CN118345098B - A corn ACCase mutant gene with herbicide resistance and its application - Google Patents
A corn ACCase mutant gene with herbicide resistance and its application Download PDFInfo
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Abstract
The invention discloses a corn ACCase mutant gene with herbicide resistance and application thereof. Belonging to the technical fields of plant genetic engineering and herbicide-resistant breeding. The corn ACCase mutant gene of the invention is obtained by mutating the nucleotide numbers 5491 and 5492 of wild corn ZmACC gene from C to T. The mutant gene discovered by the invention has the tolerance of acetyl coenzyme A carboxylase inhibitor herbicides haloxyfop-methyl, quizalofop-ethyl and clethodim, and when the mutant gene is applied to corn soybean intercropping, the mutant material shows good tolerance to the herbicide.
Description
Technical Field
The invention relates to the technical fields of plant genetic engineering and herbicide-resistant breeding, in particular to a corn ACCase mutant gene with herbicide resistance and application thereof.
Background
Acetyl-coa carboxylase (ACCase) is capable of catalyzing carboxylation of Acetyl-coa to malonyl-coa, into fatty acid de novo synthesis or lipid metabolism, which is the first step in fatty acid de novo synthesis and is also the rate limiting step in fatty acid de novo synthesis. ACCase in organisms includes both prokaryotic and eukaryotic types, eukaryotic types of ACCase including a biotin carboxylase domain, a biotin carboxy carrier protein domain, and a carboxytransferase domain. The prokaryotic ACCase consists of four subunits of BC, BCCP, alpha-CT and beta-CT, wherein BC, BCCP and alpha-CT are encoded by nuclear genes accC, accB and accA, and beta-CT is encoded by plastid genes accD. The whole catalytic reaction process comprises that BCCP is combined with biotin molecules or carboxybiotin under the catalysis of BC, and further is transferred to acetyl coenzyme A under the catalysis of CT to form malonyl coenzyme A. Dicotyledonous plants have two forms of ACCase, procaryotic ACCase mainly in chloroplasts or plastids, eucaryotic ACCase mainly in cytoplasm, whereas monocotyledonous gramineous plants have only eucaryotic ACCase, whose fatty acid biosynthesis takes place in chloroplasts (plastids).
ACCase is an important target for chemical herbicides, and herbicides targeting ACCase are capable of inhibiting fatty acid synthesis in grasses. The herbicide inhibits the carboxylation reaction of eukaryotic ACCase to generate malonyl-CoA, inhibits the synthesis of fatty acid, selectively prevents and removes gramineous weeds, and is safe for broadleaf crops. The current class of inhibitors developed based on ACCase is broadly divided into three classes, namely, aryloxyphenoxypropionate (Aryloxyphenoxypropanoates, APP), aryloxyphenyl cyclohexanediones (Aryloxyphenylcy-clohexanedione, CHD) and phenylpyrazolines (phenylpyrazolin, DEN). Among them, the developed APP commercial herbicides are more than 20, and representative herbicides are quizalofop-p-ethyl, haloxyfop-methyl, etc. Commercial herbicides of the CHD class have been developed including clethodim, sethoxydim, and clethodim, among which clethodim has the most widely used. The DEN herbicide is mainly used as a post-emergence herbicide for cereal fields, and is used for preventing and killing gramineous weeds, wherein the representative herbicide is pinoxaden, and the herbicide has higher safety to wheat and barley.
In recent years, china is widely examined and used for soybean and corn banded compound planting. However, since corn is a monocotyledonous plant of the Gramineae family and soybean is a dicotyledonous plant of the broad leaf class, the stem and leaf herbicides used for both are fatal to each other, and it is an important problem how to effectively control weeds without affecting the growth of crops while working in the field.
In summary, how to provide a soybean herbicide-compatible resistant corn is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of this, the present invention provides a maize ACCase mutant gene having herbicide resistance and uses thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A maize ACCase mutant gene with herbicide resistance is obtained by mutating the nucleotide numbers 5491 and 5492 of wild maize ZmACC gene from C to T.
Further, the nucleotide sequence is shown as SEQ ID NO. 1.
An expression cassette comprising the maize ACCase mutant gene described above.
A recombinant vector comprising the maize ACCase mutant gene described above.
The corn ACCase mutant gene, the expression cassette or the recombinant vector are applied to the aspect of corn herbicide resistance.
Further, the herbicide is an ACCase inhibitor herbicide.
Further, the herbicide is quizalofop-p-ethyl, haloxyfop-methyl or clethodim.
A method for conferring herbicide resistance to corn comprising the above-described corn ACCase mutant gene.
Compared with the prior art, the mutant gene has the advantages that the mutant gene has the tolerance of acetyl-CoA carboxylase inhibitor herbicides such as haloxyfop-methyl, quizalofop-ethyl and clethodim, and the mutant gene shows good tolerance to herbicides when being applied to corn and soybean intercropping.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a phylogenetic tree of ACC proteins according to example 1 of the present invention;
FIG. 2 is a graph showing the mutant peaks of ACC protein in example 1 of the present invention;
FIG. 3 is herbicide resistance at seedling stage of ACC mutant material M1 in example 2 of the present invention;
FIG. 4 shows the results of the field test in example 3 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The materials used in the embodiment of the invention are as follows:
Plant material
The plant material used in the invention is maize KN5585 inbred line, and genetic transformation material and progeny material of the transformation material are entrusted to non-rice biotechnology (Jiangsu) limited company.
Vectors and strains
Vector the gene editing base vector pCAMBIA3301 stored by the present laboratory.
Strain Trans1-T1 PHAGE RESISTANT ChemicallyCompetent Cell (Beijing full gold Biotechnology Co., ltd., CD 501-02).
Enzymes:
high-fidelity PCR enzyme KAPAHiFi HotStart ReadyMixPCRKit (KK 2602, beggar megahua teclmique, inc.);
Homologous recombination enzyme HiFi DNAAssembly Master Mix (Beijing-Bailingke Biotechnology Co., ltd., E2621L);
HindIII restriction endonuclease (Beijing-Bailingke Biotechnology Co., ltd., R0104);
MauBI restriction endonuclease (Beijing-Bailingke Biotechnology Co., ltd., R0113S).
The kit comprises:
QIAquick Gel Extraction Kit (250) (Beijing Jiang Xinbo Rev Biotechnology Co., ltd., 28706);
AxyPrep plasmid extraction kit (Axygen, AP-MN-P-250).
Example 1
1. Acquisition of the maize ACCase Gene
The homologous gene ZmACC1 (gene number: zm00001d 004125) of corn is obtained by comparing ACC protein sequences of different species with phylogenetic tree (figure 1) and the nucleotide sequence is shown as SEQ ID NO. 2.
atgtcacagcttggattagccgcagctgcctcaaaggccttgccactactccctaatcgccagagaagttcagctgggactacattctcatcatcttcattatcgaggcccttaaacagaaggaaaagccgtactcgttcactccgtgatggcggagatggggtatcagatgccaaaaagcacagccagtctgttcgtcaaggtcttgctggcattatcgacctcccaagtgaggcaccttccgaagtggatatttcacatggatctgaggatcctagggggccaacagattcttatcaaatgaatgggattatcaatgaaacacataatggaagacatgcctcagtgtccaaggttgttgaattttgtgcggcactaggtggcaaaacaccaattcacagtatattagtggccaacaatggaatggcagcagcaaaatttatgaggagtgtccggacatgggctaatgatacttttggatctgagaaggcaattcaactcatagctatggcaactccggaagacatgaggataaatgcagaacacattagaattgctgaccaattcgtagaggtgcctggtggaacaaacaataataactacgccaatgttcaactcatagtggagatggcacaaaaactaggtgtttctgctgtttggcctggttggggtcatgcttctgagaatcctgaactgccagatgtattgaccgcaaaagggatcgtttttcttggcccacctgcatcatcaatgaatgctttgggagataaggtcggctcagctctcattgctcaagcagccggggtcccaactcttgcttggagtggatcacatgttgaagttccattagagtgctgcttagacgcgatacctgaggagatgtatagaaaagcttgcgttactaccacagaggaagcagttgcaagttgtcaagtggttggttatcctgccatgattaaggcatcctggggaggtggtggtaaaggaataagaaaggttcataatgatgatgaggttagagcgctgtttaagcaagtacaaggtgaagtccctggctccccaatatttgtcatgaggcttgcatcccagagtcggcatcttgaagttcagttgctttgtgatcaatatggtaatgtagcagcacttcacagtcgtgattgcagtgtgcaacggcgacaccagaagattattgaagaaggtccagttactgttgctcctcgtgagacagttaaagcacttgagcaggcagcaaggaggcttgctaaggctgtgggttatgttggtgctgctactgttgagtatctttacagcatggaaactggagactactattttctggaacttaatccccgactacaggttgagcatccagtcaccgagtggatagctgaagtaaatctgcctgcagctcaagttgctgttggaatgggcatacctctttggcagattccagaaatcagacgtttctatggaatggactatggaggagggtatgacatttggaggaaaacagcagctcttgctacaccatttaattttgatgaagtagattctcaatggccaaagggccattgtgtagcagttagaattactagtgaggacccagatgatggtttcaaacctactggtgggaaagtgaaggagataagttttaaaagcaagcctaatgtttgggcctacttctcagtaaagtctggtggaggcattcatgaatttgctgattctcagttcggacatgtttttgcatatgggctctctagatcagcagcaataacaaacatgactcttgcattaaaagagattcaaattcgtggagaaattcattcaaatgttgattacacagttgacctcttaaatgcttcagactttagagaaaacaagattcatactggttggctcgacaccagaatagctatgcgtgttcaagctgagaggcccccatggtatatttcagtggttgggggtgctttatataaaacagtaaccaccaatgcagccactgtttctgaatatgttagttatctcaccaagggccagattccaccaaagcatatatcccttgtcaattctacagttaatttgaatatagaagggagcaaatacacaattgaaactgtaaggactggacatggtagctacaggttgagaatgaatgattcaacagttgaagcgaatgtacaatctttatgtgatggtggcctcttaatgcagttggatggaaacagccatgtaatttatgcagaagaagaagctggtggtacacggcttcagattgatggaaagacatgtttattgcagaatgaccatgatccatcaaagttattagctgagacaccctgcaaacttcttcgtttcttggttgctgatggtgctcatgttgatgcggatgtaccatacgcggaagttgaggttatgaagatgtgcatgcctctcttgtcacctgcttctggtgtcattcattgtatgatgtctgagggccaggcattgcaggctggtgatcttatagcaaggttggatcttgatgacccttctgctgtgaaaagagctgagccatttgatggaatatttccacaaatggagctccctgttgctgtctctagtcaagtacacaaaagatatgctgcaagtttgaatgctgctcgaatggtccttgcaggatatgagcacaatattaatgaagtcgttcaagatttggtatgctgcctggacaaccctgagcttcctttcctacagtgggatgaacttatgtctgttctagcaacgaggcttccaagaaatctcaagagtgagttagaggataaatacaaggaatacaagttgaatttttaccatggaaaaaacgaggactttccatccaagttgctaagagacatcattgaggaaaatctttcttatggttcagagaaggaaaaggctacaaatgagaggcttgttgagcctcttatgaacctactgaagtcatatgagggtgggagagagagccatgcacattttgttgtcaagtctcttttcgaggagtatcttacagtggaagaactttttagtgatggcattcagtctgacgtgattgaaacattgcggcatcagcacagtaaagacctgcagaaggttgtagacattgtgttgtctcaccagggtgtgaggaacaaagctaagcttgtaacggcacttatggaaaagctggtttatccaaatcctggtggttacagggatctgttagttcgcttttcttccctcaatcataaaagatattataagttggcccttaaagcaagtgaacttcttgaacaaaccaaactaagtgaactccgtgcaagcgttgcaagaagcctttcggatctggggatgcataagggagaaatgagtattaaggataacatggaagatttagtctctgccccattacctgttgaagatgctctgatttctttgtttgattacagtgatcgaactgttcagcagaaagtgattgagacatacatatcacgattgtaccagcctcatcttgtaaaggatagcatccaaatgaaattcaaggaatctggtgctattactttttgggaattttatgaagggcatgttgatactagaaatggacatggggctattattggtgggaagcgatggggtgccatggtcgttctcaaatcacttgaatctgcgtcaacagccattgtggctgcattaaaggattcggcacagttcaacagctctgagggcaacatgatgcacattgcattattgagtgctgaaaatgaaagtaatataagtggaataagtgatgatcaagctcaacataagatggaaaagcttagcaagatactgaaggatactagcgttgcaagtgatctccaagctgctggtttgaaggttataagttgcattgttcaaagagatgaagctcgcatgccaatgcgccacacattcctctggttggatgacaagagttgttatgaagaagagcagattctccggcatgtggagcctcccctctctacacttcttgaattggataagttgaaggtgaaaggatacaatgaaatgaagtatactccttcgcgtgaccgccaatggcatatctacacactaagaaatactgaaaaccccaaaatgttgcatagggtgtttttccgaactattgtcaggcaacccaatgcaggcaacaagtttacatcggctcagatcagcgacgctgaagtaggatgtcccgaagaatctctttcatttacatcaaatagcatcttaagatcattgatgactgctattgaagaattagagcttcatgcaattaggacaggtcattctcacatgtatttgtgcatactgaaagagcaaaagcttcttgacctcattccattttcagggagtacaattgttgatgttggccaagatgaagctaccgcttgttcacttttaaaatcaatggctttgaagatacatgagcttgttggtgcaaggatgcatcatctgtctgtatgccagtgggaggtgaaactcaagttggactgtgatggccctgcaagtggtacctggagagttgtaactacaaatgttactggtcacacctgcaccattgatatataccgagaagtggaggaaatagaatcgcagaagttagtgtaccattcagccacttcgtcagctggaccattgcatggtgttgcactgaataatccatatcaacctttgagtgtgattgatctaaagcgctgctctgctaggaacaacagaacaacatattgctatgattttccgctggcctttgaaactgcactgcagaagtcatggcagtccaatggctctactgtttctgaaggcaatgaaaatagtaaatcctacgtgaaggcaactgagctagtgtttgctgaaaaacatgggtcctggggcactcctataattccgatggaacgccctgctgggctcaacgacattggtatggtcgcttggatcatggagatgtcaacacctgaatttcccaatggcaggcagattattgttgtagcaaatgatatcactttcagagctggatcatttggcccaagggaagatgcattttttgaaactgtcactaacctggcttgcgaaaggaaacttcctcttatatacttggcagcaaactctggtgctaggattggcatagctgatgaagtaaaatcttgcttccgtgttggatggtctgacgaaggcagtcctgaacgagggtttcagtacatctatctgactgaagaagactatgctcgcattagctcttctgttatagcacataagctggagctagatagtggtgaaattaggtggattattgactctgttgtgggcaaggaggatgggcttggtgtcgagaacatacatggaagtgctgctattgccagtgcttattctagggcatatgaggagacatttacacttacatttgtgactgggcggactgtaggaataggagcttatcttgctcgacttggtatacggtgcatacagcgtcttgaccagcctattattttaacagggttttctgccctgaacaagctccttgggcgggaagtgtacagctcccacatgcagcttggtggtcctaagatcatggcgaccaatggtgttgtccacctcactgttccagatgaccttgaaggtgtttccaatatattgaggtggctcagctatgttcctgcaaacattggtggacctcttcctattaccaaacctctggaccctccagacagacctgttgcttacatccctgagaacacatgcgatccacgtgcagctatctgtggtgtagatgacagccaagggaaatggttgggtggtatgtttgacaaagacagctttgtggagacatttgaaggatgggcaaaaacagtggttactggcagagcaaagcttggaggaattcctgtgggcgtcatagctgtggagacacagaccatgatgcagatcatccctgctgatccaggtcagcttgattcccatgagcgatctgtccctcgtgctggacaagtgtggttcccagattctgcaaccaagaccgctcaggcattattagacttcaaccgtgaaggattgcctctgttcatcctggctaattggagaggcttctctggtggacaaagagatctctttgaaggaattcttcaggctgggtcaacaattgtcgagaaccttaggacatataatcagcctgcttttgtgtacattcctatggctggagagcttcgtggaggagcttgggttgtggtcgatagcaaaataaatccagaccgcattgagtgttatgctgaaaggactgccaaaggtaatgttctcgaacctcaagggttaattgaaatcaagttcaggtcagaggaactccaagactgtatgggtaggcttgacccagagttgataaatctgaaagcaaaactccaagatgtaaatcatggaaatggaagtctaccagacatagaagggattcggaagagtatagaagcacgtacgaaacagttgctgcctttatatacccagattgcaatacggtttgctgaattgcatgatacttccctaagaatggcagctaaaggtgtgattaagaaagttgtagactgggaagaatcacgctcgttcttctataaaaggctacggaggaggatcgcagaagatgttcttgcaaaagaaataaggcagatagtcggtgataaatttacgcaccaattagcaatggagctcatcaaggaatggtaccttgcttctcaggccacaacaggaagcactggatgggatgacgatgatgcttttgttgcctggaaggacagtcctgaaaactacaaggggcatatccaaaagcttagggctcaaaaagtgtctcattcgctctctgatcttgctgactccagttcagatctgcaagcattctcgcagggtctttctacgctattagataagatggatccctctcagagagcgaagtttgttcaggaagtcaagaaggtccttgattga,SEQ ID NO:2.
2. Acquisition of maize ACCase mutation site and construction of plant expression vector
Nucleotide sequence 5'-GACCAGCCTATTATTTTAACAGG-3', SEQ ID NO. 3 of maize ZmACC1 gene was selected as the target site. Base editing was performed using CRISPR/Cas9 mediated precision editing.
The gene editing basic vector pCAMBIA3301 stored in the laboratory is used as a framework vector for construction.
The ZmACC-CBE base editing vector elements mainly comprise:
(1) nCas9 (D10A) fused to the protein expression cassette of evoCDA1 deaminase, which is initiated by the promoter of the maize Ubiquitin gene, uses NOS terminator as terminator. (2) A nuclear localization signal sequence NLS (base sequence: 5 'AAGCCCCGCCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAG-3' SEQ ID NO: 4) is added to the N-terminal of evoCDA1 deaminase sequence. (3) evoCDA1 deaminase is linked to a ugi (4) esgRNA (base sequence ：5'-GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTAT CAACTTGAACAAGTGGCACCGAGTCGGTGCTTTTTTcccggg-3',SEQ ID NO:6) is started by RNA polymerase class III promoter ZmU-2, ending with poly T) with a 33 amino acid linker peptide (amino acid sequence ：TDAEYVRIHEKLDIYTFKKQFSNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWVCKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMFQVKILHTTKSPAV,SEQ ID NO:5) linked to the N-terminus of nCas (D10A) and the C-terminus of nCas (D10A).
The target site sequence was inserted between ZmU-2 promoter and esgRNA backbone sequence by homologous recombination. The target site amplified fragments ZmU-2, esgRNA amplification primers were as follows:
U6-HF:5’-CGGGTCACGCTGCACTGCAGAAGCTTCTAATTGGCCCTTACAAAAT-3’,SEQ ID NO:7;
U6-HR:5’-GAAACGTTAAAATAATAGGCTGGTCGGAGCGGTGGTCGCAGCTGAAC TTATAAG-3’,SEQ ID NO:8;
esgHF:5’-GACCAGCCTATTATTTTAACGTTTCAGAGCTATGCTGGAAACAGC-3’,SEQ ID NO:9;
esgHR:5’-CCCGGGAAAAAAGCACCGACTCGGTGCC-3’,SEQ ID NO:10。
After homologous recombination, the correctly sequenced cloning plasmid was transformed into Agrobacterium EHA105, and colony PCR was used for maize immature embryo transformation after verification of correctness.
3. Maize immature embryo genetic transformation
The correct plasmid is guided into agrobacterium EHA105 strain, and the processes of embryo stripping infection, co-culture, callus induction, differentiation into seedlings and induction rooting are carried out to finally obtain positive transformed seedlings. The method comprises the following specific steps:
agrobacterium-mediated maize genetic transformation
(1) Treatment of acceptor materials
Soaking seeds of a maize inbred line KN5585 in water for 4 hours at 37 ℃, germinating the seeds in a plate at 28 ℃ for 48 hours, placing the germinated seeds in a pot filled with nutrient soil for culture, and selecting young embryos as induced callus materials 10 days after pollination. Sterilizing young corn ears with 75% (volume fraction) ethanol in an operation table for 10min, washing with sterile water, and air drying. The seed grain is cut off by a scalpel, then the young embryo is separated by forceps and placed in sterile water for standby.
(2) Callus induction and subculture
Placing young embryo in N6 culture medium (BINDER, AA 958), culturing at 28deg.C for one week, cutting the callus with vigorous growth and bright color into small pieces, placing on N6 culture medium, culturing once every two weeks, maintaining good state of callus, and selecting callus with good growth condition for use.
(3) Agrobacterium infection
Taking out the stored agrobacterium, inoculating and shaking until the OD 600 value is about 0.8, centrifuging for 10min at 5000r/min, collecting thalli, and using an infection buffer (1L infection buffer is obtained by uniformly mixing 4g of N6 Salts containing N6 vitamin, 2mg of 2,4-D,100mg of inositol, 0.7g L-proline, 68.4g of sucrose, 36g of glucose, 1mL of AgNO 3 (10 mg/mL), 1mL of As (100 mol/L) and water, pH 5.2) to suspend the thalli until the OD 600 value is about 0.5, and then oscillating for 0.5h at 28 ℃ and 150r/min to obtain an infection solution. Soaking the callus with good growth condition selected in the step (2) in an infection buffer for 1h, transferring to an infection solution containing agrobacterium for soaking for 15min, and airing to obtain the infected callus.
(4) Co-culture and recovery culture
The infected calli were placed in a co-culture medium (1L co-culture medium was obtained by mixing 4g N6 Salts containing N6 vitamin, 2mg 2,4-D,30g sucrose, 8g agar, 1mL AgNO 3 (10 mg/mL), 1mL As (100 mol/L), 3mL L-cysteine (100 mg/mL) and water, and pH 5.8), cultured at 20℃for 3 days, transferred to a recovery medium (1L recovery medium was obtained by mixing 4g N6 Salts containing N6 vitamin, 2mg 2,4-D,0.7g L-proline, 30g sucrose, 0.5g MES,4g plant gel, 1mL AgNO 3 (10 mg/mL), 1mL cephalosporin (250 mg/mL) and water, pH 5.8), and transferred to a recovery medium to which glufosinate was added for screening positive calli.
(5) Differentiation, re-differentiation, rooting and seedling hardening
The positive calli were transferred to embryoid-inducing medium (1L embryoid-inducing medium was obtained by mixing up 4.43g MS Salts containing MS VITAMIN, 0.25mg 2,4-D,30g sucrose, 5mg 6-BA,4g plant gel, 1mL Cefo (250 mg/mL) and water, pH 5.8), dark-cultured for 2 weeks, then transferred to differentiation medium (1L differentiation medium was obtained by mixing up 4.43g MS Salts containing MS VITAMIN, 30g sucrose, 4g plant gel, 1mL Cefo (250 mg/mL) and water, pH 5.8), transferred to rooting medium (1L rooting medium was obtained by mixing up 2.215g 1/2MS,30g sucrose, 51.55mg MS vitamin,4g plant gel and water, pH 5.8), rooting was performed by exposing to air for 3 days, and transplanting seedlings.
(6) Bar test paper strip screening positive plants
Taking plant leaves of about 3 cm, putting into a tube, grinding thoroughly, adding 500 μl buffer, and inserting Bar test strip (Shanghai Youlong biotechnology Co., ltd., catalog number: enviroLogixAS 03), wherein the plant with positive strip is T0 generation positive transgenic plant.
4. Acquisition and analysis of maize ACCase mutants
Obtaining T0 generation positive transgenic plants through agrobacterium-mediated maize immature embryo genetic transformation, extracting leaf DNA, respectively amplifying target site sequences, and carrying out sanger sequencing. The following mutations were found in ZmACC gene:
The nucleotide sequences of the 5491 st and 5492 th bases are mutated from C to T, and the nucleotide sequence is shown in SEQ ID NO. 1. The material obtained was designated M1 (FIG. 2) (mutation was also made at position 5487, but the amino acid was not affected by the mutation).
atgtcacagcttggattagccgcagctgcctcaaaggccttgccactactccctaatcgccagagaagttcagctgggactacattctcatcatcttcattatcgaggcccttaaacagaaggaaaagccgtactcgttcactccgtgatggcggagatggggtatcagatgccaaaaagcacagccagtctgttcgtcaaggtcttgctggcattatcgacctcccaagtgaggcaccttccgaagtggatatttcacatggatctgaggatcctagggggccaacagattcttatcaaatgaatgggattatcaatgaaacacataatggaagacatgcctcagtgtccaaggttgttgaattttgtgcggcactaggtggcaaaacaccaattcacagtatattagtggccaacaatggaatggcagcagcaaaatttatgaggagtgtccggacatgggctaatgatacttttggatctgagaaggcaattcaactcatagctatggcaactccggaagacatgaggataaatgcagaacacattagaattgctgaccaattcgtagaggtgcctggtggaacaaacaataataactacgccaatgttcaactcatagtggagatggcacaaaaactaggtgtttctgctgtttggcctggttggggtcatgcttctgagaatcctgaactgccagatgtattgaccgcaaaagggatcgtttttcttggcccacctgcatcatcaatgaatgctttgggagataaggtcggctcagctctcattgctcaagcagccggggtcccaactcttgcttggagtggatcacatgttgaagttccattagagtgctgcttagacgcgatacctgaggagatgtatagaaaagcttgcgttactaccacagaggaagcagttgcaagttgtcaagtggttggttatcctgccatgattaaggcatcctggggaggtggtggtaaaggaataagaaaggttcataatgatgatgaggttagagcgctgtttaagcaagtacaaggtgaagtccctggctccccaatatttgtcatgaggcttgcatcccagagtcggcatcttgaagttcagttgctttgtgatcaatatggtaatgtagcagcacttcacagtcgtgattgcagtgtgcaacggcgacaccagaagattattgaagaaggtccagttactgttgctcctcgtgagacagttaaagcacttgagcaggcagcaaggaggcttgctaaggctgtgggttatgttggtgctgctactgttgagtatctttacagcatggaaactggagactactattttctggaacttaatccccgactacaggttgagcatccagtcaccgagtggatagctgaagtaaatctgcctgcagctcaagttgctgttggaatgggcatacctctttggcagattccagaaatcagacgtttctatggaatggactatggaggagggtatgacatttggaggaaaacagcagctcttgctacaccatttaattttgatgaagtagattctcaatggccaaagggccattgtgtagcagttagaattactagtgaggacccagatgatggtttcaaacctactggtgggaaagtgaaggagataagttttaaaagcaagcctaatgtttgggcctacttctcagtaaagtctggtggaggcattcatgaatttgctgattctcagttcggacatgtttttgcatatgggctctctagatcagcagcaataacaaacatgactcttgcattaaaagagattcaaattcgtggagaaattcattcaaatgttgattacacagttgacctcttaaatgcttcagactttagagaaaacaagattcatactggttggctcgacaccagaatagctatgcgtgttcaagctgagaggcccccatggtatatttcagtggttgggggtgctttatataaaacagtaaccaccaatgcagccactgtttctgaatatgttagttatctcaccaagggccagattccaccaaagcatatatcccttgtcaattctacagttaatttgaatatagaagggagcaaatacacaattgaaactgtaaggactggacatggtagctacaggttgagaatgaatgattcaacagttgaagcgaatgtacaatctttatgtgatggtggcctcttaatgcagttggatggaaacagccatgtaatttatgcagaagaagaagctggtggtacacggcttcagattgatggaaagacatgtttattgcagaatgaccatgatccatcaaagttattagctgagacaccctgcaaacttcttcgtttcttggttgctgatggtgctcatgttgatgcggatgtaccatacgcggaagttgaggttatgaagatgtgcatgcctctcttgtcacctgcttctggtgtcattcattgtatgatgtctgagggccaggcattgcaggctggtgatcttatagcaaggttggatcttgatgacccttctgctgtgaaaagagctgagccatttgatggaatatttccacaaatggagctccctgttgctgtctctagtcaagtacacaaaagatatgctgcaagtttgaatgctgctcgaatggtccttgcaggatatgagcacaatattaatgaagtcgttcaagatttggtatgctgcctggacaaccctgagcttcctttcctacagtgggatgaacttatgtctgttctagcaacgaggcttccaagaaatctcaagagtgagttagaggataaatacaaggaatacaagttgaatttttaccatggaaaaaacgaggactttccatccaagttgctaagagacatcattgaggaaaatctttcttatggttcagagaaggaaaaggctacaaatgagaggcttgttgagcctcttatgaacctactgaagtcatatgagggtgggagagagagccatgcacattttgttgtcaagtctcttttcgaggagtatcttacagtggaagaactttttagtgatggcattcagtctgacgtgattgaaacattgcggcatcagcacagtaaagacctgcagaaggttgtagacattgtgttgtctcaccagggtgtgaggaacaaagctaagcttgtaacggcacttatggaaaagctggtttatccaaatcctggtggttacagggatctgttagttcgcttttcttccctcaatcataaaagatattataagttggcccttaaagcaagtgaacttcttgaacaaaccaaactaagtgaactccgtgcaagcgttgcaagaagcctttcggatctggggatgcataagggagaaatgagtattaaggataacatggaagatttagtctctgccccattacctgttgaagatgctctgatttctttgtttgattacagtgatcgaactgttcagcagaaagtgattgagacatacatatcacgattgtaccagcctcatcttgtaaaggatagcatccaaatgaaattcaaggaatctggtgctattactttttgggaattttatgaagggcatgttgatactagaaatggacatggggctattattggtgggaagcgatggggtgccatggtcgttctcaaatcacttgaatctgcgtcaacagccattgtggctgcattaaaggattcggcacagttcaacagctctgagggcaacatgatgcacattgcattattgagtgctgaaaatgaaagtaatataagtggaataagtgatgatcaagctcaacataagatggaaaagcttagcaagatactgaaggatactagcgttgcaagtgatctccaagctgctggtttgaaggttataagttgcattgttcaaagagatgaagctcgcatgccaatgcgccacacattcctctggttggatgacaagagttgttatgaagaagagcagattctccggcatgtggagcctcccctctctacacttcttgaattggataagttgaaggtgaaaggatacaatgaaatgaagtatactccttcgcgtgaccgccaatggcatatctacacactaagaaatactgaaaaccccaaaatgttgcatagggtgtttttccgaactattgtcaggcaacccaatgcaggcaacaagtttacatcggctcagatcagcgacgctgaagtaggatgtcccgaagaatctctttcatttacatcaaatagcatcttaagatcattgatgactgctattgaagaattagagcttcatgcaattaggacaggtcattctcacatgtatttgtgcatactgaaagagcaaaagcttcttgacctcattccattttcagggagtacaattgttgatgttggccaagatgaagctaccgcttgttcacttttaaaatcaatggctttgaagatacatgagcttgttggtgcaaggatgcatcatctgtctgtatgccagtgggaggtgaaactcaagttggactgtgatggccctgcaagtggtacctggagagttgtaactacaaatgttactggtcacacctgcaccattgatatataccgagaagtggaggaaatagaatcgcagaagttagtgtaccattcagccacttcgtcagctggaccattgcatggtgttgcactgaataatccatatcaacctttgagtgtgattgatctaaagcgctgctctgctaggaacaacagaacaacatattgctatgattttccgctggcctttgaaactgcactgcagaagtcatggcagtccaatggctctactgtttctgaaggcaatgaaaatagtaaatcctacgtgaaggcaactgagctagtgtttgctgaaaaacatgggtcctggggcactcctataattccgatggaacgccctgctgggctcaacgacattggtatggtcgcttggatcatggagatgtcaacacctgaatttcccaatggcaggcagattattgttgtagcaaatgatatcactttcagagctggatcatttggcccaagggaagatgcattttttgaaactgtcactaacctggcttgcgaaaggaaacttcctcttatatacttggcagcaaactctggtgctaggattggcatagctgatgaagtaaaatcttgcttccgtgttggatggtctgacgaaggcagtcctgaacgagggtttcagtacatctatctgactgaagaagactatgctcgcattagctcttctgttatagcacataagctggagctagatagtggtgaaattaggtggattattgactctgttgtgggcaaggaggatgggcttggtgtcgagaacatacatggaagtgctgctattgccagtgcttattctagggcatatgaggagacatttacacttacatttgtgactgggcggactgtaggaataggagcttatcttgctcgacttggtatacggtgcatacagcgtcttgaccagTTtattattttaacagggttttctgccctgaacaagctccttgggcgggaagtgtacagctcccacatgcagcttggtggtcctaagatcatggcgaccaatggtgttgtccacctcactgttccagatgaccttgaaggtgtttccaatatattgaggtggctcagctatgttcctgcaaacattggtggacctcttcctattaccaaacctctggaccctccagacagacctgttgcttacatccctgagaacacatgcgatccacgtgcagctatctgtggtgtagatgacagccaagggaaatggttgggtggtatgtttgacaaagacagctttgtggagacatttgaaggatgggcaaaaacagtggttactggcagagcaaagcttggaggaattcctgtgggcgtcatagctgtggagacacagaccatgatgcagatcatccctgctgatccaggtcagcttgattcccatgagcgatctgtccctcgtgctggacaagtgtggttcccagattctgcaaccaagaccgctcaggcattattagacttcaaccgtgaaggattgcctctgttcatcctggctaattggagaggcttctctggtggacaaagagatctctttgaaggaattcttcaggctgggtcaacaattgtcgagaaccttaggacatataatcagcctgcttttgtgtacattcctatggctggagagcttcgtggaggagcttgggttgtggtcgatagcaaaataaatccagaccgcattgagtgttatgctgaaaggactgccaaaggtaatgttctcgaacctcaagggttaattgaaatcaagttcaggtcagaggaactccaagactgtatgggtaggcttgacccagagttgataaatctgaaagcaaaactccaagatgtaaatcatggaaatggaagtctaccagacatagaagggattcggaagagtatagaagcacgtacgaaacagttgctgcctttatatacccagattgcaatacggtttgctgaattgcatgatacttccctaagaatggcagctaaaggtgtgattaagaaagttgtagactgggaagaatcacgctcgttcttctataaaaggctacggaggaggatcgcagaagatgttcttgcaaaagaaataaggcagatagtcggtgataaatttacgcaccaattagcaatggagctcatcaaggaatggtaccttgcttctcaggccacaacaggaagcactggatgggatgacgatgatgcttttgttgcctggaaggacagtcctgaaaactacaaggggcatatccaaaagcttagggctcaaaaagtgtctcattcgctctctgatcttgctgactccagttcagatctgcaagcattctcgcagggtctttctacgctattagataagatggatccctctcagagagcgaagtttgttcaggaagtcaagaaggtccttgattga,SEQ ID NO:1.
The amino acid at position 1831 of the mutated amino acid sequence is mutated from P to F.
T0 is subjected to test cross to obtain T1 generation, and then is subjected to further selfing to obtain the M1 mutant material which is free of transgene element insertion and homozygous in mutation site.
Example 2
Herbicide resistance of maize ACCase mutant materials
The obtained M1 mutant materials are sown in seedling pots, 75 mu mol/L, 150 mu mol/L, 300 mu mol/L and 600 mu mol/L of quizalofop-p-ethyl, haloxyfop-methyl and 100 mu mol/L, 200 mu mol/L, 400 mu mol/L and 600 mu mol/L of clethodim herbicide are sprayed respectively in two-leaf periods, and herbicide resistance of the mutant materials is observed 14 days after spraying. As shown in fig. 3.
The results show that the wild type control material died after 75. Mu. Mol/L of quizalofop-p-ethyl, haloxyfop-p-methyl and 100. Mu. Mol/L of clethodim herbicide treatment, whereas the M1 mutant material was resistant to quizalofop-p-ethyl, haloxyfop-methyl and clethodim herbicide.
Example 3
Field experiment
The obtained M1 mutant material is sown in a field, quizalofop-p-ethyl herbicide with the concentration of 300 mu mol/L is sprayed in the four-leaf period respectively, and the herbicide resistance of the mutant material is observed 14 days after spraying. As shown in fig. 4. The results show that the wild type control material died after treatment with quizalofop-p-ethyl herbicide at a concentration of 300. Mu. Mol/L, whereas the mutant material was resistant to quizalofop-ethyl herbicide.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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