Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1997-12-16
2001-06-26
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S069100, C435S468000, C435S422000, C435S419000, C536S023200, C536S023600, C800S284000, C800S319000
Reexamination Certificate
active
06252135
ABSTRACT:
BACKGROUND OF THE INVENTION
Lignin is a major part of the supportive structure of most woody plants including angiosperm and gymnosperm trees which in turn are the principal sources of fiber for making paper and cellulosic products. In order to liberate fibers from wood structure in a manner suitable for making many grades of paper, it is necessary to remove much of the lignin from the fiberilignin network. Lignin is removed from wood chips by treatment of the chips in an alkaline solution at elevated temperatures and pressure in an initial step of papermaking processes. The rate of removal of lignin from wood of different tree species varies depending upon lignin structure. Three different lignin structures have been identified in trees: p-hydroxyphenyl, guaiacyl and syringyl, which are illustrated in FIG.
1
.
Angiosperm species, such as
Liquidambar styraciflua
L. [sweetgum], have lignin composed of a mixture of guaiacyl and syringyl monomer units. In contrast, gymnosperm species such as
Pinus taeda
L. [loblolly pine] have lignin which is devoid of syringyl monomer units. Generally speaking, the rate of delignification in a pulping process is directly proportional to the amount of syringyl lignin present in the wood. The higher delignification rates associated with species having a greater proportion of syringyl lignin result in more efficient pulp mill operations since the mills make better use of energy and capital investment and the environmental impact is lessened due to a decrease in chemicals used for delignification.
It is therefore an object of the invention to provide gymnosperm species which are easier to delignify in pulping processes.
Another object of the invention is to provide gymnosperm species such as loblolly pine which contain syringyl lignin.
An additional object of the invention is to provide a method for modifying genes involved in lignin biosynthesis in gymnosperm species so that production of syringyl lignin is increased while production of guaiacyl lignin is suppressed.
Still another object of the invention is to produce whole gymnosperm plants containing genes which increase production of syringyl lignin and repress production of guaiacyl lignin.
Yet another object of the invention is to identify, isolate and/or clone those genes in angiosperms responsible for production of syringyl lignin.
A further object of the invention is to provide, in gymnosperms, genes which produce syringyl lignin.
Another object of the invention is to provide a method for making an expression cassette insertable into a gymnosperm cell for the purpose of inducing formation of syringyl lignin in a gymnosperm plant derived from the cell.
Definitions
The term “promoter” refers to a DNA sequence in the 5′ flanking region of a given gene which is involved in recognition and binding of RNA polymerase and other transcriptional proteins and is required to initiate DNA transcription in cells.
The term “constitutive promoter” refers to a promoter which activates transcription of a desired gene, and is commonly used in creation of an expression cassette designed for preliminary experiments relative to testing of gene function. An example of a constitutive promoter is 35S CaMV, available from Clonetech.
The term “expression cassette” refers to a double stranded DNA sequence which contains both promoters and genes such that expression of a given gene is acheived upon insertion of the expression cassette into a plant cell.
The term “plant” includes whole plants and portions of plants, including plant organs (e.g. roots, stems, leaves, etc.)
The term “angiosperm” refers to plants which produce seeds encased in an ovary. A specific example of an angiosperm is
Liquidambar styraciflua
(L.)[sweetgum]. The angiosperm sweetgum produces syringyl lignin.
The term “gymnosperm” refers to plants which produce naked seeds, that is, seeds which are not encased in an ovary. A specific example of a gymnosperm is
Pinus taeda
(L.) [loblolly pine]. The gymnosperm loblolly pine does not produce syringyl lignin.
SUMMARY OF THE INVENTION
With regard to the above and other objects, the invention provides a method for inducing production of syringyl lignin in gymnosperms and to gymnosperms which contain syringyl lignin for improved delignification in the production of pulp for papermaking and other applications. In accordance with one of its aspects, the invention involves cloning an angiosperm DNA sequence which codes for enzymes involved in production of syringyl lignin monomer units, fusing the angiosperm DNA sequence to a lignin promoter region to form an expression cassette, and inserting the expression cassette into a gymnosperm genome.
Enzymes required for production of syringyl lignin in an angiosperm are obtained by deducing an amino acid sequence of the enzyme, extrapolating an MRNA sequence from the amino acid sequence, constructing a probe for the corresponding DNA sequence and cloning the DNA sequence which codes for the desired enzyme. A promoter region specific to a gymnosperm lignin biosynthesis gene is identified by constructing a probe for a gymnosperm lignin biosynthesis gene, sequencing the 5′ flanking region of the DNA which encodes the gymnosperm lignin biosynthesis gene to locate a promoter sequence, and then cloning that sequence.
An expression cassette is constructed by fusing the angiosperm syringyl lignin DNA sequence to the gymnosperm promoter DNA sequence. Alternatively, the angiosperm syrinigyl lignin DNA is fused to a constitutive promoter to form an expression cassette. The expression cassette is inserted into the gymnosperm genome to transform the gymnosperm genome. Cells containing the transformed genome are selected and used to produce a transformed gymnosperm plant containing syringyl lignin.
In accordance with the invention, the angiosperm gene sequences bi-OMT, 4CL, P450-1 and P450-2 have been determined and isolated as associated with production of syringyl lignin in sweetgum and lignin promoter regions for the gymnosperm loblolly pine have been determined to be the 5′ flanking regions for the 4CL1B, 4CL3B and PAL gymnosperm lignin genes. Expression cassettes containing sequences of selected genes from sweetgum have been inserted into loblolly pine embryogenic cells and presence of sweetgum genes associated with production of syringyl lignin has been confirmed in daughter cells of the resulting loblolly pine embryogenic cells.
The invention therefore enables production of gymnosperms such as loblolly pine containing genes which code for production of syringyl lignin, to thereby produce in such species syringyl lignin in the wood structure for enhanced pulpability.
REFERENCES:
patent: 4795855 (1989-01-01), Fillatti et al.
patent: 4801545 (1989-01-01), Stuart et al.
patent: 4886937 (1989-12-01), Sederoff et al.
patent: 4957866 (1990-09-01), Gupta et al.
patent: 5034322 (1991-07-01), Rogers et al.
patent: 5034326 (1991-07-01), Pullman et al.
patent: 5107065 (1992-04-01), Shewmaker et al.
patent: 5122466 (1992-06-01), Stomp et al.
patent: 5187092 (1993-02-01), Uddin
patent: 5236841 (1993-08-01), Gupta et al.
patent: 5294549 (1994-03-01), Pullman et al.
patent: 5413930 (1995-05-01), Becwar et al.
patent: 5451514 (1995-09-01), Boudet et al.
patent: 5457041 (1995-10-01), Ginaven et al.
“cDNA cloning, sequence analysis and seasonal expression of lignin-bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase of aspen”, by Robert C. Bugos, et al, Plant Molecular Biology 17: 1203-1215, 1991.
“Genetric engineering of wood”, by Ross Whetten, et al, Forest Ecology and Management, 43 (1991) 301-316, Elsevier Science Publishers B.V., Amsterdam.
“Logger and Lumberman” from Trailer Director Portable Sawmills & Firewood Processors, Dec. 15, 1995.
Carraway Daniel T.
Chiang Vincent L.
Smeltzer Richard H.
Fox David T.
International Paper Company
Luedeka Neely & Graham P.C.
Zaghmout Ousama
LandOfFree
Production of syringyl lignin in gymnosperms does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Production of syringyl lignin in gymnosperms, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Production of syringyl lignin in gymnosperms will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2516806