Application of WAT1 gene in regulating absorption and accumulation of plants to sulfonylurea herbicides

Abstract

The invention discloses application of a WAT1 gene to regulation of absorption and accumulation of plants to sulfonylurea herbicides. It is found that after the rice OsWAT1 gene is knocked out, absorption and accumulation of the rice to bensulfuron methyl can be reduced, the bensulfuron methyl content in the rice is reduced, the bensulfuron methyl resistance is improved, and the rice with the OsWAT1 gene knocked out also has resistance to ethoxysulfuron and cinosulfuron; after the homologous gene NtWAT1 of the flue-cured tobacco K326 is knocked out, an indoor resistance verification experiment finds that the tobacco mutant also obtains bensulfuron methyl resistance, and the bensulfuron methyl resistance is also realized by reducing the absorption and accumulation of the herbicide, so that the WAT1 gene has the effect of regulating the absorption and accumulation of the plant on the sulfonylurea herbicide; the method can be used for creating new plant varieties with sulfonylurea herbicide resistance.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering, and in particular to application of a WAT1 gene in regulating the absorption and accumulation of bensulfuron-methyl by plants. Background Art

[0002] Weeds in direct-seeded rice fields are a key issue that limits mechanized direct-seeding of rice. Sulfonylurea herbicides are highly effective herbicides for rice fields, but improper use can inhibit rice seed germination and seedling growth, and can easily remain in the soil, affecting subsequent crops. Therefore, it is of great significance to cultivate crop varieties resistant to sulfonylurea herbicides, construct simple weed control in rice fields, and promote water-dry rotation.

[0003] Sulfonylurea herbicides are widely used worldwide, mainly for the control of weeds in crops. This type of herbicide has selective systemic and transmissive properties. They are absorbed into the body through the roots and leaves of plants and transmissive through the xylem and phloem. They can inhibit the biosynthesis of branched-chain amino acids in plant cells, such as valine, leucine and isoleucine. At the same time, the accumulation of butanone in plants hinders the synthesis of DNA during cell division, and cells cannot grow normally, ultimately achieving the purpose of weed control. Most sulfonylurea herbicides are easily hydrolyzed in acidic environments. In alkaline soils, this type of herbicide has the slowest degradation rate, equivalent to 1%-20% of the herbicide used, which will remain in the soil for a long time, causing damage to subsequent sensitive crops. The residual effect can last for 2-3 years after application. Most sulfonylurea herbicides can ionize and hydrolyze in water and soil, with a half-life of up to 8 weeks. Sulfonylurea herbicides have low volatility and do not show significant photodegradability. Some of them, such as chlorsulfuron and metsulfuron, can remain in the soil for a long time.

[0004] With the promotion and popularization of direct seeding technology of rice, weeds in rice fields have become one of the main obstacles to high and stable rice yields. Sulfonylurea herbicides are widely used in rice field herbicides and have good control effects on broadleaf weeds and sedges. Bensulfuron-methyl (BSM) is an ultra-high-efficiency ALS inhibitor herbicide, which is usually used as a soil sealer. However, it is not suitable for direct seeding rice fields because it inhibits rice germination, and different crops have different sensitivities to bensulfuron-methyl. Therefore, if crop varieties resistant to sulfonylurea herbicides can be cultivated, the sulfonylurea herbicide damage to rice can be avoided while reducing its residues, further reducing the threat to the next sensitive crops such as tobacco, and providing protection for the water-dry planting system.

[0005] Walls Are Thin 1 gene (WAT1) is a new auxin transporter gene in plants and integrates auxin signals in the secondary wall formation of Arabidopsis fibers. Ranocha et al. found that WAT1 is expressed in all tissues and organs of Arabidopsis, with the highest expression in stems and hypocotyls, and highly expressed in organs with a high proportion of cells with secondary walls. Genes encoding secondary wall-related NAC domain protein SND1 and NAC secondary wall thickening promoting factor NST1 play an important regulatory role in the formation of plant fiber secondary walls. Silencing these two genes using RNAi will lead to a serious reduction in the thickness of fiber secondary walls. In Arabidopsis WAT1 mutants, the expression of SND1 and NST1 and their target genes KNAT7, MYB46 and MYB103 was significantly reduced, indicating that WAT1 may be a regulatory factor of SND1 and NST1 and play an important role in the formation of plant fiber secondary walls. The Arabidopsis WAT1 mutant plants have reduced lignin content, reduced thickness of stem fiber secondary cell walls, dwarfed plants, significantly reduced auxin content in the stem, and obstructed auxin transport. At the same time, studies have also shown that WAT1 is a transporter of auxin local vacuole membranes and plays an important role in plant morphology. Ju et al. found that the expression of the WAT1 gene in the upland cotton variety 'L28' with long internodes and loose plant structure was higher than that in the variety 'XLZ77' with short internodes and compact structure, and the expression of the WAT1 gene in 'L28' increased with the growth of the internodes of the fruiting branches, indicating that the WAT1 gene plays a role in the development of upland cotton internodes. Recent studies have also found that in addition to participating in the regulation of plant growth and development, WAT1 also plays a role in plant disease defense responses. Ye et al. found that the resistance to Verticillium wilt in cotton with GhWATs silenced was significantly increased. Denancé et al. found that Arabidopsis WAT1 mutants have enhanced resistance to bacterial wilt, Verticillium wilt and some vascular pathogens. They also pointed out that WAT1 can affect Arabidopsis disease resistance by regulating salicylic acid (SA) and tryptophan metabolism. SA is a key hormone in plant stress defense response, and tryptophan metabolism can produce precursors for auxin synthesis. This shows that WAT1 plays a pivotal role in plant growth and development and stress defense response by regulating the metabolism of hormones such as auxin and SA. However, there are no reports on the role of the WAT1 gene in the absorption and accumulation of sulfonylurea herbicides in plants. Summary of the invention

[0006] The purpose of the present invention is to overcome the above defects and shortcomings in the prior art and provide an application of the WAT1 gene in regulating the absorption and accumulation of sulfonylurea herbicides by plants.

[0007] The second object of the present invention is to provide the use of WAT1 gene in creating germplasm resources with resistance to sulfonylurea herbicides.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] The present invention finds that knocking out the rice OsWAT1 gene can reduce the absorption and accumulation of BSM in rice, reduce the BSM content in rice, and improve the resistance to BSM. The rice with the OsWAT1 gene knocked out also develops resistance to ethoxysulfuron and ethoxysulfuron. After knocking out the homologous gene NtWAT1 of flue-cured tobacco K326, an indoor resistance verification experiment finds that the tobacco mutant also acquires BSM resistance, and the resistance is also achieved by reducing the absorption and accumulation of herbicides, indicating that the WAT1 gene has the function of regulating the absorption and accumulation of sulfonylurea herbicides by plants.

[0010] Specifically, the present invention screens a rice transport protein yeast enrichment library by BSM, obtains the OsWAT1 gene by sequencing and comparison, and finds that the absorption and transport amounts of tobacco leaf discs expressing the gene are significantly different from those of the empty load through a tobacco transient expression system (tobacco leaf disc absorption, tobacco transport experiment). Afterwards, through a plant root absorption experiment, it was found that rice with the OsWAT1 gene knocked out and tobacco with NtWAT1 knocked out absorbed less BSM relative to their wild-type varieties. The present invention first verifies that OsWAT1 mutant rice has higher resistance to herbicides under culture medium conditions, and then combines herbicide seed coating technology to coat rice seeds with BSM, and finds that after coating, OsWAT1 mutant rice still shows higher herbicide resistance and has a relatively good control effect on broad-leaved weeds. By simulating the closed field weeding application method, it was found that the OsWAT1 rice mutant also has a certain resistance. The tobacco seed germination rate test, hydroponic test and spray test verified that the NtWAT1 tobacco mutant also showed BSM resistance. The research results of the present invention show that the WAT1 gene in plants has the function of regulating the absorption and accumulation of sulfonylurea herbicides by plants, and can be used to create new plant varieties with sulfonylurea herbicide resistance.

[0011] Therefore, the present invention claims the following new applications of WAT1 gene or WAT1 protein in plants:

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

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

[0014] Furthermore, the invention relates to obtaining plant varieties resistant to sulfonylurea herbicides by inhibiting the expression of WAT1 gene in plants, or inhibiting the expression amount and / or activity of WAT1 protein.

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

[0016] Furthermore, the gene knockout is specifically to construct a CRISPR-Cas9 system of the WAT1 gene and transform plants to obtain plants with WAT1 gene mutations, that is, to obtain plant varieties resistant to sulfonylurea herbicides; the CRISPR-Cas9 system contains sgRNA that recognizes the WAT1 gene.

[0017] The present invention also provides the use of a reagent for knocking out the WAT1 gene in inhibiting the absorption and accumulation of sulfonylurea herbicides by plants or in cultivating plant varieties resistant to sulfonylurea herbicides.

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

[0019] Furthermore, a CRISPR-Cas9 system for the rice OsWAT1 gene is constructed and transformed to obtain plants with OsWAT1 gene mutations, that is, plant varieties resistant to sulfonylurea herbicides are obtained; the CRISPR-Cas9 system contains an 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 a nucleotide sequence that has at least 50% homology with the nucleotide sequence shown in SEQ ID NO.3 and encodes the amino acids shown in SEQ ID NO.4; or a nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO.3 under strict conditions and encodes the amino acids shown in SEQ ID NO.4; the amino acid sequence of the NtWAT1 protein is as shown in SEQ ID NO.4.

[0021] Furthermore, a CRISPR-Cas9 system for the tobacco NtWAT1 gene is constructed and the plants are transformed to obtain plants with NtWAT1 gene mutations, that is, plant varieties resistant to sulfonylurea herbicides; the CRISPR-Cas9 system contains an sgRNA that recognizes the NtWAT1 gene, and its target sequence is shown in SEQ ID NO.6.

[0022] Furthermore, the plant is a crop.

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

[0024] Preferably, the sulfonylurea herbicide includes but is not limited to benzylsulfuron-methyl, ethoxysulfuron-methyl or ethoxysulfuron-methyl.

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

[0026] The present invention provides an application of the WAT1 gene in regulating the absorption and accumulation of sulfonylurea herbicides by plants. The present invention finds that knocking out the rice OsWAT1 gene can reduce the absorption and accumulation of BSM by rice, reduce the BSM content in rice, and improve the resistance to BSM. The rice knocking out the OsWAT1 gene also develops resistance to ethoxysulfuron and ethoxysulfuron; and after knocking out the homologous gene NtWAT1 of flue-cured tobacco K326, an indoor resistance verification experiment finds that the tobacco mutant also acquires herbicide resistance, and this is also achieved by reducing the absorption and accumulation of herbicides, indicating that the WAT1 gene in the plant has the function of regulating the absorption and accumulation of sulfonylurea herbicides by the plant, and can be used to create new plant varieties with resistance to sulfonylurea herbicides. The present invention provides a good choice for the study of herbicide-resistant crops by combining the absorption and accumulation of sulfonylurea herbicides with the resistance mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0028] Figure 2 Figure 2 shows the absorption and transport of BSM content in tobacco leaf discs overexpressing OsWAT1. A: The results of the experiment on the absorption of bensulfuron-methyl by tobacco leaf discs, BC: The results of the experiment on the transport of bensulfuron-methyl by tobacco leaf discs.

[0029] Figure 3 This is a sequence alignment result of mutants of Zhonghua11 and OsWAT1 genes.

[0030] Figure 4 This is a sequence alignment result of tobacco wild type and mutant of NtWAT1 gene.

[0031] Figure 5 The results of the germination of rice with different genotypes of OsWAT1 after adding BSM to the culture medium. A: Rice phenotypes after germination of rice seeds with different genotypes for 7 days; B: Root length of rice seeds with different genotypes; C: Length of the aboveground part of rice seeds with different genotypes.

[0032] Figure 6 The results of the growth of rice seedlings of different genotypes of OsWAT1 after adding BSM to the culture medium. A: Photos of the seedlings of rice seedlings of different genotypes; B: Seedling rate of rice seedlings of different genotypes; C: Root length of rice seedlings of different genotypes; D: Length of the aboveground part of rice seedlings of different genotypes.

[0033] Figure 7 The results of BSM coating to verify the resistance and herbicidal activity of OsWAT1 mutants. A: Photos of rice phenotypes in different treatment groups; B: Plant height and fresh weight results of rice in different treatment groups on the 14th, 21st and 28th days of growth; C: Survival of rice field weeds (grass dragon and sedge).

[0034] Figure 8 The results of BSM blocking treatment to verify the resistance of OsWAT1 mutants. A: Photos of rice phenotypes in different treatment groups; BD: Plant height and fresh weight results of rice in different treatment groups on the 21st and 35th days of growth;

[0035] Fig. 9 The results of BSM absorption by roots of rice with different genotypes of OsWAT1. A: The content of roots of rice with different genotypes grown for 21 days after being cultured in buffer solutions with different concentrations of BSM for 6 hours; B: The content of stems of rice with different genotypes grown for 21 days after being cultured in buffer solutions with different concentrations of BSM for 6 hours; C: The content of whole plants of rice with different genotypes grown for 21 days after being cultured in buffer solutions with different concentrations of BSM for 6 hours; D: The absorption dynamic curve of BSM by rice with different genotypes.

[0036] Fig.10 The results of the culture dish bioassay to verify the resistance of tobacco mutants. A: Photo of 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] Fig.11 The results of adding BSM to hydroponics to verify the resistance of tobacco mutants. A: Photos 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] Fig.12 The results of BSM absorption in hydroponic culture of different tobacco genotypes. A: benzsulfuron-methyl content in roots; B: benzsulfuron-methyl content in stems.

[0039] Fig.13 The results of spraying BSM on tobacco leaves to verify mutant resistance. A: Photos 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] Fig.14 This is the result of germination of OsWAT1 rice of different genotypes after adding ethsulfuron to the culture medium.

[0041] Fig.15 The results of the growth of rice seedlings of different genotypes of OsWAT1 after adding ethsulfuron to the culture medium. A: Germination phenotype of rice seeds of different genotypes; B: Root length of rice seeds of different genotypes; C: Stem length of rice seeds of different genotypes.

[0042] Fig.16 The results of the germination of rice seeds of different genotypes of OsWAT1 after the addition of 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] Fig.17 The results of the growth of rice seedlings of different genotypes of OsWAT1 after the addition of ethoxysulfuron to the culture medium. A: Photos of the seedlings of rice seedlings of different genotypes; B: Seedling rate of rice seedlings of different genotypes; C: Root length of rice seedlings of different genotypes; D: Stem length of rice seedlings of different genotypes. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

[0046] In the examples, Escherichia coli DH5α and Agrobacterium tumefaciens EHA105 are commonly used strains and can be purchased commercially; 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 the examples were synthesized by Shenzhen BGI, and sequencing was performed at Shenzhen BGI.

[0047] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0048] Example 1 Screening of benzylsulfuron-methyl transport-related genes using a rice transporter yeast enrichment library

[0049] 1. Screening of yeast library: A yeast library containing 1452 rice transporters enriched for expression was used to screen benzylsulfuron-sensitive yeast expression strains. The yeast strains in the 96 deep-well plates stored at -80°C were copied to SD-gal solid medium using a 96-pin replicator. After culturing at 30°C for 2-3 days, they were copied again to 96 deep-well plates containing SD-gal liquid medium for continued culturing overnight. 20 μL of overnight cultured yeast was added to a new 1 mL SD-glu liquid medium. After culturing on a shaker at 30°C and 180 rpm for 1 hour, the OD value at 595 nm was measured using an ELISA reader, and the OD value of the bacterial solution in each well was adjusted to 0.01 with sterile water. The liquid medium was set as a blank control. The adjusted OD yeast was copied to a solid medium containing 20 g / L galactose and 2.5 μM and 5 μM. After the bacterial solution was dried, it was sealed with tape and placed in a 30°C incubator for 2-3 days. The results were observed and photographed every day.

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

[0051] 3. Yeast growth inhibition curve: The yeast strains screened above were cultured overnight in SD-glu liquid medium at 30°C and 200rpm in a shaker. The OD was measured with a spectrophotometer. A certain amount of liquid medium containing BSM at different concentrations was prepared, and 100 μL was taken into a 96-well plate. Each concentration was repeated 3 times. A certain amount of yeast liquid was added to 100 μL of the medium so that its initial OD value was 0.01. Then, the OD value was measured with an automatic growth curve analyzer every 1 hour. The control was taken without drug addition, and the average value was calculated 3 times. The results showed that after overexpression of OsWAT1, the corresponding yeast strains grew more slowly in the medium containing BSM and were more sensitive to BSM ( Figure 1 B).

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

[0053]

[0054] Example 2 Verification of the function of rice benzylsulfuron-methyl transporter using tobacco transient expression system

[0055] 1. Construction of pEAQ recombinant vector

[0056] The gene OsWAT1 obtained from the yeast strain was used as a template to perform PCR amplification of the OsWAT1 gene. The plasmid amplification concentration was 5ng / μL, and the following reaction mixture was used: 10μL 5× Prime star Max DNA polymerase; 0.8μL of each primer at 10μM; 1μL DNA (5ng); H2O was added to 20μL. The specific reaction procedure was as follows: enzyme thermal activation at 98℃, 1min, 1 cycle; denaturation at 98℃, 30s, annealing at 60℃, 30s, extension at 72℃, 1min30s, a total of 32 cycles.

[0057] The sequences of primers for amplifying OsWAT1 are as follows:

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

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

[0060] Agarose gel (1.0%) electrophoresis was used to identify the PCR products. Gel Extraction Kit (OMEGA) was used for gel recovery and purification.

[0061] The enzyme digestion reaction system was established: 6 μL SaI I (Takara), 8 μL 10xH buffer (Takara); 3 ug complete pEAQ plasmid; deionized H2O to a final volume of 100 μL. The enzyme digestion was carried out at 37°C for 3 h, and the resulting product was purified using a gel recovery kit (OMEGA).

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

[0063]

[0064] The correct clones were screened by colony PCR and named pEAQ-OsWAT1. The positive transformants were selected for plasmid sequencing. The correct plasmids were transferred into Agrobacterium and the bacterial solution was saved for subsequent experiments.

[0065] 2. Protein Expression by Tobacco Injection

[0066] Activate the stored Agrobacterium culture on a plate of LB solid medium (50mg / L kan+50mg / L Rif), inoculate in 1mL LB (50mg / L Kan+50mg / L Rif) liquid medium, and culture at 180rpm, 28℃ for 24h. Take 50μL of the culture solution and add it to 50mL LB (50mg / L Kan+50mg / L Rif) liquid medium, and culture at 180rpm, 28℃ for 15h. Dispense into 10mL centrifuge tubes, centrifuge at 4000rpm for 5min, discard the supernatant, add 7mL of sterile water to wash the cells, centrifuge at 4000rpm for 5min, discard the supernatant, add 7mL of resuspension solution to resuspend the cells, let stand in the dark for 3h, and adjust the OD value of the culture solution. 600 Adjust to 0.3 for injection inoculation. Select 2-3 larger leaves in the middle of the tobacco plant for injection, and inject the pEAQ vector and gene Agrobacterium solution on both sides of the tobacco leaves respectively. Use a syringe to push the bacterial injection solution into the tobacco leaves until the bacterial solution cannot spread. After the tobacco injection is completed, use a marker to mark the area where the bacterial solution has spread, wipe the Agrobacterium on the surface of the tobacco clean to prevent the surface bacterial solution from wilting the leaves, and culture it in the dark for 1 day, and then culture it under normal conditions for 1 day.

[0067] 3. Experiment on absorption of bensulfuron-methyl by tobacco leaf discs

[0068] Two days after the transient expression of pEAQ-OsWAT1 in tobacco, a small part of the tobacco leaves injected with pEAQ-GFP was observed for luminescence. The tobacco leaves with normal and uniform luminescence were selected, and the tobacco leaf discs were punched with a 1.4 cm diameter puncher. After pre-culture in the leaf disc absorption culture medium (containing BSM) at pH 5.8 for 30 minutes, 6 leaf discs were used as 1 replicate, and 3 replicates were performed for each concentration. The drug absorption time was 2 hours, and the tobacco leaves were washed 3 times with 0.5mM CaCl2 to ensure that the drugs attached to the tobacco surface were completely removed. After the absorption was completed, the tobacco leaves were ground in liquid nitrogen, and 5mL of acetonitrile was added to collect the tissue homogenate in a 10mL centrifuge tube. After vortexing for 3 minutes, ultrasonication for 30 minutes, vortexing for another 2 minutes, and extraction by centrifugation at 4000rpm for 5 minutes. Take 1mL of the supernatant, remove impurities with a 0.22μm organic filter membrane, and the filtrate is subjected to LCMS-MS determination. The results are as follows Figure 2As shown in A, the BSM content absorbed by tobacco leaf discs expressing pEAQ-OsWAT1 was higher than that of tobacco leaf discs expressing pEAQ empty vector.

[0069] 4. Experiment on the transport of bensulfuron-methyl in tobacco

[0070] Two days after the tobacco was transiently expressed with pEAQ-OsWAT1, 100 μL of 200 μM BSM was evenly applied with a pipette to the leaf area on the same leaf that was not injected with the pEAQ vector and gene. After 24 hours, the luminescence of the tobacco part injected with pEAQ-GFP was observed, and the leaves with normal and uniform luminescence were selected. Two tobacco leaf discs were punched with a 1.4 cm diameter puncher at the site where the pEAQ vector and gene were injected for each tobacco leaf. The tobacco leaves were washed three times with 0.5 mM CaCl2 to ensure that the BSM attached to the tobacco surface was completely removed. The tobacco leaves were ground with a grinder, vortexed for 5 minutes, ultrasonicated for 30 minutes, and centrifuged at 14000 rpm for 5 minutes. 1 mL of the supernatant was taken and filtered through a 0.22 μm microporous membrane for LCMS-MS determination.

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

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

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

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

[0075] (2) Construction of pCRISPR / Cas9 recombinant vector containing the above target sequence fragment

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

[0077] The designed target sequence is added with the specific sticky end adapter of the pCRISPR / Cas9 system, and the complete adapter primer is synthesized.

[0078] rice-OsWAT1-F2: 5′-TGTGTGCGCCGAGCAGGCCGTGAGC-3′;

[0079] rice-OsWAT1-R2: 5′-AAACCGTATCCGGCGAGGATGCACA-3′;

[0080] 2) Anneal the adapter primer with sticky ends to form a double-stranded small fragment with sticky ends

[0081] Dilute the F2 primer and R2 primer into a solution with a concentration of 10 μM, take 10 μL of each and mix well, and perform annealing reaction in a PCR instrument from 98°C to 22°C to allow the F2 primer and R2 primer to complement each other to form small double-stranded fragments with sticky ends.

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

[0083] The original vector pOs-sgRNA containing sg-RNA was digested with restriction endonuclease BsaⅠ to produce sticky ends that can complement the sticky ends of the target sequence. The system for digesting the original pOs-sgRNA vector with BsaⅠ is: 10×buffer 2μL, BsaⅠ enzyme 1μL, pOs-sgRNA vector 4μg, ddH2O supplemented to 20μL, and digested at 37℃ for 12h. After the size of the digested product was checked by 1% agarose gel electrophoresis, the kit (OMEGACat#D2500-02) was used to recover and purify the digested product through a column to obtain the digested pOs-sgRNA vector, add sterilized ddH2O to dissolve, and determine the concentration before use.

[0084] 4) Connect the small double-stranded fragment with sticky ends to the pOs-sgRNA vector that has been digested to form a recombinant vector containing the target sequence and sg-RNA

[0085] Use T4 ligase to connect the double-stranded small fragment in step 2) and the pOs-sgRNA vector digested in step 3) to form a complete recombinant vector containing the target sequence and sg-RNA for OsWAT1 protein. The 15μL connection system is: 10×T4ligation buffer 1.5μL, double-stranded small fragment 4μL, digested pOs-sgRNA vector 3μL, T4 DNAligase 1μL, ddH2O supplemented to 15μL, 16℃ connection for 12 hours. The ligation product is transformed into Escherichia coli DH5α, cultured overnight on a kanamycin-resistant LB plate (containing 10mg / L kanamycin), and the positive strain is selected for sequencing to obtain a recombinant vector containing the target sequence and sg-RNA that is correctly sequenced.

[0086] 5) Use LR mix to perform LR reaction recombination on the recombinant vector containing the target sequence and sg-RNA and the vector pH-Ubicas9-7 containing Cas9 to form a complete recombinant vector containing the target sequence-sg-RNA+Cas9

[0087] 6) Use LR mix (Shanghai Beino Biotechnology Co., Ltd.) to perform LR reaction recombination on the recombinant vector obtained in step 4) and the vector pH-Ubi-cas9-7 containing Cas9 (provided by BioGene Technology Co., Ltd.). LR reaction system: 25-50ng of recombinant vector containing target sequence and sg-RNA, 75ng of pH-Ubi-cas9-7 vector, 1μL of 5×LR Clonase TM buffer, 4.5μL of TE Buffer (pH8.0), and 0.5μL of LR ClonaseTM. The system was incubated at 25°C for 2h, and 2μL of 2μg / μL Proteinase K was added after the reaction. The reaction was treated at 37°C for 10min, and then 2μL of the reaction product was transferred into Escherichia coli DH5α, and the gentamicin-resistant LB plate was cultured at 37°C overnight. The positive strains were selected for sequencing to obtain the complete pCRISPR / Cas9-OsWAT1 recombinant expression vector containing the OsWAT1 protein target sequence-sg-RNA+Cas9 that was sequenced correctly.

[0088] (3) Introducing the obtained complete recombinant vector containing the OsWAT1 protein target sequence-sg-RNA+Cas9 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 the recombinant bacterium AGL1 / pCRISPR / Cas9-OsWAT1.

[0090] 2) The recombinant bacteria AGL1 / pCRISPR / Cas9-OsWAT1 was transformed into Zhonghua 11 rice callus tissue using Agrobacterium-mediated method as follows:

[0091] 3) Pick a single colony of AGL1 / pCRISPR / Cas9-OsWAT1 and inoculate it into 10 mL of Agrobacterium culture medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin), and culture it at 28°C and 180 rpm for 2-3 days. Take 4 mL of bacterial solution, centrifuge it at 4000 rpm for 3 min, pour off the supernatant, add a small amount of AAM culture medium to resuspend the cells, then add 20 mL of AAM culture medium (containing 0.1 mM acetosyringone As), and culture it at 28°C and 150 rpm in a dark incubator for 1-2 h until OD 600= about 0.4. Select rice callus tissue of Zhonghua 11 (hereinafter also referred to as wild-type rice) with good growth status and granular shape, immerse in Agrobacterium culture solution (YEP without agar), shake at 28°C and 150-200rpm for 20min, pour out the callus tissue, absorb the excess bacterial solution with sterile filter paper, spread the callus tissue on a sterile plate containing multiple layers of filter paper, blow dry on a clean bench (the callus is dispersed and not agglomerated), and then transfer the callus tissue to the co-cultivation medium and culture it in the dark for 2-3 days. The callus is transferred to NB basic medium containing 100mg / L hygromycin and 400mg / L cephalosporin for screening for 3-4 weeks (first screening). The surviving callus tissue is transferred to the second screening medium (NB basic medium containing 100mg / L hygromycin and 200mg / L cephalosporin) for screening for 3 weeks. The resistant callus tissue was transferred to a differentiation medium (containing 100 mg / L hygromycin) for differentiation. After the regenerated plants took root on the seedling medium containing 100 mg / L hygromycin (about 3-4 weeks), they were transferred to a greenhouse. Transgenic plants with completely inactivated OsWAT1 protein were obtained in the T0 generation plants.

[0092] The culture medium used in the above transformation is 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 macroelements + AA trace 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 medium (Beijing Huayueyang Biotechnology Co., Ltd.): N6 macroelements + B5 trace elements + B5 organic components + iron salts + hydrolyzed casein (300 mg / L) + proline (500 mg / L) + sucrose (30 g / L) + agar (8 g / L), pH 5.8.

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

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

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

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

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

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

[0102] Obtaining mutant plants using transgenic positive plants

[0103] 1) Identification of mutation sites

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

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

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

[0107] 2) The mutant plants were propagated, and the seeds of individual plants without hygromycin, Cas9 and other transgenic elements were detected in the T1 generation transgenic segregation population to obtain loss-of-function mutants, which were named oswat1-1 and oswat1-2. The mutation analysis results of the loss-of-function mutants and wild-type plants are shown in Figure 3 As shown, oswat1-1 has a 2 bp deletion mutation, and oswat1-2 has a 1 bp insertion mutation.

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

[0109] Construction of mutant plants of the tobacco homologous gene NtWAT1 (the nucleotide sequence of which is shown in SEQ ID NO.3 and the amino acid sequence of which is shown in SEQ ID NO.4) of rice OsWAT1 gene

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

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

[0112] (2) Construction of pCRISPR / Cas9 recombinant vector containing the above target sequence fragment

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

[0114] The designed target sequence is added with the specific sticky end adapter of the pCRISPR / Cas9 system, and the complete adapter primer is synthesized.

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

[0116] Nicotiana tabacum-NtWAT1-R5: 5′-AAACCGTATCCGGCGAGGATGCACA-3′;

[0117] The other experimental steps refer to the above OsWAT1 rice mutant plant operation. The three types of functional mutants finally obtained were named Ntwat1-1, Ntwat1-2 and Ntwat1-3. The mutant analysis results are shown in 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 Test on resistance of rice to sulfonylurea herbicides

[0119] In order to detect the resistance of the loss-of-function mutant rice obtained in Example 3 to BSM, culture medium sensitivity experiments, indoor coated pot experiments and indoor closed treatment experiments were carried out to verify the resistance.

[0120] 1. Determination of germination rate of rice seeds of different genotypes by adding benzylsulfuron-methyl to the culture medium

[0121] Germination rate experiment of OsWAT1 gene mutant

[0122] The seeds of Zhonghua 11 and OsWAT1 mutant rice were disinfected on the workbench with 75% anhydrous ethanol and 30% sodium hypochlorite solution (1-2 drops of Tween can be added to the solution) prepared with sterile water. The seeds were first washed with 75% anhydrous ethanol for 3 minutes, a total of 3 times; then washed with 30% sodium hypochlorite solution for 3 minutes, poured out and continued to be washed with 30% sodium hypochlorite for 15-20 minutes, during which the seeds can be placed in a 150rpm shaker; finally, the seeds were washed with sterile water, and then the cleaned seeds were placed on sterilized filter paper to dry, and sown in MS medium containing 0, 1.0 and 1.5 μM BSM, respectively, and cultured in an artificial climate incubator at a constant temperature of 28°C for 7 days. Three replicates were set for each treatment, and 30 seeds were tested in each replicate. The germination of the seeds was recorded every day from 1 to 5 days after sowing, and the rice phenotype was photographed and counted after 7 days. The germination rates 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 inhibition rates of the two concentrations of BSM on the mutants OsWAT1-1 and OsWAT1-2 were the lowest (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 resistance of mutants OsWAT1-1 and OsWAT1-2 to ethoxysulfuron (Table 2, Fig.14 ) and ethoxysulfuron (Table 3, Fig.16 ) resistance, and the results showed that the rice seed germination inhibition rate of each strain was: WT>OsWAT1-1>OsWAT1-2.

[0126] Table 2 Inhibition rate of sulfuron-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. Adding sulfonylurea herbicides to the culture medium to verify herbicide resistance in rice seedlings

[0132] OsWAT1 mutant seedling resistance

[0133] The rice seed disinfection method was similar to the above method. The dried seeds were sown in MS medium with 0, 0.5, 1.5 and 2.5 μM BSM and cultured in an artificial climate incubator for 14 days. After 14 days, the root length, stem length and number of rice seedlings were measured and photographed. Compared with the wild type, oswat1-1 and oswat1-2 have higher BSM tolerance, and their root length, aboveground length and seedling rate are higher than those of the wild type and the overexpression strain ( Figure 6 ). After 14 days of continuous cultivation in the medium containing ethoxysulfuron and ethoxysulfuron using the same planting method, the root length and aboveground length of the mutants OsWAT1-1 and OsWAT1-2 were longer than those of the wild-type strain ( Fig.15 , Fig.17 ).

[0134] 3. Bensulfuron-methyl seed coating to verify rice resistance and herbicidal activity

[0135] (1) OsWAT1 gene mutant coating

[0136] The seed pelleting powder (2:1, w / w) was prepared with kaolin and attapulgite, 5g / L benzsulfuron-methyl suspension prepared with benzsulfuron-methyl technical, and 1.5% (w / w) polyvinyl alcohol was used as a binder. The seeds, the powder, suspension and binder were put into a coating machine to prepare pelleted seeds with a drug-seed ratio of 1:2500. Clear water coating was used as a control.

[0137] The experiment was conducted in a plant climate chamber. Field soil was used for this experiment. The collected field soil was dried for 3-4 days before use, and then added with an appropriate amount of clean water to mix into mud and put into a bottom-proof plastic basin to smooth it for use. Zhonghua 11, oswat1-1 and oswat1-2 rice seeds with full grains were selected for BSM seed coating. The seed coating drug-seed ratio was 1:2500, and clean water coating was used as a control. The experiment was carried out in large and small plastic basins, with 3 replicates for each treatment group. When sowing, it was divided into 3 areas from left to right. Zhonghua 11, oswat1-1 and oswat1-2 rice were planted in turn. Each strain of rice was planted in 8 holes, with 2 seeds in each hole. In order to verify whether BSM seeds have herbicidal activity against broad-leaved weeds after seed coating, 2 common rice field weeds (i.e., grass dragon and sedge) were selected for testing, and an equal amount of grass seeds were sown in each plastic basin. Keep the soil moist after sowing. After 7 days, add clean water to the pot to keep the water layer 2 cm above the soil surface. Apply rice fertilizer every 7 days. The rice phenotype was photographed and counted on the 14th, 21st and 28th days of growth, and the survival of weeds was counted on the last day. The growth of seedlings was inhibited to varying degrees after BSM coating, among which the wild type Zhonghua 11 was the most severely inhibited, with its plant height shortened and fresh weight decreased. The plant height and fresh weight of the oswat1 mutant strain were significantly higher than those of Zhonghua 11 ( Figure 7 )

[0138] 4. Bensulfuron-methyl sealed treatment to verify rice resistance

[0139] The experiment was conducted in a plant climate chamber, with a blank control group and a 25 μM BSM treatment group, with 3 replicates for each treatment. In order to ensure that the soil conditions were as close to the field as possible, field soil was used for this experiment. The collected field soil was dried for 3-4 days before use, and then added with an appropriate amount of clean water to mix into mud and put into a bottom-proof plastic pot for smoothing. After breaking the dormancy of Zhonghua 11, oswat1-1 and oswat1-2 rice seeds, they were placed at 37 ° C for germination for 1 day, and rice seedlings with the same germination trend were selected and sown in pots. When sowing, they were divided into 3 areas from left to right, and Zhonghua 11, oswat1-1 and oswat1-2 rice were planted in turn. Each rice strain was planted in 8 holes, with 2 seeds in each hole; after sowing, the soil in the pot was kept moist but the water layer did not exceed the soil surface, and the pot was sealed 1 day after sowing. Clean water and 25μM BSM solution contain 0.1% Tween 80. The mixed solution is evenly sprayed in the pot. The spraying amount for each pot is 25mL. After application, the soil is kept moist but the water layer does not exceed the soil surface. After 7 days, water is added to the pot to keep the water layer 2cm above the soil surface. After that, rice topdressing is applied once every 7 days. The rice phenotypes were photographed and counted on the 21st and 35th days of growth. After BSM sealing treatment, the growth of Zhonghua 11 was severely inhibited, and its plant height and fresh weight decreased significantly. The growth of the mutant was significantly better than that of Zhonghua 11 ( Figure 8 ).

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

[0141] After taking the rice seeds out of 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 washed with clean water for 5-6 times. After the disinfection, the rice seeds were soaked in a 37°C incubator for germination. After the seeds turned white, they were hydroponically cultivated with the modified Hogland rice nutrient solution and transferred to the plant climate chamber for further cultivation. During the cultivation in the plant climate chamber, the rice hydroponic nutrient solution was replaced every 4-5 days. When the rice grew to 21 days, it was used for the BSM root absorption experiment.

[0142] (1) Root absorption of OsWAT1 mutants

[0143] 0.5mM CaCl2 buffer solution (pH 5.8) was used as the absorption culture medium. Five concentrations were set in the concentration gradient experiment: 2.5, 5, 12.5, 25, and 50μM. A group of drug-free controls was also set. Every 3 seedlings were repeated for a total of 3 replicates. Samples were collected after 6 hours of culture. When collecting samples, the parts that were in contact with the drug solution needed to be repeatedly washed with buffer to ensure that the BSM residue was minimal. After the sample was dried with filter paper, it was weighed and put into a sample bag for storage. The roots and stems of the rice samples were collected, weighed, and recorded. The samples were cut short and placed in a 5mL sample grinding tube. One grinding bead was placed in each tube. 2mL of acetonitrile was added to the root sample for extraction, and 3mL was added to the aboveground part; the lid was tightened and placed in an automatic sample rapid grinder for grinding at 70HZ for 60s; after taking out, vortexed for 3min, then ultrasonicated for 20min, and centrifuged at 4000rpm for 10min. Take 1 mL of the supernatant after centrifugation and add purification reagent; the root purification reagent formula is: 150 mg anhydrous magnesium sulfate, 25 mg PSA; the aerial part: 150 mg anhydrous magnesium sulfate, 25 mg PSA and 5 mg GCB. Vortex for 2 minutes, centrifuge at 14000 rpm for 10 minutes, take the supernatant and filter it through a 0.22 μm filter for testing. The BSM content was detected by high performance liquid chromatography-mass spectrometry. The absorption results showed that the absorption amount of oswat1-1 and oswat1-2 was at a lower level than that of the wild type, and the affinity for BSM was reduced ( Fig. 9 ).

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

[0145] 1. Determination of tobacco germination rate by culture dish method

[0146] Fifty plump and healthy tobacco seeds of each of the four genotypes were selected and spread on a culture dish covered with filter paper. 5 mL of 1 / 1000 DMSO aqueous solution, 10 μM and 100 μM BSM solution were added, respectively, with 3 replicates for each treatment. Cultured at 30°C, 2 mL of BSM aqueous solution was added every 2 days, and the germination rate was counted after six days for statistical analysis. The 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 ( Fig.10 ).

[0147] 2. Hydroponics to verify tobacco resistance

[0148] Select a number of full and healthy tobacco seeds, sow them on the nutrient soil, cover them with plastic wrap, pierce a number of small holes with a toothpick, and cultivate them in an environment of 30°C. After 5 days, lift the plastic wrap and water them appropriately every day. After 12 days, select tobacco seedlings with consistent growth and transplant them into hydroponic boxes. Set up 6 replicates for each group, add 800mL of nutrient solution to each group, cultivate them at 26°C, and replace the nutrient solution every 3 days. The hydroponic nutrient solution for tobacco uses 1 / 4 Hogland hydroponic nutrient solution. After 15 days, apply BSM treatment to tobacco, prepare 10μM and 100μM BSM solutions, and set 6 replicates for each concentration. Continue to cultivate at 26°C, and replace the culture solution every 3 days. After 7 days, collect samples, measure root length, plant height, leaf length, leaf width, fresh weight and other indicators, and do statistical analysis. After the sample is weighed and recorded, the BSM content is tested: After the tobacco sample is sampled and weighed, the sample is pre-treated in the same way as the above-mentioned rice sample, and tested using a high performance liquid chromatography-mass spectrometer.

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

[0150] 3. Verification of tobacco resistance by spraying with benzylsulfuron

[0151] Mix the nutrient soil and vermiculite in a ratio of 3:1, select tobacco seedlings with consistent growth, transplant them on the nutrient soil, and cultivate them in an environment of 30℃. After 14 days, spray the tobacco seedlings. Prepare 1mM benzylsulfuron mother 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 1μM BSM solution into the spray pot each time, and spray it evenly on the leaves of tobacco seedlings of each genotype, with 6 replicates per group. After completion, continue to cultivate at 30℃. After 12 days, collect samples, measure indicators such as plant height, fresh weight, and chlorophyll content, and perform statistical analysis.

[0152] Determination of chlorophyll content: Weigh about 2g of leaves, cut them into pieces, put them in a mortar, add a little quartz sand and calcium carbonate and 3mL 95% anhydrous ethanol, grind into a homogenate, then add 10mL 95% ethanol, and continue grinding until the tissue turns white; let it stand for 3-5min, take a piece of filter paper, put it in a funnel, moisten it with ethanol, pour the extract into the funnel along the glass rod, filter it into a 25mL brown volumetric flask, rinse the mortar, pestle and residue with 95% ethanol several times, and finally pour it into the funnel together with the residue. Use a dropper to absorb ethanol, wash all the chloroplast pigments on the filter paper into the volumetric flask, dilute to 25mL with ethanol, and shake well. Pour the chloroplast pigment extract into a colorimetric cup with a light path of 1cm, use 95% ethanol as a blank, and measure the absorbance at wavelengths of 665nm, 649nm and 470nm. To calculate the pigment content, the formula is:

[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 volume of the extract (mL), N is the dilution factor, and m is the sample mass.

[0157] The spray results showed that the plant heights of the three mutant types were significantly higher than the wild type, and the fresh weights were slightly higher than the wild type tobacco, but there was no significant difference ( Fig.13 ). The chlorophyll a content of the mutant strains was significantly higher than that of the wild type, which was 21.33%, 35.93% and 72.94% higher than that of the wild type, respectively ( Fig.11 )

[0158] The above research results of the present invention show that the WAT1 gene in plants has the effect of regulating the absorption and accumulation of sulfonylurea herbicides by plants, and can be used to create new plant varieties with resistance to sulfonylurea herbicides. By inhibiting the expression of the WAT1 gene in plants, or inhibiting the expression and / or activity of the WAT1 protein, a plant variety resistant to sulfonylurea herbicides is obtained. The method of inhibiting the expression of the WAT1 gene in plants, or inhibiting the expression and / or activity of the WAT1 protein includes but is not limited to gene saturation mutation, gene site editing, RNA interference, homologous recombination or gene knockout, etc.

[0159] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Application of WAT1 gene or WAT1 protein in regulating the absorption and accumulation of sulfonylurea herbicides in plants.

2. Application of WAT1 gene or WAT1 protein in breeding plant varieties resistant to sulfonylurea herbicides.

3. The application according to claim 2, characterized in that: By inhibiting the expression of WAT1 gene in plants, or inhibiting the expression amount and / or activity of WAT1 protein, a plant variety resistant to sulfonylurea herbicides is obtained.

4. The use according to claim 3, characterized in that: The inhibition of WAT1 gene expression in plants, or inhibition of WAT1 protein expression and / or activity is through gene saturation mutation, gene site editing, RNA interference, homologous recombination or gene knockout.

5. Use of an agent for knocking out the WAT1 gene in inhibiting the absorption and accumulation of sulfonylurea herbicides by plants or in cultivating plant varieties resistant to sulfonylurea herbicides.

6. The use according to any one of claims 1 to 5, characterized in that: The WAT1 gene is a rice OsWAT1 gene, whose nucleotide sequence is as shown in SEQ ID No.1; or a nucleotide sequence having at least 50% homology with the nucleotide sequence shown in SEQ ID No.1 and encoding the amino acids shown in SEQ ID No.2; or a nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID No.2 under strict conditions and encodes the amino acids shown in SEQ ID No.2; the amino acid sequence of the OsWAT1 protein is as shown in SEQ ID No.

2.

7. The use according to any one of claims 1 to 5, characterized in that: The WAT1 gene is the tobacco NtWAT1 gene, and its nucleotide sequence is as shown in SEQ ID No.3; or a nucleotide sequence that has at least 50% homology with the nucleotide sequence shown in SEQ ID No.3 and encodes the amino acids shown in SEQ ID No.4; or a nucleotide sequence that hybridizes with the nucleotides shown in SEQ ID No.3 under strict conditions and encodes the amino acids shown in SEQ ID No.4; the NtWAT1 protein amino acid sequence is as shown in SEQ ID No.

4.

8. The use according to claim 6, characterized in that: The invention relates to obtaining plants with OsWAT1 gene mutations by constructing a CRISPR-Cas9 system for the rice OsWAT1 gene and transforming plants, that is, obtaining plant varieties resistant to sulfonylurea herbicides; the CRISPR-Cas9 system contains an sgRNA that recognizes the OsWAT1 gene, and its target sequence is shown in SEQ ID No.

5.

9. The use according to claim 6, characterized in that: The invention relates to obtaining plants with NtWAT1 gene mutations by constructing a CRISPR-Cas9 system for tobacco NtWAT1 gene and transforming plants, that is, obtaining plant varieties resistant to sulfonylurea herbicides; the CRISPR-Cas9 system contains sgRNA that recognizes NtWAT1 gene, and its target sequence is shown in SEQ ID No.

6.

10. The use according to any one of claims 1 to 5, characterized in that: The sulfonylurea herbicide is one or more of benzylsulfuron-methyl, ethoxysulfuron-methyl or ethoxysulfuron-methyl.

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