WO2011050716A1 - Gène résistant au paraquat et son utilisation - Google Patents

Gène résistant au paraquat et son utilisation Download PDF

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Publication number
WO2011050716A1
WO2011050716A1 PCT/CN2010/078137 CN2010078137W WO2011050716A1 WO 2011050716 A1 WO2011050716 A1 WO 2011050716A1 CN 2010078137 W CN2010078137 W CN 2010078137W WO 2011050716 A1 WO2011050716 A1 WO 2011050716A1
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Prior art keywords
paraquat
plant
protein
seq
gene
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PCT/CN2010/078137
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English (en)
Chinese (zh)
Inventor
夏勉
乔琳
王丽英
孔祥凤
刘军华
李毅
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北京未名凯拓作物设计中心有限公司
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Priority to CN2010800415141A priority Critical patent/CN102548388A/zh
Publication of WO2011050716A1 publication Critical patent/WO2011050716A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the invention belongs to the technical field of transgenic plants, and in particular, the invention relates to the application of a high paraquat resistance gene in the cultivation of transgenic plants. Background technique
  • Paraquat (1,1-dimethyl-4,4'-bipyridyl, molecular weight 257.2) is the second largest herbicide after glyphosate. It is a fast-acting contact herbicide with both Suction, mainly used for weeds in orchards, mulberry gardens, tea gardens, forest belts, etc. It is estimated that future applications will increase and increase. Paraquat has been widely used in agricultural cultivation for nearly 50 years and has been widely accepted and welcomed in more than 100 countries around the world. Since the 1990s, with the demand for agricultural production, several pesticide companies in China have started to produce paraquat raw drugs on a large scale, and have continuously improved the production process. The products have been promoted and applied in various places and played a certain role in production.
  • Paraquat is a non-selective herbicide. Plants absorb it very quickly. Even if it is rained shortly after spraying, it does not affect the effect of the drug. The absorbed agent is transmitted through the reverse flow in the xylem. Under suitable conditions, a large amount of the drug is The leaves absorb and conduct to other parts, and this conduction is only a non-protoplast (xylem) conduction, so that the foliar treated paraquat usually stagnates within the treated leaves. As a typical photosynthetic system I inhibitor, the activity of the white grass is determined by light.
  • U.S. Patent Nos. 6,451,543 B1 and US Pat. No. 7,482,425 B2 disclose in SEQ ID No: 27, respectively, a protein identical to SEQ ID No: 2 of the present invention, but they are all used for integration into a lipid matrix, thereby being suitable for FRET analysis.
  • the polypeptide on the membrane does not suggest the use of the invention.
  • Jinki Jo et al. (Biochem. Biophys. Res. Commun. 285 (4), 885-890 (2001)) found a gene greatly different from the SEQ ID No: 2 structure involved in the present invention from Achromobacter (ie pqrA, GenBank: AAF86626.1), the encoded protein has some resistance to paraquat.
  • Jinki Jo et al. transferred the pqrA gene into tobacco, and when it was well expressed, it had some resistance to paraquat compared to wild-type transgenic tobacco. When the transgenic tobacco plants with pqrA gene are compared with wild-type tobacco, the wild type grows in semi-solid medium and when the concentration of paraquat is ⁇ , it will wither.
  • the amount of 20% paraquat water in the field weeding is 100-200 ml/mu, and is diluted to 25 L water according to the conventional usage.
  • 800-1600 mg/L is a commonly used field spraying concentration, and in actual use, It is used by some farmers in over-dosage, so one of the reasons for restricting its commercialization is that the tolerance of transgenic plants to P. humilis is not high, and the resistance far removed from actual use has affected the practical application.
  • the transgenic plants can be rendered tolerant to paraquat (ie, resistant) after introduction of the protein and its encoding gene as shown in SEQ ID No: 2 into plants. It reaches a high level of 3200mg/L, and at the same time makes the plant tolerance to the concentration of H. purpurea in the medium reach a high level of 50mg/L, and the high resistance is still used for a variety of plants, and the application prospect is broad. Summary of the invention
  • the technical problem to be solved by the present invention is to satisfy the requirement of practical application of paraquat in the process of preventing weeds when planting plants or weeds after weeding, and the high concentration of paraquat in the plants to be planted. The growth under the conditions is not affected, thereby specifically killing weeds.
  • the technical problem to be solved by the present invention is also a method for screening, detecting and applying genes for high paraquat resistance to plants.
  • the high paraquat-resistant pqrK2 gene of the present invention and the protein encoded thereby can not only bring the corresponding transgenic plants a paraquat concentration capable of withstanding conventional field application, but are more tolerant to 200 mg/L.
  • concentration of paraquat on the left and right makes the tolerance of redundancy in actual use more suitable for practical application; in addition, the plant adaptation of the high paraquat resistant pqrK2 gene and its encoded protein of the present invention It has a wide range of high paraquat resistance in a large number of transgenic plants that are significantly different from the tobacco genetic background.
  • the present invention provides a method of growing a plant comprising the steps of planting a high paraquat resistant transgenic plant and applying a high concentration of paraquat, wherein the transgenic plant is introduced with a code such as SEQ ID No: 2
  • the gene of the protein (designated herein as pqrK2). Due to the high paraquat resistance pqrK2 gene of the present invention and its The protein of the code can bring high paraquat resistance to the corresponding transgenic plants, and thus can tolerate high concentrations of paraquat, so that when high concentration of paraquat is applied, the growth of the corresponding transgenic plants is not affected, but specifically removed or prevented. Weeds are removed sexually.
  • the method of planting plants of the first aspect of the invention also includes a method of removing weeds, or a method of removing weeds when planting plants.
  • the plant is tobacco, corn, rice, cotton, rape or soybean, and particularly preferably corn, rice, cotton or soybean.
  • administering refers to field application, such as field spraying.
  • concentration of paraquat in this context refers to the concentration applied in the field, which is more close to the practical application than the concentration of paraquat in the medium, unless otherwise specified.
  • the inventors have also found that the high paraquat-resistant pqrK2 gene of the present invention and the protein encoded thereby have a plant tolerance of at least 50 mg/L in the medium, which is much higher than the prior art.
  • the wild pqrA gene confers a concentration tolerance of 5 mg/L in the medium. This confirms from another aspect that the pqrK2 gene of the present invention and the protein encoded thereby are highly paraquat resistant.
  • high concentration of paraquat refers to a concentration of paraquat greater than 800 mg/L unless otherwise specified.
  • the concentration of paraquat is 100 (T3200 mg/L, more preferably 1600 ⁇ 3200 mg/L, such as 1600 mg/L commonly used in the field, or 1700 mg/L, 1800 mg/L, 2000 mg/L, 2300 which may be excessively applied. Mg/L, 2600 mg/L, 2900 mg/L, or 3200 mg/L.
  • the high paraquat resistant pqrK2 gene of the present invention and the encoded protein thereof can be brought to the corresponding transgenic plants. Paraquat resistance up to 3200 mg/L. Accordingly, "high paraquat resistance” as used herein refers to the property of the corresponding plants that is tolerant to the high concentration of paraquat.
  • the "PqrK2 protein” refers to a protein having an amino acid sequence as shown in SEQ ID No: 2 or a functionally equivalent mutant protein thereof, preferably a protein having the amino acid sequence of SEQ ID No: 2.
  • pqrK2 gene herein refers to a gene encoding the above PqrK2 protein.
  • the gene of the present invention is a nucleic acid molecule which may be in the form of DNA or in the form of RNA, preferably in the form of DNA.
  • the DNA form includes natural cDNA and synthetic cDNA, and the DNA may be a coding strand or a template strand.
  • the nucleic acid molecule encoding the protein of SEQ ID No: 2 of the present invention can be easily obtained by a person skilled in the art by a conventional technique such as a PCR method, a recombinant method or a synthetic method, knowing the specific sequence or Its fragment. Once obtained, these sequences can be cloned into vectors, transformed or transfected into corresponding cells, and then propagated by conventional host cells, from which a large number of nucleic acid molecules are isolated.
  • those skilled in the art can search for encoding under stringent conditions by deleting, substituting and/or adding one or several amino acids in the protein represented by SEQ ID No: 2, or by hybridization, according to the teachings herein.
  • nucleic acid which hybridizes with the nucleic acid of the protein represented by SEQ ID No: 2 and the mutant protein and its gene which are functionally equivalent to the PqrK2 protein of the present invention and its pqrK2 gene are selected, which are also included in the scope of the present invention.
  • a nucleic acid encoding a protein deleted, substituted and/or added with the protein of SEQ ID No: 2 or a nucleic acid which hybridizes under stringent conditions to a nucleic acid encoding the protein of SEQ ID No: 2 can be introduced into paraquat-free resistance.
  • the protein equivalent to the amino acid sequence as shown in SEQ ID No: 2 can be screened. Mutant protein and its coding gene.
  • the paraquat resistant transgenic plant can also be screened for a mutant protein having the same amino acid sequence as the protein represented by SEQ ID No: 2 and a gene encoding the same.
  • a gene encoding a mutant protein functionally equivalent to the candidate PqrK2 protein can be introduced into a plant to observe whether or not the protein having the amino acid sequence as shown in SEQ ID No: 2 is functional, thereby selecting a functionally equivalent mutant. Protein and its coding gene.
  • the gene encoding the protein as set forth in SEQ ID No: 2 is introduced into the transgenic plant by a plant transformation vector, preferably the plant transformation vector is pCAMBIA1303-pqrK2.
  • a plant transformation vector preferably the plant transformation vector is pCAMBIA1303-pqrK2.
  • vector refers to bacterial plasmids, cosmids, phagemids, yeast plasmids, plant cell viruses, animal viruses, and various other viral vectors commonly used in the art.
  • “vector” can be divided into “cloning vector", “expression vector” and “transformation vector” in this context, which means that the purpose of use is to clone and verify the gene, express the corresponding gene and Corresponding gene conversion.
  • Vectors suitable for use in the present invention include, but are not limited to, a vector for expression in bacteria (prokaryotic expression vector), a vector for expression in yeast (e.g., Pichia vector, Hansenula vector, etc.), expressed in insect cells.
  • Baculovirus vector, vector for expression in mammalian cells vaccinia virus vector, retroviral vector, adenoviral vector, gland
  • the vector of the fourth aspect of the invention is a transformation vector, in particular a plant transformation vector.
  • the plant transformation vector is pCAMBIA1303-pqrK2, the map of which is shown in FIG. pCAMBIA1303-pqrK2 was constructed by first excising the approximately 2.5 kb gus-gfp fusion gene of pCAMBIA1303, retaining the 35S promoter portion, and inserting the pjrK2 gene into the target gene using Ncol and Bstpl.
  • the gene encoding the protein as shown in SEQ ID No: 2 is derived from a high paraquat resistant bacterial cell.
  • the term "cell” as used herein may be a prokaryotic cell or a eukaryotic cell, such as a bacterial cell, a yeast cell, a plant cell, an insect cell, a mammalian cell or the like.
  • Preferred high paraquat resistant bacterial cells are E. coli cells. Such high paraquat-resistant Escherichia coli can be obtained by screening methods in the following aspects.
  • Application is usually carried out according to the instructions of the operator of paraquat, as indicated by the operator's stated start time, application concentration, application range and duration.
  • the amount of 20% paraquat water in the field weeding is 100-200ml/mu, which is calculated according to the conventional method of dilution to 25L water.
  • 800-1600mg/L is the commonly used field spray concentration.
  • the start time of administration i.e., the time of starting the application, may be when weeds are found to grow.
  • the start time of application is from 3 to 60 days after the start of growth of the plant, preferably from 5 to 50 days after the start of growth of the plant, more preferably from 7 to 30 days after the start of growth of the plant.
  • the time at which the plant begins to grow refers to the time at which the plant seeds are grown under conditions suitable for plant seed growth. The inventors have found that, after too late application, weeds also grow before application and have a certain effect on the growth of the plants.
  • the invention provides a method of screening for high paraquat resistant bacteria, comprising
  • the liquid selective medium is a liquid BS inorganic salt medium containing paraquat, preferably having a content of paraquat of 200 mg/L;
  • the solid selective medium is a solid BS inorganic salt medium containing paraquat, preferably a herb
  • the dry content is 200 mg/L ;
  • the formulation of the liquid BS inorganic salt medium is: per 100 mL of the medium containing: ⁇ 3 ⁇ 4 ⁇ 0 4 ⁇ 23 ⁇ 40 7. 0g, KH 2 P04 3. 0g, NaCl 0 ⁇ 25g, MgS0 4 ⁇ 7H 2 0 0 ⁇ 3g, CaCl 2 ⁇ 2H 2 0 0 ⁇ 02g, FeCl 3 ⁇ 6H 2 0 0.
  • the formulation of the base is a formulation of a liquid BS inorganic salt medium further containing 13. 5 g of agar powder in the formulation.
  • the bacterium is Escherichia coli.
  • the present invention provides a selective medium for screening high paraquat resistant bacteria, which is a BS inorganic salt medium containing paraquat, wherein the BS inorganic salt medium contains a formulation of:
  • Each lOOOOmL of medium contains: ⁇ 3 ⁇ 4 ⁇ 0 4 ⁇ 23 ⁇ 40 7. 0g, KH 2 P04 3. 0g, NaCl 0. 25g, MgS0 4 ⁇ 7H 2 0 0. 3g, CaCl 2 ⁇ 2H 2 0 0. 02g, FeCl 3 ⁇ 6 ⁇ 2 0 0. 045g, MnS0 4 ⁇ 4 ⁇ 2 0 0. 01g, ZnS0 4 ⁇ 7 ⁇ 2 0 0.
  • the BS formulation containing an inorganic salt medium further contains agar powder 13. 5g.
  • the BS inorganic salt medium is a liquid BS inorganic salt medium, and the BS inorganic salt medium contains a formulation containing no 13. 5 g of agar powder; and the BS inorganic salt medium is a solid BS inorganic salt. 5 ⁇ The medium, the composition of the solution containing 13. 5g agar powder.
  • the invention provides a high paraquat resistant transgenic plant or a progeny thereof, a seed into which a gene encoding a protein as set forth in SEQ ID No: 2 is introduced, preferably the plant is tobacco, Corn, rice, cotton, canola or soy.
  • a seed into which a gene encoding a protein as set forth in SEQ ID No: 2 is introduced
  • the plant is tobacco, Corn, rice, cotton, canola or soy.
  • Such plants or their progeny, seeds can be obtained by the planting method of the first aspect of the invention.
  • the present invention also provides a non-reproductive agricultural product processed from the above-mentioned transgenic plants or their progeny and the usable parts of the seeds, such as food or textiles, such as tobacco, rice (rice-free rice after dehydration) Seed embryos, cotton wool, vegetable oil, etc.
  • the processing method is well known to those skilled in the art, it is entirely possible to carry out the method of processing the corresponding non-transgenic plants into corresponding
  • the present invention provides a method for identifying a high paraquat resistant transgenic plant or a progeny thereof, a seed into which a gene encoding a protein as set forth in SEQ ID No: 2 is introduced, comprising from the transgenic plant or Amplification of the sequence encoding the protein as shown in SEQ ID No: 2 was carried out in the progeny and the seed, and the amplified sequence was subjected to sequence analysis and compared with the gene encoding the protein as shown in SEQ ID No: 2. Methods for amplification therein are well known, such as PCR amplification.
  • the gene encoding the protein represented by SEQ ID No: 2 of the present invention is isolated from bacteria, the natural plant or its seed does not contain the gene if the sequence can be amplified from the plant or its seed.
  • the sequence is identical to the gene encoding the protein as set forth in SEQ ID No: 2, then the plant or its seed is a high paraquat resistant transgenic plant into which a gene encoding the protein as set forth in SEQ ID No: 2 is introduced or Its offspring, seeds.
  • the invention provides the use of a gene encoding a protein as set forth in SEQ ID No: 2 for the cultivation of high paraquat resistant transgenic plants.
  • methods for cultivating transgenic plants are precedent.
  • genes can be introduced into plants or tissues by means of Agrobacterium transformation or microprojectile bombardment or electroporation, and then the plants or tissues thereof can be cultured. And screened in the presence of paraquat.
  • the plant is tobacco, corn, rice, cotton, rape or soybean, and particularly preferably corn, rice, cotton or soybean.
  • the present invention will be described in detail below through the specific drawings and embodiments.
  • Fig. 1 is a schematic diagram showing the construction process of the pCAMBIA1303-pqrK2 vector obtained by inserting the pqrK2 gene into the vector pCAMBIA1303.
  • Figure 2 is a photograph showing the performance of transgenic tobacco plants and wild-type plants on a medium with a concentration of 50 mg/L of paraquat.
  • the resistance of the transgenic tobacco plants transformed with pCAMBIA1303-pqrK2 in the left panel is significantly better than that in the wild. Plant type.
  • Figure 3 is an electrophoresis photograph of PCR amplification of genomic DNA of pQrK2 transgenic tobacco T Q plants.
  • the lanes 1 to 10 are transgenic tobacco, the lane M is the molecular weight marker, the lane + is the positive control, and the - is the negative control.
  • Fig. 4 is a comparative photograph of 2 days after spraying 3200 mg/L of paraquat after 30 days of growth of tobacco seedlings, in which the area indicated by the arrow marked with "wild" is planted with wild-type plants, and other well-developed areas are planted with the high of the present invention. Paraquat resistant plants.
  • Fig. 5 is a comparative photograph of before and after spraying 3200 mg/L of paraquat after 7 days of growth of cotton seedlings
  • Fig. 5a is a photograph before spraying
  • Fig. 5b is a photograph of 120 hours after spraying, wherein B of Fig. 5b is wild type cotton which has been dried.
  • a in Figure 5b is the unaffected high paraquat resistant cotton of the present invention.
  • Fig. 6 is a photograph showing the results of the test of the tolerance of paraquat in the leaves of rice seedlings, wherein the area A is the high paraquat resistance rice seedling of the invention, and the B area is the wild type rice seedling.
  • Fig. 7 is a comparative photograph of 120 hours after spraying 3200 mg/L of paraquat after 12 days of growth of the corn seedling, in which the wild type corn of the B area was dried, and the high paraquat resistant corn of the present invention of the A area was not affected.
  • Fig. 8 is a comparative photograph of 120 hours after spraying 3200 mg/L of paraquat after soybean seed growth for 18 days, in which the wild type corn of the B area was dried, and the high paraquat resistant corn of the present invention of the A area was not affected. detailed description
  • the culture solution obtained by the culture was applied to a solid selective medium (1. 5 g of agar powder was added per 100 mL of the medium), and cultured at 35 ° C under constant temperature until colonies which were clearly visible were grown. Then, a single colony was picked, transferred to a liquid selective medium, and cultured at 35 ° C with shaking at 120 rpm until the cell concentration reached 10 7 to 10 8 cfu/mL. Then, the above steps of this paragraph were repeated, and the screening was repeated in a solid selective medium and a liquid selective medium to finally obtain 6 strains which were more resistant to paraquat.
  • One strain of Escherichia coli E. C0 li was named KT-q5366. It was sequenced by gene and found a gene designated pqrK2, the sequence of which is shown as Seq ID No: 1. The protein encoded by PqrK2 is Seq ID No:
  • the genomic DNA of KT-q5366 was extracted as a template, and primers pqrK2-F (5'-CATGCCATGGCAATGAACCCTTATATTTATC-3') and pqrK2-Ra2 (5'-) which respectively introduce Nco I and Bstp I restriction sites were used.
  • GGGTcACCCTTAATGTGGTGTGCTTCGT -3' was amplified by PCR and annealed at 55 °C to amplify the 333 bp PCR fragment pqrK2_333. Then, pqrK2_333 was digested with Ncol and Bstp I, and the product was recovered as a ligation fragment, and pCAMBIA1303 (available from Invitrogen) was double-digested with Ncol and Bstp I as a vector, a T4 DNA ligase ligation fragment and a vector. Finally, the clones with kanamycin resistance were screened and verified as correct plasmids. pCAMBIA1303-pqrK2o Example 3, Plant transformation and seedling screening
  • Tobacco, cotton, rice, corn, and soybean are transformed using conventional Agrobacterium transformation.
  • the constructed plant expression vector pCAMBIA1303-pqrK2 was transformed into Agrobacterium, and then the above plants were separately transformed.
  • the T Q generation (transformed contemporary) transgenic plant seedlings were screened with hygromycin (50 mg/L) resistance and a certain concentration (25-50 mg/L) of paraquat, and then obtained from the screening.
  • Genomic DNA extracted from leaves of transgenic plants T Q generation, by PCR The pqrKl gene was positive. Then, the T Q plants were selfed, and one generation of plants were obtained: 30 tobacco, 23 rice, 19 maize, 21 cotton, and 22 soybean.
  • the first-generation seeds were planted separately according to 50-100 grains per plant.
  • the leaves of the plants were taken to test the resistance of the leaves to paraquat and/or to spray the plants.
  • the process of leaf resistance to paraquat is as follows: Each single plant takes about 2 cm long in the middle of new leaves, soaked in lml (5 ⁇ paraquat, 0.025% Tween) paraquat solution for 24 hours to observe the leaves; the process of spraying experiments As follows: spray the seedlings with paraquat at concentrations of 5mg/L, 15mg/L, 100mg/L, 150mg/L., 200mg/L, and 300mg/L, and add wild type as a negative control to observe and count normal growth. Resistant seedlings. The specific results are as follows:
  • FIG. 2 shows the performance of T Q transgenic tobacco plants and wild-type plants on a medium with a paraquat concentration of 50 mg/L, indicating that certain transgenic tobacco plants have certain resistance to paraquat, and these T Q positive-positive transgenic tobacco plants
  • the genomic DNA extracted from the leaves can amplify a pqrK2 gene fragment of about 330 bp in size (see Figure 3).
  • Fig. 6 The results of the leaf resistance to paraquat test are shown in Fig. 6.
  • the leaf of the rice seedlings with positive paraquat resistance in zone A is obviously superior to the leaves of wild type plants which are conventionally planted in B zone (without adding paraquat). .
  • Plant number Number of resistant seedlings Plant number Number of resistant seedlings

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Abstract

La présente invention concerne une protéine qui possède la séquence d'acides aminés telle que représentée dans SEQ ID NO : 2. Une plante transgénique exprimant la protéine est résistante à au moins 800 mg/l de paraquat. La présente invention porte en outre sur un gène codant pour la protéine, ainsi que sur un procédé de criblage, d'essai et d'utilisation du gène. La présente invention porte également sur un procédé de culture de la plante transgénique.
PCT/CN2010/078137 2009-10-30 2010-10-27 Gène résistant au paraquat et son utilisation WO2011050716A1 (fr)

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CN2010800415141A CN102548388A (zh) 2009-10-30 2010-10-27 百草枯抗性基因及其应用

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6451543B1 (en) * 1998-08-31 2002-09-17 Gryphon Sciences Lipid matrix-assisted chemical ligation and synthesis of membrane polypeptides
US20030018169A1 (en) * 1999-08-26 2003-01-23 Kochendoerfer Gerd G. Lipid matrix-assisted chemical ligation and synthesis of membrane polypeptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6451543B1 (en) * 1998-08-31 2002-09-17 Gryphon Sciences Lipid matrix-assisted chemical ligation and synthesis of membrane polypeptides
US20030018169A1 (en) * 1999-08-26 2003-01-23 Kochendoerfer Gerd G. Lipid matrix-assisted chemical ligation and synthesis of membrane polypeptides

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MORIMYO, M. ET AL.: "Cloning and characterization of the mvrC gene of Escherichia coli K-12 which confers resistance against methyl viologen toxicity.", NUCLEIC ACIDS RESEARCH, vol. 20, no. 12, 25 June 1992 (1992-06-25), pages 3159 - 3165 *
YELIN, Y. ET AL.: "EmrE, a Small Escherichia coli Multidrug Transporter, Protects Saccharomyces cerevisiae from Toxins by Sequestration in the Vacuole.", JOURNAL OF BACTERIOLOGY., vol. 181, no. 3, 28 February 1999 (1999-02-28), pages 949 - 956 *
YERUSHALMI, H. ET AL.: "EmrE, an Escherichia coli 12-kDa Multidrug Transporter, Exchange Toxic Cations and H+ and Is Soluble in Organic Solvents.", THE JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 270, no. 12, 24 March 1995 (1995-03-24), pages 6856 - 6863 *

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