL of the 1μM BSM solution each time and spray it evenly onto the leaves of tobacco seedlings of each genotype. Set up 6 replicates for each group. After completion, continue to cultivate at 30℃. After 12 days, collect samples and measure indicators such as plant height, fresh weight, and chlorophyll content, and perform statistical analysis.

[0152] Chlorophyll content determination: Weigh approximately 2g of leaf tissue, cut it into small pieces, place it in a mortar, add a small amount of quartz sand and calcium carbonate, and 3mL of 95% anhydrous ethanol. Grind into a homogenate, then add 10mL of 95% ethanol and continue grinding until the tissue turns white. Let stand for 3-5 minutes. Take a piece of filter paper, place it in a funnel, moisten it with ethanol, and pour the extract into the funnel along a glass rod. Filter into a 25mL brown volumetric flask. Rinse the mortar, grinding rod, and residue several times with 95% ethanol, and finally pour the rinsing and residue into the funnel. Use a dropper to draw ethanol to wash all the chloroplast pigments on the filter paper into the volumetric flask. Make up to 25mL with ethanol and shake well. Pour the chloroplast pigment extract into a cuvette with a 1cm optical path. Using 95% ethanol as a blank, measure the absorbance at wavelengths of 665nm, 649nm, and 470nm. Calculate the pigment content using the formula:

[0153] C a =13.95A 665 -6.88A 649

[0154] C b =24.96A 649 -7.32A 665

[0155] C k =(1000A) 470 -2.05C a -114.8C b ) / 245,

[0156] Where C is the pigment content (mg / L), V is the extraction volume (mL), N is the dilution factor, and m is the sample mass.

[0157] The spraying results showed that the plant height of all three mutant types was significantly higher than that of the wild type, and the fresh weight was slightly higher, but there was no significant difference. Figure 13 The chlorophyll a content of the mutant lines was significantly higher than that of the wild type, exceeding it by 21.33%, 35.93%, and 72.94%, respectively. Figure 11 )

[0158] The above research results of this invention indicate that the WAT1 gene in plants plays a role in regulating the absorption and accumulation of sulfonylurea herbicides, and can be used to create new plant varieties resistant to sulfonylurea herbicides. By inhibiting the expression of the WAT1 gene in plants, or inhibiting the expression level and / or activity of the WAT1 protein, sulfonylurea-resistant plant varieties can be obtained. The methods for inhibiting the expression of the WAT1 gene in plants, or inhibiting the expression level and / or activity of the WAT1 protein, include, but are not limited to, gene saturation mutation, site-directed gene editing, RNA interference, homologous recombination, or gene knockout.

[0159] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. WAT1 The application of the gene or WAT1 protein in breeding plant varieties resistant to sulfonylurea herbicides is characterized by, By inhibiting the plant WAT1 By expressing the gene, or inhibiting the expression level and / or activity of the WAT1 protein, a plant variety resistant to sulfonylurea herbicides is obtained; the plant is rice or tobacco; the sulfonylurea herbicide is one or more of bensulfuron-methyl, ethoxysulfuron, or ethersulfuron.

2. The application according to claim 1, characterized in that, The aforementioned inhibition of plants WAT1 Gene expression, or inhibition of WAT1 protein expression and / or activity, is achieved through gene saturation mutation, site-directed gene editing, RNA interference, homologous recombination, or gene knockout.

3. The application according to claim 1 or 2, characterized in that, The WAT1 Genes of rice OsWAT1 The gene encodes the OsWAT1 protein, whose amino acid sequence is shown in SEQ ID No.

2.

4. The application according to claim 1 or 2, characterized in that, The WAT1 Genes of tobacco NtWAT1 The gene encodes the NtWAT1 protein, whose amino acid sequence is shown in SEQ ID No.

4.

5. The application according to claim 3, characterized in that, To construct rice OsWAT1 The gene was converted to the CRISPR-Cas9 system and transformed into plants to obtain... OsWAT1 Plants with gene mutations are those that yield plant varieties resistant to sulfonylurea herbicides; the CRISPR-Cas9 system contains recognition... OsWAT1 The target sequence of the gene's sgRNA is shown in SEQ ID No.

5.

6. The application according to claim 4, characterized in that, To construct tobacco NtWAT1 The gene was converted to the CRISPR-Cas9 system and transformed into plants to obtain... NtWAT1 Plants with gene mutations are those that yield plant varieties resistant to sulfonylurea herbicides; the CRISPR-Cas9 system contains recognition... NtWAT1 The target sequence of the gene's sgRNA is shown in SEQ ID No. 6.

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