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
1997-03-22
2001-05-08
Mosher, Mary E. (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...
C435S320100, C435S468000, C536S023200, C536S023600, C536S024100, C800S278000, C800S285000, C800S286000, C800S287000, C800S288000, C800S295000, C800S298000, C800S303000
Reexamination Certificate
active
06228643
ABSTRACT:
The present invention relates to promoters and to a construct comprising the same. The present invention also relates to a method for the containment of plant germplasm.
In particular, the present invention relates to the use of a promoter for the expression of a gene of interest (GOI) in a specific tissue or tissues of a plant.
More particularly, the present invention relates to promoters for cysteine proteases. The present invention also relates to the application of these cysteine protease promoters to express a GOI in a specific tissue or tissues of a plant.
Promoters control the spatial and temporal expression of genes by modulating their level of transcription. Early approaches to genetically engineered crop plants utilised strong constitutive promoters to drive the expression of foreign genes. As strategies in plant biotechnology have become more sophisticated, there are requirements for specific promoters to target transgene expression to a particular tissue or to a particular developmental stage.
Cysteine proteases are members of a large multigene family in plants (Praekelt et al., 1988; Goetting-Minesky and Mullin, 1994), animals (Wiederanders et al., 1992) and protozoa (Mallinson et al, 1994). Cysteine proteases are synthesised as an inactive precursor (Praekelt et al., 1988). The pre-pro-enzyme is targeted to the secretory pathway (Marttila et al., 1995) and post-transcriptionally processed in the vacuoles by proteolytic cleavage of the propeptide fragment to produce the active enzyme (Hara-Nishimura et al., 1993 and 1994).
Plant cysteine proteases participate in different metabolic events of physiological importance. During seed germination and plant senescence they are involved in protein degradation (Jones et al., 1995; Valpuesta et al., 1995; Smart et al., 1995) and play a key role in protein storage mobilisation during germination (Boylan and Sussex, 1987). During seed development, cysteine proteases catalyse the post-translational processing of protein precursors into their mature form (Hara-Nishimura et al, 1995). In addition, some are subjected to hormonal regulation either by giberellic acid (Koehler and Ho, 1990; Watanabe et al., 1991) or ethylene (Cervantes et al., 1994; Jones et al., 1995). Others are induced in response to stress like wounding (Linthorst et al., 1993; Lidgett et al., 1995), dehydration (Guerrero et al., 1990), cold (Schaffer and Fischer, 1988) or are implicated in plant-microbe interactions (Goetting-Minesky and Mullin, 1994).
Germination specific cysteine proteases have been characterised for barley (Marttila et al., 1995), rice (Watanabe et al., 1991), maize (Debarros and Larkins, 1994), chick-pea (Cervantes et al., 1994), vetch (Becker et al, 1994) and a cysteine protease has been described for oil seed rape (Comai and Harada, 1989). However, the published data for oil seed rape is contradictory. Furthermore, this species is difficult to study due to its amphi-diploid nature. Rather than using more conventional and laborious techniques like subtractive or differential screening of cDNA libraries or differential display techniques, potentially generating clones of unknown identity, cysteine proteinases (cysteine proteases) in oil seed rape were studied which are expressed in germinating seeds. Promoters from genes which are uniquely expressed following seed germination were isolated and characterised.
Thus, according to a first aspect of the present invention, there is provided an oil seed rape cysteine protease gene promoter of class 1, 2 or 6.
According to a second aspect of the present invention, there is provided a promoter comprising at least part of a sequence as shown in
FIG. 19
(SEQ ID NO:71),
FIG. 20
(SEQ ID NO:72) or
FIG. 21
(SEQ ID NO:73), or at least part of a sequence that has substantial homology therewith, or a variant thereof.
According to a third aspect of the present invention, there is provided a promoter having the characteristic motifs or features of promoters of the present invention.
According to a fourth aspect of the present invention, there is provided a recombinant DNA construct comprising the promoter as defined above operably linked to a gene which codes for a protein of interest.
According to a fifth aspect of the present invention, there is provided a recombinant DNA construct functional in a plant comprising a disrupter gene encoding a product capable of disrupting cell function, and a promoter as defined above, the disrupter gene being functionally linked to and controlled by an externally regulatable gene control region which includes a promoter which is inducible by the external application of a chemical inducer.
According to a sixth aspect of the present invention, there is provided DNA comprising at least part of the sequence shown in
FIG. 12
(SEQ ID NO:59),
FIG. 13
, (SEQ ID NO:60),
FIG. 14
(SEQ ID NO:62),
FIG. 16
(SEQ ID NO:64), or
FIG. 17
(SEQ ID NO:65), or at least part of a sequence that has substantial homology therewith or a variant thereof, and which codes for a cysteine protease.
According to a seventh aspect of the present invention, there is provided a recombinant DNA construct functional in a plant comprising the DNA as defined above operably linked to a promoter.
According to an eighth aspect of the present invention, there is provided an expression system for the tissue or tissues of a plant material, the expression system comprising a gene of interest fused to a gene promoter as defined above wherein the expression system is capable of being expressed in the tissue or tissues of the plant material.
According to a ninth aspect of the present invention, there is provided an expression system comprising a construct as defined above.
According to a tenth aspect of the present invention, there is provided a recombinant plant genome comprising a promoter as defined above, DNA as defined above, a recombinant DNA construct as defined above or an expression system as defined above.
According to an eleventh aspect of the present invention, there is provided a plant, plant seed or plant cell having a recombinant plant genome as defined above.
According to a twelfth aspect of the present invention, there is provided protected germplasm comprising a recombinant DNA construct as defined above.
According to a thirteenth aspect of the present invention, there is provided a plant or seed which is capable of growing to maturity comprising a recombinant DNA construct as defined above.
According to a fourteenth aspect of the present invention, there is provided the use of a gene promoter as defined above to induce expression of a gene of interest when fused to the gene promoter in the tissue or tissues of a plant material.
Preferably, the inducible promoter of the recombinant DNA construct is functionally linked to and controls a repressor protein and the disrupter gene promoter includes an operator sequence which is recognised by the repressor protein, so that in the presence of the inducer the repressor protein is produced which interacts with the operator sequence thereby disabling the second promoter and inhibiting expression of the disrupter gene.
Preferably, the disrupter gene is a nucleotide sequence, which is in sense orientation to an endogenous plant gene which is essential to plant development or a gene conferring a desired characteristic on the plant, or comprises a partial sense sequence of the endogenous plant gene.
Preferably, the disrupter gene is a nucleotide sequence which is in antisense orientation to an endogenous plant gene which is essential to plant development or a gene conferring a desired characteristic on the plant.
Preferably, the endogenous plant gene is essential to seed germination or early seedling development.
Preferably, the externally regulatable gene control region is a chemically inducible gene promoter sequence from the glutathione S-transferase system (which is the subject of our International Patent Application No. PCT/GB96/02116), the Alc system (which is the subject of our International Patent Application Nos. PCT/GB96/01883 and PCT/GB96/01846)
Greenland Andrew James
Jepson Ian
Thomas Didier Rene Philippe
Hohenschutz Liza D.
Mosher Mary E.
Zeneca Limited
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