WO2006063533A1 - Reconstitution de l’activite de la 5-enolpyruvylshikimate-3-phosphate synthase par complementation de fragments - Google Patents

Reconstitution de l’activite de la 5-enolpyruvylshikimate-3-phosphate synthase par complementation de fragments Download PDF

Info

Publication number
WO2006063533A1
WO2006063533A1 PCT/CN2005/002219 CN2005002219W WO2006063533A1 WO 2006063533 A1 WO2006063533 A1 WO 2006063533A1 CN 2005002219 W CN2005002219 W CN 2005002219W WO 2006063533 A1 WO2006063533 A1 WO 2006063533A1
Authority
WO
WIPO (PCT)
Prior art keywords
epsps
fragment
protein
plasmid
fragments
Prior art date
Application number
PCT/CN2005/002219
Other languages
English (en)
French (fr)
Inventor
Yiping Wang
Yicheng Sun
Yan Li
Original Assignee
Peking University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peking University filed Critical Peking University
Priority to DE602005024442T priority Critical patent/DE602005024442D1/de
Priority to AT05819606T priority patent/ATE486127T1/de
Priority to EP05819606A priority patent/EP1840210B1/en
Priority to US11/721,856 priority patent/US8936925B2/en
Publication of WO2006063533A1 publication Critical patent/WO2006063533A1/zh

Links

Classifications

    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • 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
    • 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

Definitions

  • the present invention relates to a method for reconstituting 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) by fragment complementation techniques. More specifically, the present invention relates to the reconstitution of Escherichia coli EPSPS and its glyphosate-resistant EPSPS obtained after mutation, and to the reconstruction of Pseudomonas putida glyphosate-resistant EPSPS.
  • EPSPS 5-enolpyruvyl-shikimate-3-phosphate synthase
  • transgenic plants There are mainly the following ways of spreading foreign genes in transgenic plants: the propagation of pollen from transgenic plants, the formation of hybrids by transgenic plants as receptors for pollen of wild relatives, and the possible spread of genes by transgenic plants.
  • methods for controlling the spread of foreign genes in transgenic plants for example, (1) physical isolation, mainly distance isolation to block the spread of foreign genes through pollen; (2) genetic control, including: (a) Male sterility; (b) genomic incompatibility (Genome)
  • the EPSPS gene is divided into two segments, which are respectively concatenated and co-expressed with the gene expressing DnaE intein, and the self-splicing function of the intein is used to form a complete EPSPS, thereby obtaining Escherichia coli or tobacco. Tolerance of phosphine.
  • introduction of the intein-encoding gene itself into a transgenic plant as a foreign gene may also pose other risks, such as becoming a transgenic gene.
  • Protein fragments produced by protease digestion or produced by gene expression can be reconstituted in vivo or in vitro into a complex with intact protein function, which is called protein fragment complementation or protein reconstitution technology (example ⁇ Mouth, Hakansson, ⁇ et al. Curr Protein Pept Sci 2002, 3, (6), 629-42; Braun, ⁇ et al. Bacteriol 2003, 185, (18), 5508-18 ⁇ ).
  • Fragment complementation studies on proteins such as aminoacyl tRNA synthetase have shown that most of the protein fragmentation sites in which fragmentation occurs are in non-conserved regions.
  • Reconstruction of the protein means that the non-covalent interaction within the protein is so specific that the protein fragment facilitates the formation of the native structure (Shiba, K. et al. Proc Natl Acad Sci USA 1992, 89, (5), 1880-4 Shiba, K. et al. J Biol Chem 1992, 267, (32), 22703-6).
  • the protein is capable of fragmentation. Complementation indicates that the protein retains a relatively stable structure even when the covalent bond is broken.
  • the various non-covalent interactions between the two complementary peptide chains (such as hydrogen bonding, salt bridges, and hydrophobic interactions) play a large role in maintaining protein structural stability (Nelson, KE et al., Complete genome sequence). And comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 2002, 4, (12), 799-808).
  • the present invention relates to a protein fragment of 5-enolpyruvylshikimate-3-enate synthase (EPSPS) which is a fragment selected from the pair of EPSPS fragments, and the two fragments constituting the pair of fragments can be ligated into one Long EPSPS, and the two fragments can re-establish EPSPS activity by complementation without any attachment structure.
  • EPSPS 5-enolpyruvylshikimate-3-enate synthase
  • the segmentation point of the pair of segments is located in a structure of the EPSPS selected from the group consisting of: a junction region between the folding units, a junction region between the alpha helix and the beta sheet, between the two beta sheets, and a beta sheet.
  • the dividing point is inside the folding unit 1, 2, 3, 4, or 5, for example, two ⁇ folding in the folding unit 3.
  • the dividing point is inside the folding unit 1, 2, 3, 4, or 5, for example, two ⁇ folding in the folding unit 3.
  • the dividing point is inside the folding unit 1, 2, 3, 4, or 5, for example, two ⁇ folding in the folding unit 3.
  • the EPSPS is a wild type EPSPS or an EPSPS active variant thereof obtained by addition, deletion, and/or substitution of one or more amino acid residues, preferably Escherichia coli wild type EPSPS (full length thereof)
  • Amino acid sequences and nucleotide sequences are known in the art, see the Sequence Listing) or its glyphosate resistant EPSPS active variant, or preferably Pseudomonas putida C3 ⁇ 4ewifowo «os /7 ⁇ // ⁇ ) Glyphosate-resistant EPSPS, such as EPSPS of Pseudomonas putida CGMCC 0739 (see Chinese Patent Application No. 02117991.3) (its full-length amino acid sequence and nucleotide sequence are shown in SEQ ID NO: 2).
  • the protein fragment of the invention is preferably a fragment of the following fragment pair selected from the group consisting of E. coli EPSPS: N67/C68, N85/C86, N104/C105, N154/C155, N182/C183, N184/C185, N218 /C219, N224/C225, N227/C228, N259/C260, N298/C299, N371/C372, N376/C377, N383/C384 (N67/C68 fragment pair described here, represented by the N-terminal fragment N67 and C-terminus A fragment pair consisting of fragment C68, wherein N67 refers to an N-terminal fragment from the N-terminus to the 67th residue in the EPSPS sequence, and C68 refers to a C from the 68th residue to the C-terminus in the EPSPS sequence.
  • N67 refers to an N-terminal fragment from the N-terminus to the 67th residue in the EPSPS sequence
  • fragments are represented by a representation, or preferably a fragment of the following fragment pair of EPSPS selected from Pseudomonas putida CGMCC 0739: N208/C209, N214/C215, N219/C220, N222/ C223, N224/C225 (wherein N208 refers to a fragment from the N-terminus to the 208th residue in the EPSPS sequence, and C209 refers to a fragment from the 209th residue to the C-terminus in the EPSPS sequence) , other fragments are similar to the notation).
  • the invention also relates to a nucleic acid molecule encoding a protein fragment of the invention, an expression vector carrying the nucleic acid molecule, a cell comprising the nucleic acid molecule or expression vector.
  • a cell is a plant cell.
  • the invention also relates to a method of reconstituting EPSPS comprising reconstituting EPSPS activity using a protein fragment or nucleic acid molecule or expression vector of the invention in the absence of any linker structure.
  • the invention also relates to a method of splitting an EPSPS or EPSPS nucleic acid molecule comprising the resolution of a protein fragment, or nucleic acid molecule of the invention, in the absence of any linker structure.
  • the party ⁇ The split point selected in the EPSPS is preferably located in the following structure of the EPSPS: in the junction region between the folding units, in the junction region between the alpha helix and the beta sheet, between the two beta sheets, in the beta sheet, or in the alpha helix Preferably, it is located in the joint zone between the folding units, for example, in the joint zone between the folding units 1 and 6, 2 and 6, 3 and 4, 4 and 5, and 3 and 5.
  • the folding unit 1, 2, 3, 4, or 5 for example, in the joint region between the two ⁇ -folds of the folding unit 3, in the ⁇ -helix of the folding unit 4, in the ⁇ -fold of the folding unit 2 , between the ⁇ -helix and the ⁇ -fold of the folding unit 1, in the joint region between the ⁇ -helix and the ⁇ -fold of the folding unit 5, or in the joint region between the two ⁇ -folds of the folding unit 5, or in the folding unit 5 In the beta fold. Still more preferably between the positions selected from the group consisting of 67-68, 85-86, 104-105, 154-155, 182-183, 184-185, 218-219, N224-C225, N227- of E.
  • coli EPSPS 208-209 214-215, 219- of glyphosate-resistant EPSPS between C228, 259-260, 298-299, 371-372, 376-377 or 383-384, or Pseudomonas putida CGMCC 0739 220, 222-223, or 224-225 between positions.
  • the invention also relates to the use of the EPSPS fragment of the invention or the method of reconstituting EPSPS activity or the method of isolating EPSPS for controlling the safety of transgenic plants. Summary of the invention
  • the present inventors Based on the structure of EPSPS, the present inventors designed a dissociation site in a structural region which may not affect the activity of EPSPS enzyme. The present inventors also constructed an expression vector expressing an EPSPS fragment, and demonstrated that the EPSPS fragment can complement EPSPS activity in E. coli. Therefore, the EPSPS segment of the present invention and the method of reconstructing EPSPS can be applied to the field of biosafety control.
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • S3P shikimic acid-3-phosphate
  • PEP phosphoenolpyruvate
  • EPSPS consists of two domains, one of which includes three symmetric protein folding units labeled 1, 2 and 6, and the other including three labeled 3, 4 and 5.
  • Symmetrical protein folding unit Each unit consists of two parallel alpha helices and four beta folds. See Stallings, W. C. et al. Proc Natl Acad Sci USA 1991, 88, (1 1), 5046-50.
  • the present inventors designed a dissociation site in a structural region which may not affect the activity of EPSPS enzyme.
  • the cleavage of the covalent bond and the insertion of methionine generally do not affect the formation of the native structure of the protein.
  • the split protein fragments rely on non-covalent interactions within the protein and various non-covalent interactions between the two peptide chains, such as hydrogen bonding, salt bridges, and hydrophobic interactions, to form a native protein structure, thereby reconstituting protein function.
  • the splitting sites are located between the folding units, and the formation of the secondary structure of the complementary fragments is generally not affected, so the fragment complementation is more likely to occur.
  • E. coli E. coli.
  • EPSPS has 7 fractionation sites located in the junction region between the folding units, and 6 pairs of protein fragments are produced to complement each other to form EPSPS activity, and their complementary activities are Better.
  • Pseudomonas putida has three fractionation sites in the junction region between the folding units, and the three pairs of fragments formed can complement EPSPS activity.
  • E. coli EPSPS has 6 fractionation sites located in the junction region between the alpha helix and the beta sheet, and 5 of the fragments can complement EPSPS activity; another 1 locus is located In the junction region between the ⁇ -sheets, the corresponding fragment can also complement EPSPS activity.
  • Pseudomonas putida has one fractionation site. In beta folding, the resulting fragment can complement EPSPS activity.
  • E. coli EPSPS has seven fractions located in the alpha helix or beta sheet, three of which are complementary to EPSPS activity. Pseudomonas putida has one fractionation site in the beta fold, and the resulting fragment can complement EPSPS activity.
  • the N-terminal peptide chain N240: and the C-terminal peptide C241 of E. coli EPSPS can form a complex and be co-purified.
  • the enzyme activity of this complex is very low, which may be due to the interaction of only one pair of hydrophobic interaction regions in the N240/C241 complex, and thus the protein structure is unstable.
  • Pseudomonas putida had tested three split sites between the two domains, and the resulting fragments were essentially incapable of complementing EPSPS activity. Therefore, the present inventors performed fractionation in the junction region between the domains of EPSPS, and the resulting fragment was not able to reconstitute EPSPS activity.
  • the inventors successfully performed 14 splitting points in E. coli EPSPS, of which 6 were in the junction between the folding units, 3 in the junction between the alpha helix and the beta, and 2 in Of the junction regions between the ⁇ -sheets, 2 are in the ⁇ -sheet and 1 is in the ⁇ -helix.
  • the E. coli EPSPS gene was split into a sputum fragment and a C fragment.
  • the corresponding N and C fragments were expressed on two compatible plasmids, respectively. N fragment thus obtained
  • the C fragment alone does not exhibit EPSPS activity, but in vivo, the corresponding N fragment and C fragment bind to complement EPSPS activity.
  • the inventors successfully performed 5 split points in the Pseudomonas putida glyphosate resistant EPSPS, 3 of which are in the junction between the folding units, 1 Of the junction regions between the alpha helix and the beta sheet, one is in the beta sheet. Obtained by this split Fragments can also complement EPSPS activity.
  • the activity of EPSPS can be reconstituted by fragment complementation, which means that the non-covalent interaction within the protein is very specific, so that the fragment facilitates the formation of a native EPSPS-functional structure without forming other structures.
  • the protein is capable of fragment complementation, which indicates that when the covalent bond of 5 is broken in some regions, the protein can still maintain structural stability, and the two peptide chains do not have to pass covalent bonds, but only through various non- Covalent interactions such as hydrogen bonding, salt bridges, and hydrophobic interactions are tightly coupled.
  • hydrophobic interactions may play a greater role. There are two strong pairs of hydrophobic interactions between the two fragments of EPSPS, forming two "hooks" that stabilize the proteins together.
  • the invention co-purified to obtain a plurality of complexes composed of corresponding split fragments, but the amount of purified protein is greatly different, which means that the fragments can be formed in vivo, but the complex is formed.
  • the difficulty level is different.
  • the cleavage of covalent bonds in some regions has a great influence on the stability of protein structure. In some regions, the cleavage of covalent bonds has little effect on the stability of protein structure.
  • the introduction of a methionine (the amino acid encoded by the initiation codon) at the C-terminal peptide chain may also affect the stability of the protein structure, resulting in the inability of protein fragment complementation to occur.
  • the complex N240/C241 of E. coli EPSPS can also be co-purified in large quantities, but it cannot complement the growth of E. coli aro mutant strain AB2829 on restrictive medium due to EPS of complex N240/C241
  • the activity is much lower than that of wild-type full-length EPSPS.
  • the loss of its activity may be due to two reasons: First, the N240/C241 fractionation site is very close to Asp242, and Asp242 plays a very important role in the structure-inducing structure of the binding substrate, introducing a C241 0 ⁇ methionine may affect the function of Asp242 and thus its enzyme activity; the second reason may be that the N240/C241 cleavage site is located in the middle of the two domains, and the complex formed by it:: only one in the body For the hydrophobic interaction zone, although the hydrophobic interaction between them is sufficient to form a complex of two protein fragments, the structure of this complex may be different from that of the wild protein due to the lack of another pair of hydrophobic regions, thereby making the enzyme activity Lost. Therefore, the choice of partition site should be based on the effect of 5 structural changes on enzyme activity. '
  • the petal precursor EPSP synthase EPS has a molecular weight of 55kDa, and the first 72 amino acid residues are the leader peptide.
  • the current EPSP synthase enters the chloroplast and is processed to shear the transport peptide to become mature EPSP.
  • the mature enzyme, mature EPSP synthase has a molecular weight of 48 kDa.
  • the leader peptide plays an important role in the entry of the precursor EPSP synthase into the chloroplast.
  • the microbial EPSP synthase has no leader peptide. Therefore, when the microbial EPSP synthase gene is transferred into plant cells, the gene front end needs to add the plant leader peptide sequence.
  • the expressed EPSP synthase cannot enter the chloroplast. application
  • the EPSPS fragment complementation technique of the present invention can be applied to transgenic plants to prevent ecological risks from the spread of glyphosate resistant genes.
  • the N-terminal fragment of EPSPS is expressed in the plant nuclear chromosome, and its C-terminal fragment is expressed in the chloroplast of the plant. None of the EPSPS fragments expressed alone have the activity of EPSPS, so there is no selective advantage in gene diffusion.
  • the genes of the two fragments are co-expressed, the two EPSPS fragments can complement each other in the chloroplast to reconstitute EPSPS activity, thereby rendering the plant resistant to glyphosate.
  • Figure 2 shows the structure of EPSPS and the selection of the separation site.
  • Fig. 2A shows the topology of EPSP synthase
  • Fig. 2B shows a structural unit of EPSP synthase.
  • FIG. 3 Schematic diagram of plasmid construction.
  • A. The C-terminal fragment of EPSPS was subcloned into the pet 2100 vector after the Tet promoter.
  • B. Subcloning the N-terminus of EPSPS to the Tet promoter of the pACYC184 vector.
  • C. The N-terminal portion of the EPSPS-encoding gene was subcloned into the plasmid vector derived from pACYC184 following the T7 promoter.
  • D. The C-terminal portion of EPSPS was constructed into the pET28a vector with a 6 His-tag at the C-terminal tail.
  • FIG. 4 Western blot analysis of EPSPS fragments.
  • Cells expressing the indicated EPSPS were dissolved in SDS sample buffer and analyzed for equal amounts of protein (from ⁇ 0.8 ⁇ wet weight of cells) and purified protein by SDS-PAGE and immunoblotting. It is shown the amount of purified protein of the following: N218 / C219 (.
  • Figure 6 Growth of E. coli GraA-deficient strain AB2829 expressing different EPSPS in a restricted medium (i.e., liquid M63 minimal medium supplemented with the indicated concentration of glyphosate).
  • a restricted medium i.e., liquid M63 minimal medium supplemented with the indicated concentration of glyphosate.
  • Figure 7 is a SDS-PAGE electropherogram of E. coli EPSPS. 1 : N218; 2: C219; 3: : EcEPSPS; 4: co-refolding N218 + c219; 5: N218 + c219 (single renaturation together); 6: molecular weight standard.
  • FIG. 7 b Native-PAGE electrophoresis of E. coli EPSPS Western blot. 1 : N218 + C219 (together after renaturation alone); 2: blank control; 3: co-refolding N218/C219; 4: blank control; 5: wild type EPSPS. detailed description
  • upstream and downstream sequences and gene works pKU2229 p 03 derived plasmid, with aroA £ra/ , upstream and downstream sequences and Wa gene work pKU2249 pET-28a-derived plasmid, with N245-£.co/i' roA, m R this work p U2250 pET-28a-derived plasmid with C246-E.
  • the ampicillin resistant strain was screened by adding Ap to 50 g/ml in the medium.
  • the kanamycin resistant strain was screened and Km was added to the medium to 25 g/ml.
  • the chloramphenicol resistant strain was screened and Cm was added to the medium to 25 g/ml.
  • Restricted M63 medium 13.6 g/L KH 2 P0 4 , 0.5 mg/L FeS0 4 -7H 2 0 , 20 mM (NH 4 ) 2 S0 4 , 0.4% glucose, 1 mM magnesium sulfate, 0.5 mg/L vitamin B1.
  • T 4 DNA ligase, TaqDNA polymerase, DNA marker were purchased from Takara Biotechnology like. Coomassie Brilliant Blue G250, enol pyruvate (sigma), shikimic acid-3-phosphate (presented by Professor Amrehin); HisTrap HP kit (Amersham Biosciences), goat anti-rabbit IgG (promega); other chemicals are analytically pure reagents .
  • Primer 11 5, - CGGGATCCAGGTCCGAAAAAAAACGCCGAC 3' and primer 12: 5' - CGGGATCCATGGAATCCCTGACGTTACA 3' using pKU2004 as a template, ⁇ "Agarage araA gene was amplified, and ligated into pET28a vector to obtain pKU2008, the EPSPS encoded by this plasmid was N A fusion protein with a ⁇ tag at the end.
  • pKU2008 was digested with Ncol and self-ligated to obtain pKU2009, and the protein encoded by this plasmid was Escherichia coli wild type EPSPS.
  • Primer 13 5 '-TGAGTGACTGACTTTAAGAAGGAGATATAC3, and primers
  • the C-terminal cause of Enterobacter's EPSPS was ligated into the Ncol and Bamffl sites of pKU2100 to obtain plasmids pKU2102, pKU2125, pKU2126, pKU2130 and pKU2262, respectively, which encode the C-terminal peptide chain of E. coli EPSPS ( Table 1, Figure 3).
  • the corresponding primers were used for PCR amplification, and the N-terminal gene encoding E. coli EPSPS was obtained.
  • the amplified product was ligated into the EcoR V and Bamffl sites of pACYC 184 to obtain plasmids pKU2101, pKU2125, pKU2126, and pKU2130, respectively.
  • pKU2263 these plasmids respectively encode the N-terminal amino acid sequence of E. coli EPSPS (Table 1, Figure 3).
  • the pBR322 series plasmid constructed in the above step b and the pACYC 184 series plasmid constructed in the c step were digested with Ncol and BamHI, respectively, and the appropriate fragment was collected and ligated into the pET28a vector to obtain the pET28a series for expressing the N-terminus and the C-terminus of the EPSPS.
  • Expression plasmid (Table 1).
  • the pET28a plasmid for expression of the N-terminus of EPSPS was digested with Bgl II and Sal I, respectively, and the desired fragment was ligated into the BamHI and sal I sites of the pACYCl 84 vector to obtain the pACYC-T7 series expression plasmid (Table 1, Figure 3). .
  • the corresponding gene was amplified by the corresponding primers to obtain the corresponding gene of E. coli EPSPS.
  • the amplified product was digested with Xhol and Ncol and ligated into pET28a vector to obtain pET28a series expression plasmid, which was encoded by this series of plasmids.
  • the C-terminus of EPSPS is fused with six histidines to facilitate purification of the protein using a nickel column ( Figure 3).
  • PCR amplification reaction was carried out with primers to obtain a fragment of about 600 bp upstream of the arok gene, which was ligated into the BamHI and Hindlll sites of pBlueScript (stratagene) to obtain plasmid pKU2223.
  • the plasmid pBlueScript was used as a template, and a PCR amplification reaction was carried out using a primer to obtain a b/ ⁇ gene of about 900 bp in length, which was ligated into the Hindlll and EcoRI sites of pBlueScript (stratagene) to obtain plasmid pKU2224.
  • the chromosome of Escherichia coli BL21 (DE3) was used as a template, and PCR amplification reaction was carried out with primers to obtain a fragment of about 500 bp downstream of the aroA gene, which was ligated into the EcoRI and Sail sites of pBlueScript (stratagene) to obtain plasmid pKU2225.
  • the fragment in pKU2225 was digested and ligated into the EcoRI and Sail sites of pKU2223 to obtain plasmid pKU2227.
  • the fragment in pKU2224 was digested and ligated into Hindlll of pKU2227.
  • Plasmid pKU2228 was obtained from the EcoRI site.
  • the fragment between BamHI and Sail in pKU2228 was digested and ligated into pK03 vector to obtain plasmid pKU2229.
  • the strain was transfected into the strain AB2829 of Escherichia coli raA gene, and then streaked on M63 « ⁇ ) 0 solid culture medium, and the growth was detected by overnight culture.
  • the E. coli ara gene strain ⁇ 2829 carrying the plasmids pKU2004, pKU2006 and pKU2007 was inoculated in LB liquid medium overnight, centrifuged at 4000 rpm for 3 minutes, suspended in 0.9% physiological saline, centrifuged again, and the supernatant was discarded. , and resuspended with physiological saline, 5 into the liquid medium containing 0, 50 and 100 mM glyphosate, the initial inoculation amount is OD 6 (X ) 0.04, overnight culture at 37 ° C, and timed light absorption ( OD 6 . . .
  • the bacteria carrying the plasmid of interest were placed in an LB liquid medium containing the corresponding antibiotic, and cultured at 37 ° C to an OD 6 . . About 0.75, IPTG was added to a final concentration of 0.5 mM, and cultured at 15 ° C overnight.
  • the bacterial cells were centrifuged at 4 Q C, 5000 rpm for 10 minutes to collect the cells, and resuspended in a volume ratio of 10:1 to buffer A (50 mM Tris-HCl (pH 7.8), 0.4 mM DTT), and the cells were disrupted by ultrasonic wave. , 4 ° C, Centrifuge at 8000 rpm for 60 minutes.
  • the supernatant after centrifugation was subjected to protein purification using HisTrap HP kit (Amersham Biosciences) according to the instructions for use.
  • the purified protein was concentrated in buffer A using Millipore Biomax membrane (10 kDa) and stored at 4 Q C.
  • the BL21(DE3) strain carrying the plasmid pKU2008 was subjected to expression and purification as described above, and finally the protein was concentrated and resuspended in PBS buffer. The protein was used as an antigen to immunize the rabbit. After four reactions, the serum was taken after a boosting reaction (this process was completed by the Institute of Genetics of the Chinese Academy of Sciences), and the antibody titer was detected by ELASA for use.
  • the concentration of the upper layer of the SDS-polyacrylamide gel electrophoresis gel is 5%, and the concentration of the lower layer of the separation gel is generally 16%.
  • the concentration of the Native-polyacrylamide gel electrophoresis gel was 10%.
  • Each curve is the average of 4 measurements.
  • the measurement temperature is 8 Q C.
  • a 1-cm pathlength cylinder quartz cuvette was used for the measurement of the near-violet dichroism, and a 0.1-mm pathlength cylinder quartz cuvette was used for the far-purple dichroism spectroscopy.
  • the N-terminus and C-terminus of EPSPS were expressed in BA- separately, and the cells were collected by centrifugation, and then ultrasonically disrupted. The electrophoresis was carried out to detect whether the target protein mainly formed inclusion bodies and accumulated in the precipitate.
  • the precipitate was collected, 5 washed three times with buffer B (50 mM Tris-HCl (pH 7.8), 1 mM EDTA, 0.05% Triton-100), then three times with 1 M sodium chloride solution, and then buffer.
  • C 50 mM Tris-HCl (pH 7.8), 1 mM EDTA, 1 M urea
  • the precipitate after washing was added to a buffer A. of 8 M urea, and the solution was fully dissolved, and the supernatant was centrifuged and transferred to a dialysis bag.
  • the cells were dialyzed overnight with 2 M urea buffer D (50 mM Tris-HCl (pH 7.8), 1 mM GSH, 0.5 mM GSSG) 0, dialyzed against buffer D for 36 hours, and finally concentrated with PEG 12000.
  • the renatured product was analyzed by electrophoresis, immunoreactivity and enzyme activity assay.
  • the gene encoding the N 5 terminal peptide of EPSPS was constructed on pACYC184 vector, and the gene encoding the C-terminal peptide of EPSPS was constructed on pBR322 vector (see the specific construction process). method). These plasmids were then transferred to the arok gene mutant strain AB2829 of Escherichia coli to determine whether the protein fragment encoded by the gene still has EPSPS activity by detecting whether it can grow on M63-restricted medium. Results; AB2829 with only the N-terminal or C-terminal EPSPS-encoding gene could not be grown on M63 medium for 0 hours.
  • plasmid pBR322 and pACYC184 replicon have pl5A and ColEl, therefore: These two plasmids may be expressed in the same bacterial CCP.
  • the streaks were cultured at 37 Q C on M63 restriction medium plates. After 16 hours, with N218/C219 (pKU2101/pKU2102) and N227/C228 (pKU2125/pKU2138) with EPSPS, respectively
  • Plasmid AB2829 can be grown on restricted media, while AB2829 with the other three pairs of plasmids pKU2126/pKU2137 (N234/C235), pKU2162/pKU2163 (N240/C241) and pKU2110/pKU2130 (N245/C246) cannot Grow on M63 medium.
  • EPSPS fragment 0 N218/C219 and N227/C228) can complement each other to form EPSPS activity when expressed separately in vivo: The other three pairs of EPSPS fragments cannot complement EPSPS activity in vivo.
  • a plasmid for expression of the EPSPS fragment was constructed, in which the N-terminus was constructed on the pACYC184 vector, and the C-terminus of the protein was constructed on the pET28a vector, both of which were transcribed and expressed by the T7 promoter, wherein the C-terminus of the EPSPS
  • the C-terminus of the peptide chain is fused with 6 histidines (see plasmid construction for see above).
  • the two protein fragments of EPSPS can be reconstituted in vivo to form the structure of natural EPSPS, then the N-terminal peptide chain of the split EPSPS will bind to the corresponding EPSPS C-terminal peptide chain, and the complex can be adsorbed by the nickel column. And a total of purified.
  • the araA gene encoding EPSPS in E. coli BL21 was replaced with the gene encoding ⁇ -lactamase to obtain the oA gene deletion strain ⁇ -.
  • the corresponding two series of plasmids were transformed into BA-strain for co-expression.
  • the co-expressed protein was purified using HisTrap HP kit, and the purified protein was subjected to SDS-polyacrylamide gel electrophoresis, and then the prepared EPSPS antibody was used for detection (see the method for the specific procedure). The results are shown in Fig. 4.
  • the purified protein contains not only the C-terminal peptide chain of EPSPS but also the N-terminal peptide chain. These protein fragments are identical in size to the predicted fragment size, indicating that fragments of EPSPS expressed in vivo can complement each other to form a complex for co-purification.
  • the co-expressed protein does not have a full-length protein either before or after purification, indicating that no recombination occurs in the body, and thus no full-length EPSPS gene is formed.
  • Activity assays also showed that the purified proteins all have EPSPS activity. The above results indicate that EPSPS activity can be formed by complementation of fragments of peptide chains expressed in vivo separately.
  • N-terminal peptide chain and the C-terminal peptide chain of EPSPS may differ in expression in vivo, for example, the expression level of N227 is much larger than that of C228, but the content of N-terminal and C-terminal protein fragments after purification is almost 1: The relationship of 1. It can also be seen from the figure that the amount of purified N234/C235 and N245/C246 is much less than that of the other three co-purified EPSPS, which may be that it cannot complement EPSPS activity in vivo to make AB2829 on restrictive medium. The cause of growth. Although N240/C241 complex purification The amount is even more than that of N218/C219, but it does not complement the growth of AB2829 on restrictive media because of its low EPSPS activity.
  • the specific activity of the enzyme was determined to be 1.0 mM for both PEP and S3P.
  • the PEP concentration was not changed to 1 mM, and the S3P concentration was from 50 to 200 ⁇ .
  • plasmids pKU2105 and pKU2333 were constructed, respectively, encoding the two N-terminal fragments N218-G96A and N227-G96A of E. coli EPSPS. Transfer them to the corresponding strains encoding C219 and C228 into the mutant strain AB2829 (aro A-, from Yale University) of the E. coli arok gene, respectively. The growth was measured in M63-restricted medium with different concentrations of glyphosate, and the results are shown in Fig. 6.
  • the strain expressing EPSPS-G96A or N227-G96A/C228 still grew well in M63 medium containing 100 mM glyphosate, while the strain expressing N218-G96A/C219 grew slightly poorly, which may be N2227- in vivo. The reason why the G96A/C228 complex is easier to form.
  • Protein fragments can complement each other to form active complexes, and it is also possible to complement each other to form active complexes in vitro.
  • the terminal peptide chain N218 and the C-terminal peptide chain C219 of EPSPS were expressed, respectively.
  • a large number of EPSPS fragments are expressed to form inclusion bodies. After the initial purification of the inclusion bodies, 'repair them together (see the method for the procedure). After the end of renaturation, it was subjected to electrophoresis analysis, and the results are shown in Fig. 7. From Figure 7a we can see that each fragment of EPSPS appears in the expected position and does not have full-length EPSPS.
  • the native-PAGE results can appear as bands with wild EPSPS.
  • the renaturation N218 and C219 are mixed together, there is no such band, which indicates the in vitro EPSPS co-refoldability. It can then complement to form a structure similar to wild EPSPS.
  • Activity experiments also showed that N218/C219 co-refolding can complement EPSPS activity, while N218 and C219, which are renatured separately, do not complement each other to form EPSPS activity.
  • Plasmid construction a. Construction of pBR322 series shield granules (encoding EPSP synthase C-terminal peptide chain)
  • the pBR322 series plasmid constructed in 3.5 a and the pACYC1 184 series plasmid constructed in b were transferred into the corresponding two plasmids and transferred into the strain AB2829 of Escherichia coli flroA gene defect, and then streaked to M63-restricted solid. On the culture medium, overnight growth was used to examine the growth.
  • N234 C235 folding unit between 3 and 5 - - - - -
  • Fragment complementation experiments were performed using the glyphosate resistant EPSPS gene of Pseudomonas putida CGMCC 0739.
  • the plasmid, medium, strain, and experimental procedure used are all described in Example 1.
  • the reconstituted enzyme activity was detected by growing on a medium containing 100 mM glyphosate.
  • ppN2333' (BamHI): 5'- CGG GAT CCT CAG GTG TAT CGT TGC ATT TTC G-3' ppC2345' (NcoI): 5,- CAT GCC ATG GTA GAA GGC GAC TGG AGC G-3'
  • ppN2363' (BamHI): 5'- CGG GAT CCT CAG CCT TCT ACG GTG TAT CGT TG-3' ppC2375' (NcoI): 5'- CAT GCC ATG GAC TGG AGC GGT GGT GCT TT-3'

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)

Description

利用片段互补技术重建
5-烯醇丙酮酰莽草酸 -3-磷酸合成酶活性
5 技术领域
本发明涉及利用片段互补技术重建 5-浠醇丙酮酰莽草酸 -3-磷酸合酶 (5 -enolpyruvyl-shikimate-3 -phosphate synthase, EPSPS)活 4生的方法。 更具体 地， 本发明涉及大肠杆菌 EPSPS及其突变后获得的草甘膦抗性 EPSPS的 重建，本发明还涉及恶臭假单胞菌草甘膦抗性 EPSPS的重建。
0
背景技术
随着转基因植物的大规模种植， 转基因植物的生物安全控制 (biological confinement)越来越受到关注。 Wruad等 . Proc Natl Acad Sci U S A 2004, 101 , (40), 14533-8观测到抗草甘膦的 CP4 EPSPS的编码基因可以从转基因植物中 5 传播到 20公里以外的作物中， 甚至传播到杂草中， 因此对转基因植物实行生 物安全控制对于防止超级杂草等的产生十分必要。
转基因植物外源基因扩散的方式主要有以下几种： 转基因植物花粉的传 播，转基因植物作为野生亲缘种花粉的受体形成杂种，转基因植物的 DNA可 能造成的基因扩散。 目前有了一些控制转基因植物外源基因扩散的方法， 例 0 如， （1)物理隔离， 主要是距离隔离以便阻断外源基因通过花粉的扩散； （2)遗 传控制， 包括： （a)雄性不育（male sterility) ; (b)基因组不相容性 (Genome
；' · incompatibility) , 即将特定的外源基因整合到作物的与杂草不相容的基因组 上； （c)母系遗传 (maternal inheritance), 其将外源基因转入植物的叶绿体中， 使这些基因进行母系遗传， 因此不会通过花粉的传播而扩散入其它物种， 该 5 方法在烟草上已经取得了初步的成功 (Daniell等， Nat Biotechnol 1998, 16, (4)， 345-8); (d)种子木育（seed sterility); (e) Transgenic mitigation (TM), 利用与目 的基因紧密连锁、 对于转基因植物有利或中性、 而对于野草生存不利 (如防止 种子散落和降低种子二次休眠等)的 TM基因防止超级杂草 (superweed)的产 生。
0 Ye, G. N.等. Plant J 2001 , 25, (3), 261-70和 Chin, H. G.等 Pwc Natl Acad
Sci USA 2003， 100， (8), 4510-5提出了一种新的控制抗草甘膦转基因的方法， 其将 EPSPS基因分成两段， 使之分别与表达 DnaE内含肽 (intein)的基因串联 并共表达， 利用内含肽的自我拼接功能形成完整的 EPSPS , 从而使大肠杆菌 或烟草获得对草甘膦的耐受性。 但是内含肽编码基因本身作为外源基因被引 入转基因植物也可能会引发其它的风险， 例如成为被扩散的转基因等。
蛋白酶消化产生或者通过基因表达生成的蛋白质片段可以在体内或体外 重建成具有完整蛋白功能的复合体， 这被称之为蛋白质片段互补 (protein fragment complementation)或蛋白重建 (protein reconstitution)技术 (例 ^口， Hakansson, Μ·等. Curr Protein Pept Sci 2002, 3, (6), 629-42; Braun, Μ·等. Bacteriol 2003, 185, (18), 5508-18·)。对氨酰 tRNA合成酶等蛋白进行的片段互 补研究表明， 发生片段互补的蛋白片段分拆位点大多发生在非保守区域。 蛋 白能够重建意味着该蛋白内的非共价作用十分特异， 从而使蛋白片段有利于 形成的天然的结构（Shiba, K.等. Proc Natl Acad Sci USA 1992, 89， (5), 1880-4; Shiba, K.等. J Biol Chem 1992, 267， (32), 22703-6)。 此外， 蛋白能够发生片段 . 互补表明， 即使共价键断开， 该蛋白仍能保持比较稳定的结构。 两个发生片 段互补的肽链之间存在的各种非共价作用（如氢键、盐桥及疏水作用）在保持蛋 白结构稳定方面起着很大的作用（Nelson, K. E.等， Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 2002, 4, (12)， 799-808)。
我们利用这种蛋白重建技术在体内和体外重建了有活性的 EPSPS , 对草 甘膦具有耐受性的 EPSPS也可以通过这种方法重建。 EPSPS的片段互补可用 于转基因植物从而增加转基因植物安全性， 降低超级杂草形成的可能性。 发明概述
本发明涉及 5-烯醇式丙酮酰莽草酸 -3-碑酸合酶 (EPSPS)的蛋白片段， 其 是选自 EPSPS片段对的一种片段， 组成所述片段对的两种片段可以连接成全 长 EPSPS , 并且这两种片段不借助任何连接结构可以通过互补而重建 EPSPS 活性。 优选， 所述片段对的分割点位于 EPSPS的选自下列的结构中： 折叠单 元之间的连接区中、 α螺旋与 β折叠之间的连接区中、 两个 β折叠之间、 β折 叠中、 或 α螺旋中， 优选位于折叠单元之间的连接区中， 例如， 在折叠单元 1和 6, 2和 6, 3和 4, 4和 5 , 以及 3和 5之间的连接区中； 更优选该分割 点是在折叠单元 1， 2 , 3 , 4， 或 5的内部， 例如， 在折叠单元 3的两个 β折 叠之间的连接区中， 在折叠单元 4的 α螺旋中， 在折叠单元 2的 β折叠中， 在折叠单元 1的 α螺旋与 β折叠之间， 在折叠单元 5的 α螺旋和 β折叠之间 的连接区中， 或者在折叠单元 5的两个 β折叠之间的连接区中， 或者在 ^叠 单元 5的 β折叠中。
在本发明的实施方案中， 所述 EPSPS是野生型 EPSPS或其添加、 缺失、 和 /或 代一或多个氨基酸残基所得的 EPSPS活性变体，优选是大肠杆菌野生 型 EPSPS (其全长氨基酸序列和核苷酸序列是本领域已知的，可以参见序列表) 或其草甘膦抗性 EPSPS 活性变体， 或者优选是恶臭假单胞菌 C¾ewifowo«os /7Μ//ί α)草甘膦抗性 EPSPS , 例如恶臭假单胞菌 CGMCC 0739(参见中国专利申 请 02117991.3)的 EPSPS (其全长氨基酸序列和核苷酸序列见 SEQ ID NO:2)。
更具体地，本发明的蛋白片段优选是选自大肠杆菌 EPSPS的以下片段对 的一种片段： N67/C68 , N85/C86 , N104/C105, N154/C155, N182/C183, N184/C185, N218/C219, N224/C225, N227/C228, N259/C260, N298/C299, N371/C372, N376/C377, N383/C384(这里所述的 N67/C68片段对， 表示由 N 端片段 N67和 C端片段 C68所组成的片段对， 其中 N67是指 EPSPS序列中 从 N末端到第 67位残基之间的 N端片段， C68是指 EPSPS序列中从第 68 位残基到 C末端之间的 C端片段。其它片段对表示法依此类推)，或优选是选 自恶臭假单胞菌 CGMCC 0739 的 EPSPS 的以下片段对的一种片段： N208/C209 , N214/C215 , N219/C220, N222/C223, N224/C225(这里所述的 其中 N208是指 EPSPS序列中从 N末端到第 208位残基之间的片段， C209 是指 EPSPS序列中从第 209位残基到 C末端之间的片段，其它片段对表示法 依此类推)。
本发明还涉及编码本发明所述蛋白片段的核酸分子， 携带该核酸分子的 表达载体， 包含所述核酸分子或表达载体的细胞。 优选所述细胞是植物细胞。 本发明还涉及一种重建 EPSPS的方法，该方法包括在无任何连接结构存 在的奈件下， 利用本发明的蛋白片段或核酸分子或表达载体来重建 EPSPS活 性。
本发明还涉及一种拆分 EPSPS或 EPSPS核酸分子的方法， 包括在无任 何连接结构存在的条件下， 拆分出本发明的蛋白片段， 或核酸分子。 该方^ 中所选择的拆分点优选位于 EPSPS的下列结构中：折叠单元之间的连接区中、 α螺旋与 β折叠之间的连接区中、 两个 β折叠之间、 β折叠中、 或 α螺旋中， 优选位于折叠单元之间的连接区中， 例如， 在折叠单元 1和 6, 2和 6， 3和 4， 4和 5， 以及 3和 5之间的连接区中。 更优选在折叠单元 1 , 2 , 3 , 4, 或 5的内部， 例如， 折叠单元 3的两个 β折叠之间的连接区中， 折叠单元 4的 α 螺旋中， 折叠单元 2的 β折叠中， 折叠单元 1的 α螺旋与 β折叠之间， 折叠 单元 5的 α螺旋和 β折叠之间的连接区中， 或者折叠单元 5的两个 β折叠之 间的连接区中， 或者在折叠单元 5的 β折叠中。 还更优选位于选自下列的位 置之间：大肠杆菌 EPSPS的 67-68、 85-86、 104-105、 154-155、 182-183、 184-185、 218-219、 N224-C225、 N227-C228、 259-260、 298-299、 371-372 , 376-377 或 383-384位置之间，或恶臭假单胞菌 CGMCC 0739的草甘膦抗性 EPSPS的 208-209 214-215、 219-220、 222-223、 或 224-225位置之间。
本发明还涉及本发明所述的 EPSPS片段或重建 EPSPS活性的方法或拆 分 EPSPS的方法在控制转基因植物安全性方面的应用。 发明内容
本发明人基于 EPSPS的结构， 在有可能不影响 EPSPS酶活性的结构区 域中设计了分拆位点。 本发明人还构建了表达 EPSPS拆分片段的表达载体， 并证明 EPSPS片段在大肠杆菌体内可以互补 EPSPS活性。 因此， 本发明的 EPSPS片段以及重建 EPSPS的方法可以应用于生物安全控制领域。
EPSPS及其结构
5-烯醇丙酮酰莽草酸 -3-磷酸合成酶 (EPSPS)是芳香族氨基酸合成-莽草酸 合成途径中的关键酶， 存在于藻类、 高等植物、 细菌、 真菌及寄生虫的 apicomplexan 中， 它催化一分子的莽草酸 -3-磷酸 (S3P)和磷酸烯醇式丙酮酸 (PEP)生成 5-烯醇式丙酮酸莽草酸 -3-磷酸 (EPSP)。
如图 2所示， EPSPS由两个结构域组成， 其中一个结构域包括标示为 1， 2和 6的三个对称的蛋白折叠单元， 另一个结构域包括标示为 3， 4和 5的三 个对称的蛋白折叠单元。 每个单元由两个平行的 α螺旋和四个 β折叠组成。 参见 Stallings, W. C.等. Proc Natl Acad Sci USA 1991 , 88, (1 1), 5046-50。
已知 EPSP合酶在无酶底物时， 形成 "开放，， （open)构象， 而与 S3P、 草 甘膦 +S3P 形成复合物晶体结构时， "开放" 构象转换成 "闭合 "(close)构象 (Schonbrunn, E.等. Proc Natl Acad Sci 2001 , 98, (4), 1376-80)。 Mcdowell 等（·/ Biomol NMR 2004, 28, (1)， 1 1-29， 2004)利用 rotational-echo double-resonace NMR技术， 结合 EPSP合酶与 S3P和草甘膦复合体的晶体结 构， 对其进行调整， 得到符合液体 NMR结果的三维结构。
通过化学修饰、基因突变、 结构分析等各种方法， EPSP合酶中底物的结 合位点和催化位点的研究现在已经比较深入 (Schonbrunn, E.等， 出处同上； Mcdowell 等， 出处同上； Anderson, K. S.等. J Biol Chem 1990， 265, (10)， 5567-72; Huynh, Q. K. 等 J Biol Chem 1988， 263，（24)， 1 1636-9; Huynh, Q. K. 等 J Biol Chem 1988, 263，（2)， 735-9; Padgette, S. R. 等 J Biol Chem 1988， 263, (4), 1798-802; Padgette, S. R. 等 Arch Biochem Biophys 1988, 266，（1), 254-62; Eschenburg, S.等 J Biol Chem 2003, 278, (49), 49215-22 ; Mizyed, S. 等 Biochemistry 2003, 42， (23), 6986-95; Shuttleworth, W. A. 等 Biochemistry 1994, 33, (23), 7062-8; Shuttleworth, W. A. 等 Arch Biochem Biophys 1996, 334, (1)； 37-42; Shuttleworth, W. A. 等 oc/zew W^y 1999, 38, (1 )， 296-302; Stauffer, M. E. 等 Biochemistry 2001 , 40, (13), 3951-7; Stauffer, M. E. 等 FEBS Lett 2001， 499, (1-2), 182-6; McDowell, L. M. 等 Biochemistry 2004, 43, (21), 6606-1 1)。
此外， Schonbrunn等提出 Lys-22 , Arg-124, Asp-313 , Arg-344, Arg-386 和 Lys-411 参与 PEP结合， Arg-27等参与 S3P结合， Arg-100, Asp-242和 Asp-384 在酶结合底物由开放构象转换成闭合构象时起着重要作用（尸 ra ra 2000， 40, (2)， 290-8； Biochemistry 2000， 39, (9)， 2164-73； Pwc Natl Acad Sci US 2000, 97, (12)， 6345-9)。
拆分位点的选择
本发明人基于 EPSPS的结构，在有可能不影响 EPSPS酶活性的结构区域 中设计了分拆位点。
当分拆位点位于蛋白折叠单元之间时， 共价键的断裂和甲硫氨酸的插入 一般不会影响蛋白质天然结构的形成。 分拆的蛋白片段依靠蛋白内的非共价 作用及两个肽链之间存在各种非共价作用如氢键、 盐桥及疏水作用等形成天 然的蛋白结构， 从而可以重建蛋白功能。 分拆位点位于折叠单元之间， 互补 片段二级结构的形成一般不会被影响， 因此更容易发生片段互补。 在实施方 案中， 大肠杆菌. EPSPS有 7个分拆位点位于折叠单元之间的连接区域中， · 拆产生的蛋白片段有 6对可以互补形成 EPSPS活性， 而且它们的互补活性都 比较好。 恶臭假单胞菌有 3个分拆位点在折叠单元之间的连接区域中， 所形 成的 3对片段都可以互补 EPSPS活性。
当分拆位点位于 α螺旋与 /或 β折叠之间的连接区域时， 共价键的断裂一 般不会影响到 α螺旋或 β折叠的形成， 而曱硫氨酸的插入对天然结构的形成 的影响一般也没有那么明显， 因而此时片段互补容易发生。 在实施方案中， . 大肠杆菌 EPSPS有 6个分拆位点位于 α螺旋与 β折叠之间的连接区域中， 其 • 中有 5对片段可以互补 EPSPS活性； 另有 1个分拆位点位于 β折叠之间的连 接区域中， 相应片段也可以互补 EPSPS活性。 恶臭假单胞菌有 1个分拆位点 在 β折叠中， 所形成的片段可以互补 EPSPS活性。
当分拆位点位于 α螺旋或 β折叠中时， 只要 α螺旋或 β折叠可以忍受共 价键的断裂和曱石克氨酸的插入， 或者此处的 α螺旋或 β折叠对蛋白的整体功 能并不是十分重要， 则 EPSPS的片段也可以通过片段互补来重建蛋白活性。 在实施方案中， 大肠杆菌 EPSPS有 7个分拆位点位于 α螺旋或 β折叠中， 其 中有 3对片段可以互补 EPSPS活性。 恶臭假单胞菌有 1个分拆位点在 β折叠 中， 所形成的片段可以互补 EPSPS活性。
当 EPSPS在两个结构域中间分拆时， 大肠杆菌 EPSPS的 N端肽链 N240 ： 和 C端肽链 C241可以形成复合体而被共纯化出来。 但是该复合体酶活性很 低， 这可能是 N240/C241复合体中只有一对疏水作用区域相互作用， 因此蛋 白结构不稳定。 恶臭假单胞菌有在两个结构域之间测试了 3个分拆位点， '所 形成的片段都基本不能互补 EPSPS活性。 因此，本发明人在 EPSPS的结构域 之间连接区中进行分拆， 所得片段没有能够重建出 EPSPS活性。
总之， 本发明人在大肠杆菌 EPSPS中成功实施了 14个拆分点， 其中 6 个在折叠单元之间的连接区中， 3个在 α螺旋与 β折叠之间的连接区中， 2个 在 β折叠之间的连接区中， 2个在 β折叠中， 1个在 α螺旋中。 通过在这些拆 分点进行拆分， 大肠杆菌 EPSPS的基因被分拆为 Ν片段和 C片段。 将相互 对应的 N片段和 C片段分别在两种相容的质粒上表达。如此得到的 N片段
C片段单独都不表现 EPSPS活性，但在体内，相应的 N片段和 C片段结合可 以互补 EPSPS活性。
类似地， 在另外的实施方案中， 本发明人在恶臭假单胞菌草甘膦抗性 EPSPS 中成功实施了 5个拆分点， 其中 3个在折叠单元之间的连接区中， 1 个在 α螺旋与 β折叠之间的连接区中， 1个在 β折叠中。 如此拆分所得到的 片段同样可以互补 EPSPS活性。
EPSPS的活性能够通过片段互补而重建， 这意味着该蛋白内的非共价作 用十分特异， 从而使片段有利于形成天然的具有 EPSPS功能的结构， 而不会 形成其它的结构。 此外该蛋白能够发生片段互补， 这表明， 断开一些区域中 5 的共价键时， 该蛋白仍旧可以保持结构稳定性， 两个肽链之间不必通过共价 键， 而仅通过各种非共价作用如氢键、 盐桥及疏水作用等紧密结合在一起。 在 EPSPS的片段互补中， 疏水作用可能起着更大的作用。 EPSPS的两个片段 之间存在很强的两对疏水作用区域， 形成两个 "钩子" 将蛋白稳定在一起。 . 本发明共纯化得到了多个由相对应的拆分片段组成的复合体， 但是纯化 10 出的蛋白量却有很大的差异， 这说明片段互补在体内都可以形成， 但是复合 体形成的难易程度是不同的。 有些区域共价键的断裂对蛋白结构的稳定性影 响比较大， 有些区域共价键的断裂对蛋白结构的稳定性影响比较小。 C 端肽 链引入一个曱硫氨酸 (起始密码子所编码的氨基酸)也有可能影响蛋白结构的 稳定性， 从而导致蛋白片段互补不能发生。 ' :
15 大肠杆菌 EPSPS的复合体 N240/C241也可以被大量的共纯化出，但是它 却不能互补大肠杆菌 aro 基因突变菌株 AB2829在限制性培养基上的生长， 这是由于复合体 N240/C241的 EPSPS的活性远低于野生型全长 EPSPS。其活 性的丢失可能有以下两个原因： 一是 N240/C241 的分拆位点与 Asp242十分 接近， Asp242在结合底物引发结构变构过程中起着非常重要的作用，在 C241 0 中引入一个曱硫氨酸可能会影响到 Asp242的功能从而使其酶活性丧失；第二 个原因可能是 N240/C241的分拆位点位于两个结构域的中间， 其形成的复合 ：： 体中只有一对疏水作用区域， 虽然它们之间的疏水作用足以使两个蛋白片段 形成复合体， 但由于缺少另外一对疏水区域， 这个复合体的结构可能与野生 蛋白的结构有所不同， 从而使酶活性丧失。 因此， 对分拆位点的选择应基于 5 结构变化对酶活性的影响来考虑。 '
EPSP合酶的在植物细胞中的定位
Bickel等 (Phytochemistry 17: 1 19-124， 1978)发现， 植物的芳香族氨基酸 在叶绿体内进行合成。 Mousdale等随后证明， 植物的 EPSP合酶定位于叶绿 体内膜上(？1& 3，1987, 170:卜6； Plant Physiol., 1987， 83 :229-231 ; J Biol Chem 0 1988 Oct 15;263(29): 15104-9; Mol Gen Genet 1994 Dec 1 ; 245(5):616-22; Mol Gen Genet 1993 Jun; 239(3):416-24)。 Della-Cioppa等 (Bio/Technology 1987(5)： 579~584)的实验表明矮牵牛前体 EPSP 合酶 (precursor EPSPS)分子量为 55kDa, 前 72氨基酸残基为前导肽， 当前体 EPSP合酶进入叶绿体后经加工 剪切运输肽而成为成熟 EPSP合酶 (mature enzyme), 成熟 EPSP合酶分子量为 48kDa。 前导肽对前体 EPSP合酶进入叶绿体起着重要作用， 微生物 EPSP合 酶因无前导肽， 故用微生物 EPSP合酶基因在转入植物细胞中时其基因前端 需加上植物前导肽序列， 否则其表达的 EPSP合酶不能进入叶绿体。 应用
本发明的 EPSPS片段互补技术可应用于转基因植物， 以防止抗草甘膦基 因的扩散带来的生态风险。 例如， 将 EPSPS的 N端片段在植物核染色体中表 达， 而将其 C端片段在植物的叶绿体中表达。 单独表达的一种 EPSPS片段都 不具有 EPSPS的活性， 因此其基因扩散没有选择优势。 当两种片段的基因共 同表达时， 两个 EPSPS片段可以在叶绿体中发生互补而重建 EPSPS活性，从 而使植物对草甘膦产生抗性。 也可以将编码两个 EPSPS片段的基因插入叶绿 体基因组的不同位置， 在这种情况中， 两种基因共同转移到染色体上的概卓 大大降低， 从而降低转基因扩散发生的概率。 关于控制植物中细胞过程的方 法可参见 WO2004/046359和 WO2004/046360。 附图说明 , 图 1显示 pKU2004的质粒图 。
图 2 EPSPS的结构示意图及分拆位点的选择。 图 2A为 EPSP合酶的拓 朴结构， 图 2B为 EPSP合酶的一个结构单元。
图 3: 质粒构建示意图。 A. 将 EPSPS的 C-末端片段亚克隆到 pKU2100 载体的 Tet启动子之后。 B. 将 EPSPS的 N-末端亚克隆到 pACYC184载体的 Tet 启动子之后。 C. 将 EPSPS 编码基因的 N-末端部分亚克隆到衍生于 pACYC184的质粒载体的 T7启动子之后。 D. 将 EPSPS的 C-末端部分构建到 pET28a载体中， 并且在 C-末端尾部带有 6个 His组成的标签。
图 4: EPSPS片段的蛋白免疫印迹分析。 将表达所示 EPSPS的细胞溶于 SDS样品緩冲液中， 经 SDS-PAGE 和免疫印迹来分析等量蛋白（来自〜 0.8μ 湿重的细胞)和纯化的蛋白。 图中示出了以下纯化蛋白的量： N218/C219 (来自. ~150 ml湿重的细胞)， N227/C228 (来自〜 20μβ湿重的细胞)， N234/C235(来自 ~500μ§湿重的细胞)， N240/C241 (来自 ~4(^g湿重的细胞)， N245/C246(来自 ~500μ 湿重的细胞)和 pACYC184/pBR322 (来自〜 50(^g湿重的细胞)。标准分 子量的位置示于图左， 单位为千道尔顿 (kd)。
图 5. EPSPS 的圆二色性光谱分析。 系列 1 为野生 EPSPS, 系列 2 为 N227/C228复合体。
图 6: 表达有不同 EPSPS的大肠杆菌 GraA基因缺陷菌株 AB2829, 在限 制性培养基 (即添加了所示浓度的草甘膦的液体 M63基本培养基)中的生长情 况。
图 7 a大肠杆菌 EPSPS的 SDS-PAGE电泳图。 1 : N218; 2: C219; 3: ： EcEPSPS; 4: 共复性 N218 + c219; 5: N218 + c219(单独复性后一起)； 6: 分 子量标准。
图 7 b大肠杆菌 EPSPS的 native-PAGE电泳 Western印迹图。 1 : N218 + C219(单独复性后一起)； 2: 空白对照； 3: 共复性 N218/C219; 4: 空白 对照； 5: 野生型 EPSPS。 具体实施方式
实施例 1 大肠杆菌 EPSPS的重建
1 材料与方法
1.1 菌株与质粒
用于本实验的菌株和质粒列在表 1中。
表 1. 用于本研究中的细菌菌株和质粒
菌株 /质粒 相关特点 来源 /参考文献 大肠杆菌菌株
DH5a supE44AlacU 169hsdR 17recA 1 gyrA96thi-】 re 1 A 1 Hanahan D.， J Mol Biol
1983; 166:557-80
AB2829 耶鲁大学
B121 (DE3) stragene
BA" BL21 (DE3) . aroA, ApR 本工作
质粒
pUC 18 ColE l , /acZ', Αρ' Norrander 等， 1983 pBluscript-SK ColE 1 , acZ ', Ap1 Stratagene pET-28a ColEl, 表达质粒， KmR Novagen
pACYC184 CmR Chang, A. C,等 1978. J
Bacteriol 134:1141-56 pBR322 ColEl, ApR Schaeffer, F.等， 1982
EMBOJ.1,99-105 p 03 CmR Link, A丄等 1997. J.
Bacteriology 179: 6228-6237 pKU2005 pUC18衍生质粒， 带有 aroA£c。/,, ApR 本工作
pKU2006 pACYC184衍生质粒， 带有 araA c„/,, CmR 本工作
p U2007 pACYC184衍生质粒， 带有 aroA^/rGQSA, CmR 本工作
p U2008 pET-28a衍生质粒， 带有 £.co/ CWOA, KmR 本工作
p U2009 pET- 28a衍生质粒， 带有 Eco" a A， KmR 本工作
p LI2010 pET-28a衍生质粒， 带有 £.co/ ijroA-G96A, mR 本工作
pKU2011 pET-28a衍生质粒， 带有 £.co/。/OA-G96A， mR 本工作
p U2100 PBR322衍生质粒， 带有
Figure imgf000011_0001
ApR 本工作 p U2101 pACYC184衍生质粒， 带有 NSl
Figure imgf000011_0002
CmR 本工作 p U2102 pBR322衍生质粒， 带有 C2】9-araA£.c。/,, ApR 本工作 pKU2107 pBR322衍生质粒， 带有 a/OA£ra/,-G96A, ApR 本工作 pKU2110 pACYC184衍生质粒， 带有 N245-araA£∞/,， CmR 本工作 pKU2125 pBR322衍生质 ¾i, 带有 C228-mOA£c。/„ ApR 本工作 p U2126 pBR322衍生质粒， 带有 C235-。OA£c。/,， ApR 本工作 p U2127 pET-28a衍生质粒， 带有 N218-£.co"<3raA, KmR 本工作 pKU2130 pBR322衍生质粒， 带有 C246-aroA£∞/,.， ApR 本工作 p U2137 pACYC184衍生质粒， 带有 N234-aAOA c,,,,, CmR 本工作 p U2138 pACYC184衍生质粒， 带有 CmR 本工作 pKU2154 pBluscript-SK衍生质粒， 带
Figure imgf000011_0003
R 本工作 p U2159 pET-28a衍生质粒， 带有 £.co// απ?Α， KmR 本工作 p U2195 pBluscript-SK衍生质粒， 带有 C219-iwoA£c„,,, ApR 本工作 pKU2203 pET-28a衍生质粒， 带有 N218-£.co/ araA， KmR 本工作 pKU2205 pET-28a衍生质粒， 带有 C218-£.co/ α/ΌΑ， KmR 本工作 p U2223 pBluscript-S 衍生质粒， 带有 <3roA£c。,,.上游部分序列 本工作 pKU2224 pBluscript-S 衍生质粒， 带有 bla基因 本工作 p U2225 pBluscript-S 衍生质粒， 带有 aro ^m下游序列 本工作 p U2227 pBluscript-S 衍生质粒， 带有 CWOA£c。/,.上下游序列 本工作 p U2228 pBluscript-S 衍生质粒， 带有 aroA w,.上下游序列及 基因 本工作 pKU2229 p 03衍生质粒， 带有 aroA£ra/,上下游序列及 Wa基因 本工作 pKU2249 pET-28a衍生质粒， 带有 N245-£.co/i' roA, mR 本工作 p U2250 pET-28a衍生质粒， 带有 C246-E. co" aroA, mR 本工作 pKU2249 pET-28a衍生质粒， 带有簡 5-£.co" aroA, mR 本工作 p U2249 pET-28a衍生质粒 , 带有 N245-£.co// aro , mR 本工作 pKU2249 pET-28a衍生质粒， 带有 N245-E.CO" arok, mR 本工作 pKU2262 pACYC 184衍生质粒， 带有 N240-araAfc..c。,„ CmR 本工作 pKU2263 pBR322衍生质粒， 带有 C24卜 araA/;.∞/,， ApR 本工作 pKU2266 pET-28a衍生质粒， 带有 N240-E.co!i arok, KmR 本工作 p U2267 pACYC184衍生质粒， 带有 T7启动子及 N218-araA£c。/,， CmR 本工作 p U2268 pACYC184衍生质粒， 带有 T7启动子及 Ν240-ί»ΌΑΛ.∞/,， CmR 本工作 p U2269 pET-28a衍生质粒， 带有 N227- .co" arok, mR 本工作 p U2274 pACYC184衍生质粒， 带有 T7启动子及 n-aw Ecolh CmR 本工作 p U2275 pACYC 184衍生质粒， 带有 T7启动子及 N234-araA£..c„,,， CmR 本工作 p U2276 pET-28a衍生质粒， 带有 C228- .co/ arok, mR 本工作 p U2277 pET-28a衍生质粒， 带有 C235-£.CO//CTOA, KmR 本工作 p U2278 pET-28a衍生质粒， 带有 C24 £.co/ arok, mR 本工作 p U2282 pET-28a衍生质粒， 带有 C246-£:. O/ arok, mR 本工作 p U2283 pET-28a衍生质粒， 带有 C219-£.co/!' aroA, mR 本工作 p U2287 pACYC184衍生质粒， 带有 T7启动子及 Ν240-α/ΌΑ 。,,, CmR 本工作 p U2289 pACYC 184衍生质粒， 带有 T7启动子及 NSAS-oroA/^,,, CmR 本工作
Ap, 氨卞青霉素； Cm, 氯霉素； Km, 卡那審素； R, 抗性； ， 删除； .·， 融合;
1.2培养基
LB培养基: 每升所含成分:
胰蛋白胨 10g 酵母提取物 5g
NaCl lOg
补足水并用 2M的 NaOH调至 pH7.0~7.5左右， 固体培养基加 1.5%的琼 脂粉。 使用前 15磅压力， 121 °C高温灭菌 20min后备用。
筛选氨苄青霉素抗性菌株在培养基中加 Ap至 50 g/ml。
筛选卡那霉素抗性菌株在培养基中加 Km至 25 g/ml。
筛选氯霉素抗性菌株在培养基中加 Cm至 25 g/ml。
限制性 M63 培养基: 13.6g/L KH2P04, 0.5mg/L FeS04-7H20 , 20mM (NH4)2S04, 0.4 %葡萄糖， ImM硫酸镁， 0.5mg/L维生素 Bl。
1.3试剂
限制性内切酶， T4DNA连接酶， TaqDNA聚合酶， DNA marker等购于 Takara生物公司。 考马斯亮蓝 G250, 烯醇式丙酮酸 (sigma), 莽草酸 -3-磷酸 (Amrehin教授赠送)； HisTrap HP kit (Amersham Biosciences), 羊抗兔 IgG (promega); 其余化学药品均为分析纯试剂。
1.4 遗传学操作
质粒 DNA 的制备、 限制性内切酶的消化、 连接反应、 Tris-硼酸 -EDTA 緩冲液水平琼脂糖电泳、聚丙烯酰胺凝胶电泳及 western杂交等按照标准方法 (Molecular Cloning: A Laboratory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.)， Cold Spring Harbor Laboratory Press, 1989)进行。
1.5 质粒构建
a. pKU2008、 pKU2009的构建
用引物 11 : 5， - CGGGATCCAGGTCCGAAAAAAAACGCCGAC 3' 和 引 物 12 ： 5' - CGGGATCCATGGAATCCCTGACGTTACA 3' 以 pKU2004为模板， ^"大肠 杆菌的 araA基因进行扩增， 连入 pET28a载体得到 pKU2008， 该质粒编码的 EPSPS为 N端带有 Ηίέ标签的融合蛋白。 将 pKU2008用 Ncol酶切， 自我連 接得到 pKU2009， 该质粒编码的蛋白为大肠杆菌野生型 EPSPS。
b. pKU2100 系列质粒 (编码 EPSPS的 C端肽链)的构建
用引物 13 : 5 '-TGAGTGACTGACTTTAAGAAGGAGATATAC3，和引物
14： 5-CGGGATCCTCACTGATTTTCAATTTCAACAC 3' 以 pKU2009为模板进行 PCR 扩增， 扩增产物经 BamHI酶切连入 pBR322的 EcoR V和 BamH I位点， 得 到质粒 pKU2100。 以 pKU2009为模板，分别以对应引物进行扩增得到编码大 肠杆菌 EPSPS的 C端的碁因 ,将扩增产物连接入 pKU2100的 Ncol和 Bamffl 位点， 分别得到质粒 pKU2102、 pKU2125、 pKU2126、 pKU2130和 pKU2262， 这些盾粒分别编码大肠杆菌 EPSPS的 C端肽链 (表 1， 图 3)。
c pACYC 184系列质粒 (编码 EPSPS的 N端肽链)的构建
以 pKU2009为模板， 分别以对应引物进行 PCR扩增， 得到编码大肠杆 菌 EPSPS的 N端的基因，将扩增产物连接入 pACYC 184的 EcoR V和 Bamffl 位点， 分别得到质粒 pKU2101、 pKU2125、 pKU2126、 pKU2130和 pKU2263 , 这些质粒分别编码大肠杆菌 EPSPS的 N端氨基酸序列 (表 1， 图 3)。
d. pET28a系列表达质粒的构建
将上述 b步骤中构建的 pBR322 系列质粒和 c步骤中构建的 pACYC 184 系列质粒分别用 Ncol和 BamHI酶切， 回收合适片段连接入 pET28a载体中， 得到用于表达 EPSPS的 N端和 C端的 pET28a系列表达质粒 (表 1)。
e. pACYC-T7系列表达质粒的构建
将用于表达 EPSPS的 N端的 pET28a质粒分别用 Bgl II和 Sal I酶切， 回 收所需片段连接入 pACYCl 84载体的 BamHI 和 sal I位点得到 pACYC-T7系 列表达质粒 (表 1， 图 3)。
f. pET28a系列表达质粒 (用于表达 C端 His-taq融合蛋白）的构建
以 pKU2009为模板，分别以对应引物进行扩增得到编码大肠杆菌 EPSPS 相应部分的基因， 将扩增产物用 Xhol和 Ncol酶切后连接入 pET28a载体中 得到 pET28a系列表达质粒， 该系列质粒所编码的 EPSPS的 C端融合有 6个 组氨酸以便于利用镍柱纯化蛋白（图 3)。
g.用于菌株突变的质粒 pKU2229的构建
以大肠杆菌 BL21(DE3)的染色体为模板， 用引物进行 PCR扩增反应， 得 到其 arok基因上游长约 600bp的片段， 将其连入 pBlueScript (stratagene)的 BamHI和 Hindlll位点得 质粒 pKU2223。 以质粒 pBlueScript为模板， 用引 物进行 PCR扩增反应， 得到长约 900bp的 b/α基因， 将其连接入 pBlueScript (stratagene)的 Hindlll和 EcoRI位点得到质粒 pKU2224。以大肠杆菌 BL21(DE3) 的染色体为模板，用引物进行 PCR扩增反应，得到其 aroA基因下游长约 500bp 的片段， 将其连入 pBlueScript (stratagene)的 EcoRI 和 Sail 位点得到质粒 pKU2225。 酶切回收 pKU2225中的片段连接入 pKU2223的 EcoRI和 Sail位 点得到质粒 pKU2227。酶切回收 pKU2224中的片段连接入 pKU2227的 Hindlll 和 EcoRI位点得到质粒 pKU2228。 酶切回收 pKU2228中的 BamHI和 Sail间 的片段连接入 pK03载体中得到质粒 pKU2229。
以上所有构建质粒均测序保证其序列正确。
1.6 突变菌株 BA-的构建.
5 利用 pK03来源质粒 pKU2229将 BL21(DE3)的 awk基因替换为导致氨. 苄青霉素抗性的 b/β基因。 具体步骤为： 将 pKU2229转化入 BL21(DE3)中， 挑取一个转化子稀释后涂布于带有 Ap和 Cm抗生素的 LB固体平板上， 43 "G : : 过夜培养， 由于 pK03 的复制子在 43 °C不能正常起始复制， 因此 pKU2229 只有重组到染色体上， 菌株才能在 43 °C含有 Ap和 Cm抗生素的平板上生长。 10 挑取一个重组后的菌落， 稀释后涂布到含有 5 %蔗糖的仅含有 Ap抗生素的 LB培养基上， 过夜培养。 由于 pK03带有 c B基因， 该基因编码的蛋白质 分解蔗糖后对细菌产生毒性，导致带有 基因的细菌不能在含有 5 %蔗糖 的培养基上生长， 因此在含有 5 %蔗糖及 Ap抗生素的 LB培养基上生长的菌 落， 又一次发生同源重组， 使 araA基因和 ^c B基因丢失。 挑取几个菌落， 15 划线于 Cm平板和 M63平板， 确定 araA基因确实被删除， 提取其总 DNA, 进行 PCR扩增反应以最终确定 b/α基因替换掉 aro 基因。
1.7体内互补实验 转入大肠杆菌 raA基因缺陷的菌株 AB2829中， 然后将其划线于 M63 «·) 0 性固体培养培养基上， 过夜培养检测其生长情况。
1.8 生长曲线实验
将带有质粒 pKU2004、 pKU2006和 pKU2007的大肠杆菌 ara 基因突 ί 菌株 ΑΒ2829接种于 LB液体培养基中过夜培养， 取菌液 4000rpm离心 3分 钟， 用 0.9%生理盐水悬浮， 再次离心， 弃去上清， 并用生理盐水重新悬浮， 5 接入含有 0、 50和 100 mM草甘膦的液体培养基中，初始接菌量为 OD6(X) 0.04, 37°C 过夜培养， 并定时测光吸收 (OD6。。)。
1.9 蛋白的表达与纯化
将带有目的质粒的 菌接入含有相应抗生素的 LB液体培养基中， 在 37°C摇床培养至 OD6。。 0.75左右， 加入终浓度为 0.5 mM的 IPTG, 15°C过夜 0 培养。 将菌液在 4QC、 5000rpm离心 10分钟收集菌体， 按体积比 10: 1重悬至 緩冲液 A(50 mM Tris-HCl (pH 7.8), 0.4 mM DTT)中， 超声波破碎菌体， 4°C, 8000rpm离心 60分钟。
将离心后的上清液用 HisTrap HP kit (Amersham Biosciences)按照使用说 明进行蛋白纯化。 纯化后的蛋白利用 Millipore Biomax membrane (10 kDa)浓 缩于緩沖液 A中， 4QC保存。
5 1.10 EPSPS多克隆抗体的制备
将带有质粒 pKU2008的 BL21(DE3)菌株按以上步骤进行表达纯化，最后 将蛋白浓缩， 重悬于 PBS緩沖液中。 以此蛋白为抗原， 对兔子进行免疫反应，. 经过四次反应， 一次加强反应后取血清 (此过程由中科院遗传所完成）， 用 ELASA检测抗体效价后备用。
10 1.11 聚丙烯酰胺凝胶电泳
SDS -聚丙烯酰胺凝胶电泳凝胶的上层浓缩胶浓度为 5 %，下层分离胶浓 度一般为 16 %。 Native -聚丙烯酰胺凝胶电泳凝胶的浓度为 10 %。 凝胶、 电 泳緩冲液的配置及电泳方法参见 Sambrook等， 出处同上。
1.12 圆二色性光谱 (CD spectra)的测定
15 远紫外和近紫外 CD光谱的测量在一台接有恒温水浴循环控制仪的 Jobin
Yvon CD6上进行。 每条曲线是 4 次测量的平均。 测量温度为 8QC。 进行近紫 夕卜圓二色性光语测定时使用 1-cm pathlength cylinder quartz cuvette, 而远紫夕卜 圆二色性光谱测定时使用 0.1 -mm pathlength cylinder quartz cuvette。
1.13 EPSPS片段的蛋白免疫印迹实验
0 转膜及免疫印迹反应方法参见 Sambrook等， 出处同上。 简要步骤为： 蛋 白经过 16 % SDS -聚丙烯酰胺凝胶电泳后转移至硝酸纤维素膜上， 将膜与 ； ' 1 :2000稀释的兔的多克隆抗体杂交， 抗体抗原复合物进一步与结合有辣根过 氧化物酶的第二抗体 -羊抗兔 IgG(promega)反应形成复合物。 显色反应采用 辣根过氧化物酶分解 DAB (华美生物工程公司）的方法， 具体操作见说明书。 5 1.14 EPSPS活性测定
酶活性的测定
(1)取 95 μΐ底液 (在反应体系中浓度为 50 mmol/L HEPE Sbuffer (pH 7.5), 1 mmol/L PEP, 1 mmol/L S3P), 于 28 °C的培养箱中预热 5 min。
(2)加 5 μΐ酶液， 在 28 °C培养箱中放置 1一 20 min (以酶量活性决定时间 0 长短)， 加 800 μΐ MG/AM/NP混合 1 min。
(3)加 100 μΐ的 34 %的柠檬酸钠溶液，混合后半小时于 660 nm的分光光 度计上测定 OD值。
1.15 体外互补实验
将 EPSPS的 N端和 C端分别在 BA-中表达， 离心收集菌体后超声波破 碎， 电泳检测目的蛋白是否主要形成包涵体从而聚集在沉淀中。 收集沉淀， 5 先用緩冲液 B(50 mM Tris-HCl (pH 7.8), 1 mM EDTA, 0.05%的 Triton-100)洗 三次， 再用 1M氯化钠溶液洗三次， 再用緩冲液 C(50 mM Tris-HCl (pH 7.8), 1 mM EDTA, 1M尿素）洗三次， 最后用蒸馏水洗三次。 将洗涤完毕后的沉淀加 入还有 8M尿素的緩沖液 A.中， 充分溶解后离心取上清转入透析袋中。 先用 还有 2M尿素的緩沖液 D(50 mM Tris-HCl (pH 7.8)， 1 mM GSH, 0.5mM GSSG) 0 透析过夜， 再用緩沖液 D透析 36小时， 最后用 PEG 12000浓缩。 复性产物通 过电泳、 免疫反应及酶活性检测加以分析。
2.大肠杆菌 EPSPS的片段互补结果
2.1 体内功能互补实验
为了检测 EPSPS能否体内表达形成片段互补， 分别将编码 EPSPS的 N 5 端肽链的基因构建在 pACYC184载体上， 而将编码 EPSPS的 C端肽链的基 因构建在 pBR322载体上 (具体构建过程见方法)。然后将这些质粒分别转入大 肠杆菌的 arok基因突变菌株 AB2829中， 通过检测其是否能在 M63限制性 培养基上生长，来确定该基因编码的蛋白片段是否仍具有 EPSPS活性。结果; 仅带有编码 N端或 C端 EPSPS编码基因的 AB2829不能在 M63培养基上生 0 长。
由于 pACYC184和 pBR322质粒分别具有复制子 pl5A and ColEl , 因此 : 这两个质粒可以在同一个细菌中共表达。我们于是将编码 EPSPS的 N端肽链 的 pACYC184系列质粒， 和在同一个位点分拆的编码 EPSPS的 C端肽链的 pKU2100系列质粒，共转化入大肠杆菌的 oA基因突变菌株 AB2829中，将 5 其划线于 M63 限制性培养基平板上 37QC培养。 16 小时后， 分别带有编码 EPSPS的N218/C219(pKU2101/pKU2102)和N227/C228 (pKU2125/pKU2138)
' ' 质粒的 AB2829可以在限制性培养基上生长， 而带有其它三对质粒 pKU2126/ pKU2137(N234/C235), pKU2162/pKU2163(N240/C241)和 pKU2110/pKU2130 (N245/C246)的 AB2829 不能在 M63 培养基上生长。 这说明， EPSPS 片段 0 (N218/C219和 N227/C228)在体内分别表达时， 可以互补形成 EPSPS活性，： 其它三对 EPSPS的片段在体内不能互补 EPSPS活性。 2.2 EPSPS片段复合体的纯化和检测
为了确定是否因为编码 EPSPS片段的基因在体内发生重组而导致合成全 长 EPSPS并恢复 EPSPS活性，将蛋白片段共纯化出来，体外检测酶活性及蛋 白片段大小， 以确定 EPSPS活性恢复确实是因为片段互补的原因。 为此， 构. 建了用于表达 EPSPS片段的质粒， 其中 N端构建于 pACYC184载体上， 而 蛋白 C端构建于 pET28a栽体上， 都由 T7启动子起始转录表达， 其中 EPSPS 的 C端肽链的 C端融合有 6个组氨酸 (质粒构建见方法见上文)。 如果 EPSPS 的两个蛋白片段能够体内重建，形成天然 EPSPS的结构，那么分拆后的 EPSPS 的 N端肽链， 将与相应的 EPSPS C端肽链结合， 并且该复合体可以被镍柱所 吸附而共纯化出来。 为了消除细菌染色体上 aroA基因编码的 EPSPS的影响， 将大肠杆菌 BL21体内编码 EPSPS的 araA基因， 替换为编码 β-内酰胺酶的 基因， 得到 oA基因删除菌株 ΒΑ―。 将相应的两个系列的质粒， 转化入 BA-菌株中共表达。 首先将其划线于涂有 IPTG的 M63平板上， 过夜培养检 测其体内互补情况。 结果与前面一致， 带有 N218/C219、 N227/C228 片段编 码基因的 BA 可以在 M63限制性培养基上生长； 带有其它三对片段编码基 因的 BA―， 不能在 M63限制性培养基上生长。 将 EPSPS的 N端肽链和 C端 肽链在 BA—中分别表达或共表达， SDS-PAGE检测， 结果表明它们均能正者 表达。 用 HisTrap HP kit对共表达的蛋白进行纯化， 纯化后的蛋白 SDS-聚丙 烯酰胺凝胶电泳后， 用制备好的 EPSPS的抗体进行检测 (具体步骤见方法)。 ' 结果如图 4所示， 纯化后的蛋白中不仅有 EPSPS的 C端肽链， 而且也有其 应的 N端肽链。 这些蛋白片段都与预计的片段大小一致， 这说明在体内表达 的 EPSPS的片段， 可以互补形成复合体从而被共纯化出来。 从图中也看出， 共表达的蛋白无论是纯化前还是纯化后都没有全长的蛋白， 这表明体内没有 发生重组， 因此没有形成全长 EPSPS基因。 活性试验也显示， 纯化出的蛋白 都具有 EPSPS活性。 以上的结果表明， EPSPS活性可以由在体内分别表达的 肽链发生片段互补而形成。
EPSPS的 N端肽链和 C端肽链虽然在体内表达量可能有所差异，如 N227 的表达量远大于 C228的表达量， 但是纯化后 N端和 C端蛋白片段的含量几 乎都是 1 : 1 的关系。 从图中也可以看出， 纯化出的 N234/C235、 N245/C246 量远远少于其它三个共纯化的 EPSPS, 这可能是其不能在体内互补 EPSPS活 性以使 AB2829在限制性培养基上生长的原因。 虽然 N240/C241复合体纯化 出的量甚至多于 N218/C219, 但是它却不能互补 AB2829在限制性培养基上 的生长， 这是因为其 EPSPS活性比较低的原因
2.3测定酶活性
为了对 N218/C219、 N227/C228这两个能重建酶活性的复合体进行进一 步的研究， 将上一步用 HisTrap HP kit纯化后 N218/C219、 N227/C228及全长 的 EPSPS用 Sephadex-G75柱进行进一步纯化， 纯化结果表明 N218/C219、 N227/C228及全长的 EPSPS具有同一个洗脱峰， 表明其分子量及结构大致相 同。 随后测定了这三个蛋白的酶活性及对底物的 Km值， 结果如表 2， 从表 中可以看出，片段互补后的 EPSPS复合体 N218/C219及 N227/C228的酶的活 力分别约为全长 EPSPS的 70%和 64 % ,而对底物的亲和力并没有太大的改变， 这表明重建的 EPSPS结构比较稳定。
表 2. EPSPS及重建后 EPSPS的酶学性质
醉 (μΜ) 比活力 b( mol min"1 mg"1) Km [PEP] \μΜ) Km [S3P] d
EPSPS 44土 2 14 ± 3 45 ± 1
218/219 31 ± 3 18 ± 3 49 ± 3
227/228 28 ± 4 19 ± 3 49 ± 6
以上结果为两次独立实验， 每次实验至少有三个平行。
酶比活力测定 PEP和 S3P的浓度均为 1.0 mM 。
c测定 Km[PEP]时， S3P浓度不变为 1 mM, PEP浓度从 50到 200 μΜ。
d测定 KJS3P]时， PEP浓度不变为 1 mM , S3P浓度从 50到 200 μΜ。
2.4 圓二色性光谱 (CD spectra)的分析
为了确定， 片段互补重建所得的 EPSPS是否与野生型 EPSPS结构一致， 对 EPSPS 及复合体 N227/C228 进行了圆二色性光谱分析。 EPSPS 与 N227/C228的远紫外 CD 光谱基本没有显著差异 (图 5) , 这意味着它们的二 级结构几乎是相同的。 但近紫外 CD 光谱之间有所差异 (图 5)， 表明重建的 EPSPS与野生型 EPSPS结构还是有所差异。
2.5 大肠杆菌突变型 EPSPS的体内互补
为了检测突变后的 EPSPS片段是否可以在体内进行功能互补， 分别构建 了质粒 pKU2105和 pKU2333， 它们分别编码大肠杆菌 EPSPS的两个 N端片 段 N218-G96A和 N227-G96A。 将它们与相应的编码 C219和 C228的质粒分 别转入大肠杆菌 arok基因的突变菌株 AB2829(aro A―， 来源于耶鲁大学))中， 在带有不同浓度草甘膦的 M63限制性培养基中检测其生长情况，结果如图 6。 从图中可以看到， 在未加草甘膦的 M63培养基中， 带有不同质粒的 AB2829 均生长良好， 没有明显的差异。 在含有 50 mM草甘膦的 M63培养基中， 表 达有突变的 EPSPS 全酶 (EPSPS-G96A)或突变的互补片段 (N227-G96A/C228 和 N218-G96A/C219)的菌株生长较好， 而表达有野生型 EPSPS或野生互补片 段的菌株生长被阻遏。 表达有 EPSPS-G96A或 N227-G96A/C228的菌株在含 有 lOO mM草甘膦的 M63培养基中仍旧生长良好，而表达有 N218-G96A/C219 的菌株生长稍差，这可能是在体内 N2227-G96A/C228复合体更容易形成的原 因。
2.6 体夕卜 EPSPS 重建
蛋白片段能在体内互补形成有活性的复合体， 那么在体外同样有可能互 补形成有活性的复合体。 为此， 分别表达了 EPSPS的 Ν端肽链 N218和 C端 肽链 C219。 EPSPS片段的大量表达形成包涵体， 对包涵体进行初步纯化后， ' 将它们一起复性 (操作步骤见方法)。复性结束后对其进行电泳分析，结果见图 7。 从图 7a中我们可以看出， EPSPS的各个片段出现在预计的位置， 并没有 全长的 EPSPS。 当将其体外共复性之后， native - PAGE结果可以出现与野 ' 生 EPSPS—致的条带， 将单独复性后的 N218和 C219混合在一起时并没有 此带， 这说明体外 EPSPS共复性后可以互补形成类似于野生 EPSPS的结构。 活性实验也表明， N218/C219共复性可以互补形成 EPSPS活性， 而单独复性 后的 N218和 C219混合在一起并不能互补形成 EPSPS活性。
3. EPSPS的片段互补与结构的关系
3.1 所用的菌株和质粒见下表。
表 3. 用于本研究中的细菌菌株和质粒 _； 菌株 /盾粒 相关特点 来源 /参考文献
大肠杆菌菌株
DH5a supE44AlacU 169hsdR 17recA 1 gyrA96thi- 1 re 1 A 1 Hanahan D., J ol Biol
1983; 166:557-80
AB2829 aroAi^ 耶鲁大学
质粒
pUC 18 ColEl , /acZ', Ap' Norrander等， 1983 pBluscript-SK ColE 1 , / cZ'( Ap' Stratagene pET-28a ColEl, 表达质粒， KmR Novagen pACYC184 CmR Chang, A. C.等 1978. J
Bacteriol 134:1141-56 pBR322 ColEl, ApR Schaeffer, F.等， 1982 EMBO
J.1,99-105
p U2005 pUC18衍生质粒， 带有 araA .c„,,， ApR 本工作
p U2006 pACYC184衍生质粒， 带有 a/OA£∞,,， CmR 本工作
pKU2007 pACYC184衍生质粒， 带有
Figure imgf000021_0001
- G96A, CmR 本工作
p U2008 pET-28a衍生质粒， 带有 £.co/ aroA, mR 本工作
pKU2009 pET-28a衍生质粒， 带有 £.co/ araA , KmR 本工作
p U2010 pET-28a衍生质粒， 带有 £co" oroA-G96A, mR 本工作
pKU2011 pET-28a衍生质粒， 带有 £.co/!' <3OA-G96A, KmR 本工作
p U2100 PBR322衍生质粒， 带有 a/OA,:.ra/,， ApR 本工作
pKU2101 pACYC184衍生质粒， 带有 N218-araAE.c。,,， CmR 本工作
p U2102 pBR322衍生质粒， 带有 CS
Figure imgf000021_0002
ApR 本工作
p U2103 pACYC184衍生质粒， 带有 N238-araA£.c。/,， CmR 本工作
p U2104 pBR322衍生质粒， 带有 C239-iJroA£∞,,， ApR 本工作
p U2110 pACYC184衍生质粒， 带有 N245-araA£c。/,， CmR 本工作
p U2111 pACYC184衍生质粒， 带有 N259-aAOA£c。„, CmR 本工作
pKU2124 pBR322衍生质粒， 带有€225-αΌΑ£. /,, ApR 本工作
p U2125 pBR322衍生质粒， 带有 C228-aroA£c。/,, ApR 本工作
p U2126 pBR322衍生质粒， 带有
Figure imgf000021_0003
ApR 本工作
pKU2129 pACYC184衍生质粒， 带有 N3卜。 TOA£i：。, ,， CmR 本工作
pKU2130 pBR322衍生质粒， 带有 C246-araA£C。/,, ApR 本工作
p U2131 pBR322衍生质粒， 带有 C260-aroA£c。/,, ApR 本工作
p U2135 pBR322衍生质粒， 带有 C299-aTOA 。/,， ApR 本工作
pKU2136 pACYC184衍生质粒， 带有 NZSS-araA/^/,, CmR 本工作
p U2137 pACYC184衍生质粒， 带有
Figure imgf000021_0004
CmR 本工作
pKU2138 pACYC184衍生质 ， 带有 Ν227-β·οΑ£∞/,, CmR 本工作
p U2139 pACYC184衍生质粒， 带有 N224-。/OA£c。,,, CmR 本工作 pKU2148 pACYC184衍生质粒， 带有 N165-。/OA£.c。,,.， CmR 本工作 pKU2149 pBR322衍生质粒， 带有 C^^aroAs.^, ApR 本工作 p U2150 pACYC184衍生质粒， 带有 NS iwoA . , CmR 本工作 pKU2151 pBR322衍生质粒， 带有 C372-a/OAt..c„/„ ApR 本工作 pKU2290 pACYC184衍生质粒， 带有 ~N6T-aroA/.coii, CmR 本工作 p U2291 pACYC184衍生质粒， 带有 73-oroAf.en//, CmR 本工作 pKU2292 pACYC184衍生质粒， 带有 N84-aroAt..c。"， CmR 本工作 p U2293 pACYC184衍生质粒， 带有 N
Figure imgf000022_0001
CmR 本工作 pKU2294 pACYC184衍生质粒， 带有 N154-aroA/j.∞/,， CmR 本工作 p U2295 pACYC184衍生质粒， 带有 W82-amA, '(,ii, CmR 本工作 p U2296 pACYC184衍生质粒， 带有 Νΐδ αΌΑ,;.™,,, CmR 本工作 p U2297 pACYC184衍生质粒， 带有 6-aroAR.coii, CmR 本工作 pKU2298 pACYC184衍生质粒， 带有 N383-ijroA£c。/,， CmR 本工作 p U2299 pB 322衍生质粒， 带有 C68-araA£∞/,:, ApR 本工作 pKU2300 pBR322衍生质粒， 带有 C74-araA c。,,.， ApR 本工作 pKU2301 pBR322衍生质粒， 带有 C86-CTOA£C。,,.， ApR 本工作 pKU2302 pBR322衍生质粒， 带有 C 105-aroAi;.cnli, ApR 本工作 p U2303 pBR322衍生质粒， 带有 C155-aroA£c。/,， ApR 本工作 pKU2304 pBR322衍生盾粒， 带有 CI 83-aOA/;.C0,, ApR 本工作 pKU2305 pBR322衍生质粒， '带有 C185-aTOA£C„,,, ApR 本工作 p U2306 pBR322衍生质粒， 带有 C377-araA£c„", ApR 本工作 p U2307 pBR322衍生质粒， 带有 C384-aroA .c„/,, ApR 本工作
Ap, 氨卞青審素； Cm, 氯審素； R，抗性。
3.2培养基
参看本实施例 1.2节。
. 3.3试剂
限制性内切酶， T4DNA连接酶， DNA聚合酶， DNA marker等购于 Takara 生物公司。 其余化学药品均为分析纯试剂。
3.4 遗传学操作
质粒 DNA的制备、 限制性内切酶的消化、 连接反应、 Tris-硼酸 -EDTA 緩沖液水平琼脂糖电泳、 按照标准方法进行 (Maniatis 等， 1982)。
3.5 质粒构建 a. pBR322 系列盾粒 (编码 EPSP合酶 C端肽链)的构建
以 pKU2009(大肠杆菌 arok基因）为模板， 分别以对应引物 (进行扩增 得到编码大肠杆菌 EPSP合酶 C端的基因， 将扩增产物连接入 pKU2100的 Ncol和 BamHI位点， 得到编码 EPSP合酶 C端肽链系列质粒 (表 3)。
b. pACYC 184系列质粒 (编码 EPSP合酶 N端肽链)的构建
以 pKU2009或 pGMO为模板， 分别以对应引物进行扩增得到编码大 肠杆菌 EPSP合酶 N端的基因， 将扩增产物连接入 pACYC 184的 EcoR V和 BamHI位点， 分别得到编码 EPSP合酶 N端肽链系列质粒 (表 3)。
以上所有构建质粒均测序保证其序列正确。
3.6体内互补实^ r
将 3.5 a中构建的 pBR322系列质粒和 b中构建的 pACYC1 184系列质粒 分别转入或相对应两个质粒共转入大肠杆菌 flroA基因缺陷的菌株 AB2829 中， 然后将其划线于 M63限制性固体培养培养基上， 过夜培养检测其生长情 况。
3.7 结果
基于大肠杆菌 EPSPS的结构一共设计了 21个分拆位点， 进行了 EPSPS 片段互补研究。 有三个分拆位点在 α螺旋上， 其中 N31/C32和 N245/C246都 不能互补大肠杆菌 araA基因突变菌株 ΑΒ2829在限制性培养基上的生长， 而 N105/C106 互补情况较差； 有三个拆分位点在 β折叠上， 其中 N73/C74和 N238/C239不能互补 EPSP酶活性， 而 N224/C225可以互补 AB2829在限制 性培养基上的生长； 有 5个在 β折叠或 α螺旋之间的连接区域上， 其中仅有 N165/C166不能互补 ΑΒ2829在 Μ63培养基上的生长，而其它四对 EPSPS的 片段都可以互补 EPSPS活性； 分拆位点在两个折叠单元之间的有 6个， 其中 仅有 N234/C235不能互补 EPSPS的活性， 其它五对片段都能互补 AB2829在 M63培养基上的生长， 而且互补情况较好。
表 4 大肠杆菌 EPSPS片段互补
EPSPS片段 分拆位点所在区域 互补情况
N端 N端 C端 N端 +
N31 C32 α螺旋
N67 C68 两个 β折叠之间
N73 C74 β折叠 N85 C86 折叠单元 3和 4之间 - - + + +
N104 C105 α螺旋 - - - + / -
N154 C155 折叠单元 4和 5之间 - - + + +
N165 C166 α螺旋与 β折叠之间 - - - -
N182 C183 α螺旋与 β折叠之间 - - +
N184 C185 α螺旋与 β折叠之间 -一 - + +
N218 C219 两个 β折叠之间 - - ― + +
N224 C225 β折叠 - - +
N227 C228 折叠单元 3和 5之间 - - ― + + +
N234 C235 折叠单元 3和 5之间 - - - -
N238 C239 β折叠 - - - -
N240 C241 两个结构戈之间 -一 - -
N245 C246 α螺旋 - - - -
N259 C260 β折叠 - - - +
N298 C299 折叠单元 2和 6之间 - - - + + +
N371 C372 折叠单元 1和 6之间 - - - + + +
N376 C377 折叠单元 1和 6之间 - - 一 + + +
N383 C384 α螺旋与 β折叠之间 - - - +
-， 不能生长； + , 可以生长<
实施例 2 恶臭假单胞菌草甘膦抗性 EPSPS的片段互补
利用恶臭假单胞菌 CGMCC 0739的草甘膦抗性 EPSPS基因进行片段互补 实验。 所用的质粒， 培养基， 菌株， 以及实验步骤都参见实施例 1。 通过在 含有 lOOmM草甘膦的培养基上生长来检测重建的酶活性。
表 5 恶臭假单胞菌草甘膦抗性 EPSPS的片段互补
EPSPS片段 分拆位置 互补情况
N208/C209 α螺旋与 β折叠之间
N214/C215 β折叠中
N219/C220 折叠单元之间
N222/C223 折叠单元之间
N224/C225 折叠单元之间 + + ,ε-OIV VVV ODV VVO丄 IVI VIO £)丄 V 300 IV - '■ (\o ^) £ZZDdd S-DVV VV丄 VVV Oil丄：) 0 ODD 丄丄 VVD丄0 丄 VD ODD - :(lH^e) ZZZ dd
+ £ZZD/ZZZ^ -DV VVO IVO丄 VI VIO IVV OOO 330 OIV 330丄 VO ~ :(IOON) (gOZZ3dd t£—0V丄 V l丄 丄 VV VV丄 VVV OLI IV 丄: IVO ODD'S :(lH^8) ,ΖόΙΖ ^
+ +0223/61
,£-00330 VVV 311丄 VI丄丄丄 OVO OLV DDD丄 V — ： (io^NO.SSRDdd -o o i 丄丄 vo 丄丄丄 ov丄 v 丄丄 v 丄： Ό丄 VO DOJ- -(m^^ zHdd -0 VVO丄 VI VV VDD IVV OVO OIV 330 IV ~ ： ( ^Νΰ^όΟΖΟ^ -D DVV VDD VVV Dll：)丄 D VDO 0V3丄 IVD 003 -'S :(lH^e) t£803Ndd
+ 603O/803M
。丄 ^^Ι ^Τ^
( -3V OVO VOX VVD VOO 0丄丄 丄丄 333 IVO ODD ~ :(lH^e)t£3dd
4 CO V3V IVV DVV丄 OV VVD丄 OV VVO丄 V OLD VOL— ' S ： 9M.^d
：土^ ^Ι ^τ^
LZZD/9ZZR nZD/ΠΖΝ.
£ZD/i£ZR
6TZZ00/S00ZN3/X3d CCSC90/900Z OAV ρρΝ2243' (BamHI): 5'- CGG GAT CCT CAA TAT ACA TTC CCG GCT TTG-3' ppC2255' (Ncol): 5，- CAT GCC ATG GAT GAA ACG AAA ATG CAA CG-3'
N233/C234
ppN2333' (BamHI): 5'- CGG GAT CCT CAG GTG TAT CGT TGC ATT TTC G-3' ppC2345'(NcoI)： 5，- CAT GCC ATG GTA GAA GGC GAC TGG AGC G-3'
N234/C235
ppN2343' (BamHI): 5，- CGG GAT CCT CAT ACG GTG TAT CGT TGC ATT TTC- ppC2355' (Ncol): 5，- CAT GCC ATG GAA GGC GAC TGG AGC GGT GG-3'
N236/C237
ppN2363' (BamHI): 5'- CGG GAT CCT CAG CCT TCT ACG GTG TAT CGT TG-3' ppC2375'(NcoI)： 5'- CAT GCC ATG GAC TGG AGC GGT GGT GCT TT-3'

Claims

权利要求书
1. 5-烯醇式丙酮酰莽草酸 -3-磷酸合酶 (EPSPS)的蛋白片段， 其是选自 5 EPSPS的蛋白片段对的一种片段， 组成所述片段对的两种片段可以连接成全 长 EPSPS, 并且这两种片段不借助任何连接结构可以通过互补而重建 EPSPS 活性。
2. 权利要求 1的片段， 其中所述片段对的分割点位于 EPSPS的选自下 列的结构中： 折叠单元之间的连接区中、 α螺旋与 β折叠之间的连接区中、 0 两个 β折叠之间、 β折叠中、 或 α螺旋中， 优选位于折叠单元之间的连接区 中。
3.权利要求 1或 2的片段，其中所述 EPSPS是野生型 EPSPS或其添加、 缺失、和 /或取代一或多个氨基酸残基所得的 EPSPS活性变体，优选是大肠杆 菌野生型 EPSPS或其草甘膦抗性 EPSPS活性变体，或者优选是恶臭假单胞菌 5 草甘膦抗性 EPSPS。
4. 权利要求 1的片段， 其是选自大肠杆菌 EPSPS的以下片段对的一种 片段： N67/C68, N85/C86, N104/C105, N154/C155, N182/C183, N184/C185, N218/C219, N224/C225, N227/C228, N259/C260, N298/C299, N371/C372, N376/C377, N383/C384, 或是选自恶臭假单胞菌 CGMCC 0739 EPSPS的以下 0 片段对的一种片段： N208/C209 , N214/C215 , N219/C220, N222/C223,
Figure imgf000027_0001
' 5. 一种核酸分子， 其编码权利要求 1 - 4之一的蛋白片段。
6. 一种表达载体， 其携带权利要求 5所述核酸分子。
7. —种细胞， 其包含权利要求 5所述核酸分子， 或者权利要求 6所述表 5 达载体。
8. 一种重建 EPSPS的方法， 包括在无任何连接结构存在的条件下， 利 用权利要求 1 - 4之一的蛋白片段， 或权利要求 5的核酸分子， 或权利要求 6 的表达载体来童建 EPSPS活性。
9. 一种拆分 EPSPS或 EPSPS核酸分子的方法， 包括在无任何连接结构 0 存在的条件下， 拆分出权利要求 1-4之一所述的蛋白片段， 或权利要求 5所 述的核酸分子。
PCT/CN2005/002219 2004-12-16 2005-12-16 Reconstitution de l’activite de la 5-enolpyruvylshikimate-3-phosphate synthase par complementation de fragments WO2006063533A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
DE602005024442T DE602005024442D1 (de) 2004-12-16 2005-12-16 Wiederherstellung der aktivität von 5-enolpyruvylshikimat-3-phosphat-synthase durch fragmentkomplementierung
AT05819606T ATE486127T1 (de) 2004-12-16 2005-12-16 Wiederherstellung der aktivität von 5- enolpyruvylshikimat-3-phosphat-synthase durch fragmentkomplementierung
EP05819606A EP1840210B1 (en) 2004-12-16 2005-12-16 Reconstitution of 5-enolpyruvylshikimate-3-phosphate synthase activity by fragment complementation
US11/721,856 US8936925B2 (en) 2004-12-16 2005-12-16 Reconstitution of 5-enolpyruvylshikimate-3-phosphate synthase activity by fragment complementation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN200410102289.4A CN1789412B (zh) 2004-12-16 2004-12-16 利用片段互补技术重建5-烯醇丙酮酰莽草酸-3-磷酸合成酶活性
CN200410102289.4 2004-12-16

Publications (1)

Publication Number Publication Date
WO2006063533A1 true WO2006063533A1 (fr) 2006-06-22

Family

ID=36587548

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2005/002219 WO2006063533A1 (fr) 2004-12-16 2005-12-16 Reconstitution de l’activite de la 5-enolpyruvylshikimate-3-phosphate synthase par complementation de fragments

Country Status (6)

Country Link
US (1) US8936925B2 (zh)
EP (1) EP1840210B1 (zh)
CN (1) CN1789412B (zh)
AT (1) ATE486127T1 (zh)
DE (1) DE602005024442D1 (zh)
WO (1) WO2006063533A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2702157A1 (en) * 2011-04-29 2014-03-05 Keygene N.V. Glyphosate resistance enhancement
PL222067B1 (pl) * 2011-08-11 2016-06-30 Inst Biotechnologii I Antybiotyków Kaseta ekspresyjna, zastosowanie kasety ekspresyjnej, wektor ekspresyjny, komórka prokariotycznego gospodarza zawierająca wektor ekspresyjny, szczep bakteryjny oraz sposób wytwarzania polipeptydu
US9540654B2 (en) * 2012-02-01 2017-01-10 Dow Agrosciences Llc Synthetic brassica-derived chloroplast transit peptides
CN104630245A (zh) * 2013-11-08 2015-05-20 北京大学 5-烯醇式丙酮酰-莽草酸-3-磷酸合酶的环式重排变体和互补片段对
CN106517702B (zh) * 2015-07-26 2021-03-12 通化鑫鸿新材料有限公司 一种去除含镉污水污染物的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2117991U (zh) 1992-03-20 1992-10-07 兰溪市金笔厂 一种弹簧复合书写笔
WO2000071701A1 (en) * 1999-05-24 2000-11-30 New England Biolabs, Inc. Method for generating split, non-transferable genes that are able to express an active protein product
WO2004046359A2 (en) 2002-11-20 2004-06-03 Icon Genetics Ag Method of controlling gene expression in plants or plant cells
WO2004046360A2 (en) 2002-11-20 2004-06-03 Icon Genetics Ag Method of controlling cellular processes in plants

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1268572A (zh) * 1999-03-31 2000-10-04 中国农业科学院生物技术研究中心 一种5－烯醇丙酮酰－莽草酸－3－磷酸合成酶基因的合成方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2117991U (zh) 1992-03-20 1992-10-07 兰溪市金笔厂 一种弹簧复合书写笔
WO2000071701A1 (en) * 1999-05-24 2000-11-30 New England Biolabs, Inc. Method for generating split, non-transferable genes that are able to express an active protein product
WO2004046359A2 (en) 2002-11-20 2004-06-03 Icon Genetics Ag Method of controlling gene expression in plants or plant cells
WO2004046360A2 (en) 2002-11-20 2004-06-03 Icon Genetics Ag Method of controlling cellular processes in plants

Non-Patent Citations (40)

* Cited by examiner, † Cited by third party
Title
"Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
ANDERSON, K. S. ET AL., JBIOL CHEM, vol. 265, no. 10, 1990, pages 5567 - 72
BICKEL, PHYTOCHEMISTRY, vol. 17, 1978, pages 119 - 124
BIOCHEMISTRY, vol. 39, no. 9, 2000, pages 2164 - 73
BRAUN, M. ET AL., JBACTERIOL, vol. 185, no. 18, 2003, pages 5508 - 18
CHEN L ET AL: "Herbicide resistance from a divided EPSPS protein: the split Synechocystis DnaE intein as an in vivo affinity domain.", GENE., vol. 263, no. 1-2, January 2001 (2001-01-01), pages 39 - 48, XP002980420 *
CHEN, L. ET AL., GENE, vol. 263, no. 1-2, pages 39 - 48
CHIN H G ET AL: "Protein trans-splicing in transgenic plant chloroplast: Reconstruction of herbicide resistance for split genes.", PNAS., vol. 100, no. 8, 15 April 2003 (2003-04-15), pages 4510 - 4515, XP002967418 *
CHIN, H. G ET AL., PROC NATL ACAD SCI USA, vol. 100, no. 8, 2003, pages 4510 - 5
DANIELL ET AL., NAT BIOTECHNOL, vol. 16, no. 4, 1998, pages 345 - 8
DELLA-CIOPPA ET AL., BIO/TECHNOLOGY, no. 5, 1987, pages 579 - 584
ESCHENBURG, S. ET AL., JBIOL CHEM, vol. 278, no. 49, 2003, pages 49215 - 22
HAKANSSON, M. ET AL., CURR PROTEIN PEPT SCI, vol. 3, no. 6, 2002, pages 629 - 42
HUYNH, Q. K. ET AL., J BIOL CHEM, vol. 263, no. 24, 1988, pages 11636 - 9
HUYNH, Q. K. ET AL., JBIOL CHEM, vol. 263, no. 2, 1988, pages 735 - 9
J BIOL CHEM, vol. 263, no. 29, 15 October 1988 (1988-10-15), pages 15104 - 9
MCDOWELL ET AL., J BIOMOL NMR, vol. 28, no. 1, 2004, pages 11 - 29
MCDOWELL, L. M. ET AL., BIOCHEMISTRY, vol. 43, no. 21, 2004, pages 6606 - 11
MIZYED, S. ET AL., BIOCHEMISTRY, vol. 42, no. 23, 2003, pages 6986 - 95
MOL GEN GENET, vol. 239, no. 3, June 1993 (1993-06-01), pages 416 - 24
MOL GEN GENET, vol. 245, no. 5, 1 December 1994 (1994-12-01), pages 616 - 22
MOUSDALE ET AL., PLANTA, vol. 170, 1987, pages 1 - 6
NELSON, K. E. ET AL.: "Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440", ENVIRON MICROBIOL, vol. 4, no. 12, 2002, pages 799 - 808, XP002968819, DOI: doi:10.1046/j.1462-2920.2002.00366.x
PADGETTE, S. R. ET AL., ARCH BIOCHEM BIOPHYS, vol. 266, no. 1, 1988, pages 254 - 62
PADGETTE, S. R. ET AL., J BIOL CHEM, vol. 263, no. 4, 1988, pages 1798 - 802
PLANT PHYSIOL., vol. 83, 1987, pages 229 - 231
PROC NATL ACAD SCI USA, vol. 97, no. 12, 2000, pages 6345 - 9
SCHONBRUNN ET AL., PROTEINS, vol. 40, no. 2, 2000, pages 290 - 8
SCHONBRUNN, E. ET AL., PROC NATL ACAD SCI USA, vol. 98, no. 4, 2001, pages 1376 - 80
SHIBA, K. ET AL., J BIOL CHEM, vol. 267, no. 32, 1992, pages 22703 - 6
SHIBA, K. ET AL., PROC NATL ACAD SCI USA, vol. 89, no. 5, 1992, pages 1880 - 4
SHUTTLEWORTH, W. A. ET AL., ARCH BIOCHEM BIOPHYS, vol. 334, no. 1, 1996, pages 37 - 42
SHUTTLEWORTH, W. A. ET AL., BIOCHEMISTRY, vol. 33, no. 23, 1994, pages 7062 - 8
SHUTTLEWORTH, W. A. ET AL., BIOCHEMISTRY, vol. 38, no. 1, 1999, pages 296 - 302
STALLINGS, W. C. ET AL., PROC NATL ACAD SCI USA, vol. 88, no. 11, 1991, pages 5046 - 50
STAUFFER, M. E. ET AL., BIOCHEMISTRY, vol. 40, no. 13, 2001, pages 3951 - 7
STAUFFER, M. E. ET AL., FEBS LETT, vol. 499, no. 1-2, 2001, pages 182 - 6
WRUAD ET AL., PROC NATL ACAD SCI USA, vol. 101, no. 40, 2004, pages 14533 - 8
YE G N ET AL: "Plastid-expressed 5-enolpyuvylshiikimate-3-phosphate synthase genes provide high level glycophosate tolerance in tobacco.", PLANT J., vol. 25, no. 3, 2001, pages 261 - 270, XP002187551 *
YE, G N. ET AL., PLANT J, vol. 25, no. 3, 2001, pages 261 - 70

Also Published As

Publication number Publication date
CN1789412A (zh) 2006-06-21
DE602005024442D1 (de) 2010-12-09
US8936925B2 (en) 2015-01-20
EP1840210A1 (en) 2007-10-03
EP1840210A4 (en) 2008-11-05
US20090155879A1 (en) 2009-06-18
CN1789412B (zh) 2011-04-20
EP1840210B1 (en) 2010-10-27
ATE486127T1 (de) 2010-11-15

Similar Documents

Publication Publication Date Title
AU2020264325A1 (en) Plant genome modification using guide rna/cas endonuclease systems and methods of use
EP0507698B1 (fr) Promoteurs d&#39;histone
US7141659B2 (en) Starch encapsulation
MX2008015741A (es) Enzima de dicamba monooxigenasa modificada y metodos para su uso.
JPH05500002A (ja) 除草剤代謝性チトクロムｐ４５０の発現
MX2013001191A (es) Cepas de agrobacterium modificadas para incrementar la frecuencia de transformacion de plantas.
US11802290B2 (en) Expression of nitrogenase polypeptides in plant cells
CN102741415A (zh) 使用hppd抑制剂作为选择剂的大豆转化
WO2006063533A1 (fr) Reconstitution de l’activite de la 5-enolpyruvylshikimate-3-phosphate synthase par complementation de fragments
JP2003505012A (ja) 活性なタンパク質産生物を発現し得る分断した非伝達性遺伝子を産生させるための方法
EP1042491A1 (fr) Promoteur h3c4 de mais associe au premier intron de l&#39;actine de riz, gene chimere le comprenant et plante transformee
AU2013271455B2 (en) Methods of improving the yield of 2,4-D resistant crop plants
US8106159B2 (en) Insecticidal toxin complex fusion protiens
US6858775B1 (en) Method for generating split, non-transferable genes that are able to express an active protein product
CN111041011A (zh) 一种草甘膦氧化酶突变体及其克隆、表达与应用
CN110272880B (zh) 一种突变型草甘膦降解酶及其克隆、表达与应用
Chitpinityol et al. Site-specific mutations of calf chymosin B which influence milk-clotting activity
Zhao et al. Photochromic chromopeptides derived from phycoerythrocyanin: biophysical and biochemical characterization
WO2006054458A1 (ja) 除草剤耐性遺伝子及びその利用
US7056743B2 (en) MtHP promoter element
WO1998044116A1 (fr) Gene codant la trehalose phosphorylase recombinee, vecteur renfermant ce gene, transformant transforme par ce gene, et procede de production de la trehalose phosphorylase recombinee au moyen de ce transformant
WO1992016641A1 (en) Process for expressing polypeptide
RU2310688C1 (ru) РЕКОМБИНАНТНАЯ ДНК, КОДИРУЮЩАЯ ФУНКЦИОНАЛЬНО АКТИВНЫЙ ГИБРИДНЫЙ БЕЛОК ОКСИДАЗЫ D-АМИНОКИСЛОТ С ХИТИНСВЯЗЫВАЮЩИМ ДОМЕНОМ (DAOcbd) РЕКОМБИНАНТНАЯ ПЛАЗМИДА pVR1, ОБЕСПЕЧИВАЮЩАЯ ЕГО СИНТЕЗ В КЛЕТКАХ Escherichia coli, И РЕКОМБИНАНТНЫЙ ШТАММ Escherichia coli С 41(DE3)/pVR1 - ПРОДУЦЕНТ DAOcbd
Ponomarenko Algae cells with deletion of the segment D210-R226 in γ subunit from chloroplast ATP synthase have lower transmembrane proton gradient and grow slowly
AU2012261523B2 (en) Novel herbicide resistance genes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2005819606

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2005819606

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 11721856

Country of ref document: US