Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Plant cell or cell line – per se – contains exogenous or...
Reexamination Certificate
1998-04-17
2001-05-15
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Plant cell or cell line, per se, contains exogenous or...
C536S023200, C536S024100, C800S278000
Reexamination Certificate
active
06232122
ABSTRACT:
The present invention relates to a method of inhibiting gene expression, particularly inhibiting gene expression in a plant. The present invention also relates to a nucleotide sequence useful in the method. In addition, the present invention relates to a promoter that is useful for expressing the nucleotide sequence.
Starch is one of the main storage carbohydrates in plants, especially higher plants. The structure of starch consists of amylose and amylopectin. Amylose consists essentially of straight chains of &agr;-1-4-linked glycosyl residues. Amylopectin comprises chains of &agr;-1-4-linked glycosyl residues with some &agr;-1-6 branches. The branched nature of amylopectin is accomplished by the action of inter alia an enzyme commonly known as the starch branching enzyme (“SBE”). SBE catalyses the formation of branch points in the amylopectin molecule by adding &agr;-1,4 glucans through &agr;-1,6-glucosidic branching linkages. The biosynthesis of amylose and amylopectin is schematically shown in
FIG. 1
, whereas the &agr;-1-4-links and the &agr;-1-6 links are shown in FIG.
2
.
It is known that starch is an important raw material. Starch is widely used in the food, paper, and chemical industries. However, a large fraction of the starches used in these industrial applications are post-harvest modified by chemical, physical or enzymatic methods in order to obtain starches with certain required functional properties.
Within the past few years it has become desirable to make genetically modified plants which could be capable of producing modified starches which could be the same as the post-harvest modified starches. It is also known that it may be possible to prepare such genetically modified plants by expression of antisense nucleotide coding sequences. In this regard, June Bourque provides a detailed summary of antisense strategies for the genetic manipulations in plants (Bourque 1995 Plant Science 105 pp 125-149).
Whilst it is known that enzymatic activity can be affected by expression of particular nucleotide sequences (for example see the teachings of Finnegan and McElroy [1994] Biotechnology 12 883-888; and Matzke and Matzke [1995] TIG 11 1-3) there is still a need for a method that can more reliably and/or more efficiently and/or more specifically affect enzymatic activity.
According to a first aspect of the present invention there is provided a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence partially or completely codes (is) an intron in a sense orientation; and wherein the nucleotide sequence does not contain a sequence that is a sense exon sequence normally associated with the intron.
According to a second aspect of the present invention there is provided a method of affecting enzymatic activity in a starch producing organism (or a cell, a tissue or an organ thereof) comprising expressing in the starch producing organism (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron in a sense orientation; wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron; and wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed.
According to a third aspect of the present invention there is provided a sequence comprising the nucleotide sequence shown as any one of SEQ.I.D. No. 1 to SEQ.I.D. No. 13 or a variant, derivative or homologue thereof.
According to a fourth aspect of the present invention there is provided a promoter comprising the sequence shown as SEQ.I.D. No. 14 or a variant, derivative or homologue thereof.
According to a fifth aspect of the present invention there is provided a construct capable of comprising or expressing the present invention.
According to a sixth aspect of the present invention there is provided a vector comprising or expressing the present invention.
According to a seventh aspect of the present invention there is provided a cell, tissue or organ comprising or expressing the present invention.
According to an eighth aspect of the present invention there is provided a transgenic starch producing organism comprising or expressing the present invention. According to a ninth aspect of the present invention there is provided a starch obtained from the present invention.
According to a tenth aspect of the present invention there is provided pBEA11 (NCIMB 40754). According to an eleventh aspect of the present invention there is provided a sense nucleotide sequence that is obtainable from &lgr;-SBE 3.2 (NCIMB 40751) or &lgr;-SBE 3.4 (NCIMB 40752) or a variant, derivative or homologue thereof.
A key advantage of the present invention is that it provides a method for preparing modified starches that is not dependent on the need for post-harvest modification of starches. Thus the method of the present invention obviates the need for the use of hazardous chemicals that are normally used in the post-harvest modification of starches.
In addition, the present invention provides inter alia genetically modified plants which are capable of producing modified and/or novel and/or improved starches whose properties would satisfy various industrial requirements.
Thus, the present invention provides a method of preparing tailor-made starches in plants which could replace the post-harvest modified starches.
Also, the present invention provides a method that enables modified starches to be prepared by a method that can have a more beneficial effect on the environment than the known post-harvest modification methods which are dependent on the use of hazardous chemicals and large quantities of energy.
An other key advantage of the present invention is that it provides a method that may more reliably and/or more efficiently and/or more specifically affect enzymatic activity when compared to the known methods of affecting enzymatic activity. With regard to this advantage of the present invention it is to be noted that there is some degree of homology between coding regions of SBEs. However, there is little or no homology with the intron sequences of SBEs. Thus, sense intron expression provides a mechanism to affect selectively the expression of a particular SBE. This advantageous aspect could be used, for example, to reduce or eliminate a particular SBE enzyme and replace that enzyme with another enzyme which can be another branching enzyme or even a recombinant version of the affected enzyme or even a hybrid enzyme which could for example comprise part of a SBE enzyme from one source and at least a part of another SBE enzyme from another source. This particular feature of the present invention is covered by the combination aspect of the present invention which is discussed in more detail later.
Thus the present invention provides a mechanism for selectively affecting SBE activity. This is in contrast to the prior art methods which are dependent on the use of for example antisense exon expression whereby it would not be possible to introduce new SBE activity without affecting that activity as well.
Preferably with the first aspect of the present invention starch branching enzyme activity is affected and/or wherein the levels of amylopectin are affected and/or the composition of starch is changed.
Preferably with the first or second aspect of the present invention the nucleotide sequence does not contain a sequence that is sense to an exon sequence.
Preferably with the fourth aspect of the present invention the promoter is in combination with a gene of interest (“GOI”).
Preferably the enzymatic activity is reduced or eliminated.
Preferably the nucleotide sequence codes for at least substantially all of at least one intron in a sense orientation.
Preferably the nucleotide sequence codes, partially or completely, for two or more introns and wher
Danisco A/S
Frommer & Lawrence & Haug LLP
Kowalski Thomas J.
Park Hankyel T.
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