Cyhalofop-butyl-resistant digitaria calorata MYB transcription factor gene and application thereof

Abstract

The invention discloses a cyhalofop-butyl-resistant digitaria calorata MYB transcription factor gene and application thereof, and relates to the field of plant genetic engineering, and the expression of the gene in a resistant digitaria calorata biological type is remarkably up-regulated. After a plant expression vector is constructed and corn is transformed, after 11.25 g a.i. Ha-1 cyhalofop-butyl is sprayed on a transgenic corn plant in a 2-leaf stage, the pesticide resistance and the survival rate are obviously enhanced, and the transgenic corn plant is obviously superior to a wild type. The invention proves that the MYB gene can endow plants with resistance to acetyl-coenzyme A carboxylase (ACCase) inhibitor herbicides such as cyhalofop-butyl and the like, provides a new gene resource for cultivating transgenic crops resisting the acetyl-coenzyme A carboxylase (ACCase) inhibitor herbicides, and has important application value.

Description

A MYB transcription factor gene of *Digitaria buergerianum* resistant to cyhalofop-butyl and its application Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and agricultural biotechnology, specifically relating to a MYB transcription factor gene of *Digitaria sanguinalis* resistant to cyhalofop-butyl and its application. Background Technology

[0002] Digitaria ciliaris is a globally prevalent and highly detrimental weed that severely damages the production of crops such as rice and corn. Cyhalofop-butyl is an aryloxyphenoxypropionate (APP) herbicide that inhibits fatty acid synthesis by suppressing ACCase activity in plants, leading to plant death. It is widely used for weed control in paddy fields and dryland fields.

[0003] However, due to the long-term and singular use of herbicides, paddy fields have evolved resistance to cyhalofop-butyl, posing a significant challenge to weed control in farmland. The mechanisms of herbicide resistance in plants are complex, with changes in gene expression regulated by transcription factors being one important mechanism. The MYB transcription factor family is widely involved in plant secondary metabolism and stress responses, and may enhance herbicide metabolism by regulating the expression of detoxification enzyme genes.

[0004] Currently, although some research has been conducted on herbicide resistance in paddy fields, there are no reports on the functional verification and practical application of specific MYB genes in paddy fields and their role in conferring herbicide resistance to crops. Cloning and identifying key resistance genes and applying them to the genetic improvement of crop varieties is of great significance for developing new herbicide-resistant crops and addressing the challenge of weed resistance. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a MYB transcription factor gene of *Digitaria sanguinalis* resistant to cyhalofop-butyl.

[0008] To solve the above technical problems, the present invention provides the following technical solution: a MYB transcription factor gene of barnyardgrass resistant to cyhalofop-butyl, characterized in that: the nucleotide sequence of the MYB transcription factor gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2;

[0009] Another object of the present invention is to overcome the shortcomings of the prior art and provide a recombinant vector, wherein: the nucleic acid molecule is contained in the present invention, and the nucleic acid molecule is linked to an expression vector with a promoter that functions in plants, thereby constructing a recombinant vector.

[0010] As a preferred embodiment of the recombinant vector of the present invention, the promoter is CaMV35S (enhanced), and the expression vector includes the plant expression vector PC3300S-FLAG, the nucleic acid sequence of which is shown in SEQ ID NO: 3.

[0011] Another object of the present invention is to overcome the shortcomings of the prior art and provide a host cell containing the MYB transcription factor gene of Dioscorea opposita or the recombinant vector;

[0012] The host cell can be a prokaryotic cell, a eukaryotic cell, or a plant cell.

[0013] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for cultivating herbicide-resistant plants, comprising introducing the nucleic acid molecule of claim 1 or the recombinant vector of claim 3 into plant cells or tissues and obtaining regenerated transgenic plants;

[0014] The transgenic plants showed higher resistance to cyhalofop-butyl than the wild-type plants.

[0015] In a preferred embodiment of the method described in this invention, the plant is a grass crop, including maize.

[0016] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of the MYB transcription factor gene of Dioscorea haematocephala in the preparation of transgenic plants for herbicides that inhibit acetyl-CoA carboxylase.

[0017] In a preferred embodiment of the application described in this invention, the acetyl-CoA carboxylase inhibitor herbicide is cyhalofop-butyl.

[0018] In a preferred embodiment of the application described in this invention, the plant is a grass crop, including corn, sorghum, etc.

[0019] Beneficial effects of this invention:

[0020] This invention provides a MYB transcription factor gene isolated from herbicide-resistant Digitaria ciliaris in paddy fields. The protein encoded by this gene confers resistance in transgenic plants to acetyl-CoA carboxylase (ACCase) inhibitors such as cyhalofop-butyl. After constructing a plant expression vector and transforming it into maize, the transgenic maize plants were sprayed with 11.25 g ai ha at the 2-leaf stage.-1 After treatment with cyhalofop-butyl, the herbicide exhibited significantly enhanced resistance and survival rate, which were markedly superior to the wild type.

[0021] This invention confirms that the MYB gene can confer resistance to cyhalofop-butyl in plants, providing a new gene resource for breeding transgenic crops resistant to acetyl-CoA carboxylase (ACCase) inhibitor herbicides, and has important application value. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0023] Figure 1 is a graph showing the relative expression levels of the MYB gene in resistant (R) and sensitive (S) paddy fields of Dioscorea opposita in this embodiment of the invention.

[0024] Figure 2 is a sequencing verification diagram of the MYB gene in an embodiment of the present invention.

[0025] Figure 3 is a schematic diagram of the plant overexpression vector structure of the MYB gene in an embodiment of the present invention.

[0026] Figure 4 shows a phenotypic comparison of transgenic maize and wild-type (WT) maize 14 days after spraying with cyhalofop-butyl (11.25 ga.i. ha⁻¹) in an embodiment of the present invention.

[0027] Figure 5 shows the expression of the MYB gene in the transgenic maize of this invention. WT represents wild-type maize, and L1-L8 represent different lines of transgenic maize.

[0028] Figure 6 shows the dose-response curves of the transgenic (MYB) maize of this invention compared with wild-type (WT) maize 20 days after spraying with different concentration gradients of cyhalofop-butyl. Detailed Implementation

[0029] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0030] Example 1

[0031] Screening and cloning of resistance-related genes:

[0032] 1. Seeds of various Digitaria species collected from paddy fields were sown separately into disposable plastic cups filled with soil for growth. When the seeds reached the 3-leaf stage, cyhalofop-butyl (105 g ai ha) was sprayed. -1Plants that survive 21 days after application of the herbicide are classified as cyhalofop-butyl-resistant (R) crabgrass populations, while those that die are classified as cyhalofop-butyl-sensitive (S) crabgrass populations.

[0033] 2. Sow resistant (R) and susceptible (S) crabgrass seeds separately into disposable plastic cups filled with soil for growth. When the seeds reach the 3-leaf stage, spray with cyhalofop-butyl (105 g ai ha). -1 The first fully expanded leaf of resistant and sensitive plants was harvested at 0, 3, 6 and 24 hours after application, with each set of 3 leaves constituting one replicate. Three replicates were harvested at each time point.

[0034] RNA was extracted from the leaves of resistant and sensitive Digitaria pilosa at each treatment time using the FastPure Universal Plant TotalIsolation Kit from Nanjing Novogene Biotechnology Co., Ltd., and sent to Novogene for transcriptome sequencing. Bioinformatics analysis showed that the relative expression level of the MYB gene was higher in the R type than in the S type (Figure 1).

[0035] 3. Design specific primers: forward primer 5'-ATGGGGAGATCTCCTTGCTGC-3' and reverse primer 5'-TCATAGGAAATCAGTGATGAAGGGGTC-3'. Using R-type Dioscorea opposita cDNA as a template, the MYB gene was amplified by PCR and the full-length CDS sequence was sequenced (Figure 2).

[0036] The nucleotide sequence of the MYB transcription factor gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0037] Example 2

[0038] Construction of plant expression vectors:

[0039] The CDS fragment of the MYB gene was recombined into the plant expression vector PC3300S-FLAG (SEQ ID NO: 3) containing the CaMV35S enhanced promoter (sequence as shown in SEQ ID NO: 4) by double digestion with BglII and XbaI, thus constructing the recombinant plasmid PC3300S-MYB-FLAG (sequence as shown in SEQ ID NO: 5) (Figure 3).

[0040] Example 3

[0041] Cyhalofop-butyl resistance identification in genetically modified maize:

[0042] 1. The recombinant plasmid PC3300S-MYB-FLAG was introduced into Agrobacterium tumefaciens EHA105 by electroporation.

[0043] The specific process of the electric shock method is as follows:

[0044] (1) Melting competent cells: Take out a tube (about 50 μL) of EHA105 competent cells from the -80℃ freezer and place it on ice immediately to allow it to thaw slowly.

[0045] (2) Mixing plasmids with cells: Add 1-5 μL (about 50-100 ng) of plasmid DNA PC3300S-MYB-FLAG to the thawed competent cells and mix gently with a pipette tip (avoiding the formation of air bubbles).

[0046] (3) Incubation on ice: Place the mixture on ice for 5-10 minutes.

[0047] (4) Electric shock:

[0048] Transfer the entire mixture to a pre-cooled, sterile electroporation cup, ensuring the liquid is at the bottom of the cup and the outer wall of the cup is dry and free of water.

[0049] Set the parameters of the electric rotary instrument:

[0050] For Agrobacterium EHA105, the commonly used parameters are:

[0051] Voltage: 2.0 - 2.5 kV;

[0052] Capacitance: 25 μF;

[0053] Resistance: 200 - 400 Ω;

[0054] Time constant: typically 4-5 milliseconds (this is the result, no need to set).

[0055] Wipe the outer wall of the electric shock cup dry, place it steadily into the slot of the electric shock device, start the electric shock, and you will hear a beep, indicating that the electric pulse has been released.

[0056] (5) Recovery:

[0057] Immediately add 500 μL - 1 mL of antibiotic-free YEB or SOC liquid culture medium to the electroshock cup;

[0058] Gently pipette the mixture to mix, and then transfer the bacterial culture to a sterile 1.5 mL centrifuge tube.

[0059] Place the centrifuge tubes in a shaker at 28°C and 100-150 rpm for 2-4 hours to allow the cells to recover and express the resistance gene.

[0060] (6) Spreading on plates: Spread the revived bacterial solution (which can be concentrated by centrifugation as appropriate) evenly on YEB solid plates containing rifampicin (used to inhibit contaminating bacteria) and plasmid-resistant antibiotics (such as kanamycin, used to screen positive transformants).

[0061] 2. Infection of maize embryonic callus tissue using Agrobacterium-mediated transformation.

[0062] Using freshly peeled corn embryos of about 1 mm as material, the peeled corn embryos were placed in a 2 ml plastic centrifuge tube containing 1.8 mL of suspension. Approximately 150 immature embryos were processed within 30 minutes.

[0063] Remove the suspension, leaving the corn germ in the tube. Then add 1.0 ml of Agrobacterium suspension and let stand for 5 min.

[0064] After suspending the embryos in the centrifuge tubes, pour them onto the co-culture medium and use a pipette to remove excess Agrobacterium tumefaciens on the surface. Co-culture at 23 °C in the dark for 3 days.

[0065] After co-culture, the immature embryos were transferred to resting medium and cultured in the dark at 28 °C for 6 days. Then, they were placed on selection medium containing diammonium phosphate and cultured for two weeks, followed by two weeks of selection culture on a new selection medium.

[0066] The resistant callus was transferred to differentiation medium and cultured at 25°C, 5000 lx, under light for 3 weeks.

[0067] The differentiated seedlings were transferred to rooting medium and cultured at 25°C, 5000 lx, under light until roots formed.

[0068] 3. Resistant callus was screened, differentiated, and rooted on a medium containing the herbicide Basta (selection marker gene) to obtain transgenic seedlings.

[0069] Live callus was screened out using N6+2,4-D+Basta, seedlings were grown using MS+6-BA+Basta, roots were grown using 1 / 2 MS+NAA, and finally transgenic seedlings were obtained.

[0070] Specifically as follows:

[0071] I. Culture medium formulation

[0072] 1. Callus induction and proliferation medium (N6 + 2,4-D)

[0073] formula:

[0074] N6 Basic Salt and Vitamins: N6 basic salt contains macro- and micro-elements. The macro-elements include potassium nitrate (2830 mg / L), ammonium sulfate (463 mg / L), potassium dihydrogen phosphate (400 mg / L), magnesium sulfate heptahydrate (185 mg / L), and calcium chloride dihydrate (166 mg / L); the micro-elements include manganese sulfate tetrahydrate (4.4 mg / L), zinc sulfate heptahydrate (1.5 mg / L), boric acid (1.6 mg / L), and potassium iodide (0.8 mg / L).

[0075] The vitamins include inositol (100 mg / L), glycine (2.0 mg / L), thiamine hydrochloride (vitamin B1, 1.0 mg / L), pyridoxine hydrochloride (vitamin B6, 0.5 mg / L), and niacin (0.5 mg / L).

[0076] 30 g / L of sucrose;

[0077] L-proline 0.7 g / L (optional, but highly recommended);

[0078] 2,4-D 1.5-2.0 mg / L;

[0079] Plant-based gel 2.5-3.0 g / L;

[0080] pH 5.8.

[0081] Function: To induce embryogenic callus from immature embryos (forming loosely structured, rapidly proliferating Type II callus) and to be used for the proliferation culture of callus before and after transformation. No selection agent is added at this stage.

[0082] 2. Selection medium for resistant callus (N6 + 2,4-D + Basta)

[0083] Formula: Add 3-5 mg / L Basta (PPT) to the "induction medium".

[0084] Function: To exert selective pressure while maintaining callus growth. Untransformed callus browns and dies, while transformed cells form pale yellow, granular, or flocculent resistant callus. Note: Maize is generally more sensitive to PPT than rice, requiring lower concentrations.

[0085] 3. Callus recovery culture medium (N6 + 2,4-D)

[0086] Formula: Exactly the same as "Induction Medium", but without Basta.

[0087] Function: Before differentiation, it allows resistant callus tissue that has undergone multiple rounds of screening to regain its vitality in a stress-free environment, which helps to improve the subsequent differentiation rate.

[0088] 4. Differentiation and seedling culture medium (MS + 6-BA + ABA + Basta)

[0089] formula:

[0090] MS basic salts and vitamins: ammonium nitrate (1650 mg / L), potassium nitrate (1900 mg / L), potassium dihydrogen phosphate (170 mg / L), calcium chloride (440 mg / L), magnesium sulfate heptahydrate (370 mg / L).

[0091] Ferric disodium EDTA (iron salt, approx. 37.3 mg / L), ferrous sulfate heptahydrate (27.8 mg / L), manganese sulfate tetrahydrate (22.3 mg / L), boric acid (6.2 mg / L), zinc sulfate heptahydrate (8.6 mg / L), potassium iodide (0.83 mg / L), sodium molybdate dihydrate (0.25 mg / L), copper sulfate pentahydrate (0.025 mg / L), and cobalt chloride hexahydrate (0.025 mg / L); inositol (100 mg / L), glycine (2.0 mg / L), thiamine hydrochloride (vitamin B1, 0.1 mg / L), pyridoxine hydrochloride (vitamin B6, 0.5 mg / L), and niacin (0.5 mg / L);

[0092] 30 g / L of sucrose;

[0093] 6-BA 1.0-2.0 mg / L;

[0094] ABA 0.5-2.0 mg / L (key component);

[0095] Basta (PPT) 3-5 mg / L;

[0096] Plant-based gel 2.5-3.0 g / L;

[0097] pH 5.8.

[0098] Function: ABA is key to the efficient differentiation of maize embryogenic callus. It inhibits the unlimited proliferation of callus, promotes its morphogenesis, and, in synergy with 6-BA, induces the formation of green spots and the development of normal seedlings. Continuous application of ABA pressure is also beneficial.

[0099] 5. Rooting and seedling strengthening medium (1 / 2 MS + NAA)

[0100] formula:

[0101] 1 / 2 MS macroelements, MS microelements and vitamins;

[0102] Sucrose 15-20 g / L (lowering the sucrose concentration is beneficial for rooting);

[0103] NAA 0.5-1.0 mg / L;

[0104] Plant-based gel 2.5-3.0 g / L;

[0105] pH 5.8; (optional) A low concentration (1-2 mg / L) of Basta may be added for final screening.

[0106] Function: Induces transgenic seedlings to produce robust adventitious roots, forming complete T0 generation plants.

[0107] II. Training Conditions

[0108] Temperature: (26 ± 1) ℃ for all stages.

[0109] Illumination: Induction, screening, and recovery stages: Dark culture.

[0110] Differentiation and rooting stage: light culture, photoperiod 16 hours light / 8 hours dark, light intensity 2000-3000 lux.

[0111] Humidity: Maintain appropriate humidity and avoid excessive condensation during the differentiation stage.

[0112] III. Detailed Operating Procedures

[0113] Phase 1: Induction and proliferation of callus tissue (preparation for transformation)

[0114] Immature maize embryos were removed and inoculated onto callus induction medium (N6 + 2,4-D) with the scutellum facing upwards.

[0115] Dark culture for approximately 2 weeks until abundant embryogenic callus is produced in the embryonic region. Select healthy Type II callus for subculture (every 2 weeks) as transformation recipients.

[0116] Phase 2: Screening of resistant callus tissue (post-transformation)

[0117] Co-culture and recovery: After co-culturing Agrobacterium (e.g., for 3 days), the explants are transferred to an induction medium containing antibiotics (e.g., cephalosporins) and cultured in the dark for 5-7 days to restore growth and remove Agrobacterium.

[0118] First round of screening: Transfer the callus tissue to resistant callus screening medium (N6 + 2,4-D + Basta) and incubate in the dark for 2-3 weeks. Remove severely browned non-converted callus tissue promptly.

[0119] Second round of screening and proliferation: Select surviving resistant callus tissues, transfer them to fresh screening medium, and continue dark culture and screening for 2-3 weeks until enough stable resistant clones are obtained.

[0120] Phase 3: Pre-differentiation recovery

[0121] The selected resistant callus tissues were transferred to recovery medium (N6 + 2,4-D) and cultured in the dark for 1-2 weeks. This step can reduce the physiological damage caused by selection stress and significantly improve the subsequent differentiation efficiency.

[0122] Fourth stage: Differentiation into seedlings

[0123] The recovered resistant callus was transferred to differentiation and seedling culture medium (MS + 6-BA + ABA + Basta). The callus could be appropriately dispersed into small pieces.

[0124] Transplant to light conditions for cultivation. After about 7-14 days, numerous green buds will appear on the surface of the callus tissue.

[0125] Continue cultivation for 3-5 weeks, and the green buds will gradually elongate and develop into robust seedlings with normal leaves.

[0126] Fifth stage: Rooting and seedling growth

[0127] Take healthy seedlings that are more than 3 cm tall and cut off any remaining callus tissue from the base.

[0128] The seedlings were inserted into the rooting and seedling strengthening medium (1 / 2 MS + NAA).

[0129] After cultivating the seedlings under light for 2-3 weeks, once the seedlings have grown several thick adventitious roots and the plants have grown significantly taller, they can be prepared for transplanting into greenhouse soil.

[0130] 4. T0 generation transgenic positive plants were identified by Basta herbicide screening strips, and T1 generation seeds were harvested.

[0131] 5. Transgenic maize T1 generation positive plants and wild-type (WT) maize were planted in a greenhouse.

[0132] 6. When the plants have grown to the 2-leaf stage, use a greenhouse spray tower to evenly spray 7.5 mL of 10% cyhalofop-butyl OD, which means the effective dose of cyhalofop-butyl is 11.25 g ai ha⁻¹.

[0133] 7. Observe and record the symptoms of damage to the plants (yellowing, wilting, death, etc.) 14 days after application of the pesticide, and calculate the survival rate.

[0134] 8. The results are shown in Figure 4: 14 days after the application of the pesticide, wild-type maize was basically dead, while the transgenic maize that overexpressed MYB had a survival rate of 100% and did not show any symptoms of pesticide damage, which was significantly better than the wild-type control.

[0135] Example 4

[0136] Expression level of this gene in transgenic maize:

[0137] T1 generation transgenic maize kernels were planted individually in disposable plastic cups containing nutrient soil, a total of 8 cups, and placed in a greenhouse for cultivation, with wild-type maize as a control.

[0138] When the maize plants reach the 3-leaf stage, leaves are harvested from individual plants. RNA is extracted from each leaf using the Novizan FastPure Universal Plant Total RNA Isolation Kit, and the RNA is reverse transcribed into cDNA using the HiScript II Q RT SuperMix for qPCR kit. Using the cDNA as a template, MYB gene-specific primers (forward primer 5'-AGTACATCCAGAAGAACGG-3' and reverse primer 5'-CTCTTGCCGCATCTGTTC-3') and GAPDH internal reference gene primers (forward primer 5'-TCATGCCATCACTGCCACACAG-3' and reverse primer 5'-CACGGAAGGACATACCAGTGTGC-3') are designed, and quantitative real-time PCR (qPCR) analysis is performed using a Roche LightCycler 96 instrument.

[0139] qPCR results showed that, compared with wild-type maize, 5 out of 8 transgenic maize plants had high expression of the MYB gene, with expression folds ranging from 828 to 1462 fold (Figure 5).

[0140] Example 5

[0141] Resistance levels of genetically modified maize to cyhalofop-butyl:

[0142] Thirty-one seeds each of T1 generation transgenic maize and their corresponding wild-type maize seeds were individually planted in disposable plastic cups filled with nutrient soil, totaling 300 cups, and placed together in an artificial climate greenhouse for cultivation. When the maize reached the 2-3 leaf stage, 240 cups each of the uniformly growing transgenic and wild-type maize were selected. Cyhalofop-butyl was sprayed at doses of 0, 1.788, 2.861, 4.578, 7.324, 11.719, 18.750, and 30.000 g ai ha⁻¹, and the transgenic maize was sprayed at doses of 0, 2.861, 4.578, 7.324, 11.719, 18.750, 30.000, and 48.000 g ai ha⁻¹, with each treatment applied to 10 plants, and the treatment was repeated three times. Twenty days after application, the above-ground parts of the plants were collected, with 10 plants for each treatment placed in a large envelope. The above-ground parts of the plants for each treatment were placed in an oven for blanching at 105°C for 30 min and drying at 75°C for 72 h, and then weighed.

[0143] Data processing method: Used to calculate the herbicide concentration (GR) that reduces aboveground biomass by 50%. 50 The logistic regression model for y is as follows (implemented using SigmaPlot 15.0 software): y = C + (D - C) / [1 + (x / GR)] 50 )^b]. Where y represents the percentage reduction in aboveground biomass, x is the herbicide dosage, C and D represent the lower and upper asymptotes of the model, respectively, and b is the GR. 50 The slope of the nearby curve. The resistance index (RI) is measured using the GR (grafting rate) of genetically modified maize. 50 Compared with wild-type corn GR 50 The ratio was calculated. The results are shown in Figure 6, showing the GR of transgenic maize (MYB) to cyhalofop-butyl. 50 is 42.19 ga.i. ha -1 Wild-type maize (WT) to cyhalofop-butyl GR 50 5.96 g ai ha -1 The resistance index (RI) of genetically modified corn is 7.1 times.

[0144] The sequences in this embodiment of the invention are as follows:

[0145] SEQ ID NO: 1:

[0146] ATGGGGAGATCTCCTTGCTGCGACGAGAGTGGCCTCAAGAAGGGCCCGTGGACGCCGGAGGAGGACGAGAAGCTGCTGCAGTACATCCAGAAGAACGGCCATGGCAGCTGGAGGACCCTCCCAAGACTCGCCGGGCTGAACAGATGCGGCAAGAGCTGCAGGCTGCGGTGGACCAACTACCTGCGTCCGGACATCAAGCGTGGCAAGTTCTCGCAGGAGGAGGAGCAGACCATCCTCCACCTCCACTCCATGCTCGGCAACAAGTGGTCGGCGATCGCGACGCACCTGCCGGGCCGGACGGACAACGAGATCAAGAACTTCTGGAACACGCACCTCAAGAAGCGGCTCATCCAGATGGGCTTCGACCCCATGACGCACCGCCCCCGCACCGACTTCTTCGCCGCGCTGCCGCAGCTCATCGTGCTCGCCGCGCTCCGGGACCAGCTCGCCGGCGCCGGCGACCCGGCAGCGAACGCGCAGCTCCAGGCTGGCGCCGGCGCCGGCGTCGACGTTGCCATCCAGGCCGCCAAGCTCCAGTATCTCCAGTGCCTCCTCCAGTCGGCAGCCACCACCATCGCTTCAACCGCTGGCGCCGCCACGTCGGACGCCGAGGTGGCGGCTCTCGGCGCCCTCTGGTCGCCGCAGGGGACGCATGACAGTGCTACTCCGGTGTCTGCGGCCGGCGGGCAGCTACCTTCTTGCACGACGTTCCCTGAGGCGGCCGTTAGTAGCGGCGAAGGCAACCAAGACGTCAGCTTTGGCGCTGACGTGGACATGTTCGCGTGCCACGATGGTGGTGGCTCGTTGCCGCCACTGACCGACCTCTCCGATGCCGCGACGAACAACCCCGGCAGCGCCACGGCGTCGTCTAGCTTTGGCGGCGGCGGCGGGGCGAGCAGCCCGCTCCCTTGGCCGGAGTTCTTCCCCGACGACCCCTTCATCACTGATTTCCTATGA

[0147] SEQ ID NO: 2

[0148] MGRSPCCDESGLKKGPWTPEEDEKLLQYIQKNGHGSWRTLPRLAGLNRCGKSCRLRWTNYLRPDIKRGKFSQEEEQTILHLHSMLGNKWSAIATHLPGRTDNEIKNFWNTHLKKRLIQMGFDPMTHRPRTDFFAALPQLIVLAALRDQLAGAGDPAANA QLQAGAGAGVDVAIQAAKLQYLQCLLQSAATTIASTAGAATSDAEVAALGALWSPQGTHDSATPVSAAGGQLPSCTTFPEAAVSSGEGNQDVSFGADVDMFACHDGGGSLPPLTDLSDAATNNPGSATASSSFGGGGGASSPLPWPEFFPDDPFITDFL

[0149] In summary, this invention is the first to clone and functionally verify the novel function of the MYB gene derived from resistant paddy field barnyardgrass in conferring herbicide resistance to crops, providing important genetic resources and technical means for the genetic improvement of crop herbicide resistance.

[0150] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A MYB transcription factor gene for *Digitaria buergerianum* resistant to cyhalofop-butyl, characterized in that: The nucleotide sequence of the MYB transcription factor gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO:

2.

2. A recombinant vector, characterized in that: A recombinant vector is constructed by linking the nucleic acid molecule of claim 1 to an expression vector along with a promoter that functions in plants.

3. The recombinant vector as described in claim 2, characterized in that: The promoter is a constitutive promoter, and the expression vector includes the plant expression vector PC3300S-FLAG, whose nucleic acid sequence is shown in SEQ ID NO:

3.

4. A host cell, characterized in that: It contains the MYB transcription factor gene of Dioscorea opposita as described in claim 1 or the recombinant vector as described in claim 3; the host cell is a prokaryotic cell, a eukaryotic cell or a plant cell.

5. A method for cultivating herbicide-resistant plants, characterized in that: This includes introducing the nucleic acid molecule of claim 1 or the recombinant vector of claim 3 into plant cells or tissues, and obtaining regenerated transgenic plants; the transgenic plants exhibit higher resistance to cyhalofop-butyl than wild-type plants.

6. The method as described in claim 5, characterized in that: The plants in question are grasses, including maize.

7. The application of the MYB transcription factor gene of *Digitaria sanguinalis* resistant to cyhalofop-butyl as described in claim 1 in the preparation of transgenic plants resistant to acetyl-CoA carboxylase inhibitor herbicides.

8. The application as described in claim 7, characterized in that: The acetyl-CoA carboxylase inhibitor herbicide is cyhalofop-butyl.

9. The application according to claim 7 or 8, characterized in that: The plants mentioned are grasses that are sensitive to cyhalofop-butyl, including corn and sorghum.

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