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WO2003002727A2 - Ribozyme en tete de marteau, specifique a la stearoyl-acp desaturase de differentes plantes oleagineuses - Google Patents

Ribozyme en tete de marteau, specifique a la stearoyl-acp desaturase de differentes plantes oleagineuses Download PDF

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Publication number
WO2003002727A2
WO2003002727A2 PCT/EP2002/006585 EP0206585W WO03002727A2 WO 2003002727 A2 WO2003002727 A2 WO 2003002727A2 EP 0206585 W EP0206585 W EP 0206585W WO 03002727 A2 WO03002727 A2 WO 03002727A2
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WIPO (PCT)
Prior art keywords
ribozyme
stearoyl
sequence
seq
line
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Ceased
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PCT/EP2002/006585
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English (en)
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WO2003002727A3 (fr
Inventor
Caterina D'ambrosio
Adriana Lucia Stigliani
Ivana Marino
Leonarda Salfi
Francesco Cellini
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Metapontum Agrobios SCARL
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Metapontum Agrobios SCARL
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Priority to AU2002345041A priority Critical patent/AU2002345041A1/en
Publication of WO2003002727A2 publication Critical patent/WO2003002727A2/fr
Publication of WO2003002727A3 publication Critical patent/WO2003002727A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition

Definitions

  • the present invention relates to a Hammerhead ribozyme capable of modulating the expression of the gene encoding the stearoyl-ACP desaturase enzyme (also called ⁇ 9 desaturase) of different oleaginous plants, a vector containing the sequence which encodes said ribozyme and its use for the production of transgenic plants with an improved con- tent of stearic acid.
  • a Hammerhead ribozyme capable of modulating the expression of the gene encoding the stearoyl-ACP desaturase enzyme (also called ⁇ 9 desaturase) of different oleaginous plants
  • a vector containing the sequence which encodes said ribozyme and its use for the production of transgenic plants with an improved con- tent of stearic acid also called ⁇ 9 desaturase
  • lipids consist of saturated fatty acids, such as palmitic and stearic acid, and mono- and poly- unsaturated fatty acids, such as oleic acid, linoleic acid and linolenic acid. Their characterization is linked to the percentage with which each of these fatty acids is present. Lipids which are solid at room temperatures are generally defined as fatty and are characterized by a high percentage of saturated fatty acids .
  • fats are particularly requested in the confectionery industry and in all food preparation processes in which fats are subjected to high temperatures .
  • Oils are mainly used in the food industry and, those with a high content of poly-unsaturated fatty acids, mainly in the cosmetic, soap, detergent, lubricant, paint, plastic industries, etc.
  • Oils of a vegetable origin currently form about 85% of the production of food oils and fats and represent an es- sential element in human nutrition as they provide up to 25% of caloric buildup (Broun P. et al . , 1999, Genetic Engineering of plant lipids. Ann. Rev. Nutr. 19:197-216).
  • This process allows the melting point of the oil to be increased and gives a better aroma and greater resistance to oxidative reactions, to which fatty acids, and particularly linolenic acid, are susceptible.
  • the oil thus modified is used for the production of margarines which are often used as analogs of butter in the confectionery and food industries .
  • Catalytic hydrogenation processes however, not only have an economic repercussion but also cause the formation of trans-isomers of fatty acids which, when digested, cause an increase in the cholesterol level in the blood.
  • the modulation of the expression of an endogenous gene requires an interaction of the stoichiometric type between transgene and the target sequence.
  • Ribozymes are circular RNA capable of catalyzing their own fragmentation in the absence of proteins and in correspondence with particular nucleotidic sequences.
  • Hammerhead ribozymes represent a specific group of ribo- zymes and have the catalytic domain structure characterized by the presence of 3 helixes (I, II, III) of varying lengths ( Figure 1) .
  • ribozymes consist of :
  • an objective of the present invention relates to a Hammerhead ribozyme capable of modulating the expression of the gene encoding the stearoyl-ACP desaturase enzyme of different oleaginous plants characterized in that the two regions flanking the catalytic site are not perfectly complementary with any of the target sequences .
  • a further objective of the present invention relates to a DNA sequence which encodes said ribozyme.
  • Another objective of the present invention relates to an expression vector in plants containing the DNA sequence which encodes said ribozyme.
  • Yet another objective of the present invention relates to a method for increasing the content of stearic acid in different oleaginous plants which uses said ribozyme.
  • An additional objective of the present invention relates to transgenic plants transformed with said ribozyme.
  • the central box encloses the catalytic domain of the ribozyme (helix II) ; the two side boxes form, from left to right, helix III and helix I of the ribozyme.
  • the Xs indi- cate the nucleotides of the target sequence which are paired with the two helixes I and III of the ribozyme.
  • the Y present on the target sequence can be a G or an A depending on the target sequence on which the ribozyme has been designed (see Figure 2) . This nucleotidic base associated with the U and C forms the cleavage triplet indicated in bold letters.
  • Figure 2 this indicates a comparison between the target sequence defined in the present invention and the target sequences of some oleaginous plants .
  • the central ATC in bold letters and underlined, represent the cleavage triplet; the bases in bold letters, indicated in the sequences of the different species, are the bases different from the target sequence.
  • Figure 3 indicates the photo of the gel relating to the catalytic test on the transcript of the stearoyl-ACP desaturase enzymes of various species.
  • Line 1 Flax; Line 2: Flax + ribozyme; Line 3: Rape; Line 4: Rape + ribozyme; Line 5: Sad 6 (Sunflower); Line 6: Sad 6 (Sunflower) + ribozyme; Line 7: Sad 17 (Sunflower) ; Line 8: Sad 17 (Sunflower) + ribozyme; Line 9: Olive; Line 10: Olive + ribozyme; Line 11: Castor oil plant; Line 12: Castor oil plant + ribozyme.
  • Figure 4 indicates the photo of the gel relating to the catalytic test on the transcript of the stearoyl-ACP de- saturase enzymes of various species.
  • Line 1 Olive + ribozyme
  • Line 2 Olive
  • Line 3 Castor oil plant + ribozyme
  • Line 4 Castor oil plant
  • Line 5 Rape + ribozyme
  • Line 6 Rape
  • Line 7 Flax + ribozyme
  • Line 8 Flax
  • Line 9 Sad 17 (Sunflower) + ribozyme
  • Line 10 Sad 17 (Sunflower) .
  • the first product in the synthesis process of these acids is palmitate (16:0), whose carbon atom chain is lengthened to stearate (18:0) on which the stearoyl-ACP desaturase enzyme (commonly called ⁇ 9 desaturase) acts, when it is still inside the plastid.
  • This enzyme adds a double cis bond in position 9/10 of the chain, converting the stearoyl-ACP into oleoyl-ACP (stearate to oleate) .
  • the stearoyl-ACP desaturase enzymes of these plants were aligned and compared and a cleavage site (ATC) was identified in a highly preserved region.
  • target sequence refers to the sequence on which helixes I and III which flank the catalytic domain of the ribozyme and which are complementary to the target sequence itself, are designed.
  • the regions flanking the cleavage triplet are not perfectly complementary with any of the target RNA and the Xs can be substituted by any of the four nucleotides. It is this particular characteristic which allows the ribozyme to exert its catalytic action on a larger number of RNA.
  • a target sequence was defined, on which helixes I and III which flank the catalytic domain of the ribozyme Luna 9, were synthesized. More specifically, the differences between helix I of the ribozyme and the target gene sequence are: 0 for Linum usitatis- simum, 1 for Brassica napus and Olea europaea, 2 for He- lianthus annuus, Ricinus communis and Simmondsia chinensis, 3 for Carthamus tinctorius and Sesamum indicum.
  • helix III The differences between helix III and the target gene sequence are: 1 for Linum usitatissimu and Brassica napus, 2 bases for Carthamus tinctorius, Helianthus annuus, Ricinus communis and Simmondsia chinensis and 3 bases for Olea europaea and Sesamum indicum.
  • the oli- gonucleotides complementary to helixes I and III were then synthesized, which, after pairing, were cloned in a vector for in vitro transcription.
  • the cloning was effected following the standard proce- dures (Sambrook J. et al. (1989) Molecular cloning, A laboratory Manual, Cold Spring Harbor Laboratory Press, New York) .
  • the DNA encoding the ribozyme was sequenced to verify that there were no point muta- tions .
  • the transcribed ribozyme was used in catalytic tests (cleavage) in vitro to determine the cut efficiency on the stearoyl-acyl-carrier protein desaturase coming from various species (flax, rape, Castor oil plant, sunflower, olive) .
  • the sequence encoding the ribozyme can be put under the control of regulating regions which allows its expression in eukaryotic cells .
  • regulating regions include promoters of the constitutive type or of viral origin (CaMV 35S, TMV) or so-called housekeeping genes (ubiquitina, actina, tubu- lina) associated with their termination sequence or with a heterologous one .
  • CaMV 35S, TMV constitutive type or of viral origin
  • housekeeping genes ubiquitina, actina, tubu- lina
  • tissue promoters such as those of the genes involved in the biosynthesis of fatty acids (ACPs, acyltransferase, desaturase, lipid transfer protein genes) or those of the genes of seed reserve proteins (zein, napin, cruciferin, conglycin) associated with their termination sequence or with a different one .
  • ACPs acyltransferase, desaturase, lipid transfer protein genes
  • seed reserve proteins zein, napin, cruciferin, conglycin
  • Inducible promoters associated with their own termination sequences can also be used.
  • the plant expression cassette described above, can be transferred to a vector which also contains a gene marker for the selection of transformed vegetable cells (for example genes for resistance to hygromycin, kanamycin, metotrexate, phosphinotrycin) .
  • a gene marker for the selection of transformed vegetable cells for example genes for resistance to hygromycin, kanamycin, metotrexate, phosphinotrycin.
  • the marker for the selection is under the control of a constitutive promoter.
  • the vector is constructed so that both the transgene and the gene marker are both transferred into the plant genome.
  • the vegetable tissue used in the transformation can be obtained from any oleaginous plant .
  • These plants can be of the following kinds: Brassica, Helianthus, Carthamus, Sesamum, Glycine, Arachis, Gossyp- ium, Ricinus, Linum, Cuphea, Euphorbia, Limnanthes, Crambe,
  • tissue for the transformation examples include leaves, hypocotyls, cotyledons, stems, calluses, single cells and protoplasts.
  • the transformation techniques which can be used are those well known in literature on the transformation of plants and include the transformation mediated by Agrobac- terium, electroporation, polyethylene glycol (PEG) and the use of the Particle Gun. All these systems allow insertion of the transgene and selection marker into the plant genome.
  • the transformed calluses can be selected by developing them on a selective substrate, such as, for example, a medium which contains the toxic chemical substance whose resistance gene has been transferred into the plant as gene marker.
  • Samples of tissues are removed from the regenerated plants, on which molecular analyses (Southern or PCR) are effected to determine the clones in which the transgene has been integrated in the genome .
  • the positive plants for the transgene can be seeded and the seeds are analyzed to determine the composition in fatty acids of the extracted oil .
  • the characteristics of new fatty acids are determined by comparing the composition of transgenic genes with those of parental plants .
  • sequences of the stearoyl-ACP desaturase examined are those of Linum usitatissimum, Brassica napus, Ricinus communis, Helianthus annuus, Carthamus tinctorius, Simmondsia chinensis, Olea europaea and Sesamum indicum.
  • the stearoyl-ACP ⁇ 9 desaturase were compared using Clustal R multiple alignment software and a cleavage site (ATC) was identified in a highly preserved region.
  • the target sequence which is not equal to any of the desaturase indicated, was then defined.
  • the sequences of the two helixes (I and III) which flank the catalytic domain of the ribozyme and which are complementary to the target sequence ( Figure 2) were then determined.
  • helix I of the ribozyme SEQ. ID Nr. 2
  • sequence of the target gene are: 0 for Linum usitatissimum, 1 for Brassica napus and Olea europaea, 2 for Helianthus annuus, Ricinus communis and Simmondsia chinensis and 3 for Carthamus tinctorius and Sesamum indicum.
  • Helix III has the difference of 1 base for Linum usitatissimum and Brassica napus, 2 bases for Carthamus tinc- torius, Helianthus annuus, Ricinus communis and Simmondsia chinensis . and 3 bases for Olea europaea and Sesamum indicum.
  • the length of the ribozyme, called Luna 9, is 48 bp
  • the length of the helixes I and III is 12 bases.
  • the two helixes of complementary DNA were synthesized as oligonucleotides by Roche Diagnostics SpA.
  • the two oligonucleotides (10 ⁇ g) were paired in 60 ⁇ l of buffer having the following composition: 10 mM TrisHCl pH 7.4, 100 mM NaCl, 0.25 mM EDTA, pH 8.
  • the solution was maintained at 68°C for 5 minutes and then left to cool slowly.
  • the two oligonucleotides thus paired were precipitated with 2 volumes of ethanol at 100% and 1/10 of sodium acetate 3M pH 5.3, resuspended in 20 ⁇ l of water and 5 ⁇ g were subsequently digested with BamHI .
  • the digestion product after being charged onto aga- rose gel at 2% was purified from gel with the GeneCleanTM kit (BIO 101 Inc, USA) .
  • About 10 ng of DNA thus isolated were ligated to 50 ng of plasmid pGem3Zf (Promega) previously digested with the enzyme BamHI in 10 ⁇ l of reaction mixture, in the presence of 2 units of T4 DNA ligase, at 16°C for a night.
  • the plasmid DNA extracted from 5 positive clones was subjected to sequence analysis to verify the nucleotidic correspondence with the ribozyme indicated in Seq. ID Nr. 2.
  • the ribozyme was obtained by SP6 polymerase transcription of the SEQ ID Nr. 2 cloned in Smal linearized pGem3Zf .
  • the sense transcripts of the desaturase genes were obtained by linearizing the different plasmids in which they were cloned with an enzyme situated at 3 ' of the gene and at least .100 bp after the cleavage site (Table 1) .
  • Table 1 List of the ⁇ 9 desaturase used in the in vitro tests, the enzymes used for the linearization, the polymer- ase used for the transcription. The last two columns indicate the dimensions of the whole transcript and those of the fragments generated by the catalytic action of the ribozyme.
  • the Ambion Kit called MEGAscriptTM was used for the transcription.
  • the reaction mixture prepared as described by the producer, consists of:
  • the mixture was incubated for 2 hours at 37°C and the plasmid DNA was subsequently degraded by adding 1 ⁇ l of KNase I Rnase-free and incubating for a further 15 minutes at 37°C.
  • the non-incorporated nucleotides were removed by means of precipitation with LiCl 2.5 M (final concentration).
  • An estimate of the quantity of marked transcript obtained was effected by reading on the scintillator 1 ⁇ l of the transcription immediately before precipitation with LiCl (A) and 1 ⁇ l of the transcript resuspended in an equal volume after precipitation (B) .
  • the ratio between B/A multiplied by 100 expresses the incorporation percentage of the marked nucleotide.
  • the incorporation percentage multiplied by the maximum theoretical quantity of RNA which can be synthesized expresses the quantity of transcript obtained.
  • the maximum quantity of synthesizable RNA is 198 ⁇ g, in the case of SP6 polymerase, it is 132 ⁇ g.
  • the concentration of each of the four ribonucleotides is equal to 7.5 mM (in the case of SP6 polymerase, it is equal to 5 mM) in a 20 ⁇ l reaction, this means that if all the nucleotides present in the mixture were used for the formation of RNA, the maximum quantity obtainable would be equal to 198 ⁇ g, in the case of SP6 polymerase, it is 132 ⁇ g.
  • the concentration of each of the four ribonucleotides is equal to 7.5 mM (in the case of SP6 polymerase, it is equal to 5 mM) in a 20 ⁇ l reaction, this means that if all the nucleotides present in the mixture were used for the formation of RNA, the maximum quantity obtainable would be equal to 198
  • the gel was dried and exposed using a phosphor screen of Molecular Dynamics which has the capacity of capturing the images com- ing from radio-active samples.
  • the screen was then read using a Storm R 860 scanner of Molecular Dynamics.
  • the cleavage percentage was estimated as the relative intensity of the autoradiographic signal determined by the use of Storm 860 and with the aid of Image-Quant software.
  • the Phosphorimager R and annexed software it was possible to attribute a value directly correlated with the signal intensity to each band present on the gel .
  • This value can be indicated as a percentage considering the sum of all the autoradiographic signals inside each line as equal to 100.
  • the bands inside each line can be attributed to the transcript of the target gel and its possible cleavage products, it is thus possible to express as a percentage the part of the target that has been cut .
  • the catalytic efficiency of the ribozyme obtained transcribing the sequence of the present invention is indicated in Tables 2 , 3 and 4 and was determined as described above.
  • the line entitled "Line” refers to the path in which the marked gene transcript and the ribozyme transcript were charged contemporaneously.
  • the lines in which the gene transcript alone was charged, were not taken into consideration, as in this case there was no cut.
  • the values of the column “intensity %” refer to the relative intensity of the signal of the fragments present in the lines; whereas the column “cleavage %” indicates the sum of the percentages relating to the intensity of the bands produced by the cleavage .
  • the sum of the % referring to the peaks generated by the cleavage bands represents an estimate of the cut efficiency.
  • Table 2. Catalytic efficiency of the ribozyme on the stearoyl-ACP desaturase of flax, rape, sunflower and Castor oil plant .
  • Table 4 Cut efficiency on the stearoyl-ACP desaturase of Castor oil plant, rape, flax and sunflower.

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Abstract

L'invention concerne un ribozyme en tête de marteau, pouvant moduler l'expression du gène codant l'enzyme de stéaroyl-ACP désaturase dans différentes plantes oléagineuses, un vecteur contenant la séquence codant ledit ribozyme, et son utilisation pour la production de plantes transgéniques à teneur améliorée en acide stéarique.
PCT/EP2002/006585 2001-06-28 2002-06-13 Ribozyme en tete de marteau, specifique a la stearoyl-acp desaturase de differentes plantes oleagineuses Ceased WO2003002727A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002345041A AU2002345041A1 (en) 2001-06-28 2002-06-13 Hammerhead ribozyme specific for the stearoyl-acp desaturrase of different oleaginous plants

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT2001MI001364A ITMI20011364A1 (it) 2001-06-28 2001-06-28 Robozima hammerhead specifico per la stearoyl-acp desaturesi di piante oleaginose differenti
ITMI01A001364 2001-06-28

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WO2003002727A2 true WO2003002727A2 (fr) 2003-01-09
WO2003002727A3 WO2003002727A3 (fr) 2003-11-20

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AU (1) AU2002345041A1 (fr)
IT (1) ITMI20011364A1 (fr)
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RO114469B1 (ro) * 1987-12-15 1999-04-30 Gene Shears Pty Ltd Compus oligoribonucleotidic, procedeu de preparare si metoda de inactivare
EP0922767A1 (fr) * 1997-12-03 1999-06-16 Gene Shears Pty Limited Ribozymes capables de conférer une résistance à une infection par Potyvirus, et plantes exprimant ces ribozymes

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ITMI20011364A1 (it) 2002-12-28
ITMI20011364A0 (it) 2001-06-28
WO2003002727A3 (fr) 2003-11-20
AU2002345041A1 (en) 2003-03-03

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