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WO2002006319A2 - Molecule d'acide nucleique codant une amp-desaminase vegetale - Google Patents

Molecule d'acide nucleique codant une amp-desaminase vegetale Download PDF

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Publication number
WO2002006319A2
WO2002006319A2 PCT/EP2001/007767 EP0107767W WO0206319A2 WO 2002006319 A2 WO2002006319 A2 WO 2002006319A2 EP 0107767 W EP0107767 W EP 0107767W WO 0206319 A2 WO0206319 A2 WO 0206319A2
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Prior art keywords
nucleic acid
plant
amp deaminase
acid sequence
protein
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German (de)
English (en)
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WO2002006319A3 (fr
Inventor
Arno Schulz
Wolfgang Streber
Christiane Hanke
Frank Schmidt
Andreas Schubel
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Bayer CropScience AG
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Aventis CropScience GmbH
Bayer CropScience AG
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Priority to AU2001272525A priority Critical patent/AU2001272525A1/en
Priority to EP01951656A priority patent/EP1303618A2/fr
Publication of WO2002006319A2 publication Critical patent/WO2002006319A2/fr
Publication of WO2002006319A3 publication Critical patent/WO2002006319A3/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)

Definitions

  • Nucleic acid mole oil which codes for a plant AMP deaminase
  • AMP deaminase (adenosine monophosphate deaminase) (EC 3.5.4.6) catalyzes the irreversible hydrolytic deamination of the amino group of AMP (adenosine monophosphate) to equimolar amounts of IMP (inosine monophosphate) and ammonia in eukaryotes.
  • AMP deaminase (AMP-DA) can be found in all eukaryotic organisms (Meyer et al., Biochemistry, 28 (1989), 8734-8743; Thakkar et al., Biochem. J., 290 (1993), 335- 341).
  • the enzyme is regarded as a key enzyme in the control of the energy balance and the purine metabolism of eukaryotic cells.
  • the AMP deaminase plays an important role in the regulation of the energy status of the cell due to its function in the breakdown of AMP in plants (Lowenstein J.M., Int. J. Sports Med., 11 (1990), 37-46).
  • the plant AMP deaminase has been recognized for the first time by Dancer (Dancer et al., Plant Physiol., 114 (1997), 119-129) because of its role in plant purine metabolism as a herbicidal site of action.
  • a known inhibitory molecule of AMP deaminase is the carbocyclic derivative of coformycin, polygocidine. This is a natural product from the microorganism Saccharothrix spp. (Dancer et al., Plant Physiol., 114 (1997), 119-129). The effectiveness of polygocidin has been shown in various mono- and dicotyledon weeds.
  • the amount of active ingredient required for control is less than 32 g / ha in the post-emergence process in order to achieve a herbicidal action (Bush et al., Phytochemistry, 32 (1993), 737-739).
  • Symptoms observed on the plant after application of coformycin are, for example, the impairment of plant growth as a result of Bleaching and necrosis on apica bucket plants.
  • Dancer (Dancer et al., Plant Physiol., 114 (1997), 119-129) was able to show that in planta the herbicidal action of carbocyclic coformycin on the inhibition of AMP deaminase activity by the in vivo phosphorylated form (phosphorylated carbocyclic Coformycin).
  • AMP deaminase The consequence of the inhibition of AMP deaminase is presumably an accumulation of AMP, which is converted to ADP (adenosine diphosphate) by the activity of the adenylate kinase.
  • a measurable consequence of the application of the herbicide is the increase in the ATP concentration, since the synthesis of ATP (adenosine triphosphate) is determined by the availability of ADP and free phosphate (Dancer et al., Plant Physiol., 114 (1997), 119- 129).
  • Plant AMP deaminases could be partially purified from tissues of various plants (Yoshino & Murakami, Z. Plant Physiology, 99 (1980), 331-338; Lo Floc'h & Lafleuriel, Physiol. Veg., 21, (1982), 15- 17, Yabuki & Ashihara, Phytochemistry, 31 (1992) 1905-1909, Dancer et al., Plant Physiol., 114 (1997), 119-129). So far, however, no peptide sequences have been obtained from partially purified AMP deaminase fractions from plants, so that oligonucleotides for the detection of corresponding gene sequences could have been derived from this.
  • the availability of required amounts of an enzyme with high purity is advantageous for such a test system.
  • the availability of a recombinantly produced plant AMP deaminase would greatly facilitate the process of finding inhibitors of the enzyme.
  • larger quantities could be produced more easily and inexpensively, and on the other hand, its use would prevent the influence of disruptive side activities that can occur or must be taken into account when using raw plant extracts.
  • the ability to produce a recombinant enzyme requires the provision of a DNA molecule or cDNA molecule that has the nucleic acid sequence encoding such a plant AMP deaminase.
  • a cDNA molecule is a double-stranded DNA molecule, which is synthesized by synthesis of a DNA strand complementary to the mRNA, the so-called cDNA first strand, and subsequent synthesis of the first strand complementary second strand of DNA is obtained.
  • This method is a method known to the person skilled in the art in order to convert sequence information present at the mRNA level into a more manipulable and clonable nucleic acid, the DNA.
  • the isolated nucleic acid molecules thus obtained and the sequence information contained on them can then be used to implement an in vitro test system (screening) which is based on the use of the proteins encoded and expressed by at least one cDNA sequence.
  • the cDNA obtained is cloned into a suitable expression system which serves to express the protein encoded by the cDNA, in this case the AMP deaminase, in a suitable host organism.
  • This recombinant protein can then be obtained in a sufficiently pure form from the expression batch and is available for biochemical analysis or else directly for an in vitro test system in which the effectiveness of substances, for example from different chemical libraries, is tested based on precisely this protein can be.
  • the invention relates to an isolated nucleic acid molecule containing a nucleic acid sequence which codes for a protein which has the biological activity of a plant AMP deaminase, the nucleic acid sequence being selected from the group consisting of:
  • nucleic acid sequences which are fragments, derivatives or allelic variants of the nucleic acid sequences mentioned under (a) to (c).
  • the term derivative denotes changes which either have no influence on the amino acid sequence, as is the case, for example, through changes in the third base (the so-called wobble base) of a triplet codon of a nucleic acid sequence, or but which have an influence on the amino acid composition, but the total protein then still has the biochemical characteristics and / or enzymatic properties of an AMP deaminase.
  • allelic variants of the nucleic acid sequence given under SEQ ID NO 1 can be obtained from the same organism from which the nucleic acid molecule according to (a), (b) or (c) originates. In general, allelic variants of this type can be found either by searching for it directly with the entire nucleic acid sequence according to SEQ ID NO 1 or fragments thereof in corresponding gene banks, or by searching on the basis of probably conserved regions of the amino acid sequence shown in SEQ ID NO 2 PCR primers (starter molecules for the polymerase chain reaction) developed that are degenerate at several positions.
  • Nucleic acid molecules which hybridize with nucleic acid molecules which contain the nucleic acid sequence according to SEQ ID NO 1 or fragments thereof can be isolated, for example, from genomic or from cDNA libraries of different organisms. Such nucleic acid molecules can be identified and isolated from plants or other organisms using the nucleic acid molecules according to the invention (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). For example, nucleic acid molecules which have exactly or essentially the sequence or fragments of this sequence indicated under SEQ ID NO 1 can be used as the hybridization sample.
  • the nucleic acid molecules used as hybridization samples can also be act synthetic nucleic acid molecules which have been produced with the aid of the usual synthetic techniques and whose sequence essentially hybridizes with the sequence according to the invention. If RNA molecules are used as a hybridization sample, they are generally produced with the aid of in-v / ⁇ ro transcription from a DNA molecule which is integrated into a corresponding ⁇ ro-transcription vector known to the person skilled in the art.
  • a nucleic acid sequence which has significant homology to the coding nucleic acid sequence SEQ ID NO 1 or to its complementary sequence is characterized in that the coding sequence is generally at least 70%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95%, particularly preferably at least 98%, or most preferably at least 99% sequence identity to the nucleic acid sequence SEQ ID NO 1 or its complement over a length of at least 20, preferably at least 30, particularly preferably at least 40, very particularly preferably at least 60, in particular preferably has at least 100 bases in a continuous sequence, and most preferably has the full length of SEQ ID NO 1. Any combination of the above degrees and minimum sizes can be used to define the nucleic acid sequence of the invention, with more stringent conditions being the preferred choice.
  • both a nucleic acid sequence with at least a sequence identity of 90% over 20, preferably over 30 nucleotides is a subject of the invention, just as is the case with a nucleic acid sequence with 95% sequence identity over a length of 40 nucleotides.
  • the invention furthermore relates to nucleic acid molecules or fragments, derivatives or allelic variants thereof which hybridize specifically with the nucleic acid molecule according to the invention or fragments, derivatives or allelic variants thereof.
  • the corresponding fragments, derivatives or allelic variants can be by insertion, deletion, addition, substitution or inversion have arisen.
  • the minimum length of a hybridizing nucleic acid molecule is 15 nucleotides.
  • the term “specifically hybridize” means that under conventional hybridization conditions, preferably under stringent conditions, no significant cross-hybridization with nucleic acid sequences which code for proteins other than the protein according to the invention can be observed.
  • nucleic acid molecules have a total length of at least 20 nucleotides, preferably a length of at least 50 nucleotides and particularly preferably a length of at least 100 nucleotides, such molecules can be used as PCR primers or as hybridization samples
  • the coding nucleic acid sequence SEQ ID NO 1 can be changed by nucleotide substitutions, for example by 1, 2, or 3 to 10, 25, 50, 100 or 300 exchanges.
  • primers for example as PCR primers (polymerase chain reaction primers), as primers for alternative amplification procedures, as detection molecules with radioactive or non-radioactive labeling or for cloning in suitable vectors.
  • PCR primers polymerase chain reaction primers
  • detection molecules detection molecules with radioactive or non-radioactive labeling or for cloning in suitable vectors.
  • detection molecules or other fragments are preferably at least 10, at least 15, at least 20, at least 25, at least 30 or at least 40 nucleotides long.
  • nucleotides typically they are, for example, 40, 50, 60, 70, 100 or 150 nucleotides long or, for example, 200, 300, 400, 500, 600 or 700 nucleotides long or only a few nucleotides such as 5 or 10 nucleotides shorter than the entire coding sequence according to SEQ ID NO 1.
  • Another object of the invention is a method for producing a cDNA which codes for a plant AMP deaminase, preferably for an AMP deaminase from Arabidopsis thaliana, and which has been isolated from an mRNA from plant tissue.
  • the method includes the following steps, but is not limited to them, such as: (a) isolating an mRNA population from plant tissue; and
  • the invention furthermore relates to vectors which contain the nucleic acid sequence according to the invention, the non-homologous host organism being, for example, a bacterium, a yeast cell, an insect cell or a mammalian cell.
  • vector means a naturally occurring or artificially created nucleic acid molecule which serves for the uptake, multiplication, expression or transfer of further nucleic acid molecules such as the inventive nucleic acid sequences with the nucleic acid sequences contained on them.
  • the invention further relates to host cells which have the nucleic acid molecules according to the invention or vectors which contain the nucleic acid sequences according to the invention.
  • the host cell can be a prokaryotic, for example bacterial, or eukaryotic cell, for example a plant cell, a yeast cell, an insect cell or a mammalian cell.
  • the invention relates to a vector which contains the nucleic acid sequence according to the invention, the non-homologous host organism E. coli or Saccharomyces cerivisiae, or an insect cell, in particular a Schneider cell of the SL-3 line.
  • Another object of the invention is the use of one or more nucleic acid molecules containing the nucleic acid sequences according to the invention thereof for the transformation of prokaryotic or eukaryotic cells.
  • transformation in the case of eurkaryotic cells, and here in particular plant cells, means a stable integration of a nucleic acid sequence that does not occur at a certain point in the genome of the host organism and which either already exists at least once in the organism in an identical or similar form itself, or else was taken from another organism.
  • transformation means the transfer and stable replication of extrachromosomal vectors such as, for example, of plasmids or viruses.
  • the transformed nucleic acid molecule contains the nucleic acid sequence according to the invention, as well as the corresponding regulatory elements necessary for the start and stop of transcription.
  • the orientation of the integrated nucleic acid sequence with respect to that surrounding it can. Regulatory elements in sense orientation, i.e., the nucleic acid sequence potentially codes for a corresponding polypeptide, or in antisense orientation, i.e., the nucleic acid sequence codes exactly for a polypeptide.
  • Another object of the invention is the production of a recombinant protein which has the biochemical characteristics of an AMP deaminase, the production being characterized, for example, in that:
  • nucleic acid molecule which contains the nucleic acid sequence according to SEQ ID NO 1 or fragments, derivatives or allelic variants thereof is integrated into a suitable expression vector;
  • Expression vector according to (a) is transferred into a corresponding host cell; and (c) the expressed protein is optionally selectively isolated from the host organism or the medium surrounding it;
  • the protein is preferably enzymatically active even after its isolation.
  • fusion proteins have several advantages. If the foreign protein is linked to a host cell's own protein domain, proteolytic degradation is often slowed down considerably, or the toxicity of the foreign protein to the host organism is reduced, which means that the yield of the desired foreign protein can be significantly increased. In addition, the fusion of a hydrophobic protein which tends to form inclusion bodies with a hydrophilic protein often prevents aggregation. A recombinant protein can be purified significantly more efficiently if a protein domain is added by the gene fusion which has been carried out, which purification by means of affinity chromatography A distinction is made here between real fusion proteins, which consist of heterologous proteins or protein domains, from proteins which have so-called “tags”. These are short terminal amino acid sequences.
  • Such a “tag” can be, for example, a poly His tag, the fusion protein having this “tag” then being purified using Ni 2+ complexation becomes.
  • tags such as, for example, the poly-Arg tag, the Ply-Glu tag or the Asp-Tyr-Lys-Asp 4 -Lys (FLAG) are known.
  • ⁇ -galactosidase, protein A come as fusion partner proteins , Streptavidin, glutathione-S-transferase or the maltose binding protein in question, to name but a few.
  • fusion protein in this form can then be purified in two successive steps.
  • yeast cells or insect cells as host cells has proven particularly advantageous in the production of recombinant proteins which are based on eukaryotic amino acid sequences.
  • yeast cell already has many features of an expression system of higher eukaryotes, it is largely possible to use this expression system to obtain authentic eukaryotic proteins which in some cases carry all post-translational modifications of the original non-recombinant protein.
  • the recombinant protein can either be present in the cytosol of the insect cells or secreted into the endoplasmic reticulum (ER).
  • ER endoplasmic reticulum
  • Another object of the invention is the use of the recombinant protein, which is based on the nucleic acid sequence according to the invention and is encoded by this, for the discovery of new inhibitors.
  • the recombinant protein which is based on the nucleic acid sequence according to the invention and is encoded by this, for the discovery of new inhibitors.
  • a recombinant protein is to be understood as a protein which is synthesized in a heterologous host system, for example in the case of the vegetable AMP deaminase in a bacterial cell, a yeast cell, an insect cell or in an acid cell, and this either in its Amino acid composition corresponds to a sequence according to SEQ ID NO 2, or the amino acid composition of which differs from the amino acid composition given in SEQ ID NO 2, but then still has the biochemical characteristics and / or enzymatic properties of a plant AMP deaminase.
  • a recombinant protein can also have changes / additions at the amino-terminal and / or carboxy-terminal end which, for example, facilitate purification or increase the purification yield and preferably do not influence its enzymatic properties, or if so, only insignificantly.
  • the invention furthermore relates to the use of the recombinant protein, which is based on the nucleic acid sequence according to the invention, to find new herbicidal inhibitors.
  • the invention furthermore relates to the use of the recombinant protein, which is based on the nucleic acid sequence according to the invention, for the biochemical or structural characterization of inhibitors.
  • the nucleic acid molecules according to the invention it is also possible, using genetic engineering methods, to produce plants which contain at least one further gene coding for a homologous or heterologous AMP deaminase and which also have an expression of these homolologous or heterologous AMP deaminase.
  • nucleic acid sequence coding for an AMP deaminase is introduced into the plant with the aid of genetic engineering methods, which precisely did not already exist in the plant genome into which it is now integrated.
  • the nucleic acid molecules according to the invention are linked to regulatory elements which ensure transcription and translation in plant cells and introduced into plant cells in order to ensure optimal and stable transcription and translation in the plant cells of the target organism.
  • any promoter which is functional in a plant cell can be used for the expression of the various nucleic acid sequences according to the invention described in plants.
  • the promoter can be homologous or heterologous with respect to the plant species used.
  • the 35S promoter of the Cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812) is suitable, which ensures constitutive expression in all tissues of a plant.
  • promoters can also be used which lead to an expression of subsequent nucleic acid sequences only at a point in time determined by external influences or in a specific tissue of the plant (Stockhaus et al., EMBO J. 8 (1989), 2245-2251).
  • nucleic acid segments for initiating the transcription can also contain nucleic acid sequences which ensure a further increase in the transcription activity, for example so-called enhancer elements.
  • regulatory elements can also include termination signals which serve for the correct termination of the transcription and the addition of a poly-A tail to the transcript, which is assigned a function in stabilizing the transcripts.
  • termination sequences are the 3'-untranslated regions which contain the polyadenylation signal of the nopaline synthase gene (NOS gene) or the octopine synthase gene (Gielen et al., EMBO J.
  • the invention also relates to transgenic plant cells which contain a nucleic acid molecule according to the invention, this being linked to regulatory elements which ensure transcription in plant cells.
  • the regulatory elements are preferably heterologous with respect to the nucleic acid molecule according to the invention, i.e. they have no natural biological connection with the nucleic acid sequence according to the invention.
  • the invention further relates to vectors which contain the nucleic acid molecules according to the invention, in particular those in which the nucleic acid molecules described are linked to regulatory elements which ensure the transcription of the nucleic acid sequences according to the invention in plant cells.
  • the nucleic acid molecules according to the invention are preferably introduced into plant cells using plasmids. Plasmids are preferably used for this, which ensure stable integration into the plant genome.
  • plasmids which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells.
  • examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc.
  • the nucleic acid molecule that the Desired nucleic acid sequence can be introduced into the vector at an appropriate restriction site.
  • the plasmid obtained is used for the transformation of £ .co // cells. Transformed E.co// cells are cultivated in a suitable medium, then harvested and lysed.
  • the plasmid is recovered using standard methods. Restriction analyzes and sequence analyzes are generally used as the analysis method for characterizing the plasmid DNA obtained. After each manipulation, the plasmid DNA can be cleaved and the resulting fragmented nucleic acid molecules can be linked to other nucleic acid molecules in selected or unselected form.
  • the invention relates to the use of one or more nucleic acid molecules which contain the nucleic acid sequence according to the invention for the production of transformed plants, the transformed plants having an increased resistance to one or more AMP deaminase inhibitors to plants which have not been transformed but are otherwise genetically identical ,
  • Another object of the invention is a transformed cell, preferably a transformed plant cell which contains the nucleic acid sequence according to the invention, wherein, in the case of the transformed plant cell, the chromosomally stable integrated nucleic acid sequence is particularly preferably understood.
  • the invention further relates to transformed plant tissue and fertile transformed plants which contain the nucleic acid sequence according to the invention.
  • the invention furthermore relates to the seed which is obtained from the transformed plants according to the invention.
  • a particularly preferred object of the invention are transformed plant cells, plants and plant seeds of crop plants such as alfalfa, cotton, barley, oats, potatoes, maize, rice, rye, soy, summer rape, sunflower, tomato, wheat, winter rape or sugar beet.
  • the transgenic plant cells can be regenerated to complete plants using the techniques known to those skilled in the art.
  • the plants obtainable by regeneration of the transgenic plant cells according to the invention are also the subject of the present invention.
  • the invention furthermore relates to plants which contain the transgenic plant cells described above.
  • the transgenic plants can in principle be plants of any plant species, ie both monocot and dicot plants. They are preferably useful plants, in particular useful plants such as alfalfa, cotton, oats, potatoes, corn, rice, rye, soybeans, summer rape, sunflower, tomato, wheat, winter rape or sugar beet.
  • a variety of techniques are available for the introduction of nucleic acid molecules in naked form or integrated into a cloning vector in a plant host cell. These techniques include transforming plant cells with T-DNA using Agrobacte um tumefaciens or Agrobacterium rhizogenes as transformation agents (Hernalsteens et al., Nature, 287 (1980), 654-658; Fraley et al., Crit. Rev. Plant Sei ., 4 (1985, 1-46; An et al., EMBO J., 4 (1985), 277-287; Bevan et al., Nucleic Acids Research, 12 (1984), 8711-8721; Chan et al. , Plant Mol.
  • Another common method is the introduction of nucleic acid molecules n using the biolistic method (particle bombardment technique) (Christou, Curr. Opinion Biotechnol., 4 (1993), 135-143; Christou et al., Bio / Technology, 9: 957-962 (1991); Iglesias et al., 1994, Planta, 192: 84-91; Russell et al., In Vitro Cell Div. Biol, 28P (1992) 97-100; Pereira and Erickson, Plant Cell. Rep., 1995, 14: 290-293; Becker et al., The Plant Journal, 5 (1994), 299-307).
  • Methods for plant transformation are also known, such as injection or electroporation.
  • plasmids In the case of injection and electroporation as well as in the protoplast transformation of nucleic acid molecules in plant cells, there are no special requirements for the plasmids used. Simple plasmids such as pUC derivatives (Vieira and Messing, Gene, 19 (1982), 259-268) can be used. However, if entire plants are to be regenerated from cells transformed in this way, the presence of a selectable marker gene is necessary.
  • nucleic acid sequences may also be necessary in addition to the nucleic acid sequences according to the invention.
  • the Ti plasmid from Agrobactenum tumefaciens
  • the Ri plasmid from Agrobactenum rhizogenes
  • the Ti plasmid must be used, at least the right boundary, but often the right and left boundary of the Ti and Ri plasmid T-DNA as a flank area with the genes to be introduced.
  • the nucleic acid molecule to be introduced must be cloned into special plasmids, either in an intermediate vector or in a so-called binary vector.
  • the intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria by homologous recombination on the basis of nucleic acid sequences which are homologous to nucleic acid sequences in the T-DNA. This also contains the vir region necessary for the transfer of the T-DNA.
  • Intermediate vectors cannot replicate in agrobacteria.
  • the intermediate vector can be transferred to Agrobaterium tumefaciens by means of a helper plasmid (conjugation).
  • Binary vectors can replicate in both E.
  • coli and agrobacteria contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holster et al., Mol. Gen. Genet. 163 (1978), 181-187). The one for the transformation of agrobacteria
  • the plasmids used also contain a selection marker gene, for example the NPT II gene, which allows the selection of transformed bacteria.
  • the agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present.
  • the agrobacterium transformed in this way is used to transform plant cells.
  • the use of T-DNA for the transformation of plant cells has been intensively investigated and sufficient (Hoekema, In: The Binary Vector System Offsetdrukkerrij Kanters B ⁇ /., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Plant. Sei. , 4: 1-46 and An et al., EMBO J. 4 (1985), 277-287).
  • Binary vectors are partly commercially available, for example pBIN19 (Clontech Laboratories, Ins., USA).
  • plant explants can expediently be cultivated with Agrobactenum tumefaciens or Agrobactenum rhizogenes.
  • Whole plants can then be regenerated from the infected plant material (e.g. leaf pieces, stem segments, roots, but also protoplast or suspension-cultivated plant cells) in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells.
  • the plants thus obtained can then be examined for the presence of the introduced nucleic acid sequences.
  • the nucleic acid sequence that has been introduced is integrated into the genome of the plant cell, it is generally stable there and is also retained in the progeny of the originally transformed cell. It usually contains a selection marker that gives the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin and others. taught.
  • the individually selected marker should therefore allow the selection of transformed cells from cells that lack the nucleic acid sequence introduced.
  • the transformed cells grow within the plant in the usual way (see also McCormick et al., Plant Cell Reports 5 (1986), 81-84).
  • the resulting plants that have the same transformed genetic makeup or other genetic makeup are crossed.
  • the resulting hybrid individuals have the corresponding phenotypic properties. Two or more generations should be attracted to ensure that the phenotypic trait is stably maintained and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other characteristics have been preserved.
  • Green leaf material was frozen in liquid nitrogen and ground into a plant powder in a mortar. Plant material was resuspended in the GTC buffer and incubated for 20 min with gentle shaking. The suspension was centrifuged and the aqueous supernatant applied to a CsCI cushion (5.7 M) and centrifuged. The ultracentrifugation took place at 35000 rpm, for 18 h and at 20 ° C. (Method according to Chirgwin et al., Biochem. 18 (1979), 5294-5299).
  • RNA pellet obtained was taken up in DEPC H 2 O (water treated with diethyl pyrocarbonate) and precipitated with 96% ethanol pa The precipitated RNA was dissolved in DEPC (diethyl pyrocarbonate) treated H 2 O and stored at -80 ° C until further use. 5 ⁇ g of the total RNA isolated were used in the reverse transcription.
  • AMP deaminase cDNAs of various species were used as starting sequences. Homology was found at the protein level between the AMP deaminase described in mouse and the EST (Expressed Sequence Tag) T21250 from A.thaliana. For full-length cloning of the EST T21250, a Lambda UNI-ZAP (Stratagene) Arabidopsis thaliana cDNA bank was examined for further homologous sequences by means of PCR.
  • a volume of 1 ⁇ l from the cDNA bank was used for the PCR amplification and for this purpose with 5 ⁇ l PCR-Taq buffer (Qiagen), 1 ⁇ l T3 primer (25 pmol), 1 ⁇ l GA3 / GA4 primer (25 pmol), 1 ⁇ l dNTPs (25mmol), 10 ul 5 x Q solution and 30 ul H 2 0 and 1 ul Taq polymerase [5U / ul] (Qiagen) added.
  • the PCR program carried out comprised the following cycles:
  • Approx. 400 bp (pL2) and approx. 500 bp (pL13) PCR fragments could be amplified.
  • the two PCR fragments were cloned into the pCRII TOPO (Invitrogen) vector using the topoisomerase according to the manufacturer's instructions (Invitrogen).
  • the resulting pCRII TOPO vector which contained the cloned PCR fragments, was sequenced from both directions with the commercially available standard primers "T7" and "SP6".
  • the sequence "pL13 fill EST.seq” resulted from overlapping of the nucleic acid sequences pl2, pH 3 and the EST T21250. This nucleic acid sequence contains the postulated 3 'region of the AMP deaminase (stop codon) and parts of the poly A tail).
  • T3 primer (SEQ ID NO 3)
  • the following peptide partial sequence of the AMP deaminase from Arabidopsis thaliana is obtained by translating the pL13fuh EST.seq.
  • the putatively translated peptide sequence consists of 292 amino acids. SEQ ID NO .: 18
  • the marathon cDNA amplification kit used is divided into two parts. a) Reverse transcription (first and second strand synthesis) and ligation of the adapters, and b) 2.PCR amplifications
  • the mRNA was isolated from the total RNA isolated according to Example 1 using the Oiigotex mRNA kit (Qiagen).
  • the Arabidopsis thaliana mRNA thus obtained was used as the starting material for the first-strand synthesis.
  • 4 ⁇ l mRNA (1 ⁇ g) and 1 ⁇ l cDNA Synthesis Primer (10 ⁇ M) were incubated for 2 min at 70 ° C and then for 2 min on ice and with 2 ⁇ l 5x first strand buffer; 1 ul dNTP (deoxy nucleotide triphosphate) mix (10mM); 1 ⁇ l H 2 O; 1 ⁇ l AMV reverse transcriptase (20 units / ⁇ l) filled up. The subsequent incubation was carried out for 1 hour at a temperature of 42 ° C.
  • the nucleic acid was concentrated from the aqueous phase by means of ethanol precipitation.
  • the cDNA pellet resulting from the ethanol precipitation was dissolved in 10 ⁇ l sterile H 2 O. 5 ⁇ l of this was used for the subsequent adapter ligation.
  • 2 ⁇ l marathon cDNA adapter (10 ⁇ M), 2 ⁇ l 5 ⁇ DNA ligation buffer, 1 ⁇ l T4-DNA ligase (1 ⁇ / ⁇ l) were added to the ligation mixture. This was followed by incubation for 12 hours at a temperature of 16 ° C.
  • the ligation mixture was resuspended in 250 ⁇ l tricin-EDTA buffer and used as a DNA template for the PCR amplifications.
  • a volume of 2 ⁇ l of the total amount of cDNA was used for PCR amplification and for this purpose with 5 ⁇ l PCR buffer, 1 ⁇ l I 3 * AMP-DA4 primer (10 pmol), 1 ⁇ l
  • AP1 primer (10 pmol), 1 ⁇ l dNTPs (25mmol), 2 ⁇ l MgCl2 (25mmol) and 37 ⁇ l H 2 O as well as 1 ⁇ l Taq polymerase [5U / ⁇ l] (Boehringer) were added.
  • the PCR program carried out comprised the following cycles:
  • a volume of 1 ⁇ l DNA was removed from the DNA generated in the first PCR reaction (1st PCR amplification) and with 5 ⁇ l PCR buffer; 1 ⁇ l 3 ⁇ MP-DA5 primer (10pmol), 1 ⁇ l AP2 primer (10pmol), 1 ⁇ l dNTPs (25mmol), 2 ⁇ l MgCI 2 (25mmol) and 37 ⁇ l H20 as well as 1 ⁇ l Taq polymerase [5U / ⁇ l] mixed.
  • the PCR program carried out comprised the following cycles: 1 min, 94 ° C; 1 cycle; 1 min, 94 ° C; 1min, 54 ° C; 3 min, 72 ° C; 5 cycles; 1 min, 94 ° C; 1min, 54 ° C; 3 min, 70 ° C; 5 cycles; 1 min, 94 ° C; 1min, 58 ° C; 3 min, 68 ° C; 25 cycles.
  • PCR batches were separated on a 1% TBE agarose gel (Sambrook et al., (1989) Molecular Cloning - A Laboratory Manual; Cold Spring Harbor Laboratory Press, New York).
  • a PCR amplification product of approx. 1200-1300 bp was obtained.
  • the resulting PCR product was introduced into the cloning plasmid pT-Adv (Clontech) via the T / A cloning site.
  • V G, A or C
  • N G, A, C or T
  • the 3 primers named above come from the Marathon TM cDNA Amplification Kit (Clontech) 3 'AMP-DA4 primer (1261-1246) (SEQ ID NO 9) ⁇ '-GCA-CTA-GGT-CTC-AGC-GAA-TAT-TAC-TAC-CTC-ATC-G-S'
  • the two gene-specific primers (DA4 and DA5) were derived from the postulated cDNA of AMP-Deminase. The position of the primers on the corresponding DNA sequence is given in brackets.
  • the primers were synthesized using the cyanoethylphosphoamidite method (Gibco BRL).
  • the resulting vector pT-AdV which contained the cloned AMP deaminase cDNA, was sequenced from both directions using the commercially available standard primers “M13 forward” and “M13 reverse”.
  • the full-length cDNA of AMP deaminase was amplified using a polymerase chain reaction (PCR) and cloned into the expression vector PASK IBA3 (IBA).
  • PCR polymerase chain reaction
  • PASK IBA3 The cDNA produced from Arabidopsis thaliana, as described in Example 2, was used as template.
  • a volume of 2 ⁇ l of the total amount of cDNA was used for the PCR amplification and with 5 ⁇ l PCR buffer, 1 ⁇ l 5 ′ AMP-DA1 primer (25 pmol), 1 ⁇ l 3 ′ AMP-DA1 primer (25 pmol), 1 ⁇ l dNTPs (25mmol), 2 ⁇ l MgCl 2 (25mmol) and 37 ⁇ l H 2 O as well as 1 ⁇ l native Pfu polymerase [2.5U / ⁇ l] (Stratagene) were added.
  • the PCR program carried out comprised the following cycles: 1 min, 94 ° C; 1 cycle;
  • the corresponding theoretical size of the nucleic acid sequence for AMP deaminase from Arabidopsis thaliana is 2520 bp (coding reading frame).
  • the resulting PCR fragment had a size of approximately 2500 bp.
  • This fragment was isolated and integrated into the bacterial expression vector PASK IBA3 (IBA) according to the manufacturer's instructions.
  • the vector pASK IBA3 was cut with oa 1 / Bsa I and the PCR amplificate with Nhe I and Bsa I.
  • the vector was isolated on a preparative TBE agarose gel.
  • the appropriately restricted digested PCR amplificate was purified via a PCR purification column (Qiagen) and integrated into the bacterial expression vector PASK IBA 3 (IBA).
  • the ligation product was used to transform competent Ash chia coli DH10B cells (Gibco).
  • the vector was named PASK IBA3 AMP-DA.
  • the resulting vector which contained the cloned AMP deaminase cDNA, was sequenced with the standard primers "PASK IBA3 forward" and "PASK IBA3 reverse” from both directions.
  • PASK IBA3 AMP-DA the stop codon of AMP deaminase (TAA) has been replaced by a spacer (CCTCCT).
  • the spacer results in an additional pro-pro amino acid sequence at the carboxy-terminal end.
  • the spacer is followed by a nucleic acid sequence which codes for a so-called strep tag (IBA) and which ends with a stop codon, so that the carboxy-terminal end is clearly defined.
  • IBA strep tag
  • the cDNA clone coding for the AMP deaminase from Arabidopsis thaliana which corresponds to SEQ ID NO 1, was used in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure in the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH deposited.
  • the assigned DSMZ catalog number is: DSMZ Plasmid PASK IBA3-AMP-Deaminase - DSM 13440
  • RNA samples which had previously been subjected to ethanol precipitation, were pelleted and dried.
  • the RNA was resuspended in 30 ⁇ l denaturation buffer, heated to 70 ° C. for 15 minutes and mixed with 3 ⁇ l application buffer before application. The electrophoresis was carried out at 100 V for 5 hours.
  • the nylon membrane Hybond-NX (Amersham Lifescience) was used for the Northern blot. The transfer took place according to the method of Sambrook, Fritsch, Maniatis (Molecular Cloning, A Laboratory Manual). 20 ⁇ SSC (sodium salt citrate, tri-sodium citrate) was used as transfer medium; the transfer from the gel to the filter membrane took place over a total time of 24 hours. The membrane was then washed with 4 x SSC. The RNA was fixed covalently (UV crosslink) with the Stratalinker II (Stratagene) on the membrane. The membrane was prehybridized with 50 ml Herby buffer in a roller hybridization oven at 60 ° C. for 4 hours.
  • the nucleic acid fragments were labeled with the Random Primer Labeling System Kit (Gibco BRL) according to the manufacturer's instructions.
  • the radiolabelled, heat-denatured probe was then added to the hybridization mix. Hybridization was carried out at a temperature of 60 ° C for 16 hours.
  • the nylon membrane was washed once with wash buffer 1 for 10 minutes at 60 ° C. This was followed by successive further washing steps for 10 minutes each at 60 ° C. with washing buffer 2 and washing buffer 3 until sufficient stringency was achieved. Sufficient stringency means that non-specific hybridizations of the Arabidopsis ffta / Zana hybridization probe on the Filter membrane could no longer be detected. Only then was the filter exposed in the presence of an X-ray film.
  • RNA in Arabidopsis thaliana which corresponds to the length of the full-length cDNA clone.
  • a signal which had been isolated from Zea mays and Brassica napus, a signal, albeit a weaker hybridizing one, could be detected which corresponded to the hybridizing Arabidopsis thaliana-RHA in its running distance. Because in all cases the same starting quantity of the total RNA applied to the gel, the weaker hybridization signal can be interpreted in two directions in the case of Zea mays and Brassica napus.
  • RNA coding for the AMP deaminase is present in Zea mays and Brassica napus in a smaller amount than in Arabidopsis thaliana based on the total amount of RNA, or the sequence of the AMP deaminase RNA from Zea mays and Brassica napus has a certain heterology compared to the hybridization probe used from Arabidopsis thaliana.
  • a combination of both effects is also possible, so that a less expressed AMP deaminase RNA, which additionally also has a certain sequence heterology to the hybridization probe obtained from Arabidopsis thaliana, gives a weaker signal overall.
  • the present full-length cDNA of AMP deaminase from Arabidopsis thaliana was converted into the vectors pRMHa-N (day at the N-terminus) and pRMHa-C (day at the C-terminus), which are described in Benting J. et al., Anal Biochem, 278 (2000), 59-68.
  • the full-length cDNA of the AMP-DA was amplified with the aid of PCR and then cloned into the corresponding interfaces of the insect expression vectors pRMHa-N or pRMHa-C.
  • the His tag (corresponds to 10 histidine residues) is located at the N-terminus of the AMP deaminase
  • the His tag (corresponds to 10 histidine residues) is located at the C-terminus of the AMP deaminase
  • the primers were selected so that the SEQ ID NO 1 after the PCR into the two vectors could be inserted that there was a complete open reading frame (His tag plus the AMP deaminase encoded by the cDNA).
  • the plasmid PASK IBA3-AMP-DA was used as template for the cDNA of the AMP deaminase.
  • 1 ⁇ l (100ng / ⁇ l) of the plasmid was used and mixed with 5 ⁇ l PCR buffer, 1 ⁇ l 5'AMP-DA pRN-Ntag (25pmol), 1 ⁇ l 3'AMP-DA pRN-Ntag (25 pmol), or 1 ⁇ l 5'AMP-DA pRN-Ctag (25pmol), 1 ⁇ l 3'AMP-DA pRN-Ctag (25 pmol), 1 ⁇ l dNTPs (25mmol) and 40 ⁇ l H 2 O as well as 1 ⁇ l native Pfu polymerase [2 , 5U / ⁇ l] (Stratagene).
  • the PCR program carried out comprised the following cycles:
  • PCR batches were separated on a 1% TBE agarose gel using the standard method (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). A PCR amplificate of approximately 2500 bp was obtained. The resulting PCR product was integrated into the expression vectors pRMHa-N and pRMHA-C via the restriction sites Barn Hl and Kpnl.
  • SL-3 cells were sown 1 day before the transfection in a concentration of 2x10 ⁇ cells / 3 cm dish. The cells were washed with serum-free SF-900II medium. A transfection was carried out with 1.5 ⁇ g pRMHa-N-AMP-DA / pRMHa-C-AMP-Da and 0.5 ⁇ g PIZ ⁇ 5-His (Invitrogen) and 6 ⁇ l FuGene ⁇ (Röche) in SF-900II (without addition of fetal Calf serum and antibiotics) in a total volume of 1ml. Two days after the transfection, the cells were placed under selection (1 mg / ml Zeocin) (Invitrogen). An aliquot was then removed for a gel electrophoretic analysis (Western blot) and for an enzymatic activity test.
  • the Western blot was carried out according to the customary methods (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • the detection of the expressed recombinant AMP deaminase was carried out exactly as described by Benting (Benting J. et al., Anal Biochem, 278 (2000), 59-68).
  • the expected band of approximately 90 KDa was clearly detected, which corresponds to the expected size of the recombinant protein to be expressed, the AMP deaminase.
  • test buffer 60 mM Na citrate (Merck), 100 mM KCI (Merck), 0.02% Triton X-100 (Merck), pH 7.1.
  • the reaction was started by adding 50 ⁇ l of substrate solution (0.6 mM AMP (Sigma), 1 mM ATP (Sigma), 1 ⁇ M diadenosine pentaphosphate (Sigma), dissolved in test buffer). After an incubation of 2 h at 25 ° C., the reaction was stopped by adding 50 ⁇ l phenol solution.

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Abstract

L'invention concerne l'isolation et le clonage d'une séquence d'acide nucléique codant l'AMP-désaminase issue d'Arabidopsis thaliana. La molécule d'acide nucléique isolée contenant la séquence d'acide nucléique permet l'expression et l'isolation d'une AMP-désaminase végétale très pure qui permet de détecter des inhibiteurs herbicides de cette AMP-désaminase. Des plantes transgéniques peuvent en outre être obtenues à l'aide de cette AMP-désaminase isolée, lesdites plantes manifestant une résistance accrue vis-à-vis d'inhibiteurs d'AMP-désaminase.
PCT/EP2001/007767 2000-07-17 2001-07-06 Molecule d'acide nucleique codant une amp-desaminase vegetale Ceased WO2002006319A2 (fr)

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AU2001272525A AU2001272525A1 (en) 2000-07-17 2001-07-06 Nucleic acid molecule which codes for a plant amp deaminase
EP01951656A EP1303618A2 (fr) 2000-07-17 2001-07-06 Molecule d'acide nucleique codant une amp-desaminase vegetale

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DE2000135084 DE10035084A1 (de) 2000-07-17 2000-07-17 Nucleinsäuremolekül, das für eine pflanzliche AMP-Deaminase codiert
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103773792A (zh) * 2014-01-28 2014-05-07 江南大学 鼠灰链霉菌amp脱氨酶基因的原核表达方法及其表达产物的应用
CN106460052A (zh) * 2014-05-14 2017-02-22 海德堡鲁普雷希特卡尔斯大学 双链核酸的合成

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* Cited by examiner, † Cited by third party
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CA2325054A1 (fr) * 1998-04-01 1999-10-07 Basf Aktiengesellschaft Adenylique-desaminase
AU6509200A (en) * 1999-07-30 2001-02-19 E.I. Du Pont De Nemours And Company Purine metabolism genes in plants

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103773792A (zh) * 2014-01-28 2014-05-07 江南大学 鼠灰链霉菌amp脱氨酶基因的原核表达方法及其表达产物的应用
CN106460052A (zh) * 2014-05-14 2017-02-22 海德堡鲁普雷希特卡尔斯大学 双链核酸的合成
US10988795B2 (en) 2014-05-14 2021-04-27 Ruprecht-Karls-Universitat Heidelberg Synthesis of double-stranded nucleic acids
EP3143139B1 (fr) * 2014-05-14 2021-12-15 Ruprecht-Karls-Universität Heidelberg Synthèse d'acides nucléiques bicaténaires

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