WO2019086051A1 - 一种含k85突变的植物epsps突变体及其编码基因和应用 - Google Patents

一种含k85突变的植物epsps突变体及其编码基因和应用 Download PDF

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WO2019086051A1
WO2019086051A1 PCT/CN2018/121331 CN2018121331W WO2019086051A1 WO 2019086051 A1 WO2019086051 A1 WO 2019086051A1 CN 2018121331 W CN2018121331 W CN 2018121331W WO 2019086051 A1 WO2019086051 A1 WO 2019086051A1
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epsps
plant
mutant
epsps mutant
amino acid
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PCT/CN2018/121331
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French (fr)
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陈容
邓龙群
候青江
卢远根
欧倩
冯小容
李玲
黄鑫
胥南飞
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四川天豫兴禾生物科技有限公司
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Priority to CA3081378A priority Critical patent/CA3081378A1/en
Priority to US16/761,146 priority patent/US11591580B2/en
Publication of WO2019086051A1 publication Critical patent/WO2019086051A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1092—3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8275—Glyphosate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01019—3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase

Definitions

  • the present application relates to the field of genetic engineering technology, and in particular to a plant EPSPS mutant containing a K85 mutation, a gene encoding the same, and an application thereof.
  • Glyphosate is one of the most widely used herbicides in the world and has been used for nearly forty years. Glyphosate inhibits the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS catalyzes the synthesis of EPSP by PEP and shikimate-3-phosphate in the shikimate pathway, and finally synthesizes the aromatic amino acids tryptophan, phenylalanine and tyrosine. Glyphosate causes the synthesis of aromatic amino acids to be blocked, which in turn affects the normal growth of plants and eventually causes death.
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • the main method for breeding glyphosate resistant varieties is to use genetic engineering methods to introduce the glyphosate-resistant genes from bacteria into crops, thereby cultivating new varieties of transgenic glyphosate resistant crops. Since its inception in 1996, the planting area has increased rapidly. By 2015, the global glyphosate-tolerant GM crop planting area has reached 150 million hectares, accounting for 83% of the total area of GM crops, which has brought huge benefits to agricultural production and the environment. .
  • CP4EPSPS glyphosate-resistant gene in agriculture
  • Agrobacterium tumefaciens CP4 strain many glyphosate-resistant EPSPS genes have been discovered from microorganisms, these genes have not been widely used in crops.
  • the application of these microbial glyphosate resistant genes in crops is to express these genes, such as CP4EPSPS, in crops using transgenic methods.
  • the resulting transgenic crops have a large promotion area, due to the reversal of the genetic wave, the acceptance of GM crops is still low in the world.
  • the transgenes are mainly limited to corn. , soybeans, cotton and other crops.
  • the object of the present application is to provide a plant EPSPS mutant (i.e., a 5-enolpyruvylshikimate-3-phosphate synthase mutant) derived from a plant which, after mutation, has glyphosate resistance.
  • EPSPS mutant i.e., a 5-enolpyruvylshikimate-3-phosphate synthase mutant
  • Another object of the present application is to provide a coding gene which encodes a plant EPSPS mutant as described above.
  • Another object of the present application is to provide a vector comprising the above-described coding gene.
  • Another object of the present application is to provide a cell containing the above vector.
  • Another object of the present application is to provide the use of the above plant EPSPS mutant.
  • a recombinant cell containing the above vector A recombinant cell containing the above vector.
  • the plant EPSPS mutant provided by the present invention has an amino acid sequence of the plant EPSPS mutant corresponding to the 85th position of E. coli EPSPS having a mutation such as a mutation T or I, compared to E. coli EPSPS.
  • the mutation site corresponds to the 100th position of rice EPSPS, the 94th position of maize EPSPS and the 98th amino acid residue of soybean EPSPS. This site mutation significantly increased the resistance of different mutants of EPSPS to glyphosate while maintaining their own bioenzyme catalytic activity.
  • Plants or recombinant strains of the plant EPSPS mutant provided by the present application can grow normally in the presence of glyphosate, and the plant EPSPS mutant can be applied not only to the cultivation of transgenic crops but also to the cultivation of glyphosate-resistant non-transgenic plants.
  • glyphosate-resistant non-transgenic plants For example, rice, tobacco, soybeans, corn, wheat, cotton and sorghum have broad application prospects.
  • Example 1 is a diagram showing the growth results of Escherichia coli encoding the rice EPSPS mutant coding gene provided in Transformation Example 1 and Example 2 in Example 5 of the present application on a medium containing different glyphosate concentrations;
  • FIG. 2 is a diagram showing the growth results of Escherichia coli encoding the maize EPSPS mutant gene provided in Transformation Example 3 in Example 6 of the present invention on a medium containing different glyphosate concentrations;
  • Figure 3 is a diagram showing the growth results of Escherichia coli encoding the soybean EPSPS mutant-encoding gene provided in Example 7 of Example 7 on a medium containing different glyphosate concentrations;
  • Example 4 is a schematic structural view of a pADV5 vector in Example 5 of the present application.
  • Example 5 is a partial result of amino acid sequence alignment of Escherichia coli EPSPS, rice EPSPS mutant II and wild type rice EPSPS in Example 2 of the present application;
  • Figure 7 is a partial result of amino acid sequence alignment of Escherichia coli EPSPS, soybean EPSPS mutant and wild type soybean EPSPS in Example 4 of the present application.
  • Figure 8 is a schematic view showing the structure of the pGVP1-EPSPS vector provided in Experimental Example 8 of the present application.
  • the application provides a plant EPSPS mutant (ie, a mutant of 5-enolpyruvylshikimate-3-phosphate synthase), the amino acid sequence of the plant EPSPS mutant compared to E. coli EPSPS
  • the site corresponding to position 85 of E. coli EPSPS has a mutation. Mutations at this site can significantly increase glyphosate resistance in plant EPSPS mutants.
  • the amino acid sequence of the plant EPSPS mutant corresponds to a mutation in position E of position 85 of E. coli EPSPS to T or I; ie, K>T or K>I.
  • the amino acid sequence of the plant EPSPS mutant is aligned with the amino acid sequence of E. coli EPSPS, and the amino acid sequence of the plant EPSPS mutant corresponding to the 85th position of E. coli EPSPS is K-mutated to T (abbreviated as K85T), or there is a K mutation to I (abbreviated as K85I).
  • amino acid sequence of E. coli EPSPS is set forth in SEQ ID NO:25.
  • amino acid sequence of the plant EPSPS mutant is aligned with the amino acid sequence of E. coli EPSPS as shown in SEQ ID NO: 25, and the amino acid sequence of the plant EPSPS mutant corresponds to the sequence shown in SEQ ID NO:
  • the amino acid residue at position 85 is mutated from K to T or has a K mutation to I.
  • the amino acid sequence of the plant EPSPS mutant corresponds to E. coli EPSPS further having a combination of one or more of the following mutations: G96A, P101S, and K194R.
  • the mutation G96A can be understood as: the amino acid sequence of the plant EPSPS mutant is aligned with the amino acid sequence of E. coli EPSPS, and the amino acid sequence of the plant EPSPS mutant corresponds to the position of the 96th position of E. coli EPSPS. A;
  • the mutation P101S can be understood as: the amino acid sequence of the plant EPSPS mutant is aligned with the amino acid sequence of E. coli EPSPS, and the amino acid sequence of the plant EPSPS mutant corresponds to the position of the 101st position of E. coli EPSPS. For S.
  • Mutation K194R can be understood as: the amino acid sequence of the plant EPSPS mutant is aligned with the amino acid sequence of E. coli EPSPS, and the amino acid sequence of the plant EPSPS mutant corresponds to the amino acid residue at position 194 of E. coli EPSPS by K mutation. For R.
  • the plant EPSPS mutant is derived from, but not limited to, the following plants: wheat, rice, barley, oats, corn, sorghum, millet, buckwheat, alfalfa, mung bean, broad bean, Peas, lentils, sweet potatoes, potatoes, cotton, soybeans, rapeseed, sesame, peanuts, sunflowers, radishes, carrots, turnips, beets, cabbage, mustard, kale, broccoli, kale, cucumber, zucchini, pumpkin, melon, bitter gourd, Loofah, melon, watermelon, melon, tomato, eggplant, pepper, kidney bean, kidney bean, edamame, leeks, green onions, onions, leeks, spinach, celery, leeks, lettuce, sage, day lily, grapes, strawberries, beets, sugar cane, Tobacco, alfalfa, pasture, turfgrass, tea and cassava.
  • the plant EPSPS mutant provided by the present application is obtained by the above-described mutation of wild type EPSPS derived from plants such as rice, tobacco, soybean, corn, wheat, cotton, rape, and sorghum, which retains plant-derived characteristics.
  • the coding gene encoding the plant EPSPS mutant can be applied to the cultivation of glyphosate resistant crop varieties, for example, by genetic modification or genetic modification.
  • the plant-derived glyphosate-resistant gene can be directly transformed or used as a template to edit the genome, which has more reliable biosafety and is beneficial to grass resistance.
  • the plant EPSPS mutant is from rice (Oryza sativa), and the amino acid sequence of the plant EPSPS mutant is set forth in SEQ ID NO:4.
  • the plant EPSPS mutant shown in SEQ ID NO: 4 is a rice EPSPS mutant I.
  • the rice EPSPS mutant I has a mutation of K100(85)I relative to the amino acid sequence of wild-type rice EPSPS shown in SEQ ID NO: 2.
  • the K100(85)I mutation is understood to mean that the amino acid residue of the rice EPSPS mutant in SEQ ID NO: 2 is mutated from K to I relative to the wild type rice EPSPS, and this position corresponds to the large intestine. 85th place of Bacillus EPSPS.
  • the plant EPSPS mutant is from rice, the amino acid sequence of the plant EPSPS mutant having the following mutations corresponding to E. coli EPSPS: K85I, G96A and P101S.
  • amino acid sequence of the plant EPSPS mutant is set forth in SEQ ID NO: 8.
  • the plant EPSPS mutant shown in SEQ ID NO: 8 is a rice EPSPS mutant II.
  • the rice EPSPS mutant II has three mutations K100(85)I, G111(96)A and P116(101)S relative to the amino acid sequence of the wild-type rice EPSPS shown in SEQ ID NO: 2.
  • the G111(96)A mutation is understood to mean that the amino acid residue 111 of the rice EPSPS mutant II in SEQ ID NO: 2 is mutated from G to A relative to wild-type rice EPSPS, and this position corresponds to the large intestine. 96th position of Bacillus EPSPS;
  • the P116(101)S mutation is understood to mean that the amino acid residue of rice EPSPS mutant II in SEQ ID NO: 2 is mutated from P to S relative to wild-type rice EPSPS, and this position corresponds to the large intestine.
  • the plant EPSPS mutant is derived from maize (Zea mays), the amino acid sequence of the plant EPSPS mutant having the following mutations corresponding to E. coli EPSPS: K85I, G96A, P101S, and K194R .
  • amino acid sequence of the plant EPSPS mutant is set forth in SEQ ID NO: 16.
  • the plant EPSPS mutant shown in SEQ ID NO: 16 is a maize EPSPS mutant.
  • the maize EPSPS mutant has K94 (85) I, G105 (96) A, P110 (101) S, and K207 (194) R four relative to the amino acid sequence of wild type maize EPSPS shown in SEQ ID NO: 10. A mutation.
  • the K94(85)I mutation is understood to mean that the amino acid residue at position 94 of SEQ ID NO: 10 is mutated from K to I relative to wild-type maize EPSPS, which corresponds to the large intestine. 85th place of Bacillus EPSPS;
  • the G105(96)A mutation is understood to mean that the amino acid residue of the maize EPSPS mutant in SEQ ID NO: 10 is mutated from G to A relative to wild-type maize EPSPS, and this position corresponds to Escherichia coli. 96th place in EPSPS;
  • the P110(101)S mutation is understood to mean that the amino acid residue of the maize EPSPS mutant at position 110 in SEQ ID NO: 10 is mutated from P to S relative to wild-type maize EPSPS, and this position corresponds to Escherichia coli. 101st in EPSPS;
  • the K207(194)R mutation is understood to mean that the amino acid residue at position 207 of the maize EPSPS mutant in SEQ ID NO: 10 is mutated from K to R relative to wild-type maize EPSPS, which corresponds to E. coli. 194th in EPSPS.
  • the plant EPSPS mutant is from soybean (Glycine max (Linn.) Merr.), the amino acid sequence of the plant EPSPS mutant corresponding to E. coli EPSPS also having the following mutations: K85T and P101S.
  • amino acid sequence of the plant EPSPS mutant is set forth in SEQ ID NO:24.
  • the plant EPSPS mutant shown in SEQ ID NO: 24 is a soybean EPSPS mutant.
  • the wheat EPSPS mutant has two mutations, K98(85)T and P114(101)S, relative to the amino acid sequence of wild-type soybean EPSPS shown in SEQ ID NO:24.
  • the K98(85)T mutation can be understood as: the soybean EPSPS mutant is mutated from K to T at position 98 of the amino acid residue of SEQ ID NO: 18, which corresponds to Escherichia coli. 85th place in EPSPS;
  • the P114(101)S mutation can be understood as: the soybean EPSPS mutant is mutated from P to S in the 114th amino acid residue in SEQ ID NO: 18 relative to wild-type soybean EPSPS, and this position corresponds to Escherichia coli. 101st in EPSPS.
  • the application provides a coding gene encoding a plant EPSPS mutant as described above.
  • the nucleotide coding sequence of the EPSPS mutant can be easily obtained based on the amino acid sequence of the above plant EPSPS mutant. Any gene encoding a plant EPSPS mutant described above is within the scope of the present application.
  • the above-described coding gene is set forth in SEQ ID NO: 3, 7, 15, or 23.
  • SEQ ID NO: 3 is a rice EPSPS mutant I encoding gene encoding the rice EPSPS mutant I set forth in SEQ ID NO: 4.
  • SEQ ID NO: 7 The sequence shown in SEQ ID NO: 7 is a rice EPSPS mutant II encoding gene, encoding the rice EPSPS mutant II shown in SEQ ID NO: 8;
  • sequence set forth in SEQ ID NO: 15 is a maize EPSPS mutant encoding gene encoding a maize EPSPS mutant set forth in SEQ ID NO:16;
  • Maize EPSPS Mutant Maize EPSPS Mutant
  • the sequence set forth in SEQ ID NO: 23 is a soybean EPSPS mutant encoding gene encoding the soybean EPSPS mutant set forth in SEQ ID NO: 24.
  • the application provides a vector comprising the above-described coding gene.
  • the vector may be a cloning vector or an expression vector.
  • the expression vector may be a prokaryotic expression vector such as a pADV5 vector, or may be Eukaryotic expression vector.
  • the eukaryotic expression vector is a plant expression vector, such as a pBI121 vector.
  • the application provides a recombinant or recombinant cell comprising the vector described above.
  • the recombinant bacteria may be a cocci, a bacterium such as Escherichia coli or a Helicobacter; or an aerobic bacterium or an anaerobic bacterium.
  • the recombinant cell may be a prokaryotic cell or a eukaryotic cell; further, in some embodiments of the present application, the eukaryotic cell may be an animal cell or a plant cell; Further, in some embodiments of the present application, the plant cell may be a dicot plant cell or a monocot plant cell.
  • the application provides the use of the above plant EPSPS mutant for cultivating a glyphosate resistant plant.
  • the above application comprises: transforming a vector into a plant of interest, the vector comprising a coding gene encoding the plant EPSPS mutant.
  • a vector containing the rice EPSPS-encoding gene represented by SEQ ID NO: 7 can be transformed into, for example, rice callus, cultured to differentiate into a whole rice plant, and transgenic glyphosate resistant rice can be cultivated.
  • the above application comprises: modifying an endogenous EPSPS gene of a plant of interest to encode the plant EPSPS mutant.
  • EPSPS coding gene represented by SEQ ID NO: 7 is used as a template to modify the endogenous EPSPS gene of the rice genome, thereby producing non-transgenic rice.
  • the above application comprises: mutagenizing a plant cell, tissue, individual or population to encode the plant EPSPS mutant.
  • the rice material can be mutagenized, such as chemical, radiation mutagenesis, etc., thereby cultivating the rice with the endogenous EPSPS gene mutation.
  • the coding sequence of the endogenous EPSPS gene can also be modified to the base sequence of the coding gene (SEQ ID NO: 7) by CRISPR/Cas9 technology, and the rice EPSPS mutant provided by the present application is encoded in the plant of interest (SEQ ID NO: 8) The same protein, making the plant of interest have glyphosate resistance.
  • the plant of interest is any one of plants such as rice, tobacco, soybean, corn, wheat, cotton, canola, and sorghum.
  • the plant EPSPS mutant provided by the present application has an amino acid sequence corresponding to E. coli EPSPS corresponding to the 85th position of E. coli EPSPS having a mutation such as K>T or K>I.
  • the position of this mutation site corresponds to position 100 of wild type rice EPSPS (SEQ ID NO: 2); corresponds to position 94 of wild type maize EPSPS (SEQ ID NO: 10); corresponds to wild type soybean EPSPS (SEQ ID NO: 18) 98th.
  • This site mutation can significantly improve the resistance of glyphosate to different mutants of EPSPS of various plants, while maintaining the biological enzyme catalytic activity of the mutant itself. Plants or recombinant strains that transform the EPSPS mutant of the plant can grow normally.
  • the EPSPS mutant of the plant can be used not only for the cultivation of genetically modified crops, but also for the cultivation of glyphosate-resistant non-transgenic plants such as rice, tobacco, soybean, corn, wheat, Cotton and sorghum have broad
  • This embodiment provides a plant EPSPS mutant derived from rice and is a rice EPSPS mutant I obtained by mutating a wild type rice EPSPS (amino acid sequence as shown in SEQ ID NO: 2), and the amino acid sequence thereof is as follows. SEQ ID NO: 4 is shown.
  • the rice EPSPS mutant I provided in the present example has a K100(85)I mutation relative to the amino acid sequence of the wild type rice EPSPS shown in SEQ ID NO: 2.
  • the 100th amino acid residue of rice EPSPS mutant I was mutated from G to A relative to wild type rice EPSPS, and this position corresponds to position 85 of E. coli EPSPS.
  • This example also provides a rice EPSPS mutant I encoding gene encoding the above rice EPSPS mutant I, the nucleotide sequence of which is shown in SEQ ID NO: 1.
  • the rice EPSPS mutant I coding gene and the rice EPSPS mutant I provided in the examples of the present application can be obtained by chemical synthesis.
  • This embodiment provides a plant EPSPS mutant which is derived from rice and is a rice EPSPS mutant II which is obtained by mutating a wild type rice EPSPS (amino acid sequence as shown in SEQ ID NO: 2), and the amino acid sequence thereof is as follows. SEQ ID NO:8.
  • the rice EPSPS mutant II has three mutations of K100(85)I, G111(96)A and P116(101)S relative to the amino acid sequence of wild-type rice EPSPS shown in SEQ ID NO: 2.
  • FIG. 5 shows partial results of amino acid sequence alignment of Escherichia coli EPSPS, rice EPSPS mutant II and wild type rice EPSPS, in which: Ec-EPSPS WT represents E. coli EPSPS; Os -EPSPS M stands for rice EPSPS mutant II; Os-EPSPS WT stands for wild type rice EPSPS), the 100th amino acid residue of rice EPSPS mutant II is changed from K to I, compared to wild type rice EPSPS. The point corresponds to position 85 of E. coli EPSPS (SEQ ID NO: 25); the 111th amino acid residue is mutated from G to A, which corresponds to position 96 of E. coli EPSPS; the 116th amino acid residue consists of The P mutation is S, which corresponds to position 101 of E. coli EPSPS (as indicated by the arrow in Figure 5).
  • This example also provides a rice EPSPS mutant II encoding gene encoding the above rice EPSPS mutant II, the nucleotide sequence of which is shown in SEQ ID NO: 7.
  • Both the rice EPSPS mutant II encoding gene and the rice EPSPS mutant II provided in the examples of the present application can be obtained by chemical synthesis.
  • This example provides a plant EPSPS mutant derived from maize and is a maize EPSPS mutant obtained by mutating wild type maize EPSPS (amino acid sequence as shown in SEQ ID NO: 10), and the amino acid sequence thereof is SEQ. ID NO: 16 is shown.
  • the maize EPSPS mutant provided in the present example has K94 (85) I, G105 (96) A, P110 (101) S, and K207 relative to the amino acid sequence of wild type maize EPSPS shown in SEQ ID NO: 10. 194) R four mutations.
  • FIG. 6 shows partial results of amino acid sequence alignment of E. coli EPSPS, corn EPSPS mutant and wild type corn EPSPS, in which: Ec-EPSPS WT represents E. coli EPSPS; Zm- EPSPS M stands for maize EPSPS mutant; Zm-EPSPS WT stands for wild-type maize EPSPS), compared to wild-type maize EPSPS, the 94th amino acid residue of maize EPSPS mutant is mutated from K to I, which corresponds to the large intestine 85th position of Bacillus EPSPS; the 105th amino acid residue is mutated from G to A, which corresponds to position 96 of E.
  • the present application also provides a maize EPSPS mutant encoding gene encoding the above maize EPSPS mutant, the nucleotide sequence of which is shown in SEQ ID NO: 15.
  • Both the maize EPSPS mutant coding gene and the maize EPSPS mutant provided in the examples of the present application can be obtained by chemical synthesis.
  • the present embodiment provides a plant EPSPS mutant which is derived from soybean and is a soybean EPSPS mutant which is obtained by mutating wild type soybean EPSPS (amino acid sequence as shown in SEQ ID NO: 18), and the amino acid sequence thereof is SEQ. ID NO: 24 is shown.
  • soybean EPSPS mutant has two mutations, K98(85)T and P114(101)S, relative to the amino acid sequence of wild-type soybean EPSPS shown in SEQ ID NO: 18.
  • FIG. 7 shows partial results of amino acid sequence alignment of Escherichia coli EPSPS, soybean EPSPS mutant and wild type soybean EPSPS, in which: Ec-EPSPS WT represents E. coli EPSPS; Gm- EPSPS M stands for soybean EPSPS mutant; Gm-EPSPS WT stands for wild-type soybean EPSPS), compared with wild-type soybean EPSPS, the 98th amino acid residue of soybean EPSPS mutant is mutated from K to T, which corresponds to the large intestine The 85th position of Bacillus EPSPS (SEQ ID NO: 25); the 114th amino acid residue was mutated from P to S, which corresponds to position 101 of E. coli EPSPS (as indicated by the arrow in Figure 7).
  • the present application also provides a soybean EPSPS mutant encoding gene encoding the above soybean EPSPS mutant, the nucleotide sequence of which is shown in SEQ ID NO: 23.
  • soybean EPSPS mutant coding gene and the soybean EPSPS mutant provided in the examples of the present application can be obtained by a chemical synthesis method.
  • Escherichia coli encoding the rice EPSPS mutant I coding gene (SEQ ID NO: 3) provided in Example 1 was used as experimental group 1 (with only K100 (85) I mutation);
  • a gene encoding a rice EPSPS mutant having only the G111(96)A and P116(101)S mutations without the K100(85)I mutation is experimental group 2;
  • Escherichia coli encoding the rice EPSPS mutant II encoding gene (SEQ ID NO: 7) provided in Example 2 was used as experimental group 3 (the encoded rice EPSPS mutant II has K100(85)I, G111(96)A and P116(101)S three mutations);
  • Escherichia coli transformed with the wild type rice EPSPS encoding gene (SEQ ID NO: 1, which encodes wild type rice EPSPS shown in SEQ ID NO: 2) was used as a control group.
  • M9 basic medium can be prepared by the following methods:
  • 5 ⁇ M9 salt solution Weigh 6.78 g of Na 2 HPO 4 , 3.0 g of KH 2 PO 4 , 0.5 g of NaCl, 1.0 g of NH 4 Cl, add ddH 2 O to 200 ml, and autoclave at high temperature;
  • 200 ml of M9 basal medium was prepared by adding 200 ml of 5 x M9 salt solution, 20 ml of 20% glucose, 2 ml of 1.0 M MgSO 4 and 0.1 ml of 1.0 M CaCl 2 , and adding ddH 2 O to 1000 ml.
  • the Escherichia coli used is a double-knocked Escherichia coli, ie, EPSPS-deficient Escherichia coli (E. coli DH5 ⁇ after knocking out the EPSPS gene and the CP Lyase gene, and is named EDCE.
  • EDCE EPSPS-deficient Escherichia coli
  • the detection method is a well-known method in the art, and is briefly described as follows: a chemical synthesis method is used to synthesize a rice EPSPS mutant coding gene, and a restriction enzyme site (Pac1 and Sbf1) is introduced at both ends, and after enzyme digestion, the ligase is ligated.
  • the expression vector (for example, pADV5 vector, whose structure is shown in Figure 4) after the same digestion treatment, and then transformed into double-knocked Escherichia coli, after verification, pick positive clones and inoculate glyphosate-containing Grow on the medium and observe the growth.
  • the EPSPS-deficient E. coli of the control group and the experimental group 1-3 were able to grow on the medium containing 0 mM glyphosate, indicating the EPSPS protein of the experimental group and the control group (SEQ ID NO: 2, SEQ ID NO: 4).
  • SEQ ID NO: 6, SEQ ID NO: 8) all have normal EPSPS enzyme activity;
  • Escherichia coli transformed with the wild type maize EPSPS encoding gene (SEQ ID NO: 9, encoding wild type maize EPSPS shown in SEQ ID NO: 10) was used as a control group;
  • a gene encoding a maize EPSPS mutant having only three mutations of G105(96)A, P110(101)S and K207(194)R without K94(85)I mutation (nucleotide sequence is SEQ ID NO: 13 shows that the amino acid sequence of the encoded triple mutant maize EPSPS mutant is as shown in SEQ ID NO: 14) is experimental group 2;
  • the coding gene (SEQ ID NO: 15) of the maize EPSPS mutant (having four mutations of K94 (85) I, G105 (96) A, P110 (101) S and K207 (194) R) provided in Transformation Example 3 Escherichia coli was experimental group 3.
  • the Escherichia coli used is a double-knocked Escherichia coli (E. coli DH5 ⁇ after knocking out the EPSPS gene and the C-P Lyase gene, and is named EDCE, and the preparation method thereof can be referred to the Chinese invention patent No. 2016103256926).
  • the experimental group and the control group were able to grow normally on the medium containing 0 mM and 1 mM glyphosate, indicating that the EPSPS protein of the experimental group and the control group had normal EPSPS enzyme activity; however, the growth of the experimental group 1 (Z1) was significantly better.
  • the control group (Z0), and the experimental group 3 (Z3) E. coli growth was significantly better than the experimental group 2 (Z2), indicating that the K94 (85) I mutation provides higher EPSPS enzyme activity.
  • Escherichia coli transformed with the wild-type soybean EPSPS encoding gene (SEQ ID NO: 17, encoding wild-type soybean EPSPS shown in SEQ ID NO: 18) was used as a control group;
  • a gene encoding a single mutant soybean EPSPS mutant having a K98(85)T mutation and having no P114(101)S mutation (SEQ ID NO: 19, the amino acid sequence of the single mutant soybean EPSPS mutant encoded as SEQ ID NO: 20) Escherichia coli is experimental group 1;
  • a gene encoding a single mutant soybean EPSPS mutant having only the P114(101)S mutation without a K98(85)T mutation (nucleotide sequence as shown in SEQ ID NO: 21, encoding a single mutant soybean EPSPS mutant) Escherichia coli having the amino acid sequence shown in SEQ ID NO: 22 is experimental group 2;
  • the Escherichia coli used is a double-knocked Escherichia coli (E. coli DH5 ⁇ after knocking out the EPSPS gene and the C-P Lyase gene, and is named EDCE, and the preparation method thereof can be referred to the Chinese invention patent No. 2016103256926).
  • Fig. 3 The results are shown in Fig. 3 (in the figure: G0 stands for the control group, G1 stands for the experimental group 1, G2 stands for the experimental group 2, and G3 stands for the experimental group 3): the experimental group and the control group are both on the medium containing 0 mM glyphosate. Normal growth, indicating that the EPSPS protein of the experimental group and the control group has normal EPSPS enzyme activity;
  • control group (G0) could not grow basically, and the growth of the experimental group (G1) was significantly better than that of the control group (G0, indicating that the soybean EPSPS mutant with K98(85)T single mutation was resistant to grass.
  • the ability of glyphosate is significantly better than wild type soybean EPSPS;
  • the G1 (SEQ ID NO: 20), G2 (SEQ ID NO: 22) and G3 (SEQ ID NO: 24) soybean EPSPS mutants provided in Example 7 were tested, and the R1 rice EPSPS mutant provided in Example 5 (SEQ ID NO: 4), and the glyphosate resistance of the Z1 maize EPSPS mutant (SEQ ID NO: 12) provided in Example 6 in transgenic rice, as follows:
  • the plasmid (containing the EPSPS mutant gene) of the monoclonal resistant strain of pADV5-EPSPS in Examples 1-7 was extracted in a conventional manner. This was digested with Pac1 and BstEII, and a small fragment was recovered, and then the small fragment was ligated to the pGVP1 vector after the same double digestion with T4 DNA ligase to obtain a pGVP1-EPSPS vector, and its structure is shown in FIG.
  • the pGVP1-EPSPS vector was transformed into EHA105 (Agrobactrium tumefaciens) competent cells, and the monoclonal antibody was picked for colony PCR detection to obtain a positive strain; then the positive strain was inoculated into 1 mL of 50 ⁇ g ⁇ mL -1 Kanamycin.
  • the protein was expanded in YEP culture with 50 ⁇ g ⁇ mL -1 rifampicin, stored at -80 ° C, or used in subsequent experiments.
  • Disinfection and pre-culture Take the seeds of mature rice (Nipponbare), artificially shell the seeds, select the seeds with full sterile spots, and disinfect according to the following steps: Put the seeds into a 50ml sterile centrifuge tube, add 70% alcohol for 30 seconds, and pour Remove alcohol and wash once with sterile water; add 10-20ml of 2.6% sodium hypochlorite solution and soak for 20 minutes. Pour the sodium hypochlorite solution and soak it in sterile water for 6-7 times for 3 minutes each time.
  • Induction and subculture The seeds are blotted on sterile filter paper, and the mature embryos are placed in the induction medium, 12 per dish. After the operation, the culture dishes are sealed with a sealing film and cultured in the dark at 30 ° C for 21-28 days. The callus was transferred to fresh medium and cultured for about 7-14 days. A spherical callus of 1-2 mm size was taken as an infusion receptor.
  • the callus is placed in a centrifuge tube or a culture cup, and the prepared Agrobacterium suspension is added to the suspension for 10 minutes, and shaken several times; the bacterial solution is removed, the callus is taken out, and placed on a sterile filter paper to be blotted. Surface bacterial solution (about 30 minutes); the callus was placed on a sterile filter paper in a Petri dish and incubated for 2-3 days at 25 ° C in the dark.
  • Recovery culture The co-cultured callus was inoculated on a recovery medium and cultured in the dark at 30 ° C for 5-7 days.
  • the first round of screening the callus was transferred to screening medium 1 (S1), and cultured in the dark at 30 ° C for 14 days.
  • the first round of differentiation the screened resistant callus was transferred to a differentiation medium, irradiated at 30 ° C for 19 hours, and cultured for about 21 days.
  • the second round of differentiation the newborn young shoots were selected and moved to a new differentiation medium, and the cultivation was continued for about 21 days.
  • the new seedlings grow to about 2 cm, they are transferred to rooting medium and cultured at 30 ° C for 16 to 8 hours for 3 to 4 weeks.
  • the roots are induced and the seedlings grow to 7 to 10 cm, they are taken out from the medium and washed.
  • the medium contaminated with the net roots was transferred to a nursery tray and continued to be cultured for about 10 days before being transferred to a greenhouse or a field.
  • Induction medium NB BasaL Medium [N6 mass + MS iron salt solution + B5 trace and organic] + hydrolyzed casein 0.3g / L + L-proline + 2.787g / L + sucrose 30g / L + 2, 4-D 4mg / L+ agar 8 g/L, pH 5.8.
  • Dip-dyeing medium NB BasaL Medium [N6 mass + MS iron salt solution + B5 trace and organic] + L-proline 0.7g / L + sucrose 68.4g / L + glucose 36g / L + 2,4-D 2mg / L pH5.2 (small pot 115 ° C), AS 20mg / L is currently used.
  • Co-culture medium NB BasaL Medium [N6 mass + MS iron salt solution + B5 trace and organic] + hydrolyzed casein 0.3g / L + L-proline 2.787g / L + sucrose 30g / L + 2,4-D 1mg / L pH 5.2, AS 20mg / L is currently used.
  • Screening medium 1 (S1): NB BasaL Medium [N6 mass + MS iron salt solution + B5 trace and organic] + glutamine 0.2 g / L + L-proline 2.787g / L + sucrose 30g / L + 2,4- D 2 mg / L + agar 8 g / L, pH 5.8.
  • Cefotaxime cephalosporin
  • Timentin agar
  • Vancomycin vancomycin
  • Screening medium 2 (S2): NB BasaL Medium [N6 mass + MS iron salt solution + B5 trace and organic] + glutamine 0.2 g / L + L-proline 2.787g / L + sucrose 30g / L + 2,4- D 2 mg / L + agar 8 g / L, pH 5.8.
  • Cefotaxime cephalosporin
  • Timentin agar
  • Vancomycin vancomycin
  • Differentiation medium MS BasaL Medium [MS mass, iron salt solution, trace and organic] + glutamine 0.2 g / L + sucrose 30 g / L + sorbitol 30 g / L + agar 8 g / L pH 5.8. After sterilization, add Cefotaxime (cemium) 200mg / L + KT 2mg / L + NAA 0.02mg / L + GLyphosate 1-5mg / L.
  • Cefotaxime cemium
  • Rooting medium 1/2 MS BasaL Medium [MS mass, iron salt solution, trace and organic] + inositol 0.1 g / L + sucrose 30 g / L + agar 8 g / L, pH 5.8. After sterilization, add Cefotaxime (cephalosporin) 100mg/L, NAA 0.2mg/L
  • the rice plants transfected with the EPSPS mutant gene were detected by PCR, and the primers were detected according to the pGVP1-EPSPS vector sequence and the rice reference gene design.
  • the sequences are as follows:
  • CaMV15 5'-GGTGGCTCCTACAAATGCCATC-3';
  • CTS3 5'-GAGCCAATTAACGTCATCCCAC-3'; the amplified fragment size is 452 bp;
  • OsF 5'-GCTTCTGACCAGCCCATTATTCTGC-3';
  • OsR 5'-CCCTCAAGGGTAAGCTCATCTCTCTTC-3'; the amplified fragment size was 629 bp.
  • the genomic DNA of the rice plants transfected with the pGVP1-EPSPS gene was separately extracted and homogenized to 100 ng/ ⁇ L.
  • PCR detection system 10 ⁇ L 2 ⁇ TsINGKe, 2 ⁇ L of primer mixture (10 ⁇ mol/L of OsF, OsR, CaMV15, CTS3, 0.5 ⁇ L each), 1 ⁇ L of genomic DNA template (100 ng/ ⁇ L), and 7 ⁇ L of ddH 2 O.
  • PCR detection procedure 94 ° C, 3 min; 94 ° C, 30 s; 62 ° C, 30 s; 72 ° C, 45 s; 30 cycles; 72 ° C, 10 min;
  • the PCR amplification product was electrophoresed on a 1.5% agarose gel, and the transgenic positive tobacco plants were ligated at the 452 bp position and the 629 bp position.
  • This example demonstrates glyphosate resistance of EPSPS mutants in rice transgenic plants.
  • the experimental method is as follows:
  • Transplanted transgenic rice seedlings were evenly discharged in the same experimental area (avoiding leaf overlap).
  • the area occupied by the experimental group and the control group was calculated, and glyphosate was sprayed at a dose of 1X according to the area of 1060 g/ha (0.106 g/m2).
  • the 2X dose was 2120 g/ha
  • the 5X dose was 5300 g/ha
  • the 20X dose was 21200 g/ha.
  • the statistical standard for glyphosate resistance is: if the plant does not have any glyphosate damage, the growth is normal, and the glyphosate-resistant plants are represented by "+++”; if the leaves of the plants are somewhat yellowish, Growth is slightly slow, it is a medium glyphosate-resistant plant, expressed by "++”; if some leaves of the plant are withered, the growth is very slow, and it is a low glyphosate-resistant plant, which is represented by "+”; if the plant is withered, Death, a non-glyphosate resistant plant (without glyphosate resistance), is indicated by "-".
  • soybean EPSPS mutant G2 and soybean EPSPS mutant G3 were resistant to 1X glyphosate, but at 2X dose, all rice seedlings containing soybean EPSPS mutant G2 died, while soybean EPSPS mutant G3 was included. 19.4% of the rice seedlings were medium-resistant, indicating that the soybean EPSPS mutation at the K98(85)T locus could significantly improve the glyphosate resistance based on the glyphosate resistance provided by the P114(101)S site mutation. Sex.
  • the plant EPSPS mutant provided by the present application (the single mutant rice EPSPS mutant represented by SEQ ID NO: 3, compared to the wild type plant EPSPS, such as rice wild type EPSPS, corn wild type EPSPS, soybean wild type EPSPS, The single mutant maize EPSPS mutant shown in SEQ ID NO: 12, the single mutant soybean EPSPS mutant shown in SEQ ID NO: 20, and the multiple mutant rice EPSPS mutant shown in SEQ ID NO: 8, SEQ ID NO: 16.
  • the multi-mutant maize EPSPS mutant shown, the multi-mutant soybean EPSPS mutant shown in SEQ ID NO: 24, and the gene encoding the same have higher glyphosate resistance and intact bioenzyme activity;
  • the plant EPSPS mutants and coding genes thereof provided by the present application are derived from rice, corn, soybean varieties themselves rather than microorganisms, and are suitable for transforming various plant varieties such as rice, tobacco, soybean, corn, cotton, sorghum, wheat. Plants, which have a wider range of applications. Meanwhile, according to the sequence of the plant EPSPS mutant-encoding gene provided by the present application, it can be used for cultivating a new glyphosate resistant rice variety (non-transgenic method), obtaining a glyphosate-resistant non-transgenic conventional variety, and improving The public acceptance of new varieties.

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Abstract

提供了一种含K85突变的植物EPSPS突变体及其编码基因和应用。该植物EPSPS突变体，与大肠杆菌EPSPS相比，该植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第85位的位点具有突变K85T或者K85I。该位点的突变可提高多种植物的EPSPS的不同突变体对草甘膦的抗性，同时保持自身的生物酶催化活性。转化该植物EPSPS突变体的植株或重组菌均能够在草甘膦存在的条件下正常生长。该植物EPSPS突变体可用于转基因作物培育。

Description

一种含K85突变的植物EPSPS突变体及其编码基因和应用
相关申请的交叉引用
本申请要求于2017年11月2日提交中国专利局的申请号为201711062251.2、名称为“一种含K85突变的植物EPSPS突变体及其编码基因和应用”以及2018年9月13日提交中国专利局的申请号为201811070065.8、名称为“一种含K85突变的植物EPSPS突变体及其编码基因和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及基因工程技术领域，具体而言，涉及一种含K85突变的植物EPSPS突变体及其编码基因和应用。
背景技术
草甘膦是目前世界上使用的最为广泛的除草剂之一，至今已使用了近四十年。草甘膦抑制5-烯醇丙酮莽草酸-3-磷酸合成酶(EPSPS)的活性。EPSPS在莽草酸途径中催化PEP和shikimate-3-phosphate合成EPSP，最终合成芳香族氨基酸色氨酸、苯丙氨酸和酪氨酸。草甘膦导致芳香族胺基酸合成受阻，进而影响植物正常生长，终致死亡。
目前培育抗草甘膦品种的主要方法是应用基因工程手段将来自细菌的抗草甘膦基因导入农作物中，从而培育出转基因抗草甘膦作物新品种。1996年开始推广以来，种植面积迅速增加，至2015年，全球抗草甘膦转基因作物种植面积已达1.5亿公顷，占转基因作物种植总面积的83％，给农业生产和环境带来了巨大利益。
但是，目前农业上应用最广的抗草甘膦基因是来源于农杆菌(Agrobacterium tumefaciens)CP4菌株的CP4EPSPS。虽然先后从微生物中发现了许多能抗草甘膦的EPSPS基因，但这些基因在农作物中尚未得到广泛应用。这些微生物抗草甘膦基因在农作物中的应用是将这些基因如CP4EPSPS，用转基因的方法在农作物中表达。由此而产生的转基因作物虽然有相当大的推广面积，但由于反转基因浪潮，转基因作物在全世界的接受程度任然较低，即使在转基因作物种植面积最大的美洲，转基因也主要局限于玉米、大豆、棉花等几个作物。
发明内容
本申请的目的在于提供一种植物EPSPS突变体(即5-烯醇丙酮莽草酸-3-磷酸合成酶突变体)，其来自于植物，经突变后，具有了草甘膦抗性。
本申请的另一目的在于提供一种编码基因，其可编码上述的植物EPSPS突变体。
本申请的另一目的在于提供含有上述的编码基因的载体。
本申请的另一目的在于提供含有上述载体的细胞。
本申请的另一目的在于提供上述植物EPSPS突变体的应用。
本申请是这样实现的：
一种植物EPSPS突变体，其与大肠杆菌EPSPS相比，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第85位的位点具有突变。
一种编码基因，其编码上述植物EPSPS突变体。
含有上述编码基因的载体。
含有上述载体的重组细胞。
上述植物EPSPS突变体在培育抗草甘膦植物中的应用。
本申请具有以下有益效果：
本申请提供的植物EPSPS突变体，其与大肠杆菌EPSPS相比，该植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第85位的位点具有突变例如突变为T或I。该突变位点对应为水稻EPSPS的第100位、玉米EPSPS的第94位和大豆EPSPS的第98位的氨基酸残基。该位点突变明显提高了多种植物的EPSPS的不同突变体对草甘膦的抗性，同时保持了自身的生物酶催化活性。转化本申请提供的植物EPSPS突变体的植株或重组菌均能够在草甘膦存在的条件下正常生长，该植物EPSPS突变体不仅转基因作物培育外，也可应用于培育抗草甘膦非转基因植物例如水稻、烟草、大豆、玉米、小麦、棉花和高粱等，具有广阔的应用前景。
附图说明
为了更清楚地说明本申请实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本申请的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本申请实施例5中的转化实施例1和实施例2提供的水稻EPSPS突变体编码基因的大肠杆菌在含不同草甘膦浓度的培养基上的生长结果图；
图2为本申请实施例6中的转化实施例3提供的玉米EPSPS突变体编码基因的大肠杆菌在含不同草甘膦浓度的培养基上的生长结果图；
图3为本申请实施例7中的转化实施例4提供的大豆EPSPS突变体编码基因的大肠杆菌在含不同草甘膦浓度的培养基上的生长结果图；
图4为本申请实施例5中的pADV5载体的结构示意图；
图5为本申请实施例2中的大肠杆菌EPSPS、水稻EPSPS突变体II以及野生型水稻EPSPS三者的氨基酸序列比对的部分结果；
图6为本申请实施例3中的大肠杆菌EPSPS、玉米EPSPS突变体以及野生型玉米EPSPS三者的氨基酸序列比对的部分结果；
图7为本申请实施例4中的大肠杆菌EPSPS、大豆EPSPS突变体以及野生型大豆EPSPS三者的氨基酸序列比对的部分结果。
图8为本申请实验例8提供的pGVP1-EPSPS载体的结构示意图。
具体实施方式
为使本申请实施例的目的、技术方案和优点更加清楚，下面将对本申请实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本申请提供的一种含K85T和K85I突变的植物EPSPS突变体及其编码基因和应用进行具体说明。
一方面，本申请提供了一种植物EPSPS突变体(即5-烯醇丙酮莽草酸-3-磷酸合成酶的突变体)，其与大肠杆菌EPSPS相比，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第85位的位点具有突变。该位点的突变可以明显提高植物EPSPS突变体的草甘膦抗性。
进一步地，在本申请的一些实施方案中所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第85位的位点K突变为T或者I；即K>T或者K>I。
或者可理解为：将植物EPSPS突变体的氨基酸序列与大肠杆菌EPSPS的氨基酸序列进行比对，植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第85位的位置的氨基酸残基由K突变为T(简写为K85T)，或者是有K突变为I(简写为K85I)。
进一步地，在本申请的一些实施方案中，大肠杆菌EPSPS的氨基酸序列如SEQ ID NO:25所示。
也就是说，将植物EPSPS突变体的氨基酸序列与如SEQ ID NO:25所示的大肠杆菌EPSPS的氨基酸序列进行比对，植物EPSPS突变体的氨基酸序列对应于SEQ ID NO:25所示的第85位的位置的氨基酸残基由K突变为T，或者是有K突变为I。
进一步地，在本申请的一些实施方案中，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS还具有以下突变中的一种或多种的组合：G96A、P101S以及K194R。
突变G96A可理解为：将植物EPSPS突变体的氨基酸序列与大肠杆菌EPSPS的氨基酸序列进行比对，植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第96位的位置的氨基酸残基由G突变为A；
突变P101S可理解为：将植物EPSPS突变体的氨基酸序列与大肠杆菌EPSPS的氨基酸序列进行比对，植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第101位的位置 的氨基酸残基由P突变为S。
突变K194R可理解为：将植物EPSPS突变体的氨基酸序列与大肠杆菌EPSPS的氨基酸序列进行比对，植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第194位的位置的氨基酸残基由K突变为R。
G96A、P101S以及K194R中的一种或几种突变的组合与第85位的突变结合例如K85T或K85I突变的结合，可以加强植物EPSPS突变体的草甘膦抗性，且可保持植物EPSPS突变体的生物酶活性。
进一步地，在本申请的一些实施方案中，所述植物EPSPS突变体来自于包括但不限于以下植物：小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、绿豆、蚕豆、豌豆、扁豆、甘薯、马铃薯、棉花、大豆、油菜、芝麻、花生、向日葵、萝卜、胡萝卜、芜菁、甜菜、白菜、芥菜、甘蓝、花椰菜、芥蓝、黄瓜、西葫芦、南瓜、冬瓜、苦瓜、丝瓜、菜瓜、西瓜、甜瓜、番茄、茄子、辣椒、菜豆、豇豆、毛豆、韭菜、大葱、洋葱、韭葱、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甜菜、甘蔗、烟草、苜蓿、牧草、草坪草、茶和木薯。
也就是说，本申请提供的植物EPSPS突变体由来自于水稻、烟草、大豆、玉米、小麦、棉花、油菜和高粱等植物的野生型EPSPS经上述突变后得到，其保留了植物来源特性。可将编码该植物EPSPS突变体的编码基因应用于培育抗草甘膦的农作物品种，例如可采用转基因或基因修饰的方法实现培育目的。现对于现有的转化来自微生物的抗草甘膦的基因的方式，直接用植物来源的抗草甘膦基因转化或以此为模板进行编辑基因组，具有更可靠的生物安全性，有利于抗草甘膦新品种的推广和应用，提高公众接受程度。
优选地，在本申请的一些实施方案中，所述植物EPSPS突变体来自水稻(Oryza sativa)，所述植物EPSPS突变体的氨基酸序列如SEQ ID NO:4所示。SEQ ID NO:4所示的植物EPSPS突变体，为水稻EPSPS突变体I。相对于SEQ ID NO:2所示的野生型水稻EPSPS的氨基酸序列而言，该水稻EPSPS突变体I具有K100(85)I一个突变。
K100(85)I突变可理解为：相对于野生型水稻EPSPS而言，该水稻EPSPS突变体于SEQ ID NO:2中的第100位氨基酸残基由K突变为I，该位点对应于大肠杆菌EPSPS的第85位。
优选地，在本申请的一些实施方案中，所述植物EPSPS突变体来自水稻，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS具有以下突变：K85I、G96A和P101S。
进一步优选地，在本申请的一些实施方案中，所述植物EPSPS突变体的氨基酸序列如SEQ ID NO:8所示。
SEQ ID NO:8所示的植物EPSPS突变体，为水稻EPSPS突变体II。相对于SEQ ID NO:2所示的野生型水稻EPSPS的氨基酸序列而言，该水稻EPSPS突变体II具有K100(85)I、G111 (96)A和P116(101)S三个突变。
G111(96)A突变可理解为：相对于野生型水稻EPSPS而言，水稻EPSPS突变体II于SEQ ID NO:2中的第111位氨基酸残基由G突变为A，该位点对应于大肠杆菌EPSPS的第96位；
P116(101)S突变可理解为：相对于野生型水稻EPSPS而言，水稻EPSPS突变体II于SEQ ID NO:2中的第116位氨基酸残基由P突变为S，该位点对应于大肠杆菌EPSPS的第101位。
进一步地，在本申请的一些实施方案中，所述植物EPSPS突变体来自玉米(Zea mays)，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS具有以下突变：K85I、G96A、P101S以及K194R。
进一步地，在本申请的一些实施方案中，所述植物EPSPS突变体的氨基酸序列如SEQ ID NO:16所示。
SEQ ID NO:16所示的植物EPSPS突变体，为玉米EPSPS突变体。相对于SEQ ID NO:10所示的野生型玉米EPSPS的氨基酸序列而言，该玉米EPSPS突变体具有K94(85)I、G105(96)A、P110(101)S以及K207(194)R四个突变。
K94(85)I突变可理解为：相对于野生型玉米EPSPS而言，玉米EPSPS突变体于SEQ ID NO:10中的第94位氨基酸残基由K突变为I，该位点在对应于大肠杆菌EPSPS的第85位；
G105(96)A突变可理解为：相对于野生型玉米EPSPS而言，玉米EPSPS突变体于SEQ ID NO:10中的第105位氨基酸残基由G突变为A，该位点对应于大肠杆菌EPSPS的第96位；
P110(101)S突变可理解为：相对于野生型玉米EPSPS而言，玉米EPSPS突变体于SEQ ID NO:10中的第110位氨基酸残基由P突变为S，该位点对应于大肠杆菌EPSPS的第101位；
K207(194)R突变可理解为：相对于野生型玉米EPSPS而言，玉米EPSPS突变体于SEQ ID NO:10中的第207位氨基酸残基由K突变为R，该位点对应于大肠杆菌EPSPS的第194位。
进一步地，在本申请的一些实施方案中，所述植物EPSPS突变体来自大豆(Glycine max(Linn.)Merr.)，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS还具有以下突变：K85T和P101S。
进一步地，在本申请的一些实施方案中，所述植物EPSPS突变体的氨基酸序列如SEQ ID NO:24所示。
SEQ ID NO:24所示的植物EPSPS突变体，为大豆EPSPS突变体。相对于SEQ ID NO:24 所示的野生型大豆EPSPS的氨基酸序列而言，该小麦EPSPS突变体具有K98(85)T和P114(101)S两个突变。
K98(85)T突变可理解为：相对于野生型大豆EPSPS而言，大豆EPSPS突变体于SEQ ID NO:18中的第98位氨基酸残基由K突变为T，该位点对应于大肠杆菌EPSPS的第85位；
P114(101)S突变可理解为：相对于野生型大豆EPSPS而言，大豆EPSPS突变体于SEQ ID NO:18中的第114位氨基酸残基由P突变为S，该位点对应于大肠杆菌EPSPS的第101位。
另一方面，本申请提供了一种编码基因，其编码上述的植物EPSPS突变体。
根据密码子的简并性，在上述植物EPSPS突变体的氨基酸序列基础上能够很容易地得到该EPSPS突变体的核苷酸编码序列。只要是能够编码出上述植物EPSPS突变体的编码基因即属于本申请的保护范围。
进一步地，在本申请的一些实施方案中，上述编码基因如SEQ ID NO:3、7、15或23所示。
SEQ ID NO:3所示的为水稻EPSPS突变体I编码基因，编码SEQ ID NO:4所示的水稻EPSPS突变体I。
SEQ ID NO:7所示的序列为水稻EPSPS突变体II编码基因，编码SEQ ID NO:8所示的水稻EPSPS突变体II；
SEQ ID NO:15所示的序列为玉米EPSPS突变体编码基因，编码SEQ ID NO:16所示的玉米EPSPS突变体；
玉米EPSPS突变体玉米EPSPS突变体SEQ ID NO:23所示的序列为大豆EPSPS突变体编码基因，编码SEQ ID NO:24所示的大豆EPSPS突变体。
本领域技术人员，根据密码子的简并性，容易在上述编码基因的序列基础进行一个或多个核苷酸的替换，得到相应的衍生序列，以使编码出本申请提供的植物EPSPS突变体。因此，在上述编码基因的序列基础进行一个或多个核苷酸的替换，得到相应的编码出本申请提供的植物EPSPS突变体的衍生序列也属于本申请的保护范围。
另一方面，本申请提供了含有上述编码基因的载体。
进一步地，在本申请的一些实施方案中，载体可以是克隆载体，也可以是表达载体，进一步地，在本申请的一些实施方案中，表达载体可以是原核表达载体例如pADV5载体，也可以是真核表达载体。进一步地，在本申请的一些实施方案中，真核表达载体是植物表达载体例如pBI121载体。
容易理解，本领域技术人员根据需要，可以选择合适的载体，作为运载上述编码基因的工具，其均属于本申请的保护范围。
另一方面，本申请提供了含有上述载体的重组菌或重组细胞。
进一步地，在本申请的一些实施方案中，重组菌可以是球菌、杆菌例如大肠杆菌或者是螺旋菌；也可以是自氧菌或者是异氧菌。
进一步地，在本申请的一些实施方案中，重组细胞可以原核细胞也可以是真核细胞；进一步地，在本申请的一些实施方案中，真核细胞可以是动物细胞，也可以是植物细胞；进一步地，在本申请的一些实施方案中，植物细胞可以是双子叶植物细胞，也可以是单子叶植物细胞。
容易理解，本领域技术人员根据需要，可以选择合适的细菌或细胞，作为上述编码基因的宿主，其均属于本申请的保护范围。
另一方面，本申请提供了上述植物EPSPS突变体在培育抗草甘膦植物中的应用。
进一步地，在本申请的一些实施方案中，上述应用包括：将载体转化目的植物，所述载体含有编码所述植物EPSPS突变体的编码基因。
例如，将含有SEQ ID NO:7所示的水稻EPSPS编码基因的载体转化例如水稻愈伤组织，培养使其分化形成完整水稻植株，即可培育出转基因抗草甘膦水稻。
进一步地，在本申请的一些实施方案中，上述应用包括：修饰目的植物的内源EPSPS基因，使其编码所述植物EPSPS突变体。
例如，以SEQ ID NO:7所示的水稻EPSPS编码基因的部分或全部为模板，对水稻基因组的内源EPSPS基因进行修饰，进而可培育出非转基因水稻。
进一步地，在本申请的一些实施方案中，上述应用包括：诱变植物细胞、组织、个体或群体，使其编码所述植物EPSPS突变体。
例如，以SEQ ID NO:7所示的为水稻EPSPS编码基因为指导，对水稻材料进行诱变，如化学、辐射诱变等，进而可培育出内源EPSPS基因突变的水稻。例如还可以通过CRISPR/Cas9技术将内源EPSPS基因的编码序列修饰为该编码基因的碱基序列(SEQ ID NO:7)，在目的植物中编码出与本申请提供的水稻EPSPS突变体(SEQ ID NO:8)一样的蛋白，使得目的植物具有草甘膦抗性。
进一步地，在本申请的一些实施方案中，目的植物为水稻、烟草、大豆、玉米、小麦、棉花、油菜和高粱等植物中的任意一种。
容易理解，本领域技术人员根据需要，可以选择需要培育的农作物品种，只要是应用了本申请提供的植物EPSPS突变体和/或其编码基因即属于本申请的保护范围。
需要说明的是，本申请所涉及的蛋白序列比对所使用的比对方式为Clustal在线比对，其网站地址为：http://www.ebi.ac.uk/Tools/msa/clustalo/。采用其他的序列比对工具(例如DNAMAN，相关参数设置按默认设置)所得到结果与Clustal在线比对得到的结果基本一 致。
综上，本申请提供的植物EPSPS突变体，其与大肠杆菌EPSPS相比，该植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第85位具有突变例如K>T或K>I。该突变位点的位置对应于野生型水稻EPSPS(SEQ ID NO:2)的第100位；对应于野生型玉米EPSPS(SEQ ID NO:10)的第94位；对应于野生型大豆EPSPS(SEQ ID NO:18)的第98位。该位点突变可明显提高多种植物的EPSPS的不同突变体对草甘膦的抗性，同时保持突变体自身的生物酶催化活性。转化该植物EPSPS突变体的植株或重组菌均能够正常生长，该植物EPSPS突变体不仅转基因作物培育外，也可应用于培育抗草甘膦非转基因植物例如水稻、烟草、大豆、玉米、小麦、棉花和高粱等，具有广阔的应用前景。
以下结合实施例对本申请的特征和性能作进一步的详细描述。
实施例1
本实施例提供一种植物EPSPS突变体，其来源于水稻，为水稻EPSPS突变体I，其由野生型水稻EPSPS(氨基酸序列如SEQ ID NO:2所示)经突变后得到，其氨基酸序列如SEQ ID NO:4所示。
相对于SEQ ID NO:2所示的野生型水稻EPSPS的氨基酸序列而言，本实施例提供的水稻EPSPS突变体I具有K100(85)I一个突变。
即，相对于野生型水稻EPSPS而言，水稻EPSPS突变体I的第100位氨基酸残基由G突变为A，该位点对应于大肠杆菌EPSPS的第85位。
本实施例还提供了编码上述水稻EPSPS突变体I的水稻EPSPS突变体I编码基因，其核苷酸序列如SEQ ID NO:1所示。
本申请实施例提供的水稻EPSPS突变体I编码基因和水稻EPSPS突变体I均可通过化学合成的方法获得。
实施例2
本实施例提供一种植物EPSPS突变体，其来源于水稻，为水稻EPSPS突变体II，其由野生型水稻EPSPS(氨基酸序列如SEQ ID NO:2所示)经突变后得到，其氨基酸序列如SEQ ID NO:8所示。
相对于SEQ ID NO:2所示的野生型水稻EPSPS的氨基酸序列而言，该水稻EPSPS突变体II具有K100(85)I、G111(96)A和P116(101)S三个突变。
即，如图5所示(图5显示了大肠杆菌EPSPS、水稻EPSPS突变体II以及野生型水稻EPSPS三者的氨基酸序列比对的部分结果，图中：Ec-EPSPS WT代表大肠杆菌EPSPS；Os-EPSPS M代表水稻EPSPS突变体II；Os-EPSPS WT代表野生型水稻EPSPS)，相对于野生型水稻EPSPS而言，水稻EPSPS突变体II的第100位氨基酸残基由K突变为I，该位 点对应于大肠杆菌EPSPS(SEQ ID NO:25)的第85位；第111位氨基酸残基由G突变为A，该位点对应于大肠杆菌EPSPS的第96位；第116位氨基酸残基由P突变为S，该位点对应于大肠杆菌EPSPS的第101位(如图5中箭头所指位点)。
本实施例还提供编码上述水稻EPSPS突变体II的水稻EPSPS突变体II编码基因，其核苷酸序列如SEQ ID NO:7所示。
本申请实施例提供的水稻EPSPS突变体II编码基因和水稻EPSPS突变体II均可通过化学合成的方法获得。
实施例3
本实施例提供一种植物EPSPS突变体，其来源于玉米，为玉米EPSPS突变体，其由野生型玉米EPSPS(氨基酸序列如SEQ ID NO:10所示)经突变后得到，其氨基酸序列如SEQ ID NO:16所示。
相对于SEQ ID NO:10所示的野生型玉米EPSPS的氨基酸序列而言，本实施例提供的玉米EPSPS突变体具有K94(85)I、G105(96)A、P110(101)S以及K207(194)R四个突变。
即，如图6所示(图6显示了大肠杆菌EPSPS、玉米EPSPS突变体以及野生型玉米EPSPS三者的氨基酸序列比对的部分结果，图中：Ec-EPSPS WT代表大肠杆菌EPSPS；Zm-EPSPS M代表玉米EPSPS突变体；Zm-EPSPS WT代表野生型玉米EPSPS)，相对于野生型玉米EPSPS而言，玉米EPSPS突变体第94位氨基酸残基由K突变为I，该位点对应于大肠杆菌EPSPS的第85位；第105位氨基酸残基由G突变为A，该位点对应于大肠杆菌EPSPS的第96位；第110位氨基酸残基由P突变为S，该位点对应于大肠杆菌EPSPS的第101位；第207位氨基酸残基由K突变为R，该位点对应于大肠杆菌EPSPS的第194位(如图6中箭头所指位点)。
本申请实施例还提供了编码上述玉米EPSPS突变体的玉米EPSPS突变体编码基因，其核苷酸序列如SEQ ID NO:15所示。
本申请实施例提供的玉米EPSPS突变体编码基因和玉米EPSPS突变体均可通过化学合成的方法获得。
实施例4
本实施例提供一种植物EPSPS突变体，其来源于大豆，为大豆EPSPS突变体，其由野生型大豆EPSPS(氨基酸序列如SEQ ID NO:18所示)经突变后得到，其氨基酸序列如SEQ ID NO:24所示。
相对于SEQ ID NO:18所示的野生型大豆EPSPS的氨基酸序列而言，该大豆EPSPS突变体具有K98(85)T和P114(101)S两个突变。
即，如图7所示(图7显示了大肠杆菌EPSPS、大豆EPSPS突变体以及野生型大豆EPSPS 三者的氨基酸序列比对的部分结果，图中：Ec-EPSPS WT代表大肠杆菌EPSPS；Gm-EPSPS M代表大豆EPSPS突变体；Gm-EPSPS WT代表野生型大豆EPSPS)，相对于野生型大豆EPSPS而言，大豆EPSPS突变体第98位氨基酸残基由K突变为T，该位点对应于大肠杆菌EPSPS(SEQ ID NO:25)的第85位；第114位氨基酸残基由P突变为S，该位点对应于大肠杆菌EPSPS的第101位(如图7中箭头所指位点)。
本申请实施例还提供了编码上述大豆EPSPS突变体的大豆EPSPS突变体编码基因，其核苷酸序列如SEQ ID NO:23所示。
本申请实施例提供的大豆EPSPS突变体编码基因和大豆EPSPS突变体均可通过化学合成的方法获得。
实施例5
以转化实施例1提供的水稻EPSPS突变体I编码基因(SEQ ID NO:3)的大肠杆菌为实验组1(仅具有K100(85)I突变)；
以转化不具有K100(85)I突变仅具有G111(96)A和P116(101)S突变的水稻EPSPS突变体的编码基因(核苷酸序列如SEQ ID NO:5所示，编码的水稻EPSPS突变体的氨基酸序列如SEQ ID NO:6所示)的大肠杆菌为实验组2；
以转化实施例2提供的水稻EPSPS突变体II编码基因(SEQ ID NO:7)的大肠杆菌为实验组3(编码的的水稻EPSPS突变体II具有K100(85)I、G111(96)A和P116(101)S三个突变)；
以转化野生型水稻EPSPS编码基因(SEQ ID NO:1，其编码SEQ ID NO:2所示的野生型水稻EPSPS)的大肠杆菌为对照组。
通过检测被转化的大肠杆菌在含不同草甘膦浓度(0mM、1mM、10mM、50mM)的培养基(以M9为基础培养基，再添加一定浓度的抗生素Spec(Spectinomycin、奇放线菌素)、Gen(Gentamycin、庆大霉素)、Amp(Ampicillin、氨苄青霉素)以及不同浓度的草甘膦得到的培养基)上的生长情况，验证水稻EPSPS突变体I编码基因及其编码的水稻EPSPS突变体I在大肠杆菌中的抗草甘膦能力，以及水稻EPSPS突变体II编码基因及其编码的水稻EPSPS突变体II在大肠杆菌中的抗草甘膦能力。
其中，M9基础培养基可参考如下方法配制：
5×M9盐溶液：称取6.78g Na 2HPO 4，3.0g KH 2PO 4，0.5g NaCl，1.0g NH 4Cl，加ddH 2O至200ml，高温高压灭菌处理；
20％葡萄糖：称取葡糖糖20g，加80ml ddH 2O溶解，定容至100ml，过滤灭菌处理；
1.0M MgSO 4：称取24.6g MgSO 4-7H 2O，用80ml的ddH 2O溶解，定容至100ml，灭菌处理；
1.0M CaCl 2：称取11.1g CaCl 2，用80ml的ddH 2O溶解，定容至100ml，灭菌处理；
用200ml 5×M9盐溶液，20ml 20％葡萄糖，2ml 1.0M MgSO 4和0.1ml 1.0M CaCl 2，加ddH 2O补足至1000ml，即配制成1000ml M9基础培养基。
其中，所用大肠杆菌为双敲除的大肠杆菌即EPSPS缺陷型大肠杆菌(敲除EPSPS基因和C-P Lyase基因后的大肠杆菌DH5α，命名为EDCE，其制备方法可参考申请号为2016103256926的中国发明专利)。
检测方法为本领域公知的方法，简要描述为：采用化学合成的方法合成水稻EPSPS突变体编码基因，两端引入酶切位点(Pac1和Sbf1)，酶切后，在连接酶的作用下连接至经相同酶切处理后的表达载体(例如pADV5载体，其结构如图4所示)上，然后转化双敲除的大肠杆菌，经验证后，挑取阳性克隆，接种至含草甘膦的培养基上生长，观察生长情况。
结果如图1所示(图中：R0为对照组，R1为实验组1，R2为实验组2，R3为实验组3)。
在含0mM草甘膦的培养基上，对照组和实验组1-3的EPSPS缺陷型大肠杆菌均能生长，表明实验组和对照组的EPSPS蛋白(SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8)都具有正常EPSPS酶活力；
在含1mM草甘膦的培养基上，对照组(R0)生长缓慢，实验组1(R1)生长明显优于对照组(R0)，说明含K100(85)I的单突变的EPSPS突变体抗草甘膦的能力明显优于野生型EPSPS；
实验组2(R2)和实验组3(R3)均能在含10mM草甘膦的培养基上生长，但实验组3(R3)的生长明显优于实验组2(R2)，只有实验组3(R3)能在在含50mM草甘膦的培养基上，说明K100(85)I位点突变能够在G111(96)A和P116(101)S位点变异提供的草甘膦抗性基础上进一步大幅度提高草甘膦抗性。
上述结果表明，单突变K100(85)I能够赋予水稻EPSPS突变体草甘膦抗性，且其与G111(96)A和P116(101)S突变结合后，具有草甘膦抗性加强的特性。
实施例6
以转化野生型玉米EPSPS编码基因(SEQ ID NO:9，编码SEQ ID NO:10所示的野生型玉米EPSPS)的大肠杆菌为对照组；
以转化仅具有K94(85)I单突变的玉米EPSPS突变体I的编码基因(SEQ ID NO:11，其编码SEQ ID NO:12)的大肠杆菌为实验组1；
以转化不具有K94(85)I突变仅具有G105(96)A、P110(101)S以及K207(194)R三个突变的玉米EPSPS突变体的编码基因(核苷酸序列如SEQ ID NO:13所示，编码的三突变玉米EPSPS突变体的氨基酸序列如SEQ ID NO:14所示)的大肠杆菌为实验组2；
以转化实施例3提供的玉米EPSPS突变体(具有K94(85)I、G105(96)A、P110(101)S以及K207(194)R四个突变)的编码基因(SEQ ID NO:15)的大肠杆菌为实验组3。
检测转化的大肠杆菌在含不同草甘膦浓度(0、1、20、50mM)的培养基上的生长情况，验证玉米EPSPS突变体III编码基因及其编码的玉米EPSPS突变体III在大肠杆菌中的抗草甘膦能力。其中，所用大肠杆菌为双敲除的大肠杆菌(敲除EPSPS基因和C-P Lyase基因后的大肠杆菌DH5α，命名为EDCE，其制备方法可参考申请号为2016103256926的中国发明专利)。
结果如图2所示(图中：Z0代表对照组，Z1代表实验组1，Z2代表实验组2，Z3代表实验组3)：
在含0mM和1mM草甘膦的培养基上实验组和对照组均能正常生长，说明实验组和对照组的EPSPS蛋白具有正常的EPSPS酶活力；但实验组1(Z1)的生长明显优于对照组(Z0)，且实验组3(Z3)的大肠杆菌生长明显优于实验组2(Z2)，说明K94(85)I突变提供了更高的EPSPS酶活性。
在含1mM草甘膦的培养基上，对照组(Z0)生长微弱，实验组1(Z1)生长明显优于对照组(Z0)，说明含K94(85)I的单突变EPSPS突变体III抗草甘膦的能力明显优于野生型EPSPS；
实验组2(Z2)和实验组3(Z3)均能在含20mM草甘膦的培养基上生长，但实验组3(Z3)的生长明显优于实验组2(Z2)，只有实验组3(Z3)能在在含50mM草甘膦的培养基上，说明K94(85)I位点突变能够在G105(96)A、P110(101)S以及K207(194)R位点变异提供的草甘膦抗性基础上进一步大幅度提高草甘膦抗性。
上述结果进一步说明，单突变K94(85)I与G105(96)A、P110(101)S以及K207(194)R突变结合，具有草甘膦抗性加强的特性。
实施例7
以转化野生型大豆EPSPS编码基因(SEQ ID NO:17，编码SEQ ID NO:18所示的野生型大豆EPSPS)的大肠杆菌为对照组；
以转化仅具有K98(85)T突变，不具有P114(101)S突变的单突变大豆EPSPS突变体的编码基因(SEQ ID NO:19，其编码的单突变大豆EPSPS突变体的氨基酸序列如SEQ ID NO:20所示)的大肠杆菌为实验组1；
以转化不具有K98(85)T突变仅具有P114(101)S突变的单突变大豆EPSPS突变体的编码基因(核苷酸序列如SEQ ID NO:21所示，编码的单突变大豆EPSPS突变体的氨基酸序列如SEQ ID NO:22所示)的大肠杆菌为实验组2；
以转化实施例4提供的大豆EPSPS突变体(具有K98(85)T和P114(101)S两个突变) 的编码基因(SEQ ID NO:23)的大肠杆菌为实验组3；
检测大肠杆菌在含不同草甘膦浓度(0、1、10mM)的培养基上的生长情况，验证大豆EPSPS突变体编码基因及其编码的大豆EPSPS突变体在大肠杆菌中的抗草甘膦能力。其中，所用大肠杆菌为双敲除的大肠杆菌(敲除EPSPS基因和C-P Lyase基因后的大肠杆菌DH5α，命名为EDCE，其制备方法可参考申请号为2016103256926的中国发明专利)。
结果如图3所示(图中：G0代表对照组，G1代表实验组1，G2代表实验组2，G3代表实验组3)：在含0mM草甘膦的培养基上实验组和对照组均能正常生长，表明实验组和对照组的EPSPS蛋白具有正常的EPSPS酶活力；
在含1mM草甘膦的培养基上，对照组(G0)基本不能生长，实验组(G1)生长明显优于对照组(G0，说明含K98(85)T单突变的大豆EPSPS突变体抗草甘膦的能力明显优于野生型大豆EPSPS；
实验组2(G2)和实验组3(G3)均能在含20mM草甘膦的培养基上生长，但实验组3(G3)的生长优于实验组2(G2)，说明K98(85)T位点突变能够在P114(101)S位点变异提供的草甘膦抗性基础上进一步提高草甘膦抗性。
该结果进一步说明，单突变K98(85)T具有草甘膦抗性，且其与P114(101)S突变结合后，具有草甘膦抗性加强的特性。
实验例8
检测实施例7提供的G1(SEQ ID NO:20)、G2(SEQ ID NO:22)和G3(SEQ ID NO:24)大豆EPSPS突变体，实施例5提供的R1水稻EPSPS突变体(SEQ ID NO:4)，和实施例6提供的Z1玉米EPSPS突变体(SEQ ID NO:12)在转基因水稻中的草甘膦抗性，方法如下：
按常规方法提取实施例1-7中的pADV5-EPSPS的单克隆抗性菌的质粒(含EPSPS突变基因基因)。将其用Pac1和BstEII双酶切，回收小片段，然后用T4DNA连接酶将小片段连接至经同样双酶切后的pGVP1载体上，得到pGVP1-EPSPS载体，其结构如图23所示。
将pGVP1-EPSPS载体转化EHA105(根癌农杆菌、Agrobactrium tumefaciens)感受态细胞，挑取单克隆进行菌落PCR检测，得到阳性菌株；再将阳性菌株接种到1mL的含50μg·mL -1卡那霉素和50μg·mL -1利福平的YEP培养中扩繁，保存于-80℃，或用于后续实验。
水稻转化：
把-80℃保存的含目的基因载体的菌种400ul加进有YEP+50μg/mL利福平+50μg/mL卡那的固体培养基的培养皿中，28℃暗中培养24小时，再将该菌加进浸染培养基，调该菌液到OD＝0.2作为浸染液。
消毒和预培养:取成熟水稻(日本晴)种子，人工脱壳，挑选饱满无菌斑的种子，按以 下步骤消毒：将种子放入50ml无菌离心管中，加入70％酒精消毒30秒,倒去酒精，使用无菌水清洗一次；加入10-20ml的2.6％次氯酸钠溶液，浸泡消毒20分。倒去次氯酸钠溶液，用无菌水浸泡清洗6-7次，每次3分。
诱导与继代培养:种子放在无菌滤纸上吸干，置成熟胚于诱导培养基中，每皿12颗；操作完毕用封口膜封好培养皿，在30℃暗中培养21-28天,将愈伤组织转接到新鲜培养基上,继续培养7-14天左右.,取1-2mm大小的球型愈伤组织,作为浸染受体。
浸染与共培养：
将愈伤组织接入离心管或培养杯中，加入调制好的农杆菌悬浮液浸染10分钟，期间摇动数次；倒去菌液，将愈伤组织取出，置于无菌的滤纸上吸干表面菌液(30分钟左右)；将愈伤组织置于培养皿中无菌滤纸上，25℃暗中培养2-3天。
恢复培养：将共培养后的愈伤组织接种于恢复培养基上，30℃暗中培养5-7天。第一轮筛选：将愈伤转入筛选培养基1(S1),30℃暗中培养14天.
第二轮筛选：然后将愈伤转到筛选培养基2(S2),30℃暗中培养14天。
第一轮分化：将经过筛选后的抗性愈伤组织转接到分化培养基上，30℃光照19小时，培养21天左右。第二轮分化：挑选新生的幼小嫩芽移至新的分化培养基，继续培养21天左右。
待新生幼苗长至2cm左右时，移至生根培养基中，30℃光照(16/8h)培养3～4周，待诱导出根并且幼苗长至7～10cm时，从培养基中取出，洗净根部沾染的培养基，移至育秧盘中，继续培养10d左右后移入温室或大田。
培养基配方：
诱导培养基：NB BasaL Medium【N6大量+MS铁盐溶液+B5微量及有机】+水解酪蛋白0.3g/L+L-proline+2.787g/L+蔗糖30g/L+2,4-D 4mg/L+琼脂8g/L，pH5.8。
浸染培养基：NB BasaL Medium【N6大量+MS铁盐溶液+B5微量及有机】+L-proline0.7g/L+蔗糖68.4g/L+葡萄糖36g/L+2,4-D 2mg/L pH5.2(小锅115℃)，AS 20mg/L现用现加。
共培养培养基：NB BasaL Medium【N6大量+MS铁盐溶液+B5微量及有机】+水解酪蛋白0.3g/L+L-proline 2.787g/L+蔗糖30g/L+2,4-D 1mg/L pH5.2，AS 20mg/L现用现加。
恢复培养基：NB BasaL Medium【N6大量+MS铁盐溶液+B5微量及有机】+谷氨酰胺0.2g/L+L-proline 2.787g/L+蔗糖30g/L+2,4-D 4mg/L+琼脂8g/L，pH5.8。灭菌后加Cefotaxime(头孢100mg/L)+Timentin(特美汀)100mg/L+Vancomycin(万古霉素)50mg/L。
筛选培养基1(S1)：NB BasaL Medium【N6大量+MS铁盐溶液+B5微量及有机】+谷氨酰胺0.2g/L+L-proline 2.787g/L+蔗糖30g/L+2,4-D 2mg/L+琼脂8g/L，pH5.8。灭菌后加Cefotaxime(头孢)100mg/L+Timentin(特美汀)100mg/L+Vancomycin(万古霉素)50mg/L。加筛选剂(Glyphosate 400mg/L,or hygromycin 50mg/L)。
筛选培养基2(S2)：NB BasaL Medium【N6大量+MS铁盐溶液+B5微量及有机】+谷氨酰胺0.2g/L+L-proline 2.787g/L+蔗糖30g/L+2,4-D 2mg/L+琼脂8g/L，pH5.8。灭菌后加Cefotaxime(头孢)100mg/L+Timentin(特美汀)100mg/L+Vancomycin(万古霉素)50mg/L。加筛选剂(Glyphosate 50-400mg/L,or hygromycin 30mg/L)。
分化培养基(F)：MS BasaL Medium【MS大量、铁盐溶液、微量及有机】+谷氨酰胺0.2g/L+蔗糖30g/L+山梨醇30g/L+琼脂8g/L pH5.8。灭菌后加Cefotaxime(头孢)200mg/L+KT 2mg/L+NAA 0.02mg/L+GLyphosate 1-5mg/L.
加筛选剂(Glyphosate 1-5mg/L,or hygromycin 20mg/L)。
生根培养基：1/2MS BasaL Medium【MS大量、铁盐溶液、微量及有机】+肌醇0.1g/L+蔗糖30g/L+琼脂8g/L,pH5.8。灭菌后加Cefotaxime(头孢)100mg/L,NAA 0.2mg/L
转基因植株的检测：
采用PCR法检测出转EPSPS突变体基因的水稻植株，根据pGVP1-EPSPS载体序列和水稻内参基因设计正反向检测引物，序列如下：
针对载体部分序列：
CaMV15:5’-GGTGGCTCCTACAAATGCCATC-3’；
CTS3：5’-GAGCCAATTAACGTCATCCCAC-3’；扩增片段大小为452bp；
针对水稻内参基因：
OsF:5’-GCTTCTGACCAGCCCATTATTCTGC-3’；
OsR：5’-CCCTCAAGGGTAAGCTCATCTCTCTTC-3’；扩增片段大小为629bp。
分别提取转pGVP1-EPSPS基因的水稻植株的基因组DNA，均一化至100ng/μL。
PCR检测体系：10μL 2×TsINGKe，2μL引物混合物(10μmol/L的OsF、OsR、CaMV15、CTS3，各0.5μL)，1μL基因组DNA模板(100ng/μL)，7μL ddH 2O。
PCR检测程序：94℃，3min；94℃，30s；62℃，30s；72℃，45s；30个循环；72℃，10min；12℃保持。
PCR扩增产物1.5％琼脂糖凝胶电泳，在452bp位置和629bp位置有条带的则为转基因阳性烟草植株。
本实施例验证EPSPS突变体在水稻转基因植株中的草甘膦抗性。实验方法如下：
将移栽后的转基因水稻苗均匀地排放在同一实验区域(避免叶片重叠)。计算实验组和对照组的占地区域面积，根据区域面积，按1060克/公顷(0.106g/m2)为1X剂量来喷施草甘膦。2X剂量为2120克/公顷，5X剂量为5300克/公顷，20X剂量为21200克/公顷。
用市售的41％草甘膦铵盐农达，根据上述的喷洒浓度，取相应体积的草甘膦铵盐农达，然后用20倍体积水稀释后，均匀喷施在实验组和对照组的植株上。待叶面干燥后，将植株移入温室或室外培养。
草甘膦抗性的统计标准为：如果植株没有任何的草甘膦损伤现象，生长正常，为高草甘膦抗性植株，用“+++”表示；如果植株的叶片颜色有些发黄，生长略为缓慢，为中草甘膦抗性植株，用“++”表示；如果植株的部分叶片枯萎，生长非常缓慢，为低草甘膦抗性植株，用“+”表示；如果植株枯萎、死亡，为非草甘膦抗性植株(不具有草甘膦抗性)，用“-”表示。
喷洒1X草甘膦后，于第10天观察、记录各组植株的生长状况，并对存活的植株喷洒2X草甘膦。再过10天观察、记录各组植株的生长状况，并对存活的植株喷洒5X草甘膦。再过10天观察、记录各组植株的生长状况，并对存活的植株喷洒20X草甘膦。再过10天观察、记录各组植株的生长状况，结果见表一，其中-、+、++和+++为相应的植株数，“％++&+++”为中、高抗草甘膦植株占观察植株总数的百分比。结果见表1。
表1
Figure PCTCN2018121331-appb-000001
根据表1的结果可看出：
在喷施1X草甘膦后，转化野生型大豆EPSPS G0的水稻全部无抗性，均死亡，但转化 大豆EPSPS突变体G1的水稻苗的草甘膦抗性明显优于G0，有6.4％为中抗或高抗，在2X草甘膦剂量下G1的水稻苗仍有存活，说明含K98(85)T的大豆EPSPS单突变体抗草甘膦的能力明显优于野生型；
转化大豆EPSPS突变体G2、大豆EPSPS突变体G3的水稻均对1X草甘膦有一定抗性，但在2X剂量下，含大豆EPSPS突变体G2的水稻苗全部死亡，而含大豆EPSPS突变体G3的水稻苗有19.4％为中抗，说明K98(85)T位点的大豆EPSPS突变能够在P114(101)S位点突变提供的草甘膦抗性基础上很明显地进一步提高草甘膦抗性。
在喷施1X草甘膦后，转化野生型水稻EPSPS R0的水稻全部无抗性，除一株严重受损外，其余全部死亡，但转化水稻EPSPS突变体R1的水稻苗的草甘膦抗性明显优于R0，有88.9为中抗或高抗，即使在2X和5X草甘膦剂量下R1仍有27.8％和11.1％为中抗或高抗，说明含K100(85)I的水稻EPSPS单突变体抗草甘膦的能力明显优于野生型；
在喷施1X草甘膦后，转化野生型玉米EPSPS Z0的水稻苗虽有少数存活，但严重受损，没有中、高抗的植株，但转化玉米EPSPS突变体Z1的水稻苗的草甘膦抗性明显优于Z0，有10.5％为中抗或高抗，在2X和5X草甘膦剂量下，转化野生型玉米EPSPS Z0的水稻没有一株存活，但转化玉米EPSPS突变体Z1的水稻苗均有存活，说明含K94(85)I的玉米EPSPS单突变体抗草甘膦的能力明显优于野生型；
以上结果充分说明，与大肠杆菌EPSPS序列相比，植物EPSPS突变体的氨基酸序列在对应于大肠杆菌EPSPS的第85位具有突变K85I可以赋予或提高植物EPSPS突变体在植物中的草甘膦抗性。
综上，相较于野生型植物EPSPS例如水稻野生型EPSPS、玉米野生型EPSPS、大豆野生型EPSPS，本申请提供的植物EPSPS突变体(SEQ ID NO:3所示的单突变水稻EPSPS突变体、SEQ ID NO:12所示的单突变玉米EPSPS突变体、SEQ ID NO:20所示的单突变大豆EPSPS突变体以及SEQ ID NO:8所示的多突变水稻EPSPS突变体、SEQ ID NO:16所示的多突变玉米EPSPS突变体、SEQ ID NO:24所示的多突变大豆EPSPS突变体)及其编码基因具有更高的草甘膦抗性和完整的生物酶活性；
此外，本申请提供的植物EPSPS突变体及其编码基因来源于水稻、玉米、大豆品种本身而不是微生物，其可适于转化各种植物品种例如水稻、烟草、大豆、玉米、棉花、高粱、小麦等植物，其具有更广阔的适用范围。同时，根据本申请提供的植物EPSPS突变体编码基因的序列，可以将其用于培育新的抗草甘膦的水稻品种中(非转基因方法)，获得抗草甘膦的非转基因常规品种，提高新品种的公众接受程度。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、 等同替换、改进等，均应包含在本申请的保护范围之内。

Claims (19)

  1. 一种植物EPSPS突变体，其特征在于，其与大肠杆菌EPSPS相比，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第85位的位点具有突变。
  2. 根据权利要求1所述的植物EPSPS突变体，其特征在于，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS的第85位的位点K突变为T或者I。
  3. 根据权利要求1所述的植物EPSPS突变体，其特征在于，大肠杆菌EPSPS的氨基酸序列如SEQ ID NO:25所示。
  4. 根据权利要求1、2或3所述的植物EPSPS突变体，其特征在于，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS还具有以下突变中的一种或多种的组合：G96A、P101S以及K194R。
  5. 根据权利要求1、2或3所述的植物EPSPS突变体，其特征在于，所述植物EPSPS突变体来自水稻、烟草、大豆、玉米、小麦、棉花、油菜和高粱中的任意一种。
  6. 根据权利要求1、2或3所述的植物EPSPS突变体，其特征在于，所述植物EPSPS突变体来自水稻，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS具有以下突变：K85I、G96A和P101S。
  7. 根据权利要求6所述的植物EPSPS突变体，其特征在于，所述植物EPSPS突变体的氨基酸序列如SEQ ID NO:8所示。
  8. 根据权利要求1、2或3所述的植物EPSPS突变体，其特征在于，所述植物EPSPS突变体来自玉米，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS具有以下突变：K85I、G96A、P101S以及K194R。
  9. 根据权利要求8所述的植物EPSPS突变体，其特征在于，所述植物EPSPS突变体的氨基酸序列如SEQ ID NO:16所示。
  10. 根据权利要求1、2或3所述的植物EPSPS突变体，其特征在于，所述植物EPSPS突变体来自大豆，所述植物EPSPS突变体的氨基酸序列对应于大肠杆菌EPSPS具有以下突变：K85T和P101S。
  11. 根据权利要求10所述的植物EPSPS突变体，其特征在于，所述植物EPSPS突变体的氨基酸序列如SEQ ID NO:24所示。
  12. 一种编码基因，其特征在于，其编码权利要求1-11中任一项所述的植物EPSPS突变体。
  13. 含有权利要求12所述的编码基因的载体。
  14. 含有权利要求13所述的载体的重组菌或重组细胞。
  15. 权利要求1-11任一项所述的植物EPSPS突变体在培育抗草甘膦植物中的应用。
  16. 根据权利要求15所述的应用，其特征在于，其包括：将载体转化目的植物，所述载体含有编码所述植物EPSPS突变体的编码基因。
  17. 根据权利要求16所述的应用，其特征在于，其包括：修饰目的植物的内源EPSPS基因，使其编码所述植物EPSPS突变体。
  18. 根据权利要求15所述的应用，其特征在于，其包括：对植物细胞、组织、个体或群体进行诱变、筛选，使其编码所述植物EPSPS突变体。
  19. 根据权利要求17-18任一项所述的应用，其特征在于，目的植物为小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、绿豆、蚕豆、豌豆、扁豆、甘薯、马铃薯、棉花、大豆、油菜、芝麻、花生、向日葵、萝卜、胡萝卜、芜菁、甜菜、白菜、芥菜、甘蓝、花椰菜、芥蓝、黄瓜、西葫芦、南瓜、冬瓜、苦瓜、丝瓜、菜瓜、西瓜、甜瓜、番茄、茄子、辣椒、菜豆、豇豆、毛豆、韭菜、大葱、洋葱、韭葱、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甜菜、甘蔗、烟草、苜蓿、牧草、草坪草、茶和木薯中的任意一种。
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