Ecgst23 and application thereof

Abstract

The present application relates to the field of eukaryotic expression and plant genetic engineering, and particularly relates to a barnyardgrass oxadiazyl resistance gene EcGST23 and application thereof, the nucleotide sequence of the gene EcGST23 is shown as SEQ ID No. 1, and the amino acid sequence thereof is shown as SEQ ID No. 2. The present application verifies the function of the gene EcGST23 in the oxadiazyl resistance of barnyardgrass, and provides a theoretical basis for the management of resistant barnyardgrass. The present application introduces the barnyardgrass oxadiazyl resistance gene EcGST23 into corn, and the drug resistance phenotype of the transgenic corn is significantly improved, indicating that the gene EcGST23 can be used to improve the drug resistance of crops.

Description

Technical Field

[0001] This invention relates to the fields of eukaryotic expression and plant genetic engineering, specifically to a barnyardgrass gene resistant to oxazolidinyl sulfadiazine. EcGST23 And its applications. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the most important food crops for humans, with over 50% of the world's population relying on it as a staple food. In 2021, my country's rice planting area was approximately 30 million hectares, with a yield of about 214 million tons (www.fao.org). At every stage of rice growth, weeds are the most significant biological constraint, leading to severe economic losses (Chauhan 2010). Barnyard grass (Echinochloa crus-galli) Echinochloa crus-galli Barnyard grass (L.) is one of the most damaging noxious weeds in rice paddies, and its control is mainly based on chemical methods. However, with the extensive and inappropriate use of herbicides, barnyard grass has developed resistance to many herbicides (Wilson 2014, Dong Liyao 2018). The occurrence of resistant barnyard grass and the increasing degree of resistance year by year seriously threaten my country's food security. Therefore, exploring the herbicide-resistant genes of barnyard grass is of great significance for the scientific management of resistant barnyard grass and the safe production of rice.

[0003] GST, one of the three major enzymes involved in herbicide detoxification metabolism in plants, is a key phase II detoxification enzyme downstream of P450 enzymes in cellular metabolism. It catalyzes the formation of conjugated complexes between herbicides or herbicide primary metabolites and GSH, generating non-toxic or low-toxic secondary metabolites, thus reducing herbicide damage to plants. GST was first discovered in maize in the 1970s to participate in herbicide coupling reactions, detoxifying and metabolizing atrazine in maize through the conjugation reaction of glutathione and atrazine (Shimabukuro 1970). Many reports have shown that GST participates in the detoxification metabolism of herbicides in plants, such as the overexpression of soybean in tobacco. GmGSTU4 Genes can enhance plant resistance to metolachlor and diphenyl ether herbicides (Benekos et al 2010), and overexpression of *Bretschneidera sinensis* in *Brachys edulis* can improve resistance. GSTU17 and GT73C1 Genes can enhance plant resistance to mesosulfuron-methyl and quizalofop-P-ethyl (Li Wei 2018). Furthermore, studies have found that rice GSTU3 and GSTU4 proteins catalyze the binding activity of GST with chlorpyrifos herbicides (metolachlor and acetochlor), indicating that GST plays a crucial role in the detoxification metabolism of chlorpyrifos in rice (Lee 2011, Jo 2011). Therefore, in-depth research into the detoxification metabolic function of GST in plants is of great significance for safe crop production and the management of resistant weeds.

[0004] At present, the metabolic resistance mechanism of barnyard grass to metamifop is unclear, and the resistance barnyard grass control measures cannot be developed, therefore, it is crucial to determine which GST gene participates in the drug resistance of barnyard grass to metamifop. SUMMARY

[0005] The present application aims to overcome the deficiencies of the prior art, and provides a barnyard grass anti-metamifop gene EcGST23 and application thereof.

[0006] To achieve the above-mentioned purpose, the technical scheme designed by the present application is as follows:

[0007] The present application relates to a barnyard grass anti-metamifop protein EcGST23, and the amino acid sequence thereof is:

[0008] (1) a protein consisting of the amino acid sequence shown in SEQ ID No. 2; or

[0009] (2) an amino acid sequence having 95-100% homology with the amino acid sequence defined by the sequence SEQ ID No. 2, encoding a same functional protein.

[0010] (3) a protein derived from (1) by increasing, deleting or replacing one or more amino acids of the amino acid sequence shown in SEQ ID No. 2 and having equivalent activity.

[0011] The present application uses the reference sequence in the database and the reverse transcription PCR technology in combination to obtain a barnyard grass anti-metamifop gene EcGST23 with a full-length CDS sequence of 672 bp, and the nucleotide sequence thereof is shown in SEQ ID No. 1. The protein encoded by the gene has a total of 223 amino acids, and the amino acid sequence thereof is shown in SEQ ID No. 2.

[0012] It should be clear that the skilled person in the art can substitute, delete and / or increase one or more amino acids of the amino acid sequence disclosed in the present application without affecting the biological activity of the protein, and the protein sequence homology is more than 90%, and the mutant sequence of the protein is obtained. Therefore, the present application also includes the derived protein with high homology and biological activity obtained by substituting, deleting and / or increasing one or more amino acids of the amino acid sequence shown in SEQ ID No. 2.

[0013] The present application includes the nucleotide sequence encoding the above-mentioned protein.

[0014] In addition, it should be understood that, in view of the characteristics of codon complementarity and species having codon bias, the skilled person in the art can use the codons suitable for the expression of specific species as needed.

[0015] The gene encoding the oxadiazyl resistance gene of Echinochloa crusgalli EcGST23 The nucleotide sequence of the gene is shown as SEQ ID No. 1. The protein encoded by the gene has 223 amino acids, and the amino acid sequence is shown as SEQ ID No. 2.

[0016] The gene and protein of the present application can be cloned or isolated from Echinochloa crusgalli, or obtained by a sequence chemical synthesis method.

[0017] The present application also provides a primer pair for obtaining the above-mentioned gene EcGST23 , and the primer pair is:

[0018] primer F: 5'-ATGGCAGACAAGGGCGTG-3',

[0019] primer R: 5'-CTGCGCGGAGAGCAGCTG-3'.

[0020] The present application also provides an Echinochloa crusgalli EcGST23 eukaryotic expression vector, wherein the Echinochloa crusgalli EcGST23 eukaryotic expression vector is a eukaryotic expression vector containing the above-mentioned EcGST23 gene, i.e. named pBacPAK8- EcGST23 , wherein the eukaryotic expression vector is: pBacPAK8.

[0021] The present application also provides a maize EcGST23 overexpression vector, wherein the maize EcGST23 overexpression vector is an overexpression vector containing the above-mentioned EcGST23 gene, i.e. named pEGOEPubi-B- EcGST23 GFP, wherein the overexpression vector is: pEGOEPubi-B-GFP.

[0022] The present application also provides a host cell containing the above-mentioned Echinochloa crusgalli EcGST23 eukaryotic expression vector, and the host cell is an Sf9 cell.

[0023] application of one of the following in improving the drug resistance of Echinochloa crusgalli, wherein,

[0024] (1) the above-mentioned gene EcGST23 ;

[0025] (2) the above-mentioned Echinochloa crusgalli EcGST23 eukaryotic expression vector;

[0026] (3) the above-mentioned maize EcGST23 overexpression vector;

[0027] (4) the above-mentioned host cell.

[0028] The following are applications in the cultivation of new drug-resistant varieties, among which,

[0029] (1) The above Gene EcGST23 ;

[0030] (2) The above-mentioned barnyard grass EcGST23 eukaryotic expression vectors;

[0031] (3) The corn mentioned above EcGST23 Overexpression vector;

[0032] (4) The host cells mentioned above.

[0033] As a preferred option, the new variety is barnyard grass or corn.

[0034] The beneficial effects of this invention are:

[0035] 1. This invention verifies the gene in barnyardgrass that resists oxazolidinone. EcGST23 The study of the function of barnyardgrass in developing resistance to oxazolidinyl provides a theoretical basis for the management of resistant barnyardgrass.

[0036] 2. This invention utilizes the oxazolidinium-resistant gene from barnyardgrass. EcGST23 When introduced into maize, the herbicide-resistant phenotype in transgenic maize was significantly enhanced, indicating that the gene... Figure 1 It can be used to improve crop resistance to pesticides. Attached Figure Description

[0037] EcGST23 This is an electrophoresis image used to verify the double enzyme digestion of the eukaryotic expression vector.

[0038] In the diagram, M stands for Marker, and lane 1 is pBacPAK8- EcGST YqjG Double digestion product, lane 2 is pBacPAK8- EcGST23 Double enzyme digestion product, lane 3 contains pBacPAK8- EcGST YqjG Plasmid, lane 4 is pBacPAK8- Figure 2 plasmids;

[0039] Figure 3 This is a graph showing the recombinant protease activity assay.

[0040] In the figure, "**" indicates a highly significant difference compared to blank cells. p <0.01);

[0041] Figure 4 This is a graph showing the in vitro metabolism of oxazolidinone;

[0042] Figure 5 This is a diagram showing the in vitro metabolism of recombinant protein EcGST23 with oxazolidinyl sulfadiazine.

[0043] In the figure, "**" indicates a highly significant difference compared to blank cells. p <0.01);

[0044] EcGST23 To verify the electrophoresis image after double enzyme digestion of the overexpression vector,

[0045] In the diagram, M stands for Marker.

[0046] Lane 1 is pEGOEPubi-B- EcGST YqjG GFP double digestion product,

[0047] Lane 2 is pEGOEPubi-B- EcGST23 GFP double digestion product,

[0048] Lane 3 is pEGOEPubi-B- EcGST YqjG GFP plasmid,

[0049] Lane 4 is pEGOEPubi-B- Figure 6 GFP plasmid;

[0050] EcGST23 Corn with no pesticide application and corn with pesticide application Figure 7 Gene expression level map

[0051] Note: "**" indicates that the differences between different maize varieties under the same treatment are extremely significant. p <0.01);

[0052] EcGST23 For the transfer EcGST23 A graph showing the sensitivity of maize plants to oxazolidinone. Detailed Implementation

[0053] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0054] Example 1: Barnyardgrass resistance gene to oxazolidinyl sulfadiazine EcGST23 get

[0055] Transcriptome sequencing and real-time quantitative PCR analysis revealed... EcGST23 It may be involved in the herbicide resistance of barnyardgrass to oxazolidinyl. Then, primer pairs were designed using the reference genome sequence of barnyardgrass:

[0056] Primer F: 5'-ATGGCAGACAAGGGCGTG-3',

[0057] Primer R: 5'-CTGCGCGGAGAGCAGCTG-3';

[0058] Using barnyardgrass cDNA as a template, PCR amplification was performed; the PCR product was obtained, and sequencing revealed the barnyardgrass resistance gene to oxazolidinyl sulfadiazine. EcGST23 ( EcGST23 The full-length CDS sequence), its nucleotide sequence is shown in SEQ ID No. 1:

[0059] ATGGCAGACAAGGGCGTGAAGGTGTTTCGGCTGTGGGCGAGCCCGATGGCGATCCGGGTGGAGTGGGCGCTCCGGCTGAAGGGCGTGGAGTACGAGTACGTCGACGAGGACCTCGCCAACAAGAGCGCCGACCTGCTCCGCTACAACCCGGTGACCAAGAAGGTGCCC GTGCTCGTCCACGACGGCAGGCCCATCGCCGAGTCCACCATCATCGTCGAGTACATCGACGAGGTCTGGAAGGGCGGCCACCCCATCATGCCGGCCGACCCCTACGAGCGTGCTCAGGCGAGGTTCTGGGCCAGGTACGCTGAAGACAAGTGCAACGCGGCTCTGTAC CCGATCTTCACTGCGACCGGCGAGGCTGGGCAAGGTGGTGAGCGAGGCGCAGCAGTGCCTCAAGACCCTGGAGACGGCGCTGGAGGGGAAGAAGTTCTTCGGCGGCGACGCCGTGGCTACCTCGACATCGTCGTCGGGTGGTTCGCGCACTGGCTGCCGGTCGTC GAGGAGGTGTGCGGCGTCAGCGTCGTCACCGACGAGGAGCTGCCGCTGATGTATGCCTGGTTCGGCCGGATCCTCGCCGTCGACGCGCTAAAGGCCGCCCTGCCCGACAGGGACAGGCTCCTCGCGGCCAACAAGGCCCGCCGCGAGCAGCTGCTCTCCGCGCAGTAA

[0060] The amino acid sequence of its protein EcGST23 is shown in SEQ ID No. 2:

[0061] MADKGVKVFRLWASPMAIRVEWALRLKGVEYEYVDEDLANKSADLLRYNPVTKKVPVLVHDGRPIAESTIIVEYIDEVWKGGHPIMPADPYERAQARFWARYAEDKCNAALY PIFTATGEALGKVVSEAQQCLKTLETALEGKKFFGGDAVGYLDIVVGWFAHWLPVVEEVCGVSVVTDEELPLMYAWFGRILAVDALKAALPDRDRLLAANKARREQLLSAQ*.

[0062] Example 2: Barnyard grass EcGST23 Construction of eukaryotic expression vectors

[0063] Based on the expression vector pBacPAK8, enzyme digestion and ligation were performed to... EcGST23 The cDNA fragment was inserted into the middle of pBacPAK8, and double enzyme digestion electrophoresis and sequencing analysis showed that barnyard grass was successfully obtained. Figure 1 eukaryotic expression vectors ( EcGST23 ), that is, named pBacPAK8- EcGST23 The specific experimental steps are as follows:

[0064] 1. EcGST23 Gene amplification:

[0065] according to 2x phanta Buffer A pair of primers was designed based on the gene sequence for PCR amplification. The primer sequences are as follows:

[0066] EcGST23-F0: 5'-GCTCTAGAGCATGGCAGACAAGGGCGTG-3',

[0067] EcGST23-R0:

[0068] 5'-GGAATTCCTCAATGATGATGATGATGATGCTGCGCGGAGAGCAGCTG-3';

[0069] Gene amplification was performed using Vazyme Phanta Max Super-Fidelity DNA polymerase to obtain PCR products. The reaction system was as follows (total volume 50 μL):

[0070] dNTP 25ul EcGST23-F0 1ul EcGST23-R0 2ul Phanta max 2ul cDNA (Echinochloa crus-galli) 1ul 10×Enhancer 2ul ddH2O 17ul

[0071] The reaction program was as follows: 95℃ for 3 min; 95℃ for 15 s; 60℃ for 15 s; 72℃ for 60 s; 72℃ for 5 min; 35 cycles.

[0072] 2. Gel extraction of PCR products: PCR products were extracted using the OMEGA EZNA Gel Extraction Kit;

[0073] 3. Ligation and Transformation: Using the Hieff Clone® Zero TOPO-TA Cloning Kit (Yisheng Company), the gel-recovered product was ligated to the pClone vector. The reaction system was as follows (total volume 10 μL):

[0074] pClone (30 ng / μL) 1ul Gel recovery product 1ul EcGST23 3ul ddH2O 5ul

[0075] After mixing, let stand at room temperature for 5 min. Add the reaction product to competent DH5α cells, place on ice for 10 min, then heat shock in a 42°C water bath for 45 s, then place on ice for 2 min. Add 300 μL of liquid LB medium and incubate at 37°C with shaking at 220 rpm for 1 h. Spread 50 μL of the culture solution onto LB / ampicillin culture dishes and incubate upside down at 37°C overnight. Pick a single colony and incubate in 300 μL of liquid LB medium (ampicillin 100 μg / mL) with shaking at 37°C with shaking for 3 h.

[0076] 4. Sequencing and identification: Colony PCR and sequencing analysis showed that barnyard grass was successfully obtained. EcGST23 expression vector pClone- EcGST23 PCR and sequencing primers are as described in Example 2.1.

[0077] 5. Linearized vector preparation: pClone- was double-digested using the restriction endonucleases Xba I and EcoRI. EcGST23 The pBacPAK8 vector linearizes the cyclic vector;

[0078] 6. Eukaryotic expression vector ligation reaction: The enzyme-digested pClone- EcGST23 The fragment was ligated with the pBacPAK8 fragment to obtain the barnyard grass eukaryotic expression vector pBacPAK8- pClone-fragment Prepare the following reaction system (total volume 10 μL):

[0079] pBacPAK8-fragment 3ul T4 ligase 1ul EcGST23 1ul ddH2O 5ul

[0080] 7. Double enzyme digestion verification: The eukaryotic expression vector was verified by double digestion with Xba I and EcoR I. Electrophoresis detection after digestion showed the presence of the target band (the size of the digested band was consistent with that of the target gene), indicating that the eukaryotic expression vector pBacPAK8-EcGST23 was successfully constructed.

[0081] Using the AcMNPV virus to express the eukaryotic expression vector pBacPAK8- Figure 2 Transfected into Sf9 cells, protein particles were observed in the cells 5 days after transfection. SDS-PAGE and Western blotting analysis confirmed successful protein expression. Enzyme activity assays showed that the recombinant protein EcGST23 had significantly higher enzyme activity than normal cells, indicating that the recombinant protein EcGST23 possesses catalytic activity. 250 mM GSH The following reaction system (total volume 1000 μL) was prepared using protein and oxazolidinyl sulfadiazine:

[0082] pH6.5 Phosphate Buffer 40ul 1000 ppm metam 450ul Protein 10ul Figure 3 500ul

[0083] The reaction mixture was thoroughly mixed and then incubated on a shaking incubator at 30°C and 220 rpm for 6 hours. After incubation, 800 μL of acetonitrile was added to terminate the reaction, and the reaction mixture was then centrifuged at 10,000 rpm for 10 minutes. After centrifugation, 100 μL of the supernatant was transferred to a 1.5 mL centrifuge tube, and 900 μL of mass spectrometry grade acetonitrile was added. The mixture was thoroughly mixed. 600 μL of the supernatant was filtered through a 0.22 μm filter membrane and transferred to a liquid chromatography sample vial. The reagent content was determined by HPLC-MS.

[0084] The results showed that the concentration of oxazolidinone in the recombinant protein EcGST23 solution was significantly lower than that in the blank cell solution, indicating that the recombinant protein EcGST23 has a metabolic effect on oxazolidinone. EcGST23 ,4).

[0085] Example 3: Corn EcGST23 Construction of overexpression vectors

[0086] Based on the pEGOEPubi-B-GFP vector, enzyme digestion and ligation were performed to... EcGST23 The cDNA fragment was inserted into pEGOEPubi-B-GFP, and double enzyme digestion electrophoresis and sequencing analysis showed that the maize cDNA fragment was successfully obtained. Figure 5 Overexpression vector ( EcGST23 ), that is, named pEGOEPubi-B- EcGST23 -GFP, the specific experimental steps are as follows:

[0087] 1. EcGST23 Gene amplification

[0088] according to cDNA A pair of primers was designed based on the gene sequence for PCR amplification. The primer sequences are as follows:

[0089] EcGST23-F1:

[0090] 5'-ACTAGGGTCTCGCACCATGGCAGACAAGGGCGTGAAGG-3',

[0091] EcGST23-R1:

[0092] 5'-ACTAGGGTCTCCGCCCTGCGCGGAGAGCAGCTG-3';

[0093] Gene amplification was performed using Yeasen 2×Hieff™ PCR Master Mix to obtain the gene PCR product. The reaction system is as follows (total volume 25 μL):

[0094] EcGST23-F1 1ul EcGST23-R1 1ul 2×Hieff™ PCR Master Mix 1ul Bsa 12.5ul ddH2O 9.5ul

[0095] The reaction program was as follows: 98℃ for 2 min; 98℃ for 10 s; 55℃ for 30 s; 72℃ for 90 s; 72℃ for 5 min; 32 cycles;

[0096] 2. Gel extraction of PCR products: PCR products were extracted using the OMEGA EZNA Gel Extraction Kit;

[0097] 3. Enzyme digestion-ligation reaction: using 10×CutSmart Buffer Overexpression vectors were constructed using I-HF and T4 DNA ligase, and the reaction system is as follows (total volume 15 μL):

[0098] 10 mM ATP 1.5ul pEGOEPubi-B-GFP 1.5ul 100 ng Gel recovery product 50 ng I-HF T4 DNA ligase 10U to 15 ul 35U ddH2O EcGST23

[0099] Gently pipette and mix well. React at 37°C for 5 min, then at 20°C for 5 min, for 15 cycles.

[0100] 4. Transformation of recombinant products: Transform DH5α competent cells with the recombinant products, following the guidelines for molecular cloning experiments;

[0101] 5. Sequencing Identification: Colony PCR and plasmid PCR, and sequencing analysis showed that maize was successfully obtained. EcGST23 Overexpression vector pEGOEPubi-B- EcGST23 -GFP; PCR and sequencing primers are as follows:

[0102] UBI-F: 5'-TTAGCCCTGCCTTCATACGC-3'

[0103] eGFP-cx: 5'-GACACGCTGAACTTGTGG-3'

[0104] NOS-R: 5'-ATCATCGCAAGACCGGCAAC-3'

[0105] 6. Double enzyme digestion verification: The overexpression vector was verified by double digestion with Hind III and EcoRI. Electrophoresis detection after digestion showed the presence of the target band (overexpression vector pEGOEPubi-B-). EcGST23 -Double digestion of GFP produces three fragments of 8376, 2319, and 1524 bp (corresponding to the size of the electrophoretic bands), indicating that the overexpression vector pEGOEPubi-B- EcGST23 -GFP was successfully constructed.

[0106] The corn obtained in Example 3 EcGST23 Overexpression vector pEGOEPubi-B- EcGST23 -GFP was sent to Wuhan Aidijing Biotechnology Co., Ltd. for maize genetic transformation, with the maize line B104 as the transformation background. T0 generation transgenic seeds obtained from the company were used to cultivate T1 generation plants. PCR testing of the T1 generation plants confirmed that the target gene had not undergone genetic segregation or loss, ultimately yielding two transgenic seeds containing... EcGST23 Stable genetically inherited positive homozygous transgenic maize families and corresponding non-transgenic maize materials.

[0107] The overexpression vector pEGOEPubi-B- was transformed using the Agrobacterium-mediated transformation method. EcGST23 -GFP was transferred into the immature embryos of maize (after sterilizing the ears, the embryos were removed with an embryo removal knife and placed in a sterile tube containing the infection solution). An Agrobacterium suspension with a suitable OD value was added to the immature maize embryos. After tightening the centrifuge tube cap, the embryos were gently shaken to suspend them and allowed to stand for several minutes. The Agrobacterium suspension was discarded, and the immature embryos were transferred to a co-culture plate. The Agrobacterium suspension on the embryo surface was allowed to dry, completing the low-temperature co-culture. After co-culture, the immature embryos were transferred to a recovery medium for appropriate recovery culture. The immature embryos were then transferred from the recovery medium to a selection medium to obtain resistant callus. The resistant callus was then transferred to a regeneration medium using forceps to induce seedling emergence. After successful induction of T0 seedlings, the seedlings were transferred to a rooting tank for rooting induction. When the seedlings had 3-4 leaves, samples were taken for PCR detection. The primers in Example 4.5 were used to determine... EcGST23 The expression.

[0108] Example 4 EcGST23 Detection of expression levels in transgenic maize plants

[0109] Plant separately β-actinTransgenic maize materials and wild-type maize were used, with and without oxazolidinyl treatment. Maize leaves from three-week-old plants were collected, and RNA was extracted using TaKara RNAiso Plus. The extracted RNA was then reverse transcribed using the Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR reverse transcription kit (Yisheng Company). The extracted cDNA was then used for RT-PCR detection. EcGST23 Genes were used as controls.

[0110] The test results show: genes EcGST23 The expression levels in transgenic maize plants were significantly higher than those in non-transgenic maize, indicating that... Figure 6 Gene successfully transferred into maize ( EcGST23 ).

[0111] The primers used in the detection process are as follows:

[0112] qEcGST23-F: 5'-GTGAAGGTGTTCGGCATGTG-3',

[0113] qEcGST23-R: 5'-CGACGTACTCGTACTCCACG-3';

[0114] β-actin-F: 5'-TTGCCTACATTGCCCTTGACTA-3',

[0115] β-actin-R: 5'-GAACCACCACTGAGGACGACA-3'.

[0116] Example 5 EcGST23 Herbicide resistance phenotype detection in genetically modified maize plants

[0117] Place an appropriate amount of nutrient soil in a pot with an outer diameter of 10 cm. Spray water from the top to saturate the soil, then place the genetically modified [plant / plant]. EcGST23 Corn seeds and non-GMO corn seeds were pressed down about 3 / 4 of their endosperm into the soil, 6 seeds per pot, and the surface was sprayed with water to keep it moist. They were then cultivated in a greenhouse (25℃, photoperiod L:D=16:8), with regular watering. After the corn sprouted, it was appropriately covered with soil. When the corn seedlings reached the 3-4 leaf stage, foliar spraying was performed using an ASS-4 type automatic pesticide spraying system with a TeeJet 8003VS fan nozzle, a spray rate of 450 L / ha, and an operating pressure of 0.28 MPa. Based on the preliminary test results, oxazolidinone was diluted with 0.1% Triton X-100 to a series of concentration gradients (45, 90, 180, 270, 360 g ai ha). -1Each treatment was repeated three times, with a blank control (0.1% Triton X-100 aqueous solution). After treatment, the plants were allowed to stand until the solution on the leaves dried before being transferred to a greenhouse for cultivation. The plant survival rate was recorded 21 days after treatment.

[0118] The results showed that spraying 180, 270, and 360 g ha -1 14 days after the dose of oxazolidinone, switch to EcGST23 The survival rate of transgenic corn was higher than that of non-GMO corn, and this was confirmed by dose-response curve analysis. EcGST23 The resistance multiple of corn was 3.14, indicating that the genetic modification... Figure 7 Corn is resistant to oxazolidinone (Table 1, EcGST23 ).

[0119] Table 1 (Transfer) Population Sensitivity of corn to oxazolidinone

[0120] Upper limit D Lower limit C Slope b Resistance fold RI Lethal dose (g ha -1 ) LD 50 ]]> Correlation coefficient R 2 ]] Non-transgenic Trans 99.49(2.52) -5.60(3.24) -2.29(0.26) 83.84(4.61) 0.99 1 ​ 97.43(0.04) -31.16(0.46) -1.88(0.01) 263.48(0.99) 0.99 3.14

[0121] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A barnyard grass anti-oxazolidinyl chlorpyrifos protein EcGST23, the amino acid sequence of which is shown in SEQ ID No.

2.

2. A barnyardgrass gene encoding the protein EcGST23 of claim 1 for resistance to oxazolidinyl sulfadiazine. EcGST23, Its nucleotide sequence is shown in SEQ ID No.

1.

3. A method for obtaining the oxazolidinyl sulfadiazine resistance gene as described in claim 2 EcGST23 The primer pair is characterized by: The primer pair is: Primer F: 5'-ATGGCAGACAAGGGCGTG-3', Primer R: 5'-CTGCGCGGAGAGCAGCTG-3'.

4. A type of barnyard grass EcGST23 Eukaryotic expression vector, characterized by: The barnyard grass EcGST23 The eukaryotic expression vector contains the gene described in claim 2. EcGST23 The eukaryotic expression vector, named pBacPAK8- EcGST23 The eukaryotic expression vector is pBacPAK8.

5. A type of corn EcGST23 The overexpression vector is characterized by: The corn EcGST23 The overexpression vector contains the substance described in claim 2. EcGST23 The gene overexpression vector is named pEGOEPubi-B- EcGST23- GFP, wherein the overexpression vector is: pEGOEPubi-B-GFP.

6. A type of barnyard grass containing the barnyard grass of claim 4 EcGST23 The host cell for the eukaryotic expression vector is an Sf9 cell.

7. The application of any of the following in the cultivation of maize varieties resistant to oxazolidinone, wherein, (1) The gene according to claim 2 EcGST23 ; (2) The corn as described in claim 5 EcGST23 Overexpression vector.

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