WO2009156496A1

WO2009156496A1 - Plant improvement method

Info

Publication number: WO2009156496A1
Application number: PCT/EP2009/058016
Authority: WO
Inventors: Cédric RIBOULET; Yves Barriere; Françoise FABRE
Original assignee: Genoplante-Valor
Priority date: 2008-06-27
Filing date: 2009-06-26
Publication date: 2009-12-30
Also published as: FR2933103A1; AR072369A1

Abstract

The present invention relates to the field of plant improvement, in particular the field of improving the digestibility and ingestibility of maize, and improving the degradability thereof for the production of biofuels, by inhibition of the expression of the SBP1 enzyme.

Description

METHOD OF IMPROVING PLANTS

The present invention relates to the field of plant breeding, in particular the improvement of digestibility (proportion of a digested food in the digestive tract) and ingestibility (quantity of a feed distributed ad libitum which is spontaneously ingested by the animal) corn fodder. This invention also relates to the field of production of biofuels (bio fuels or biogas) second generation, since what makes the degradability of the lignocellulosic plant substrate is similar for both purposes. Maize is particularly used as feed for ruminants, and the nutritional value of silage maize can be improved by reducing the lignin content, by modifying the monomeric composition of lignins and the types of linkages between monomers, or by modifying the relationships between parietal components (in particular lignin x hemicellulose bonds and hemicellulose chain linkages). However, improving digestibility by modifying the lignin biosynthetic pathway can be counterbalanced by plants with poor agronomic qualities (such as lodging problems, or greater susceptibility to pathogens). Another way would consist in modifying the friability of the wall, in particular by modifying the bonds between lignins and sugars (cellulose and hemicelluloses).

Corn silage is an interesting feed: field yield is relatively high, harvesting and storage are easy, palatability and energy value are stable, and this forage can easily be supplemented with protein by other forage silage. fodder or especially oilseed meal (soybean, rapeseed, sunflower). Different experiments (Emile 1995, Annales de zootechnie, Barriere et al., J Dairy Sci 2004 May, 87 (5): 1439-45) have shown that feeding livestock with a more ingestible and more digestible corn can increase the level of milk production permitted by the corn ration of one to four kilograms per cow per day, with equal or greater weight gain, compared to conventional elite maize feeding. Thus, the more the variety is digestible and ingestible, the lower the energy concentrate costs for a given level of milk production, without changing the weight recoveries. Optimizing the qualities of corn silage thus consists in to increase the net ingested energy provided by this type of diet by improving its digestibility and its qualities, and in particular by modifying the biochemical and physical characteristics of the lignocellulosic polymers.

Given the qualitative importance of lignins in the digestibility and ingestibility properties, an increase, decrease or modification of lignin structure can have important industrial and agronomic consequences. The present invention, however, partly questions this paradigm, since it makes it possible to improve the ingestibility and the digestibility of maize by modulating the expression of a gene coding for an enzyme which is not, a priori, directly related to lignin synthesis.

Lignins form with cellulose and hemicelluloses the major components of the plant wall of grasses. The plant wall mainly offers the cell a natural barrier against the outside. Many agronomic or industrial sectors have their yields directly related to the content and / or composition of lignins in the wall. Among them, it is possible to mention the paper industries, the production of second-generation biofuels or the production of silage or hay.

Thus, the selection or obtaining of corn plants more ingestible and more digestible, including the pathway of biosynthesis of lignins and phenylpropanes is modified is one of the preferred areas for improving maize. However, selected plants should have good yields and be insensitive to various stresses (mechanical, watery, biotic ...).

The pathway of biosynthesis of phenylpropanes whose lignins is a complex pathway, involving a large number of possible enzymatic reactions (Dixon et al, 2001, Phytochemistry, 57 (7), 1069-1084, Barrier et al, 2007 Genes, Genomes and Genomics 1, 133-15), but whose paths leading in vivo to each component are still imperfectly elucidated. It is thus difficult to know exactly how to modify the lignin biosynthesis pathway and to predict what the consequences of the modifications will be. Lignins are insoluble polymers of 3 monomers of p-hydroxycinnamic alcohols, or monolignols, with the alcohol?-Coumaryl (H subunits), the coniferyl alcohol (G subunits, which are associated with ferulic acids and diflerulics) and sinapyl alcohol (S subunits, to which is associated with the acid? - coumaric). The three monolignols and ferulic and p-coumaric acids are derived from the phenylpropanoid pathway (Neish, 1968, Constitution and Biosynthesis of Lignin, eds New York, Springer Verlag 1-43). Each type of monolignol can form a variety of bonds with others and thus constitute lignins. Other strategic links with respect to the biodegradability of the walls are also established between parietal compounds (ferulate bridge between hemicellulose chains and between lignins and hemicelluloses) to form a complex, rigid network, and little accessible to microbial enzymes.

For several years attempts have been made to modify the content and composition of lignin plants by overexpressing or expressing one or more genes in the lignin biosynthetic pathway (Anterola and Lewis, 2002, Phytochemistry 61, 221-294). A number of strategies have been devised, as described in particular in patent applications WO 9924561, EP0516958, WO9305160, WO9305159, WO9712982. However, the possibilities offered by the overexpression or the under expression of one or more enzymes of the phenylpropane pathway are far from having been completely imagined and tested, partly because of having chosen the good target genes, and do not give always consistent and predictable results.

The object of the present application is to provide a person skilled in the art with a plant, and particularly a maize which does indeed have improved ingestibility and digestibility, by deregulation of the expression of the enzyme SBP1, and in particular by setting point of a favorable allele of the enzyme SBPl (called SBPl-L), which contains a deletion of a glutamine codon in the first exon, an insertion of twelve nucleotides in the intron. The gene represented by SEQ ID No. 1 corresponds to the genomic DNA for this enzyme SBP1 in line F2. SEQ ID NO: 4 corresponds to the genomic DNA of SBPl-L.

The sequence of a cDNA (GenBank NM OO1 112606) is represented by SEQ ID NO: 2, the coding portion extending from nucleotides 31 to 1222 for this sequence. It is clear that these sequences are given only as examples, and that one skilled in the art is able to identify itself the genomic and / or mRNA sequences of the enzyme SBP1 for different varieties. of corn. It is anticipated that such sequences have a percentage of identity as described below with SEQ ID No. 2.

The maize according to the invention exhibits a quantitative and / or qualitative alteration of the synthesis of lignins and parietal phenylpropanes. By "quantitative alteration of lignin synthesis" is meant a decrease in the amount of lignins in the modified maize according to the invention compared to a normal maize (unmodified control according to the invention), evaluated, for example by measuring Klason lignin or lignin obtained by acid detergent (lignin detergent acid) according to methods well known in the art (see for example Jung et al, J. Agric Food Chem 1999 May; 47 (5): 2005 Jung et al., J. Dairy Sci., 1997 Aug; 80 (8): 1622-8).

By "qualitative alteration of the parietal phenylpropane synthesis" is meant a modification in the composition and structure of lignins and crosslinking between polymers of the plant modified according to the invention compared to a control plant (unmodified according to US Pat. invention), for example a variation of the ratio between H / S / G subunits, the methods of qualitative analysis of the lignins being also known in the art. We can notably mention the NMR. The structural modifications of the lignin polymer are also related to the frequency of acylation of the S units by β-coumaric acid. Furthermore, the intensity of the crosslinking of the walls by ferulic acid and diferulic acid bridges is also involved in the structural quality of the wall. The p-hydroxycinnamic acids are not dosed with conventional methods, in particular with HPLC (Mechin et al., J. Sci Food Agric., 80, 574-580).

The inventors have in fact located the SBPl enzyme of maize and established that there is a co-localization between this gene and QTL linked to digestibility. It has also been shown that this gene is under-expressed in more ingestible and more digestible maize.

The SBP1 gene has been identified by Scott-Craig et al (Plant Physiol., (1998) 116: 1083-1089) who have described it as a protein for resistance to certain herbicides, although they have noted a similarity with O-methyl transferases. However, the authors of this publication have stated that this protein does not have a pattern found in other OMTs involved in O- methylation of phenolic compounds. However, homology research suggests that this gene encodes an OMT. In particular, it shares 30% identity and 54% similarity to the corn COMT sequence (these are the highest percentages). The authors conclude that this enzyme does not seem to catalyze the transfer reaction of a methyl group of S-adenosyl methionine to catechol or caffeic acid, COMT catalysing a reaction on 5OH-coniferaldehyde.

A genomic DNA sequence of SBP1 is represented by SEQ ID No. 1. The corresponding polypeptide sequence is represented in the attached sequence listing under the number SEQ ID No. 3.

The coding region generally consists of a 79 base pair (bp) intron and 2 exons. In an ingestible and digestible line, for Pallèle SBPl-L, the length of the intron is 91 bp. The genomic DNA sequence of SBP1 obtained from the high digestibility corn line, containing the insertion of 12 nucleotides into the intron is represented by SEQ ID NO: 4.

The discovery by the inventors of the involvement of SBPl in digestibility provides a set of means for obtaining corn having increased digestibility and ingestibility.

The subject of the present invention is thus a process for improving the digestibility and the ingestibility of a plant, and preferably a grass, and more preferably a maize, characterized in that the kinetics of expression are modified, and / or totally or partially inhibits the expression and / or activity of the enzyme

SBP1 of said plant.

Said plant is in particular maize, rice, sorghum, millet, wheat and other straw cereals, or a forage plant.

Here, the term "SBP1 enzyme" is used to define any protein whose polypeptide sequence has at least 75%, preferably at least 85%, or at least

90%, advantageously at least 94%, and most preferably at least

95%, more preferably at least 97% identity with the SEQ ID sequence

# 3 on a widest possible comparison window, corresponding to preferably, the whole of the sequence SEQ ID NO: 3. Unless otherwise specified, the percentages of identity indicated herein are established using the BLAST2 software (ALTSCHUL et al., Nucleic Acids Res., 25, 3389-3402, 1997 ) using the default parameters (BLOSUM62 matrix for protein comparisons, with penalties of 11 for an open gap, 1 for an expansion gap, 50 for a gap x dropoff, a value of random wait of 10, a word size (word size) of 3, and no filter).

Total or partial inhibition of the expression and / or activity of the SBP1 enzyme can be obtained in various ways known to those skilled in the art.

Preferably, the expression of the gene coding for SBP1 is reduced by mutagenesis or by inhibition or modification of its transcription or translation.

Mutagenesis of the gene coding for SBP1 can take place at the level of the coding sequence or expression regulation sequences, in particular of the promoter. For example, it is possible to delete all or part of said gene and / or to insert an exogenous sequence. By way of example, mention may be made of insertional mutagenesis: a large number of individuals derived from an active plant for the transposition of a transposable element (AC element or Mutator in maize) are produced and selected by example by PCR, the plants in which an insertion was carried out in the SBP1 gene, or in the vicinity with consequent effect on translation or expression.

Inhibition of the SBP1 gene can be achieved by any means known in the art. Thus, one can proceed to the mutation of the genes by insertion of a transposable element or a transfer DNA (T-DNA). It is also possible to carry out a physical or chemical mutagenesis, in particular by the use of EMS, X-rays or ultraviolet radiation (see in particular McCallum et al., Plant Physiol., 123, 439-442,

2000). The mutations of interest have the consequence of shifting the reading frame and / or of introducing a stop codon into the sequence and / or of modifying the level of transcription and / or translation of the gene and / or of making the enzyme less active than the wild-type protein. The plants thus mutated are screened for example by PCR, using primers located in the target gene. However, other screening methods, such as Southern blots or AIMS screening described in WO 99/27085 (for detecting insertions) can also be used, using probes specific for the target genes, or methods of screening. detection of point mutations or small insertions / deletions using particular endonucleases (CeI I, Endo I) as described in WO 2006/010646.

In another embodiment, the inhibition is achieved by transformation of the plant with a vector containing a sense or anti-sense construct of the target gene. These two methods are known to allow, under certain conditions, the inhibition of the target gene. The method of RNA interference (RNAi) is also used which is particularly effective for the extinction of genes in plants. This method is well known to those skilled in the art and consists of transforming the plant with a construct producing, after transcription, a double-stranded duplex of RNA, one of whose strands is complementary to the mRNA of the gene. target (for review on post-transcriptional inhibition techniques, Chicas and Macino, EMBO reports, 21 (11), 992-996,2001, for review on the use of interfering RNAs, Hannon, Nature, 418,244-251, 2002).

The present invention thus particularly relates to the use of at least one polynucleotide chosen from: a) a polynucleotide encoding an SBPl enzyme as defined above, and represented by SEQ ID No. 3; b) a polynucleotide complementary to a polynucleotide a) above; c) a fragment of at least 12 consecutive nucleotides, preferably at least 15, advantageously at least 20, and most preferably at least 50 consecutive nucleotides, specific for a polynucleotide a) or b) above, or capable of selectively hybridizing with said polynucleotide, d) a polynucleotide containing a polynucleotide X containing at least 12 nucleotides, preferably at least 15, advantageously at least 20, and most preferably at least 50 nucleotides capable of selectively hybridize with a polynucleotide defined in a), as well as a complementary polynucleotide Y of said polynucleotide X, to obtain a plant with increased digestibility and ingestibility.

A polynucleotide containing the genetic information for the synthesis of the SBP1 protein represented by SEQ ID No. 3, or of a protein having at least 94%, or 95% or 97% identity, is defined herein as the polynucleotide encoding SBP1. with SEQ ID No. 3.

This includes, in particular, genomic DNA, for example the sequence SEQ ID No. 1 represented in the appendix, as well as the corresponding cDNA (SEQ ID No. 2).

A specific fragment of polynucleotide a) or b) above is a fragment of said polynucleotide whose sequence is not found in other genes of the same plant.

A polynucleotide capable of hybridizing selectively with polynucleotide a) or b) above, is a polynucleotide which when hybridized under stringent conditions with a nucleic acid library of the same plant (especially a DNA library genomic, or cDNA) produces a detectable hybridization signal at least 2 times higher, and preferably at least 5 times higher than the background with said polynucleotide, but produces no detectable signal with other sequences of said library . Those skilled in the art are able to define the stringency of the hybridization conditions, which depend on the size and base composition of the polynucleotide concerned, as well as the composition of the hybridization mixture (in particular pH and ionic strength). Generally, stringent conditions for a polynucleotide of size and sequence data are obtained by operating at a lower temperature of about 5 ⁰ C to 10 ⁰ C above the melting temperature (Tm) of the hybrid formed, in the same reaction mixture, with this polynucleotide and its complement.

The subject of the present invention is in particular a method for increasing the digestibility and the ingestibility of a plant, by total or partial inhibition of the endogenous SBP1 enzyme of said plant, characterized in that it comprises the transformation of said plant with a recombinant DNA construct comprising a polynucleotide as defined above, placed in a sense orientation or in antisense orientation, or can be transcribed into double-stranded RNA, under transcriptional control of a suitable promoter.

The present invention also relates to recombinant DNA constructs comprising a polynucleotide as defined above. These constructs are in particular expression cassettes, comprising a polynucleotide as defined above, under transcriptional control of a suitable promoter. These expression cassettes may also contain other regulatory elements, in particular transcriptional regulatory elements such as terminators, amplifiers. Recombinant vectors which comprise polynucleotides as defined according to the invention or an expression cassette according to the invention are also objects of the invention.

Such vectors may also comprise other elements, for example one or more selection markers, and may especially be used for obtaining transgenic plants.

As non-limiting examples of promoters that may be used in the context of the present invention, mention may be made of the constitutive promoters, such as the 35S promoter of cauliflower mosaic virus (CaMV), or its derivatives, the promoter Cassava vein mosaic virus (CsVMV) (WO 97/48819), ubiquitin promoter or Actin-Intron-actin promoter, rice (McElroy et al., Mol. Gen. Genet., 231). , 150-160, 1991; GenBank S 44221).

Inducible or tissue-specific promoters can also be used. This makes it possible to induce inhibition of SBP1 only at certain stages of the plant's development, under certain environmental conditions, or in certain target tissues, such as, for example, stems, leaves, seeds, spathes, the cortex or xylem. It is thus possible to use the promoters of genes involved in the synthesis of lignins such as the COMT promoters, CCoAOMT, C4H, etc.

Advantageously, other transcriptional regulatory elements such as terminators, and in particular the 3'NOS terminator of nopaline synthase (Depicker et al., J. Mol Appl., Genet., 1, 561-573, 1982) are also advantageously used. or the 3'CaMV terminator (Franck et al., 21,285-294,1980, GenBank V00141). The marker genes of selection that may be used in the context of the present invention are, in particular, genes conferring resistance to an antibiotic such as hygromycin, kanamycin, bleomycin or streptomycin, or to a herbicide (EP 0 242 246) such as glufosinate, glyphosate or bromoxynil. The nptll gene which confers resistance to kanamyine can thus be used.

Plant transformation can be carried out by many methods, known in themselves to those skilled in the art.

For example, it is possible to transform plant cells, protoplasts or explants, and to regenerate an entire plant from the transformed material. The transformation can thus be carried out by transfer of the vectors according to the invention into protoplasts, in particular after incubation of the latter in a solution of polyethylene glycol (PG) in the presence of divalent cations (Ca 2+) according to the method described in the article. Krens et al. (Nature, 296, 72-74, 1982) or by electroporation notably according to the method described in the article by Fromm et al. (Nature, 319, 791-793, 1986). It is also possible to use a gene gun which can be used to project, at very high speed, metal particles covered with the DNA sequences of interest, thereby delivering genes inside the cell nucleus, in particular according to the technique described in article by Finer et al. (Plant

CeIl Report, 11,323-328,1992) or cytoplasmic or nuclear microinjection. The method of transformation with

Agrobacterium tumefaciens, in particular according to the method described by Ishida et al.

(1996, Nature Biotechnol 14, 745-50).

The subject of the present invention is also the plant cells and the transgenic plants that can be obtained by a method according to the invention, and the cells and plants containing a recombinant polynucleotide or an expression cassette as defined above. The invention also relates to the plant cells and to the plants obtained after mutation of the gene coding for SBP1 so that this results in an inhibition of the expression and / or the activity of said enzyme.

Of course, the present invention encompasses plants derived from plants according to the invention, which are in particular descendants, in particular hybrids resulting from a cross involving at least one plant according to the invention, obtained by planting or by vegetative propagation, plants according to the invention or obtained directly or indirectly by a method according to the invention.

The invention also includes plant cells and tissues, as well as organs or parts of plants, including leaves, stems, roots, flowers, fruits, and / or seeds obtained from a plant according to the invention.

Preferably, the invention applies to corn and cells and tissues derived from corn.

The present invention also relates to a maize selection process, characterized in that it comprises the search for an allele of the SBPl gene having a mutation resulting in an improvement in the digestibility and ingestibility of said maize. In a preferred case, said mutation results in a total or partial inhibition of the expression and / or the activity of the SBP1 protein.

Said allele can be searched by direct detection of the mutation responsible for the inhibition; it can also be sought by detecting the allelic form associated with this mutation of a polymorphism in linkage disequilibrium therewith. For example, the presence of the 12 nucleotides inserted into the intron of SBPl can be detected directly.

It is thus possible to use pairs of primers which selectively amplify the intron of the SBP1 gene, and detect the insertion of the 12 nucleotides by comparison of the size of the amplified fragment with the fragment obtained from a corn that does not contain these nucleotides.

The digestibility-friendly mutant alleles thus identified can then be introgressed in selected lines, and especially in elite lines, namely lines with significant agronomic and commercial potential.

The invention thus also relates to a particular allele of the SBPl gene, which is associated with a better digestibility. This allele is called SBPl-L.

The invention thus relates in particular to a corn plant, or a corn grain having said SBPL-L allele, and methods for obtaining them. In contrast, the invention relates to a plant or maize grain having a mutated SBPl allele, said mutation resulting in inhibition of the expression and / or activity of SBP1 (relative to a quasi-isogenic corn not having the mutation), and not being SBPl-L.

Preferably, the corn according to the invention is an "elite" corn. Those skilled in the art are familiar with the definition of elite maize. Elite maize is corn intended to produce hybrids intended to be marketed by crossing with another elite maize. An elite maize is defined as such in relation to the territory envisaged for marketing, as well as the agronomic character (s) sought for hybrid offspring. This includes a corn that can be included in a reference catalog. Thus, if the offspring is intended for animal feed, one will look for example a high and regular yield in tonnage of dry matter per hectare and a good ingestibility and digestibility, when one evaluates the nature "elite corn" . If the offspring is destined for the production of biofuels, such as biofuels or biogas, we will look for the maize with the best energy efficiency after processing.

In order to determine the elite character of a maize, hybrids obtained from it are compared to commercial reference hybrids (sold for the same purpose in the same region), field trials, survey surveys, and control measures. agronomic traits appropriate to the objective sought. A corn is defined as elite if the results obtained parameters studied for a hybrid obtained by crossing said maize are greater than 90% to the results recorded for the same parameters of the reference hybrids. In the context of the present invention, the digestibility character and the wall components (measured by NIRS (near infrared spectroscopy), for example) are notably studied. Near-infrared reflectance spectroscopy (NIRS) is the measurement of the reflectance intensity of near-infrared light by a sample in the 800 nm - 2.5 μm (12,500-4,000 cm-1) domains. This spectroscopy is typically used for quantitative measurements of organic functional groups, in particular O-H, N-H and C = O. This method is commonly used in the analysis of the digestibility and biochemical composition of plant samples.

Thus, an elite maize is a corn gathering the maximum of agronomic characteristics necessary for an economic penetration of the target market. The Since the corn market is now a hybrid market, the evaluation of the elite character of maize is also done by the ability of the maize to combine / produce hybrids.

Thus, the implementation of the present invention makes it possible to obtain an elite maize intended for the marketing of hybrids for animal feed and silage, or the production of biofuels presenting Pallèle SBPl-L. This elite maize is homozygous for the SBPl-L allele.

In another embodiment, the invention makes it possible to obtain a hybrid maize obtained by crossing two homozygous parent lines, said hybrid maize having an SBPL-L allele. This hybrid maize can be homozygous (if each homozygous parent has the SBPL-L allele) or heterozygous for the allele

SBPL-L.

By the implementation of the invention, it is also possible to generate a maize or maize grain containing one or more transgenes in addition to the SBPL-L allele. Mention may be made of transgenes conferring male sterility, male fertility, resistance to a herbicide (in particular glyphosate, glufosinate, imidazolinone, sulfonylurea, L-phosphinotricin, triazine, benzonitrile), insect resistance (especially a transgene encoding a toxin of Bacillus thuringiensis), tolerance to water stress. These maize can be obtained by crossing a corn containing the SBPL-L allele with a corn containing said transgene. The implementation of backcrossing followed by self-fertilization yields homozygous elite maize for the SBPL-L and the trangene allele. Thus, a hybrid maize containing both the SBPL-L allele and the transgene can be obtained by following the teaching of the invention.

The present invention also provides those skilled in the art with the means to select corn plants having improved fungal pathogen tolerance characteristics. It suffices to carry out a PCR, or a Southern blot (genomic DNA hybridization on membrane) to monitor the presence of the insertion of 12 nucleotides in the first intron of the gene coding for SBP1. Those skilled in the art can easily determine the primers and probes for identifying the presence of the SBPL-L allele. The invention thus also relates to a method for monitoring the SBPL-L allele by means of molecular biology techniques, and in particular via PCR using the primers mentioned in the examples.

The invention also relates to a method for obtaining a maize line having a better ingestibility and digestibility, comprising the step of introgression of the SBPL-L allele, in a reference line, having a quality agronomic character. The introgression of the character is notably carried out by selection, according to methods known in the art (crosses and selfing). Plants are selected in particular using molecular markers.

The principle is recalled below:

A series of backcrosses is performed between the elite line and the line bearing the SBPL-L allele.

During backcrossing, individuals carrying the SBPL-L allele can be selected, and recombining the smallest fragment of the donor line around this allele. Indeed, thanks to the molecular markers, one selects the individuals having for the markers close to the gene, the genotype of the elite line.

In addition, it is also possible to accelerate the return to the elite parent through molecular markers distributed throughout the genome. At each backcross, the individuals with the most fragments from the recurrent elite parent will be selected.

With good implementation, from the fourth generation, it is possible to obtain an almost isogenic line of the elite line, that is to say identical to the original elite line, but having integrated the locus carrying the SBPL-allele. L. The integrated fragment carries the SBPL-L allele as well as flanking chromosomal regions derived from

F116.

The invention thus relates to a method for obtaining corn having increased digestibility and ingestibility, comprising introgressing the SBPL-L allele in said maize, comprising the steps of: a) crossing a first corn line exhibiting the SBPL-L allele with a second maize not having the said allele, b) genotyping the progeny obtained and selecting the progeny with the SBPL-L allele, c) backcrossing said descendants with said second elite maize line usable for the production of hybrids, d) repeating if necessary steps b) and c) until obtaining an isogenic line of said second maize, having the SBPL allele -L, e) optionally, perform a self-fertilization to obtain a homozygous plant for the SBPl-L allele.

The genotyping of step b) is preferably carried out using molecular markers (microsatellite markers for example), making it possible to define the share of each of the two parents in the offspring. Maize, which has the appropriate genetic character with respect to the SBPL-L allele, is conventionally selected in the offspring by conventional molecular biology methods (such as PCR or Southern Blot). Optionally, in step b), the offspring with the best genome ratio are selected for the second maize. Maize obtained from step d) or e) can be used to create hybrids, or multiplied as parents. The descendants (hybrids or descendants by self-fertilization or other crosses) are thus maize derived maize obtained by the selection process mentioned above.

The increased digestibility and ingestibility of maize according to the present invention is understood to be relative to a quasi-isogenic corn that does not include the SBPL-L allele, and is measured by NIRS, as described above or by the decrease in crosslinking ferulic acid and the amount of lignins (Lignin Acid Detergent) after isolation of the walls (Neutral Detergent Fibers), according to methods known to those skilled in the art. One can also directly measure the improvement of the digestibility of the organic matter of the stem + leaves part of the plant, in percentage of digested organic matter, or the percentage of lignins and the digestibility of the walls, and the ferulic acid crosslinking content according to the methods known in the art.

In addition, the agronomic results observed after multiple backcrosses do not show any difference between the isogenic lines presenting the SBPL-L allele and the quasi-isogenic plants. Finally, the invention relates to the use of a maize according to the invention, obtained by a process according to the invention, or derived from such a maize (in particular a hybrid maize of which at least one of the two parents is a corn according to the invention or has been obtained by a process according to the invention) for the preparation of a composition intended for animal feed, a method for the preparation of a composition intended for animal feed comprising silage of a corn according to the invention, as well as the composition intended for animal feed, thus obtained. In particular, said maize is particularly useful for feeding cattle, or for the production of biofuels, including biofuels or biogas.

The nonlimiting examples which follow illustrate the invention and the implication of SBP1 in digestibility, and its use for obtaining plants having improved digestibility and ingestibility and the production of biofuels such as biofuels or biogas.

EXAMPLES

Example 1 Comparison of the Expression of the SBPl Gene Between Two Corn Varieties Having Different Digestibilities

Corn lines called Fl 16 (more digestible) and F2 (less digestible) were used. The plants were kept in a greenhouse. The stems were collected just after emergence of the panicles, 4 hours after sunrise. Samples of the three biological repeats were mixed for RNA extraction. The samples were maintained at -80 ^° C. in liquid nitrogen.

Total RNA was isolated from 2 g of each sample, and the material resuspended in the extraction buffer (100 mM Tris, pH 8.0, 100 mM LiCl, 10 mM EDTA, 1% SDS, 1% PVPP, 1% PVP, 770 mg / l DTT) and extracted twice with phenol: chloroform: isoamyl alcohol (25: 24: 1). Total RNA was precipitated with 2 M LiCl at 4 ° C overnight and centrifuged (45 min at 12,000 rpm and 4 ° C). The pellet was resuspended in sterile distilled water. The total RNA was then purified with the RNeasy Midi kit (Qiagen, Ref 75144), and subjected to DNA digestion with the RNase-free DNase kit (Qiagen, Ref 79254) for 45 min at 30 ^° C. RNA was measured at 260 nm with a Biophotometer (Eppendorf) and visualized on 1.5% agarose gels. The RNA concentration was adjusted to 1 μg / μl for future use.

A membrane containing labels specific for 691 genes (gene-specific tags (GSTs)), described by Guillaumie et al (Plant Physiol, 2007 Jan, 143 (I): 339-63), was used. The synthesis of cDNA probes, membrane hybridization and data analysis were performed according to Pesquet et al. (Plant Physiol 2005 Dec 139 (4): 1821-39) and Guillaumie et al. (op cit). Each gene was deposited twice on two membranes, corresponding to four replicates (20 x 20 cm Nytran Super-Charge Membrane Nylon, Schleicher and Schuell, Keene, NH, USA). Two independent hybridizations were performed for each sample. The reproducibility of the gross signal intensity on the membranes was checked for each membrane and between the membranes for each gene. Outliers were not taken into account. Normalization was performed on the basis of background without hybridization, non-specific hybridization and positive controls (Tris- EDTA pH8, pBluescriptII, ubiquitin and kanamycin-nptll). The standard deviation was calculated for each gene after normalization. According to Pesquet et al. (2005) and Guillaumie et al. (2007), genes that show a hybridization difference of more than twice compared to mean signals are considered differentially expressed. The normalized level of hybridization at 3000 is similar to the level observed on a membrane without hybridization, the minimum level of significant hybridizations was set at a normalized value of 6000 (2 times the value of the blank).

It has been shown that the SBP1 gene is differentially significantly differentiated. This gene is under-expressed in line FI16 with an intensity ratio F116 / F2 equal to 0.39. This gene has been cloned by Scott-Craig et al. (Plant Physiol (1998) 116: 1083-1089) in corn line B73, and was believed to be involved in plant protection mechanisms against chloroacetanilide and thiocarbamate herbicide-related injuries. The normal role of SBP1 in maize metabolism has not been elucidated, although some studies have linked it to possible involvement in lignification (Scott-Craig et al., 1998). The inventors have also shown its involvement in the biosynthesis of ferulic acid and probably S units. The same RNA samples were also used to perform a study on a DNA chip. This chip contains 57,000 corn-specific oligonucleotides. RNA samples, which were labeled with a Cy3 and Cy5 system with Ambion Amino Allyl MessageAmp kit (ref 1752, www.ambion.com), were reverse transcribed. The samples were hybridized to the slides and incubated overnight at 37 ° C on a Slide Booster (Advalytix, www.advalytix.com). After hybridization, the slides were scanned. Each hybridization produces a pair of images that have been processed with Arrayvision software (Imaging Research Inc), which allows the segmentation and correction of background noise and produces fluorescence intensity pairs for each oligonucleotide on each slide with R = red for Cy5 and G = green for Cy3.

For data normalization, the loess method has been used, and allows for A-dependent local normalization.

Of the 57,000 oligonucleotides deposited on the slide, 175 were identified as genes potentially involved in cell wall synthesis. Of these 175 genes, 14 are under-expressed, 27 over-expressed, and 134 show no expression differences between F2 and F116. This study has shown that the SBPl gene is under-expressed in

F116 compared to F2, with an expression ratio F116 / F2 being close to 0.4.

These two studies show that the SBP1 gene is under-expressed in a more digestible corn line compared to another corn line.

Example 2 Identification of a Specific Allele of a Digestible Variety

Sequencing of the SBP1 gene was performed on four amplification products with the following primer pairs: SBP 110 forward 5'-GAGCACACTTGACTAGTC-3 '(SEQ ID NO: 6), SBP 110 reverse 5'-CAGAAGCCATTGCT AGAG -3 '(SEQ ID NO: 7); SBPl I forward 5'-CACATACACAAGCACAAGCG-3 '(SEQ ID NO ^: 8), SBP I I reverse 5'-

TATCTCGATGCCGAAGTTGTC-3 '(SEQ ID NO ^: 9); SBP1 21 forward 5'-GGTGC AATCTC ACGGAT AGAC-3 '(SEQ ID NO: 10), SBP 1 21 reverse 5'- GGTGC AATCTC ACGGAT AGAC-3 '(SEQ ID NO: 11); SBP1 31 forward 5'-CCTGAAGGAATTTGGAGCTC-3 '(SEQ ID NO ^: 12), SBP 31 reverse 5'-CGGTAGTGAT AGTAGTAAGC A-3' (SEQ ID NO: 13).

The PCRs were carried out in a total volume of 50 μl with 30 ng of genomic DNA, buffer Taq polymerase, 3 mM MgCl ₂ , 0.2 mM of each DNTP, 0.4 μM of each primer, 0.5 Platinium Taq DNA polymerase (Invitrogen Ref 10966-018). The conditions were as follows: 94 ° C for 2 min; 40 cycles at 94 ° C for 1 min, 56 ° C for 1 min, 72 ° C for 1 min 30; and a final extension at 72 ° C for 7 min. The amplification products were checked on a 1% agarose gel and the sequencing performed.

A polymorphism study was performed on both FIl 6 and F2 lines. With respect to the F2 allele, the F116 allele has a deletion of a codon encoding a glutamic acid (GAG) in the first exon, and a 12 bp insertion into the intron. These elements are quite original and specific to F116 (according to a comparison with other lineages). An insertion of 12 bp in the 3 'untranslated region (3'UTR) in F116, absent in F2, was also detected. It should be noted, however, that these 12 nucleotides are present in the genome of line B73, studied by Scott-Craig et al., Or in the genome of other lines. The specific allele of F116 (SEQ ID NO: 4) is called SBP1-L. Without being bound by this interpretation, it is possible that the addition of nucleotides in the SBPL-L allele leads to greater instability of the messenger RNA, and to significant degradation. It is also possible that the translated protein (SEQ ID No. 5) has a lower activity than the protein obtained for the allele described by SEQ ID No. 1, due to the disappearance of a glutamic acid.

Example 3 Introduction of the SBPL-L Allele in the F2 Line

To assess the effect of the allele SBPL-L was developed 140 recombinant inbred lines (RIL, Recombinant Inbred Lines) between lines F2 and wire 6 until the 7 ^th generation. These lines were tested for their digestibility. It has been shown that the SBPl gene, located on bin 2.04 co localizes with several QTLs linked to ferulic acid. The presence of the SBI-L allele from FIl 6 in quasi-isogenic lines (which differ globally only in the presence or absence of this allele results in a ferulic acid content etherified lower than the observed content for the line containing the allele from F2, and better digestibility in vitro.

From these data, it is concluded that the SBPL-L allele allows itself to modify the ferule (and possibly S) content and to improve the digestibility and ingestibility of maize.

Example 4 Subexpression of SBPl by Transgenesis

Corn plants were transformed by Agrobacterium tumefaciens by a partial sequence of the SBP1 gene (Plant Physiol 1998: 116: 1083-1089) in order to specifically inhibit the expression of this gene in order to increase their digestibility.

The partial sequence of SBP1 corresponds to nucleotides 597-1047 of

SEQ ID No. 1. This fragment was obtained by PCR on genomic DNA and cloned into the vector pGEMT-easy in order to obtain the plasmid named pBIOS1428. This PCR fragment was then cloned into pENTR-1A by cleaving pBIOS1428 with SpeI (cohesive ends) and NotI and cleaving pENTR-1A with XmnI and NotI.

After ligation, plasmid pBIOS1451 was obtained. The ligation reaction between pBIOS1451 and pBIOS893 made it possible to obtain the plasmid pBIOS1461 (FIGURE 1) which bears the partial sequence of SBP1 in sense and antisense orientation, the two fragments being separated by an intron in order to favor the meeting of the complementary 2 strands. to form a double-stranded RNA during transcription. This construct makes it possible to inhibit the expression of the SBPl gene by RNAi.

Plasmid pBIOS893 is a derivative of pSB1 (Komari et al., Plant J. 10,

165-174, 1996) which contains, in particular, marker genes for the selection of transformed maize, a constitutive CsVMV promoter (Verdaguer et al., Plant Mol

Biol 6, 1129-1139, 1996) followed by the intron of the rice actin gene (McElroy et al.

Plant Cell, (2) 163, 1990), and a Sac66 polyadenylation site (Jenkins et al., Plant.

This is about. 22, 159-167, 1999). pBIOS1461 was transferred to the Agrobacterium strain LBA4404 (pSB1) according to the provisions of Komari et al (1996, op cit) and the A188 line was transformed with the Agrobacterium strain as described by Ishida et al. . (1996, op cit). Transformants are analyzed by Southern Blot to determine the number of copies and if possible the number of loci.

Seedlings on the T1 generation are made to collect mesocotyl and hypocotyl. These samples are intended to extract the RNAs in order to measure the residual expression of SBP1 in these tissues. Transcriptomic data are thus obtained to select transformants in which the expression of SBP1 is reduced and those will be used for further analyzes.

It is then a question of phenotyping these plants via biochemical analyzes in order to measure the content of ferulic acid, which intervenes in the crosslinking of the walls, and to verify the ingestibility and the digestibility of the plants thus obtained.

Claims

claims

A method for improving the digestibility and ingestibility of a plant, as well as its degradability for the production of biofuels, characterized in that the expression and / or activity in said plant is totally or partially inhibited, an enzyme hereinafter referred to as SBP1, the polypeptide sequence of which has at least 75% identity with the sequence SEQ ID No. 3.

2. Method according to claim 1, characterized in that said plant is corn.

3. Use of at least one polynucleotide selected from: a) a polynucleotide encoding an SBP1 enzyme as defined in claim 1; b) a polynucleotide complementary to a polynucleotide a) above; c) a fragment of at least 12 consecutive nucleotides, a polynucleotide a) or b) above, or capable of hybridizing selectively with said polynucleotide; d) a polynucleotide allowing the production of a duplex that can be used for the interference of RNA (RNAi), said polynucleotide containing a polynucleotide X containing at least 12 nucleotides, preferably at least 15, advantageously at least 20, and at least 50 nucleotides capable of selectively hybridizing with a polynucleotide defined in a) and a complementary polynucleotide Y of said polynucleotide X for the implementation of a process according to any one of claims 1 or 2.

4. Method according to any one of claims 1 or 2, characterized in that the inhibition of the expression and / or activity of the enzyme SBP1 is obtained by mutagenesis of the gene encoding said enzyme.

5. Method according to any one of claims 1 or 2, characterized in that it comprises the transformation of said plant with a recombinant DNA construct comprising a polynucleotide as defined in claim 3, under transcriptional control of a suitable promoter .

An expression cassette comprising a polynucleotide as defined in claim 3, under transcriptional control of a suitable promoter.

Recombinant vector containing an expression cassette according to claim 6.

8. Corn obtainable by the method according to claim 4, and having a mutation in the gene encoding the SBP1 enzyme whose sequence has at least 75% identity with SEQ ID No. 2, said mutation resulting in an inhibition of the expression and / or the activity of SBP1.

9. Genetically modified corn obtainable by a process according to claim 5.

10. A method of selecting maize, characterized in that it comprises the search for an allele of the SBPl enzyme gene having a mutation resulting in an improvement in the digestibility of said maize.

11. The method of claim 10, characterized in that said allele is represented by SEQ ID No. 4.

A method for obtaining corn having increased digestibility, comprising introgressing the SBPL-L allele in said maize, comprising the steps of: a) crossing a first maize line having the SBPL-L allele with a second maize does not have said allele, b) genotyping the progeny obtained and selecting the progeny with Pallele SBP1-L, and optionally having the best genome ratio for said second maize, c) backcrossing said progeny with said second elite maize line usable for the production of hybrids, d) if necessary repeat steps b) and c) until an isogenic line of the second maize with the SBPL-L allele is obtained, e) optionally, perform self-fertilization in order to to obtain a homozygous plant for the SBPL-L allele.

13. Use of a maize according to claim 8 or 9, obtained by a process according to claim 12 or derived from such maize, for the preparation of a composition intended for animal feed or the preparation of biofuels.

14. A method for the preparation of a composition for animal feed comprising silage corn according to claim 8 or 9, obtained by a process according to claim 12 or derived from such corn.

15. Method for the preparation of biofuels comprising the degradation of a corn according to claim 8 or 9, obtained by a process according to claim 12 or derived from such corn.