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WO2009001398A2 - Système de confinement transgénique par l'inhibition récupérable de la germination dans des graines transgéniques - Google Patents

Système de confinement transgénique par l'inhibition récupérable de la germination dans des graines transgéniques Download PDF

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Publication number
WO2009001398A2
WO2009001398A2 PCT/IT2008/000441 IT2008000441W WO2009001398A2 WO 2009001398 A2 WO2009001398 A2 WO 2009001398A2 IT 2008000441 W IT2008000441 W IT 2008000441W WO 2009001398 A2 WO2009001398 A2 WO 2009001398A2
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Prior art keywords
sequence
genie
construct
promoter
construct according
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PCT/IT2008/000441
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WO2009001398A3 (fr
Inventor
Martin Kater
Lucia Colombo
Monica Colombo
Simona Masiero
Raffaella Battaglia
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Universita' Degli Studi Di Milano
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Publication of WO2009001398A2 publication Critical patent/WO2009001398A2/fr
Publication of WO2009001398A3 publication Critical patent/WO2009001398A3/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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/829—Female sterility
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8217—Gene switch
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]

Definitions

  • the present invention relates in general to a transgenic containment system through the recoverable inhibition of the germination in transgenic seeds.
  • the invention concerns a genie construct that can be inserted into the genome of a crop and which is able to block in a reversible and controllable way a determined fundamental function for the development of a new generation.
  • transgenic plants which are ingegnerized for the production of pharmaceutics, biofuel, specific chemicals are arising a great interest from a commercial point of view.
  • transgene containment system through the recoverable inhibition of germination of transgenic seeds would block spontaneous seed germination allowing a safe co-existence between genetically modified and non-genetically modified crops.
  • terminal a first system named “terminator” (terminator) (US 5,723,765) is based on the seed-suicide mechanism. This system uses an exogenous stimulus (tetracycline) to control the activation of a site-specific recombinase which will in turn allow the expression of the cytotoxic ribosome-inhibitor protein (RIP) specifically in the seed. Seeds of transgenic plants not treated with tetracycline are capable of germinating under natural conditions while treated seeds dye during late embryogenesis.
  • exogenous stimulus tetracycline
  • RIP cytotoxic ribosome-inhibitor protein
  • receptor DNA cassettes receptor DNA cassette
  • chemical ligands of constructs activation For example, unrecoverable blocks (arrest) of embryo development have been obtained in Brassica napus by the expression of the exotoxin A of Pseudomonas aeruginosa under the control of the napin promoter.
  • the above mentioned RBF system solves the problem of unrecoverability since the blocked function is not recovered under natural conditions, but is capable of being recovered only when an external controllable treatment or intervention is applied. Nevertheless, experimental data show that this, system works well in tobacco but it does not work as efficiently in other species. Moreover, the BARNASE-BARSTAR strategy is highly dependent on a tightly controlled activity of the constructs promoters since even a minimal transcriptional level can lead to the precocious expression of the BARNASE gene and to cell death. There derives that the success of this approach depends on how well the block and control construct are regulated and on whether they show a balanced expression, as an expression from moderated to high is necessary for the BARNASE activity to be inhibited by the recover construct.
  • the technical problem at the base of the present invention is to provide a transgene containment system which works through the recoverable inhibition of germination in transgenic seeds in a safe way without the danger of transgene dissemination in crop of no interest and without the creation of uncontrollable fertile hybrids. Another problem is that of providing a system working in a really effective way.
  • a first object of the invention is therefore to make available a genetic construct comprising a genie block element and a genie recoverable element operably associated.
  • a second object is to offer a method for the transgene containment in sexual reproducing genetically modified plants . Further characteristics, objects and advantages of the present invention will be more evident in the following detailed description of a realization provided purely as a non limitating example with reference to the figures.
  • FIGURES - Figure 1 represents the nucleotide sequence of the AGL23 gene from Arabidopsis thaliana
  • FIG. 2 represents the nucleotide sequence of the PHERES promoter (pPHERES);
  • Figure 3 represents the nucleotide sequence of the LEC2 promoter (pLEC2)
  • Figure 4 represents the nucleotide sequence of the precursor of the artificial micro RNA amiR-BnAGL23;
  • Figure 5 represents the sequence of the BnAGL23 gene;
  • Figure 6 represents the sequence of the mutated BnAGL23 gene;
  • Figure 7 represents the sequence of the Pheres promoter:: amiR-BnAGL23 construct;
  • Figure 8 represents the sequence of the LEC2::amiR-
  • Figure 10 represents the pBGW.O vector
  • Figure 11 represents the NOB1 plasmid
  • Figure 12 represents the NOB2 plasmid
  • Figure 13 represents the nucleotide sequences of the Ve49 e
  • Figure 14 represents the NOB4 plasmid
  • Figure 15 represents the pPHERES::GUS construct
  • Figure 16 represents the pLEC2::GUS construct
  • FIG. 17 represents GUS sequence
  • Figure 18 represents the NOB7 plasmid
  • Figure 19 represents the NOB8 plasmid
  • Bnp2, Bnp3, Bnp4 oligos Bnp2, Bnp3, Bnp4 oligos
  • Figure 22 represents the NOB10 plasmid
  • Figure 23 represents the recombination between NOB 419 plasmid and NOB 10 plasmid which gives the NOB1 1 plasmid;
  • Figure 24 represents the NOB5 plasmid;
  • Figure 25 represents the transformation of the NOB11 plasmid with the genie element reported in figure 7 to give the NOB12 plasmid;
  • - Figure 26 represents NOB 13 plasmid;
  • Figure 27 represents the NOB 6 plasmid
  • Figure 28 represents the NOB14 plasmid
  • Figure 29 represents the NOB16 plasmids
  • Figure 30 represents the pPHERES::ami23 construct (of Seq. ID n. 29).
  • the idea at the base of the invention is to contrive a transgene containment system of the type of a recoverable block of the function by using a function of some genes which does not involve the above mentioned problems linked to the known systems.
  • MADS-box a group of plant transcription factors named MADS-box and identified also in even very distantly related species have been performed. These transcription factors are responsible for the control of a wide range of developmental processes, including the flowering time, the transition from the vegetative to the reproductive phase, as well as floral meristem identity, floral organs identity and ovule identity. The choice fell on MADS-box transcription factors in the light of their known role in many developmental processes.
  • Arabidopsis thaliana MADS-box genes comprise 107 identified members; among which only for about 20 of them the function is known.
  • Said genes have been subjected to mutations in order to allow a permanent block of their function.
  • a genie expression construct has been ideated and generateda wherein various genie elements have been associated to each other in order to block seeds maturation in a crop.
  • AtAGL23 gene from Arabidopsis thaliana (At1G65360), having the sequence SEQ. I. D. N.1 reported in figure 1 , plays a role in the regulation of megagametogenesis and in embryo development.
  • AGL23 gene plays a key role in the regulation of chloroplast biogenesis during embryo development: in fact the destruction of AGL23 provokes the formation of a seed containing an albino embryo.
  • the agl23/ag/23 homozygous embryos cannot complete the chloroplast biogenesis and dye.
  • the first object of the present invention is therefore a genie construct for the recoverable inhibition of the germination of transgenic seeds, comprising: - a genie element which blocks the embryo development S
  • a vegetal embryo promoter operably associated to a nucleotide sequence capable of inactivate a gene involved in such embryo development
  • a genetic element for the recovery of embryo development 5 comprising an inducible promoter operably associated to a sequence of said gene involved in embryo development which is mutated in order to result insensitive to such sequence capable of inactivating a gene involved in embryo development but active in the function of embryo development;
  • the construct of the invention can comprise one or more genes or transgenic nucleotidic sequences of interest.
  • genes or sequences can be represented by genetic elements encoding for a genetic product having a desired function like a protein, an enzyme including metabolites, hormones, antibiotics or toxins.
  • these genetic elements are operably 15 associated in the construct in order to be contained between its right and left ends. In particular, they will be located in the proximity of the genetic block element.
  • the genetic element for the block of embryo development is represented by one or more nucleotidic sequences operably associated 20 together so that their expression might cause the death or at least the alteration of the phenotype or of the physiology of the natural host organism.
  • This construct therefore allows the containment of seeds which have been possibly spread in the environment during harvest and/or transportation, since such seeds are not able to originate adult and vital plants. 25 Also the seeds produced from random crossings between enginerized plants and wild varieties (or non-enginerized) would be unable modified crops and wild relative (non genetically modified) would not be able to germinate and, as a consequence, also the dispersion of the pollen produced by transgenic individuals would be controlled. This results has been obtained, as it will be described in detail in the following, allowing the activation of the blocking genie constructs under normal conditions, natural, i.e. without any external interventions.
  • the genetic element for blocking the embryo development comprises a vegetal embryo specific promoter like, for instance, the PHERES promoter, the LEC2 promoter or the napin promoter (Josefsson, 1987).
  • the promoter is pPHERES or pLEC2 which nucleotide sequences SED I. D. N.2 and SEQ I. D. N. 3 are represented in figure 2 and 3 respectively.
  • the promoter has been chosen on the base of its capability of activating only during the embryo development of a plant, therefore, without interfering with female and male gametophytes maturation.
  • Said promoter is then operably associated to a nucleotide sequence able to inactivate a gene involved in a plant embryo development.
  • sequence may be any sequences designed in order to express in turn a nucleotide sequence which is able to bind to an active site of a gene involved in the embryo development to inactivate it,, thus blocking the normal embryo development of a plant.
  • said sequence is a sequence which hybridizes with a
  • the sequence able to inactivate said target sequences or genes is a sequence which encodes for the expression of an artificial micro RNA (amiRNA) or for a small interference RNA (siRNA).
  • amiRNA artificial micro RNA
  • siRNA small interference RNA
  • the plant MADS-box sequences represent those preferred for the present invention.
  • the plant MADS-box sequences represent those preferred for the present invention.
  • a mutation of the AGL23 gene is responsible of the embryo development.
  • the destroy of the AGL23 gene with the agl23-1 mutation due to the insertion of a T-DNA element (SaIk line_147048, ecotype CoI-O (Alonso et al., 2003) causes the formation of an albino embryo unable to germinate and to develop an adult plant.
  • the non mutated gene AGL23 is responsible for the chloroplast biogenesis.
  • the sequence ables to inactivate the target gene preferably is a sequence amiRNA SEQ I. D. N. 30 ( Figure 4a), projected to hybridize with the AGL23 gene of Arabidopsis thaliana and to inactivate it through breakage.
  • SEQ I. D. N. 30 can be used.
  • Such variants can be represented by sequences having an identity of at least 70%, preferably at least 80%, with said sequence SEQ I. D. N. 30. Examples of such variants are: ⁇ '-TTAATGGAGACTTACTCGGCC-S' (SEQ ID 31 ), and ⁇ '-TGAACACGTATCATACAGCTT-S" (SEQ ID 32).
  • the construct of the invention comprises also a genetic element to recover the embryo development which is able to recover the blocked function, thus it is able to reactivate the normal embryo development, following a specific external stimulus.
  • this element has an inducible system which is able to react to said external stimulus activating the transcription of the nucleotidic sequence operably associated to it. Inducible systems are known in literature (Wang et al., 2003) and in general each of these systems can be used in the present invention.
  • the inducible system is chosen among the system mediated by glucocorticoids (positively regulated by dexametason), the heat shock induced system (which were already used with success in the model species Arabidopsis thaliana (Sablowski e Meyerowitz, 1998) and the ethanol inducible system. It is preferred the use of the ethanol inducible system, which comprises an inducible pALC promoter which is activated by the ALCR protein, which is present in the cell but in an inactive form, which is in turn activated by the external stimulus represented by the ethanol.
  • the just described system is known in literature (Roslan et al., 2001; Laufs et al., 2003).
  • the genetic recovery element can comprise a constitutive promoter operably associated to a gene which sinthetizes a protein which, when activated by the external stimulus, is able to bind to the inducible promoter and to activate in turn its transcription.
  • the constitutive promoter can be chosen among the constitutive promoters known in literature, like for instance the 35S-CaMV, the ubiquitin promoter, the actin and tubulin promoter.
  • the constitutive promoter is the 35S-CaMV, which regulates the production of the ALCR protein, which responds to the action of the ethanol. Treatment with ethanol induces a conformation modification in ALCR and it becomes able to bind to the pALCA promoter.
  • pALCA regulates the transcription of the sequence of the gene involved in embryo development mutated in order to be insensitive to the action of the sequence which inactivates the genes which control the embryo development. Nevertheless, the mutated version is an active version in relation to the embryo development promoting function.
  • the sequence of genes involved in embryo development mutated in order to result insensitive to the above said sequence capable to inactivate the genes involved in embryo development but active in its function is downstream associated to said inducible promoter.
  • sequence has been projected by creating a mutation, alteration, deletion or other modification in nucleotides so that the sequence of the genetic block element is no more able to bind it, hybridizing and carrying out the in activation function.
  • the created modification is such to make no more recognizable the sequence of recovery of the blocked function from the inactivation sequence.
  • modification will be made by the technician in the field by adopting known procedures such those available in the recombinant DNA technology, for example for obtaining a point mutation.
  • the modification has to be projected in order to maintain the functionality of the original sequence thus to recover the natural function.
  • This result can be achieved considering the degeneracy of the genetic code, therefore, the last nucleotidic base of a triplet encoding for an amino acid can be substituted without altering the correct translation. Its alteration is useful in order to provoke the non-recognition by part of the blocking sequence.
  • a system particularly useful to make a sequence insensitive to a blocking sequence but maintaining its natural function is the one which uses of the amiRNA or siRNA technology above hinted and following detailed.
  • the recovery sequence is modified even through the substitution of a sole nucleotide, the blocking sequence is no more able to hybridize, i.e. to recognize its target.
  • the original sequence of the AGL23 gene from Brassica napus SEQ. I. D. N. 5 (BnAGL23) ( Figure 5) has been preferably modified by introducing a mutation in position 405 from A to G 1 , as it will be described in details in the following.
  • sequence of the genes involved in the embryo development mutated in order to be insensitive sequence of in activation of such genes but active in its natural function is therefore preferably represented by the mutated sequence BnAGL23 SEQ. LD. N. 6 (figure 6).
  • the method for the preparation of a genetic construct for the recoverable inhibition of the germination in transgenic seeds as previously described comprises the steps of:
  • step 12 recombinating the product of step 11) in a suitable vector
  • step 17 integration of the product from step 15) into the plasmid of step 16);
  • step 18) ligating the insert of the vector of step 12) and the linearized plasmid from step 16) in order to obtain a plasmid containing the genetic element of recovery of the embryo development; 19) recombinating the products from step 12) and step 18) to obtain the genetic construct for the recoverable inhibition of the germination in transgenic seeds.
  • the genetic construct previously described is inserted into a suitable host which is able, when in contact with the target organism of interest or part of it like its gametes, to infect and thus to transfer the construct.
  • the method comprises the steps of: a) providing a genetic construct for the recoverable inhibition of the germination in transgenic seeds as above described; b) inserting said construct into an host organism which is able to transform a target organism or parts of it.
  • the genetic construct can be cloned and transferred into vectors for the heterologous expression, like binary vectors, using the known techniques in the field.
  • it can be used the Gateway system
  • Agrobacterium tumefaciens as a host to transform Brassica napus.
  • the blocking construct is necessary for the degradation of the endogenous RNA messenger produced by the gene BnAGL23. It is composed of an embryo-specjfic promoter that drives the transcription of the artificial micro-RNA specifically directed toward BnAGL23 mRNA (the artificial micro- RNA will be from now onwards indicated as amiR-BnAGL2). Ami-RNAs are ,short sequences 21 nucleotides long complementary to the endogenous RNA messenger to be desiderably degraded, therefore amiR-BnAGL23 causes the , specific degradation of BnAGL23 mRNA.
  • the bases in position 10-11 are strategic for the recognition and the pairing to the endogenous messenger which is desired to be silenced, if a mis-match is introduced in the above said positions the degradative property is irremediably lost (Schwab et al., 2006).
  • Two blocking constructs have been prepared which differ for the use because there have been used two embryo-specific promoters active in two different phases of the embryo development.
  • the promoter which drives the trascription of the gene PHERES (pPHERES) is active in the initial phases of the embryo development, from phases immediately successive to the fertilization up to the globular embryo stage (Khoeler et al., 2003).
  • the promoter of the gene LEC2 (pi_EC2) on the contrary is active during the final phases of the embryo development, from the globular embryo stage up to the torpedo stage (Kroj et al., 2003).
  • the blocking construct with the amiR- BnAGL23 regulated by pPHERES will be indicated as PHERES::amiR-AGL23 (SEQ. I. D. N.7 represented in figure 7), while the construct amiR-BnAGL23 regulated by pLEC2 will be named LEC2::amiR-AGL23 (SEQ. I.D. N.8 represented in figure 8).
  • Amplification of the promoters The promoters pPHERES and pLEC2 have been amplified by the genomic DNA extracted from Arabidopsis thaliana.
  • pPHERES has been amplified with the Rrimers Atp1552 of SEQ ID n.
  • Atp1553 of SEQ ID n.10 (figure 9)
  • pLEC2 has been amplified with Atp1554 of SEQ ID n 11 and Atp1555 of SEQ ID n. 33 (figure 9).
  • Atp1552 and Atp1554 contain a Xbal restriction site, while Atp 1553 and Atp 1555 possess a site recognized by Aatll. The introduction of the above said restriction sites has been necessary for the cloning of the two promoters in the pBGW plasmid.
  • the PCR reaction has been performed using the Taq phusion (Finnzymes), taq polymerase with high fidelity and high processivity, indeed the error rate of this polymerase has been determined in 4.4 x 10 "7 and also thanks to its exonucleasic activity it is able to correct possible inserted mis-matches.
  • the amplification 10 ng of genomic DNA of Arabidopsis thaliana have been used.
  • the denaturation is performed at 98°C.
  • the Taq phusion (Finnzymes) has a processivity of 4 kilo-bases/minute. 35 cycles of amplification have been performed.
  • the PCR products after having been checked on 1% agarose gel, have been purified with the employment of the "QIAquick PCR purification kit (Qiagen)" following the enclosed instructions.
  • the PCR products have been then digested with Xbal-Aatll (Roche), the double digestion has been carried out in buffer A (33 mM Tris-acetate. 66mM K-acetate, 10 mM Mg-acetate, 0,5 mM dithiothreitolo, pH 7.9) following the instructions supplied by Roche.
  • the reaction has been incubated at 37°C for 2 hours.
  • the pBGW plasmid has been digested with Xbal-Aatll (Roche).
  • pBGW is a gateway vector (Karimi et al., 2002) with a polilinker placed upstream the gateway cassette (figure 10). Subsequently, the plasmid digested with Xbal-Aatll and the two PCR fragments (pPHERES and pLEC2) digested with Xbal-Aatll have been purified from gel employing .the QIAquick Gel Extraction Kit (Qiagen). For each ligation reaction there have been used 10 ng of pBGW, digested with Xbal-Aatll and then purified, and about 20 ng of the two inserts (pPHERES and pLEC2) they also digested and purified. For the reaction there have been used the T4 ligase produced by Promega, incubating at 16°C for 12 hours.
  • NOB1 pPHERES::pBGW
  • NOB2 pLEC2::pBGW
  • the gene BnAGL23 (the homologous of AtAGL23) in Brassica napus has been amplified using the primers Atp983 of SEQ ID n. 12 and Atp984 of SEQ ID n. 13 (figure 9).
  • the PCR product to be conveniently sequenced has been cloned in the vector pGEM®-T Easy.
  • the vector pGEM®-T Easy has been prepared by the Promega supplier by cutting the vector pGEM®-5Zf with EcoR V and adding a terminal thymine to the two 3' ends. This vector allows an easy cloning of the templates if the Taq employed for the production of the fragment of interest adds some "Adenines (A)" to the ends of the fragments.
  • the gene BnAGI23 has been amplified with the enzyme Taq phusion (Finnzymes), unable to add the A to the ends of our amplified, these have been added in a subsequent phase through incubation at 72 0 C for 20' with the Taq polymerase supplied by Roche.
  • the PCR product has then been purified with the "QIAquick PCR purification kit" and then ligated in pGEM®-T Easy following the indications provided by Promega. In particular the reaction has been performed in a final volume of 20 ⁇ l and incubated for 2 hours at room temperature.
  • the precursor of amiR-BnAGL23 is generated with 4 subsequent and overlapped cycles of amplifications.
  • the first reaction forsees the use of the primers Ve50 of SEQ ID n. 21 (figure 13) with Atp 1559 of SEQ ID n. 17 (figure 9) (the amplified fragment is designated Te1 ); the starting template is the vector pRS300.
  • the template is always the pRS300 plasmid, but it is amplified with Atp 1557 of SEQ ID n. 15 and Atp 1558 of SEQ ID n. 16 (figure 9) (the product is named Te2).
  • pBRS300 is amplified with Ve50 and Atp 1556 of SEQ ID n. 14 (the product is named Te3).
  • Te3 the product is named Te3
  • Te4 the final template is named Te4
  • Te4 the final template is named Te4
  • Te4 has been then cloned in pENTRTM/SD/D-TOPO (Invitrogen) generating the Nob4 represented in figure 14. Indeed, Ve49 possesses the extension (CACC) necessary for Te4 to enter the pTOPO.
  • the reaction has been prepared in a final volume of 10 ⁇ l containing 10 ng of the purified PCR product. The reaction mixture has been then incubated at 22°C for 30 minutes and 2 ⁇ l have been used for the subsequent electroporation of the cells of E. coli.
  • pPHERES::amiR-BnAGL23 and pLEC2::amiR- BnAGL23 have been produced through a Gateway recombination of LR type between NOB4 and the vectors previously described NOB1 (pPHERES::pBBGW) and NOB2 (pLEC2::BBGW).
  • the Gateway® system (Invitrogen) is a cloning system based on the bacteriophage ⁇ site-specific recombination in Escherichia coli.
  • the Gateway® system is a cloning system based on the bacteriophage ⁇ site-specific recombination in Escherichia coli.
  • the bacteriophage infects E. coli bacterial cells it can undertake the lytic or the lysogenic cycle.
  • the bacteriophage integrates itself in E. coli genome thanks to a site-specific recombination process and it is transmitted to the daughter cells from generation to generation, until when there not verify suitable conditions for its excision from the genoma.
  • the bacteriophage begins the lytic stadium of its lifecycle. The processes through which the bacteriophage integrates and excises itself from E.
  • the four att. v sites are recombination sites of the bacteriophage ⁇ and they contain the recognition sites for the proteins involved in the processes of integration and excision.
  • the reaction attB X attP mediates the process of integration
  • the reaction attL X attR mediates the process of excision from the genome.
  • a LR recombination reaction has been set in a final volume of 10 ⁇ l.
  • the reaction has been incubated at 25 0 C for about 3 hours.
  • 1 ⁇ l has been used for submitting E. coli to electroporation.
  • the final plasmids have been checked, extracted by E. coli and introduced through electroporation in Agrobacterium tumefaciens, the microorganism that will be then used for transforming Brassica napus with the purpose to check the goodness of the construct.
  • Electroporator 2510 Eppendorf
  • a potential difference of 1500 V Promoter activity and tissue specific control
  • pPHERES and pLEC2 are regulating sequences of Arabidopsis thaliana, it is necessary to check that these two promoters are able to drive the trascription of the genes also in Brassica napus embryos.
  • pPHERES and pLEC2 have been cloned upstream the GUS reporter gene (two constructs have been therefore produced, pPHERES::GUS (SEQ. I.D. N. 22) and pLEC2::GUS (SEQ. I.D. N. 23) (figures 15 and 16) just to verify that the product of the GUS gene SEQ. I.D. 24 (figure 17) is tracked in Brassica developing embryos.
  • the coding sequence of the GUS gene has been amplified with Atp
  • NOB7 has been then recombined through a gateway LR recombination reaction (see above) with both NOB1 and NOB2, generating respectively pPHERES::GUS (NOB8 represented in figure 19) and pLEC2::GUS (NOB9, represented in figure 20).
  • the recovery genie element contains an ethanol inducible promoter which drives the expression of a BnAGL23 version insensitive to the action of amiR-BnAGL23 because, by means of site-specific mutagenesis, a point mutation has been introduced in bases 10-11 recognized by amiR-BnAGL23 and fundamental for the silencing of the gene. When induced, this construct allows to recover the damages caused to the seed by amiR-BnAGL23 action and therefore vital plants are produced only when desired and after the treatment with the ethanol of the recovery construct.
  • the BnAGL23 mutated version has been obtained introducing a point mutation through PCR, using a couple of primers (which introduce the mutation) complementary between them, each specific for a filament (sense and antisense).
  • the primers Bnp2 of SEQ ID n. 26 and Bnp3 which change the position 405 of the BnAGL23 sequence from A to G.
  • Bnp2 is used in combination with Bnp1 of SEQ ID n. 25 (figure 21) to generate a first emi-fragment of BnAGL23 (Producti , PM), while in a second PCR reaction Bnp3 of SEQ ID n. 27 and Bnp4 of SEQ ID n.
  • pDNR there has been used NOB10 (figure 22), also known as pDONR201 (Invitrogen).
  • BP reaction has been performed in 10 ⁇ l incubating at 22°C for at least 12 hours.
  • NOB10 schematic 22
  • pDONR201 Invitrogen
  • ALCR can bind to the pALCA promoter, nevertheless such binding is possible only in the presence of ethanol. Therefore, any gene which is cloned downstream of pALCA will be transcripted and translated if the plant will be subjmitted to an ethanol treatment.
  • the recovery system contains 35S::ALCR, while pALCA regulates the transcription and therefore the translation of the BnAGL23 mutated version
  • NOB419 was kindly provided by T. Laufs and it is described in Roslan et al.
  • the gateway cassette (frameA) (Life Technologies) has been inserted in pL4 (Syngenta Ltd) exploiting the Smal site between the pALCA promoter and the terminator.
  • the pALCA promoter, the gateway cassette and the terminator have been excised from the plasmid through Xbal digestion and inserted in the pBIN19 (Clontech).
  • the ultimate purpose is to bring the blocking and the recovery construct on the same T-DNA in order to be inserted in linkage in the transgenic plants. This is necessary to avoid their segregation in the subsequent generations.
  • NOB10 and the NOB419 (figure 23).
  • the final vector has been called NOB11 (figure 23) and therefore BnAGL23mutated has been produced (BnAGL23mut) under the control of the pALCA inducible promoter (NOB11 : pALCA::BnAGL23mut).
  • PHERES: :amiR-BnAGL23 (the reaction template was the NOB5 represented in figure 24) has been amplified (with Taq phusion, Finnzymes) using the primers Atp1552 and Ve50. These primers introduce a XBAI restriction site, then the PCR product has been digested with this restriction enzyme (Roche, the reaction has been done using the buffer H, composition of the stock 1OX: 10 mM Tris-HCI, 0.1 M NaCI, 5 mM MgCI2, 1 mM 2-Mercaptoethanol, pH 8), purified (QIAquick PCR purification kit) and finally ligated with Promega T4 DNA ligase in the vector NOB11. Also NOB11 had been previously digested with Xbal and purified. The vector thus obtained has been named NOB12 (figure 25).
  • NOB12 has been digested at this point with the Roche restriction enzyme HINDIII (the digestion has been performed at 37 0 C with the buffer B, composition of the stock 10X: 0,5 M Tris-HCI, 1 M NaCI 1 0.1 M MgCI2, 10 mM DTT, pH 7.5), digestion which allows to take off a fragment containing pALCA::BnAGI23mut,PHERES::amiR-BnAGL23. The digestion has been separed' on an agarose gel and the fragment corresponding to the above- mentioned sequence has been extracted from the gel using the QIAquick Gel Extraction Kit.
  • HINDIII Roche restriction enzyme
  • NOB13 This fragment has been then inserted in the NOB 425 (containing the T-DNA with 35S::ALCR) also digested with Hindlll (Roche). The ligation reaction has been incubated at 16°C for 12 hours. This is the first final construct which we have called NOB13 (figure 26).
  • the LEC2::amiR-BnAGL23 fragment has been amplified using as template the NOB6 (figure 27) and the following primers have been used for the PCR reaction: Atp1554 and Ve50. These two primers introduce a restriction site recognized by the endonucleases Xbal, therefore after having purified the PCR product it has been digested and subsequently introduced in the NOB1 1 (see above) also digested with Xbal.
  • NOB11 + LEC2::amiR-BnAGL23 has been called NOB14 (figure 28).
  • NOB 14 is digested with Hindlll, the fragment containing pALCA::BnAGL23 mut;LEC2::amiR-BnAGL23 is eluted from the gel and ligated in the NOB 425 previously cut with Hindlll.
  • This final vector is digested with Hindlll, the fragment containing pALCA::BnAGL23 mut;LEC2::amiR-BnAGL23 is eluted from the gel and ligated in the NOB 425 previously cut with Hindlll.
  • NOB16 (figure 29).
  • NOB14 and NOB16 like some of the previous vectors, have been introduced through electroporation in Agrobacterium tumefaciens.
  • the Agrobacterium modified strains have been used for the transformation of B.napus.
  • B. napus transformation is a long procedure which foresses some in vitro culturing steps, carried out according to what is reported by Bade Damm (1995).
  • Plant Cell. 18, 1121 -33. o Drivele-Calzada, J. P., Baskar, R. and Grossniklaus, U. (2000). Delayed activation of the paternal genome during seed development. Nature 404, 91-94. o Wang, R., Zhou, X., and Wang, X. (2003). Chemically regulated expression systems and their applications in transgenic plants, Transgenic Res. 12, 529-540.

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Abstract

La présente invention porte d'une manière générale sur un système de confinement transgénique par l'inhibition récupérable de la germination dans des graines transgéniques. En particulier, l'invention concerne un produit de construction par génie génétique qui peut être introduit dans le génome d'une culture et qui est capable de bloquer de façon réversible et contrôlable une fonction fondamentale déterminée pour le développement d'une nouvelle génération.
PCT/IT2008/000441 2007-06-28 2008-06-30 Système de confinement transgénique par l'inhibition récupérable de la germination dans des graines transgéniques WO2009001398A2 (fr)

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ITMI20071291 ITMI20071291A1 (it) 2007-06-28 2007-06-28 Sistema di contenimento transgenico attraverso l'inibizione recuperabile della germinazione in semi transgenici.

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2499252A4 (fr) * 2009-11-11 2013-04-10 Univ Trobe Stérilité mâle de plante transgénique
WO2013063487A1 (fr) * 2011-10-28 2013-05-02 E. I. Du Pont De Nemours And Company Procédés et compositions pour le silençage de gènes utilisant des microarn artificiels

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IL101592A (en) * 1991-04-16 1998-12-27 Mogen Int Recombinant male sterile polynucleotide plant containing it and method of production of the plant
ATE476513T1 (de) * 1998-12-22 2010-08-15 Dow Agrosciences Llc Verfahren zum limitieren des herauskreuzens und unerwünschten genflusses in nutzpflanzen
US6849776B1 (en) * 2000-07-14 2005-02-01 Unicrop Ltd Molecular control of transgene segregation and its escape by a recoverable block of function (RBF) system

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2499252A4 (fr) * 2009-11-11 2013-04-10 Univ Trobe Stérilité mâle de plante transgénique
WO2013063487A1 (fr) * 2011-10-28 2013-05-02 E. I. Du Pont De Nemours And Company Procédés et compositions pour le silençage de gènes utilisant des microarn artificiels
US20130111634A1 (en) * 2011-10-28 2013-05-02 E I Du Pont De Nemours And Company And Pioneer Hi-Bred International Methods and compositions for silencing genes using artificial micrornas
CN103890179A (zh) * 2011-10-28 2014-06-25 纳幕尔杜邦公司 使用人工微rna沉默基因的方法和组合物

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