Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1998-07-24
2001-04-03
Hutzell, Paula K. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S069100, C435S070100, C435S071100, C435S466000, C435S410000, C435S418000, C435S419000, C435S320100, C536S023100, C536S023600, C800S284000, C800S288000, C800S290000, C800S295000, C800S298000, C800S320100
Reexamination Certificate
active
06211432
ABSTRACT:
The present invention relates to the use of DNA sequences which code for a cinnamoyl-CoA reductase (CCR) in plants, or any fragment of these sequences, or also any sequence derived from the latter, or their complementary sequences, in the context of carrying out processes for regulating the level of lignin in plants.
Lignin is a complex heterogeneous aromatic polymer which renders impermeable and reinforces the walls of certain plants cells.
Lignin is formed by polymerization of free radicals derived from monolignols, such as paracoumaryl, coniferyl and sinapyl alcohols (Higuchi, 1985, in Biosynthesis and degradation of wood components (T. Higuchi, ed.), Academic Press, Orlando, Fla. pp. 141-160).
Lignins have a wide variation in their relative content of monolignols, as a function of the species and the various tissues within the same plant
This variation is probably caused and controlled by different activities and specificities of substrates, the enzymes necessary for biosynthesis of lignin monomers (Higuchi, 1985, loc. cit.).
Beyond its role in the structure and development of plants, lignin represents a major component of the terrestrial biomass and assumes a major economic and ecological significance (Brown, 1985, J. Appl. Biochem. 7, 371-387; Whetten and Sederoff, 1991, l orest Ecology and Management, 43, 301-316).
At the level of exploitation of the biomass, it is appropriate first to note that lignin is a limiting factor of the digestibility and nutritional yield of fodder plants. In fact, it is clearly demonstrated that the digestibility of fodder plants by ruminants is inversely proportional to the content of lignin in these plants, the nature of the lignins also being a determining factor in this phenomenon (Buxton and Roussel, 1988, Crop. Sci., 28, 553-558; Jung and Vogel, 1986, J. Anim., Sci., 62, 1703-1712).
Among the main fodder plants in which it would be of interest to reduce the lignin contents there may be mentioned: lucerne, fescue, maize, fodder used for silaging . . . .
It should also be noted that high lignin contents are partly responsible for the limited quality of sunflower cake intended for feeding cattle, and for the reduction in germinative capacities of certain seeds in the horticultural sector.
It may also be emphasized that the intense lignification which results during preservation of plant components after harvesting rapidly renders products such as asparagus, yam, carrots etc . . . unfit for consumption.
Furthermore, it is also appropriate to note that more than 50 million tonnes of lignin are extracted from ligneous material each year in the context of production of paper pulp in the paper industry. This extraction operation, which is necessary to obtain cellulose, is costly in energy and, secondly, causes pollution through the chemical compounds used for the extraction, which are found in the environment (Dean and Eriksson, 1992, Holzforschung, 46, 135-147: Whetten and Sederoff, 1991, loc. cit.).
To reduce the proportions of lignins (which make up to 20 to 30% of the dry matter, depending on the species) to a few per cent (2 to 5%) would represent an increase in yield and a substantial saving (chemical products), and would contribute to improving the environment (reduction in pollution). Given the scale of use of ligneous material, these decreases would have extremely significant repercussions. In this case, the species concerned could be poplar, eucalyptus,
Acacia magnium
, the genus Casuarina and all the angiosperms and gymnosperms used for the production of paper pulp.
It is clear that in the two sectors under consideration, the reduction in the levels of lignins must be moderated to preserve the characteristics of rigidity and the normal architecture of the plant (or the tree), since the lignins which strengthen the cell walls play a significant role in maintaining the erect habit of plants.
The natural variations in the lignin contents observed in nature for the same species (deviations which can be up to 6-8% of the dry matter among individuals) justify the reductions suggested above.
The resistance to degradation of lignin, like the difficulties encountered in the context of its extraction, are probably due to the complex structure of this polymer, which is made up of ether bonds and carbon-carbon bonds between the monomers, as well as to the numerous chemical bonds which exist between the lignin and the other components of the cell wall (Sarkanen and Ludwig, 1971, in Lignins: Occurrence, Formation, Structure and Reactions (K. V. Sarkanen and C. H. Kudwig ed.) New York: Wiley—Interscience, pp. 1-18).
Starting from cinnamoyl-CoA, the biosynthesis of lignins in plants is effected in the following manner:
An approach to attempt to reduce the level of lignins in plants by genetic engineering would consist of inhibiting the synthesis of one of the enzymes in the biosynthesis chain of these lignins indicated above.
A particularly suitable technique in the context of such an approach is to use antisense mRNA which is capable of hybridizing with the mRNA which codes for these enzymes, and consequently to prevent, at least partly, the production of these enzymes from their corresponding mRNA.
Such an antisense strategy carried out with the aid of the gene which codes for the CAD in tobacco was the subject matter of European Patent Application no. 584 117, which describes the use of antisense mRNA which is capable of inhibiting the production of lignins in plants by hybridizing with the mRNA which codes for the CAD in these plants.
The results in the plants transformed in this way demonstrate a reduction in the activity of the CAD, but paradoxically the contents of lignins show no change. Complementary studies indicate that the lignins of transformed plants are different from control lignins, since the cinnamylaldehydes are incorporated directly into the lignin polymer.
One of the aims of the present invention is specifically that of providing a process which allows effective regulation of the contents of lignins in plants, either in the sense of a considerable reduction in these contents with respect to the normal contents in plants, or in the sense of an increase in these contents.
Another aim of the present invention is to provide tools for carrying out such a process, and more particularly constructions which can be used for the transformation of plants.
Another aim of the present invention is to provide genetically transformed plants, in particular fodder plants which can be digested better than non-transformed plants, or also transformed plants or trees for the production of paper pulp, from which the extraction of lignins would be facilitated and less polluting than in the case of non-transformed trees.
Another aim of the present invention is that of providing transformed plants which are more resistant to attacks from the environment, in particular to parasitic attacks, than the non-transformed plants are, or also transformed plants of a larger size, or of a smaller size (than that of the non-transformed plants).
The present invention relates to the use of recombinant nucleotide sequences containing one (or more) coding region(s), this (these) coding region(s) being made up of a nucleotide sequence chosen from the following:
the nucleotide sequence represented by SEQ ID NO 1 which codes for an mRNA, this mRNA itself coding for the cinnamoyl-CoA reductase (CCR) of lucerne represented by SEQ ID NO 2,
the nucleotide sequence represented by SEQ ID NO 3 which codes for an mRNA, this mRNA itself coding for the CCR of maize represented by SEQ ID NO 4,
a fragment of the nucleotide sequence represented by SEQ ID NO 1, or of that represented by SEQ ID NO 3, this fragment coding for a fragment of the CCR represented by SEQ ID NO 2 or for a fragment of the CCR represented by SEQ ID NO 3 respectively, this CCR fragment having an enzymatic activity equivalent to that of the two abovementioned CCRs,
the nucleotide sequence complementary to that represented by SEQ ID NO 1 or SEQ ID NO 3, this complementary sequence coding for an antisense mRNA whi
Beckert Michel
Boudet Alain-Michel
Briat Jean-François
Gamas Pascal
Grima-Pettenati Jacqueline
Centre National de la Recherche Scientifique
Hutzell Paula K.
Nixon & Vanderhye P.C.
Zaghmout Ousama
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