Patent Application
20120192312
The present invention relates to a method for selecting or producing plants having a reduced lignin content.
Lignins are insoluble polymers which are located in plant walls and are the result of the polymerization of 3 phenolic monomers (or monolignols), deriving from the phenylpropanoid pathway (Neish, Constitution and Biosynthesis of Lignin, publisher New York: Springer Verlag, 1-43, 1968). Their biosynthetic pathway is complex and comprises various steps, one part of which is carried out in the cytoplasm (monolignol synthesis) and another part in the wall (polymerization). p-Coumaryl, coniferyl and sinapyl alcohols are the respective precursors for the p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units constituting lignins. These precursors are oxidized to phenolic radicals which spontaneously couple via various linkages, thereby resulting in the formation of lignins. Among the inter-unit linkages, a distinction is made between labile linkages, called β-O-4 linkages, and resistant linkages. During polymerization, other linkages can also be established with other wall compounds (polysaccharides and proteins) in order to form a complex three-dimensional network. The formation of the phenolic radicals is thought to be catalyzed by oxidases, such as peroxidases, laccases or other oxidases. A large number of these enzymes in combination with regulatory proteins is thought to be necessary for assembly of the H, G and S units (Boudet, Plant Physiol. Biochem., 38, 81-96, 2000; see for review: Ralph et al., Lignins, Encyclopedia of Life Sciences, John Wiley & Sons, 2007). However, these enzymes are still poorly identified since they belong to multigene families (Barrière et al., Genes, Genomes and Genomics; Global Science Books, 2007, Review).
Although the mechanisms involved in vivo in lignin biosynthesis are not completely elucidated, it is generally considered that laccases could be volved in the first steps of polymerization, for the formation of dimers or trimers, while peroxidases could make it possible to obtain a greater degree of polymerization from the dimers and trimers (Ros Barcelo, International Review of Cytology, 176, 87-132, 1997).
The lignin content of plants has a major influence on their industrial uses. For example, it affects the nutritive value of plants intended for animal feed, and also the performance levels of papermaking processes (yield and quality of the paper pulp obtained) and the production yield for biofuel. Indeed:
In this context, the quantitative modification of the lignins in plants is the subject of numerous research studies. The qualitative modification of lignins (modification of their structure or of their interactions with the other wall polymers) is also greatly studied. For example, lignins rich in S units or in β-O-4 linkages are much easier to remove during the chemical production of paper pulp.
One of the preferred routes for decreasing the lignin content in plants concerns production by genetic engineering of plants. It has thus been proposed to act on the enzymes of the lignin biosynthesis pathway, such as laccases (International Application WO 97/45549), peroxidases (International Application WO 2004/080202), cinnamoyl CoA reductase (CCR; International Applications WO 97/12982 and WO 98/39454), caffeic acid O-methyl transferase (COMT; International Application WO 94/23044; Oba and Allen, J. Dairy Sci., 82, 135-142, 1999), cafeoyl coenzyme A 3-O-methyl transferase (CCoAOMT; Application EP 0516958; Guo et al., Transgenic Res. 10, 457-464, 2001), cinnamyl alcohol dehydrogenase (CAD; Lapierre et al., Plant
Physiol., 119, 153-164, 1999), and 4-coumarate:coenzyme A ligase (4CL; Hu et al., Nat. Biotech. 17, 808-812, 1999).
With regard more particularly to the laccases, International Application WO 97/45549 describes a tobacco laccase (the sequence of which is, moreover, described by Kiefer-Mayer et al., Gene, 178, 205-207, 1996), and proposes increasing or reducing the amount of lignins produced by a plant by overexpressing said laccase (or a protein having at least 50% of amino acids homologous to those of said laccase), or by inhibiting its expression.
In Arabidopsis thaliana, the laccase multigene family comprises 17 members, 7 of which are expressed in the stems, the stem being the most lignified organ. The genes most strongly expressed are LAC4 (At2g38080), LAC17 (At5g60020) and LAC2 (At2g29130). The LAC2 and LAC17 genes belong to the same subclass, and the LAC4 gene belongs to a subclass which is close according to the phylogenetic trees published by Pourcel et al. (Plant Cell, 17, 2966-2980, 2005) and Caparros-Ruiz et al. (Plant Science, 171, 217-225, 2006). The LAC17 gene encodes the LAC17 protein (AtLAC17), the sequence of which is available under accession number NM—125395 in the Genbank database, and is also reproduced in the appended sequence listing under the identifier SEQ ID No. 2. The LAC4 gene encodes the LAC4 protein (AtLAC4), the sequence of which is available under accession number NM—129364 in the Genbank database, and is also reproduced in the appended sequence listing under the identifier SEQ ID No. 4. The LAC2 gene encodes the LAC2 protein (AtLAC2), the sequence of which is available under accession number NM—128470 (GI:186503951) in the Genbank database. In Arabidopsis, AtLAC17 is expressed in the interfascicular fibers.
The inventors have thus demonstrated that the proteins that are orthologs of the AtLAC17 protein exhibit at least 60% identity or at least 75% similarity with said protein and comprise, from the N-terminal end to the C-terminal end, at least one of the 4 consensus peptide domains of sequence:
By way of nonlimiting examples of orthologs of the A. thaliana LAC17 protein, mention will in particular be made of the laccases of:
The table represented in
The LAC17 protein exhibits 55.2% identity with the LAC4 protein, and 67.1% identity with the LAC2 protein, but the latter does not comprise the consensus peptide domain of sequence SEQ ID No. 14, and 54.6% identity with the tobacco laccase described in International Application WO 97/45549; the LAC4 protein exhibits 54.0% identity with the LAC2 protein and 75.8% identity with the tobacco laccase described in International Application WO 97/45549 (it appears that said tobacco laccase is the ortholog of the AtLAC4 protein), the percentages of identity being calculated over the entire length of the sequences by means of the needle program (Needleman and Wunsch, J. Mol. Biol., 48, 443-453, 1970) using the default parameters: “Matrix”: EBLOSUM62, “Gap penalty”: 10.0 and “Extend penalty”: 0.5. In Arabidopsis, AtLAC4 is expressed in the vessels of the xylem and in the interfascicular fibers.
By way of nonlimiting examples of orthologs of the A. thaliana LAC4 protein, mention will in particular be made of the laccases of:
A. thaliana lines of the SALK collection in the Col0 accession (Columbia) exhibiting T-DNA insertions in the LAC17 (SALK—016748 line), LAC4 (SALK—051892 line) and LAC2 (SALK—025690 line) genes have been identified. The mutants lac4 and lac2 have in particular been described by Brown et al. (Plant Cell, 17, 2281-2295, 2005). These mutants exhibit a greatly reduced or zero expression of the mutated gene, but do not exhibit any particular phenotype under glass.
The inventors have investigated whether these mutations have an effect on the amount of lignins of the mutated plants, and their qualitative (structural) properties. They have noted that the lac2 mutant does not exhibit any notable difference compared with the Col0 wild-type line, but that, on the other hand, the lac4 and lac17 mutants contain an amount of lignins (determined on mature dry stems) that is reduced by 6 to 8% and exhibit a cellulolysis yield that is increased by 17% in the case of lac17 and by 52% in the case of lac4, compared with the Col0 wild-type line (cellulolysis carried out without acid pretreatment).
In addition, the inventors have obtained lac4/lac17 double mutants from the lac4 and lac17 mutants by crossing. They have noted that these double mutants exhibit a very reduced amount of lignins (reduced by approximately 19% compared with the Col0 wild-type line), and a better cellulolysis yield compared with the Col0 wild-type line (+25% to +42%) and with the lac17 single mutant (+6% to +21%) approximately.
Consequently, the subject of the present invention is a method for reducing the lignin content of a plant and increasing the cellulolysis of the walls of said plant, characterized in that the expression and/or the activity in said plant:
The term “lignin content” is intended to mean the Klason lignin content. This content can be measured by assaying the acid-insoluble lignin (ASL) fraction present in the parietal residue (PR) of a plant, as described in Example 3 below.
The term “laccase” is intended to mean a copper-comprising enzyme (EC 1.10.3.2) which catalyzes the oxidation of a phenolic substrate using dioxygen as final electron acceptor.
Unless otherwise specified, the percentages of identity indicated herein are established, as indicated above, by means of the needle program using the default parameters.
The present invention applies to dicotyledonous or monocotyledonous plants. By way of nonlimiting examples, it can apply to corn, wheat, barley, rye, triticale, oats, rice, sorghum, sugar cane, poplar and pine.
By way of nonlimiting examples of laccases, as defined in paragraph a) above, mention may be made, in corn (Zea mays), of the peptide sequences SEQ ID Nos. 5, 6 and 42 to 47, in sugar cane (Saccharum officinarum), of the sequence SEQ ID No. 7, in sorghum (Sorghum bicolor), of the peptide sequences SEQ ID Nos. 8 to 11, in Brachypodium, of the peptide sequences SEQ ID Nos. 48 to 55, in rice, of the peptide sequences SEQ ID Nos. 56 to 62, and in poplar, of the peptide sequences SEQ ID Nos. 63 to 68.
By way of nonlimiting examples of laccases, of which the polypeptide sequence has at least 60% identity or at least 75% similarity with the sequence SEQ ID No. 2 and comprises, from its N-terminal end to its C-terminal end, the 4 consensus peptide domains of sequence SEQ ID Nos. 12, 13, 14 and 15 respectively, mention may be made, in corn, of the peptide sequences SEQ ID Nos. 5, 6, 42, 43, 44 and 45, in sugar cane, of the peptide sequence SEQ ID No. 7, in sorghum, of the peptide sequences SEQ ID Nos. 8 (partial sequence of the protein), 9, 10 and 11, in Brachypodium, of the peptide sequences SEQ ID Nos. 50 and 51, in rice, of the peptide sequence SEQ ID No. 58, and in poplar, of the peptide sequences SEQ ID Nos. 63, 67 and 68.
By way of nonlimiting examples of laccases, of which the polypeptide sequence has at least 65% identity with the sequence SEQ ID No. 4, mention may be made, in Brachypodium, of the peptide sequence SEQ ID No. 69, in rice, of the peptide sequence SEQ ID No. 70, and in poplar, of the peptide sequences SEQ ID Nos. 71 to 74.
The total or partial inhibition of the expression and/or of the activity of a laccase as defined above can be obtained in various ways, by methods known per se.
Particularly advantageously, this inhibition can be obtained by intervening upstream of the production of said laccase, by mutagenesis of the gene encoding this protein, or else by inhibition or modification of the transcription or of the translation of this laccase.
The mutagenesis of the gene encoding said laccase can take place at the level of the coding sequence or of the expression-regulating sequences, in particular of the promoter. It is, for example, possible to delete all or part of said gene and/or to insert an exogenous sequence. By way of example, for corn, mention will be made of insertional mutagenesis: a large number of individuals deriving from a plant that is active in terms of the transposition of a transposable element (AC or mutator element) are produced, and the plants in which an insertion has taken place in the gene of said laccase are selected, for example by PCR. This exogenous sequence can also be a T-DNA (fragment of the Agrobacterium tumefaciens Ti plasmid).
It is also possible to introduce one or more point mutations with physical agents (for example radiation) or chemical agents. These mutations result in the reading frame being shifted and/or in a stop codon being introduced into the sequence and/or in the level of transcription and/or of translation of the gene being modified and/or in the enzyme being made less active than the wild-type protein. The mutated alleles of the gene of said laccase can be identified, for example, by PCR using primers specific for said gene.
In this context, techniques of “TILLING” type (Targeting Induced Local Lesions IN Genomes; McCallum et al., Plant Physiol., 123, 439-442, 2000) can in particular be used.
It is also possible to carry out a site-directed mutagenesis targeting the gene encoding said laccase. The inhibition or the modification of the transcription and/or the translation can be obtained via the expression of sense, antisense or double-stranded RNAs derived from the gene of said laccase, or of the cDNA of this protein, or else through the use of interfering RNAs (for review on antisense inhibition techniques see, for example: Watson and Grierson, Transgenic Plants: Fundamentals and Applications (Hiatt, A, ed) New York: Marcel Dekker, 255-281, 1992; Chicas and Macino, EMBO reports, 21, 992-996, 2001; for review concerning more specifically the use of interfering RNAs, see Hannon, Nature, 418, 244-251, 2002).
The subject of the present invention is also a recombinant DNA construct comprising one or more polynucleotides capable of inhibiting the expression of the two laccases as defined above. By way of nonlimiting examples, said polynucleotides can encode antisense RNAs, interfering RNAs (noncoding double-stranded RNAs approximately 21 to 25 nucleotides in length), micro-RNAs (noncoding, single-stranded RNAs approximately 21 to 25 nucleotides in length) (Ossowski et al., The Plant Journal, 53, 674-690, 2008; Schwab et al., Methods Mol Biol., 592, 71-88, 2010; Wei et al., Funct Integr Genomics., 9, 499-511, 2009) or ribozymes targeting the gene encoding a laccase as defined above.
Preferably, said polynucleotides capable of inhibiting the expression of the LAC4 and LAC17 laccases as defined above are micro-RNAs, such as the micro-RNAs miR397 and miR408, preferably miR397. By way of nonlimiting examples of such micro-RNAs, use may be made of those having the following sequences:
According to one preferred embodiment of the invention, the recombinant DNA construct is chosen from:
According to one particular embodiment of the invention, said recombinant DNA construct comprises a fragment of at least 15 consecutive nucleotides, preferably at least 20, and entirely preferably at least 50 consecutive nucleotides of a polynucleotide of sequence SEQ ID No. 1 or SEQ ID No. 3, or of a polynucleotide complementary to a polynucleotide of sequence SEQ ID No. 1 or SEQ ID No. 3.
These constructs can in particular be:
Recombinant DNA constructs in accordance with the invention can also comprise other elements, for example one or more selectable markers.
Those skilled in the art have at their disposal a very wide choice of elements that can be used for obtaining recombinant DNA constructs in accordance with the invention.
By way of nonlimiting examples of promoters that can be used in the context of the present invention, mention will be made of:
By way of nonlimiting examples of other regulatory elements for transcription that can be used in the context of the present invention, mention will be made of terminators, such as the NOS 3′ terminator of nopaline synthase (Depicker et al., J. Mol. Appl. Genet., 1, 561-573, 1982), or the CaMV 3′ terminator (Franck et al., Cell, 21, 285-294, 1980; GenBank accession number V00141).
By way of nonlimiting examples of selectable marker genes that can be used in the context of the present invention, mention will in particular be made of genes which confer resistance to an antibiotic (Herrera-Estrella et al., EMBO J., 2, 987-995, 1983) such as hygromycin, kanamycin, bleomycin or streptomycin, or to a herbicide (EP 0 242 246) such as glufosinate, glyphosate or bromoxynil, or the NPTII gene which confers resistance to kanamycin (Bevan et al., Nucleic Acid Research, 11, 369-385, 1984).
The plants can be transformed using numerous methods, known in themselves to those skilled in the art.
It is, for example, possible to transform plant cells, protoplasts or explants and to regenerate a whole plant from the transformed material. The transformation can thus be carried out, by way of nonlimiting examples:
Use may also be made of Agrobacterium tumefaciens, in particular according to the methods described in the articles by Bevan et al. (Nucleic Acid Research, 11, 369-385, 1984) and by An et al. (Plant Phydiol., 81, 86-91, 1986), or else Agrobacterium rhizogenes, in particular according to the method described in the article by Jouanin et al. (Plant Sci., 53, 53-63, 1987). For example, the plant cell transformation can be carried out by transfer of the T region of the Agrobacterium tumefaciens tumor-inducing extrachromosomal circular plasmid Ti, using a binary system (Watson et al., publisher De Boeck University, 273-292, 1994). Agrobacterium tumefaciens can also be used on whole plants, for example by depositing at the level of the wound of a monocotyledonous plant, the bacterium harboring the DNA to be transferred, in the presence of substances released at the level of the wound of a dicotyledonous plant.
The subject of the present invention is also a plant cell comprising an expression cassette as defined above or a recombinant vector as defined above.
The subject of the present invention is also the plants which can be obtained by means of a method in accordance with the invention, with the exception of the A. thaliana mutants SALK_016748 and SALK_051892. Said plants can carry mutations which inhibit the LAC4 and LAC17 laccases as defined above or express one or more polynucleotides capable of inhibiting the expression of said LAC4 and LAC17 laccases as defined above. Of course, the present invention encompasses the descendants, in particular the hybrids resulting from crossing involving at least one plant according to the invention, which are obtained by sowing or by vegetative multiplication of the plants directly obtained by means of the method of the invention.
The plant material, such as protoplasts, cells, calluses, leaves, stems, roots, flowers, fruits, cuttings and/or seeds, obtained from the plants in accordance with the invention (with the exception of the A. thaliana mutants SALK—016748 and SALK—051892), are also part of the subject of the present invention.
The subject of the present invention is also the use of the plants in accordance with the invention or of plant material obtained from said plants, for producing fodder plants, biofuels or paper pulp.
The present invention will be understood more clearly by means of the further description which follows, which refers to nonlimiting examples illustrating the reduction in lignin content and the increase in cellulolysis of the walls of a plant in which the expression of the LAC17 and/or LAC4 laccases is inhibited, and also the appended figures:
1) Selection of the A. thaliana Laccase Mutants
A. thaliana lines of the SALK collection in the Col0 accession exhibiting T-DNA insertions in the LAC17, LAC4 and LAC2 genes (respectively, the SALK—016748, SALK—051892 and SALK—025690 lines) were identified and characterized.
The SALK—016748 (lac17) mutant contains two T-DNAs inserted in inverted tandem into the promoter of the gene encoding LAC17, 146 base pairs from the ATG start codon.
The SALK—051892 (lac4) mutant contains one T-DNA inserted into the promoter of the gene encoding LAC4, 127 base pairs from the ATG start codon.
The SALK—025690 (lac2) mutant contains one T-DNA inserted into its coding sequence.
2) Genotyping of the A. thaliana lac17 (SALK—016748), lac4 (SALK—051892) and lac2 (SALK—025690) Mutants
a) Materials and Methods
Primers for the LAC17, LAC4 and LAC2 genes were defined using the OLIGO 4 software (National Biosciences Inc., Plymouth, USA). Their sequences (5′->3′) are represented in Table 1 hereinafter:
The DNA of the plants was extracted according to the protocol described by Edwards et al. (Nucleic Acid Research, 19, 1349, 1991).
The PCRs were carried out in 25 μl on 30 ng of genomic DNA, with 2 mM of MgCl2, 0.4 mM of each dNTP, 0.4 mM of each primer, and 1.25 units of Taq DNA polymerase (Invitrogen).
The PCR cycles for the genotyping of the lac17 mutants are (95° C. 30 sec, 50° C. 30 sec, 72° C. 1 min) 28 times, with a final extension of 10 min at 72° C.
The PCR cycles for the genotyping of the lac4 mutants are (95° C. 30 sec, 58° C. 30 sec, 72° C. 30 sec) 30 times, with a final extension of 10 min at 72° C.
The PCR cycles for the genotyping of the lac2 mutants are (95° C. 30 sec, 54° C. 30 sec, 72° C. 1 min 30 sec) 30 times, with a final extension of 10 min at 72° C.
The PCR products are then separated on 1% agarose gels in TAE buffer.
b) Results
2 plants of the SALK—016748 line, 3 plants of the SALK_051892 line and 1 plant of the SALK—025690 line were tested.
The genotyping results are represented in
3) Laccase Expression in the lac17 and lac4 Mutants
The expression profile of Arabidopsis laccases expressed in the floral scape in the wild-type line (Columbia), the lac17 mutant (SALK—016748) and the lac4 mutant (SALK—051892) was determined by RT-PCR.
a) Materials and Methods
A 26-cycle RT-PCR was performed on cDNAs obtained from 1 μg of RNA extracted using an RNeasy kit (Qiagen), that were treated with a DNase and reverse-transcribed with the SSRTII reverse transcriptase (Invitrogen).
The primers used are described in Table 2 hereinafter:
The pairs of primers termed “FST” (for Flanking Sequence Tag) were designed on either side of the T-DNA; they were used for amplifying on gDNA (genomic DNA). The pairs of primers termed “RT” were defined in the coding sequence and make it possible to amplify on cDNAs.
The RT-PCR cycles on the lac17 and lac4 mutants and the wild-type line are (95° C. 30 sec, 50° C. 30 sec, 72° C. 1 min 30) 26 times, with a final extension of 10 min at 72° C. for the laccases 2, 4, 6, 12 and 17 and the tubulins.
The RT-PCR cycles on the lac17 and lac4 mutants and the wild-type line are (95° C. 30 sec, 55° C. 30 sec, 72° C. 1 min 30) 26 times, with a final extension of 10 min at 72° C. for the laccases 5 and 10.
The RT-PCR cycles on the lac17 and lac4 mutants and the wild-type line are (95° C. 30 sec, 58° C. 30 sec, 72° C. 1 min 30) 26 times, with a final extension of 10 min at 72° C. for the laccase 11.
The RT-PCR products are then separated on 1% agarose gels in TAE buffer in order to visualize a difference in intensity of the fragments amplified.
b) Results
The results are represented in
4) Cytological Analysis of the lac17 Mutant
a) Materials and Methods
Sections of primary scape of 20 cm were stained with phloroglucinol-HCl according to the protocol described by Sibout et al. (Plant Cell, 17, 2059-2076, 2005). The red coloration observed corresponds to the lignified cell walls.
b) Results
The cytological observation results are represented in
a) Materials and Methods
Plants of the SALK—016748 line (lac17) were crossed with plants of the SALK—051892 line (lac4) in order to obtain a lac4/lac17 double mutant (hereinafter referred to as Kim mutant).
The lac4/lac17 double mutant was then characterized by genotyping according to the protocol described in Example 1.a) and using the lac4 FST dir, lac4 FST rev, irx12 FST dir and irx12 FST rev primers.
b) Results
The results of the genotyping for the Kim mutant are represented in
The presence of two T-DNAs in the promoter of the LAC17 gene and of one T-DNA in the promoter of the LAC4 gene was confirmed by amplification of the sequences adjacent to the T-DNAs and sequencing of the amplicons.
a) Materials and Methods
i) Assaying of Lignin Content
The assaying of the lignins was carried out on the stems collected at maturity, ground and subjected to thorough extraction with the solvents ethanol/toluene (2/1, v/v), ethanol, and then water; extractions carried out in a Soxhlet apparatus. The material extracted and dried represents the “parietal residue” or PR (since it consists of the plant walls). The removal of the soluble compounds by extraction with solvent is essential before any assaying of lignins (these compounds possibly interfering with gravimetric or spectrometric assays).
The lignin content was measured by assaying the acid-insoluble lignin fraction present in the PR and called Klason lignin (KL). This KL fraction, assayed by gravimetric analysis by treating the parietal residue with concentrated sulfuric acid (which makes it possible to hydrolyze the polysaccharides and to leave a KL residue which is rinsed, dried and weighed), represents most of the parietal lignins. However, a very small fraction of the lignins may be solubilized during the treatment with sulfuric acid: it is the fraction called acid-soluble lignin (ASL), which is evaluated by measuring the absorbance of the sulfuric supernatant in the ultraviolet range.
These measurements were carried out using the T222 om-83 method, known to those skilled in the art, and developed for wood and derivatives thereof by TAPPI (Technical Association of the Pulp and Paper Industry) (C. W. Dence, The determination of lignin; in: S. Y. Lin, and C. W. Dence, (Eds.). Methods in Lignin Chemistry. Springer-Verlag, pp. 33-61, 1992).
ii) Study of Lignin Structure
The structure of the lignins was evaluated by thioacidolysis. Thioacidolysis of the lignins releases thioethylated monomer products H, G or S from the p-hydroxyphenyl (H), guaiacyl (G) or syringyl (S) units linked only via β-O-4 linkages (major inter-unit linkages in native lignins). These products were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) of their trimethylsilyl (TMS) derivatives. The trimethylsilyl H, G or S monomers were assayed using chromatograms reconstructed respectively on the 239, 269 or 299 ions (the most intense ions of their mass spectrum obtained by electron impact).
The protocol that was used is similar to that described by Lapierre et al. (Res. Chem. Interm., 21, 397-412, 1995) and by Mir Derikvand et al. (Planta 227, 943-956, 2008). The H monomers are most commonly minor (less than 1% of the total monomers) and were therefore not considered (except in the case of mutant plants affected in the formation of the G and S units, or in the case of stress lignins).
iii) Measurement of the Enzymatic Degradability by Cellulolysis in Vitro
The susceptibility of the parietal polysaccharides to enzymatic hydrolysis was evaluated in vitro, by subjecting the walls (i.e. the parietal residue PR) to a preparation of cellulase and hemicellulase enzymes.
The protocol that was used is described by Hoffmann et al. (Plant Cell 16, 1446-1465, 2004), which is a protocol adapted from the method by Rexen (Anim. Feed Sci. Technol., 2, 205-218, 1977). Said enzymatic preparation used was the Cellulase Onozuka R10 preparation extracted from Trichoderma viride, 096i/mg (Serva Electrophoresis GmbHD, Germany).
b) Results
The results are given in Table 3 (giving the mean values between the 2 analytical repeats and the mean deviation between these repeats, except for the percentage PR for which a single measurement was carried out) hereinafter, in which:
It should be noted that the SALK—025690 mutant (lac2) does not exhibit any decrease in the amount of lignins compared with the wild-type line (Col0).
It emerges from the above results that:
Two genetic constructs are prepared in order to overexpress a micro-RNA (called miR397, SEQ ID No. 77) capable of inhibiting the expression of poplar (Populus) laccases LAC17 and LAC4, under the control either of the CaMV 2x35S constitutive promoter or of the “lignin-specific” promoter of Eucalyptus cinnamyl alcohol dehydrogenase 2 (CAD2) (called EuCAD).
The Gateway pMDC32 binary vector (Curtis and Grossniklaus, Plant Physiology, 133, 462-469, 2003) is used to overexpress the transgenes under the control of the 2x35S constitutive promoter.
For expression under the control of the EuCAD promoter, the 2x35S promoter is excised from the pMDC32 plasmid above by digestion with the HindIII-KpnI enzymes (unique restriction sites) and replaced with the “lignin-specific” EuCAD promoter.
The genetic transformation of the poplar is carried out according to the method described in Leplé et al. (Plant Cell Rep. 11, 137-141, 1992), i.e. by coculture of explants of poplar stems with agrobacteria containing a binary vector for the expression of miR397, isolation of transgenic calluses and regeneration of transformed seedlings.
Transgenic calluses are selected for the regeneration step. Seedlings are regenerated from these different calluses, therefore corresponding to different transformation events. Each seedling is cloned by multiplication.
An example of each transgenic line is used to:
These first phenotyping analyses make it possible to identify the lines which exhibit reductions in the amount of lignins in the wood.
Lines are then analyzed for the expression of the transgenes and the genes encoding LAC17 and LAC4. These expression analyses are carried out by quantitative RT-PCR (qRT-PCR).
Gateway binary vectors which are compatible and specific for the transformation of monocotyledons, containing a sequence encoding a micro-RNA of sequence SEQ ID No. 78 or 79, under the control either of a constitutive promoter, for example the maize ubiquitin promoter (ZmUbi), or a “lignin-specific” promoter, and a selectable gene such as the pat gene (which confers resistance to basta), are used for the genetic transformation of Brachypodium distachyon.
The analysis of the structure and of the lignin content of the transgenic plants obtained can be carried out using the same methods as for the analysis of the transgenic poplars above.
A vector as described for the genetic transformation of Brachypodium can be used for the genetic transformation of corn.
It is also possible to use the integrative vector L1038 (represented by the sequence SEQ ID No. 80), which contains an expression cassette comprising a herbicide resistance gene (Basta resistance gene), an expression cassette comprising a gene encoding a fluorescent protein for following the transgene without genotyping (gene encoding a GFP under the control of an Actin promoter), and a “triple Gateway” cassette attR4-ccdB-attR3 (where attR4 and attR3 are recombination sites and ccdB is a negative selection gene), which makes it possible to recombine a promoter of choice (attL4-attR1 ends), a gene of choice (attL1-attL2 ends) (in the case in point, a micro-RNA miR397) and a mock (attR1-attL3) (see the instruction manual published by Invitrogen, “MultiSite Gateway Pro”, Version B, Oct. 3, 2006).
| Number | Date | Country | Kind |
|---|---|---|---|
| 0902812 | Jun 2009 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB10/52590 | 6/10/2010 | WO | 00 | 4/4/2012 |
