Patent Grant
9556422
This application is the U.S. National Stage of International Application PCT/CN2012/070440 filed Jan. 17, 2012, which designates the U.S and was published by the International Bureau in Chinese on Jul. 26, 2012, and which claims the benefit of Chinese Application No. 201110009329.0, filed Jan. 17, 2011, both of which are hereby incorporated by reference in their entirety.
The instant invention falls into the field of plant genetic engineering, and particularly, the instant invention relates to a method for modifying glyphosate-resistant gene, glyphosate-resistant genes and encoded proteins thereof, and a method for obtaining transgenic glyphosate-resistant plant using these modified glyphosate-resistant genes. The invention can be applied to breeding crops and screening plant cell cultures.
Glyphosate is a very important herbicide, which inhibits an important enzyme, i.e. 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), for the biosynthesis of aromatic amino acids in plants. Glyphosate is a herbicide with extremely wide spectrum, which is also lethal to crops. Hence, to perform selective weeding during the growth of crops, the crops have to obtain glyphosate resistance.
Glyphosate can inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of the biosynthesis of aromatic amino acids in majority of bacteria. However, it has been discovered that, EPSPS of partial bacteria has resistance to glyphosate, and the EPSPS of these bacteria has been obtained by separation. Plants can obtain glyphosate resistance by transgenic expression of resistant EPSPS. The resistance obtained by expression of EPSPS of Agrobacterium (Agrobacterium tumefaciens sp CP4) and Salmonella typhimurium CT7 in plants has already been applied in production (U.S. Pat. Nos. 453,590, 4,769,061 and 5,094,945). However, to raise the resistance level of transgenic crops and increase the diversity of resistant gene, new glyphosate-resistant genes and transgenic glyphosate-resistant plants based thereon are still in demand in application in production.
The EPSPS obtained from Deinococcus radiodurans R1 has high glyphosate resistance (Chinese Patent 200910098129.x). The invention “A glyphosate-resistant gene and application thereof” filed in 2009 under the application No. 200910098129.x provides a glyphosate-resistant gene, wherein the amino acid sequence of the protein encoded by G1174 gene is as shown in SEQ ID NO: 2 in the patent. Although the glyphosate-resistance of EPSPS of D. radiodurans R1 is relative high, it is still very necessary to obtain modified genes of EPSPS with higher resistance. The resistance level conferred to transgenic plants can raise the application dosage of glyphosate, thereby retarding the development of resistant weeds and avoiding hazard caused by dosage problem during the application process.
The instant patent aims to solve the technical problem of providing a method for modifying glyphosate-resistant EPSPS gene and providing a glyphosate-resistant gene. The resistant gene has higher glyphosate resistance than natural EPSPS gene of D. radiodurans R1. The gene can be used for the production of transgenic glyphosate-resistant plants, and can also be used as screening marker in plant cell cultures.
In order to solve the above technical problems, the instant invention is achieved by the following technical solution: the instant invention provides a method for obtaining highly glyphosate-resistant mutated G1174 gene (highly glyphosate-resistant mutated gene) through mutagenesis, wherein two or more amino acid mutations are introduced in the amino acid sequence from position 95 to position 114 (Pro Gly Asn Ala Gly Ala Val Ala Arg Phe Leu Met Gly Val Ala Ala Leu Thr Ser Gly), (SEQ ID NO:31), and the highly glyphosate-resistant mutants are selected. Particularly, two or more mutations at the following positions are introduced: 1) glycine at position 99; 2) alanine at position 100; 3) phenylalanine at position 104; 4) methionine at position 106; and 5) glycine at position 107.
The instant invention also provides a glyphosate-resistant mutated G1174 gene (highly glyphosate-resistant mutated gene), wherein the encoded protein thereof contains at least two mutations at the following positions: 1) mutation of amino acid at position 99 from glycine to alanine; 2) mutation of amino acid at position 100 from alanine to phenylalanine, or threonine, or methionine, or serine; 3) mutation of amino acid at position 104 from phenylalanine to leucine or serine; 4) mutation of amino acid at position 106 from methionine to leucine or serine; and 5) mutation of amino acid at position 107 from glycine to alanine.
The instant invention further provides a glyphosate-resistant mutated G1174 gene (highly glyphosate-resistant mutated gene), wherein the amino acid sequence from position 95 to position 114 of the encoded protein thereof is mutated to at least one of the following sequences: 1) SEQ ID NO: 4, 2) SEQ ID NO: 5, 3) SEQ ID NO: 6, 4) SEQ ID NO: 7, 5) SEQ ID NO: 8, 6) SEQ ID NO: 9, 7) SEQ ID NO: 10, 8) SEQ ID NO: 11, 9) SEQ ID NO: 12, 10) SEQ ID NO: 13, 11) SEQ ID NO: 14, 12) SEQ ID NO: 15, 13) SEQ ID NO: 16, 14) SEQ ID NO: 17, 15) SEQ ID NO: 18, 16) SEQ ID NO: 19, 17) SEQ ID NO: 20, 18) SEQ ID NO: 21, 19) SEQ ID NO: 22, 20) SEQ ID NO: 23, and 21) SEQ ID NO: 24.
The instant invention also provides a highly glyphosate-resistant mutated gene, which has the nucleotide sequence of any one of SEQ ID NO: 26 to SEQ ID NO: 30.
The instant invention also provides a vector containing the above highly glyphosate-resistant mutated gene.
The instant invention further provides a use of the above highly glyphosate-resistant mutated gene: conferring a plant with glyphosate resistance through expression of the transgene in the plant. The plant is rice, maize, cotton, wheat, barley, sorghum, rape, soybean, turf grass or forage grass.
The EPSPS genes originated from Deinococcus deserti VCD115 and Deinococcus geothermalis (Gene bank: YP_002785524 and YP_604470, polypeptides SEQ ID NO: 4 and SEQ ID NO: 5 provided by the patent application 200910098129.x) have relatively high homology to the G1174 used in the instant invention, and compared with G1174, the amino acid sequences of the encoded proteins thereof are identical to that of G1174 at corresponding positions from position 95 to position 114. The instant invention also provides a method for modifying these homologous genes and other glyphosate-resistant polypeptides with at least 85% similarity to the amino acid sequence of G1174. To obtain proteins with higher glyphosate resistance by screening, at least two of the following mutations can be introduced to these polypeptides at the following positions corresponding to G1174: 1) mutation of amino acid at position 99 from glycine to alanine; 2) mutation of amino acid at position 100 from alanine to phenylalanine, or threonine, or methionine, or serine; 3) mutation of amino acid at position 104 from phenylalanine to leucine or serine; 4) mutation of amino acid at position 106 from methionine to leucine or serine; and 5) mutation of amino acid at position 107 from glycine to alanine.
The instant invention further provides representative nucleotide sequences encoding the highly resistant EPSPS protein mutants: SEQ ID NO: 26, or SEQ ID NO: 27, or SEQ ID NO: 28, or SEQ ID NO: 29, or SEQ ID NO: 30. These genes have DNA fragment encoding chloroplast signal peptide on their 5′-end.
The amino acid sequence of the glyphosate-resistant gene provided by the instant invention can be used in the design and synthesis of nucleotide sequence beneficial to the expression in plants (Campbell and Gowri, Plant Physiol. 92: 1-11), and further constructed to artificial gene expression cassette capable of expressing in plants. The artificial gene expression cassette capable of expressing in plants includes a promoter, a glyphosate-resistant gene with chloroplast signal peptide, and a terminator. The promoter, chloroplast signal peptide and terminator required for the expression and production of glyphosate resistance in plants are known to the prior art. For example, by the transformation of monocotyledon, the promoter can be Ubiqutin-1 promoter of maize or Actin promoter of rice; the terminator can be the terminator from Agrobacterium tumefaciens (Nos) or other terminators; and the signal peptide guiding the glyphosate-resistant protein into the chloroplast can be the signal peptide of subunit of Rubisco (de Castro Silva Filho, 1996, Plant Mol Biol. 30: 767-780), signal peptide of plant EPSPS gene (Archer, 1990, J. Bioenerg. Biomemb. 22 (b): 789-810), and etc. The DNA fragment encoding the chloroplast signal peptide is linked to the 5′-end of a glyphosate-resistant gene and is in an expression cassette. The expression construction can be integrated to a plant genome via Agrobacterium (Agrobacterium strain LAB4404), gene gun method or other methods, and expressed to obtain a glyphosate-resistant plant. The technologies and methods for plant transformation are known and established. The transformation method and procedure are different for different plants. However, generally Agrobacterium and gene gun are used for the introduction into immature embryo, mature embryo, undifferentiated callus or protoplast of a plant. Then a medium containing 1-5 mM glyphosate is used for screening culture. Then transformed seedlings are obtained via differentiation, and transgenic seedlings that can be planted are obtained via culturing in a rooting medium. Furthermore, glyphosate-resistant transgenic plant can be screened by spraying glyphosate.
The instant invention further provides a plasmid containing the above glyphosate-resistant nucleotide sequence molecule, and functionally linked to the nucleotide sequence controlling the expression in a plant to form an expression cassette.
The instant invention further provides a method for modifying plants, including: introducing the above glyphosate-resistant gene expression cassette to plant cells using plant gene transformation technique, and subjecting the cells to a differentiation culture to obtain corresponding transgenic plants. The obtained plants have glyphosate resistance. These plants can be rice, maize, cotton, wheat, soybean, rape, sorghum, barley, turf grass or forage grass. The transformation technique for these plants are known in prior art.
The instant invention also provides the use of the nucleotide sequence molecule of the glyphosate-resistant gene for use as screening marker in plant transgenic cell culture.
Another aspect of the instant invention is to provide glyphosate-resistant gene for use as screening marker for gene transfer in plants. The above glyphosate-resistant artificial gene capable of expressing in plant cells can be constructed together with a target gene expression cassette in the same DNA transformation fragment of the same plant transformation plasmid. The target gene can be any valuable gene. The plant expression plasmid can be introduced to a plant tissues with gene gun, Agrobacterium-mediated method or other methods, while a culture medium containing appropriate concentration of glyphosate (for e.g. 1-5 mM) can be used for selective killing of plant cells without the introduced DNA transformation fragment, so as to select plant cells containing the target gene.
The nucleotide sequence of the glyphosate-resistant gene of the instant invention can have many different mutations including but not limited to: 1) different nucleotide sequences obtained by using different codons of the same amino acid, wherein these nucleotide sequences encode protein polypeptides with the same activity; 2) nucleotide sequences obtained by introducing other mutations, wherein these nucleotide sequences still encode proteins with glyphosate resistance. These mutations can be random mutations, or targeted site mutations, or insertion or deletion mutations. One of ordinary skill in the art can generate the above mutations via molecular biological methods.
The instant invention can be applied to all plants including dicotyledon and monocotyledon.
The following specific embodiments are provided for further description of the present invention, but the protection scope of the present invention is not limited thereto.
The molecular biological and biochemical methods applied in the following embodiments are known in the prior art. Detailed descriptions can be found in references such as Ausubel, “Current Protocols in Molecular Biology”, John Wiley and Sons Company; J. Sambrookequi et al., “Molecular Cloning: A Labortory Manual” 3rd ED, Cold Spring Harbor Laboratory Press (2001); and etc.
The EPSPS gene of Deinococcus, radiodurans (nucleotide sequence SEQ ID NO: 1) was obtained from Sangon Biotech (Shanghai) Co., Ltd through artificial synthesis, and the encoded amino acid sequence is SEQ ID NO: 2. The gene was named G1174, and xmaI and KpnI restriction sites were designed on both ends of the nucleotide fragment of its nucleotide sequence encoding the amino acid from position 95 to position 114.
Vector PMD19 (purchased from TAKARA Company) was used for the expression of G1174 to determine its glyphosate resistance. The G1174 was cloned into PMD19 with general biological method and plasmid vector PMD19-M-1174 was obtained, wherein SEQ ID NO: 3 was the whole sequence of the plasmid vector and sequence of the inserted G1174 gene is 2218-3531. The Escherichia coli TG-1 cells transformed with PMD19 and PMD19-M-1174 were shake-cultured in a LB medium (yeast extract 5.0 g, peptone 10.0 g, sodium chloride 10.0 g, agar 15.0 g, and water to 1.0 L, pH 7.0) at 37° C. for 16 h respectively, then the Escherichia coli cells were collected by centrifugation. SDS-PAGE separation was conducted and Western blot was conducted with anti-G1174 antibody. The results showed that no signal was detected by PMD-19, while an obvious signal was detected by PMD19-M-1174, which indicated that the vector can express G1174 protein.
To screen EPSPS mutants with higher resistance level, the nucleotide fragment of the amino acid sequence from position 95 to position 114 encoded by G1174 was mutated. The following two degenerate primers were synthesized:
The Tris-HCl solutions of the above two nucleotide degenerate primers were mixed, subjected to 5′-end phosphorylation with T4 polynucleotide kinase, then incubated at 65° C. for 20 min, and then cooled to 22° C. to obtain a double-chained DNA fragment capable of linking with a vector restrictively digested with SmaI and KpnI. The DNA fragment was further mixed with SmaI- and KpnI-digested PMD19-M-1174, linked thereto with DNA ligase, and introduced into Escherichia coli TG-1 to obtain a mutant library containing different G1174 mutants.
A M63 medium containing 60 mM or 100 mM glyphosate was prepared. The formula of the M63 medium was as followed (per L): KH2PO4 13.6 g, (NH4)2SO4 2 g, FeSO4-7H2O 0.5 mg, MgCl2 2.4 mg, glucose 10 g, thiamine-HCl 10 mg and L-proline 25 mg, agar 40 g and distilled water in balance, pH 7.0. After that the M63 medium was sterilized under high temperature and high pressure, and as the temperature was decreased to 55° C., glyphosate was added to a final concentration of 60 mM or 100 mM.
The mutant library containing different G1174 mutants was applied to the M63 medium containing 60 mM glyphosate and cultured. Growth condition was observed 72 h later. The growth of the original G1174 gene on the M63 medium containing 60 mM glyphosate was very slow, and no obvious colony could be observed 72 h later. However partial G1174 mutants were able to grow to visible colonies after cultured for 72 h.
The glyphosate-resistant mutant clones were further cultured in the M63 culture medium containing 100 mM glyphosate. The following clones were able to grow to colonies at 72 h, and they are: G1174-A, G1174-B, G1174-C, G1174-D, G1174-E, G1174-F; G1174-G, G1174-H, G1174-1, G1174-J, G1174-K, G1174-L, G1174-M, G1174-N, G1174-O, G1174-P, G1174-Q, G1174-R, G1174-S, G1174-T, G1174-U, G1174-V and G1174-W.
The clones that were able to grow normally on 100 mM glyphosate could contain mutants with higher glyphosate resistance than the original G1174 gene. Therefore, nucleotide sequence of the mutation region of the glyphosate-resistant clones obtained from example 3 was determined. The results showed that, the mutation results of these genes were as followed (amino acid from position 95 to position 114):
The mutations of these highly glyphosate-resistant mutants included: 1) mutation of amino acid at position 99 from glycine to alanine; 2) mutation of amino acid at position 100 from alanine to phenylalanine, or threonine, or methionine, or serine; 3) mutation of amino acid at position 104 from phenylalanine to leucine or serine; 4) mutation of amino acid at position 106 from methionine to leucine or serine; and 5) mutation of amino acid at position 107 from glycine to alanine. Particularly, these mutations generally contained two or more amino acid mutations.
The previous investigations have discovered that mutation of the amino acid of EPSPS of Escherichia coli at position 96 (corresponding to the amino acid of G1174 of the invention at position 99) from glycine to alanine can increase glyphosate resistance, however the activity of the mutated enzyme was reduced (GenBank accession no. X00557; Kishore. et al. 1986; Padgette et al. 1991). To investigate whether high glyphosate resistance can be obtained barely through mutating the amino acid of G1174 gene at position 99 from glycine to alanine, the method of point mutation was applied in the instant invention to obtain a G1174 mutant gene G1174G99A, the amino acid of which is mutated from glycine to alanine at position 99.
To compare the glyphosate resistance of G1174-G99A with G1174-A, G1174-F, G1174-V, and etc. of the instant invention, PMD19 vectors expressing the above mutants were introduced to Escherichia coli TG-1, and cultured in an LB medium at 37° C. for 16 h to obtain single colony. Five clones of each mutant were transferred to the M63 medium containing 100 mM glyphosate and cultured at 37° C. for 48 h, and the growth was observed. The results showed that, the growth of the clone expressing G1174-G99A was perspicuously slower than that of the clones expressing G1174-A, G1174-F and G1174-V. At the same time, these expression clones were transferred to the M63 medium containing 100 mM glyphosate (without agarose, other components were the same as the M63 medium), and shake-cultured at 37° C. for 48 h. OD600 was measured, and the results showed that, the OD600 value of G1174-G99A was 0.24, of G1174-A was 0.73, of G1174-F was 0.65 and of G1174-V was 0.53. Hence, the glyphosate-resistant mutants provided by the instant invention showed higher resistance than the sole mutation from glycine to alanine at position 99.
A codon optimized artificial gene of G1174 suitable for expression in monocotyledon (SEQ ID NO: 25, comprising nucleotide sequence encoding chloroplast signal peptide on the 5′-end) was artificially synthesized. The DNA fragment of the artificial gene has a BamHI site on the 5′-end and a xhoI site on the 3′-end. Corresponding mutations were introduced respectively with prior art mutagenesis method, and G1174 mutant genes containing mutation sites of G1174-A, G1174-C, G1174-L, G1174-O and G1174-V were obtained. (The nucleotide sequences were SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30 respectively.)
The promoter for initiating the expression of G1174 mutant genes was ubiquitin-1 promoter of maize. The promoter was obtained from maize genome by PCR. The primers for PCR were zmUbiF (GCGAAGCTTGCATGCCTACAGTGCAGCGTGACCCGGTCGTGC, a HindIII site was added, which is represented by the underlined letters) (SEQ ID NO: 35), and zmUbiR (GTGGGATCCTCTAGAGTCGACCTGCA GAAGTAACACCAAACAACAG, a BamHI site was added, which is represented by the underlined letters) (SEQ ID NO: 36).
The G1174 mutant genes were restrictively digested with xhoI and BamHI, the ubiquitin-1 promoter of maize obtained by PCR was restrictively digested with HindIII and BamHI, and the both were simultaneously linked to the pCamlbial300 vector restrictively digested with HindIII and xhoI to obtain T-DNA vectors: pCAM1300-1174A, pCAM1300-1174C, pCAM1300-1174L, pCAM1300-11740 and pCAM1300-1174V. The terminator the G1174 mutant genes in the vectors were from the terminator of 35S gene of CaMV from the vectors themselves.
The prior art (L U, Xiongbin and GONG, Zuxun, 1998, Life Science 10: 125-131; LIU, Fan, et al., 2003, Molecular Plant Breeding 1: 108-115) is adopted in the method for obtaining the transgenic plants. The hulls of mature and full seeds of “Xiu-shui 110” were removed, and calli for use as transformation materials were generated through induction. Agrobacteria containing the vectors pCAM1300-1174A, pCAM1300-1174C, pCAM1300-1174L, pCAM1300-11740 and pCAM1300-1174V were streaked on plates respectively, and single colonies were isolated and inoculated to prepare Agrobacteria for use in transformation. The to-be-transformed calli were placed into an Agrobacteria solution (containing acetosyringone) with appropriate concentration to allow the attachment of Agrobacteria on the surface of the calli, then the calli were transferred to a co-culture medium and co-cultured for 2-3 days. The transformed calli were washed with bacteria-free water, transferred to a selection medium containing appropriate amount of antibiotics, and subjected to a selection culture (2 mM glyphosate) for two months (subculture for once in the middle of the time). The calli with good growth activity after screening were transferred to a pre-differentiation medium and cultured for about 20 days, then the pre-differentiated calli were transferred to a differentiation medium and illuminated for 14 h for differentiation and germination. Two to three weeks later, the resistant regenerated plants were transferred to a rooting medium for hardening seedlings and rooting, and finally the regenerated plants were washed to remove agar and planted in a greenhouse for use as identification materials.
The transformation method of maize is well established, for e.g. Frame et al. described the transformation method of maize using Agrobacterium (Plant Physiol, 2002, 129: 13-22). Agrobacteria containing the vectors pCAM1300-1174A, pCAM1300-1174C, pCAM1300-1174L, pCAM1300-11740 and pCAM1300-1174V were streaked on plates respectively, and single colonies were isolated and inoculated to prepare Agrobacteria for use in transformation. Hi-II corn cobs were collected 8-10 days after pollination. All immature embryos (1.0-1.5 mm in size) were collected. Agrobacteria containing T-DNA vector were bread and cultured together with the immature embryos for 2-3 days (22° C.). The immature embryos were transferred to a callus induction medium (200 mg/L Timentin for killing Agrobacteria) and cultured at 28° C. in the dark for 10-14 days. All calli were transferred to a selection culture medium containing 2 mM glyphosate and cultured at 28° C. in the dark for 2-3 weeks.
All tissues were transferred to a fresh culture medium containing glyphosate and cultured at 28° C. in the dark for 2-3 weeks. Then all embryonic tissues survived from the selection were transferred to a regeneration medium and cultured at 28° C. in the dark for 2-3 weeks with each dish containing one strain. The embryonic tissues were transferred to a fresh regeneration medium and cultured at 26° C. in the light for 10-14 days. All full-developed plants were transferred to a rooting medium, cultured at 26° C. in the light until full development of the roots, then transferred to a greenhouse for single plant cultivation, and the herbicide resistance of the transgenic maize was detected.
Ten different transgenic rice strains were selected from transgenic rice obtained from pCAM1300-1174A, pCAM1300-1174C, pCAM1300-1174L, pCAM1300-11740 and pCAM1300-1174V respectively, and planted and cultured in a greenhouse (15-25° C.) together with a non-transgenic strain of “Xiu-shui 110” of the same variety. As the seedlings grew to 10 cm, glyphosate (0.4% w/v) was sprayed at 40 mL/m2. Tests were conducted 8 days later, and the results were as in Table 1. The results showed that, the glyphosate resistance of these mutated genes were good.
Ten different transgenic rice strains were selected respectively from transgenic maize obtained from pCAM1300-1174A, pCAM1300-1174C, pCAM1300-1174L, pCAM1300-11740 and pCAM1300-1174V, and planted and cultivated in a greenhouse (15-25° C.) together with a non-transgenic strain of “Xiu-shui 110” of the same variety. During the seedling stage around 5-6 leaves, glyphosate (0.4% w/v) was sprayed at 40 mL/m2. Tests were conducted 8 days later, and the results were as in Table 2. The results showed that, the glyphosate resistance of partial transformed strains of the transgenic maize obtained by using these mutated genes had already reached the level for application in production.
Finally, it should be noted that, what listed above are only several particular embodiments of the invention. Obviously, the invention is not limited to the above embodiments, and numerous variants are possible thereto. Any variations that can be directly derived or conceived from the content disclosed by the invention by one of ordinary skill in the art, are intended to fall within the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011 1 0009329 | Jan 2011 | CN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN2012/070440 | 1/17/2012 | WO | 00 | 9/12/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2012/097720 | 7/26/2012 | WO | A |
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| Number | Date | Country | |
|---|---|---|---|
| 20140298533 A1 | Oct 2014 | US |
