An anti-herbicide goosegrass glutathione-s-transferase gene ecgstt1 and application thereof

Abstract

The application discloses an anti-herbicide Echinochloa crusgalli glutathione-S-transferase gene EcGSTT1, and the nucleotide sequence is SEQ ID NO:1, or a sequence with not less than 80% homology compared with SEQ ID NO:1. The application introduces the resistance gene glutathione-S-transferase gene EcGSTT1 from Echinochloa crusgalli into plants sensitive to high-efficiency fluazifop, so that the tolerance of the sensitive plants to high-efficiency fluazifop and other AOPP herbicides is significantly enhanced, thereby making the herbicide originally having a sterilization effect on gramineous crops and unable to be safely used in gramineous crop fields be able to be used for precise prevention and control of gramineous weeds in the fields of transgenic crops into which the resistance gene is introduced. The application provides a new gene for cultivating new germplasm of anti-herbicide crops, so as to expand the application range of AOPP herbicides and enrich the gene resource reserves of anti-herbicide crops in China.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology. More specifically, this invention relates to a herbicide-resistant barnyard grass glutathione-S-transferase gene EcGSTT1 and its application. Background Technology

[0002] With the widespread application of direct seeding technology in agricultural production, weed infestation has become a significant constraint on crop yields. Chemical weeding, due to its high efficiency and cost-effectiveness, occupies an indispensable position in modern agriculture. However, traditional breeding and domestication processes have not addressed the selection of herbicide tolerance traits, resulting in a severe shortage of resistance genes in natural germplasm resources. Currently, the main approaches to developing herbicide-resistant crop germplasm resources include: utilizing natural or induced mutations of herbicide target genes to screen and identify endogenous resistant materials; and introducing heterologous resistance genes through transgenic technology to confer specific herbicide resistance traits on crops. For example, acetolactate synthase (ALS) gene mutants obtained through EMS mutagenesis have been successfully applied to rice varieties such as "Jinjing 818," demonstrating significant weed control advantages in production. However, most resistance sites of known major herbicide target genes such as EPSPS, ACCase, ALS, and HPPD are protected by patents from foreign commercial companies, limiting the technological application space for the domestic seed industry. Therefore, discovering novel herbicide resistance genes with independent intellectual property rights, especially gene resources that confer resistance through metabolic detoxification pathways, is of great scientific significance and application value for breeding herbicide-resistant crop varieties.

[0003] The mechanism of action of aryloxyphenoxypropionic acid (AOPP) herbicides lies in inhibiting the activity of acetyl-CoA carboxylase (ACCase). When ACCase is inhibited, malonyl-CoA synthesis is blocked, leading to fatty acid deficiency, membrane structure damage, increased permeability, and ultimately cell death. Mutations at the ACCase target sites in some gramineous crops (such as wheat and rice) can reduce their sensitivity to AOPP herbicides. Transgenic crops expressing these mutant proteins can acquire resistance, and related technologies are protected by patents (Chinese patents: CN112410308A, CN109355264B). However, existing resistance sites are mostly covered by patents of foreign commercial companies, and target mutation resistance is often limited to a single or a few herbicide varieties. Therefore, discovering metabolic detoxification resistance genes with independent intellectual property rights is of great practical significance for increasing the diversity of resistance genes, improving resistance levels, and meeting the diversified needs of production.

[0004] Glutathione S-transferases (GSTs) are a class of multifunctional detoxification enzymes widely found in plants, animals, and microorganisms. Studies have shown that increased GST activity is one of the important mechanisms by which weeds develop metabolic resistance. In recent years, several GST genes involved in herbicide detoxification have been identified from crops such as rice, corn, and wheat, as well as resistant weeds such as barnyard grass and sedge. For example, a recent study reported that the EpGST1 gene cloned from barnyard grass (Echinochloa phyllopogon) conferred resistance to penflusulfonamide in transgenic rice by metabolizing it. However, GST genes capable of efficiently metabolizing AOPP herbicides such as haloxyfop-R-methyl and conferring stable resistance in crops still need to be discovered, especially the GST gene resources derived from barnyard grass, a noxious weed in rice paddies, which have not yet been fully developed and utilized. Summary of the Invention

[0005] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0006] To achieve these objectives and other advantages of the present invention, a barnyardgrass glutathione-S-transferase gene EcGSTT1 resistant to herbicides is provided, with the nucleotide sequence of SEQ ID NO:1, or a sequence having at least 80% identity with SEQ ID NO:1.

[0007] A protein polypeptide encoded by the barnyardgrass glutathione-S-transferase gene EcGSTT1 as described above, having the amino acid sequence SEQ ID NO:2, or a sequence having at least 80% identity with SEQ ID NO:2.

[0008] A plasmid containing the nucleotide sequence described above.

[0009] A recombinant strain is obtained by transforming the plasmid described above into Agrobacterium.

[0010] Application of the barnyardgrass glutathione-S-transferase gene EcGSTT1 as described above, or the protein polypeptide as described above, or the plasmid as described above, or the recombinant strain as described above in plant herbicide resistance.

[0011] One application, as described above, involves expressing genetically modified genes in plants to endow them with herbicide resistance, or as a screening gene for plant genetic modification.

[0012] Application of the barnyardgrass glutathione-S-transferase gene EcGSTT1 as described above, or the protein polypeptide as described above, or the plasmid as described above, or the recombinant strain as described above in the breeding of herbicide-resistant transgenic plants.

[0013] Preferably, the plant is cotton, corn, soybean, rice, wheat, sorghum, turfgrass, or pasture.

[0014] Preferably, the herbicide is an aryloxyphenoxypropionate herbicide.

[0015] Preferably, the aryloxyphenoxypropionate herbicide is at least one of the following: haloxyfop-R-methyl, quizalofop-P-ethyl, haloxyfop-R-methyl, quizalofop-P-ethyl, clodinafop-P-methyl, and cyhalofop-P-ethyl.

[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 haloxyfop-R-methyl protein polypeptide sequence provided by this invention, a nucleic acid sequence can also be artificially synthesized, and further used to construct an artificial gene capable of being expressed in plants. 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 transgenic plants resistant to haloxyfop-R-methyl. 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 common practice to introduce immature embryos, mature embryos, undifferentiated callus tissue, or protoplasts of plants using Agrobacterium or a gene gun. 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, haloxyfop-R-methyl transgenic plants can be screened by spraying with haloxyfop-R-methyl.

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

[0021] The present invention has at least the following beneficial effects: This invention provides a herbicide-resistant gene with independent intellectual property rights. Specifically, by introducing the glutathione-S-transferase gene EcGSTT1, derived from barnyard grass, into plants sensitive to haloxyfop-P-ethyl (such as rice and other gramineous crops), the tolerance of these sensitive plants to AOPP herbicides such as haloxyfop-P-ethyl is significantly enhanced. This allows herbicides that are normally non-selective and cannot be safely used in gramineous crop fields to be used for precise control of gramineous weeds in transgenic crops incorporating this resistance gene. This invention provides a new gene for cultivating new herbicide-resistant crop germplasm, expanding the application scope of AOPP herbicides and enriching my country's genetic resource reserves of herbicide-resistant crops.

[0022] This invention takes a novel approach, starting from the metabolic resistance mechanism of weeds, and deeply analyzes the metabolic resistance mechanism of barnyard grass to AOPP herbicides such as haloxyfop-R-methyl. It has successfully cloned and identified a glutathione S-transferase theta subfamily gene EcGSTT1.

[0023] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0024] Figure 1This is a comparison of the growth status of resistant (R) and susceptible (S) barnyardgrass populations in Example 1 of the present invention under the same dosage of highly effective flupyridine; Figure 2 This is an electrophoresis diagram of the PCR products in Example 2 of the present invention; Figure 3 This is a schematic diagram illustrating the construction of the transgenic overexpression vector in Example 3 of the present invention; Figure 4 This image shows the growth status of rice callus tissues transfected with GFP and EcGSTT1 genes in Example 3 of the present invention on N6D medium containing different concentrations of high-efficiency flupyridine. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0026] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0027] Example 1 Resistance determination of barnyardgrass populations resistant to highly effective flupyradifurone: Barnyard grass is one of the most serious weeds in cotton fields in my country. In order to clarify the resistance level of barnyard grass to the commonly used herbicide fluroxypyr, the sensitivity level of collected barnyard grass biotypes to fluroxypyr was studied by whole-plant assay. Figure 1 This invention compares the growth status of resistant (R) and susceptible (S) barnyard grass populations under the same dosage of haloxyfop-R-methyl in Example 1. The results showed that the median fresh weight inhibition dose (GR50) of haloxyfop-R-methyl for the resistant (R) barnyard grass biotype was 29.45 g ai ha⁻¹, while the GR50 for the susceptible (S) barnyard grass biotype was 4.87 ga.i. ha⁻¹, representing a relative resistance fold of 6.05 times. Therefore, the resistant (R) barnyard grass biotype may contain genes resistant to haloxyfop-R-methyl.

[0028] Example 2 Cloning of the barnyard grass glutathione S-transferase gene EcGSTT1: Transcriptome sequencing technology successfully obtained transcriptome data from two barnyard grass populations (R and S). Using the barnyard grass genome database as a reference genome, the sequencing quality was statistically evaluated, revealing that the Q30 values ​​were all above 95%, indicating highly reliable sequencing quality. Quantitative real-time PCR was used to verify candidate resistance genes, revealing that the glutathione-S-transferase gene EcGSTT1 was consistently highly expressed in the resistant population R compared to population S. Total RNA was extracted from the leaves of the barnyard grass biotype resistant to flupyradifurone and reverse-engineered into cDNA. Based on the CDS sequence of the EcGSTT1 gene (ID: scaffold135.72.gene) in the barnyard grass genome, two specific primers (F: ATGACGCCCATCAAGGTGTACG, R: TCAGAGCCTCGATGTGATTTTGA) were designed. Full-length amplification was performed using a high-fidelity 2×PrimeSTAR Max Premix (TaKaRa) under the following amplification conditions: condition: Figure 2 This is an electrophoresis image of the PCR product in Example 2 of the present invention. The target PCR product was purified, recovered, cloned, and sequenced, and the nucleotide sequence (SEQ ID NO:1) of the barnyard grass glutathione-S-transferase gene EcGSTT1 and its encoded amino acid sequence (SEQ ID NO:2) were successfully obtained. SEQ ID NO: 1, details are as follows: ATGACGCCCATCAAGGTGTACGCCCACCGGTTGTCGCAGCCCTCCCGCGCCGTCATCATCTTCTGCAGGGTGAACCGGATCGATTTCGAGGAGGTCACGGTAGATCTGTTCAAGAAACAGCATCTCGCCCCTGAATTCAAAAAGGTGAACCCAATGGGCCAAGTCCCAGCAATTATTGATGGAAGGTTTAAACTGTTCGAAAGTCATGCCATTTTGAGGTATCTTGCATCAGTCTTCCCTGGAGTTGCAGATCACTGGTACCCTGCAGACTTGTTTGCCAGAGCCAAAATTGAGTCAATCTTGGATTGGCATCACTCGAATTTGCGACGTGGTGCAGTAACCATTGTATTGAACACTGCATTGGCTCCTTTTCTTGGTCTTACACCAAATCTAGATGCTGCAAAACAAGCAGAAAAACTGCTGGTGCGGTCACTGGCTACAATTGAATCCGTGTGGCTCAAAGGTGAAGCCAAGTTTTTACTTGGTAGCCCTCAGCCTTCAATTGCAGATCTGAGCCTTGTCTGTGAGATAATGCAGTTGGAGGTCCTTGGTGACGATACACGAGACAGATTTCTGGGAGGTCATGAGAAGATCCTCACTTGGATGAGCAACGTGAAGAAAGCCACGAGTCCTTATTTTGAGGAAGCCCACATGTTCCTCTTTGAAGTTAAGGGTAAGATGCAAATCAAGGCAGCAGCTGCAGCAGCAGCAGAAAAACATGATGGTTCTGAGCCAAGCTCAAAGCTCAAAATCACATCGAGGCTCTGA; SEQ ID NO: 2, specifically as follows: MTPIKVYAHRLSQPSRAVIIFCRVNRIDFEEVTVDLFKKQHLAPEFKKVNPMGQVPAIIDGRFKLFESHAILRYLASVFPGVADHWYPADLFARAKIESILDWHHSNLRRGAVTIVLNTALAPFLGLT PNLDAAKQAEKLLVRSLATIESVWLKGEAKFLLGSPQPSIADLLSLVCEIMQLEVLGDDTRDRFLGGHEKILTWMSNVKKATSPYFEEAHMFLFEVKGKMQIKAAAAAAAEKHDGSEPSSKLKITSRL.

[0029] Example 3 Validation of herbicide resistance function of barnyardgrass glutathione S-transferase gene EcGSTT1 expressed in rice callus: (1) Construction of EcGSTT1 gene overexpression vector. Figure 3 This is a schematic diagram illustrating the construction of the transgenic overexpression vector in Example 3 of the present invention. Specific primers (POX-F: 5'-TGTTACTTCTGCAGGGTACCATGACGCCCATCAAGGTGTACG-3', POX-R: 5'-CGGATCCATAACGCGTTCAGAGCCTCGATGTGATTTTGA-3') were designed. Using the correctly cloned and sequenced EcGSTT1 from Example 2 as a template, PCR amplification and purification were performed using a high-fidelity 2×PrimeSTAR Max Premix (TaKaRa, Inc.) to obtain the EcGSTT1 fragment with the homologous arm of the POX vector. The POX vector was double-digested using Thermo Fisher Scientific's fast digesters (KpnI + MluI). The specific steps were as follows: 2 μL of 10× FastDigest Green Buffer, 1 μL of FastDigest KpnI, 1 μL of FastDigest MluI, 1000 ng of PHG plasmid were added to a PCR tube, and sterile water was added to bring the volume to 20 μL. After incubation at 37°C for 5 minutes, the linearized vector was obtained. The purified target fragment and linearized vector were recombined and ligated using the Takara In Fusion 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-EcGSTT1 containing the full-length expression sequence of the EcGSTT1 gene.

[0030] (2) EcGSTT1 gene overexpression vector transformed into Agrobacterium. Agrobacterium EHA105 was used to transform the overexpression vector into rice callus for overexpression in order to test whether EcGSTT1 has herbicide resistance function. The method for transforming Agrobacterium with the overexpression vector is as follows: ① Disinfect the electroporation cuvette and lid by soaking them in 75% alcohol for 2 hours. Remove them from the laminar flow hood and place them on clean filter paper to drain the water. After the alcohol has completely evaporated, pre-cool them in ice. ② Take EHA105 Agrobacterium competent cells (Coolaber, Beijing) from a -80℃ freezer. Gently squeeze the competent cells with your fingers until they partially melt, then place them on ice to allow them to completely melt. ③ Pipette 1000 ng of recombinant plasmid POX-EcGSTT1 into the EHA105 competent cells. Gently aspirate and mix, then transfer the mixture completely into the electroporation cuvette and cover it. ④ Set the parameters of the electroporator (Eppendorf, Germany) to 2.5 kV and 5 ms. Wipe the water off the electroporation cuvette and quickly place it in the electroporation tank for electroporation. After electroporation, quickly place it on ice and add 700 ml of... ⑤ Collect bacterial cells by centrifugation at 4000 rpm for 1 minute, reserving approximately 100 μL of supernatant. Mix this supernatant with the bacterial cells and spread it onto YEB medium containing 50 μg / mL kanamycin and 20 μg / mL rifampin. Incubate at 28°C for 2-3 days. ⑥ Perform colony PCR on single colonies. Determine if plasmid transfer was successful and select positive transformants for later use.

[0031] (3) Induction of rice callus. ZH11 rice seeds were dehulled, and mature and healthy seeds were selected for disinfection and induction. The specific steps are as follows: ① Place the rice seeds in a 50mL centrifuge tube, add an appropriate amount of 75% ethanol, shake for about 60s, pour out the ethanol, and wash with sterile water 5-7 times; ② Add 2.5% sodium hypochlorite to the 50mL centrifuge tube, add 1 drop of Tween 20, shake for 15 minutes, pour out the sodium hypochlorite, and wash with sterile water 5-7 times; ③ Add an appropriate amount of 2.5% sodium hypochlorite again, shake for 15 minutes, pour out the sodium hypochlorite, and wash with sterile water 7-10 times; ④ Take out the seeds and place them on filter paper to absorb the moisture, place them on N6D induction medium, and induce at 30℃ in the dark for 10-12 days.

[0032] (4) Agrobacterium infection of callus and co-culture. The positive EHA105 transformant in step (2) was shaken in YEP liquid medium until turbid, and then added to 50 mL of YEP liquid medium at a ratio of 1:50 and shaken vigorously until OD600≈0.6. The bacterial cells were collected and diluted with AAM medium to OD600=0.1. The dense and hard callus in step (3) was selected and added to it. After soaking for 2 minutes, it was taken out and placed on filter paper to absorb the water. A filter paper was placed on 2N6 medium, 500 μL of AAM medium was added, and the above-mentioned callus that had been absorbed of water was placed on the filter paper. It was cultured in the dark at 30℃ for 3 days.

[0033] (5) Screening of positive callus and determination of sensitivity to haloxyfop-R-methyl. After co-culturing for 3 days in step (2), the callus tissue was washed several times with sterile water and then dried with filter paper. It was then placed in N6D medium for subculture. Antibiotics were added to the medium: 1 mL of hygromycin (50 mg / L) + 1 mL of thiazomycin (250 mg / L) + 0.6 mL of termethin (200 mg / L) to 1 L of medium. Determination of herbicide resistance of transgenic callus tissue: The co-cultured callus tissue was placed on a selection medium containing hygromycin (50 mg / L) and cultured in the dark for 14 days. Then it was transferred to a freshly prepared selection medium and screened for another 14 days. From the resistant callus tissue that grew after two rounds of screening, the milky yellow and dense resistant callus tissue was selected for herbicide tolerance test. Using transgenic rice callus overexpressing POX-GFP as a control, the resistance of transgenic rice callus overexpressing POX-EcGSTT1 to AOPP herbicide (quizalofop-p-ethyl) was observed.

[0034] Figure 4 This image shows the growth status of rice callus transfected with GFP and EcGSTT1 in Example 3 of this invention on N6D medium containing different concentrations of haloxyfop-R-methyl. The results showed that, compared with GFP, rice callus overexpressing EcGSTT1 exhibited better growth at haloxyfop-R-methyl concentrations of 25, 50, 100, 200, and 400 nM. This indicates that EcGSTT1 can confer resistance to haloxyfop-R-methyl in rice callus.

[0035] The culture medium scheme used in this invention: ①YEP liquid culture medium formulation: ②YEB solid culture medium formula: ③ N6D culture medium formulation: ④ 2N6 culture medium formula: ⑤AAM culture medium formula: Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A herbicide-resistant barnyardgrass glutathione-S-transferase gene, EcGSTT1, characterized in that, The nucleotide sequence is SEQ ID NO:1, or a sequence that has at least 80% identity with SEQ ID NO:

1.

2. A protein polypeptide encoded by the barnyardgrass glutathione-S-transferase gene EcGSTT1 as described in claim 1, characterized in that, The amino acid sequence is SEQ ID NO:2, or a sequence that has at least 80% identity with SEQ ID NO:

2.

3. A plasmid, characterized in that, It comprises the nucleotide sequence described in claim 1.

4. A recombinant bacterial strain, characterized in that, The plasmid described in claim 3 was transformed into Agrobacterium to obtain a recombinant strain.

5. The application of the barnyardgrass glutathione-S-transferase gene EcGSTT1 as described in claim 1, or the protein polypeptide as described in claim 2, or the plasmid as described in claim 3, or the recombinant strain as described in claim 4 in plant herbicide resistance.

6. The application as described in claim 5, characterized in that, Genes are expressed in plants through transgenic technology, thereby enabling plants to acquire herbicide resistance, or serving as screening genes for plant transgenes.

7. The application of the barnyardgrass glutathione-S-transferase gene EcGSTT1 as described in claim 1, or the protein polypeptide as described in claim 2, or the plasmid as described in claim 3, or the recombinant strain as described in claim 4 in the breeding of herbicide-resistant transgenic plants.

8. The application as described in claim 5 or 7, characterized in that, The plants mentioned are cotton, corn, soybeans, rice, wheat, sorghum, turfgrass, or pasture grass.

9. The application as described in claim 5 or 7, characterized in that, The herbicide is an aryloxyphenoxypropionate herbicide.

10. The application as described in claim 9, characterized in that, The aryloxyphenoxypropionate herbicide is at least one of the following: haloxyfop-R-methyl, quizalofop-P-ethyl, haloxyfop-R-methyl, quizalofop-P-ethyl, clodinafop-P-methyl, and cyhalofop-P-ethyl.

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