Patent Grant
7303917
The present invention relates to the expression of proteins within plant tissues. More specifically, this invention relates to the expression of proteins in floral cells including those associated with anther and pistil.
Pollen production is essential to the sexual reproductive success of the flowering plant. Male gametogenesis is a highly regulated developmental process that occurs within the diploid sporophytic tissue of the anther. It comprises three major phases: the differentiation of the sporogenous cells and meiosis; the development of the free uninucleate microspores; and the pollen maturation following microspore mitosis and ending with the formation of mature pollen (Scott, R., Hodge, R., Paul, W., Draper, J. Plant Sci. 80:167-191 (1991)). Typically, pollen captured by a receptive stigma of the pistil will germinate and the pollen tube will grow extracellularly through the stigma and style until it reaches the ovule where it releases its nuclei that effect double fertilization. Similarly to a seed, the pollen accumulates reserves that enable it to germinate on a receptive stigma.
Normal pollen development is dependent upon the tapetum, a cellular layer lining the locular space of the anther. The tapetum provides the developing microspores with nutrients and other necessary products such as enzymes and structural components (Pacini, E., Franchi, G. G., Hesse, M. Plant Syst. Evol. 149:155-185 (1985)). In Brassica, the secretory tapetum is made up of cells which are metabolically very active until about microspore mitosis at which time they degenerate (Grant, I., Beversdorf, W. D., Peterson, R. L. Can. J. Bot. 64:1055-1068 (1986); Murgia, M., Charzynska, M., Rougier, M., Cresti, M. Sex. Plant Reprod. 4:28-35 (1991); Polowick, P. L., Sawhney, V. K. Sex. Plant Reprod. 3:263-276 (1990)). When the tapetal cells degenerate they release their cellular contents into the anther locule where they are thought to contribute to the formation of the external pollen coat (Evans, D. E., Taylor, P. E., Singh, M. B., Knox, R. B. Planta 186:343-354 (1992); Heslop-Harrison, J. New Pytol. 67:779-786 (1968)). The pollen coat (sporoderm) consists of two layers, the exine (outer wall) and the intine (inner wall). The exine can be further subdivided into the nexine and sexine layers and is often elaborately sculptured and patterned (Scott, R. J. In: Molecular and Cellular Aspects of Plant Reproduction (eds) Scott, R. J., Stead, M. A. 55:49-81 (1994)).
The interstices of the exine contain various substances including proteins, enzymes, lipids and allergens (Knox, R. B. In: Embryology of Angiosperms, (ed) Johri, B. M. pp. 197-271 (1994)) many of which are of tapetal origin. The lipidic and proteinaceous layer coating the exine is also called the tryphine. The mature pollen grain released upon anther dehiscence is dry and the drying process causes the tryphine to retract into the exine cavities. Numerous pollen enzymes have been identified (Brewbaker, J. L. In: Pollen: Development and Physiology (ed) Heslop-Harrison, J. pp. 156-170 (1971); Hiscock, S. J., Dewey, F. M., Coleman, J. O. D., Dickinson, H. G. Planta 193:377-383 (1994); Knox, R. B. In: Pollen: Development and Physiology (ed) Heslop-Harrison, J. pp. 171-173 (1971); Lavithis, M., Bhalla, P. L. Sex. Plant Reprod. 8:289-298 (1995); Travis, J., Whitworth, T., Matheson, N., Bagarozzi, D. Acta Biochim. Pol. 43:411-418 (1996)). Many of these enzymes are located in the pollen coat especially in the intine layer and are readily elutable from the pollen grain. Some of these enzymes such as pectate lyases and ribonucleases have been shown to correspond to pollen allergens (Knox, R. B., Suphioglu, C. Sex. Plant Reprod. 9:318-323 (1996)).
Recently, genes encoding some of the pollen coat proteins have been isolated. The PCP7 gene encodes a pollen coat peptide from Brassica oleracea that has been shown to interact with S-locus glycoproteins (Doughty, J., Hedderson, F., McCubbin, A., Dickinson, H. Proc. Natl. Acad. Sci. USA 90:467-471 (1993); Hiscock, S. J., Doughty, J., Willis, A. C., Dickinson, H. G. Planta 194:367-374 (1995)). The PCP1 gene encodes a cysteine-rich protein which may be involved in pollen-stigma interactions in Brassica oleracea and which belongs to a family of 30 to 40 genes (Stanchev, B. S., Doughty, J., Scutt, C. P., Dickinson, H., Croy, R. R. D. Plant J. 10:303-313 (1996)). This gene was shown to be expressed gametophytically and its product is released from the pollen protoplast into the surface coating.
There have also been numerous genes isolated which show expression in the tapetum, yet the function of the proteins they encode (Schrauwen, J. A. M. Acta Bot. Neerl. 45:1-15 (1996)), and whether they associate with the pollen coat is largely unknown. However, genes encoding β-1,3-glucanase have been shown to be expressed in the tapetum and these enzymes are involved in breaking down the callose wall surrounding the tetrads releasing the microspores (Bucciaglia, P. A., Smith, A. G. Plant Mol. Biol. 24:903-914 (1994); Hird, D. L., Worrall, D., Hodge, R., Smartt, S., Paul, W., Scott, R. Plant J. 4:1023-1033 (1993)). There are also some examples of tapetal-specific genes (ie. expressed sporophytically) whose products were shown to be localized to the pollen coat. The related genes Satap35 and Satap44 from Sinapis alba are associated with the exine of the developing microspore and may be involved in sporopollenin formation and/or deposition (Staiger, D., Kappeler, S., Müller, M., Apel, K. Planta 192:221-231 (1994)). Recently the pollen coat localized male determinant of self-incompatibility has been identified in B. campestris (B. rapa) and B. oleracea as the S-locus cysteine rich protein SCR/SP11 which is expressed in the tapetum (Schopfer, C. R., Nasrallah, M. E., Nasrallah, J. B. Science 296:1697-1700 (1999); Takayama, S., Shiba, H., Iwano, M., Asano, K., Hara, M., Che, F. -S., Watanabe, M., Hinata, K., Isogai, A. Proc. Natl Acad. Sci. USA 97: 1920-1925 (2000); Kachroo, A., Schopfer, C. R., Nasrallah, M. E., Nasrallah, J. B. Science 293: 1824-1826 (2001); Shiba, H., Takayama, S., Iwano, M., Shimosato, H., Funato, M., Nakagawa, T., Che, F. -S., Suzuki, G., Watanabe, M., Hinata, K., Isogai, A. Plant Physiol. 125: 2095-2103 (2001); Shiba, H., Iwano, M., Entani, T., Ishimoto, K., Shimosato, H., Che, F. -S., Satta, Y., Ito, A., Takada, Y., Watanabe, M., Isogai, A., Takayama, S. Plant Cell 14: 491-504 (2002)). Similarly, the predominant protein on the surface of maize pollen is an endoxylanase synthesized by the tapetum (Bih, F. Y., Wu, S. S. H., Ratnayake, C., Walling, L. L., Nothnagel, E. A., Huang, A. H. C. J. Biol. Chem. 274: 22884-22894 (1999)).
The Sta 41-2 and Sta 41-9 genes from Brassica napus encode proteins that possess a hydrophobic domain similar to that of the seed oleosins (Robert, L. S., Gerster, J. L., Allard, S., Cass, L., Simmonds, J. Plant J. 6:927-933 (1994a)). Sequence similarity among the Sta 41-2 and Sta 41-9 genes, and seed oleosin genes from Brassica napus (Murphy, D. J., Prog. Lipid Res. 32:247-280 (1993)) are limited to the relatively small hydrophobic domain and show levels of 30-36% identity. These tapetally expressed genes have now been demonstrated to belong a large family of related anther oleosin-like genes in Brassica (Ross, J. H. E., Murphy, D. J. Plant J. 9:625-637 (1996); Ruiter, R. K., Van Eldik, G. J., Van Herpen, R. M. A., Schrauwen J. A. M., Wullems, G. J. Plant Cell 9:1621-1631 (1997)). Unlike the other tapetally expressed pollen coat localized proteins mentioned above, the oleosin-like proteins do not possess a signal peptide and are thought to be released passively into the anther locule upon tapetum degeneration by association with lipids released from the tapetum or found as part of the tryphine of the pollen coat. Without wishing to be bound by theory, the hydrophobic region of the tapetal oleosin-like protein may be required for localization upon the pollen coat by association with lipids. The tapetal oleosin-like proteins constitute the major protein of the Brassica pollen tryphine and they occur as post-translationally cleaved protein products (Ross, J. H. E., Murphy, D. J. Plant J. 9:625-637 (1996)). The function of the tapetal oleosin-like proteins is unknown but they may play a role in the interaction between the pollen and the stigma the specialized part of the pistil that receives the pollen.
The stigma is responsible for capturing and selecting compatible pollen grains and for facilitating their germination. Angiosperm stigmas have been classified morphologically as ‘dry’ stigmas having an extracuticular proteinaceous pellicle but no free-flowing secretion or ‘wet’ stigmas which are covered by a secretion at the receptive stage (Heslop-Harrison, Y., Shivanna, K. R. Ann. Bot. 41:1233-1258 (1977)). In Brassica, the dry stigma is the site of the sporophytic self-incompatibility (SI) response with incompatible pollen being unable to grow through the stigmatic papillar cells or failing to germinate altogether.
A number of genes have been shown to be preferentially expressed in the Brassica stigma and most of these genes correspond to genes associated with SI: SLG (S-locus glycoprotein), SRK (S receptor kinase; U.S. Pat. No. 5,484,905) or SLR (S-locus-related; WO94/25613) genes (for review: Nasrallah, J. B., Nasrallah, M. E. Plant Cell 5:1325-1335 (1993)). The products of the SLG and SRK genes are believed to be involved in a signal pathway modulating the SI reaction in response to a ligand carried by the pollen grain. WO94/25613 is directed to pistil-, and anther-specific gene expression. It discloses the cloning of several SLG's genes and the isolation of the SLG1 promoter region, and the preparation of transcriptional fusion products using the promoters from the SLG genes. Furthermore, U.S. Pat. No. 5,585,543 discloses several genes related to the S-locus.
Another example of a gene highly expressed in the Brassica stigma is Pis 63 (Robert, L. S., Allard, S., Gerster, J. L., Cass, L., Simmonds, J. Plant Mol. Biol. 26:1217-1222 (1994b)). The promoter obtained from the genomic clone PISG 363, which contains gene Pis 63-2 was shown to direct the expression of the marker gene β-glucuronidase transiently in B. napus stigmas and stably in the stigmas of transformed tobacco plants (Robert, L. S., Lévesque-Lemay, M., Gerster, J. L., Hong, H. P., Keller, W. Plant Cell Rep. 18:357-362 (1999)).
The SI response in Brassica provides an example that a molecular based interaction between the pollen grain and the stigmatic papillae exists and that such an interaction can be modified or mimicked by targeting polypeptides to the appropriate part of the pollen and/or stigma. It is thought that localization of the SLG proteins arrises as a result of the appropriate signal peptide directing the protein extracellularly, following expression.
The preparation of plants with female sterility based on a style-stigma specific “STMG” gene and derived constructs using PSTMG promoter cassettes is disclosed in U.S. Pat. No. 5,633,441. These constructs include transcriptional fusions comprising barnase, papain or RNAse. In U.S. Pat. No. 5,652,354, the use of stamen-selective promoters useful in driving expression in anther, pollen, or filament cells, especially in the tapetum or anther epidermal cells is disclosed. U.S. Pat. No. 5,571,904 is directed to male flower specific gene sequences. Genomic clones of pMS10, 14 and 18 were obtained and promoter cassettes were constructed using MS10. There is also evidence presented where pMS14 expression has been localized within the tapetal cell layer. Other publications also disclose floral-specific genes and associated regulatory elements. For example, U.S. Pat. No. 5,633,438 discloses microspore-specific regulatory element, Bnm1; U.S. Pat. No. 5,545,546 discloses the cloning of W2247, a pollen specific promoter obtained from maize (inbred maize line W22); U.S. Pat. No. 5,659,124 teaches use of existing anther specific promoters to produce male sterile plants; WO92/13957 is directed to the cloning of CA444 which is a stamen/anther specific gene; WO97/13401 discloses the cloning of a rice tapetal specific gene RTS2; WO93/25695, is directed to the preparation of male sterile plants using tapetal specific promoters such as those from the TA29 gene or PT72; CA 2,099,482, teaches the disruption of the formation of viable pollen resulting in male sterile plants using an anther specific promoter; CA 2,106,718 is directed to the disruption of normal pollen development using anther specific promoters driving chimeric constructs that disrupt pollen development; Worrall, D., Hird, D. L., Hodge, R., Paul, W., Draper, J., Scott, R. Plant Cell 4:759-771 (1992)) teaches the use of a tapetal specific promoter to drive the expression of callase which prematurely degrades the callose wall surrounding the developing tetrad of microspores thereby releasing the microspores into the anther locule. This premature release of microspores leads to male sterility. CA 2,165,934 discloses the use of a polygalacturonase promoter to drive a chimeric construct within microspores of Brassica napus plants. However, there is no teaching of modifying the extracellular domain of a free microspore or pollen grain.
Based upon the review of the prior art, there are two known mechanisms that exist for the targeting of a protein onto the pollen coat, either by deposition following tapetal degradation, or as a result of extracellular targeting either from the tapetal cells or microspore cells via a signal peptide. Similarly, extracellular targeting of pistil-derived gene products, for example the SLG gene product, appears to involve the use of a signal peptide. However, none of the prior art publications disclose modifying the protein composition of the microspore/pollen coat or the interactions of these proteins with the stigma or pistil. Rather, most of the published literature is directed to producing sterile plants through the disruption of pollen development, although this disruption does not occur by modifying the extracellular compartment. Nor does the prior art teach a similar modification of the stigma cells using chimeric gene constructs that would affect the interaction between these cells and a modified pollen grain, while all of these cell types could remain viable. The approach described herein is primarily directed at modifying pollen or stigma function, and in some instances affects the interaction between pollen and stigma.
There is no teaching of the preparation of transcriptional or translational fusion proteins specifically designed to localize on the exterior of a pollen or stigma cell. For example, but not limited to, comprising hydrophobic domains of pollen coat proteins and the like, to direct the translocation of the fusion product to the exterior surface of the pollen. Furthermore, beyond uses that are directed to pollen disruption for the production of sterile plants, the prior art does not disclose methods that provide for peptide display, antibody production, altering the food value of pollen for human consumption, the use of treating insects, or alleviating allergenic responses by specifically targeting protein products to the surface of the appropriate floral cell.
This invention relates to a method of modifying the extracellular compartment of floral cells, including targeting proteins, protein fusions, fragments thereof, or peptides to this extracellular domain. Methods using chimeric gene constructs that allow targeting of proteins, fusion proteins or peptides of interest to cells of the pistil, microspore, or to the pollen coat to modify floral functions or interactions are disclosed and exemplified.
The present invention relates to the expression of proteins within plant tissues. More specifically, this invention relates to the expression of proteins within the extracellular compartment of floral cells including those associated with anther and pistil.
According to the present invention there is provided a method for modifying the extracellular compartment of a floral cell of a plant, the method comprising, expressing a construct comprising a gene of interest within an anther or stigma cell, the gene of interest encoding a protein, fusion protein or peptide, or a fragment of said protein, fusion protein or peptide; the protein, fusion protein or peptide, or a fragment of the protein, fusion protein or peptide capable of modifying the composition of the extracellular compartment of the floral cell and altering either the function, use or development of the floral cell, or modifying the interaction of the floral cell with other cells. This invention relates to the above method wherein the gene of interest is native, or non-native, to the plant, or wherein the construct is a chimeric gene construct.
This invention relates to the method as defined above wherein the floral cell is a pollen grain, and the protein, fusion protein or peptide, or a fragment of the protein, fusion protein or peptide is released into a locule of an anther thereby associating with the extracellular compartment of the pollen grain. This invention also embraces the above method, wherein the floral cell is either a pollen grain or a stigma cell, and the construct comprises a translated sequence capable of directing the extracellular localization of said protein, fusion protein or peptide, or a fragment of the protein, fusion protein or peptide on the floral cell. Preferably the translated sequence is selected from the group consisting of a signal peptide, a hydrophobic domain, or a combination thereof, or the translated sequence is a protein, or fragment thereof, known to be targeted to the extracellular compartment of a floral cell, for example but not limited to a oleosin-like sequence, an extracellular lipase sequence, and a pollen polygalacturonase sequence.
The present invention also provides a method (A) for modifying the extracellular compartment of a pollen grain or an anther cell of a plant, the method comprising,
The invention also includes the method (A) as described above, wherein the first amino acid sequence, encoded by the nucleotide sequence, or the amino acid sequence encoded by the alternated nucleotide sequence, is selected from the group consisting of an oleosin-like sequence, an extracellular lipase sequence, and a pollen polygalacturonase sequence. Furthermore, the oleosin-like sequence can be a tapetal oleosin-like sequence. The tapetal oleosin-like sequence may be selected from the group consisting of BnOlnB;1, BnOlnB;2, BnOlnB;3 BnOlnB;4, BnOlnB;5, BnOlnB;6, BnOlnB;7, BnOlnB;8, BnOlnB;9, BnOlnB;10, BnOlnB;11 and BnOlnB;12; AtOlnB;1, AtOlnB;2, AtOlnB;3 AtOlnB;4; BoOlnB;1 and BrOlnB;1, BrOlnB;2, BrOlnB;3 BrOlnB;4, and BrOlnB;5.
The present invention is directed to the method (A) as defined above wherin the plant is selected from the group consisting of Brassica, Raphanu., Arabidopsis, Triticum, Hordeum, Avena, Niciotiana, Glycine, Pisum, Acer, Agropyron, Medicago, Malus, Aster, Phaseolus, Beta, Betula, Vicia, Bromus, Daucus, Cedrus, Citrus, Gossypium, Populus, Cucurbita, Helianthus, Lactuca, Lilium, Lycopersicon, Allium, Prunus, Capsicum, Pinus, Picea, Ambrosia, Secale, Tsuga, Zea, Oryz and Solanum.
The present invention pertains to a microspore or a pollen, or combination thereof, prepared using the method (A) as decribed above.
According to the present invention there is provided a method (B) for localizing a protein, fusion protein, or peptide of interest, or a fragment of the protein, fusion protein, or peptide, on the extracellular compartment of a a pollen grain or an anther cell of a plant, comprising:
Also included is a method for localizing a protein, fusion protein, or peptide of interest, or a fragment of the protein, fusion protein, or peptide, within the extracellular compartment of a floral cell, comprising:
Furthermore, this invention includes the method as just described, wherein the translated sequence of step c) is selected from the group consisting of a signal peptide, a hydrophobic domain, or a combination thereof.
This invention is also directed to a method (C) of chemically linking a protein or peptide of interest to the pollen coat comprising:
This invention embraces a pollen grain prepared by the method (C) as described above.
Furthermore, this invention includes a microspore or pollen, or a combination thereof, prepared using the method (B) as described above. This invention is also directed to a transgenic plant cell, a transgenic plant comprising the microspore or pollen, or combination thereof prepared using the method (B) as described above, and to seeds obtained from the transgenic plant.
This invention also embraces a method (D) of modifying pollen-pistil interaction or function comprising, producing a microspore, pollen, or pistil cell, or combination thereof, within a plant using the method (B) as described above, so that the microspore, pollen, or pistil, or combination thereof comprise an extracellular protein, fusion protein, or peptide of interest, or a fragment of the protein, fusion protein, or peptide, that modifies pollen and pistil interaction or function. This invention also embraces a method (D), wherein the extracellular protein, fusion protein, or peptide of interest, or a fragment of the protein, fusion protein, is localized to the microspore, or pollen, or to the pistil cell, or combination thereof.
This invention also provides for a method (D) wherein the pollen-pistil interaction or function produces, mediates, or prevents self-compatibility, self-incompatibility, out-crossing, in-crossing or a combination thereof.
This invention also relates to the method (D) as described above, wherein the protein, fusion protein, or peptide of interest, or a fragment of the protein, fusion protein, or peptide, is selected from the group consisting of protease, glucosidase, glycanase, nuclease, lipase, hydrolyase, toxin and antibody, or an active portion thereof.
This invention also embraces a vector comprising:
This invention also embraces a vector comprising a construct, the construct comprising either:
The present invention includes the vector as just defined, wherein the first amino acid sequence encoded by the nucleotide sequence, or the amino acid sequence encoded by the alternate nucleotide sequence, is selected from the group consisting of an oleosin-like sequence, an extracellular lipase sequence, and a pollen polygalacturonase sequence. The oleosin-like sequence may be a tapetal oleosin-like sequence. The tapetal oleosin-like sequence can be selected from the group consisting of BnOlnB;1, BnOlnB;2, BnOlnB;3 BnOlnB;4, BnOlnB;5, BnOlnB;6, BnOlnB;7, BnOlnB;8, BnOlnB;9, BnOlnB;10, BnOlnB;11 and BnOlnB;12; AtOlnB;1, AtOlnB;2, AtOlnB;3 AtOlnB;4; BoOlnB;1 and BrOlnB;1, BrOlnB;2, BrOlnB;3 BrOlnB;4, and BrOlnB;5.
The present invention provides a method (E) for modifying pollen-pistil interaction in a plant, comprising:
Furthermore, the preent invention provides a method for modifying pollen-pistil interaction in a plant, comprising:
This invention also embraces the method (B) as described above, wherein the protein, fusion protein, or peptide of interest, or a fragment of the protein, fusion protein, or peptide, is localized on the surface of a pollen grain for the purpose of peptide display.
Also included within the present invention is a method (B) defined above, wherein the protein, fusion protein, or peptide of interest, or a fragment of the protein, fusion protein, or peptide, is localized on the surface of pollen and it is an antibody or antigen, or it exhibits properties beneficial to human or animal nutrition, or health, or it is effective in controlling insect growth, behaviour, feeding, development, or reproduction, or a combination thereof, or it is capable of alleviating allergenic responses within a human.
The present invention also pertains to a method for modifying a pollen coat of a plant, comprising:
Also included in the present invention is the method as just definedwherein the first amino acid sequence, encoded by the nucleotide sequence, or the amino acid sequence encoded by the alternated nucleotide sequence, is selected from the group consisting of an oleosin-like sequence, an extracellular lipase sequence, and a pollen polygalacturonase sequence. The oleosin-like sequence can be a tapetal oleosin-like sequence. The tapetal oleosin-like sequence can be selected from the group consisting of BnOlnB;1, BnOlnB;2, BnOlnB;3 BnOlnB;4, BnOlnB;5, BnOlnB;6, BnOlnB;7, BnOlnB;8, BnOlnB;9, BnOlnB;10, BnOlnB;11 and BnOlnB;12; AtOlnB;1, AtOlnB;2, AtOlnB;3 AtOlnB;4; BoOlnB;1 and BrOlnB;1, BrOlnB;2, BrOlnB;3 BrOlnB;4, and BrOlnB;5.
This invention is directed to modifying the protein composition of the extracellular domain of a microspore, or pollen coat, or the interactions of these proteins with the stigma, pistil, or other cells of interest, while possibly maintaining the pollen, and the cells of the stigma, in a viable state. Furthermore, this invention relates to modifying the protein composition of the extracellular domain of stigma cells in order to affect the interaction between these cells and either unaltered or modified pollen grains, wherein each of these cell types could remain in a viable state. The prior art is directed to producing sterile plants through the disruption of pollen development. However, this disruption does not occur by modifying the extracellular domain of the pollen. The approach described herein is primarily directed at modifying pollen or stigma cell function, and in some instances affects the interaction between pollen and stigma. However, the methods disclosed within this invention are not necessarily disruptive to pollen development as is the case within the prior art, nor are they necessarily disruptive to pistil development.
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
d shows a schematic representation of the construction of plant transformation vector TOG-3, a Brassica napus tapetal oleosin-like Sta 41-9/E. coli β-glucuronidase translational fusion (Example 22; SEQ ID NO:28). The GUS gene in TOG-3 is positioned adjacent to the C-terminal end of the STA 41-9 hydrophobic domain and replaces the C-terminal domain.
The present invention relates to the expression of proteins within plant tissues. More specifically, this invention relates to the expression and localization of proteins within the extracellular compartment of floral cells including those associated with pollen, anther and pistil.
The following description is of a preferred embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.
Methods and compositions are provided for the targeting of proteins or peptides to the extracellular domain of a microspore, or pollen or pistil cells. The methods include preparing chimeric DNA constructs encoding a polypeptide, or a fusion polypeptide consisting of a microspore, pollen coat or pistil protein and a coding sequence for a polypeptide of interest. Inserting this DNA construct in a plant genome, and regenerating transgenic plants that produce pollen, stigmas, or both, with the polypeptide or fusion polypeptide.
For example, the present invention provides a method for modifying the extracellular compartment of a pollen grain or an anther cell of a plant, the method comprising,
Furthermore, the present invention provides a construct comprising:
As used herein “pollen function” includes processes associated with development of pollen, dispersal of the pollen, recognition, interaction and adhesion of the pollen to any cell, for example, but not limited to a stigma cell, natural or artificial substrates, pollen tube germination and pollen tube growth, and fertilization.
By “pistil function” it is meant processes associated with development of the pistil, interactions with pollen, including pollen capture, permitting or preventing pollen germination, pollen tube growth, fertilization, or a combination thereof, and nurturing zygote development.
By “extracellular compartment” or “extracellular domain” it is meant the region of the cell that includes, and lies outside, the plasmalemma. However, the extracellular compartment, or domain, may be associated with the cell in some manner. This compartment may comprise proteins that are anchored within the plasmalemma and that are displayed toward the outside of the cell, or proteins that are localized, via excretion or deposition, within the apoplast, cell wall or outer regions of the cell wall such as the surface of the cell, or that are released within the locule. For example, in the case of pollen (40; see
By “directing extracellular localization” it is meant using a chimeric gene construct comprising motifs capable of targeting a protein or protein fusion or peptide of interest passively or actively to the extracellular compartment. For example, which is not to be considered limiting in any manner, such motifs responsible for actively directing extracellular localization may include sequences encoding signal peptides, or hydrophobic domains, for example fragments obtained from the tapetal oleosin-like protein, or a hydrophobic domain obtained from a seed oleosin or tapetal oleosin-like protein. Motifs responsible for passively targeting extracellular localization upon tapetal degradation may include, but are not limited to, protein primary structure or protein modifications affecting affinity to the extracellular domain. This localization may also comprise a transient association between the protein, fusion protein, or peptide of interest and the extracellular domain, such as enzyme substrate interactions, for example glycosidase-carbohydrate or protease-protein reactions.
By “oleosin-like protein” it is meant a protein, or a fragment thereof comprising a hydrophobic domain (see Huang, A. H. C. (1996) Plant Physiol. 110: 1055-1061; WO 96/21029, both of which are incorporated herein by refrence, for examples of oleosin-like proteins), and characterized as having a conserved region known as a Proline Knot Motif (PKM; Abell, B. M., Holbrook, L. A., Abenes, M., Murphy, D. J., Hills, M. J., Moloney, M. M. (1997) Plant Cell 9: 1481-1493, which is incorporated herein by reference). This group includes but is not limited to seed oleosin proteins and tapetal-oleosin-like proteins. An oleosin like protein or a fragement thereof comprising the PKM and neighbouring amino acids as known in the art, is capable of directing of the protein, or a translational fusion of the oleosin-like protein with a protein of interest, to the pollen coat using the methods described herein.
By “tapetal oleosin-like protein” it is meant a protein, or a fragment of a protein comprising a hydrophobic domain, a PKM, or both a hydrophobic domain and a PKM, or other sequence, that directs targeting of the protein, or a translational fusion of the protein with a protein of interest, to the pollen coat. Non-limiting examples of tapetal oleosin-like proteins include STA 41-2 and STA 41-9 (renamed BnOlnB;3 and BnOlnB;4 respectively, Robert, L. S., Gerster, J., Allard, S., Cass, L., Simmonds, J. Plant J. 6:927-933 (1994a)), PUTG1, ATGRP-6, -7, and -8 (renamed AtOln;B1, AtOln;B2, AtOln;B3, AtOln;B4 de Oliveira, D. E., Franco, L. O., Simoens, C., Seurinck, J., Coppieters, J., Botterman, J., Van Montagu, M. Plant J 3:495-507 (1993)), 13 (renamed BnOlnB;1 Roberts, M. R., Robson, F., Foster, G. D., Draper, J., Scott, R. J. Plant Mol. Biol. 17:295-299 (1991)), C98 (renamed BnOlnB;2, Hodge, R., Paul, W., Draper, J., Scott, R. Plant J 2:257-260 (1992)), POL3 (renamed BnOlnB;5, Roberts, M. R., Hodge, R., Scott, R. Planta 195:469-470 (1995)), BOPC4 (renamed BoOlnB;1, Ruiter, R. K., Van eldik, G. J., Van Herpen, R. M. A., Wullems, G. J, Schrauwen, J. A. M. Plant Cell 9:1621-1631 (1997)), BrOlnB1, 2,3,4 and 5 (Lim et al. (1994) EMBL Acc. No. L33510, L33543, L33564, L33603, L33618), BnOlnB;6, 7, 8, 9, 10, 11 and 12 (Ross, J. H. E., Murphy, D. J. Plant J. 9:625-637 (1996)). These OlNB proteins comprise conserved hydrophobic domains, and any of these proteins, or a fragment of any of these proteins may be used for targeting a protein of interest to the pollen coat, using the methods as described herein.
By “gene of interest” or “coding region of interest” it is meant a nucleic acid sequence that encodes a protein. The gene of interest, or coding region of interest may be of native origin, in that it is obtained from the same species of plant within which it is to be reintroduced, or it may be of non-native origin, i.e. it is obtained from a plant that is different from the plant to which it is to be introduced, or it is obtained from another source, i.e. bacterial, viral, animal etc. A coding region of interest may further be operatively linked to regulatory regions such as promoters, enhancers, terminator sequences and the like that are endogenous to the coding region of interest with which they are isolated. By “operatively linked” it is meant that the particular sequences interact either directly or indirectly to carry out an intended function, such as mediation or modulation of gene expression. The interaction of operatively linked sequences may, for example, be mediated by proteins that interact with the operatively linked sequences. A coding region of interest may also be introduced within a vector along with other sequences, typically heterologous, to produce a chimeric construct.
By “chimeric DNA construct” or “chimeric construct” it is meant a nucleic acid molecule comprising-regions of DNA sequences not normally associated with the gene of interest. These regions may be homologous or heterologous with respect to the gene of interest, and may be obtained from native or non-native sources. For example, a chimeric construct that results in a translational fusion product may include a native or heterologous enhancer region, a native or heterologous promoter region, followed by regions comprising a portion of a native or heterologous 5′ coding region including such motifs as signal peptides, or hydrophobic domains as required, a native or heterologous DNA sequence capable of encoding a protein or peptide of interest, followed by 3′ motifs that may also be involved in extracellular targeting or regulatory functions, or both, and a terminator region. It is to be understood that a range of 5′ or 3′ regions of the chimeric construct may be used in order to optimize synthesis of the final gene construct, expression of the gene product, and localization of the gene product within the extracellular compartment. Furthermore, a chimeric construct that results in a transcriptional fusion product may comprise a native or non-native enhancer and promoter region operationally fused with an optional signal peptide and the protein or peptide of interest, followed by a 3′ regulatory, or terminator region, or a region comprising both a regulatory and terminating function as defined above.
By “modified gene” or “modifed coding region” it is meant a gene or coding region whose sequence has been altered using methods known in the art such as but not limited to site-directed, or random mutagenesis, deletions, rearrangements, or fusions and the like.
By “fusion protein” it is meant proteins synthesized from chimeric DNA constructs. These proteins may comprise a portion of a native protein along with a heterologous protein comprising the protein of interest. Such a fusion protein may comprise a signal peptide, or hydrophobic domain, or other motif that permits targeting of the protein of interest to the extracellular compartment, for example, but not limited to, motifs obtained from an oleosin-like protein, STA 41-2 or STA 41-9, STA 44, SLGWS1 or PIS 63. It is to be understood that increases in protein expression levels may also be obtained by protein modification that affects protein stability or by multimeric constructs involving multiple copies of the protein or peptide of interest.
By “expression cassette” it is meant a chimeric DNA molecule that includes transcriptional and translational regulatory sequences of DNA capable of expressing a chimeric gene whose product is subsequently targeted to the extracellular compartment of a floral cell. For example, an expression cassette may comprise promoter and regulatory sequences controlling the expression of genes, and the targeting of the encoded products within the tapetum or the pollen. However, this is not to be considered limiting in any manner as other constructs may also be directed to other extracellular compartments as previously defined. In the case of tapetal expression, the gene product may be expressed in the tapetum and subsequently translocated to the pollen or developing microspores, for example callase or oleosin-like proteins, or the protein may be expressed within the pollen and re-located to the microspore or pollen coat during development or germination, for example, pectate lyase or PCP1.
By “promoter” or “regulatory region”, it is meant a region typically within a genomic sequence that has the property of controlling the expression of a DNA sequence that is operably linked with the regulatory region. Such regulatory regions may include promoter or enhancer regions, and other regulatory elements recognized by one of skill in the art. Typically this region comprises nucleotide sequences at the 5′ end of a coding region, or fragment thereof that contain all the signals essential for the initiation of transcription and for the regulation of the rate of transcription. The promoters used to exemplify the present invention may be selected to ensure expression of a desired gene within the tissue of interest, or during appropriate stages of development, for example, but not limited to the organ-specific promoters regulating the following genes:
These and other promoters are, or would be, known to those of skill in the art. If desired, constitutive promoters may also be used such as, but not limited to, the CaMV 35S (Timmermans, M. C. P., Maliga, P., Vieira, J., Messing, J. J. Biotechnol. 14: 333-344 (1990)), ubiquitin (Holtorf, S., Apel, K., Bohlmann, H. Plant Mol. Biol. 29: 637-646 (1995)), actin (An, Y. Q., McDowell, J. M., Huang, S., McKinney, E. C., Chamblis, S., Meagher, R. B. Plant J. 10:107-121(1996)), rice actin 1 (Zhang, W., McElroy, D., Wu, R. Plant Cell, 3:1155-1165 (1991)), triosephosphate isomerase 1 (Xu, Y., Yu, H., Hall, T. C. Plant Physiol. 106:459-467 (1994)), the maize ubiquitin 1 (Cornejo, M. J., Luth, D., Blankenship, K. M., Anderson, O. D., Blechl, A. E. Plant Mol. Biol. 29:637-646 (1993)), tobacco t-CUP (WO/99/67389; U.S. Pat. No. 5,824,872), the HPL (WO 02/50291), and the tobacco translational initiation factor 4A (Mandel, T., Fleming, A. J., Krahenbuhl, R., Kuhlemeier, C. Plant Mol. Biol. 29:995-1004 (1995)) promoter.
Also included are inducible promoters which may also be used in order to regulate the expression of the gene following the induction of expression by providing the appropriate stimulus for inducing expression. The inducer can be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound, or a physiological stress imposed directly by heat, cold, salt, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus. For example, a plant or plant cell containing an inducible regulatory element may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating or similar methods. Inducible elements may be derived from either plant or non-plant genes (e.g. Gatz, C. and Lenk, I. R. P. Trends Plant Sci. 3:352-358 (1998); which is incorporated by reference). Examples, of potential inducible promoters include, but are not limited to, tetracycline-inducible promoter (Gatz, C. Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108 (1997); which is incorporated by reference), steroid inducible promoter (Aoyama, T. and Chua, N. H. Plant J. 2:397-404 (1997); which is incorporated by reference) and ethanol-inducible promoter (Salter, M. G., et al. Plant J. 16:127-132 (1998); Caddick, M. X., Greenland, A. J., Jepson, I., Krause, K. P., Qu, N., Riddel,1 K. V., Salter, M, G., Schuch, W., Sonnewald, U., Tomsett, A. B. Nature Biotech. 16:177-180 (1998), which are incorporated by reference), cytokinin inducible IB6 and CK11 genes (Brandstatter, I. and Kieber, J. J. Plant Cell 10:1009-1019 (1998); Kakimoto, T. Science 274:982-985 (1996), which are incorporated by reference) and the auxin inducible element, DR5 (Ulmasov, T., Murfett, J., Hagen, G., Guilfoyle, T. J. Plant Cell 9:1963-1971 (1997), which is incorporated by reference).
The chimeric gene constructs of the present invention can farther comprise a 3′ untranslated region. A 3′ untranslated region refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing, mRNA stability, or gene expression. The polyadenylation signal is usually characterized by effecting the addition of polyadenylic acid tracks to the 3′ end of the mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5′ AATAAA-3′ although variations are not uncommon. Examples of suitable 3′ regions are the 3′ transcribed non-translated regions containing a polyadenylation signal of Agrobacterium tumour inducing (Ti) plasmid genes, such as the nopaline synthase (Nos gene) and plant genes such as the soybean storage protein genes and the small subunit of the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene. The 3′ untranslated region from the structural gene of the present construct can therefore be used to construct chimeric genes for expression in plants.
The gene constructs of the present invention can also include further enhancers, either translation or transcription enhancers, as may be required. These enhancer regions are well known to persons skilled in the art, and can include the ATG initiation codon and adjacent sequences. The initiation codon must be in phase with the reading frame of the coding sequence to ensure translation of the entire sequence. The translation control signals and initiation codons can be from a variety of origins, both natural and synthetic. Translational initiation regions may be provided from the source of the transcriptional initiation region, or from the structural gene. The sequence can also be derived from the promoter selected to express the gene, and can be specifically modified so as to increase translation of the mRNA.
To aid in identification of transformed plant cells, the constructs of this invention may be further manipulated to include plant selectable markers. Useful selectable markers include enzymes that provide for resistance to an antibiotic such as gentamycin, hygromycin, kanamycin, and the like. Similarly, enzymes providing for production of a compound identifiable by colour change such as GUS (β-glucuronidase), or luminescence, such as luciferase are useful.
By “transformation” it is meant the stable transfer of genetic information. The constructs of the present invention can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, micro-injection, electroporation, etc as would be known to those of skill in the art. For reviews of such techniques see for example Weissbach and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988); Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988); and Miki, B. and Iyer, V. Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2d Ed. Dennis, D. T., Turpin, D. H., Lefebrve, D. D., Layzell, D. B. (eds), Addison Wesly, Langmans Ltd. London, pp. 561-579 (1997). For transformation of Arabidopsis see Clough, S. J. and Bent, A. F. Plant J. 16: 735-743 (1998)). Also reference is made to the Examples of the present application for additional transformation protocols.
Also considered as part of the present invention are transgenic plants containing the chimeric gene construct as described herein. Plants include, but are not limited to members of the Brassica-family, for example, which is not to be considered limiting, canola, Brasica napus, B. carinata, B. nigra, B. oleracea, B. chinensis, B. cretica, B. incana, B. insularis, B. japonica, B. atlantica, B. bourgeaui, B. narinosa, B. juncea, B. rapa, Raphanus species, Arabidopsis species however, other plants may also be modifed using the methods described herein for example but not limited to, Zea species, Oryza species, Triticum species, Hordeum species, Avena species, Niciotiana species, Glycine species, Pisum species, Acer species, Agropyron species, Medicago species, Malus species, Aster species, Phaseolus species, Beta species, Betula species, Vicia species, Bromus species, Daucus species, Cedrus species, Citrus species, Gossypium species, Populus species, Cucurbita species, Helianthus species, Lactuca species, Lilium species, Lycopersicon species, Allium species, Prunus species, Capsicum species, Pinus species, Picea species, Ambrosia species, Secale species, Tsuga species, and Solanum species. Methods of regenerating whole plants from plant cells are known in the art, and the method of obtaining transformed and regenerated plants is not critical to this invention. In general, transformed plant cells are cultured in an appropriate medium, which may contain selective agents such as antibiotics, where selectable markers are used to facilitate identification of transformed plant cells. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be used to establish repetitive generations, either from seeds or using vegetative propagation techniques.
By “sporophytically expressed pollen coat protein” it is meant a protein synthesised within sporophytic tissue, and via subsequent processing and translocation, is deposited upon the outer surface of the pollen coat. For example, the Sta 41-2 and Sta 41-9 gene products are expressed within tapetal tissue, and, even though not comprising a signal peptide, these gene products are deposited on the exterior of the pollen coat during development. Furthermore, other gene products comprising signal peptides that are tapetally expressed can be targeted to the pollen coat, e.g. SCR, SATAP35 and SATAP44.
By “gametophytically expressed pollen coat proteins” it is meant, proteins that are synthesised within gametophytic tissue, such as microspore, pollen, or ovary cells, and are translocated to the extracellular compartment of these cells. For example, the PCP1 pollen coat protein is synthesized in the pollen cell and relocated to the pollen coat.
The methods of this invention allow for the localization of a protein or peptide of interest within the extracellular compartment of pollen or stigma cells, including enzymes, receptors, antigens, antibodies, ligands, substrates, inhibitors and peptides which may modify normal male or female reproductive tissues (including but not limited to pollen, microspore, pollen tube, stigma, ovary, or egg) interaction, function, or both. When expressed on the surface of pollen or pistil, the fusion peptides or proteins can be used:
Furthermore, it is contemplated that the linkage of peptides to the pollen coat may also be achieved chemically. This may be achieved by a variety of ways known to those of skill in the art, for example, using lectin concavalin A to covalently link the proteins to sugar residues on the pollen exine, or using tannic acid to covalently link proteins of interest to naturally occurring proteins expressed on the pollen coat. Such methods may either be used to prepare modified coat walls of pollen for the purposes disclosed within this invention, or for evaluating the feasibility, or function of desired proteins of interest located to the extracellular compartment of pollen, prior to designing, preparing and transforming plants with appropriate constructs and vectors leading to the expression of the desired protein.
In accordance with the subject invention, the method for modifying the protein composition of the extracellular compartment of a floral cell of a plant comprises expressing a construct comprising a gene of interest within an anther or pistil cell. The gene of interest encodes a protein, fusion protein or peptide, or a fragment thereof, and this protein or fragment thereof is capable of modifying the composition of the extracellular compartment of the floral cell. By altering the composition of the extracellular compartment of the floral cell, the function, development or use of the floral cell, or the interaction of said floral cell with other cells is modified while possibly maintaining the pollen, and the cells of the pistil or stigma, in a viable state. However, it is not necessary that these cells remain viable. For example, expression of a protease on the surface of a stigma cell may or may not kill the stigma cell depending upon the protease selected and the concentration of protease expressed. If the stigma cell is killed by the protease, pollen (especially if expressing protease inhibitor) may still germinate on the surface of the stigma, however, a female sterile plant may be obtained if germination is prevented as a result of protease expression or disruption of the stigma cells. Similarly, pollen may or may not remain viable following the modification of the extracellular compartment as described herein.
This invention also relates to a method for expressing a protein of interest on the surface of a pollen or pistil cell which includes preparing an expression cassette containing a construct comprising one or more regulatory sequences and a gene of interest which encodes a polypeptide or derivatives thereof, along with motifs that ultimately direct the expression of the protein extracellularly, so that when produced in a transgenic plant the protein is localized extracellularly, that is that the protein is located on the surface of the pollen or pistil cell. The peptide of interest may be a novel protein not normally found on the pollen or pistil cell surface, however, it is also contemplated that it may be desired to modify the composition of the extracellular domain or the abundance or the properties of a native protein within the extracellular compartment using the method of this invention. For example a protein from the following list, which is not to be considered limiting, may be used for the preparation of chimeric constructs:
Proteins localized extracellularly can be used to modulate pollen function and for example prevent normal fertilization. The pollen from the genetically modified plant can also be used as a carrier for various polypeptides. This provides a novel protein expression, production and purification system.
Example 17 describes the results of the translational fusion of a coding region of interest, for example but not limited to, the E. coli β-glucuronidase reporter gene, at the C-terminus of the full-length sequence of the tapetal oleosin-like gene Sta 41-9 (including the N-terminal, central hydrophobic and C-terminal domains). The N-terminal domain of the tapetal oleosin-like proteins are highly variable in length (ranging from 6 to 67 amino acid residues) and sequence, and the central hydrophobic domain is variable in sequence and slightly variable in length (Ross, J. H. E. and Murphy, D. J. Plant J. 9: 625-637 (1996)). The C-terminal domain is variable in length and sequence; the different types of sequence found in the C-terminal domains consist of varying classes of repeated domains which are rich in various amino acids, such as glycine, lysine and glycine, or proline and, alanine (Ross, J. H. E. and Murphy, D. J. Plant J. 9: 625-637 (1996)). Both the N- and C-terminal domains contain sequences that differ considerably among the varying classes of tapetal oleosin-like genes (Ross, J. H. E. and Murphy, D. J. Plant J. 9: 625-637 (1996)). In B. napus and Arabidopsis, a common characteristic of the tapetal oleosin-like proteins is their localization to the pollen coat, despite their sequence variability (Murphy, D. J. and Ross, J. H. E. Plant J. 13: 1-16 (1998); Mayfield, J. A., Fiebig, A., Johnstone, S. E., Preuss, D. Science 292:2482-2485 (2001)).
Therefore, strategies for the construction of translational fusions of targeting proteins such as, but not limited to, STA 41-9, to target proteins of interest to the pollen coat include, (also see
In order to obtain a sporophytically expressed pollen coat protein, the tapetal oleosin-like proteins can be fused translationally to a polypeptide of interest. For example, which is not to be considered limiting, the nucleotide and deduced amino acid sequences of a tapetum-expressed oleosin-like gene Sta 41-9 (Robert, L. S., Gerster, J., Allard, S., Cass, L., Simmonds, J. Plant J. 6:927-933 (1994a); also termed BnOlnB;4) is used for the preparation of a number of possible translational fusions. The polypeptide of interest can be fused to the C-terminal end of the protein Sta 41-9 (
Also considered within the scope of this invention is the expression of a gene in the tapetum whose product could modify a protein that is subsequently targeted to the extracellular domain of pollen. Similarly, the expression of a gene encoding a protein that is targeted to the extracellular domain of a floral cell, for example, but not limited to, an oleosin-like protein, may be inhibited using methods known within the art, for example but not limited to, antisense RNA, ribozymes, RNA interference or co-suppression. In this manner a reduction of a protein within the extracellular domain of a pollen grain results and modifies pollen stigma interaction.
Gametophytically expressed pollen coat proteins are also used in translational fusions with the polypeptide of interest or this polypeptide is directed to the microspore or pollen coat by transcriptional or translational fusion to a promoter directing pollen expression. These can be part of the coat of the pollen grain or can be released extracellularly. As an example, which is not to be considered limiting in any manner, a translational fusion is made to the cysteine rich B. oleracea PCP1 pollen coat protein (Stanchev, B. S., Doughty, J., Scutt, C. P., Dickinson, H., Croy, R. R. D. Plant J. 10:303-313 (1996)) that is synthesized in the pollen cell and relocated to the pollen coat.
It is also contemplated that a chimeric DNA construct encoding a polypeptide of interest can be prepared so that the polypeptide is synthesised within gametophytic tissue, and released at a later time, for example within pollen, and released upon pollen germination. In this case, the protein of interest is either fused translationally to B. napus pollen polygalacturonase STA 44 (Robert, L. S., Allard, S., Gerster, J. L., Cass, L., Simmonds, J. Plant Mol. Biol. 23:1273-1278 (1993)), or fused transcriptionally to the promoter of Sta 44G(2) (Hong, H. P., Gerster, J. L., Datla, R. S. S., Albani, D., Scoles, G., Keller, W., Robert, L. S. Plant Cell Rep. 16:373-378 (1997a); U.S. patent application Ser. No. 08/577,463). These fusion proteins are then produced within the pollen grain and released upon germination.
Translational fusions comprising a tapetal oleosin-like protein, for example, but not limited to, Sta 41-91/Onchocerca volvulus protease inhibitor OV7 fusion (TOPI-1;
Similar results are also observed during flower development in transgenic Nicotiana tabacum (
With reference to
To further demonstrate the targeting of a protein of interest to the pollen coat, a tapetal oleosin-like gene was translationally fused to the protein of interest and expressed within developing anthers. In this example, which is not to be considered limiting in any manner, the tapetal oleosin-like gene BnOlnB;4 (also known as Sta 41-9; including the promoter and the entire coding region with the exception of the TGA stop codon), isolated from B. napus anthers (Robert, L. S., Gerster, J. L., Allard, S., Cass, L., Simmonds, J., Plant J. 6:927-933 (1994a)), was translationally fused to GUS to produce TOG (see
Northern analysis (using either a GUS or BnOlnB;4 probe) revealed that TOG-2 mRNA was detectable during anther development, first in anthers isolated from about 3 mm flower buds, reaching its maximum level in anthers from 5 mm buds, and decreasing in anthers from buds 6-7 mm in length, correlating with the onset of tapetal degeneration. By the late pollen maturation stage (8 mm buds), TOG-2 mRNA was no longer detectable in anthers (
To determine whether a translational fusion is necessary to target proteins from the tapetum to pollen grains, expression of a GUS transcriptional fusion to the tapetum-specific BnOlnB;4 promoter (BnOlnB;4-GUS (also called Sta 41-9-GUS) which lacks the coding region of BnOlnB;4) was analyzed in transgenic B. napus. GUS expression directed by the BnOlnB;4 promoter was previously shown to be specific to the tapetum in anthers isolated from 3-5 mm flower buds of B. napus (Hong, H. P., Ross, J. H. E., Gerster, J. L., Rigas, S., Datla, R. S. S., Hatzopoulos, P., Scoles, G., Keller, W., Murphy, D., Robert, L. S. Plant Mol. Biol. 34:549-555 (1997b)). The approximately 2 kb BnOlnB;4 promoter is the same as that used to direct expression of the TOG-2 translational fusion. GUS mRNA from the BnOlnB;4-GUS construct was detected in transgenic B. napus prior to tapetal degradation in 4-5 mm flower buds (anther; consistent with earlier reports, Hong, H. P., Ross, J. H. E., Gerster, J. L., Rigas, S., Datla, R. S. S., Hatzopoulos, P., Scoles, G., Keller, W., Murphy, D., Robert, L. S. Plant Mol. Biol. 34:549-555 (1997b)) and undetectable in pollen isolated from 6-7 mm buds (pollen) after the tapetum had degenerated just prior to floral opening (BnOlnB;4-GUS;
Western blot analyses with an anti-GUS antibody detected the GUS protein, of BnOlnB;4-GUS, in anthers from 4-5 mm buds (anther;
A GUS transcriptional fusion to a B. napus polygalacturonase promoter (Sta 44-GUS) that directs high levels of expression within pollen late in development (Hong, H. P., Gerster, J. L., Datla, R. S. S., Albani, D., Scoles, G., Keller, W., Robert, L. S. Plant Cell Rep. 16: 373-378) was also examined (
Collectively, these data indicate that a protein of interest, for example but not limited to GUS, relocates to the pollen coat upon tapetal degeneration if fused translationally to a tapetal oleosin-like protein, for example but not limited to, BnOlnB;4 (also known as STA 41-9). Furthermore, the coding region of interest is active when associated with pollen. Transcriptional fusion of the GUS gene to promoters driving its expression in either the tapetum or the pollen grain is not sufficient for pollen coat targeting.
Subcellular localization of native tapetal oleosin-like proteins (
GUS histochemical staining with pollen from 5 mm bud anthers obtained from TOG-2 plants is negligible (
To indicate whether the GUS activity localized to pollen of TOG-2 transgenic plants was indeed the result of sporophytic expression rather than gametophytic expression, GUS histochemical analysis was performed on TOG-2 lines containing single copy insertions. In 9 GUS-positive progeny of each of two self-pollinated T0 plants, GUS histochemical staining of pollen from 8 mm flower buds typically revealed about 98±0.2% GUS positive pollen grains. In comparison, a mix of stained and unstained pollen grains are observed by GUS histochemical staining of T1 progeny of a self-pollinated B. napus transgenic line containing a single copy of the pollen-expressed Sta 44-GUS construct (
Therefore, as demonstrated herein (Example 17), the composition of the pollen coat protein can be altered by targeting a protein of interest synthesized in the tapetum to the pollen coat. Targeting to the pollen coat can be achieved with a translational fusion between a tapetal oleosin-like protein gene, for example but not limited to, BnOlnB;4, and a protein of interest, for example but not limited to the uidA gene encoding GUS (TOG-2, see
Tapetal oleosin-like proteins, which lack a signal peptide, use a unique targeting pathway to move from the tapetum into the locule and ultimately to the pollen. The TOG-2 proteins are localized to tapetosomes within the tapetum, remain associated with the tapetosomes following tapetal degradation and then become localized to the pollen coat of mature pollen (e.g. see
Furthermore, as demonstrated, for example in Example 17, targeting of a protein of interest to the pollen coat is not affected by the addition of a lengthy translational fusion of the protein of interest, at the C-terminus of a tapetal oleosin-like protein.
An alternate example of an oleosin-like sequence that may be used to target a coding region of interest to the pollen coat, in a manner as demonstrated above using Sta 41-9 (BnOlnB;4, as exemplified with TOG-2), includes an oleosin-like sequence, Atgrp 19 (see
Similar targeting may also be effected using other proteins, or portions thereof, for example extracellular lipase. A non-limiting example of such a protein is an A. thaliana exl 4 extracellular lipase, as shown in Example 25, used in the preparation of EXLG-1+ (see
Further evidence that chimeric constructs of the present invention, for example comprising POV-1 (see
Co-expression of POP-1 (protease) and POV-1 (protease inhibitor) in pollen demonstrated that loss of pollen viability observed in pollen obtained from plants expressing POP-1 (e.g. see Example 21, Table 4) may be overcome in the presence of POV-1 (see Table 5, Example 21). In this example, plants expressing POV-1, that were shown to exhibit protease inhibitior activity (
An alternate example of a construct comprising a polygalacturonase fusion, includes POG-2 (
Therefore, the present invention provides a method for modifying the extracellular compartment of a pollen grain or an anther cell of a plant comprising, expressing a construct comprising a coding region of interest within the pollen grain or the anther cell, where the coding region of interest encodes a protein, a fusion protein, a peptide, or a fragment of the protein, fusion protein or peptide. Furthermore, the protein, fusion protein or peptide, or a fragment of the protein, fusion protein or peptide comprises a tapetal oleosin-like sequence that directs the protein, fusion protein or peptide, or a fragment of the protein, fusion protein or peptide to the extracellular compartment of the pollen grain or anther cell.
The method of this invention can be used for the purposes of altering pollen function; preventing self-pollination, allowing self-pollination; preventing cross-pollination; peptide display, antigen delivery, altering nutritional value of pollen; treatment or control of insect populations; or alleviating allergenic responses. These are discussed in more detail below:
1. Alter Pollen or Pistil Function.
Normal pollen function necessitates specific inter- and intra-molecular interactions with constituents of the stigma, style and ovary. Modifying pollen or pistil function, or these interactions could result in the failure or allowance of pollen germination, pollen tube growth and/or fertilization of the egg. Modification can be achieved by specific catalytic enzymes (eg. proteases, glucosidases, glycanases, nucleases, lipases, hydrolases etc.), toxins (eg. Diphteria toxin A chain), antibodies, lectins etc. localized on the surface of the pollen or pistil cell, or released from the pollen, or pollen tube.
As an example of this embodiment, which is not to be considered limiting in any manner, the tapetally expressed STA 41-9 protein or the gametophytically expressed STA 44 is fused to a cysteine protease from Sitophilus, (Matsumoto, I., Emori, Y., Abe, K, Arai, S. J. Biochem. 121: 464-476 (1997)). It is also contemplated that transcriptional fusions may also be used for protease expression. Once targeted to the microspore/pollen coat, these enzymes digest proteins important to pollen germination or pollen-stigma interactions and therefore prevent pollen germination or pollen tube growth (see
As further examples of this embodiment, chimeric gene constructs could comprise the stigma-expressed gene encoding the PIS 63 protein fused to the Sitophilus protease (see
2 Permitting Hybrid Seed Production by Preventing Self-Pollination.
Heterosis (hybrid vigour) corresponds to the increase in productivity and vigour that results from the genetic differences in parental lines. The advantage of growing hybrid crop varieties becomes evident when the benefits from the yield increase due to heterosis significantly outweigh the cost of seed production. Values reported for heterosis in Brassica napus seed yield have been greater than 50% (Grant I. and Beversdorf W. D., Can. J Genet. Cytol. 27: 472-478 (1985)). Although heterosis is observed in nearly every crop, the use of hybrids is mostly limited to crops for which there is an economically viable and effective means of pollination control. Many major crops, such as Canola, have small bisexual flowers that render manual emasculation impractical and thus hybrids cannot be produced commercially without using methods of interfering with pollen development and causing male sterility (Williams M. E., TIBTECH 13: 344-349 (1995)). Cytoplasmic male sterility (CMS) has been used for this purpose in oilseed rape, but this approach suffers from the breakdown of sterility in certain environments and from undesirable agronomic traits linked to the restorer genes (Feistritzer, W. P. and Kelly, A. F., (eds) Hybrid Seed Production of Selected Cereal Oil and Vegetable Crops, FAO (1987)). Genetically engineered nuclear-encoded male sterility may represent a viable alternative approach for pollination control in crops such as Canola (Stefansson, B. R. and Downey, R. K. in: Harvest of Gold: The History of Field Crop Breeding in Canada, Slinkard, A. E. and Knott, D. R. (Eds) Saskatoon: University Extension Press (1995)).
Several stages of plant reproduction including gamete development, pollination and fertilization depend on both gametophytic and sporophytic gene expression. Male or female sterility can result from mutations disrupting normal gene expression in haploid (eg. pollen) or diploid cells (eg. tapeturm) throughout these developmental stages. Examples of molecular approaches used successfully to generate transgenic male sterile plants have been reported. Such approaches can involve the use of cytotoxic genes (Mariani, C., De Beuckeleer, M., Truettner, J. Leemans, J., Goldberg, R. B. Nature 347: 737-741 (1990); Koltunow, A. M., Truettner, J., Cox, K. H., Wallroth, M., Goldberg, R. B. Plant Cell 2: 1201-1224 (1990)), antisense versions of essential pollen genes (Muschietti, J., Dircks, L., Vancanney, G., McCormick, S. Plant J 6: 321-338 (1994); Xu, H., Knox, R. B., Taylor, P. E., Singh, M. B. Proc. Natl. Acad. Sci. USA 92: 2106-2110 (1995)) or genes encoding enzymes involved in pollen development (Worrall, D., Hird, D. L., Hodge, R., Paul, W., Draper, J., Scott, R. Plant Cell 4: 759-771 (1992)). These approaches have been the subject of patent applications such as WO 90/08828 and WO 92/18625. However, none of these approaches are aimed at modifying the protein composition of the microspore/pollen coat or the interactions of these proteins.
The targeting to the microspore/pollen coat of proteases, antigens, enzymes, inhibitors, ligands, or peptide(s) which interact with endogenous or supplemented stigma constituents (eg. stigma-expressed protease inhibitors, antibodies, receptors, protein binding polypeptides, proteolytic enzymes) modulate the activity of the microspore/pollen coat or'stigma (see
This approach can also be used to prevent out-crossing (cross-pollination). In this example, the same plant has both the pollen coat protease and a stigmatic inhibitor of the protease thereby allowing self-fertilization (
This approach can also be used to modify self-compatibility or self-incompatibity for example when a self-incompatible plant contains an introduced S-locus cysteine rich (SCR) protein fused translationally to STA 41-9 (
It is also contemplated that localization of a tapetal oleosin-like/anti-S-locus glycoprotein antibody fusion to the pollen coat could be used to disrupt normal pollen or pistil development. For example, an antibody to a S-locus glycoprotein could be raised and the immunoglobulin heavy chain and light chain variable regions fused into a single chain antibody fragment (ScFv). This fragment could be cloned and expressed on the pollen coat as described in Examples 1-3. When expressed on the pollen coat of a self-incompatible Brassica species for example, this antibody could interfere with the normal interactions with the stigma and abolish self-incompatibility. Other examples of polypeptides that could also disrupt or improve normal pollen or pistil development include: lectins such as avidin, inhibitor peptides obtained by affinity screening and arabinogalactan proteins (Gerster, J., Allard, S., Robert, L. S. Plant Physiol. 110: 1231-1237 (1996)).
3. Targeting Proteins or Peptides to the Pollen Coat for the Purposes of Peptide Display, for Protein Production and Utilization.
The display of a protein on the surface of a pollen grain would permit a ready source of the protein for further purification or for utilization, for example as an immobilized enzyme. It is conceived that the construct employed for protein production further comprises a site that permits cleavage of the protein from the rest of the fusion protein. Such a site may be a proteolytic site and susceptible to cleavage using a protease, or a site susceptible to chemical cleavage. Proteins or peptides produced in plants have the advantage of being properly glycosylated as opposed to prokaryotic expression systems. Any peptide that could be produced by this method may be selected for use in this application, including therapeutic, or nutritive peptides such as leptin or any other useful polypeptide such as avidin, interleukin, interferon etc.
Immobilized enzymes may also be prepared using the method of this invention. For example, the enzyme β-glucuronidase (GUS) when targeted to the pollen coat could be utilized as an immobilized enzyme. Other examples of enzymes which could be attached to the pollen coat include invertase, xylanase, β-1,3-glucanase, cysteine protease (Sitophilus), cysteine protease inhibitor (Onchocerca), oxidases, chitinases, invertases, endo-β-1,4-xylanases, callases, triacylglycerol lipases, phytases, glucan 1,3 β-glucosidases, endo 1,3-1,4β-glycanases, N-glycosidases, trypsin inhibitors, caspases (e.g. ICE cysteine proteases), aspartic protease lactases (intestinal), cellulases, xylanases, fructosyl amino acid oxidases, polygalacturonidases, pectate lyases, pectin methylesterases, chalcone synthases, alginate lyases, D-amino acid oxidases, β-glucuronidase.
Tapetal oleosin-like protein fusions may be used to produce, utilize or purify recombinant polypeptides. For example, the peptide tripticin can be produced on the surface of pollen and used directly as a bacteriostatic agent. Alternatively, the recombinant peptide can be proteolytically cleaved by introducing a cleavage site, such as the one used by thrombin, between the tapetal oleosin-like protein and the peptide of interest. Other peptides which could be produced in this way include, for example, leptin fragment, lectin (e.g. avidin), arabinogalactans, canine parvovirus coat protein, thaumatin, Pin-I and Pin-II, protease inhibitors.
4. Antigen Delivery for Antibody Production.
To generate anti-peptide or anti-protein antibodies, the peptides are often prepared by chemical synthesis using solid phase techniques (Merrifield, R. Science 85: 2149-2154 (1963)) and coupled to a carrier. Since many small peptides (haptens) are not highly immunogenic, they require a means of increasing their antigenicity such as chemical coupling to keyhole limpet haemocyanin (KLH), or bovine serum albumin (BSA). By expressing a peptide on the surface of, or within pollen grains, these peptides can be released or presented directly to the animal immune system. The benefits include multiple copies of the antigen displayed on the surface of each pollen grain, the elimination of chemical coupling to carrier molecules, the production of large quantities of pollen and therefore antigen, and the possibility to administer the immunogen orally or. nasally, and therefore stimulate the mucosal immune system. The large size of the pollen grain may also alleviate the need for adjuvants and therefore be useful in immunization.
This approach is also an economical means of producing and presenting oral vaccines and therapeutic agents, since plants are not known to be contaminated with any animal viruses or pathogens. Recombinant proteins and therapeutics may be expressed in transgenic plants and packaged on intact pollen grains with little processing or purification in some cases. Irradiation of pollen grains prior to administration or use could eliminate the possibility of pollen escape.
Antigens for antibody or vaccine production may also be prepared using the method of this invention. For example, the antigen or vaccine could be fused to the tapetal oleosin-like protein and pollen coated with this fusion protein could be administered for example: intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously and nasally (nasal spray). Any protein or peptide now used for the production of vaccines could be utilized in this way, for example canine parvovirus (Dalsgaard, K., Uttenthal, A., Jones, T., Xu, F., Merryweather, A., Hamilton, W., Langeveld, J., Boshuizen, R., Kamstrup, S., Lomonossoff, G., Porta, C., Vela, C., Casal, I., Meloen, R., Rodgers, P. Nature Biotech. 15: 248-252 (1997)).
This approach can also be used to raise antibodies to poorly immunogenic antigens such as small peptides due to the method of presentation on the pollen grain.
5. Pollen Coat of Protein(s) or Peptide(s) for Animal Consumption.
Pollen is consumed by humans for its potential health benefits. Pollen is also consumed as a component of certain foods such as broccoli. Targeting novel proteins and/or peptides to the pollen coat could enhance these health benefits or add value to food. For example, value-added components that can be expressed on the surface of pollen grains include sweeteners (eg. thaumatin, brazzein, monellin) to increase palatability, peptides beneficial to human health (eg. antibiotics, hormones such as endorphins, enzymes such as lactase, peptides high in essential amino acids). Transgenic plants producing pollen with these novel characteristics can be planted in a greenhouse or similarly confined growth environments where pollen can be collected manually or by using bees and hive pollen traps.
6. Treatment or Control of Insect Populations.
Beneficial insects such as honeybees can be beneficially treated by providing transgenic pollen comprising antibiotics or food supplements (eg. synthetic proteins or peptides rich in insect-essential amino acids, especially the aromatic amino acids). The treatment of harmful, destructive or phytophagous insects such as pollen beetles with antifeedants or antibiotics (protease inhibitors, Bt toxins, lectins) represents a novel and efficient control method of these insects. Examples of protease inhibitors that may be used in this manner include the serine class of protease inhibitors, Pin-I and Pin-II, (Johnson, R., Narvaez, J., An, G., Ryan C. Proc. Nat. Acad. Sci. USA 86: 9871-9875 (1989)) and cysteine protease inhibitors such as onchocystatin (OV7; Lustigman, S., Brotman, B., Huima, T., Prince, A. M. Mol. Biochem. Parasitol. 45: 65-76 (1991)). The effects of the inhibitors can be measured by monitoring weight loss of insects feeding on modified pollen. An alternative strategy is to employ pollen coated with protease inhibitor inducing factors (PIIF) that induce the systemic induction of protease inhibitors and defensive compounds in distal organs of plants (McGurl, B. F., Pearce, G., Orozco-Cardenas, M., Ryan, C. A. Science 255: 1570-1573 (1992)). In a similar manner, pollen coat targeting of insecticidal toxins from Bacillus thuringiensis (Bt toxins) in plants such as maize (Zea mays) which sheds high quantities of pollen over foliar, silk, and floral surfaces represents a novel method to deliver insecticidal proteins. This method of delivery is also useful for the dissemination of antifungal, antiviral, and antibacterial peptides and proteins over the vegetative and floral surfaces of plants. Such proteins could consist of pectinases, oxidases and chitinases.
7. Alleviation of Allergenic Responses.
Many pollen coat proteins have been implicated as potential allergens for eg. pathogenesis-related proteins, cysteine-rich proteins of unknown function and cell wall associated enzymes (Knox, R. B. and Suphogliu, C. Sex. Plant Reprod. 9: 318-323 (1996)). Therefore, targeting proteins that bind, interact, inactivate or mask these allergenic proteins of the pollen coat could potentially alleviate allergic reactions. Examples of proteins that could be used to interfere with the allergenic proteins include antibodies specific to an allergenic protein, binding proteins (for eg. selected by phage display), protease inhibitors, endopolygalacturonase-inhibiting protein (PGIP; Toubart, P., Desiderio, A., Salvi, G., Cervone, F., Daroda, L., De Lorenzo, G. Plant J. 2: 367-373 (1992)) or enzymes that would interfere or destroy the pollen allergen (eg. proteases). Such pollen could be used to de-sensitize allergic responses by repeated exposure to pollen grains with attenuated allergenicity. Pollen or fractions thereof could be collected from transgenic plants and administered directly as mentioned in example 4 above. Furthermore, transgenic plants producing pollen with reduced or non-allergenic properties could be made available, for example, for nursery stocks of cedars or birch.
The above description is not intended to limit the claimed invention in any manner, furthermore, the discussed combination of features might not be absolutely necessary for the inventive solution.
The present invention will be further illustrated in the following examples. These examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any manner.
The following sequences are used in order to exemplify the present invention:
SEQ ID NO's: 1 and 2 are oligonucleotide sequence (KSB-3): TAG GTA CCG AGC TCG GGG GAT CC; (SEQ ID NO:1) corresponding to the plus strand of the KSB adapter, and oligonucleotide sequence (KSB-4): TAG GAT CCC CCG AGC TCG GTA CC (SEQ ID NO:2) corresponding to the minus strand of the KSB adapter (see Example 2).
SEQ ID NO:3 is the nucleotide sequence of the translational fusion in plasmid TOG-1 (see Example 2;
SEQ ID NO:4 is the nucleotide sequence of the translational fusion in plasmid TOP-1 (see Example 3,
SEQ ID NO:5 is the nucleotide sequence of the translational fusion in plasmid TOPI-1 (see example 4;
SEQ ID NO's: 6 and 7 are oligonucleotide sequence (BKX-1): TCG AGG GGA TCC GGT ACC TCT AGA (SEQ ID NO:6); corresponding to the plus strand of the BKX 12 adapter, and oligonucleotide sequence (BKX-2): TCG ATC TAG AGG TAC CGG ATC CCC (SEQ ID NO:7) corresponding to the minus strand of the BKX 12 adapter (see Example 5).
SEQ ID NO:8 is the nucleotide sequence of the translational fusion in plasmid SPF-1 (see Example 5,
SEQ ID NO:9 is the nucleotide sequence of the translational fusion in plasmid SPIF-1 (see Example 6,
SEQ ID NO's: 10 and 11 are oligonucleotide sequence SLG26 (7): ATA GAG CTC CGA TGA AAG GCA TAA GAA (SEQ ID NO:10), and oligonucleotide sequence SLG 26 (8): TAT GGT ACC TTC TTC AGA AGA CAA AGT G (SEQ ID NO:11), see Example 7.
SEQ ID NO:12 is the nucleotide sequence of the translational fusion in plasmid SPOV-1 (see Example 7,
SEQ ID NO's: 13 and 14 are oligonucleotide sequence (EXK-1): CGA ATT CTC TAG AGG TAC CGC ATG (SEQ ID NO:13); corresponding to the plus strand of the EXK 12 adapter, and oligonucleotide sequence (EXK-2): CGG TAC CTC TAG AGA ATT CGC ATG (SEQ ID NO:14); corresponding to the minus strand of the EXK 12 adapter (see Example 8).
SEQ ID NO:15 is the nucleotide sequence of the translational fusion in plasmid POV-1 (see Example 8a). This fusion consists of the 5′ upstream and the partial coding region including the signal peptide of Brassica napus genomic clone Sta 44G(2) which encodes a pollen-expressed polygalacturonase gene (Robert, L. S., Allard, S., Gerster, J. L., Cass, L., Simmonds, J. Plant Mol. Biol. 23: 1273-1278 (1993); Hong, H. P., Gerster, J. L., Datla, R. S. S., Albani, D., Scoles, G., Keller, W., Robert, L. S. Plant Cell Rep. 16: 373-378 (1997a); U.S. patent application Ser. No. 08/577,463) fused to the Onchocerca volvulus protease inhibitor OV7 coding region.
SEQ ID NO's: 16 and 17 are a forward primer (GUSsense-1): GGA ATT CAC CGC GTC TTT GAT CGC (SEQ ID NO:16), and reverse primer (nos #2): GCG CGC GAT AAT TTA TCC (SEQ ID NO:17) that anneal to the GUS gene and nopaline synthase terminator, respectively (see Example 13).
SEQ ID NO's: 18 and 19 are primer (NptII-121): GGG CGC CCG GTT CTT TTT (SEQ ID NO:18) and primer (NptIl-B): CAG CAA TAT CAC GGG TAG CCA ACG C (SEQ ID NO:19), see Example 13.
SEQ ID NO's: 20 and 21 are primers used for detection of the Sitophilus protease gene (see Example 3): forward primer (P1): GCG CGG ATC CTT GCC TGA TAC TGT TGA C: (SEQ ID NO:20) and reverse primer (P2): GCG CGA ATT CAA GCT TCT AAA CCA AAG GAT AAC TAG C (SEQ ID NO:21).
SEQ ID NO's: 22 and 23 are primers used for TOG-2 construction (see Example 17): (XK+): GAT CCT CTA GAG GTA CCG (SEQ ID NO:22) and (KX−): GAT CCG GTA CCT CTA GAG (SEQ ID NO:23).
SEQ ID NO:24 comprises the first 20 amino acid residues of the C-terminal domain of BnOlnB;4 (Sta 41-9), LGIPESIKPS NIIPESIKPS.
SEQ ID NO:25: is the nucleotide sequence of the translational fusion in plasmid POP-1 (see Example 8;
SEQ ID NO's: 26 and 27 are primer pairs for TOG-3 construction (see Example 22): (Sta 41 ATG): CTA GGA TCC AGA CCA CAC AAC TCC TTC (SEQ ID NO:26) and (Sta 41-13R): GAGA GGATTC CAA CAG AGA TAG GGA TGG C (SEQ ID NO:27).
SEQ ID NO:28 is the nucleotide sequence of the translational fusion in plasmid TOG-3 (see Example 22;
SEQ ID NO's29 and 30: primer pairs for GUS amplification (Example 22): primers (GUS-6F): TAG AGG ATC CCC GGG TGG TCA GTC (SEQ ID NO:29) and (GUS-7R): GAG AGA GCT CAG ATC TTT GTT TGC CTC CCT GCT GCG GT (SEQ ID NO:30).
SEQ ID NO's: 31 and 32: primer pairs for amplifying Sta 41-9 (Example 22): primers (STA 41-14): GAG AAG ATC TAT GAG AAA CGA AAT TCA AAA CGA AAC (SEQ ID NO:31) and (STA 41-15R): GAG AGA GCT CAT ATG TGT TTA CCA CCA CTC CCA (SEQ ID NO:32).
SEQ ID NO:33: TOG-4 (see Example 22;
SEQ ID NO's: 34-36: primers for SCR 13 isolation (see Example 23): primers (GAGA T18-2): GAG AGA GAG AGA CTC GAG TTT TTT TTT TTT TTT TTT A/C/G (SEQ ID NO:34), (SCR13-3F): AAC AAG AAT TTG CTG CGA GTA AAA GAG AAT (SEQ ID NO:35) and (SCR13-4R): ATT TTG ACT AAG ACG AAT TTT GGA ATG ATT (SEQ ID NO:36).
SEQ ID NO's: 37 and 38: primers to amplify the coding region corresponding to the mature SCR protein (see Example 23): primers (SCR13-7F): GAG AAT TAA TAA ATC TGA TGA TGC CTT GTG G (SEQ ID NO:37) and (SCR13-8R): CTG CAG AAC CAA CGC GTT GGA GCT CCT AAC ACA ATT TAC AAT CAC AAG (SEQ ID NO:38).
SEQ ID NO:39: TOS13-1 (Example 23,
SEQ ID NO's: 40-42: primers for amplifying the A. thaliana Atgrp 19 gene (Example 24,
SEQ ID NO:43: is the nucleotide sequence of the translational fusion in plasmid TOG-2 (see Example 17;
SEQ ID NO:44: is the nucleotide sequence of the translational fusion in plasmid ATOG-3 (see Example 24;
SEQ ID NO:45: is the nucleotide sequence of the translational fusion in plasmid ATOG-4+ (see Example 24;
SEQ ID NO's:46 and 47: primers for amplifying the A. thaliana exl 4 gene (Example 25,
SEQ ID NO:48: is the nucleotide sequence of the translational fusion in plasmid EXLG-1+ (Example 25,
SEQ ID NO's:49-51: Primers for amplifying the Brassica napus Sta 44 gene (Example 26,
The cDNA clones Sta 41-2 and Sta 41-9 encoding tapetal oleosin-like proteins were isolated by differential screening of a flower cDNA library from Brassica napus (Robert, L. S., Gerster, J. L., Allard, S., Cass, L., Simmonds, J., Plant J. 6:927-933 (1994a)). The genomic clone Sta 41G(10) corresponding to cDNA clone Sta 41-9 was also isolated and the region upstream of the coding region shown to direct expression of a marker gene to the tapetum of transgenic Brassica napus plants (Hong, H. P., Ross, J. H. E., Gerster, J. L., Rigas, S., Datla, R. S. S., Hatzopoulos, P., Scoles, G., Keller, W., Murphy, D., Robert, L. S. Plant Mol Biol. 34:549-555 (1997b); U.S. patent application Ser. No. 08/595,937).
The genomic clone Sta 41G(10) was used for the construction of translational fusions to polypeptides of interest for targeting to the pollen coat. Other tapetal oleosin-like genes are also known (Ross, J. H. E., Murphy, D. J. Plant J. 9:625-637 (1996); Ruiter, R. K., Van Eldik, G. J., Van Herpen, R. M. A., Schrauwen, J. A. M., Wullems, G. J. Plant Cell 9:1621-1631 (1997); de Oliveira, D. E., Franco, L. O., Simoens, C., Seurinck, J., Coppieters, J., Botterman, J., Van Montagu, M. Plant J 3:495-507 (1993); Lim et al. EMBL Acc. No. L33510, L33543, L33564, L33603, L33618 (1994)) and can be used for gene fusions aimed at targeting polypeptides to the pollen coat.
The Sta 41 G(10) Sac I subclone mp 101 is digested with Bam HI and Hind III releasing a fragment containing the 5′ upstream region of the tapetal oleosin-like gene and cloned into the Bam HI and Hind III sites of pBSK+ (Stratagene) to generate plasmid BH-1. Plasmid BH-1 is digested with Nde I, blunt ended with the Klenow fragment of DNA polymerase I and religated, effectively destroying the Nde I site and generating plasmid NB-6. Plasmid NB-6 is digested with Bgl II and Kpn 1 and used to replace the Bgl II and Kpn 1 fragment of mp 101. This effectively reconstructs the Sta 41 G(10) Sac I subclone (without the Nde I site within the promoter while preserving the Nde I site just upstream of the stop codon) and generates plasmid KB-1 (
KSB-3: TAG GTA CCG AGC TCG GGG GAT CC (SEQ ID NO:1) and
KSB-4: TAG GAT CCC CCG AGC TCG GTA CC (SEQ ID NO:2)
generating plasmid OFC-1 (
Fusion of the Brassica napus Tapetal Oleosin-Like Gene to the Sitophilus SCPc 1 Protease Gene (TOP-1)
A fragment of a cysteine protease from Sitophilus, from plasmid pSCPc1, was amplified by PCR (polymerase chain reaction). This fragment corresponds to the SCPc1 cDNA fragment cloned into pBSK-(Matsumoto, I., Emori, Y., Abe, K, Arai, S. J Biochem. 121: 464-476 (1997)). The oligonucleotide primers used in the PCR reaction are:
forward primer: P1: GCGCGGATCCTTGCCTGATACTGTTGAC (SEQ ID NO:20)
and reverse primer: P2: GCGCGAATTCAAGCTTCTAAACCAAAGGATAACTAGC (SEQ ID NO:21).
These primers permit the amplification of the mature cysteine protease coding sequence and introduce a Bam HI site (bold) at the 5′ end of the amplified DNA fragment and Eco RI and Hind III sites (bold) at the 3′ end.
The PCR fragment is digested with Bam HI and Hind III, and subcloned into the Bam HI and Hind III sites of pGEM 4Z (Promega) generating plasmid pSCPc1BH. Plasmid pSCPc1BH is digested with Bam HI and Hind III and ligated into the Bam HI and Hind III sites of pGEM 7Z generating plasmid SCP-2. Plasmid SCP-2 is digested with Bam HI and Sma I and the fragment containing the protease coding sequence ligated into the Bam HI and Sma I of Binter (this corresponds to the binary vector Bin 19 (Bevan, M. Nucleic Acids Res. 12:8711-8721 (1984)) into which the nopaline synthase terminator polyadenylation signal is subcloned as a Sac I and Eco RI fragment) generating plasmid BS-2. The Bam HI fragment of OFC-1 containing the tapetal oleosin promoter and coding sequence referred to in Example 2 is ligated into the Bam HI site of BS-2 generating a translational fusion between the tapetal oleosin-like gene and the Sitophilus protease gene in plasmid TOP-1 (
Plasmid pGEXOV7 (Lustigman, S., Brotman, B., Huima, T., Prince, A. M. Mol. Biochem. Parasitol. 45: 65-76 (1991)) containing the cDNA clone coding for the Onchocerca protease inhibitor (PI) is digested with Eco RI releasing a 582 bp fragment which is blunt ended by filling in with the Klenow DNA polymerase I fragment and subcloned into the Sma I site of pGEM 4Z generating plasmid pGEMOV7. This fragment contains the coding sequence for the mature Onchocerca PI. Plasmid pGEMOV7 is digested with Bam HI and Eco RI, and ligated into the Bam HI and Eco RI sites of pGEM 7Z to generate plasmid OV-71. Plasmid OV-71 is digested with Sac I and ligated into the Sac I of Binter generating plasmid BO-3. The Kpn I fragment of OFC-1 containing the tapetal oleosin promoter and coding sequence referred to in Example 2 is ligated into the Kpn I site of BO-3 generating a translational fusion between the tapetal oleosin-like gene and the Onchocerca OV7 gene of plasmid TOPI-1 (
A fragment of genomic clone Pis G363 containing the promoter and the Pis 63 coding region was cloned into pGEM 4Z resulting in plasmid Bg2. Plasmid Bg2 was digested with Kpn I and Xba I, and the insert cloned into the corresponding sites of pGEM 7Z to yield plasmid KX-1 (
BKX-1: TCG AGG GGA TCC GGT ACC TCT AGA (SEQ ID NO:6) and
BKX-2: TCG ATC TAG AGG TAC CGG ATC CCC (SEQ. ID NO:7)
introducing additional Bam HI, Kpn I and Xba I sites within the 3′ coding region and resulting in plasmid XX-1. The Bam HI fragment of plasmid XX-1 containing the Pis G363 promoter and partial coding region was cloned into the Bam HI site of BSP-1 (Example 3) resulting in a translational fusion to the SCPc 1 protease in plasmid SPF-1 (
The Kpn I fragment of plasmid XX-1 described above containing the Pis G363 promoter and partial coding region was cloned into the Kpn I site of BOP-1 (Example 4) resulting in a translational fusion to the OV7 protease inhibitor in plasmid SPIF-I (
The region coding for the signal peptide of the Brassica napus SLGWS1 gene was obtained by PCR amplification from plasmid SLG 26 using oligonucleotide primers SLG 26(7) and SLG 26(8) (SEQ ID NO's:10 and 11). These primers introduced respectively Sac I and Kpn I sites flanking the signal peptide. The signal peptide was digested with Sac I and Kpn I, and introduced into the Sac I and Kpn I sites of pGEMOV7 to yield plasmid SP-1 (
a) Fusion of the Brassica napus Pollen Polygalacturonase Sta 44G(2) Promoter and Signal Peptide to the Onchocerca OV7 Protease Inhibitor (POV-1).
Plasmid HP-9 containing the Sta 44G(2) promoter and signal peptide was digested with Sph I and ligated to EXK-12 adaptor:
EXK-1: CGA ATT CTC TAG AGG TAC CGC ATG (SEQ ID NO:13) and
EXK-2: CGG TAC CTC TAG AGA ATT CGC ATG (SEQ ID NO:14)
introducing additional Sph I, Eco RI, Xba I and Kpn I sites downstream of the signal peptide and yielding plasmid SS-1 (
b) Fusion of the Brassica napus Pollen Polygalacturonase Sta 44G(2) Promoter and Signal Peptide to the Sitophilus SCPc 1 Protease (POP-1).
Plasmid SS-2 (
Plasmid T28 containing the tapetal oleosin-like Sta 41 G(10) promoter fragment (Hong, H. P., Ross, J. H. E., Gerster, J. L., Rigas, S., Datla, R. S. S., Hatzopoulos, P., Scoles, G., Keller, W., Murphy, D., Robert, L. S. Plant Mol. Biol. 34:549-555 (1997b)) was digested with Bam HI and Hind III, and the fragment containing the promoter was cloned into Camter III a derivative of the binary vector Bin 19 (Bevan, M., Nucleic Acids Res. 12:8711-8721 (1984)) containing the nopaline synthase polyadenylation signal. The resulting plasmid was called T1. The tapetal oleosin-like cDNA clone Sta 41-2 was partially digested with Eco RI and cloned in the antisense orientation into the Eco RI site of T1 to generate the plant transformation vector SAS-1 (
Pollen grains may be conjugated to a protein of interest, for example, the enzyme horseradish peroxidase, the protease papain, or the protease inhibitor potato multicystatin, through conjugation to sugar residues on the exine. However, residues of the protein may also be modified prior to conjugation.
Pollen grains from opened flowers of Brassica napus cv. Westar are collected and “activated” by suspension in phosphate-buffered saline (PBS) solution containing 10 mM Ca+2, 10 mM Mn+2, 10 mM Mg+2, 0.5% Triton X-100, and 10 μg/ml Concanavalin A (Sigma C2010) for 1 h at room temperature. Pollen grains are washed repeatedly in PBS solution by centrifugation at 800×g in 1.5 ml centrifugation tubes for 5 min. Conjugation of purified enzymes (e.g. horseradish peroxidase, papain, or potato multicystatin, a cysteine protease inhibitor) is performed by incubating the selected protein at 50 μg/ml in PBS solution with the activated pollen grains for 1 h at room temperature. Pollen grains are then washed five times with PBS solution and centrifuged for 5 min.
Germination of pollen grains is evaluated by plating isolated pollen grains on 1 ml of filter sterile Brewbaker medium (10% (w/v) sucrose, 100 ppm H3BO3, 300 ppm Ca(NO3)2.4H2O, 200 ppm MgSO4.7H2O, 100 ppm KNO3) in sterile petri dishes 15×60 mm diameter at 25° C. for 24 h. The percentage of germinating pollen grains is determined under an inverted microscope, in the presence or absence of proteases or protease inhibitors.
Detection of the conjugated enzymes is measured by placing the pollen grains in enzyme substrate solutions. For horseradish peroxidase, pollen grains are incubated in 50 mM sodium acetate pH 5.0 containing 0.01% H2O2, 200 ug/ml amino ethyl carbazole, and the blue pollen grains are recorded under an inverted microscope. Papain activity is recorded on conjugated pollen grains by incubating pollen grains in microtitre plates containing the chromogenic substrate Pyr-Phe-Leu-pNA at 2 mM in Tris.Cl pH 6.5 at 37° C. for 30 min and reading the absorbance of the substrate solution at 410 nm in a microtitre plate reader. Papain inhibitory activity is measured on conjugated pollen grains by incubating pollen grains in a solution containing 10 μg/ml papain in Tris.Cl at pH 6.5 for 10 min at 37° C., and then adding the chromogenic papain substrate Pyr-Phe-Leu-pNA at 2 mM for 30 min at 37° C. The reactions are quantitated by measuring the absorbance at 410 nm.
Immune response of conjugated proteins to pollen grains is quantified by washing fresh pollen grains of canola with PBS solution for 5 min and centrifugation at 800×g. Pollen grains are suspended in PBS at 5% (vol/vol) and an equal volume of freshly prepared 0.005% tannic acid solution is added and mixed. This mixture is incubated at 37° C. for 15 min with gentle agitation, before removing the tannic acid solution by centrifugation. The pollen grains are incubated in either purified papain, horseradish peroxidase, or potato multicystatin in PBS solution for 15 min at 37° C. with gentle shaking. The pollen grains are then washed in PBS by centrifugation three times prior to immunization of mice. Pollen grains coated with either antigen are used to immunize Balb/c mice. Approximately 100 μg of coated pollen grains are suspended in Freund's incomplete adjuvant (100 μL) and used to inject into the foot pads of mice. A boost is administered after 10 days with the same concentration of antigen, and the same route of injection. Trial bleeds are examined for the titre of antibodies specific for the antigen coated on the surface of pollen grains, and compared to pre-immune serum.
To demonstrate that the protease inhibitor OV7 from Onchocera vovulus (Lustigman, S., Brotman, B., Huima, T., Prince, A. Mol. Biochem. Parasitol. 45: 65-76 (1991)) is capable of normal function as a fusion protein, it is cloned into the phagemid pCantab 5E (Pharmacia Inc.) and expressed on the surface of filamentous phage as a fusion to the gene 3 phage coat protein.
A SacI and Nco I digest of the coding region of the protease inhibitor clone OV7 (Lustigman et al 1991), is ligated to the pCantab 5E vector (Pharmacia Biotech) Sac I and Nco I sites. This phagemid is transformed into E. coli TG1 cells by electroporation. An overnight culture of 10 μL of these cells are grown in 5 ml of SOBAG medium containing 10 μl of carbenicillin (50 mg/mL) at 28° C. The next day, 0.5 ml of this culture is used to inoculate 25 ml of 2×YT medium, containing 50 μl of carbenicillin (50 mg/ml) and 1.56 ml of 2M glucose and grown for 1.25 h at 37° C., with shaking at 250 rpm. At this point 62.5 μl of helper phage strain M13K07 (9.0×10 pfu/ml) is added and the cells were incubated at 37° C. for 30 min with shaking at 150 rpm, and for another 30 min at 250 rpm. Recombinant phage particles are harvested by centrifugation and PEG precipitation. Phage are used in an ELISA assay to determine the specificity of binding to the cysteine protease papain, the serine protease trypsin, and to rabbit polyclonal antibodies directed against a potato cysteine protease inhibitor which also recognize the native OV7 protease inhibitor (anti-OV7 antibodies). Wells of a Nunc microtitre plate (maxisorb) are coated with either papain, anti-cysteine protease inhibitor antibodies, trypsin, or anti M13 antibodies in 100 μl Na 2CO3 buffer.
After incubating the phage indicated (OV7-those displaying the cysteine protease inhibitor on their surface, M13-wild type or control phage not displaying any foreign protein, and a no phage control) for 2 h, the wells are washed in PBS with 0.5% Tween-20. Phage particles that bound to the antigens are detected with a 1:5000 dilution of anti-M13 antibody/HRP conjugate (Pharmacia). Detection is with the HRP substrate ABTS solution (Pharmacia) for 30 min and absorbance was measured at 410 nm.
The results (Table 1) demonstrate that the phage displaying the OV7 protease inhibitor clearly binds specifically to the cysteine protease papain and to the anti-cysteine protease inhibitor antibodies at high levels. The O.D. values for the negative control phage binding (either M13 phage not expressing the protease inhibitor bound to papain, or the phage expressing the cysteine protease inhibitor bound to trypsin a serine protease) are very low.
Phage display demonstrates that the cysteine protease inhibitor OV7 is capable of functioning as a fusion protein when bound to the phage coat protein 3 and phage particles. This protease inhibitor is capable of binding specifically to papain (cysteine protease) and does not bind to trypsin (serine protease).
It has also been demonstrated that proteases can be expressed on the surface of bacteriophage and that they too can function as fusion proteins (Corey, D. R., Shiau, A. K., Yang, Q., Janawski, B. A., Craik, C. S. Gene 128: 129-134 (1993)).
Pollen from Nicotiana tabacum cv. Delgold was harvested by placing 5 dehiscent anthers in 1 ml of sterile Brewbaker and Kwack medium (Brewbaker, J. and Kwack, B. Amer. J. Bot. 50: 859-865 (1963)) containing various concentrations of cylcoheximide, or papain, or casein. The medium consisted of 10% w/v sucrose, 100 ppm H3BO3, 300 ppm Ca (NO3)2.4H2O, 200 ppm MgSO4.7H2O, and 100 ppm KNO3 with either 42 μM papain (twice crystallized, Sigma), with or without 30 μM recombinant onchocystatin, or 71 μM cycloheximide (Sigma), or 1 mg/ml casein. Anthers were vortexed in medium for 30 sec followed by centrifugation at 2,000 rpm for 2 min. Pollen was cultured in 50 μl drops in sterile 15×60 mm petri dishes at 25° C. The percentage of pollen grains germinated after 3 h of culture was measured and the average pollen tube length was recorded using an ocular micrometer on an inverted microscope. The protease papain at 42 μM inhibited the germination and stunted the growth of pollen tubes. The addition of 30 μM of the protease inhibitor onchocystatin (OV7) to the 42 μM papain treatment restored the germination and pollen tube growth. Cycloheximide at 71 μM also inhibited the germination and elongation of pollen tubes in vitro (Table 2,
These in vitro germination assays demonstrate that pollen germination can be affected by enzymes and antibiotics. Furthermore, the reduction in pollen germination caused by a protease can be restored by a protease inhibitor.
Plant transformation vectors were introduced separately into Agrobacterium tumefaciens strain EHA 105 following the protocol supplied with the Pharmacia Agrobacterium cells (product: #27-1535). To prepare the Agrobacterium competent cells, 5 ml of YEP medium (10 g yeast extract, 10 g peptone, 5 g sodium chloride per liter, pH 7.0) with 25 μg/ml chloramphenicol was inoculated with a loopful of a glycerol stock of Agrobacterium tumefaciens and cultured at 28° C. by shaking at 250 rpm approximately 15 h. Two ml of the culture was added to 50 ml of fresh YEP medium and grown at 28° C. to an O.D. of 0.5-1.0 at 600 nm. The culture was then chilled on ice for 10 min and centrifuged at 5,000 rpm. The cells were resuspended in 1 ml of cold 20 mM CaCl2 These competent cells were dispensed into pre-chilled 1.5 ml Eppendorf tubes in 100 μl aliquots and frozen at −80° C.
The Agrobacterium EHA 105 cells were transformed as follows. One μg of uncut plasmid DNA in water was added to 100 μl of Agrobacterium competent cells and incubated on ice for 30 min. The cells were then frozen in liquid nitrogen and thawed quickly at 37° C. for 5 min and 1 ml of YEP medium was added to the cell/DNA mixture and incubated at 28° C. for 2 h with gentle shaking (100 rpm). Cells were then centrifuged in a microfuge for 30 s, the supernatant was poured out and the pellet resuspended in the remaining supernatant (50-100 μl). The resuspended cells were spread on a YEP plate with 25 μg/ml chloramphenicol and 50 μg/ml kanamycin, and incubated at 28° C. for 2-3 days.
Plasmid DNA from individual Agrobacterium colonies was digested and analyzed by agarose gel electrophoresis to verify the integrity of the vector. Individual colonies that contained the desired recombinant plasmid were selected and grown overnight in 10 ml LB medium (10 g/L bacto-tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.0) with 25 μg/ml chloramphenicol and 50 μg/ml kanamycin. One ml of overnight culture was centrifuged at 13,000 rpm for 5 min and the cells resuspended in MMO medium (4.6 g/L MMO, GIBCO BRL) to an O.D. of 0.1.
Agrobacterium-mediated transformation of tobacco cv. Delgold was performed as follows. Pieces of fresh young tobacco leaves were sterilized 1-2 min in 70% ethanol, 5 min in Javex and then rinsed in sterile water for 2 min 3 times. Leaf discs were obtained with a 5 mm cork borer. Leaf discs were transferred to a dish containing the Agrobacterium cell suspension and placed at 25° C., 16 h light/8 h dark with lights to 70-100 μE for 2-3 days. The co-cultivated discs were transferred to TTK plates (4.56 g/L MMO, 1.0 mg/L benzyl adenine (BA), 0.1 mg/L α-naphthaleneacetic acid (NAA), 3% sucrose, pH 5.8; 300 μg/ml timentin and 25 μg/ml kanamycin added after autoclaving) and incubated at 25° C., 16 h light/8 h dark with lights to 70-100 μE for 2 weeks. Regenerated shoots were transferred to Magenta GA-7 vessels containing B5 rooting medium (23.2 g/L Gamborg's B5 medium (GIBCO BRL), 7.5 g/L phytagar, pH 5.7; 300 μg/ml timentin and 100 μg/ml kanamycin added after autoclaving). Once a good root system had developed, the plantlets were removed from the vessels, most of the agar was removed from the roots and the plantlets transferred to moist potting soil.
Kanamycin resistant Nicotiana tabacum plants were demonstrated to be transformed by PCR analysis following transformation with TOG-1 (SEQ ID NO: 3).
Forward Primer
GUSsense-1: GGA ATT CAC CGC GTC TTT GAT CGC-3′ (SEQ ID NO:16); and Reverse Primer
nos #2: GCG CGC GAT AAT TTA TCC-3′ (SEQ ID NO:17),
that anneal in the GUS gene and nopaline synthase terminator, respectively, were used to amplify a 513 base pair fragment. Transformation of tobacco plants with plasmids TOP-1 (SEQ ID NO:4) and TOPI-1 (SEQ ID NO:5) was also confirmed by PCR using primers
NptII-121: GGG CGC CCG GTT CTT TTT-3′ (DNA SEQ ID:18)
and NptII-B: CAG CAA TAT CAC GGG TAG CCA ACG C-3′ (SEQ ID NO:19).
Plant transformation vectors were introduced separately into Agrobacterium tumefaciens strain GV3101:pMP90 following the protocol supplied with the Pharmacia Agrobacterium cells (product: #27-1535). To prepare the Agrobacterium competent cells, 5 ml of YEP medium (10 g yeast extract, 10 g peptone, 5 g sodium chloride per liter, pH 7.0) with 150 μg/ml rifampicin and 100 μg/ml gentamycin was inoculated with a loopful of a glycerol stock of Agrobacterium tumefaciens and cultured at 28° C. by shaking at 250 rpm approximately 15 h. Two ml of the culture was added to 50 ml of fresh YEP medium and grown at 28° C. to an O.D. of 0.5-1.0 at 600 nm. The culture was then chilled on ice for 10 min and centrifuged at 5,000 rpm. The cells were resuspended in 1 ml of cold 20 mM CaCl2. These competent cells were dispensed into pre-chilled 1.5 ml Eppendorf tubes in 100 μl aliquots and frozen at −80° C.
The Agrobacterium cells were transformed as follows. One μg of uncut plasmid DNA in water was added to 100 μl of Agrobacterium competent cells and incubated on ice for 30 min. The cells were then frozen in liquid nitrogen and thawed quickly at 37° C. for 5 min, and 1 ml of YEP medium was added to the cell/DNA mixture and incubated at 28° C. for 2 h with gentle shaking (100 rpm). Cells were then centrifuged in a microfuge for 30 s, the supernatant was poured out and the pellet resuspended in the remaining supernatant (50-100 μl). The resuspended cells were spread on a YEP plate with 150 μg/ml rifampicin, 100 μg/ml gentamycin and 50 μg/ml kanamycin, and incubated at 28° C. for 2-3 days.
Plasmid DNA from individual Agrobacterium colonies was digested and analyzed by agarose gel electrophoresis to verify the integrity of the vector. Colonies that contained the desired recombinant plasmid were selected and grown overnight in 5 ml AB minimal medium with 50 μg/ml kanamycin and 50 μg/ml gentamycin. The overnight culture was centrifuged at 4500 rpm for 15 min and the cells resuspended in 1 ml of double distilled water or 10 mM MgSO4.
Agrobacterium-mediated transformation of B. napus cv. Westar was performed according to the method of Moloney M. M., Walker, J. M., Sharma, K. K. Plant Cell Rep. 8:238-242 (1989) with modifications. Seeds were sterilized by brief wetting in 95% ethanol then 70% commercial bleach (Javex) with a drop of detergent (Tween 20) for 15 min with occasional agitation; 0.025% mercuric chloride with a drop of Tween 20 for 10 min and finally rinsed well with sterile distilled water at least 3 times. Thirty to forty seeds were plated on ½ strength hormone-free MS medium (Sigma) with 1% sucrose in 15×60 mm petri dishes. They were then placed, with the lid removed, into sterilized Magenta GA-7 vessels and kept at 25° C., with 16 h light/8 h dark and a light intensity of 70-80 μE.
Cotyledons were excised from 4-day old seedlings by gently grasping both petioles just above the point where they join the hypocotyl. The cotyledons were soaked in BASE solution (4.3 g/L MS (GIBCO BRL), 10 ml 100×B5 Vitamins (0.1 g/L nicotinic acid, 1.0 g/L thiamine-HCl, 0.1 g/L pyridoxine-HCl, 10 g/L m-inositol), 2% sucrose, 1 mg/L 2,4-D, pH 5.8; 1% DMSO and 200 μM acetosyringone added after autoclaving) containing Agrobacterium cells with the recombinant plant transformation vector. Most of the BASE solution was removed and the cotyledons were incubated at 28° C. for 2 days in the dark. The dishes containing the cotyledons were then transferred to 4° C. for 3-4 days in the dark. Cotyledons were transferred to plates containing MS B5 selection medium (4.3 g/L MS, 10 ml 100×B5 Vitamins, 3% sucrose, 4 mg/L benzyl adenine (BA) ph 5.8; timentin (300 μg/ml) and kanamycin (20 μg/ml) were added after autoclaving) and left at 25° C., 16 h light/8 h dark with lighting to 70-100 μE. Shoots were transferred to Magenta GA-7 vessels containing MS B5 selection medium without BA. When shoots were sufficiently big they were transferred to Magenta GA-7 vessels containing rooting medium (4.3 g/L MS, 5.0 ml 100×B5 Vitamins, 3% sucrose, 0.1 mg/L α-naphthaleneacetic acid (NAA), pH 5.8; 300 μg/ml timentin and 20 μg/ml kanamycin were added after autoclaving). Once a good root system had developed, the plantlets were removed from the vessels, most of the agar was removed from the roots and the plantlets transferred to moist potting soil.
Plant transformation vectors were introduced separately into Agrobacterium tumefaciens strain GV3101:pMP90 following the protocol supplied with Pharmacia Agrobacterium cells (product: #27-1535). To prepare the Agrobacterium competent cells, 5 ml of YEP medium (10 g yeast extract, 10 g peptone, 5 g sodium chloride per liter, pH 7.0) with 150 μg/ml rifampicin and 100 μg/ml gentamycin was inoculated with a loopful of a glycerol stock of Agrobacterium tumefaciens and cultured at 28° C. by shaking at 250 rpm approximately 15 h. Two ml of the culture was added to 50 ml of fresh YEP medium and grown at 28° C. to an O.D. of 0.5-1.0 at 600 nm. The culture was then chilled on ice for 10 min and centrifuged at 5,000 rpm. The cells were resuspended in 1 ml of cold 20 mM CaCl2. These competent cells were dispensed into pre-chilled 1.5 ml Eppendorf tubes in 100 μl aliquots and frozen at −80° C.
The Agrobacterium cells were transformed as follows. One μg of uncut plasmid DNA in water was added to 100 μl of Agrobacterium competent cells and incubated on ice for 30 min. The cells were then frozen in liquid nitrogen and thawed quickly at 37° C. for 5 min and 1 ml of YEP medium was added to the cell/DNA mixture and incubated at 28° C. for 2 h with gentle shaking (100 rpm). Cells were then centrifuged in a microfuge for 30 s, the supernatant was poured out and the pellet resuspended in the remaining supernatant (50-100 μl). The resuspended cells were spread on a YEP plate with 150 μg/ml rifampicin, 100 μg/ml gentamycin and 50 μg/ml kanamycin, and incubated at 28° C. for 2-3 days.
Plasmid DNA from individual Agrobacterium colonies was digested and analyzed by agarose gel electrophoresis to verify the integrity of the vector. Individual colonies that contained the desired recombinant plasmid were selected and grown for 2-3 days in 5 ml of LB medium (10 g/L bacto-tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.0) with 50 μg/ml kanamycin with shaking at 28° C. A 50 μl aliquot of this culture was used to inoculate 5 ml of fresh LB medium containing 50 μg/ml kanamycin and incubated as above to an O.D. of 0.1 at 660 nm.
The B. carinata (Ethiopian mustard, B. carinata A. Braun, breeding line C90-1163, obtained from Dr. K. Falk, Saskatoon Research Centre, AAFC) seeds were sterilized in 2% PPM (Plant Preservative Mixture, Plant Cell Technology Inc.) for 4 h with gentle stirring and rinsed with 1L of sterilized water. Twenty seeds were plated on fresh seed germination medium (½ strength MS pH 5.6 (GIBCO BRL), 1% sucrose and 0,7% phytagar) in a 60×20 mm petri dishes fitted inside GA-7 Magenta vessels. They were incubated at 25° C. for 3-4 days under a 16 h light/8 h dark photoperiod.
Brassica carinata plants were transformed as described by Babic, V., Datla, R. S., Scoles, G. J., Keller, W. Plant Cell Reports, 17, 183-188 (1998) with modifications. Healthy green cotyledons were cut at the point where they join the hypocotyl. The petiole of each explant was dipped into the Agrobacterium suspension and then transferred to 100×25 mm petri dishes with Whatman No. 1 filter paper covering the regeneration medium (MS pH 5.8, 3% sucrose; 2 mg/L BA, 0.05 mg/L NAA and 0.7% phytagar). The explants were incubated at 25° C. for 2 days under a 16 h light/8 h dark photoperiod. The explants were then transferred to 100×25 mm petri dishes containing the selection medium (MS, 2% sucrose; 2 mg/L BA; 0.05 mg/L NAA; 5 mg/L AgNO3; 500 mg/L soluble PVP-10; 500 mg/L MES pH 5.8 and 0.7% phytagar supplemented with 25 μg/ml kanamycin and 300 μg/ml timentin) and incubated for 2 weeks as above.
Regenerated shoots were transferred to shoot elongation medium (½ MS pH 5.8; 2% sucrose; 0.05 mg/L BA; 0.03 mg/L (gibberellic acid) GA 3; 150 mg/L phloroglucinol; 0.9% phytagar supplemented with 25 μg/ml kanamycin and 300 μg/ml timentin) in 60×20 mm petri dishes fitted in GA-7 Magenta vessels and incubated for two weeks as above. Shoots were transferred to rooting media (½ MS; 1% sucrose; 0.05 mg/L NAA and 0.7% phytagar supplemented with 25 μg/ml kanamycin and 300 μg/ml timentin) and when healthy roots appeared the plantlets were transferred to soil.
Anther Development in B. carinata Flowers:
The tapetum is present within anthers of flower buds from 2-6 mm in length but is absent in 7 mm buds. Flowers open and release mature pollen once the buds have reached approximately 8 mm in length. Anther development in B. carinata parallels that of B. napus, in which flower bud length can be correlated to developmental events within the anther (Scott, R., Dagless, E., Hodge, R., Paul, W., Soufleri, I., Draper, J. Plant Mol. Biol. 17: 195-207 (1991)). However, B. carinata buds tend to be somewhat larger than those of B. napus at similar developmental stages. In B. napus, tapetal degeneration occurs at about 5 mm bud length and floral opening occurs by approximately 6-7 mm bud length.
Anthers were carefully dissected from buds at different developmental stages corresponding to the length of the bud (measured in mm) from the base to the tip of the closed sepals. Anther development was determined by staining resin-embedded sections of buds at different lengths with Toluidine Blue.
TOPI-1 Expression in B. carinata
B. carinata plants were transformed, as described in Example 15, with TOPI-1 (SEQ ID NO:5, Example 4).
Anthers were dissected from B. carinata flower buds, frozen in liquid nitrogen and ground to a fine powder. Proteins were extracted from the frozen powder by mixing and sonicating at 6 μl/mm length of flower bud with 1.2×SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) loading buffer (75 mM Tris-HCl pH 6.8; 2.4% SDS; 12% glycerol; 6% 2-mercaptoethanol; 0.01% bromophenol blue). The extract was centrifuged for 5 min at 13,000 rpm, the supernatant transferred to a fresh tube and heated to 50° C. before loading on the gel.
The SDS PAGE was performed according to the Laemmli, U. K. Nature 227: 680-685 (1970) using a 10% acrylamide gel. The gel was blotted electrophoretically (Hoeffer) to a PVDF membrane (Millipore) in 50 mM Tris-HCl, 380 mM glycine, 0.1% SDS and 20% methanol. The membrane was blocked with 5% skim milk powder, 3% bovine serum albumin (BSA) in TBS (50 mM Tris-HCI pH 7.5, 150 mM NaCl) for 1 h at 20° C. The blocked PVDF membrane was incubated with the anti-CPI (cysteine protease inhibitor) antibody diluted 1/12,500 in 0.5% blocking solution (Roche Molecular Biochemicals). The membrane was washed twice in TBST (TBS+0.1% Tween 20) for 10 min and twice more in 0.5% blocking solution. The membrane was incubated with the secondary antibody, anti-rabbit horseradish peroxidase conjugate, diluted to 1/2000 in 0.5% blocking solution for 30 min at 20° C. The membrane was then washed 4 times 15 min in TBST at 20° C. Proteins were detected using the Chemiluminescence Blotting Substrate (POD, Roche Molecular Biochemicals) and the membranes were exposed to X-ray film (Kodak, X-Omat).
Western blot analysis was performed on transgenic Brassica carinata plants containing TOPI-1 (SEQ ID NO:5). The full-length Brassica napus tapetal oleosin-like STA 41-9/Onchocerca volvulus OV7 protease inhibitor fusion (expected molecular weight 57 kDa) and the smaller processed version where the N-terminal end of the oleosin-like protein is cleaved (expected molecular weight 47 kDa) were both detected in anther protein extracts from 4 mm flower buds obtained from different transgenic B. carinata plants (
With reference to
Pollen used for pollen coat fractionation were isolated by gently squeezing anthers from open flowers just prior to anther dehiscence in an Eppendorf tube with a disposable blue pestle to release the pollen grains and suspend them in extraction buffer (100 mM HEPES pH7.5, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.4 M sucrose, 0.01% Triton X-100). The pollen grains were centrifuged at 1000×g for 3 min and the pollen pellet was washed in extraction buffer. Pollen coats were prepared by drying suspended pollen by centrifugation in a glass fibre-plugged filter basket at 20,000×g for 20 sec and then extracting the pollen coats by centrifuging cyclohexane through the dried pollen on the filter into a new tube. Cyclohexane was evaporated under a stream of nitrogen gas leaving the pollen coats as a residue. Proteins were solubilized from the pollen coat residue by mixing with 0.2 μl per anther 2×SDS-PAGE loading buffer described above. The extracts were centrifuged for 1 to 5 min at 13,000 rpm, the supernatant transferred to a fresh tube and heated to 50° C. or 95° C. for 4 min before loading on the gel.
TOPI-1 Expression in N. tabacum
N. tabacum plants were transformed, as described in Example 13, with TOPI-1 (SEQ ID NO:5, Example 4).
With reference to
These results demonstrate that the oleosin-like STA 41-9/Onchocerca volvulus OV7 protease inhibitor fusion protein while expressed specifically in the tapetum can re-locate to the pollen grain.
TOP-1 Expression in B. carinata
B. carinata plants were transformed, as described above with TOP-1 (SEQ ID NO:4, Example 3).
With reference to
Western blot analysis (
This example provides data that demonstrates that a protein of interest, for example but not limited to the GUS protein, translationally fused to a tapetal oleosin-like protein, for example but not limited to, BnOlnB;4 (also known as Sta 41-9) is targeted to the pollen coat.
Pollen isolation: Anthers were gently squeezed in an Eppendorf tube with a disposable blue pestle (Eppendorf) to release and suspend the pollen in extraction buffer (100 mM HEPES pH 7.5, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.4 M sucrose, 0.01% Triton X-100). The pollen grains were centrifuged at 1000×g for 3 min and the pollen pellet was washed in extraction buffer.
Pollen coat purification: Suspended pollen was dried by centrifugation in a glass fibre-plugged filter basket at 20,000×g for 20 sec and the pollen coats extracted by similarly centrifuging cyclohexane through the dried pollen on the filter into a new tube. Cyclohexane was evaporated under a stream of nitrogen gas leaving the pollen coats as a residue. Pollen viability was determined using a 1:1 mixture of two viability staining solutions containing malachite green, acid fuchsin and orange G (Alexander, M. P. Stain Technol. 44: 117-122 (1969); Alexander, M. P. Stain Technol. 55: 13-18 (1980)).
Northern analysis: Total RNA was isolated from anthers and pollen grains using Trizol (Gibco BRL) according to the manufacturer's instructions. Five to ten micrograms of total RNA were electrophoresed on 1-1.5% (w/v) agarose/formaldehyde gels and transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech). Membranes were hybridized in a modified Church aqueous phosphate buffer (Amersham Pharmacia Biotech) at 65° C. with random-primed 32P-labelled GUS (2 kb Bam HI/Sac I fragment of pBI121 (Clontech)) or BnOlnB;4 (0.2 and 1.1 kb Eco RI fragments of the BnOlnB;4 cDNA clone Sta 41-9; see Robert, L. S., Gerster, J. L., Allard, S., Cass, L., Simmonds, J., Plant J. 6: 927-933 (1994a)) probes. Blots were washed in 2×SSC, 0.1% SDS at 65° C., 3 times for 30 min, and exposed to X-ray film. Equal loading was assessed by A260 of the sample and by ethidium bromide staining of rRNA bands.
GUS enzymatic assays: GUS fluorogenic assays of tissue samples from stem, leaf, pistil, anther and pollen were performed (Jefferson, R. A. Plant Mol. Biol. Reporter, 5: 387-405 (1987)). Extracts were centrifuged to remove debris and the supernatant was assayed for GUS activity and protein concentration using a modified Bradford assay (BioRad). Fluorescence at timed intervals was measured with excitation at 320-390 nm and emission at 415-650 nm using a Hitachi F-2000 Fluorescent Spectrophotometer and the slope was determined. The specific activity of the GUS enzyme was calculated as pmol 4-methyl umbelliferone (MU) min-1 mg-1 total protein. GUS activity was estimated from the average of at least three replicate assays. GUS histochemical staining of pollen was performed using a method modified from that of Jefferson, R. A. Plant Mol. Biol. Reporter, 5: 387-405 (1987) in a solution of 50 mM NaPO4 pH 7.0, 10 mM EDTA, 0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6], 0.1% sarcosyl, 0.1% β-mercaptoethanol, 0.1% Triton X-100, 1 mg/ml X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid) at 37° C. overnight and imaged using a Zeiss Axioplan 2 microscope. For the analysis of segregating progeny of single-copy TOG-2 lines, 400 to 800 pollen grains were counted per GUS-positive plant
SDS-PAGE and western blotting: Protein samples were extracted directly in 2× loading buffer and separated by SDS-PAGE according to the methods of Laemmli, U. K. Nature, 227: 680-685 (1970). Protein concentrations were determined by a modified Lowry RCDC Protein assay (BioRad). Proteins were transferred to PVDF membranes (BioRad) and blocked in 3% bovine serum albumin, 5% skim milk powder in TBS (10 mM Tris pH 8.0, 150 mM NaCl). A polyclonal anti-GUS rabbit IgG (Molecular Probes) was used at a 1:4000-5000 dilution in 0.5% blocking solution (Roche Molecular Biochemicals). A polyclonal anti-BnOlnB;4 rabbit IgG was generated using a synthesized 20-mer peptide (LGIPESIKPS NIIPESIKPS; SEQ ID NO:24; Syngentia), corresponding to the first 20 residues of the C-terminal domain of BnOlnB;4, conjugated to Keyhole limpet haemocyanin (KLH). The anti-BnOlnB;4 IgG was used at a 1:3000 dilution. Proteins were detected using a 1:15000 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase (Sigma) using BM chemiluminescence blotting substrate (Roche Molecular Biochemicals)
Preparation of TOG-2
The tapetal oleosin-like BnOlnB;4 gene, isolated from B. napus anthers (also known as Sta 41-9; (Robert, L. S., Gerster, J. L., Allard, S., Cass, L., Simmonds, J., Plant J. 6: 927-933 (1994a)) was used to construct a translational fusion between the BnOlnB;4 gene, including the promoter and the entire coding region with the exception of the TGA stop codon, and the uidA gene encoding β-glucuronidase (TOG-2;
TOG-2 was constructed by ligating an adapter of two annealed oligos containing Kpn I sites:
Construction of the control plasmids BnOlnB;4-GUS (pOB4G) and the B. napus Sta 44-GUS transcriptional fusions and Brassica napus transformations were described elsewhere (Hong, H. P., Gerster, J. L., Datla, R. S. S., Albani, D., Scoles, G., Keller, W., Robert, L. S. Plant Cell Rep. 16:373-378 (1997a); Hong, H. P., Ross, J. H. E., Gerster, J. L., Rigas, S., Datla, R. S. S., Hatzopoulos, P., Scoles, G., Keller, W., Murphy, D., Robert, L. S. Plant Mol. Biol. 34:549-555 (1997b)).
The TOG-2 translational fusion construct was introduced into Ethiopian mustard (B. carinata, A. Braun, breeding line C90-1163, obtained from Dr. K. Falk, Saskatoon Research Centre, AAFC) grown in growth cabinets or the greenhouse typically at 15° C. day/10° C. night or 20° C. day/15° C. night under natural and/or artificial light), by Agrobacterium-mediated transformation (Agrobacterium strain EHA 105) performed essentially as described by Babic, V., Datla, R. S., Scoles, G. J., Keller, W. Plant Cell Reports, 17, 183-188 (1998); see Example 15.
TOG-2 containing transgenic B. carinata plants were screened histochemically for GUS enzymatic activity of the fusion protein in anthers isolated from 5 mm flower buds. Nine TOG-2 lines exhibited GUS activity at various levels. GUS expression in anthers isolated from 5-mm buds was analysed in all nine GUS-positive TOG plant lines (Nos. 5, 8, 11, 15, 19, 20, 22, 24, 28) by fluorogenic analysis (
To confirm that expression of the TOG-2 construct was restricted to the tapetum, GUS fluorogenic assays were performed on various plant tissues of two high expressing TOG-2 transformants Nos. 5 and 22. GUS activity (pmol 4-methyl umbelliferone (MU) min-1 mg-1 total protein) was not detected in any tissue tested (leaf, stem, emasculated floral bud and pistil) except anthers (
Transmission of the TOG-2 phenotype (the ability to target GUS enzyme activity to pollen grains) to the progeny was confirmed by GUS histochemical staining of anthers from self-progeny of TOG-2 plant line No. 22 (Table 3).
All self-progeny plants tested, along with the parent plant TOG-2 plant line No. 22, exhibited a positive reaction for GUS activity on the pollen grains (+), unlike the non-transformed control B. carinata plant which failed to exhibit any GUS activity (−). Consistent with the high frequency of transmission of the TOG-2 phenotype to progeny, TOG-2 plant line No. 22 was shown to contain approximately 5 copies of the TOG-2 transgene by Southern blot hybridization of restriction enzyme digested genomic DNA with a GUS coding region probe (results not shown).
Based on these data, TOG-2 lines were selected as described below for detailed analyses.
The TOG-2 Fusion is Expressed During Anther Development
Two transgenic TOG-2 lines showing high levels of GUS activity were selected for further analysis during anther development. Results are presented for line 22. A northern blot hybridized with a GUS probe revealed a transcript of approximately 3 kb, consistent with the predicted size of the TOG-2 construct (
The pattern of TOG-2 protein accumulation differed from that of the TOG-2 transcript. Western blot analysis with an anti-GUS primary antibody revealed a protein at approximately 125 kDa, consistent with the molecular weight predicted for a full-length fusion protein between BnOlnB;4 and GUS (
Western blot analysis with an anti-BnOlnB;4 primary antibody revealed the same proteins as described for the anti-GUS antibody (data not shown). In addition to the TOG-2 proteins, lower molecular weight native tapetal oleosin-like proteins were detected with the anti-BnOlnB;4 antibody in both transgenic TOG-2 and non-transformed B. carinata plants. Two proteins were detected of about 54 and 62 kDa in 4-7 mm flower buds and two proteins of about 38 and 46 kDa persisted during late anther development (data not shown). The multiple forms of the native tapetal oleosin-like protein detected in B. carinata with the anti-BnOlnB;4 antibody were reminiscent of the multiple forms which have been identified in B. napus with an anti-BnOlnB;3 antibody (Murphy, D. J. and Ross, J. H. E. Plant J. 13: 1-16 (1998)). The detection of multiple forms in B. napus was thought to occur due to the similarity of amino acid sequence between BnOlnB;3 and BnOlnB;4, as well as the alternative splicing of tapetal oleosin like genes (Murphy, D. J. and Ross, J. H. E. Plant J. 13: 1-16 (1998)).
GUS enzymatic activity was also examined throughout anther development (
The TOG-2 Fusion Protein is Targeted to the Pollen
Expression of the TOG-2 gene fusion was examined in isolated pollen grains at the late pollen maturation stage (8 mm flower buds;
To determine whether a translational fusion is necessary to target proteins from the tapetum to pollen grains, expression of a GUS transcriptional fusion to the tapetum-specific BnOlnB;4 promoter (BnOlnB;4-GUS; lacking the coding region of BnOlnB;4) was analysed in transgenic B. napus. GUS expression directed by the BnOlnB;4 promoter was previously shown to be specific to the tapetum in anthers isolated from 3-5 mm flower buds of B. napus (Hong, H. P., Ross, J. H. E., Gerster, J. L., Rigas, S., Datla, R. S. S., Hatzopoulos, P., Scoles, G., Keller, W., Murphy, D., Robert, L. S. Plant Mol. Biol. 34:549-555 (1997b)). The approximately 2 kb BnOlnB;4 promoter is the same as that used to direct expression of the TOG-2 translational fusion.
Like the TOG-2 RNA, the GUS mRNA (approx. 2 kb) transcriptionally fused to the BnOlnB;4 tapetal oleosin-like promoter (and lacking the coding region of BnOlnB;4) was detected in transgenic B. napus prior to tapetal degradation in 4-5 mm flower buds (anther) as previously reported (Hong, H. P., Ross, J. H. E., Gerster, J. L., Rigas, S., Datla, R. S. S., Hatzopoulos, P., Scoles, G., Keller, W., Murphy, D., Robert, L. S. Plant Mol. Biol. 34:549-555 (1997b)). However, BnOlnB;4-GUS RNA was undetectable in pollen isolated from 6-7 mm buds (pollen) after the tapetum had degenerated just prior to floral opening (BnOlnB;4-GUS;
Western blot analyses with an anti-GUS antibody detected the GUS protein, of BnOlnB;4-GUS, in anthers from 4-5 mm buds (anther;
Collectively, these data demonstrate that a coding region of interest, for example but not limited to GUS, that is produced in the tapetum associates with pollen when it is fused translationally to a tapetal oleosin-like protein, for example but not limited to, BnOlnB;4. Furthermore, the coding region of interest is active when associated with pollen.
A GUS transcriptional fusion to a B. napus polygalacturonase promoter (Sta 44-GUS) that directs high levels of expression within pollen late in development (Hong, H. P., Gerster, J. L., Datla, R. S. S., Albani, D., Scoles, G., Keller, W., Robert, L. S. Plant Cell Rep. 16: 373-378 (1997a)) was also examined. GUS mRNA was present in 4-5 mm buds of B. napus transformed with Sta 44-GUS and was also detected at high levels in pollen isolated from 6-7 mm buds (
The TOG-2 Fusion Protein is Localized to the Pollen Coat
To determine whether the TOG-2 translational fusion protein was targeted to the pollen coat, pollen coats were purified by cyclohexane solubilization and western blot analyses were performed using the anti-GUS antibody.
The mature 115 kDa TOG-2 protein was found in pollen coats purified from pollen of open flowers immediately prior to anther dehiscence (
Pollen coats purified from open flowers of transgenic B. napus containing the transcriptional fusion constructs BnOlnB;4-GUS (comprising only the promoter region of BnOlnB;4 also known as Sta 41-9), or Sta 44-GUS (construct expressed within pollen) did not exhibit detectable proteins cross-reacting with the anti-GUS antibody (
Collectively, these data indicate that a protein of interest, for example but not limited to GUS, relocates to the pollen coat upon tapetal degeneration only if fused translationally to a tapetal oleosin-like protein, for example but not limited to, BnOlnB;4 (also known as STA 41-9).
Immunogold Localization
Anthers were fixed in 0.8% glutaraldehyde, 4% paraformaldehyde, 0.1 M NaPO4 buffer pH 7.2. After washing in 0.1 M NaPO4 pH 7.2, tissues were dehydrated in an ethanol series and infiltrated with LR White acrylic resin (London Resin Co., London) over several days at 25° C. Following polymerisation of the resin at 50° C. overnight, ultra-thin sections (approx. 100 nm) were cut using a Reichert ultra-microtome and collected on nickel grids. Sections were incubated in 1% glycine in PBS (0.01 M NaPO4 pH 7.4, 0.85% NaCl) for 30 min to inactivate residual aldehydes and blocked in 1% ovalbumin in PBS for 10 min. Antibody incubations were carried out with anti-GUS (1:1000 dilution) or anti-BnOlnB;4 (1:100) primary antibodies in 0.01% ovalbumin in PBS followed by re-blocking in 1% ovalbumin in PBS and then with 10-15 nm-diameter gold-conjugated goat anti-rabbit secondary antibody (EY Laboratories, CA, USA) in 0.01% ovalbumin in PBS for 1 h at 25° C. Three 5 min washes in PBS were performed between each incubation or blocking step. After the procedure, residual salts were removed by washing in water. As a control, samples were incubated with pre-immune rabbit serum. Samples were observed in a Zeiss EM902A transmission electron microscope.
Immunogold localization was used to determine the subcellular localization of native tapetal oleosin-like proteins (
Native tapetal oleosin-like proteins were assessed in non-transformed B. carinata with the anti-BnOlnB;4 antibody, which cross-reacted with the tapetosome lipid bodies of anthers from 5 mm flower buds (
The anti-GUS antibody cross-reacted with the tapetosomes within the tapetum of anthers isolated from 5 mm flower buds, but not elsewhere within the anther obtained from transgenic TOG-2 plants (
Immunogold localizations were also performed with transgenic B. napus containing the tapetal-expressed BnOlnB;4-GUS (lacking the coding region of BnOlnB;4:
Collectively, these data confirm that a protein of interest, for example but not limited to the GUS protein, requires a translational fusion to a tapetal oleosin-like protein, for example but not limited to, BnOlnB;4 for localization to the tapetosomes and ultimately to the pollen coat.
In agreement with the immunolocalization of the TOG-2 protein to pollen, GUS histochemical staining was negligible with pollen from 5 mm bud anthers (
To indicate whether the GUS activity localized to pollen of TOG-2 transgenic plants was indeed the result of sporophytic expression rather than gametophytic expression, GUS histochemical analysis was performed on TOG-2 lines containing single copy insertions. In 9 GUS-positive progeny of each of two self-pollinated T0 plants, GUS histochemical staining of pollen from 8 mm flower buds typically revealed about 98±0.2% GUS positive pollen grains. In comparison, a mix of stained and unstained pollen grains could be observed by GUS histochemical staining of T1 progeny of a self-pollinated B. napus transgenic line containing a single copy of the pollen-expressed Sta 44-GUS construct (
These data demonstrate that the protein composition of the pollen coat can be modified by the targeting of a translational fusion protein from the tapetum. A tapetal oleosin-like protein, for example but not limited to BnOlnB;4 (also known as STA 41-9), provides an effective translational fusion partner to shuttle proteins from the tapetum to the pollen coat. Furthermore, a protein targeted to the pollen coat can remain active. Significantly, the activity of the GUS enzyme used in this demonstration persisted for weeks after dehiscence.
B. carinata and N. tabacum plants were transformed, as described above, with SPOV-1 (SEQ ID NO:12,
With reference to
B. carinata plants were transformed, as described in Example 15, with POV-1 (SEQ ID NO:15,
With reference to
These results demonstrate that the use of a signal sequence, fused to target a gene of interest, for example the protease inhibitor protein, is also targeted to the pollen grain.
Protease Inhibition Assays of Transgenic B. carinata Plants Containing the POV-1 Construct.
Mature pollen was isolated by gently squeezing anthers from open flowers just prior to anther dehiscence in a microfuge tube with a pestle to release the pollen grains and suspend them in extraction buffer (100 mM HEPES pH 7.5, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.4 M sucrose, 0.01% Triton X-100). Pollen grains were filtered through 0.44 μm mesh to remove debris, pelleted by centrifugation and frozen in liquid nitrogen. Pollen grains were ground to a fine powder, mixed with 1.4 μl per anther of extraction buffer (100 mM MES pH 6.5, 1% Triton X-100) and centrifuged at 4° C. for 5 min. Protein contents of extracts were determined using the BioRad Protein Assay.
For the protease inhibition assay, 5 μg of protein extract in 50 μl of extraction buffer described above was added to a microtiter plate followed by 20 ng of papain in 50 μl of 50 mM MES pH6.5 and incubated for 15 min at room temperature. Then 100 μl of substrate solution (1 mM Pyr-Phe-Leu-pNA (BACHEM) in activation buffer (30% DMSO, 1 mM EDTA,.1 mM DTT in 50 mM MES pH 6.5)) was added and the plate incubated in the dark at room temperature. The plate was read after 2 to 3 h of incubation on a plate reader at 410 nm. Results were calculated as the percentage of papain protease activity present in the mature pollen protein extract of the non-transformed control plant±standard deviation. Inhibition controls (purified protease inhibitors E-64 and OV7-GST), protease controls (purified papain without extract), negative controls (non-transformed control plant) and background controls (plant protein extract without substrate) were included. Results are shown in
The protease inhibition controls of purified protease inhibitors E-64 and OV7-GST exhibited 3% and 2%, respectively, of the papain protease activity exhibited by the non-transformed plant, whereas the protease control of purified papain without plant extract exhibited 145% of the protease activity of the non-transformed control plant. The background absorbance of proteins extracted from mature pollen, in the absence of added substrate, were included in the assay and subtracted from the readings.
Isolated pollen grains from three transgenic B. carinata plant lines containing the POV-1 construct (Nos. 20, 25 and 21) exhibited 53% to 61% of the papain activity in the non-transformed control plant (
Arabidospsis thaliana plants were transformed according to the method of Clough, S. J. and Bent, A. F. Plant J. 16: 735-743 (1998).
Arabidopsis thaliana seeds were germinated 5-6 weeks prior to transformation. The seeds were cold treated before sowing by placing 50 seeds per ml 0.1% agarose per pot in the refrigerator overnight. Three pots per construct were prepared by overfilling with moistened soil, covering with fibreglass screen and autoclaving. Pots were allowed to imbibe overnight, the tops of the pots were re-watered and 1 ml of the seeds/cold agarose was distributed onto the top of the soil. The pots were transferred to a growth cabinet set at 22° C. constant temperature, 16 h day/8 h night. Plants were thinned to a density of 10 to 12 plants per pot. The plants were grown until 5-10 plants per pot were bolting and the bolts were about 1-15 cm tall (about 4-5 weeks).
Four ml 2YT (or LB) liquid medium containing 50 mg/L kanamycin and 50 mg/L gentamycin was inoculated with Agrobacterium strain GV3 101 (containing the construct of interest) in a 50 ml Falcon tube and grown overnight at 28° C. with shaking at 250 rpm. Four ml overnight culture was used to inoculate 500 ml 2YT (or LB) containing 50 mg/L kanamycin, 30 mg/L gentamycin and 50 mg/L rifampicin, and bacteria grown overnight at 28° C. at 250 rpm until the culture reached an optical density at 600 nm of 1.2 to 1.5. (O.D. 600˜8×108 cells/ml). The culture was centrifuged at 5000×g for 7-10 min, the medium decanted and the cells resuspended in 300 ml, total, of ½×MS+5% sucrose+0.05% Silwet L77.
Prior to dipping, opened A. thaliana flower buds and seed pods were trimmed. The plants were soaked by inverting the bolts into the Agrobacterium solution for 30-45 seconds. The pots were incubated on their sides, covered and without watering, for 2 nights at room temperature and then transferred to the growth cabinet (22° C.) 16 h day/8 h night for an additional 24 h. The plants were partially uncovered for another 24 h and then the lid were removed completely. The plants were wrapped with acetate sheets to keep them separate while setting seed. After about 4 weeks, the plants started to form some seed pods and turn yellow. The plants were removed from the cabinet and the seeds harvested after one week at room temperature.
T0 seeds (100 μl per pot) in Eppendorf tubes were surface sterilized by vortexing for 2 min at maximum speed at room temperature in 70% ethanol. The ethanol was discarded, 50% bleach added and the seeds vortexed for 1 min at maximum speed followed by shaking for 9 min. The bleach was removed and the seeds rinsed well with autoclaved Milli Q water by vortexing for 10-30 sec. The water was removed and the washes repeated 4 more times. One ml 0.1% agarose was added to each tube and the seeds resuspended. The seeds were then placed at 4° C. overnight before plating onto selection medium (½×MS salts, 1% sucrose, 0.8% agarose, pH 6.0 containing 250 mg/L timentin and 30-50 mg/L kanamycin). The plates were incubated at 22-23° C. for 16 h day/8 h night. Within 7-10 days, the putative transformed plants appeared bright green whereas the non-transformed plants were a lighter green and then turned white by two weeks. Two weeks after being plated on selection medium, the seedlings were transferred onto recovery medium (½×MS salts, 1% sucrose, 0.8% agarose, pH 6.0) and incubated at 22° C. for 16 h day/8 h night for two weeks. The plants were then transferred to soil.
Staining of Pollen Grains from Transgenic B. carinata, A. thaliana and N. tabacum Plants Transformed with the POP-1 Construct
Transgenic B. carinata (20 plants), A. thaliana (54 plants) and N. tabacum (13 plants) containing the POP-1 construct (transformed as indicated in Examples 15, 20 and 13, respectively; see Example 8,
B. carinata POP-1 transgenic plant lines were also subjected to northern blot analysis of RNA isolated from anthers to verify the expression of POP-1. Some plants exhibited a partial loss of viability phenotype (see Table 4; B. carinata POP-1 plant line Nos. 2, 5, 12; A. thaliana POP-1 plant lines Nos. 7, 8, 10, 21, 24, 25, 27, 30, and N. tabacum plant lines Nos. 32, 33, 37, 40, 42, 55) indicating an alteration in the function of pollen grains as a result of expression of the POP-1 construct. However, most plant lines exhibited higher levels of steady state POP-1 mRNA than plant lines Nos. 2 and 12 (which exhibited the most severe loss of viability phenotype) indicating that expression of POP-1 also occurred in viable pollen (results not shown).
Brassica
Arabidopsis
Nicotiana
carinata
thaliana
tabacum
Interaction Between POV-1 and POP-1 when Co-expressed in F1 B. carinata Plants.
Pollen of POV-1 primary transgenic B. carinata line Nos. 20 or 25, which exhibited enhanced protease inhibitor activity (
POP-1 and POV-1 expression in anthers was analyzed by northern blot analysis and western blot analysis as described above. Several F1 plants contained and expressed both POP-1 and POV-1 constructs (Table 5). When dehisced anthers from these F1 plants were subjected to Alexander staining as described above, they exhibited a pollen viability that was higher, than that compared to the parent POP-1 plants (see Table 5: POP-1 No. 2×POV-1 No. 20 progeny Nos. 2 and 3; and POP-1 No. 12×POV-1 No. 20 progeny Nos. 1 and 2).
There were other instances when POV-1 was expressed at a level insufficient to counteract the protease activity from the POP-1 expression found in F1 plants containing both POP-1 and POV-1 (see Table 5: POP-1 No. 12 ×POV-1 No. 20 progeny Nos. 1 and 4). In the absence of POV-1, POP-1 causes loss of viability, whereas in the absence of POP-1, POV-1 fails to affect viability as determined by Alexander staining.
Translational Fusion Eliminating the C-Terminal Domain of STA 41-9 (TOG-3)
A translation fusion between E. coli β-glucuronidase and the tapetal oleosin-like gene Sta 41-9 was made which eliminated the C-terminal domain of STA 41-9 and positioned the β-glucuronidase coding region at the C-terminus. Plasmid pGEMTOG-2 (
Sta 41 ATG: CTA GGA TCC AGA CCA CAC AAC TCC TTC (SEQ ID NO:26) and
Sta 41-13R: GAGA GGATTC CAA CAG AGA TAG GGA TGG C (SEQ ID NO:27)
to amplify the region corresponding to nucleotides 1 to 400 of Sta 41-9 (GenBank Acc. No. L33283). Primer Sta 41-13R includes a Bam HI restriction site to allow in frame translational fusion of GUS at the end of the STA 41-9 hydrophobic domain (Ross, J. H. E. and Murphy, D. J. Plant J. 9: 625-637 (1996)). The resulting PCR product was then digested with Hind III and Bam HI, and ligated to the Hind III and Bam HI fragment of BB-2 (
Translational Fusion with Protein of Interest at the N-Terminal Domain of STA 41-9 (TOG-4)
A translation fusion between E. coli β-glucuronidase (GUS) and the full-length sequence of the B. napus tapetal oleosin-like gene Sta 41-9 was made in which the β-glucuronidase protein was positioned at the N-terminus of STA 41-9. The GUS gene was PCR amplified from pGEMTOG-2 (
GUS-6F encompasses the Bam HI and Sma I sites upstream of the GUS gene. GUS-7R removes the stop codon TGA from the 3′ end of the GUS gene and adds Bgl II and Sac I restriction sites. The PCR product was digested with Bam HI and Sac I, and ligated to Bam HI and Sac I digested plasmid pGEMNosTer (
Sta 41-14F contains the ATG methionine translation initiation codon of Sta 41-9, includes an upstream Bgl II site and maintains the reading frame with GUS. Sta 41-15R encompasses the Nde I site and TGA stop codon of Sta 41-9 and adds a Sac I site. The PCR product was digested with Bgl II and Sac I, and ligated into Bgl II and Sac I digested plasmid SS-5 creating an N-terminal translational fusion of GUS to STA 41-9 as shown in plasmid SS-6 (
It is well known that self-incompatibility (SI) in the Brassicaceae is sporophytically determined. Recently the male determinant of self-incompatibility has been identified in B. campestris (B. rapa) and B. oleracea as the S-locus cysteine rich protein SCR/SP11 (Schopfer, C. R., Nasrallah, M. E., Nasrallah, J. B. Science 296:1697-1700 (1999); Takayama, S., Shiba, H., Iwano, M., Asano, K., Hara, M., Che, F. -S., Watanabe, M., Hinata, K., Isogai, A. Proc. Natl Acad. Sci. USA 97: 1920-1925 (2000); Kachroo, A., Schopfer, C. R., Nasrallah, M. E., Nasrallah, J. B. Science 293: 1824-1826 (2001); Shiba, H., Takayama, S., Iwano, M., Shimosato, H., Funato, M., Nakagawa, T., Che, F. -S., Suzuki, G., Watanabe, M., Hinata, K., Isogai, A. Plant Physiol. 125: 2095-2103 (2001); Shiba, H., Iwano, M., Entani, T., Ishimoto, K., Shimosato, H., Che, F. -S., Satta, Y., Ito, A., Takada, Y., Watanabe, M., Isogai, A., Takayama, S. Plant Cell 14: 491-504 (2002)).
Similar genes have also been found in related species, for example, B. napus and A. thaliana (Acc. No. AJ250857, AJ250856; Nasrallah, M. E., Liu, P., Nasrallah, J. B. Science 297: 247-249 (2002)). It has also been demonstrated that expression of an SCR gene transcriptionally fused to an SCR promoter was sufficient to confer a new SI phenotype specifically on pollen grains (the stigma phenotype was unaltered) when transformed into B. oleracea or B. campestris having a different S haplotype than the introduced gene ((Schopfer, C. R., Nasrallah, M. E., Nasrallah, J. B. Science 296:1697-1700 (1999); Shiba, H., Takayama, S., Iwano, M., Shimosato, H., Funato, M., Nakagawa, T., Che, F. -S., Suzuki, G., Watanabe, M., Hinata, K., Isogai, A. Plant Physiol. 125: 2095-2103 (2001)).
A B. oleracea SCR13 (Ace. No. AF195626) gene was isolated by RT-PCR performed on total RNA from 2 to 4 mm length flower buds from an S13 haplotype plant line using primer:
GAGA T18-2: GAG AGA GAG AGA CTC GAG TTT TTT TTT TTT TTT TTT A/C/G (SEQ ID NO:34)
and Superscript II (Invitrogen) for the first strand cDNA synthesis, and gene specific primers:
SCR13-3F: AAC AAG AAT TTG CTG CGA GTA AAA GAG AAT (SEQ ID NO:35) and
SCR13-4R: ATT TTG ACT AAG ACG AAT TTT GGA ATG ATT (SEQ ID NO:36)
and Taq DNA polymerase (Invitrogen) for the PCR according to the manufacturer's protocols.
The amplified region corresponds to a 367 bp fragment beginning in the 5′ untranslated region, contains the entire SCR13 coding region and ends in the 3′ untranslated region of SCR13. The resulting PCR product was ligated into pGEM T-Easy (Promega) and transformed into E. coli strain DH5FT (Invitrogen) according to the manufacturer's protocols to create plasmid SCR13-1. The portion of SCR13 corresponding to the mature SCR peptide was amplified from SCR13-1 using primers:
Primer SCR13-7F introduces a unique Ase I site and an additional nucleotide immediately upstream of the first nucleotide encoding the putative mature peptide of SCR13 to facilitate in frame translational fusion with the Sta 41 G(10) promoter and coding region at its Nde I restriction site. Primer SCR13-8R introduces unique Sac I and Bst XI sites immediately 3′ of the stop codon of the SCR13 coding sequence. The PCR product was digested with Ase I and Bst XI, and ligated to SS-4 (
A genomic sequence including the A. thaliana Atgrp 19 oleosin-like gene (Ace. No. AF362478) was PCR amplified using oligonucleotides SEQ ID NO's:40-42, all derived from the A. thaliana BAC clone T2I1 sequence (Ace. No. AL163912).
Atgrp19-F1: AAT GGT ACC GAA TAA GTG AGT CTT GCA CAC TGG
(SEQ ID NO:40) is used to amplify the plus strand of the Arabidopsis thaliana genomic sequence containing the Atgrp 19 oleosin-like gene (Ace. No. AL163912). This oligonucleotide introduces a unique Kpn I site at the 5′ end of the genomic fragment.
Atgrp 19-R1: TAT GGA TCC GAC GCC GGA ACC TGC TGG GTT AG (SEQ ID NO:41) is used to amplify the minus strand of the Arabidopsis thaliana genomic sequence containing the Atgrp 19 oleosin-like gene (Ace. No. AL163912). This oligonucleotide introduces a unique Bam HI site at the 3′ end of the genomic fragment.
Atgrp19-R2: TAT AGA TCT ACC ATG ACG CCG GAA CCT GCT GGG TTA G (SEQ ID NO:42) is used to amplify the minus strand of the Arabidopsis thaliana genomic sequence containing the Atgrp 19 oleosin-like gene (Ace. No. AL163912). This oligonucleotide introduces a unique Bgl II site at the 3′ end of the genomic fragment.
The amplified region consists of 978 bp of sequence upstream of the initiation ATG codon and the complete coding sequence (including the intron) up to, but excluding the stop codon. Oligonucleotide SEQ ID NO:40 introduces a unique Kpn I site at the 5′ end of the genomic fragment, whereas oligonucleotide SEQ ID NO:41 introduces a unique Bam HI site at the 3′ end of the Atgrp 19 coding region thus allowing an in-frame fusion with the GUS coding region by replacing the Kpn I/Bam HI fragment containing the Brassica oleosin-like gene in TOG-2 (
The genomic sequence including the A. thaliana exl 4 extracellular lipase gene (Acc. No. AY028612) was PCR amplified from genomic DNA using oligonucleotides:
Atexl 4-F1: ATA GGT ACC TTA ACA TTC TTG TAG TTA GGG C (SEQ ID NO:46)
and
Atexl 4-R1: TAT CCA TGG CAA GGC CAT TCT TGA TAT CCT GG (SEQ ID NO:47)
derived from the A. thaliana BAC clone T4012 (Acc. No. AC007396). Atexl 4-F1 is used to amplify the plus strand of the Arabidopsis thaliana genomic sequence containing the exl 4 extracellular lipase gene (Acc. No. AC007396). This oligonucleotide introduces a unique Kpn I site at the 5′ end of the genomic fragment. Atexl 4-R1 is used to amplify the minus strand of the Arabidopsis thaliana genomic sequence containing the Atgrp 19 oleosin-like gene (Acc. No. AC007396). This oligonucleotide introduces a unique Nco I site at the 3′ end of the genomic fragment.
The amplified region consists of 438 bp of sequence upstream of the initiation ATG codon and the complete coding sequence except the stop codon. Oligonucleotide SEQ ID NO:46 introduces a unique Kpn I site at the 5′ end of the genomic fragment, whereas oligonucleotide SEQ ID NO:47 introduces a unique Nco I site at the 3′ end of the exl 4 coding region to allow an in-frame fusion with the GUSPlus™ gene in the binary vector pCambia 1305.1 (see cambia.org) and generate binary vector EXLG-1+ (
The genomic sequence including the Brassica napus Sta 44 pollen polygalacturonase gene (Robert, L. S., Allard, S., Gerster, J. L., Cass, L., Simmonds, J. Plant Mol. Biol. 23: 1273-1278 (1993)) was PCR amplified from genomic clone Sta 44G(2) using oligonucleotides:
Sta44G2(2): ATA GGT ACC GAC AGT ATA CAT AAT TTA GAG AGA G (SEQ ID NO:49) and
Sta44-4(2): TAT GGA TCC CTC TTT GCC AGG AGC CTT GAC CAC (SEQ ID NO:50), or
Sta44-4(3): TAT CCA TGG TCT CTT TGC CAG GAG CCT TGA CCA C(SEQ ID NO:51).
Oligonucleotide SEQ ID NO:49 is derived from the Sta 44 promoter sequence (U.S. Pat. No. 5,689,053), whereas oligonucleotides SEQ ID NO:50 and SEQ ID NO:51 are derived from the Sta 44-4 cDNA clone (Acc. No. L19879). The amplified region consists of 647 bp of sequence upstream of the initiation ATG codon and the complete coding sequence except the stop codon. Oligonucleotide SEQ ID NO:49 introduces a unique Kpn I site at the 5′ end of the genomic fragment, whereas oligonucleotide SEQ ID NO:50 introduces a unique Bam HI site at the 3′ end of the Sta 44 coding region to allow an in-frame fusion with the GUS coding region by replacing the Kpn I/Bam HI fragment containing the Brassica oleosin-like gene in TOG-2 (
All citations are herein incorporated by reference.
The present invention has been described with regard to preferred embodiments. However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as described herein.
This application is a Continuation in part of U.S. application Ser. No. 09/272,204, filed Mar. 19, 1999, abandoned, which claims priority to U.S. application Ser. No. 60/078,728, filed Mar. 20, 1998, both of which are incorporated herein by reference.
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| Number | Date | Country | |
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| Parent | 09272204 | Mar 1999 | US |
| Child | 10322656 | US |
