Ec cyp89b16 gene, polypeptide, vector and application thereof in plant resistance to herbicides and selection of herbicide-resistant transgenic plants

Abstract

This invention discloses the EcCYP89B16 gene, polypeptide, vector, and their application in the breeding of herbicide-resistant plants and herbicide-resistant transgenic plants. The nucleotide sequence of this gene is SEQ ID NO:1 or a sequence having at least 80% identity with SEQ ID NO:1. This invention also discloses the protein polypeptide encoded by the above gene, whose amino acid sequence is SEQ ID NO:2 or a sequence having at least 80% identity with SEQ ID NO:2. This invention relates to the herbicide-resistant gene and its encoded protein, vector, and its application in the breeding of herbicide-resistant transgenic plants. This P450 gene can be expressed in plants to confer resistance to aryloxyphenoxypropionic acid herbicides, thereby enabling selective weed control using herbicides.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to herbicide-resistant genes and their encoded proteins and vectors, as well as the application of these genes and vectors in the breeding of herbicide-resistant transgenic plants. Background Technology

[0002] With the widespread application of direct seeding technology in agricultural production, weed problems in farmland have become a significant factor affecting crop yields. In particular, the physiological and metabolic processes of gramineous weeds in paddy fields are highly similar to those of rice, making appropriate herbicides difficult to control. Since traditional breeding and domestication processes do not involve herbicide tolerance traits, resistant resources in natural genetic resources are extremely rare. Utilizing herbicide-inhibiting target genes and screening for and identifying endogenous resistance mutants is currently a feasible approach to developing herbicide-resistant germplasm resources for staple crops. For example, resistance resources can be obtained by EMS-induced mutagenesis of the imidazole herbicide target gene ALS. In recent years, this mutation has been widely used in varieties such as "Jinjing 818," demonstrating significant cultivation advantages in chemical weed control. However, the endogenous resistance sites of known major herbicide target genes such as EPSPS, ACCase, ALS, and HPPD are mostly protected by patents held by foreign commercial companies. Simultaneously, with repeated application of single-type herbicides, the acquired resistance of weeds in the field increases rapidly. Therefore, discovering novel herbicide resistance genes in plants and obtaining herbicide resistance traits by expressing heterologous metabolic genes through transgenic methods is essential for cultivating herbicide-resistant plants.

[0003] Aryloxyphenoxypropionate (APP) herbicides are one of the important types of herbicides for controlling grassy weeds. They were gradually developed in the 1960s by Hoechst Corporation, who replaced the phenyl group in the 2,4-D structure with a diphenyl ether and further researched and developed the compound quizalofop-P-ethyl. In 1971, the structural model of this class of herbicides was established, with the parent ring consisting of A and B. Starting in 1975, many pesticide companies modified and altered the original structural model, changing the A benzene ring to a heterocyclic or fused ring, and introducing active F atoms into the ring, resulting in a series of herbicides with higher activity and better selectivity.

[0004] APP herbicides primarily inhibit fatty acid synthesis by suppressing the activity of acetyl-CoA carboxylase (ACCase, EC6.4.1.2) in gramineous plants. Fatty acids are the main energy storage and supply substances in plants, a key component of cell membranes, and also play an important role in plant metabolism and biosynthesis.

[0005] Taking cyhalofop-butyl as an example, it is the only APP herbicide with high safety for rice. Like other herbicides in this class, it is a systemic herbicide. It is absorbed by the leaves and leaf sheaths, translocated through the phloem, and accumulates in the meristematic tissue of the plant. It inhibits acetyl-CoA carboxylase (ACCase), stopping fatty acid synthesis, disrupting normal cell growth and division, damaging lipid structures such as membranes, and ultimately leading to plant death. Cyhalofop-butyl is mainly used to control barnyard grass, barnyardgrass, crabgrass, foxtail, goosegrass, and paspalum notatum in rice paddies. The process from contact absorption of cyhalofop-butyl to weed death is relatively slow, generally requiring 1-3 weeks. Herbicide damage symptoms appear 5-7 days after application, starting with yellowing and browning from the leaf heart, gradually spreading to the entire plant, and finally causing death. However, besides rice, cyhalofop-butyl remains toxic to other crops, and its long-term, widespread use has also led to some degree of environmental toxicity, significantly limiting its application scope and duration. Therefore, identifying herbicide resistance genes and using transgenic technology is of great significance for crops to acquire herbicide resistance.

[0006] Cytochrome P450 (CYP) enzymes are a class of membrane-bound hemoglobin-based enzymes widely found in microorganisms, plants, animals, and humans, possessing various biocatalytic activities such as oxidation, epoxidation, hydroxylation, and demethylation. P450 enzymes are particularly important in drug metabolism, participating in the metabolic responses of drugs and toxins in humans, the biosynthesis of secondary metabolites in plants and microorganisms, and some microbial degradation pathways, which has attracted widespread attention from pesticide researchers. Currently, some CYP81 subfamily genes (CYP81A12, CYP81A21, CYP81A68, CYP81A10v7, CYP81Q32) have been shown to metabolize herbicides, thus conferring herbicide resistance to weeds. Among these, only the CYP81A68 gene has been shown to confer resistance to cyhalofop-butyl in weeds. However, no non-CYP81 family genes have been found to metabolize cyhalofop-butyl and confer resistance to it in weeds. Summary of the Invention

[0007] The primary objective of this invention is to provide a herbicide-resistant gene that can be efficiently expressed in plants. By transferring this gene sequence into plants, novel herbicide-tolerant transgenic plants can be cultivated.

[0008] The present invention provides a herbicide-resistant gene EcCYP89B16, the nucleotide sequence of which is SEQ ID NO:1 or a sequence having no less than 80% identity with SEQ ID NO:1.

[0009] A second aspect of the present invention also provides a protein polypeptide encoded by the above-mentioned gene SEQ ID NO:1, the amino acid sequence of which is SEQ ID NO:2 or has a sequence having not less than 80% identity with SEQ ID NO:2.

[0010] A third aspect of the present invention aims to provide the application of the aforementioned gene EcCYP89B16 in plant herbicide resistance.

[0011] Specifically, this is achieved by expressing the EcCYP89B16 gene in plants, thereby enabling the plants to acquire herbicide resistance.

[0012] The herbicide mentioned is an aryloxyphenoxypropionic acid herbicide, specifically including at least one of cyhalofop-butyl, clodinafop-propargyl, and penoxsulam.

[0013] The plants mentioned include: rice, wheat, corn, cotton, sorghum, or pasture.

[0014] A fourth aspect of the present invention also provides a plasmid comprising an expression cassette consisting of a nucleotide sequence of the gene linked with a nucleotide sequence controlling expression, the nucleotide sequence encoding the aforementioned protein polypeptide.

[0015] The fifth aspect of this invention aims to provide the application of the said gene, the said polypeptide, or the said plasmid in the breeding of herbicide-resistant transgenic plants.

[0016] The nucleotide sequence of the herbicide-resistant gene of the present invention can have a variety of different variations, including but not limited to: 1) different nucleotide sequences obtained using different codons of the same amino acid, which encode protein polypeptides with the same activity; 2) nucleotide sequences that encode proteins with herbicide resistance by introducing variations in the nucleotide sequence. Such variations can be random variations, targeted point mutations, or insertion or deletion variations. Those skilled in the art can generate the above variations using molecular biological methods.

[0017] The nucleotide sequences provided by this invention can also be used to obtain homologous genes with the same function. One method is to use the nucleic acids provided by this invention as probes to hybridize DNA libraries to obtain homologous genes; another method is to design primers based on the nucleic acid sequences provided by this invention and clone homologous genes by PCR. Furthermore, those skilled in the art can also use the nucleic acid and protein sequences provided by this invention to identify genes with high homology from genomic libraries using molecular informatics methods. For example, using the BLAST method (www.ncbi.nih.gov), genes with high homology to those provided by this invention can be found based on the nucleotide sequences and amino acid sequences of the protein polypeptides provided by this invention.

[0018] Using the herbicide resistance gene nucleotide sequence provided by this invention, an artificial gene capable of being expressed in plants can be constructed. Similarly, using the cyhalofop-butyl resistance protein polypeptide sequence provided by this invention, a nucleic acid sequence can also be artificially synthesized, and further, an artificial gene capable of being expressed in plants can be constructed. The components of the artificial gene capable of being expressed in plants include a promoter, a herbicide resistance gene, and a terminator.

[0019] The promoters and terminators required to express and produce herbicide resistance in plants are existing technologies. For example, when transforming monocotyledonous plants, the promoter can be the maize Ubiqutin-1 promoter or the rice Actin promoter; while the terminator can be the Agrobacterium tumefaciens terminator (Nos) or other terminators. This expression component can be integrated into the plant genome using Agrobacterium (such as Agrobacterium strains), gene gun methods, or other methods to obtain cyhalofop-butyl-resistant transgenic plants. The techniques and methods for plant transformation are known and well-established. The methods and steps for transformation vary among different plants. However, it is generally done by introducing Agrobacterium or gene guns into immature embryos, mature embryos, undifferentiated callus tissue, or protoplasts of plants. Differentiation then occurs to obtain transformed shoots, which, after culturing in rooting media, yield transgenic seedlings ready for planting. Herbicide-resistant transgenic plants can be screened by spraying with herbicide-resistant agents. Furthermore, cyhalofop-butyl-resistant transgenic plants can be screened by spraying with cyhalofop-butyl.

[0020] This invention is applicable to all plants, including dicotyledonous and monocotyledonous plants.

[0021] This invention first discovered a barnyardgrass variety resistant to cyhalofop-butyl, then cloned its cytochrome P450 gene EcCYP89B16 using its genome sequence information, and further demonstrated the resistance of the EcCYP89B16 gene to cyhalofop-butyl and other aryloxyphenoxypropionic acid herbicides in rice, as well as its use in breeding transgenic crops resistant to aryloxyphenoxypropionic acid herbicides. The novel cytochrome P450 protein gene EcCYP89B16 involved in this invention has never been reported to possess resistance to aryloxyphenoxypropionic acid herbicides. Attached Figure Description

[0022] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Figure 1This is a schematic diagram of the construction of plant expression vectors; the expression cassette is mainly driven by the Ubi ubiquitin promoter to overexpress EcCYP89B16, and there is also HPT (hygromycin phosphotransferase) driven by the 35S promoter, which can confer rice callus resistance to hygromycin and be used to screen positive callus. Kan confers the vector resistance to kanamycin and is used for positive screening during vector construction, etc.

[0024] Figure 2 The results of RT-qPCR identification of differentially expressed P450 gene screened in the barnyardgrass population in Example 2;

[0025] Figure 3 The results of the tolerance test of cyhalofop-butyl to barnyardgrass transgenic rice EcCYP89B16 in Example 3 are shown.

[0026] Figure 4 The results of tolerance test for cyclohexane in rice with the barnyardgrass gene EcCYP89B16 in Example 4;

[0027] Figure 5 The results of the tolerance test of penoxsulam to barnyardgrass transgenic rice EcCYP89B16 in Example 5 are shown. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. It should be understood that these embodiments are only used to illustrate the method of the present invention and are not intended to limit the scope of the present invention. All experimental methods not specifically described or otherwise specified are conventional conditions and methods well known to those skilled in the art.

[0029] Example 1: Resistance determination to cyhalofop-butyl barnyardgrass

[0030] Barnyard grass (Echinochloa crusgalli) belongs to the Poaceae family and is an annual weed. It is one of the 18 most prevalent and noxious weeds worldwide and one of the most widespread and damaging weeds in rice paddies in my country. To determine the resistance level of barnyard grass to cyhalofop-butyl, the sensitivity of collected barnyard grass biotypes to cyhalofop-butyl was studied using a whole-plant assay. The results showed that cyhalofop-butyl inhibited the fresh weight of resistant (R) barnyard grass biotypes by a certain median dose (EDT). 50 The value is 167.54ga.i.ha -1 ; while for the sensitive (S) barnyardgrass biotype ED 50 The value is 55.16g aiha -1 The relative resistance multiple was 3.05 times. Therefore, the resistant (R) barnyardgrass biotype may contain the cyhalofop-butyl resistance gene.

[0031] Example 2 Cloning of the resistance gene

[0032] A plant's resistance to cyhalofop-butyl may involve multiple mechanisms, such as possessing an ACCase gene for cyhalofop-butyl resistance; possessing enzymes capable of degrading cyhalofop-butyl; limiting cyhalofop-butyl uptake; or possessing enzymes that modify cyhalofop-butyl to inactivate it. To study the target resistance mechanism in resistant barnyardgrass populations, 20 plants from each resistant population were selected for cloning of an acetyl-CoA carboxylase (ACCase, EC6.4.1.2) fragment. The cloned gene sequence was compared with the gene sequence of a sensitive (S) barnyardgrass population. The results showed that the R population did not have a target enzyme mutation, indicating that this population lacks a target enzyme resistance mechanism. Transcriptome sequencing technology successfully obtained transcriptome data from two barnyardgrass populations (R and S). Using the barnyardgrass genome database as a reference genome, the sequencing quality was statistically evaluated, and the Q30 values ​​were all above 90%, indicating very reliable sequencing quality. Further analysis of the assembled gene expression levels revealed 351 differentially expressed P450 genes between R and S populations, with nine potential non-target resistance-related genes identified. Quantitative real-time PCR was used to validate these nine non-target resistance-related differentially expressed genes using RNA samples from transcriptome sequencing. The results showed that, compared to the susceptible population S, the cytochrome P450 gene (EcCYP89B16) was consistently highly expressed in the resistant population R. Using total RNA extracted from barnyard grass as a template, based on the EST sequence fragment of the barnyard grass EcCYP89B16 gene obtained from the transcriptome, two specific primers were designed according to the 3' and 5' splicing sequences for PCR amplification and cloning to obtain the EcCYP89B16 plasmid (empty vector: pClone007 Versatile Simple Vector K, Beijing Qingke) (primer sequences: (F: 5'-ATGGACACGCAGCTCCTACTCC-3', R: 5'-TCACCCAGAGCCAGAGGTTCT-3'). Sequencing was performed, and the full length of the barnyard grass EcCYP89B16 gene (SEQ ID NO: 1) was successfully obtained.

[0033] Example 3: Construction of EcCYP89B16 gene overexpression vector and obtaining transgenic rice resistant to cyhalofop-butyl.

[0034] 1) Construct an overexpression vector for the EcCYP89B16 gene.

[0035] Specific primers were designed (pox-F: 5'-gtgttacttctgcagggtaccATGGACACGCAGCTCCTACTCC-3', pox-R: 5'-gttatcggatccataacgcgtTCACCCAGAGCCAGAGGTTCT-3'), using the correctly cloned and sequenced EcCYP89B16 plasmid from Example 2 as a template, and a high-fidelity enzyme ( Max DNA Polymerase (purchased from TaKaRa) was used for PCR amplification and purification to obtain the EcCYP89B16 fragment with a pox homologous arm.

[0036] The pox vector was double-digested using Thermo Fisher Scientific digestive enzymes (FastDigest KpnI: 5'GGTAC↓C 3' and FastDigest MluI: 5'A↓CGCGT 3'). The specific steps were as follows: 2 μL of 10X FastDigest Green Buffer, 1 μL of FastDigest KpnI, 1 μL of FastDigest MluI, 1000 ng of pox plasmid were added to a PCR tube, and sterile water was added to bring the volume to 20 μL. The mixture was reacted at 37°C for 5 minutes and then purified to obtain the linearized vector.

[0037] The purified target fragment and linearized vector were recombined and ligated using the ClonExpressII One Step Cloning Kit. 10 μL of the recombinant product was transformed into DH5α competent E. coli cells, and single clones were picked for sequencing identification to obtain the expression vector pox-EcCYP89B16 containing the full-length expression sequence of the barnyard grass EcCYP89B16 gene.

[0038] 2) Transformation of Agrobacterium with overexpression vector

[0039] Agrobacterium EHA105 was used to overexpress the expression vector into rice callus tissue to test whether EcCYP89B16 possesses herbicide resistance. The method for transforming Agrobacterium with the overexpression vector is as follows:

[0040] The electric shock cup and lid are disinfected by soaking in 75% alcohol for 2 hours. They are then taken out in a clean bench and placed on clean filter paper to drain the water. After the alcohol has completely evaporated, they are placed in ice to pre-cool.

[0041] Take EHA105 Agrobacterium competent cells (Beijing Qingke) from a -80℃ freezer, gently pinch them with your fingers until the competent cells partially melt, then place them on ice to completely melt them;

[0042] Pipettes 1000 ng of recombinant plasmid pox-EcCYP89B16 into EHA105 competent cells, gently aspirates and mixes, then transfers it completely into an electroporation cup and covers the cup.

[0043] The parameters of the electroporator (Eppendorf, Germany) were set to 2.5 kV and 5 ms. The water on the electroporation cup was wiped dry and quickly placed into the electroporation tank for electroporation. After electroporation, the cup was quickly placed on ice. 700 μL of antibiotic-free LB medium was added, and the mixture was aspirated and mixed. The mixture was then transferred into a 1.5 mL tube and cultured on a shaker at 28 °C for 2-3 hours.

[0044] Collect bacterial cells by centrifugation at 4000 rpm for 1 minute, leaving about 100 μL of supernatant. Mix the supernatant with the bacterial cells and spread it on LB medium containing 50 μg / mL kanamycin and 20 μg / mL rifampin. Incubate at 28°C inverted for 2-3 days.

[0045] Select a single colony for colony PCR. Determine if the plasmid has been successfully transformed, and select positive transformants for later use.

[0046] 3) Induction of rice callus

[0047] ZH11 rice seeds are dehulled, and mature, healthy seeds are selected for disinfection and induction.

[0048] The specific steps are as follows:

[0049] Place rice seeds into a 50mL centrifuge tube, add an appropriate amount of 75% ethanol, shake for about 60 seconds, pour out the ethanol, and wash with sterile water 5-7 times.

[0050] Add 2.5% sodium hypochlorite to a 50mL centrifuge tube and add 1 drop of Tween 20. Shake for 15 minutes, then pour out the sodium hypochlorite and wash with sterile water 5-7 times.

[0051] Add an appropriate amount of 2.5% sodium hypochlorite again, shake for 15 minutes, then pour out the sodium hypochlorite and rinse with sterile water 7-10 times.

[0052] Remove the seeds and place them on filter paper to absorb moisture. Then place them on N6D induction medium and incubate at 30°C in the dark for 10-12 days.

[0053] 4) Agrobacterium infection of callus and co-culture.

[0054] In step 2), the positive EHA105 transformants were shaken in LB liquid medium until turbid, and then added to 50 mL of LB medium at a ratio of 1:50 and shaken vigorously until OD600≈0.6. The cells were collected and diluted with AAM+AS medium to OD600=0.1. The dense and hard callus from step 3) was selected and added to the medium. After soaking for 2 minutes, the callus was removed and placed on filter paper to absorb the moisture.

[0055] Place a piece of filter paper on 2N6 medium, add 500 μL of LAAM+AS, and place the dried callus on the filter paper. Incubate in the dark at 30°C for 3 days.

[0056] 5) Screening for positive callus and cyhalofop-butyl sensitivity test

[0057] After co-culture, the callus tissue was transferred to a 50 mL tube, washed 10 times with sterile water, and then washed 7 times with sterile water containing thiazolyl (250 mg / L) and hygromycin (50 mg / L). After being placed on filter paper to absorb the moisture, the callus tissue was transferred to N6D medium containing hygromycin (50 mg / L), thiazolyl (250 mg / L), and termethin (200 mg / L) to screen for positive callus. The callus tissue was cultured at 30°C in the dark, and after 14 days, it was transferred to a new N6D medium for subculture.

[0058] After two rounds of screening, newly grown dense and firm milky yellow positive callus was selected and transferred to differentiation medium containing hygromycin (50 mg / L). It was first cultured in the dark for 3 days, and then transferred to light conditions (30℃, light intensity 1500-2000 xl, photoperiod: 16h / 8h (light / dark)) for culture. After about 15-25 days, green spots appeared, and seedlings further differentiated after 30-40 days.

[0059] Screening of transgenic plants. When the shoots differentiated from the resistant callus tissue grow to about 2 cm, the seedlings are transferred to rooting medium and cultured for about two weeks. Seedlings about 10 cm tall with well-developed root systems are selected, the medium is washed off, and they are transplanted into soil in a greenhouse to obtain transgenic plants.

[0060] Transgenic rice was treated with cyhalofop-butyl, with transgenic rice overexpressing pox-GFP as a control, to observe its sensitivity to cyhalofop-butyl. The results showed that transgenic rice with pox-GFP overexpression (used as a sensitive control line, confirming that overexpression of EcCYP89B16 enhanced rice resistance) showed improved resistance at a cyhalofop-butyl concentration of 4200 g / L. -1 It was completely dead, while the transgenic rice carrying the barnyardgrass pox-EcCYP89B16 gene overexpression grew at 4200g aiha -1 The plant continued to grow even at concentrations of cyhalofop-butyl, leading us to conclude that overexpression of the EcCYP89B16 gene in barnyardgrass can induce resistance to cyhalofop-butyl in transgenic rice.

[0061] Example 4: Sensitivity determination of EcCYP89B16 transgenic rice to clopyralid.

[0062] Transgenic rice was treated with clodinafop-propargyl, with transgenic rice overexpressing pox-GFP serving as a control, to observe its sensitivity to clodinafop-propargyl. The results showed that transgenic rice overexpressing pox-GFP showed the best sensitivity at a clodinafop-propargyl concentration of 24 g a.i.ha. -1 The rice was completely dead, while the transgenic rice carrying the barnyardgrass pox-EcCYP89B16 gene overexpression grew to 24 ga.i.ha -1 The plant continued to grow even at concentrations of clodinafop-propargyl, leading us to conclude that overexpression of the EcCYP89B16 gene in barnyardgrass can induce transgenic rice resistance to clodinafop-propargyl.

[0063] Example 5: Sensitivity determination of EcCYP89B16 transgenic rice to penoxsulam.

[0064] Transgenic rice was treated with penoxsulam, with transgenic rice overexpressing pox-GFP as a control, to observe its sensitivity to penoxsulam. The results showed that transgenic rice overexpressing pox-GFP showed sensitivity to penoxsulam at a concentration of 300g aiha. -1 The expression of the barnyardgrass pox-EcCYP89B16 gene was severely suppressed, while transgenic rice with overexpression of the barnyardgrass pox-EcCYP89B16 gene showed a significant increase in yield at 300g aiha. -1 At concentrations of penoxsulam, there was only a slight inhibition, therefore we conclude that overexpression of the barnyardgrass EcCYP89B16 gene can induce transgenic rice resistance to penoxsulam.

[0065] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. It should be pointed out that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0066] The present invention uses culture media and formulations

[0067] N6D culture medium regimen (callus induction and growth medium):

[0068]

[0069]

[0070]

[0071] AAM medium

[0072]

[0073] 2N6-AS medium (without AS)

[0074]

[0075] Regeneration medium (differentiation medium)

[0076]

[0077]

[0078] MS hormon-free medium (rooting medium)

[0079]

[0080] Antibiotic working solution concentration:

[0081]

[0082] Hormone working solution concentration:

[0083]

[0084]

[0085]

Claims

1. A herbicide-resistant gene EcCYP89B16 Its characteristics are, The nucleotide sequence is shown in SEQ ID NO:

1.

2. The herbicide resistance gene as described in claim 1 EcCYP89B16 The encoded polypeptide is characterized by, The amino acid sequence is shown in SEQ ID NO:

2.

3. The gene according to claim 1 EcCYP89B16 Its application in plant herbicide resistance is characterized by, By EcCYP89B16 The gene is expressed in the plant, thereby giving the plant resistance to herbicides; the herbicide is at least one of cyhalofop-butyl, clodinafop-propargyl, and penoxsulam.

4. The application according to claim 3, characterized in that, The plants mentioned include: rice, wheat, corn, cotton, sorghum, or pasture.

5. A plasmid, characterized in that: An expression frame comprising the nucleotide sequence of claim 1 linked with a nucleotide sequence controlling expression.

6. The application of the plasmid according to claim 5 in plant herbicide resistance, characterized in that, The plasmid is transferred into plants for expression, thereby enabling the plants to acquire herbicide resistance; the herbicide is at least one of cyhalofop-butyl, clodinafop-propargyl, and penoxsulam.

7. The application according to claim 6, characterized in that, The plants mentioned include: rice, wheat, corn, cotton, sorghum, or pasture.

8. The application of the gene of claim 1, the polypeptide of claim 2, or the plasmid of claim 5 in the breeding of herbicide-resistant transgenic plants, characterized in that, The gene or plasmid is transferred into a plant to express the polypeptide, wherein the herbicide is at least one of cyhalofop-butyl, cyclohexane, and penoxsulam.

9. The application according to claim 8, characterized in that, The plants mentioned include: rice, wheat, corn, cotton, sorghum, or pasture.

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