Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
1998-12-14
2001-03-20
Smith, Lynette R. F. (Department: 1649)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters plant part growth
C800S298000, C800S278000, C800S295000, C536S023600, C536S024100, C435S417000, C435S468000
Reexamination Certificate
active
06204434
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to the field of modification of lignin content and composition in plants. More particularly, this invention relates to enzymes involved in the lignin biosynthetic pathway and nucleotide sequences encoding such enzymes.
BACKGROUND OF THE INVENTION
Lignin is an insoluble polymer which is primarily responsible for the rigidity of plant stems. Specifically, lignin serves as a matrix around the polysaccharide components of some plant cell walls. The higher the lignin content, the more rigid the plant. For example, tree species synthesize large quantities of lignin, with lignin constituting between 20% to 30% of the dry weight of wood. In addition to providing rigidity, lignin aids in water transport within plants by rendering cell walls hydrophobic and water impermeable. Lignin also plays a role in disease resistance of plants by impeding the penetration and propagation of pathogenic agents.
The high concentration of lignin in trees presents a significant problem in the paper industry wherein considerable resources must be employed to separate lignin from the cellulose fiber needed for the production of paper. Methods typically employed for the removal of lignin are highly energy- and chemical-intensive, resulting in increased costs and increased levels of undesirable waste products. In the U.S. alone, about 20 million tons of lignin are removed from wood per year.
Lignin is largely responsible for the digestibility, or lack thereof, of forage crops, with small increases in plant lignin content resulting in relatively high decreases in digestibility. For example, crops with reduced lignin content provide more efficient forage for cattle, with the yield of milk and meat being higher relative to the amount of forage crop consumed. During normal plant growth, the increase in dry matter content is accompanied by a corresponding decrease in digestibility. When deciding on the optimum time to harvest forage crops, farmers must therefore chose between a high yield of less digestible material and a lower yield of more digestible material.
For some applications, an increase in lignin content is desirable since increasing the lignin content of a plant would lead to increased mechanical strength of wood, changes in its color and increased resistance to rot. Mycorrhizal species composition and abundance may also be favorably manipulated by modifying lignin content and structural composition.
As discussed in detail below, lignin is formed by polymerization of at least three different monolignols which are synthesized in a multistep pathway, each step in the pathway being catalyzed by a different enzyme. It has been shown that manipulation of the number of copies of genes encoding certain enzymes, such as cinnamyl alcohol dehydrogenase (CAD) and caffeic acid 3-O-methyltransferase (COMT) results in modification of the amount of lignin produced; see, for example, U.S. Pat. No. 5,451,514 and PCT publication no. WO 94/23044. Furthermore, it has been shown that antisense expression of sequences encoding CAD in poplar leads to the production of lignin having a modified composition (Grand, C. et al.
Planta
(
Berl.
) 163:232-237 (1985)).
While DNA sequences encoding some of the enzymes involved in the lignin biosynthetic pathway have been isolated for certain species of plants, genes encoding many of the enzymes in a wide range of plant species have not yet been identified. Thus there remains a need in the art for materials useful in the modification of lignin content and composition in plants and for methods for their use.
SUMMARY OF THE INVENTION
Briefly, the present invention provides isolated DNA sequences obtainable from eucalyptus and pine which encode, or partially enccode enzymes involved in the lignin biosynthetic pathway, DNA constructs including such sequences, and methods for the use of such constructs. Transgenic plants having altered lignin content and composition are also provided.
In a first aspect, the present invention provides isolated polynucleotide coding for cinnamyl alcohol dehydrogenase (CAD) isolated from eucalyptus, and for the following enzymes isolated from pine: cinnamate 4-hydroxylase (C4H), coumarate 3-hydroxylase (C3H), phenolase (PNL), 0-methyl transferase (OMT), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl-CoA reductase (CCR), phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase (4CL) and peroxidase (POX). In one embodiment, the isolated polynucleotides comprise a nucleotide sequence selected from the group consisting of: (a) sequences recited in SEQ ID NO: 1-13 and specified portions of the sequences recited in SEQ ID NOS: 2, 4-10, and 12; (b) complements of the sequences recited in SEQ IID NO: 1-13 and complements of specified portions of the sequences recited in SEQ ID NOS: 2, 4-10 and 12; (c) reverse complements of the sequences recited in SEQ ID NO: 1-13 and reverse complements of specified portions of the sequences recited in SEQ ID NOS: 2, 4-10 and 12; (d) reverse sequences of the sequences recited in SEQ ID NO: 1-13 and reverse complements of specified portions of the sequences recited in SEQ ID NOS: 2, 4-10 and 12; and (e) variants of the sequences of (a)-(d).
In another aspect, the invention provides DNA constructs comprising a DNA sequence of the present invention, either alone or in combination with one or more of the inventive sequences, or in combination with one or more known DNA sequences, together with transgenic cells comprising such constructs.
In a related aspect, the present invention provides DNA constructs comprising, in the 5′-3′ direction, a gene promoter sequence; a polynucleotide including a sequence disclosed herein, the polynucleotide preferably comprising an open reading frame coding for at least a functional portion of an enzyme involved in a lignin biosynthetic pathway; and a gene termination sequence. The open reading frame may be orientated in either a sense or antisense direction. DNA constructs comprising a polynucleotide including a sequence disclosed herein, the polynucleotide preferably comprising a non-coding region of a gene coding for an enzyme involved in a lignin biosynthetic pathway, together with a gene promoter sequence and a gene termination sequence, are also provided. Preferably, the gene promoter and termination sequences are functional in a host plant. Most preferably, the gene promoter and termination sequences are those of the original enzyme genes but others generally used in the art, such as the Cauliflower Mosaic Virus (CMV) promoter, with or without enhancers, such as the Kozak sequence or Omega enhancer, and
Agrobacterium tumefaciens
nopalin synthase terminator may be usefully employed in the present invention. Tissue-specific promoters may be employed in order to target expression to one or more desired tissues. In a preferred embodiment, the gene promoter sequence provides for transcription in xylem. The DNA construct may further include a marker for the identification of transformed cells.
In a further aspect, transgenic plant cells comprising the DNA constructs of the present invention are provided, together with plants comprising such transgenic cells, and fruits and seeds of such plants.
In yet another aspect, methods for modulating the lignin content and composition of a plant are provided, such methods including stably incorporating into the genome of the plant a DNA construct of the present invention. In a preferred embodiment, the target plant is a woody plant, preferably selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of
Eucalyptus grandis
and
Pinus radiata
. In a related aspect, a method for producing a plant having altered lignin content is provided, the method comprising transforming a plant cell with a DNA construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.
In yet a further aspect, the present invention provides methods for modifying the ac
Bloksberg Leonard N.
Grierson Alastair
Havukkala Ilkka
Genesis Research & Development Corporation Limited
Haas Thomas
Sleath Janet
Smith Lynette R. F.
Speckman Ann W
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