WO2009059485A1 - Epsp synthase possédant une résistance élevée au glyphosate, et sa séquence codée - Google Patents

Epsp synthase possédant une résistance élevée au glyphosate, et sa séquence codée Download PDF

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WO2009059485A1
WO2009059485A1 PCT/CN2007/071071 CN2007071071W WO2009059485A1 WO 2009059485 A1 WO2009059485 A1 WO 2009059485A1 CN 2007071071 W CN2007071071 W CN 2007071071W WO 2009059485 A1 WO2009059485 A1 WO 2009059485A1
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glyphosate
epsp
epsp synthase
seq
sequence
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PCT/CN2007/071071
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French (fr)
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Min Lin
Aimin Liang
Wei Lu
Liang Li
Ming Chen
Wei Zhang
Shuzhen Ping
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Biotechnology Research Institute, The Chinese Academy Of Agricultural Sciences
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Priority to US12/741,965 priority Critical patent/US8207403B2/en
Publication of WO2009059485A1 publication Critical patent/WO2009059485A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01019—3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase
    • 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 novel highly tolerant glyphosate-containing EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase), and a nucleotide sequence encoding the same. Background technique
  • Glyphosate is the main active ingredient in Monsanto's Roundup® product. It is a broad-spectrum, endocrine-conducting excellent herbicide and one of the most used herbicides in the world. However, the herbicide is also a non-selective herbicide that also kills crops. In order to use glyphosate in agricultural production, crops with glyphosate resistance or degradation properties must be cultivated.
  • Glyphosate inhibits the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) during the metabolism of shikimic acid in plants, thereby blocking the biosynthesis of aromatic amino acids and causing plant death (SR Padgette et al. , in Herbicide- Res is ten t Crops : Agricul tural, Environmental, Economic, Regula tory, and Technical Aspec ts, S. 0. Duke, Ed. (CRC Press, Boca Raton, FL, 1996), pp. 53-84), All glyphosate-resistant GM crops currently grown commercially in the world are designed for EPSP and are the only mechanism of action for commercial transgenic glyphosate-resistant crops.
  • EPSP 5-enolpyruvylshikimate-3-phosphate synthase
  • the mechanism of drug resistance confirmed that the arok gene is the coding gene of glyphosate target EPSP synthase.
  • Companies such as Mosanto and Calegene in the United States have applied for more than 100 patents in the EPSP synthase coding gene sroA and its glyphosate-tolerant transgenic plants, and obtained a series of transgenic glyphosate resistant soybean, corn, rape, sugar beet and cotton crops. Varieties, including soybeans and other genetically modified crops have entered commercial production.
  • the object of the present invention is to discover and artificially synthesize a novel highly tolerant glyphosate-like EPSP synthase and a nucleotide sequence encoding the same, and transfer the sequence into a plant to cultivate a novel high tolerance glyphosate-resistant Transgenic plants.
  • the present invention has for the first time discovered a novel highly tolerant glyphosate EPSP synthase, such as the amino acid sequence set forth in SEQ ID NO: 1, and the nucleotide sequence encoding the synthase, such as SEQ ID NO: 2 or SEQ. ID NO: 3 is shown. Sequence structure analysis and sequence comparison analysis (see Figure 3) showed that the EPSP synthase belongs to type I EPSP synthase.
  • the invention collects soil samples in the extremely polluted environment of glyphosate, and separates the total DNA of the community level from the culture-free method, constructs a total DNA cosmid library at the community level, and screens the glyphosate-resistant transformants; the transformants are placed at 20 mM Resistant transformants were screened on M9 solid medium of glyphosate.
  • the present invention also carried out a glyphosate tolerance test, and the results showed that the above transformant had very strong glyphosate tolerance activity.
  • the present invention also performs full nucleotide sequence determination of a DNA fragment highly resistant to glyphosate.
  • the analysis showed that the inserted fragment was 3151 bp in size and contained a 1335 bp reading frame.
  • the sequence of SEQ ID NO: 2 contained a full-length nucleotide sequence of 1335 bases. Located at positions 885-2220, encoding an EPSP synthase of 445 amino acids in full length (as shown in SEQ ID NO: l).
  • the present invention artificially synthesizes the above-mentioned highly tolerant glyphosate EPSP synthase gene, and its sequence is shown in SEQ ID NO: 3.
  • the artificially synthesized 5' and 3' end cleavage sites were BomHI and HindlTL site EPSP genes for expression of a highly tolerant glyphosate-resistant EPSP synthase and construction of the corresponding gene plant expression vector.
  • the above-prepared EPSP gene was digested with BamHI and H dlll, and ligated into the same vector pET28a to obtain the recombinant plasmid pETGR-79. Transform E. coli BL21 (DE3) (Promega).
  • the present invention also carried out the enzyme activity measurement and kinetic parameter determination of EPSP, and the enzyme activity was 10.477 U/mg. Ki/K m is 2.16. According to the kinetic parameters, GR-79 EPSP not only has higher glyphosate resistance, but also maintains a strong affinity with PEP, which will provide a possibility for the cultivation of genetically modified crops.
  • the invention constructs a plant expression vector of EPSP synthase gene with high tolerance to glyphosate, and transforms and constructs glyphosate-resistant transgenic tobacco by leaf disc method.
  • the glyphosate resistance gradient experiment proves that the transgenic plant can contain 20 mM grass. Good growth on the medium of glyphosate.
  • the present invention also provides a recombinant vector comprising the DNA of SEQ ID NO: 2.
  • the present invention transforms host cells with the above recombinant vectors, including prokaryotic cells, including eukaryotic cells.
  • the present invention also provides a method for transforming SEQ ID NO: 2 into a plant using transgenic technology to increase glyphosate resistance in plants, the steps of which are as follows:
  • step (1) (2) transferring the expression vector in step (1) into a plant cell
  • the transformed cells are obtained by screening and finally the transgenic plants and their progeny are regenerated, including plant seeds and plant tissues.
  • the above "operably linked to” means that: some parts of a linear DNA sequence can affect the same linearity.
  • the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; if the ribosome binding site is placed at a position that enables translation, then it is operably linked to the coding sequence.
  • “operably linked” means adjacent, and for secretory leader sequences means adjacent in the reading frame.
  • the above vectors may be selected from various carriers known in the art, such as commercially available vectors, including plasmids, cosmids and the like.
  • the EPSP synthase encoding gene refers to a nucleotide sequence encoding a polypeptide having SEQ ID ⁇ :1 protein activity and a degenerate sequence thereof.
  • the degenerate sequence refers to a sequence in which one or more codons in the sequence are replaced by degenerate codons encoding the same amino acid. Due to the degeneracy of the codon, a degenerate sequence having a homology of less than about 89% to SEQ ID NO: 2 can also encode the sequence set forth in SEQ ID NO: 2.
  • the term also encompasses nucleotide sequences that hybridize to the nucleotide sequence of SEQ ID NO: 2 under moderately stringent conditions, more preferably under highly stringent conditions.
  • the term also encompasses nucleotide sequences having at least 89%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homology to the nucleotide sequence of SEQ ID NO:2.
  • the term also encompasses variant forms of the open reading frame sequence of SEQ ID NO: 2 which encodes a protein having the same function as the native SEQ ID ⁇ :1.
  • variants include, but are not limited to, several (usually 1-90, preferably 1-60, more preferably 1-20, optimally 1-10) nucleotide deletions. , insert and / or replace, and add a few at the 5' and / or 3' end (usually less than 60, preferably less than 30, more preferably less than 10, and most preferably less than 5 ) nucleotides.
  • the SEQ ID ⁇ :1 protein further includes a variant having the same function as SEQ ID ⁇ :1.
  • variants include, but are not limited to, a number (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions and/or Substituting, and adding one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and/or the N-terminus.
  • the function of the protein is usually not altered.
  • the addition of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the SEQ ID ⁇ :1 protein.
  • Variants of the polypeptide include: homologous sequences, EPSP synthase conserved variant polypeptides, allelic variants, natural A mutant, an inducible mutant, a protein encoded by a DNA which hybridizes to SEQ ID NO: 2 under high or low stringency conditions, and a polypeptide or protein obtained using an antiserum of the polypeptide of SEQ ID NO: 1.
  • EPSP synthase conservative variant polypeptide means having up to 10, preferably up to 8, more preferably up to 5, and optimally up to 3 amino acids compared to the amino acid sequence of SEQ ID NO: 1. Substitutes of similar or similar amino acids are substituted to form a polypeptide. These conservative variant polypeptides can be produced by amino acid substitution according to Table 1.
  • Figure 1 is a graph showing the resistance analysis of GR-79 cloned glyphosate.
  • GR-79-ER strain is partially digested with total DNA and then transformed into EPSP synthase-deficient Escherichia coli ER2799 strain (NEB) after ligation with the vector pACYC184.
  • the glyphosate resistant strain obtained after the company).
  • the CP4-ER strain is a glyphosate-resistant strain obtained by ligating the EPSP synthase gene derived from Agrobarten' m sp. cp4 to the vector pACYC184 and transforming it into EPSP synthase-deficient Escherichia coli ER2799 strain (NEB). This figure serves as a positive control.
  • pACYC184-ER is an EPSP synthase-deficient Escherichia coli ER2799 strain containing the pACYC184 plasmid (NEB). This experiment served as a negative control.
  • This figure is to connect the three strains to the restricted medium M9 containing 0, 20, 50, 80, 100, 120, 150, 200, 250, 300 mM glyphosate, after 37 ° C, 36 h
  • the absorbance value of the bacterial solution at OD600 was measured and plotted. It can be seen that the strain GR-79-ER can grow in a limiting medium containing a concentration of 250 mM glyphosate, indicating that the strain has a glyphosate resistance of 250 mM. It is indicated that the exogenous fragment carried on the plasmid can complement the defective strain ER2799.
  • the negative control strain could not grow in the restriction medium and could not complement the defective strain.
  • the positive control strain had a glyphosate resistance of 200 mM.
  • Figure 2 shows the protein expression of EPS-synthase of GR-79 at different times.
  • the EPSP synthase gene in the GR-79 strain was ligated into the pET28a vector and then transferred into BL21, and protein expression was induced under the induction of IPTG. The sampling time was separated by one hour. The sample was boiled and separated by SDS-PAGE electrophoresis. The results showed that the protein expression level of the strain reached a high level at 4 hours.
  • the expressed protein is a soluble protein. The size is about 45kD.
  • Figure 3 is a comparison of the amino acid sequence of GR-79 with the reported typical amino acid sequences of Class I and Class II. The comparison showed that the amino acid sequence of GR-79 belongs to Class I type EPSP synthase. And GR-79's EPSP synthase is a type I enzyme with glyphosate resistance. detailed description
  • Soil samples were collected from soils that had been contaminated with about 50% glyphosate for more than ten years (opening repacking point for a glyphosate production plant in a chemical company in Hebei).
  • DNA samples were purified using the Wizard spin column clean-u isolation kit.
  • the purified DNA was dissolved in a total volume of 100 ⁇ l of 10 mM Tris-EDTA (pH 8.0) buffer.
  • the soil bacterial DNA was partially digested with a 10 ⁇ l reaction system.
  • the enzyme was diluted 1:100, and digested at 37 ° C for 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, and then added 10 X loading buffer 1 ⁇ . 1
  • the reaction was terminated, and the electrophoresis was carried out to determine the optimal digestion time. Then the same system was selected and digested for 30 min for extensive digestion. After agarose gel electrophoresis, the 2 ⁇ 6 kb DNA fragment was recovered by cutting.
  • the plasmid vector pACYC184 (NEB) was completely digested with fiamHI and subjected to terminal dephosphorylation with SAP alkaline phospholipase to reduce vector autoligation.
  • the recovered soil bacterial DNA (200 ng) and the terminal dephosphorylated plasmid vector pACYC184 (150 ng) were ligated with 2 U of T4 ligase for 16 h at 4 °C.
  • the above-mentioned ligation product was transferred to E. coli ER2799 (NEB) electroshock-competent cells, coated with LB+Cm ⁇ , and then the clones grown on the LB plate were photocopied on a plate of M9 Cmr+50 mM glyphosate, and cultured at 37 ° C for 48 h. .
  • the transfected bacteria were coated on LB plates containing Cm (chloramphenicol), and after incubation at 37 ° C for 20 h, about 5,000 colonies were grown, and these colonies were photocopied on M9 plates containing en and 50 mM glyphosate. After 48 hours, three colonies grew. The three colonies were inoculated on M9 plates containing 100 mM, 150 mM glyphosate, and it was found that only one clone was able to grow on M9 plates containing 150 mM glyphosate, and the plasmid contained therein was named pACYCGR- 79.
  • the plasmid pACYCGR-79 extracted from the clone was transferred into Escherichia coli ER2799 (NEB) or Escherichia coli JM109 (Promega), and the transformants were spotted on a M9 solid medium containing 20 mM glyphosate using a sterile toothpick. Resistance, the results showed that the transformants produced by this clone all had glyphosate resistance, indicating that the glyphosate resistance was indeed caused by the transfer to pACYCGR-79.
  • Escherichia coli ER2799 (containing pACYCGR-79 plasmid carrying the new clone) was inoculated into M9 liquid medium (Cm) containing 0 ⁇ 200 mm glyphosate, and cultured at 37 ° C for 36 h on a shaker. OD600. At the same time, E. coli ER2799 with no insert plasmid was used as a negative control.
  • ER2799 (cPACYCGR-79 plasmid) was inoculated into M9 liquid medium (Cm containing 0 ⁇ 300 mM glyphosate, and cultured at 37 ° C for 36 h on a shaker, it was found that the negative control could hardly grow in M9.
  • ER2799 (pACYCGR-79) was also able to grow in M9 liquid medium containing 250 mM glyphosate (see Figure 1). This result indicates that the exogenous fragment carried on pACYCGR-79 is very resistant to glyphosate. It is active.
  • the CP4 plasmid positive control bacteria can only grow in 200 mM liquid medium.
  • the fully tolerated glyphosate DNA fragment subcloned in Example 1 was subjected to full nucleotide sequence determination. The analysis showed that the inserted fragment was 3151 bp in size and contained a 1335 bp reading frame. The sequence of the inserted fragment was as shown in sequence 1. It contained a polynucleotide sequence of 1335 bases in length and its open reading frame at 885- 2220, encoding a 445 amino acid EPSP synthase.
  • the subcloned highly tolerant glyphosate coding sequence was less homologous at the nucleotide level than the reported EPSP synthase encoding gene (araA).
  • the amino acid sequence homology analysis showed that the amino acid sequence of the GR-79 amino acid sequence and the reported typical type I EPSP synthase were higher than the amino acid sequence of the enzyme and the type II EPSP synthase, and The GR79 amino acid sequence does not contain a conserved amino acid segment typical of the type II enzyme, but contains a conserved amino acid segment similar to the type I enzyme.
  • Description GR-79 EPSP belongs to Class I EPSP.
  • the phylogenetic comparison of GR-79 EPSP with typical Type I and Type II EPSP synthases is shown in Figure 3.
  • a single-stranded oligonucleotide fragment having a sticky end of about 150-200 bp in length was synthesized from the positive and the sub-chain sequences, respectively, in eight segments.
  • Eight complementary single-stranded oligonucleotide fragments each corresponding to a single strand and a sub-strand are respectively annealed to form eight double-stranded oligonucleotide fragments with sticky ends Paragraph.
  • the double-stranded oligonucleotide fragment was ligated and assembled into a complete EPSP synthase gene by T4 DNA ligase.
  • the synthetic DNA fragment contains the nucleotide sequence of 1-35 in SEQ ID NO: 2, and the upstream and downstream ends of the synthetic gene contain BamHI and H wdlll sites. As shown in SEQ ID NO: 2.
  • the above-mentioned synthetic 5'-end 3'-end cleavage sites were BomHI and HindlU locus EPSP genes, which were digested with B rniHl and H milll and ligated into the same vector pET28a (NEB) to obtain recombinant plasmid pETGR. -79 and transformed it into E. coli BL21 (DE3) (Promega).
  • the transformants were first cultured in LB + Kn medium at JVC, 200 rpm to an OD600 value of about 0.5, added with IPTG (final concentration of 0.75 mmol/L), and transferred to 37 °C to induce protein expression, which was detected by SDS-PAGE electrophoresis.
  • E. coli BL21 (DE3) (Promega) containing pETGR-79 reached the highest value after induction with IPTG for 4 h at 37 °C.
  • the target protein is a soluble protein; the size is about 45kD, which is consistent with the predicted value (see Figure 2).
  • Inorganic phosphorus standard curve 10 mM inorganic phosphorus standard solution is diluted 1:10, respectively, take 0, 1, 2, 3 ⁇ 20 ⁇ 1 in 1.5 ml Eppendorf centrifuge tube, add milli-Q pure water to 100 ⁇ 1 and mix. Add 0.8 ml of MAT solution and mix well. After mixing for 3 minutes, add 34 ⁇ l solution of 100 ⁇ l and mix rapidly. After standing at room temperature for 20 min, measure the OD660 value. repeat three times. The inorganic phosphorus standard curve was obtained by taking the inorganic phosphorus concentration as the abscissa and the OD660 value as the ordinate.
  • Enzyme activity assay Enzyme crude extract protein was quantified using Coomassie Brilliant Blue G-250 staining (Bradford, 1976). Add the following solution to a 1.5 ml Eppendorf centrifuge tube on ice: 10 mM PEP solution 2 ⁇ l, 10 mM S3P solution 2 ⁇ l, 0.5 ⁇ HEPES solution 2 ⁇ l, ImM ( ⁇ 4 ) 6 ⁇ 0 7 0 24 ⁇ 4 ⁇ 2 0 The solution 2 ⁇ 1 and the milli-M pure-Q pure water 12 ⁇ 1 was mixed. After the temperature was incubated at 28 ° C for 5 min, each tube sample was added with 1 ⁇ 1 of the crude enzyme solution at intervals of 2 s and timed.
  • the enzyme activity of GR-79 EPSP was 10.477 U/mg, and the GR-79 EPSP assay was as shown in Table 2: Table 2 Kinetic parameters of GR-79 EPSP
  • GR-79 EPSP According to the kinetic parameters of GR-79 EPSP, GR-79 EPSP not only has higher glyphosate resistance, but also maintains a strong affinity with PEP. These characteristics will be used for GR-79 EPSP.
  • the cultivation of genetically modified crops offers the possibility.
  • pBI121 (ClonTech) Wo B pCAMBIA2301 (ClonTech) was digested with H mffll and EcoRI, pBI121 with p35S-GUS-Nos-ter fragment was ligated into pCAMBIA2301 to form intermediate vector P35S-2301-GUS;
  • the differentiated buds can be seen. After the buds grow up, they are cut and placed in rooting medium (1 / 2MS+NAA 0.5mg/l+Kan 25mg/l) for rooting, 2-7 Rooting around the day;
  • the resistant plants were confirmed to be transgenic resistant plants by Southern, Northern hybridization and Westhern blot.

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Description

高耐受草甘膦的 EPSP合酶及其编码序列
技术领域
本发明涉及一种新型高耐受草甘膦的 EPSP合酶 （5-烯醇式丙酮莽草酸 -3-磷酸合酶） ， 以及编码该合酶的核苷酸序列。 背景技术
草甘膦 （glyphosate ) 为 Monsanto公司产品 Roundup®中的主要活性成分， 该除草剂是 一种广谱灭生性、 内吸传导型优秀除草剂， 是全世界使用量最大的除草剂品种之一。 但是， 该除草剂也是一种非选择性除草剂， 对农作物同样有着杀死作用。 为在农业生产中使用草甘 膦， 须培育出具有草甘膦抗性或降解性质农作物。
草甘膦抑制植物莽草酸代谢过程中 5-烯醇式丙酮莽草酸 -3-磷酸合酶 （EPSP ) 活性， 进 而阻断芳香族氨基酸的生物合成而使植物死亡 （S. R. Padgette et al. , in Herbicide- Res is ten t Crops : Agricul tural， Environmental, Economic, Regula tory, and Technical Aspec ts, S. 0. Duke , Ed. ( CRC Press , Boca Raton , FL , 1996)， pp. 53- 84)， 当前全球商 业化种植的所有草甘膦抗性转基因作物均为针对 EPSP所设计， 是目前商业化转基因抗草甘 膦作物的唯一作用机制。 应用化学诱变细菌产生的 arcA突变体， 抗药性机理研究确证了 arok基因是草甘膦作用靶标 EPSP合酶的编码基因。美国 Mosanto和 Calegene等公司在 EPSP 合酶的编码基因 sroA及其抗草甘膦转基因植物等方面已申请了 100余份专利， 获得转基因 抗草甘膦大豆、 玉米、 油菜、 甜菜和棉花等作物系列品种， 其中大豆等多种转基因作物已进 入商品化生产。
目前尚未见到在核苷酸水平与已报道的 EPSP合酶编码基因 （aroA) 同源性较低的抗草 甘膦的 EPSP合酶。 发明内容
本发明的目的是发现并人工合成新型高耐受草甘膦的 EPSP合酶以及编码该合酶的核苷 酸序列， 并将该序列转入植物中， 培育新型的高耐受草甘膦的转基因植物。
本发明首次发现了一种新型高耐受草甘膦的 EPSP合酶， 如 SEQ ID ΝΟ: 1所示的氨基酸 序列， 以及编码该合酶的核苷酸序列， 如 SEQ ID NO:2或 SEQ ID NO:3所示。经序列结构分 析和序列比较分析 （见图 3 ) ， 显示该 EPSP合酶属于 I型 EPSP合酶。
本发明采集草甘膦极端污染环境中土壤样品， 用免培养方法从中分离群落水平总 DNA， 构建群落水平总 DNA粘粒文库， 并筛选草甘膦抗性转化子； 将转化子点于含 20mM草甘膦 的 M9固体培养基上筛选抗性转化子。 本发明还进行了草甘膦耐受实验， 结果表明上述转化 子具有非常强的草甘膦耐受活性。
本发明还进行了高耐受草甘膦的 DNA片段的全核苷酸序列测定。 分析结果表明， 插入 的片段大小为 3151bp， 其中包含了一个 1335bp的阅读框， 其序列如 SEQ ID NO:2所示， 它 包含的核苷酸序列全长为 1335个碱基， 其开放读框位于 885-2220位， 编码全长为 445个氨 基酸的 EPSP合酶 （如 SEQ ID NO: l所示） 。
本发明对上述高耐受草甘膦的 EPSP合酶基因进行了人工合成， 其序列如 SEQ ID NO: 3 所示。 将人工合成的 5'和 3'端酶切位点为 BomHI和 HindlTL位点 EPSP基因， 用于表达高 耐受草甘膦的 EPSP合酶以及构建相应的基因植物表达载体。将上述人工合成的 EPSP基因， 用 BamHI和 H dlll酶切后， 连入相同酶切的载体 pET28a得到重组质粒 pETGR-79并将其 转化大肠杆菌 BL21 (DE3) (Promega公司） 。
本发明还进行了 EPSP的酶活测定和动力学参数的测定， 酶活性为 10.477U/mg。 Ki/Km 为 2.16。 根据动力学参数可知， GR-79 EPSP不仅具有较高的草甘膦抗性， 而且还保持着与 PEP较强的亲和性， 这些特性将为用于转基因作物的培育提供可能。
本发明构建了高耐受草甘膦的 EPSP合酶基因植物表达载体， 利用叶盘法转化构建抗草 甘膦的转基因烟草， 经草甘膦抗性梯度实验证明， 转基因植物能在含 20mM草甘膦的培养基 上良好生长。
本发明还提供了一种重组载体， 它包含 SEQ ID NO:2所述的 DNA。 本发明用上述重组 载体转化宿主细胞， 这些宿主包括原核细胞， 也包括真核细胞。
本发明还提供了一种利用转基因技术将 SEQ ID NO:2转化入植物的方法，以提高植物对 草甘膦抗性， 其步骤如下：
( 1 ) 将 SEQ ID ΝΟ:1或 SEQ ID NO:2所示序列可操作地连于植物表达调控序列， 形 成植物表达载体；
(2) 将步骤 （1 ) 中的表达载体转入植物细胞；
(3) 经筛选获得转化细胞并最终再生转基因植株及其后代，包括植物种子及植物组织。 上述 "可操作地连于"表示如下情况： 即线性 DNA序列的某些部分能够影响同一线性
DNA序列其他部分的活性。 例如， 如果信号肽 DNA作为前体表达并参与多肽的分泌， 那么 信号肽 （分泌前导序列） DNA就是可操作地连于多肽 DNA; 如果启动子控制序列的转录， 那么它是可操作地连于编码序列； 如果核糖体结合位点被置于能使其翻译的位置时， 那么它 是可操作地连于编码序列。 一般， "可操作地连于"意味着相邻， 而对于分泌前导序列则意 味着在阅读框中相邻。
上述载体可选用本领域已知的各种载体， 如市售的载体， 包括质粒， 粘粒等。
在本发明中， EPSP合酶编码基因指编码具有 SEQ ID ΝΟ:1蛋白活性的多肽的核苷酸序 列及其简并序列。 该简并序列是指所述序列中有一个或多个密码子被编码相同氨基酸的简并 密码子所取代后而产生的序列。 由于密码子的简并性， 所以与 SEQ ID NO: 2同源性低至约 89%的简并序列也能编码出 SEQ ID NO:2所述的序列。 该术语还包括能在中度严谨条件下， 更佳的在高度严谨条件下与 SEQ ID NO:2核苷酸序列杂交的核苷酸序列。 该术语还包括与 SEQ ID NO:2中的核苷酸序列的同源性至少 89%， 较佳地至少 80%， 更佳地至少 90%， 最佳 地至少 95%的核苷酸序列。
该术语还包括能编码具有与天然的 SEQ ID ΝΟ:1相同功能的蛋白的 SEQ ID NO:2中开放 阅读框序列的变异形式。 这些变异形式包括（但并不限于） ： 若干个（通常为 1-90个， 较佳 地 1-60个， 更佳地 1-20个， 最佳地 1-10个）核苷酸的缺失、 插入和 /或取代， 以及在 5' 和 / 或 3' 端添加数个 （通常为 60个以内， 较佳地为 30个以内， 更佳地为 10个以内， 最佳地为 5个以内） 核苷酸。
在本发明中， SEQ ID ΝΟ:1蛋白还包括具有与 SEQ ID ΝΟ:1的相同功能的变异形式。 这些变异形式包括但并不限于若干个 （通常为 1-50个， 较佳地 1-30个， 更佳地 1-20个， 最 佳地 1-10个）氨基酸的缺失、插入和 /或取代，以及在 C末端和 /或 N末端添加一个或数个（通 常为 20个以内， 较佳地为 10个以内， 更佳地为 5个以内） 氨基酸。 例如， 在所述蛋白中， 用性能相近或相似的氨基酸进行取代时， 通常不会改变蛋白质的功能。 又比如， 在 C末端和 /或 N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括 SEQ ID ΝΟ:1 蛋白的活性片段和活性衍生物。
所述多肽的变异形式包括： 同源序列、 EPSP合酶保守性变异多肽、 等位变异体、 天然 突变体、诱导突变体、在高或低的严谨条件下能与 SEQ ID NO: 2 杂交的 DNA所编码的蛋白、 以及利用 SEQ ID NO: l多肽的抗血清获得的多肽或蛋白。
上述 "EPSP合酶保守性变异多肽"指与 SEQ ID NO: l的氨基酸序列相比， 有至多 10个， 较佳地至多 8个， 更佳地至多 5个， 最佳地至多 3个氨基酸被性质相似或相近的氨基酸所替换 而形成多肽。 这些保守性变异多肽可以根据表 1进行氨基酸替换而产生。
表 1 氨基酸替换表
Figure imgf000005_0001
附图说明
图 1是 GR-79克隆草甘膦抗性分析图， 图中： GR-79-ER菌株是土壤总 DNA部分酶切 后与载体 pACYC184连接后转化入 EPSP合酶缺陷型大肠杆菌 ER2799菌株（NEB公司）后 获得的草甘膦抗性菌株。
CP4-ER菌株是将来源于 Agrobarten' m sp. cp4的 EPSP合酶基因与载体 pACYC184连接 后转化入 EPSP合酶缺陷型大肠杆菌 ER2799菌株 （NEB公司） 后获得的草甘膦抗性菌株。 本图中作为阳性对照。
pACYC184-ER是含有 pACYC184质粒（NEB公司）的 EPSP合酶缺陷型大肠杆菌 ER2799 菌株。 本实验中作为阴性对照。
本图是将三株菌株分别接入到含有 0， 20， 50， 80， 100， 120， 150， 200， 250， 300mM 草甘膦浓度的限制性培养基 M9中， 经过 37 °C、 36h的摇床培养后， 测定菌液在 OD600时的 吸光度值， 所绘制而成。 图中可见菌株 GR-79-ER在含有 250mM浓度的草甘膦的限制性培养基中能够生长， 说 明该菌株草甘膦抗性能达 250mM。说明质粒上携带的外源片段能够对缺陷型菌株 ER2799进 行功能互补。而阴性对照菌株不能在限制性培养基中生长，不能对缺陷型菌株进行功能互补。 阳性对照菌株草甘膦抗性达 200mM。
图 2是 GR-79的 EPSP合酶在不同时间的蛋白表达。
GR-79菌株中的 EPSP合酶基因与 pET28a载体连接后转入 BL21中， 在 IPTG的诱导下 进行蛋白表达， 取样时间分别间隔一小时。样品经过煮沸后经 SDS-PAGE电泳分离。 结果显 示该菌株在 4小时的蛋白表达量就已经达较高程度。表达的蛋白为可溶性蛋白。大小约 45kD。
图 3是 GR-79氨基酸序列与报道的 Class I和 Class II典型类型的氨基酸序列的比较。 比较结果显示 GR-79的氨基酸序列属于 Class I类型的 EPSP合酶。 并且 GR-79的 EPSP 合酶是一种具有草甘膦抗性的 I型酶。 具体实施方式
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于举例说明本发明 的方法， 而不用于限制本发明的范围。 凡未注明具体实验条件的， 均为按照本领域技术人员 熟知的常规条件。 实施例 1 高耐受草甘膦的 DNA片段克隆
1、 草甘膦极端污染环境中土壤样品的采集
从被 50%左右草甘膦污染达十年以上的土壤中（河北某化工有限公司草甘膦生产工厂开 放式分装点） 采集土壤样品。
2、 采用免培养方法从草甘膦极端污染土壤样品中分离群落水平总 DNA
称取草甘膦污染土壤样品 2克，加入 0.6g 细玻璃珠（d<0.11mm)， 4000转 /分振荡 2 次。 加入 300 μ 1 2 SDS + 12%苯酚 Tris缓冲液 （pH8.0)溶液冰上 1小时， 加入等量苯酚 Tris缓 冲液， pH8.0 (约 700ml) ， 充分混匀， 经 4 °C， 13， OOOrpm离心 5分钟。 上层溶液加入 0.1 倍体积的 3M NaAc pH5.2, 混匀后加入 0.6倍体积异丙醇混匀。 DNA沉淀溶于 200 μ 1 ΙχΤΕ (II DNA) 。 称 lOOmg氯化铯置于一个新的 1.5ml Epp.离心管中， 加入 100 μ 1粗 DNA 轻 轻混匀， 室温黑暗条件下静置 1-3小时。 室温， 13， OOOrpm, 离心 20分钟。 上清液中加入 400 μ ΐ无菌去离子水和 300 μ ΐ异丙醇， 室温静置 30分钟。室温， 13， OOOrpm, 离心 20分钟。 沉淀溶于 100 μ ΐ ΙχΤΕ禾卩 40 μ 1 8Μ醋酸钾 （KAc) ， 室温静置 15分钟。 4°C， 13, OOOrpm 离心 15分钟。 上清液加入 0.6倍体积异丙醇混匀。 室温静置 30分钟。 室温， 15， OOOrpm离 心 20分钟。 DNA沉淀溶于 100 μ 1 1χΤΕ。
采用 Wizard spin column clean-u 分离试剂盒纯化 DNA样品。 纯化 DNA溶于总体积为 100 μ 1的 10mM Tris-EDTA (pH8.0) 缓冲液中。
3、 群落水平总 DNA粘粒文库的构建
土壤细菌 DNA用 在 10 μ 1反应体系进行部分酶切试切， 酶按 1:100稀释， 37°C，分别酶切 10min， 20min, 30min， 40min， 50min, 60min后加入 10 X loading buffer 1 μ 1终止反应，电泳检测最适酶切反应时间。而后选择相同的体系酶切 30 min进行大量酶切。 经琼脂糖凝胶电泳后切胶回收 2〜6kb DNA片段备用。 质粒载体 pACYC184 (NEB公司）用 fiamHI完全酶切后用 SAP碱性磷酸脂酶进行末端去磷酸化， 以减少载体自连。 上述回收后 的土壤细菌 DNA (200ng)和末端去磷酸化的质粒载体 pACYC184 ( 150ng)用 2U的 T4 ligase 在 4°C下连接 16h。 上述连接产物转入 E.coli ER2799 (NEB公司） 电击感受态细胞， 涂布 LB+Cm^ 然后 将 LB板上生长的克隆影印到 M9十 Cmr+50mM草甘膦的平板， 37°C培养 48h。 将平板上生 长的菌经 LB平板划线培养后， 将这些菌落再接种到含不同草甘膦浓度的 M9平板上 （100， 150mM 草甘膦） 。 将这些重组菌中的质粒抽提后重新转化 ER2799后涂布 M9十 Cm平板验 证 （ER2799十 pACYC184为对照） ， 同时进行重组质粒酶切验证。
4、 筛选草甘膦抗性转化子
将转染细菌涂布含 Cm (氯霉素） 、 的 LB平板上， 37°C培养 20h后， 约有 5000个菌落 生长，将这些菌落影印到含 en 和 50mM草甘膦的 M9平板上培养 48h后，有三个菌落生长。 将这三个菌落接种到含 100mM、 150mM的草甘膦的 M9平板上培养， 发现只有 1个克隆能 在含 150mM的草甘膦的 M9平板上生长， 其所含的质粒被命名为 pACYCGR-79。 从该克隆 抽提的质粒 pACYCGR-79转入大肠杆菌 ER2799 (NEB公司）或大肠杆菌 JM109 (Promega 公司） 中， 将转化子用无菌牙签点于含 20mM草甘膦的 M9固体培养基上检验抗性， 结果证 明这个克隆所产生的转化子均具有抗草甘膦特性， 表明抗草甘膦特性确实是由于转入 pACYCGR-79引起的。
5、 草甘膦耐受实验
将大肠杆菌 ER2799 (含携带有新克隆的 pACYCGR-79质粒） 接种到含 0〜200mm草 甘膦的 M9液体培养基 （Cm ) 中， 经过 37°C、 36h的摇床培养后， 测定培养物的 OD600。 同时以无插入片段的质粒的大肠杆菌 ER2799为阴性对照。
结果： 将 ER2799 (携带 pACYCGR-79质粒）接种到含 0〜300mM草甘膦的 M9液体培 养基 （Cm 中， 经过 37°C、 36h的摇床培养后， 发现阴性对照在 M9中几乎不能生长； 而 ER2799 (pACYCGR-79) 在含有 250mM草甘膦的 M9液体培养基中还能生长 （见图 1 ) 。 这一结果说明 pACYCGR-79上携带的外源片段具有非常强的草甘膦耐受活性。 而带有 CP4 质粒阳性对照菌只能在 200mM的液体培养基中生长。 实施例 2 高耐受草甘膦的 DNA片段的序列分析及其 EPSP合酶功能验证
1、 高耐受草甘膦的 DNA片段的序列分析
对实施例 1中所亚克隆的高耐受草甘膦 DNA片段进行全核苷酸序列测定。 分析结果表 明， 插入的片段大小为 3151bp， 其中包含了一个 1335bp的阅读框， 其序列如序列 1所示， 它包含的多核苷酸序列全长为 1335个碱基， 其开放读框位于 885-2220位， 编码全长为 445 个氨基酸的 EPSP合酶。
将所亚克隆的高耐受草甘膦编码序列与已报道的 EPSP合酶编码基因（araA) 比较， 在 核苷酸水平同源性较低。
氨基酸序列同源性分析结果表明， GR-79氨基酸序列与已报道的典型的 I型 EPSP合酶 的氨基酸同源率均高于该酶与 II型 EPSP合酶的氨基酸序列的同源率， 并且 GR79氨基酸序 列中不含有 II型酶中典型的保守氨基酸区段， 而含有的保守氨基酸区段类似于 I型酶。 说明 GR-79 EPSP属于 I类 EPSP。 GR-79 EPSP与典型的 I型和 II型 EPSP合酶的系统发育比较结 果， 如图 3所示。 实施例 3 高耐受草甘膦的 EPSP合酶基因的人工合成
根据已完成的含 1335bp编码区的核苷酸序列， 首先分 8个区段分别根据正链和副链序 列， 分别合成出长度约 150-200bp、 具有粘性末端的单链寡核苷酸片段。 将正链和副链各一 一对应的 8个互补的单链寡核苷酸片段分别退火， 形成 8个带有粘性末端的双链寡核苷酸片 段。 混合双链寡核苷酸片段， 经 T4 DNA连接酶催化组装成一个完整的 EPSP合酶基因。 该 合成的 DNA片段含有 SEQ ID NO:2中 1-335位的核苷酸序列， 并且合成基因的上下游两端 含 BamHI和 H wdlll位点。 如 SEQ ID NO:2所示。
将上述人工合成的 5' 和 3' 端酶切位点为 BomHI和 H dlll位点 EPSP基因， 用于表达高耐 受草甘膦的 EPSP合酶以及构建相应的基因植物表达载体。 实施例 4 高耐受草甘膦的 EPSP表达
上述人工合成的 5' 禾卩 3'端酶切位点为 BomHI和 HindlU位点 EPSP基因， 用 B rniHl和 H milll酶切后， 连入相同酶切的载体 pET28a (NEB公司） 得到重组质粒 pETGR-79并将其 转化大肠杆菌 BL21 (DE3 ) (Promega公司）。将转化子先在 LB + Kn 培养基中 JVC， 200rpm 培养至 OD600值约 0.5， 加入 IPTG (终浓度为 0.75mmol/L) 后转入 37°C诱导蛋白表达， SDS-PAGE电泳检测。
经 SDS-PAGE 电泳检测， 含有 pETGR-79的大肠杆菌 BL21 (DE3 ) (Promega公司） 在 37°C经 IPTG诱导 4h后表达量即达到最高值。 目的蛋白为可溶性蛋白； 大小约 45kD， 与 预测值相符 （见图 2) 。 实施例 5 EPSP的酶活测定和动力学参数的测定
1、 测定方法
无机磷标准曲线： 10mM无机磷标准液按 1:10稀释， 分别取 0、 1、 2、 3〜20 μ 1于 1.5ml Eppendorf离心管中， 加入 milli-Q纯水至 100 μ 1混匀， 加入 MAT溶液 0.8ml混匀， 计时三 分钟后加入 34% SC溶液 100 μ 1迅速混匀， 室温静置 20min后测定 OD660值。 重复三次。 以无机磷浓度为横坐标， OD660值为纵坐标作图得到无机磷标准曲线。
1 ) 酶活测定： 酶粗提物蛋白定量采用考马斯亮蓝 G-250染色法 （Bradford, 1976) 。 在冰上于 1.5ml Eppendorf离心管中加入以下溶液： 10mM PEP溶液 2 μ 1， 10mM S3P溶液 2 μ 1， 0.5Μ HEPES溶液 2 μ 1， ImM (ΝΗ4) 6Μ07024· 4Η20溶液 2 μ 1禾卩 milli-Q纯水 12 μ 1 混匀， 于 28°C温浴 5min后各管样品间隔 2S加入 1 μ 1粗酶液并计时， 2min后再间隔 2S依 次加入 200 μ 1 MAT溶液， 显色 3min后再间隔 2S依次加入 20 μ 1 34% SC溶液迅速混匀， 室 温显色 20min后测定 OD660值。对照除不加酶液外，其余同样品管。样品管与对照管的 OD660 值相减后， 对照无机磷标准曲线即可求得反应释放出的无机磷摩尔量， 再除以反应时间和酶 蛋白量就得到该酶的酶活力 （U/mg) 。
2)半抑制剂量 (IC50)测定：上述反应液中添加 0、 10-3、 10-2、 10-1、 1、 10、 100、 500mM 草甘膦， 所得酶比活力数据以草甘膦浓度为 X轴， 采用对数坐标， 以反应速度 V (U/mg) 为 Y轴作图。
3) Km (PEP)测定： 将 S3P溶液浓度恒定于 lmM， 在不同 PEP浓度（0.05、 0.067、 0.1、 0.2、 0.5、 LOmM) 下按上述反应体系测定酶反应速度， 所测数值按 V-v/[S] (Eadic-Hofstee) 法作图。
K; ( glyphosate) 测定： 在不同草甘膦浓度 （0、 10、 50、 100 μ Μ) 下测定 PEP浓度为 66.7、 100、 200、 500 μ Μ时 EPSP的酶反应速度。 采用双对数作图， 得到 1/V-1/[S]直线， 再 将各直线的斜率作为纵坐标， 草甘膦浓度作为横坐标得到一条新的直线， 该直线与 X轴的交 点即为 Ki (glyphosate) 值。
2、 结果
GR-79 EPSP的酶活性为 10.477U/mg， GR-79 EPSP测定如表 2所示： 表 2 GR-79 EPSP的动力学参数
Figure imgf000009_0001
根据 GR-79 EPSP的动力学参数可知， GR-79 EPSP不仅具有较高的草甘膦抗性， 而且还 保持着与 PEP较强的亲和性，这些特性将为将 GR-79 EPSP用于转基因作物的培育提供可能。 实施例 6 高耐受草甘膦的 EPSP合酶基因植物表达载体的构建
高耐受草甘膦的 EPSP合酶基因植物表达载体构建的具体方法如下：
A. pBI121 (ClonTech公司） 禾 B pCAMBIA2301 (ClonTech公司） 用 H mffll和 EcoRI双 酶切， 将 pBI121 带有 p35S-GUS- Nos-ter 的片段连入 pCAMBIA2301， 形成中间载体 P35S-2301-GUS;
B. 用 Xbal和 ^cl双切 p35S-2301-GUS和上述人工合成的 EPSP基因， 用 EPSP置换 P35S-2301-GUS相应酶切位点的 GUS, 从而获得高耐受草甘膦的 EPSP合酶基因植物表达载 体。 再将其转入农杆菌中， 用于转化模式植物烟草。 实施例 7 利用叶盘法转化构建抗草甘膦的转基因烟草
( 1 )用无菌牙签挑取 YPE选择平板上的实施例 5中制备的阳性克隆， 接种于 2MLYPE 液体 (Smr， Kanr) ， 28°C， 200rpm振荡培养 24-36小时；
(2) 室温下 4， 000g离心 10分钟；
(3 )弃上清，菌体用 1 I 2MS液体培养基悬浮，稀释到原体积的 5-20倍，使菌体的 OD600 在 0.5左右；
(4)取生长两周左右的烟草的无菌叶片， 去掉其主叶脉， 将其剪成约 lcm2见方的小叶 片；
(5 )将叶片放入制备好的菌液中， 浸泡 2-5分钟， 在无菌滤纸上吸干菌液； 把经浸染的 叶片放于 MS培养基上， 28°C暗培养 48小时；
(6)将叶片转到愈伤培养基 （MS+6-BA 1.0mg/l+NAA 0.1mg/l+Kan 50mg/l+羧苄青霉素 250mg/l) 上， 25-28°C光照下培养， 7-15天可见愈伤组织的形成；
(7 )约 20天后可见分化芽长出， 待芽长大后， 切下， 置于生根培养基 （1 / 2MS+NAA 0.5mg/l+Kan 25mg/l) 上进行生根培养， 2-7天左右生根；
( 8 ) 待根系发达后， 将植株取出， 用无菌水洗净附着着的固体培养基， 移入土壤中， 刚开始几天用玻璃罩罩几天， 待植株健壮后再取下玻璃罩， 转移至在含 10mM的草甘膦的固 体培养基中筛选草甘膦抗性的植株。
(9)抗性植株经 Southern 、 Northern杂交以及 Westhern blot验证为转基因的抗性植株。
( 10) 在温室中经草甘膦抗性梯度实验证明， 转基因植物能在含 20mM草甘膦的培养基 上良好生长。

Claims

权利要求
1. 一种高耐受草甘膦的 EPSP合酶的氨基酸序列， 如 SEQ ID NO:l所示。
2. 编码权利要求 1所述氨基酸序列的 DNA序列， 如 SEQ ID NO:2或 SEQ ID NO:3 所示。
3. 包含权利要求 2所述 SEQ ID NO:2或 SEQ ID NO:3的 DNA序列的重组载体。
4. 用权利要求 3所述的重组载体转化的宿主细胞， 包括原核细胞和真核细胞。
5. 权利要求 2所述的 DNA序列用于培育高耐受草甘膦植物的用途。
PCT/CN2007/071071 2007-11-09 2007-11-16 Epsp synthase possédant une résistance élevée au glyphosate, et sa séquence codée WO2009059485A1 (fr)

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